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Crosstalk between somatostatin receptor subtypes and cannabinoid receptor 1 in excitotoxicity Zou, Shenglong 2017

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CROSSTALK BETWEEN SOMATOSTATIN RECEPTOR SUBTYPES AND CANNABINOID RECEPTOR 1 IN EXCITOTOXICITY  by  Shenglong Zou  B.Sc., Wuhan University, China 2010  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pharmaceutical Sciences)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  December 2017  © Shenglong Zou, 2017 ii  Abstract Both somatostatin (SST) and cannabinoid receptor 1 (CB1R) are critical components modulating neurotransmission in the central nervous system (CNS), with sharing functional properties in various neurological activities. To explore their potential crosstalk, first we investigated the expression of SST and CB1R in rat brain hypothalamus and hippocampus. The distributional patterns and colocalization of CB1R and SST were selective and region specific. Neuronal population expressing either SST or CB1R alone, as well as colocalization were seen in various intensities in different regions, suggesting a possible interaction. SST exerts its biological effects via binding to somatostatin receptors (SSTRs). Both SSTRs and CB1R belong to G-protein coupled receptor family that are known to function as oligomers. Accordingly, we investigated the colocalization of CB1R and SSTR5 in rat brain and HEK-293 cells cotransfected with hCB1R and hSSTR5. Our results showed that CB1R and SSTR5 colocalized in rat brain regions. In cotransfected HEK-293 cells, SSTR5 and CB1R existed in a constitutive heteromeric complex under basal condition. Agonist treatments lead to the disruption of CB1R/SSTR5 heterodimer, along with preferential formation of SSTR5 homodimer and dissociation of CB1R homodimer. cAMP and ERK1/2 signaling was modulated in a SSTR5-dominant manner in co-transfected cells.  To explore pathological significance of such interaction, we further expanded our study in HD transgenic mice and Huntingtin (Htt) knock-in striatal neuronal cells. We observed significant loss of neuronal subpopulation displaying colocalization between SSTRs and CB1R with selective sparing of SSTR positive neurons in cortex and hippocampus but not in striatum of 11-week-old R6/2 mice, in comparison to wild-type and 7-week-old R6/2 mice. Using mHtt knock-in (STHdhQ111/111) and wild-type (STHdhQ7/7) striatal cells, we discovered that STHdhQ111/111 cells iii  were more vulnerable to QUIN and displayed suppressed cell survival signalings. Receptor-specific agonist protected cells against QUIN-induced toxicity and selectively activated ERK1/2 in both STHdh cells. Co-activation of SSTR subtypes and CB1R resulted in diminished protective effects, delayed ERK1/2 phosphorylation and altered receptor complex composition, with more pronounced effects in STHdhQ111/111 cells than STHdhQ7/7 cells.  Taken together, our results provide evidence for functional interaction between SSTR and CB1R, emphasizing its therapeutic potentials in excitotoxicity and associated neurological disorders.  iv  Lay Summary Somatostatin receptors (SSTRs) are the receptors responsible for inhibiting growth hormone release. Cannabinoid receptor 1 (CB1R) is the receptor responsible for the neurological effects of marijuana. The goal of my study is to find out whether an interaction exists between these two receptor families. We anticipate that such an interaction could serve as novel target for drug development to increase the beneficial effects and decrease side effects of cannabinoids. Results presented here demonstrate that SSTR5 and CB1R interact with each other, form a complex, and function differently than single receptors. Both SSTR subtypes and CB1R rescue neuronal cells from toxicity-induced cell death, whereas activation of both receptors simultaneously masks such protective effect. These findings reveal a possible regulatory crosstalk between SSTRs and CB1R, shedding light on future drug development on neurological disorders and energy balance. v  Preface All of the work presented in this dissertation was conducted in the Faculty of Pharmaceutical Sciences at the University of British Columbia. Part of Chapter 1 (mainly Section 1.3) is now under consideration for publication. Chapter 2, 3 and 4 has been published in peer-reviewed journals. Chapter 5 is currently unpublished and will be submitted for publication soon. Slight modifications were made to the contents of the abovementioned chapters from the originally versions to maintain the consistency of the dissertation. The contribution of individual author to each chapter are listed below. Protocols regarding animal care were followed in compliance with principles of the Canadian Council on Animal Care and were approved by the University Animal Care Committee (Protocol #A06-0419). Chapter 1. Zou S, Kumar U. Cannabinoid receptors and endocannabinoid system: signaling and function in the central nervous system. I wrote the manuscript and Dr. Kumar edited it. Chapter 2. Zou S, Kumar U (2015) Colocalization of cannabinoid receptor 1 with somatostatin and neuronal nitric oxide synthase in rat brain hippocampus. Brain Res 1622:114-126. I performed the experiments, analyzed the data, and wrote the manuscript. Dr. Kumar designed the study and edited the manuscript. Chapter 3. Zou S, Somvanshi RK, Paik S, Kumar U (2015) Colocalization of cannabinoid receptor 1 with somatostatin and neuronal nitric oxide synthase in rat brain hypothalamus. J Mol Neurosci 55:480-491. I performed the experiments, analyzed the data, and wrote the manuscript. Dr. Kumar designed the study and edited the manuscript. Dr. Somvanshi and Mr. Paik edited the manuscript. Chapter 4. Zou S, Somvanshi RK, Kumar U (2017) Somatostatin Receptor 5 is a Prominent Regulator of Signaling Pathways in Cells with Coexpression of Cannabinoid Receptors 1. vi  Neuroscience 340:218-231. Dr. Kumar and me designed the study and wrote the manuscript. I performed the experiments and analyzed the data. Dr. Somvanshi conducted confocal microscopy. Chapter 5. Zou S and Kumar U. Role of Somatostatin and cannabinoid receptor type 1 in protection of striatal neurons and regulation of signaling pathways. I designed the study, performed the experiment and wrote the manuscript. Dr. Kumar edited the manuscript. vii  Table of Contents  Abstract .......................................................................................................................................... ii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ........................................................................................................................ vii List of Figures .............................................................................................................................. xii List of Abbreviations and Symbols .......................................................................................... xvi Acknowledgements ................................................................................................................... xxii Dedication ................................................................................................................................. xxiii Chapter 1: Introduction ................................................................................................................1 1.1 G protein-coupled receptors (GPCRs) ........................................................................ 1 1.2 Somatostatin (SST) ..................................................................................................... 3 1.2.1 Somatostatin receptors (SSTRs) ......................................................................... 7 1.2.2 SSTR localization ............................................................................................... 8 1.2.3 SSTR signaling ................................................................................................... 9 1.3 Cannabinoids............................................................................................................. 12 1.3.1 CBRs ................................................................................................................. 13 1.3.2 Endocannabinoid system .................................................................................. 14 1.3.3 Endocannabinoid-mediated signaling ............................................................... 16 1.3.4 CBR localization ............................................................................................... 18 1.3.5 CBR signaling ................................................................................................... 21 1.3.6 Physiological and pathological roles of CB1R ................................................. 25 viii  1.4 GPCR oligomerization .............................................................................................. 28 1.4.1 SSTR oligomerization ....................................................................................... 32 1.4.2 CB1R oligomerization ...................................................................................... 34 1.5 Excitotoxicity ............................................................................................................ 36 1.5.1 Huntington’s disease ......................................................................................... 38 1.5.2 SST in excitotoxicity and HD ........................................................................... 40 1.5.3 CB1R in excitotoxicity and HD ........................................................................ 41 1.6 STHdh cell lines ........................................................................................................ 43 1.7 Summary of background ........................................................................................... 45 1.8 Hypothesis................................................................................................................. 46 1.9 Specific Aims ............................................................................................................ 46 Chapter 2: Colocalization of CB1R with SST and nNOS in rat brain hypothalamus ..........47 2.1 Background ............................................................................................................... 47 2.2 Materials and Methods .............................................................................................. 49 2.2.1 Animals ............................................................................................................. 49 2.2.2 Materials ........................................................................................................... 49 2.2.3 Immunocytochemistry ...................................................................................... 50 2.2.4 Western Blot Analysis ...................................................................................... 50 2.2.5 Quantitative Analysis ........................................................................................ 51 2.3 Results ....................................................................................................................... 52 2.3.1 Specificity of immunoreactivity and colocalization ......................................... 52 2.3.2 CB1R and nNOS are coexpressed in hypothalamus ......................................... 57 2.3.3 Colocalization of SST and nNOS in hypothalamus, cortex and striatum ......... 59 ix  2.4 Discussion ................................................................................................................. 63 Chapter 3: Colocalization of CB1R with SST and nNOS in rat brain hippocampus ...........67 3.1 Background ............................................................................................................... 67 3.2 Materials and Methods .............................................................................................. 69 3.2.1 Animals ............................................................................................................. 69 3.2.2 Materials ........................................................................................................... 69 3.2.3 Immunocytochemistry ...................................................................................... 70 3.2.4 Quantitative analysis ......................................................................................... 70 3.3 Results ....................................................................................................................... 71 3.3.1 SST in hippocampus is expressed in selective CB1R positive neurons ........... 72 3.3.2 Colocalization of CB1R and nNOS in rat brain hippocampus ......................... 76 3.3.3 In hippocampus not all SST positive neurons express nNOS........................... 79 3.3.4 Quantitative analysis ......................................................................................... 83 3.4 Discussion ................................................................................................................. 84 Chapter 4: SSTR5 is a prominent regulator of signaling pathways in cells with coexpression of CB1R .........................................................................................................................................90 4.1 Background ............................................................................................................... 90 4.2 Materials and Methods .............................................................................................. 93 4.2.1 Animals ............................................................................................................. 93 4.2.2 Materials ........................................................................................................... 93 4.2.3 Cell culture and transfection ............................................................................. 94 4.2.4 Immunofluorescence immunohistochemistry ................................................... 94 4.2.5 Co-IP ................................................................................................................. 94 x  4.2.6 Microscopic photobleaching FRET (pbFRET) analysis ................................... 95 4.2.7 Receptor internalization .................................................................................... 96 4.2.8 Coupling to AC ................................................................................................. 96 4.2.9 Western blot analysis ........................................................................................ 97 4.2.10 Statistical analysis ............................................................................................. 97 4.3 Results ....................................................................................................................... 97 4.3.1 SSTR5 and CB1R are coexpressed in rat brain regions ................................... 97 4.3.2 CB1R is expressed in SSTR5 immunoprecipitates from rat brain tissue ....... 100 4.3.3 SSTR5 and CB1Rs exist as constitutive heterodimers in HEK-293 cells ...... 102 4.3.4 Agonist-dependent suppression of SSTR5 and CB1Rs heterodimerization ... 103 4.3.5 Agonist-dependent internalization of CB1R and SSTR5 in cotransfected HEK-293 cells…… ...................................................................................................................... 104 4.3.6 Inhibition of cAMP in cells cotransfected with SSTR5/CB1R is prominently regulated by SSTR5 ............................................................................................................ 107 4.3.7 PKA is regulated in cAMP-dependent manner ............................................... 110 4.3.8 Receptor-mediated ERK1/2 signaling is altered in cotransfected cells .......... 110 4.3.9 PI3K Phosphorylation is regulated in time and receptor dependent manner .. 112 4.4 Discussion ............................................................................................................... 113 Chapter 5: Role of SSTRs and CB1R in protection of striatal neurons and regulation of signaling pathways .....................................................................................................................120 5.1 Background ............................................................................................................. 120 5.2 Materials and Methods ............................................................................................ 124 5.2.1 Animals ........................................................................................................... 124 xi  5.2.2 Materials ......................................................................................................... 124 5.2.3 Cell cultures .................................................................................................... 125 5.2.4 Double-labeled fluorescence Immunohistochemistry ..................................... 125 5.2.5 MTT assay ...................................................................................................... 126 5.2.6 Co-IP ............................................................................................................... 126 5.2.7 Western Blot ................................................................................................... 127 5.2.8 Statistical analysis ........................................................................................... 128 5.3 Results ..................................................................................................................... 128 5.3.1 Colocalization of SSTR2, SSTR5 and CB1R in R6/2 mice ........................... 128 5.3.2 Expression of SSTR2, SSTR5 and CB1R in STHdh cells changes upon receptor activation….. ....................................................................................................................... 131 5.3.3 Receptor agonist-induced changes in signaling pathways in STHdh cells ..... 137 5.3.4 Role of SSTRs and CB1R in protection against QUIN induced toxicity in STHdh cells………. ........................................................................................................................ 145 5.3.5 Effects of combined treatment on QUIN-induced cell death, receptor interaction and ERK1/2 signaling ......................................................................................................... 146 5.4 Discussion ............................................................................................................... 151 Chapter 6: Concluding Remarks ..............................................................................................157 6.1 Overall Discussion .................................................................................................. 157 6.2 Overall Conclusion ................................................................................................. 166 6.3 Future Study ............................................................................................................ 166 Bibliography ...............................................................................................................................168  xii  List of Figures Figure 1.1 Action sites of SST in human body. [Reprinted with permission from (Kumar and Grant, 2010)] ................................................................................................................................... 4 Figure 1.2 Major distribution sites and functions of CB1R function in human body. ................. 20 Figure 1.3 GPCR dimerization and property changes. ................................................................. 31 Figure 2.1 Photomicrographs illustrating the specificity of CB1R antibody and immunofluorescence in HEK-293 cells, hypothalamus section and tissue lysate. ....................... 53 Figure 2.2 Representative confocal photomicrographs illustrating the colocalization of SST and CB1R in hypothalamus. ................................................................................................................ 56 Figure 2.3 Quantitative analysis of neurons coexpressing SST and CB1R in three hypothalamic nuclei. ............................................................................................................................................ 56 Figure 2.4 Confocal photomicrographs depicting colocalization of CB1R with nNOS positive neurons in rat brain hypothalamus. ............................................................................................... 59 Figure 2.5 Representative histograms showing quantitative analysis of neurons coexpressing CB1R and nNOS in PeVN, PVN and VMH. ................................................................................ 59 Figure 2.6 Representative confocal photomicrographs showing colocalization between SST and nNOS in rat brain hypothalamus, cortex and striatum. ................................................................. 62 Figure 2.7 Quantitative analysis of neurons coexpressing SST and nNOS in three hypothalamic nuclei. ............................................................................................................................................ 62 Figure 3.1 Photomicrographs illustrating the specificity of CB1R antibody. ............................... 72 Figure 3.2 Representative confocal photomicrographs illustrating the colocalization of SST and CB1R in CA1-3 areas of hippocampus. ........................................................................................ 74 xiii  Figure 3.3 Representative confocal photomicrographs illustrating the colocalization of SST and CB1R in dentate gyrus. ................................................................................................................. 75 Figure 3.4 Confocal photomicrographs depicting subcellular distribution and colocalization of CB1R with nNOS positive neurons in CA1-3 areas of hippocampus. ......................................... 78 Figure 3.5 Double labelled immunofluorescence confocal photomicrographs depicting colocalization of CB1R with nNOS positive neurons in dentate gyrus. ....................................... 79 Figure 3.6 Representative confocal photomicrographs of hippocampus showing colocalization between SST and nNOS in CA1-3 regions. .................................................................................. 82 Figure 3.7 Representative double label immunofluorescence confocal photomicrographs illustrating colocalization between SST and nNOS in dentate gyrus. .......................................... 83 Figure 3.8 Quantitative analysis of the expression of CB1R in SST and nNOS positive neurons and SST in nNOS positive neurons in hippocampus. ................................................................... 84 Figure 4.1 Representative confocal photomicrographs showing the colocalization of SSTR5 and CB1R in rat brain. ......................................................................................................................... 99 Figure 4.2 Co-IP analysis illustrating CB1R expression in SSTR5 immunoprecipitates. .......... 101 Figure 4.3 Co-IP analysis displaying constitutive SSTR5/CB1R heterodimerization in cotransfected cells. ...................................................................................................................... 102 Figure 4.4 Representative histograms displaying relative FRET efficiency of homo- and heterodimerization in cotransfected cells. ................................................................................... 104 Figure 4.5 Agonist-induced internalization of SSTR5 and CB1R in cotransfected HEK-293 cells...................................................................................................................................................... 106 Figure 4.6 cAMP is predominantly regulated by SSTR5 in cotransfected cells. ....................... 108 Figure 4.7 The phosphorylation of PKA in cotransfected cells is in parallel to cAMP. ............ 109 xiv  Figure 4.8 Changes in the phosphorylation status of ERK1/2 upon agonist treatment in cotransfected cells. ...................................................................................................................... 111 Figure 4.9 Time-dependent modulation of PI3K phosphorylation upon SSTR5 and CB1R activation. .................................................................................................................................... 113 Figure 5.1 Representative confocal photomicrographs showing colocalization of CB1R and SSTR2 in regions of wt and HD transgenic R6/2 mice (7- and 11-week-old) mice brain. ........ 129 Figure 5.2 Double labelled immunofluorescence confocal photomicrographs depicting expression and colocalization of CB1R and SSTR5 in brain regions of wt and R6/2 mice. ...... 130 Figure 5.3 Representative photomicrographs showing colocalization of SSTR2 and CB1R in STHdhQ7/7 and STHdhQ111/111 cells. ............................................................................................. 133 Figure 5.4 Representative confocal images showing colocalization of SSTR5 and CB1R in STHdhQ7/7 and STHdhQ111/111 cells. ............................................................................................ 134 Figure 5.5 Representative immunoblots showing receptor expression in both STHdhQ7/7 and STHdhQ111/111 cells. ..................................................................................................................... 135 Figure 5.6 Agonist-induced changes of receptor expression in STHdhQ7/7 and STHdhQ111/111 cells...................................................................................................................................................... 136 Figure 5.7 Time- and agonist-dependent changes in ERK1/2 phosphorylation. ........................ 138 Figure 5.8 Agonist and/or QUIN-induced changes in ERK1/2 phosphorylation in STHdhQ7/7 and STHdhQ111/111 cells. ..................................................................................................................... 140 Figure 5.9 PI3K activity was increased upon agonist treatment in the presence of QUIN in STHdhQ7/7 cells. .......................................................................................................................... 142 Figure 5.10 Changes in PTEN expression upon agonist and/or QUIN treatment in both STHdhQ7/7 and STHdhQ111/111 cells. ............................................................................................ 144 xv  Figure 5.11 Striatal cells with expression of mHtt are more susceptible to QUIN. ................... 145 Figure 5.12 Combined treatment failed to protect cells from QUIN-induced toxicity. .............. 147 Figure 5.13 Loss of pERK1/2 in STHdhQ111/111 cells upon prolonged receptor activation. ....... 148 Figure 5.14 CB1R is expressed in SSTR2 and SSTR5 immunoprecipitates. ............................. 150 Figure 6.1 Schematic illustration summarizing the crosstalk between SSTRs and CB1R. ........ 164  xvi  List of Abbreviations and Symbols 2-AG  2-Arachidonoylglycerol 3-NP  3-Nitropropionic acid A2AR  Adenosine A2A receptor AC  Adenylyl cyclase AD  Alzheimer’s disease Akt  Protein kinase B AMPA  α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid AN  Arcuate nucleus AR grade Analytical reagent grade AR  Adrenergic receptor AT1R  Angiotensin II receptor 1 ATP  Adenosine triphosphate BBB  Blood brain barrier BDNF  Brain-derived neurotrophic factor BRET  Bioluminescence resonance energy transfer C  Celsius Ca2+  Calcium cAMP  Cyclic adenosine monophosphate CB1R  Cannabinoid receptor 1 CBR  Cannabinoid receptor cGMP  Cyclic guanosine monophosphate CHO-K1 Chinese hamster ovary-K1 cells xvii  CNS  Central nervous system Co-IP  Co-immunoprecipitation CREB  cAMP response element-binding protein CRF  Corticotropin releasing factor CRF  Corticotropin releasing factor C-terminus Carboxyl terminus Cy3  Cyanine 3 D2R  Dopamine receptor 2 DAG  Diacylglycerol DAGL  DAG lipase DMEM Dulbecco’s modified Eagle’s medium DNA  Deoxyribonucleic acid  DSE  Depolarization-induced suppression of excitation DSI  Depolarization-induced suppression of inhibition ER  Endoplasmic reticulum ERK1/2 Extracellular signal-regulated kinase 1 and 2 FAAH  Fatty acid amide hydrolase FITC  Fluorescein isothiocyanate FRET  Fluorescence resonance energy transfer FSK  Forskolin GABA  γ-aminobutyric acid GDP  Guanosine diphosphate GH  Growth hormone xviii  GHRH  Growth hormone-releasing hormone GI tract Gastrointestinal tract GIRK  G-protein-coupled inwardly rectifying K+ channel GPCR  G protein-coupled receptor GRK  G protein-coupled receptor kinase GTP  Guanosine triphosphate h  Hour HA  Hemagglutinin HD  Huntington’s disease HEK-293 Human embryonic kidney-293 cells HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid hSSTR  Human somatostatin receptor Htt  Huntingtin protein IRK  Inwardly rectifying K+ channel JNK  c-Jun N-terminal kinase K+  Potassium kDa  Kilodalton LTD  Long-term depression LTP  Long-term potentiation M  Molar MAGL Monoacylglycerol lipase MAPK  Mitogen-activated protein kinase ME  Median eminence xix  mHtt  Mutant Huntingtin protein min  Minute mL  Milliliter mM  Millimolar mRNA  Messenger ribonucleic acid MS  Multiple sclerosis MSN  Medium-sized spiny neuron NAPE  N-acyl-phosphatidylethanolamine NAPE-PLD NAPE-specific phospholipase D NGS  Normal goat serum NHE  Sodium/hydrogen exchanger nM  Nanomolar NMDA N-methyl-D-aspartate NMDAR N-methyl-D-aspartate receptor nNOS  Neuronal nitric oxide synthase NO  Nitric oxide NPY  Neuropeptide Y N-terminus Amino terminus OR   Opioid receptor p  Phospho PAGE  Polyacrylamide gel electrophoresis pbFRET photobleaching fluorescence resonance energy transfer PBS  Phosphate buffered saline xx  PD  Parkinson’s disease PeVN  Periventricular nucleus PI3K  Phosphatidylinositol-4, 5-bisphosphate 3-kinase PKA  Protein kinase A PLC  Phospholipase C PNS  Peripheral nervous system PolyQ  Polyglutamine POMC  Proopiomelanocortin PPSST  Preprosomatostatin PSD95  Postsynaptic density protein 95 PSST  Prosomatostatin PTEN  Phosphatase and tensin homologue PTPase Phosphotyrosine phosphatase PTX  Pertussis toxin PVN  Paraventricular nucleus QUIN  Quinolinic acid RET  Resonance energy transfer RIPA  Radioimmune precipitation assay SDS  Sodium dodecyl sulfate SE  Standard error SRIF  Somatotropin-release inhibiting factor SST  Somatostatin SSTR  Somatostatin receptor xxi  t  Total TBS  Tris buffered saline THC  Δ9-Tetrahydrocannabinol TrkB  Tropomyosin receptor kinase B TRPV1 Transient receptor potential cation channel subfamily V member 1 TSH  Thyroid-stimulating hormone VMH  Ventromedial hypothalamic nucleus VP  Vasopressin WIN  WIN 55,212-2 wt  Wild-type α  Alpha   β  Beta γ  Gamma δ/ Δ  Delta µ  Mu µg  Microgram µL  Microliter μm  Micrometer µM  Micromolar  xxii  Acknowledgements I would like to thank my supervisor, Dr. Ujendra Kumar, for his generous help and kind guidance throughout my Ph.D. journey. I am grateful for his support in my research and his patience with my progress. His dedication to work and research inspired me a lot. I would definitely like to thank my committee members, Dr. Frank Abbott, Dr. Sastry Bhagavatula, Dr. Adam Frankel, and Dr. Urs Hafeli. Their suggestions and critiques helped me to build up my research in a better shape. Their kindness supported me through all these years. I would like to express my gratitude to all the past and present lab members for all the support and company. I would like to give my special thanks to Dr. Rishi Somvanshi, who helped me tremendously since the first day I arrived. I would also like to thank Dr. Sajad War, for his generous help whenever I approached him. I sincerely acknowledge the University of British Columbia for financial support in Tuition and Travel Awards. I am also thankful to our previous secretary Ms. Rachel Wu and current secretary Ms. Shirley Wong for their timely help and endless support throughout my graduate studies.  Last but definitely not the least, I would like to give my gratitude to my family and friends back at home. It would be impossible for me to make to this point without you. Thank you for all the support throughout these years.  xxiii  Dedication To my family and friends. 1  Chapter 1: Introduction 1.1 G protein-coupled receptors (GPCRs) GPCRs represent one of the largest and most diverse protein superfamily in mammalian genome, with more than 800 members been identified so far. GPCRs share a common structure of 7-transmembrane helices, generally with an extracellular amino terminus and an intracellular carboxyl terminus. It is now well established that both extracellular and intracellular segments exhibit significant variations in length and peptide sequences. Based on their amino acid sequence similarities, GPCR superfamily is further divided into six different classes (Foord et al., 2005, Ghosh et al., 2015). The majority of GPCRs belong to the largest family, which is Class A (Rhodopsin-like receptor) family that comprises 672 members (Millar and Newton, 2010). The rest of GPCRs are divided into another five smaller families, including Class B (the secretin receptors), Class C (metabotropic glutamate receptors), Class D (fungal mating pheromone receptors), Class E (cyclic adenosine monophosphate (cAMP) receptors) and Class F (Frizzled and Smoothened receptors) (Ghosh et al., 2015). GPCRs respond to a variety of extracellular signals, including hormones, neurotransmitters, tastes, light, odorants, and conduct these stimuli into the cells through activation of heterotrimeric G proteins composed of G alpha(α), G beta(β), and G gamma(γ) subunits (McCudden et al., 2005). G proteins are closely linked to the intracellular faces of GPCRs and serve as their partners. In resting state, Gα subunit is bound to guanosine diphosphate (GDP) and the three subunits are kept as a trimer associated with GPCR. Upon agonist binding, GPCRs switch the conformation to an active state that allows the replacement of GDP by guanosine triphosphate (GTP) and the subsequent dissociation of G protein trimers into Gα subunit and Gβγ complex. The Gβγ dimer then diffuses and interacts with several downstream signaling proteins including ion channels (McCudden et al., 2005). The GTP-bound Gα subunit can, 2  depending on Gα isoform, interact with various effectors such as adenylyl cyclase (AC), which is able to convert cAMP from adenosine triphosphate (ATP). As a second messenger, cAMP interacts with a variety of signaling cascades, including protein kinase A (PKA) and ion channels, transducing external stimulus to internal signals. Free Gα subunit has an intrinsic GTPase activity that will hydrolyze GTP to GDP, bring Gα subunit back to inactive state and thus terminate the signaling. On the other hand, following agonist-induced activation, GPCR can be phosphorylated by GPCR kinase (GRK), resulting in the recruitment of β-arrestin, which will desensitize GPCR by preventing G protein from binding to it. β-arrestin can then recruit adaptor protein-2 and clathrin, which will initiate the process of receptor internalization, ending up with receptor recycled back to the membrane or directed to lysosomes for degradation. Besides the crucial role in receptor desensitization and internalization, β-arrestin is also able to mediate a variety of other signaling pathways in a G protein-independent manner. Among them, extracellular signal-regulated kinase 1 and 2 (ERK1/2) from the mitogen-activated protein kinase (MAPK) family is the best characterized. In addition to agonist-dependent signaling, many GPCRs display constitutive activity in the absence of an agonist, as a result of the spontaneous structural transition between inactive and active states (Seifert and Wenzel-Seifert, 2002, Bouvier, 2013). The constitutive activity of GPCRs has been implicated to play a critical role in drug actions and various physiological and pathological conditions, including food intake, growth hormone (GH) synthesis, and drug withdrawal symptoms (Seifert and Wenzel-Seifert, 2002, Bouvier, 2013, Meye et al., 2014). Moreover, oligomerization of GPCR has been proven to be mandatory for proper function of Class C GPCR and widely reported for other GPCRs (Kniazeff et al., 2011, Ferre et al., 2014) (discussed in details later). Although the structural properties and structural-functional relationship of GPCR 3  oligomers have not been fully elucidated, the changes of downstream signaling and novel pharmacological properties have been highly appreciated and applied in search of new drugs and therapies (Ferre et al., 2014).   1.2 Somatostatin (SST) SST was first isolated from ovine hypothalamus a tetradecapeptide capable of inhibiting GH release at nanomolar scale, thus was given the name “somatotropin-release inhibiting factor (SRIF)”, which later turned out to be just one aspect of many diverse roles SST plays in physiological and pathological conditions (Brazeau et al., 1973). Analysis of porcine intestinal SST revealed that this peptide exists in two bioactive forms: SST-14 that is previously identified in hypothalamus, and its congener with an extra 14 amino acid extension at amino terminus (N-terminus), named SST-28 (Pradayrol et al., 1978, Pradayrol et al., 1980). Both SST-14 and SST-28 are produced from a large inactive precursor molecule, preprosomatostatin (PPSST), containing 116 amino acids (Patel, 1999). PPSST is processed enzymatically to yield 92-amino-acid prosomatostatin (PSST), which is then processed by certain enzymes that belong to a class of serine proteinase called precursor convertases, primarily at carboxyl terminus (C-terminus) to generate SST-14 and SST-28 (Galanopoulou et al., 1993, Galanopoulou et al., 1995). These two bioactive forms of SST are synthesized differently in the human body depending on the precursor processing (Patel et al., 1981, Baskin and Ensinck, 1984). In the brain, SST-14 is the predominant form mostly confined in hypothalamus, whereas SST-28 only accounts for 20-30% of total SST-like immunoreactivity (Patel et al., 1981, Baskin and Ensinck, 1984). SST-14 is also expressed dominantly in several other peripheral tissues, including pancreatic islets, stomach, and neural tissues (Patel et al., 1981, Baskin and Ensinck, 1984). Furthermore, studies have also demonstrated 4  that in retina, peripheral nerves, and enteric neurons, SST-14 is the only form identified. SST-28, however, is mainly synthesized as the terminal product of PSST processing in intestinal mucosal cells, which provides the majority of SST-28 in peripheral tissues (Patel et al., 1981, Baskin and Ensinck, 1984, Patel, 1999).  Figure 1.1 Action sites of SST in human body. [Reprinted with permission from (Kumar and Grant, 2010)]  SST-producing cells are found in high densities throughout the central (CNS) and peripheral nervous systems (PNS), endocrine pancreas, and the gut (Figure 1.1) (Patel, 1999). Small numbers of cells producing SST are also present in the thyroid, adrenals, submandibular glands, kidneys, prostate, placenta, blood vessel walls, and lymphoid organs such as spleen and thymus (Aguila et 5  al., 1991, Patel, 1999). It has been shown in rat that gut is the major source of SST, accounting for approximately 65% of total body SST, followed by the brain and pancreas, accounting for 25% and 5% respectively, and the remaining 5% is distributed among other organs (Patel and Reichlin, 1978). SST provides tonic inhibition to the release of GH through direct interaction in pituitary or indirectly via the feedback loop of GH-releasing hormone (GHRH), which is the counterpart of SST, as it stimulates GH release (Katakami et al., 1988, Tannenbaum et al., 1990). Moreover, the inhibitory effect of SST is not exclusive to the release of GH. Studies have shown that prolactin, thyroid-stimulating hormone (TSH), majority of hormones in the gastrointestinal (GI) tract, even SST itself in hypothalamus are subjected to its negative regulation (Katakami et al., 1988, Tannenbaum et al., 1990). As a neurotransmitter and neuromodulator, SST has been reported to inhibit the release of dopamine from midbrain, norepinephrine from hypothalamus, glutamate from cerebral cortex and γ-aminobutyric acid (GABA) in basal forebrain (Patel, 1999, Grilli et al., 2004, Momiyama and Zaborszky, 2006). On the other hand, SST receives the regulatory input from a broad range of secretagogues, including neuropeptides, neurotransmitters, hormones, growth factors, cytokines and even daylight length (Reichlin, 1983a, b, Patel et al., 2001, Barnett, 2003, Dulcis et al., 2013). For instance, studies have shown that SST secretion from hypothalamus is promoted by dopamine, substance P, glucagon, neurotensin, acetylcholine, vasoactive intestinal polypeptide, but inhibited by glucose (Chihara et al., 1979, Berelowitz et al., 1982, Reichlin, 1983a, b). A similar mechanism has also been proposed in the regulation of TSH secretion from hypothalamus (Reichlin, 1983a, Patel, 1999).  The concept of SST being a promising anti-cancer agent dates back to early 1980s, when octreotide, an analog of SST, was proven to be beneficial in the treatment of hypersecreting tumors 6  of pancreas, intestine, and pituitary, and has drawn great attention ever since (Lamberts et al., 1996). Clinical evidence obtained in subsequent studies supported the effect of SST on the control of hypersecreting adenomas (Susini and Buscail, 2006, Grozinsky-Glasberg et al., 2008). Decades of research reveal that SST exerts a profound inhibitory effect on tumor growth through both direct and indirect actions (Susini and Buscail, 2006, Kumar and Grant, 2010). Directly, the cytostatic and cytotoxic effects of SST lead to cessation of cell division and activation of programmed cell death. Indirectly, SST inhibits the secretion of growth factors and hormones necessary for tumor growth, restricts blood supply to growing tumors by inhibiting angiogenesis, and modulates the immune system to suppress tumor growth and metastasis (Susini and Buscail, 2006).  In addition to its well-known role as an inhibitory peptide and hormone, SST functions as a neurotransmitter and neuromodulator in the brain and exerts profound effects on cognition, locomotion, sensation, and autonomic functions (Schindler et al., 1996). Most importantly, the critical roles of SST implicated in several neurological disorders have been widely reported (Kumar and Grant, 2010). Reduction of SST messenger ribonucleic acid (mRNA) expression and immunoreactivity in multiple brain regions has been observed in Alzheimer’s disease (AD) and Parkinson’s disease (PD) patients respectively (Davies et al., 1980, Beal et al., 1988, Kumar and Grant, 2010). In contrast, SST-like immunoreactivity is increased in basal ganglia of Huntington’s disease (HD) patients, and SST-positive medium-sized aspiny interneurons are preferentially spared during the early stage of striatal atrophy (Aronin et al., 1983, Ferrante et al., 1985, Reiner et al., 1988). In temporal lobe epilepsy, despite massive loss of hilar SST interneurons, the surviving few sprout axons form new synapses with granule cells, as a compensatory mechanism (Sundstrom et al., 2001, Zhang et al., 2009). Due to its robust effect on inhibition of epileptiform activity, SST has been recognized as an endogenous antiepileptic peptide (Tallent and Qiu, 2008). 7  Several previous studies have suggested a relation between SST and excitotoxicity (Kumar, 2004, 2008). SST has been shown to protect the blood brain barrier (BBB) from cytokines, suggesting a reduced level of SST in multiple sclerosis (MS) may contribute to the damage of BBB (Sorensen, 1987, Basivireddy et al., 2013). The reduced SST level in brain is also a common feature in mood disorders, such as major depression and schizophrenia (Lin and Sibille, 2013). These observations emphasize the role of SST in neurological activities and disorders.  1.2.1 Somatostatin receptors (SSTRs) In target tissues, SST exerts direct and indirect effects. Various biological effects of SST are mediated through the binding of five different receptor subtypes, namely SSTRs. The first pharmacological characterization of high-affinity plasma membrane receptors for SST was described in 1978 by using a whole-cell binding assay in GH4C1 rat pituitary tumor cells (Schonbrunn and Tashjian, 1978). Three years later, the existence of multiple members in the SSTR family was unraveled by the distinct binding affinities and potencies for SST-14 and SST-28 in brain, pituitary and islet cells (Mandarino et al., 1981, Srikant and Patel, 1981). Yet it took another decade for the first SSTR sequence to be successfully cloned, followed by the identification of all five SSTRs known as SSTR1-5 today [reviewed in (Patel, 1999)]. The genes encoding the five SSTRs are located on different chromosomes and are intronless, except SSTR2, which bears an intron at the 3’-end and thus gives rise to two spliced variants, SSTR2A and SSTR2B, differing only in the length of their C-terminus [reviewed in (Patel, 1999)]. Among the five SSTRs, the sequence identity is 39-57% and the encoded proteins range in size from 356 to 391 amino acid residues [reviewed in (Patel, 1999)]. All SSTRs display a typical GPCR structure of seven α-helical transmembrane segments, in which they share highest sequence similarity, 8  whereas the N- and C-terminal regions bear the maximum divergence. As a result, the five SSTRs show distinct pharmacological properties. Based on their binding affinities to SST hexapeptide and octapeptide analogs, the five receptors are grouped into two categories: SSTR2, 3, and 5 as SRIF I, which bind to these SST analogs, whereas SRIF II, constituted by SSTR1 and 4, are insensitive to SST analogs (Tran et al., 1985). In the case of endogenous SST, all five SSTRs are activated by both isoforms at similar nanomolar (nM) affinity, with SST-14 usually higher than SST-28, and the only exception being SSTR5 that binds to SST-28 with a 5-10 fold higher affinity than SST-14 [reviewed in (Patel, 1999)].  1.2.2 SSTR localization The distribution of SSTRs has been characterized in different systems, including human and rodent tissues as well as several tumor cell lines (Bruno et al., 1993, Patel, 1999, Kumar and Grant, 2010). The distributional pattern turns out to be widespread, yet subtype-specific, tissue-selective, and species-specific. In rat, SSTR1 is predominant in the brain and also expressed in islets and adrenal glands. Comparatively SSTR2 has less expression in the brain, comparable expression in islets and more expression in adrenal glands. SSTR3 is preferentially expressed in cerebellum, while highly expressed in spleen, kidneys, and liver. SSTR4 has the lowest expression in the brain but is abundant in the heart and moderately expressed in the lungs and islets. SSTR5 is sporadically expressed throughout the brain, but is the principle subtype in pituitary, and also has high expression in islets. Expression of SSTRs in human brain, pituitary, and peripheral tissues is broadly similar to that in rat. Colocalization of two or more SSTRs in one particular region is also frequently reported in both rodents and human, suggesting a cooperation/crosstalk between SSTR subtypes (Patel, 1999, Moller et al., 2003, Kumar and Grant, 2010). 9  All human SSTRs (hSSTRs) except SSTR1 subject to agonist-stimulated desensitization and internalization, with SSTR3 showing highest internalization rate, followed by SSTR5, 4 and 2, whereas SSTR1 is translocated from the subcellular compartments to the plasma membrane, yielding a higher expression when expressed in Chinese hamster ovary-K1 cells (CHO-K1) and human embryonic kidney-293 cells (HEK-293) (Hukovic et al., 1996, Hukovic et al., 1999). Prolonged treatment with agonist leads to the upregulation of SSTR1, as well as SSTR2 and 4, although to a much lower extent, whereas almost no change is observed in SSTR3 and 5 expression (Hukovic et al., 1996). The process is rapid, and time- and temperature-dependent. Further investigation revealed that agonist-induced upregulation of hSSTR1 at cell surface does not require new protein synthesis and is heavily dependent on its C-terminus (Hukovic et al., 1999). However, it is important to note that agonist-induced receptor internalization is species- and cell type-specific. For instance, SSTR1 of rat origin expressed in rat pancreatic insulinoma and HEK-293 cells displays efficient internalization upon agonist treatment, whereas rat SSTR4 transfected in HEK-293 cells turns out to be the only subtype resistant to agonist-induced internalization (Roosterman et al., 1997, Roth et al., 1997). Human SSTR1 transfected to COS-7 cells exhibits considerable internalization instead of upregulation following agonist treatment (Nouel et al., 1997). Moreover, SSTR2 of mouse origin internalizes three times higher in COS-7 cells than hSSTR2 transfected in CHO-K1 cells (Hukovic et al., 1996, Nouel et al., 1997).  1.2.3 SSTR signaling All SSTRs belong to the GPCR superfamily, and are coupled to G proteins to transduce signals. Multiple second-messenger systems are modulated by SSTRs, including AC, phosphotyrosine phosphatases (PTPases), MAPKs, Protein kinase B (Akt)/Phosphatidylinositol-4, 5-bisphosphate 10  3-kinase (PI3K), phospholipase C (PLC), phospholipase A2, and calcium (Ca2+) and potassium (K+) ion channels and sodium/hydrogen exchangers (NHEs). The outcome of SSTR activation is affected largely by multiple factors, including ligand, receptor subtype, receptor crosstalk, and cell type [reviewed in (Patel, 1999, Kumar and Grant, 2010)]. All five SSTRs negatively modulate the activity of AC by coupling to pertussis toxin (PTX)-sensitive Gi protein, resulting in the inhibition of cAMP production in a concentration-dependent manner [reviewed in (Kumar and Grant, 2010)]. Studies have shown a bell-shape curve of the inhibition of forskolin (FSK)-stimulated cAMP formation in response to different concentrations of SST (Moller et al., 2003). At very low concentration [10-17-10-13 molar (M)], SST increases cAMP, which is associated with SST-induced GH release at corresponding concentration in cultured primary pituitary cells of porcine and primate origins (Ramirez et al., 2002, Cordoba-Chacon et al., 2012). It is not yet well understood whether the effect is due to the switching of G protein coupling from Gi to Gs, as seen in the case of other GPCRs, such as α2A-adrenergic receptor (AR) and cannabinoid receptor 1 (CB1R) (Vilardaga et al., 2003, Jarrahian et al., 2004). So far, there is no evidence supporting SSTRs coupling to Gs.  Several types of K+ channels have been shown to be modulated by SSTRs, including the delayed rectifier, inward rectifier, ATP-sensitive K+ channels, and large-conductance Ca2+-activated big K+ channels [reviewed in (Kumar and Grant, 2010)]. It is suggested that the modulation is via Gi3 or Gβγ proteins (Takano et al., 1997). The regulation of high-voltage-dependent Ca2+ channels via Go2 has also been reported (Ikeda and Schofield, 1989, Kleuss et al., 1991). Aside from G proteins, in chick ciliary ganglion neurons, SST has been shown to inhibit Ca2+ current via a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (Meriney et al., 1994). NHE is another target of SSTR subtypes to modulate intracellular ion homeostasis, 11  affecting features as cell adhesion, migration, and proliferation (Ye et al., 1999). SSTR1, 3 and 4 have been shown to be involved in the regulation of NHE, but not SSTR2 and 5 (Lin et al., 2003). Modulation of MAPK signaling has been reported for all five SSTRs in various systems, both positively and negatively. The outcome of SSTR-mediated MAPK activation/inhibition is receptor subtype- and cell type-specific. Activation of SSTR1 has been reported to induce cell cycle arrest through hyperactivation of ERK1/2 (Florio et al., 1999). SSTR2 inhibits GH-induced MAPK activation in C6 glioma cells and in human neuroblastoma SH-SY5Y cells in a cAMP-independent manner, rendering the anti-proliferative effect of SST (Pola et al., 2003, Barbieri et al., 2008). However, it has also been shown that SSTR2 activates MAPK via Ras, B-Raf and Rap1 to decrease cell proliferation in transfected CHO cells (Sellers et al., 2000, Lahlou et al., 2003). Activation of SSTR3 and 5 has been reported to inhibit cell proliferation via blockade of ERK1/2 (Reardon et al., 1996, Florio et al., 2003). In contrast, SSTR4 is the only subtype showing proliferative effect via stimulating MAPK (Sellers, 1999). The SSTR-induced activation/inhibition of MAPK has been reported to involve PTPases, PI3K and cGMP (Kumar and Grant, 2010). Similarly, SSTRs modulate PI3K signaling pathway in a receptor- and cell type-specific manner. SSTR2 activates PI3K in heterologous systems, whereas inhibition of PI3K has been observed in tumor cells where SSTR2 is endogenously present or overexpressed (Lahlou et al., 2003, Bousquet et al., 2006, Kumar and Grant, 2010). A recent study has shown that SSTR2 regulates PI3K activity in a way distinct from other SSTRs (Bousquet et al., 2006, Najib et al., 2012). SSTR2 physically associates with the regulatory subunit of PI3K, p85, and contributes to the basal level of PI3K activity in the absence of agonist (Bousquet et al., 2006, Najib et al., 2012). However, this association is disrupted upon SSTR2 activation, resulting in subsequent inhibition 12  of PI3K (Najib et al., 2012). In addition, studies have shown that PI3K is also positively involved in SSTR1- and 4-mediated ERK1/2 activation (Florio et al., 1999, Smalley et al., 1999).  1.3 Cannabinoids The plant Cannabis sativa, better known as marijuana, has long been used for medical purpose in human history. The first record can be traced back to ancient China around 5,000 years ago, where the extracts of the plant were used for relief of cramps and pain (Mechoulam, 1986). The widely-documented usages of marijuana include anti-nociception, anti-inflammation, anticonvulsion, antiemesis, as well as recreation, which has largely limited its medical application (Mechoulam, 1986, Iversen, 2000). Not until half a century ago, the first light was shed on the myth of versatile functions of marijuana by the discovery of Δ9-tetrahydrocannabinol (THC), the main psychoactive component of approximately 70 phytocannabinoids identified in the plant (Gaoni and Mechoulam, 1964, Elsohly and Slade, 2005). The milestone discovery led to the generation of a variety of synthetic cannabinoids with similar or distinct structures to phytocannabinoids, which finally led to the identification and successful cloning of CB1R (Devane et al., 1988, Matsuda et al., 1990, Pertwee et al., 2010). Not long after that, another cannabinoid receptor (CBR) was identified and cloned, later termed as CB2R (Munro et al., 1993). Despite dispute on a putative “CB3R”, only CB1R and CB2R are widely-acknowledged as CBRs (Howlett et al., 2002, Ryberg et al., 2007, Kano et al., 2009). Meanwhile, N-arachidonoyl-ethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG) have been discovered to serve as endogenous agonists of CBRs, namely endocannabinoids (Devane et al., 1992, Mechoulam et al., 1995, Sugiura et al., 1995). These two compounds are the first to be identified and remain as the best-studied endocannabinoids, which are all derivatives of arachidonic acid (Pacher et al., 2006). In 13  recent years, much attention has been drawn to utilizing marijuana extracts in medicine (Izzo et al., 2009). Due to the beneficial clinical application of marijuana and the non-psychoactive nature of most phytocannabinoids except THC, the therapeutic potentials of these compounds have been greatly appreciated (Izzo et al., 2009). Although this area of research is quite controversial and debatable, several phytocannabinoids, especially cannabidiol, have been suggested to exert beneficial effects in various pathological conditions, including inflammation, cancer, and epilepsy (Izzo et al., 2009, Nagarkatti et al., 2009, Hill et al., 2012, dos Santos et al., 2015, Patil et al., 2015).  1.3.1 CBRs  Due to the lipophilic nature of cannabinoids, it was initially thought that these compounds exert various biological effects by disrupting cell membrane nonspecifically (Lawrence and Gill, 1975). However, the identification of THC as the psychoactive component in the plant and subsequent emerging of several chemically synthesized cannabinoids made it possible to map and pharmacologically characterize the binding sites of cannabinoid in the brain, which, in turn, revealed the existence of a putative CBR and its similarity to GPCR. Its properties matched with a previously known orphan GPCR that was then renamed as CB1R (Gaoni and Mechoulam, 1964, Devane et al., 1988, Matsuda et al., 1990, Pertwee et al., 2010).  CB1R is encoded by gene CNR1 and consists of 472 amino acids in human (473 amino acids in rat and mouse, showing 97-99% amino acid sequence identity among these species). Two recent studies have described the crystal structure of CB1R independently (Hua et al., 2016, Shao et al., 2016). Several variations of CNR1 have been associated with cannabis dependence (Agrawal and Lynskey, 2009, Hartman et al., 2009, Schacht et al., 2012). In addition to the canonical long form 14  of CB1R, two additional isoforms with shorter N-terminus have been reported, both resulting from alternative splicing (Shire et al., 1995, Ryberg et al., 2005). Recently, the different expression patterns of these three isoforms have been characterized at mRNA level in human brain, skeletal muscle, liver and islet (Gonzalez-Mariscal et al., 2016). Full length CB1R dominates in the brain and skeletal muscle, whereas CB1Rb (with 33 amino acid deletion at N-terminus) shows a higher expression level in liver and islet where it is involved in metabolism (Gonzalez-Mariscal et al., 2016). The pharmacological and physiological properties of the two splice variants are yet to be explored, as current studies accomplished in non-human model revealed discrepancy (Ryberg et al., 2005, Xiao et al., 2008, Straiker et al., 2012).  CB2R is encoded by gene CNR2, consisting of 360 amino acid in human. It shares only 44% sequence homology with CB1R at protein level (Pertwee et al., 2010). CB2R also has greater species differences among human and rodents in comparison to CB1R, as the amino acid sequence homology is slightly above 80% between human and rodents (Liu et al., 2009b, Zhang et al., 2015). In human, two isoforms of human CB2R have been identified, with one predominantly expressed in testis and at lower levels in brain reward regions, whereas the other is mainly expressed in spleen and at even lower levels in the brain (Liu et al., 2009b). The testis isoform has a promoter at 45 kb upstream from the spleen isoform (Liu et al., 2009b). So far, four rat CB2R isoforms and two mouse isoforms have been discovered (Liu et al., 2009b, Zhang et al., 2015).  1.3.2 Endocannabinoid system The successful identification and cloning of CB1R prompted the discovery of its first endogenous agonist, anandamide, in 1992 (Devane et al., 1992). The fact that anandamide cannot fully reproduce the effects induced by THC led to the discovery of another important 15  endocannabinoid, 2-AG (Smith et al., 1994, Mechoulam et al., 1995, Sugiura et al., 1995). Today, most studies on endocannabinoid system focus on these two endocannabinoids, despite a series of arachidonic acid derivatives generating endocannabinoid-like effects and the CB1R-interacting peptides found recently (Bisogno et al., 2005, Di Marzo and De Petrocellis, 2012). These well-documented two endocannabinoids, as pharmacologically characterized later, possess distinct properties. Anandamide turns out to be a high-affinity, partial agonist of CB1R, and almost inactive at CB2R; whereas 2-AG acts as a full agonist to both CBRs with moderate-to-low affinity (Stella et al., 1997, Di Marzo and De Petrocellis, 2012). Interestingly, both anandamide and 2-AG have been reported to interact with various receptors, among which the transient receptor potential cation channel subfamily V member 1 (TRPV1) activated by anandamide is the best-documented for its significant role in synaptic transmission and pain regulation, whereas the interaction of 2-AG and non-CBRs have been emerging only recently (Di Marzo and De Petrocellis, 2012). Although anandamide and 2-AG have significant differences in receptor selectivity, both of these two endocannabinoids are produced on demand (although controversies exist in the case of 2-AG), in response to an increased intracellular Ca2+ concentration (Kano et al., 2009, Alger and Kim, 2011). However, anandamide and 2-AG are synthesized, transported and inactivated in respective target tissues differently. In brief, anandamide is catalyzed from N-acyl-phosphatidylethanolamine (NAPE) by NAPE-specific phospholipase D (NAPE-PLD) or other routes not involving NAPE-PLD (Liu et al., 2008). On the other hand, 2-AG is produced from diacylglycerol (DAG) by either DAG lipase (DAGL) α or β, although most if not all 2-AG mediating synaptic transmission in adult brain is generated by DAGL- α (Tanimura et al., 2010). After release into the intracellular space, due to the uncharged hydrophobic nature, endocannabinoids are unable to diffuse freely like other neurotransmitters. Several models have been proposed to elucidate the transportation of 16  anandamide: simple diffusion driven by concentration gradients generated from enzymatic degradation, endocytosis involving caveolae/lipid rafts, through certain carrier proteins like fatty acid binding proteins and heat shock proteins 70 (Kaczocha et al., 2009, Kano et al., 2009, Oddi et al., 2009). 2-AG may be sharing the same transport system with anandamide, but it is not well understood yet (Huang et al., 2016). Once endocannabinoids are taken up by the cells, they can be degraded through hydrolysis and/or oxidation (Vandevoorde and Lambert, 2007). Anandamide is uniquely degraded by fatty acid amide hydrolase (FAAH) into free arachidonic acid and ethanolamine, whereas 2-AG is mostly hydrolyzed by monoacylglycerol lipase (MAGL) into arachidonic acid and glycerol, while other enzymes could be involved as well (Blankman et al., 2007, Vandevoorde and Lambert, 2007). Oxidation of both anandamide and 2-AG can involve cyclooxygenase-2 and several lipoxygenases (Rouzer and Marnett, 2011).  1.3.3 Endocannabinoid-mediated signaling Given the fact that basal level of 2-AG is approximately 1000 times higher than anandamide in the brain and pharmacological manipulations of 2-AG metabolism, but not anandamide, exert remarkable effects in endocannabinoid-mediated retrograde signaling, it is proposed that 2-AG is the primary endogenous ligand for CBR in the CNS [reviewed in (Katona and Freund, 2008)]. However, anandamide has been shown to activate TRPV1, inhibit L-type Ca2+ channel independently, as well as negatively regulate 2-AG biosynthesis and physiological effects in striatum, underscoring its essential role in the regulation of synaptic transmission (Maccarrone et al., 2008, Di Marzo and De Petrocellis, 2012). The first conclusive evidence supporting retrograde endocannabinoid signaling came from the observation of depolarization-induced suppression of inhibition (DSI)/excitation (DSE) (Kreitzer 17  and Regehr, 2001, Ohno-Shosaku et al., 2001, Wilson and Nicoll, 2001). Later, it was discovered that endocannabinoid system is involved not only in short-term depression, but also in long-term depression (LTD) at both excitatory and inhibitory synapses (Gerdeman et al., 2002, Marsicano et al., 2002, Robbe et al., 2002, Chevaleyre and Castillo, 2003). Since then, endocannabinoid system has become the most-studied retrograde signaling system in the brain. In most cases, endocannabinoid-mediated retrograde signaling starts with the production of 2-AG, in response to increased intracellular Ca2+ concentration and/or activated Gq/11-coupled receptors [reviewed in (Kano et al., 2009, Castillo et al., 2012)]. 2-AG is then released into extracellular space, traveling across via a mechanism not fully elucidated yet, then arrives at the presynaptic terminal and binds to CB1R. Activated CB1R suppresses the release of neurotransmitter through two ways: first, inhibiting voltage-gated Ca2+ channel to reduce presynaptic Ca2+ influx; second, inhibiting AC and subsequent cAMP/PKA pathway, which is more involved in LTD [reviewed in (Kano et al., 2009)]. The termination of signaling requires the degradation of 2-AG by MAGL, which is expressed in selective synaptic terminals and glial cells [reviewed in (Castillo et al., 2012)].  Anandamide has been shown to contribute to endocannabinoid-mediated synaptic transmission in several ways. Anandamide is a full agonist of TRPV1, which is proposed to participate in endocannabinoid signaling (De Petrocellis and Di Marzo, 2010). Anandamide-mediated LTD has been reported in several studies, likely in a TRPV1-dependent manner [reviewed in (Castillo et al., 2012)]. The differential recruitment of 2-AG and anandamide by different types of presynaptic activity has been described in the extended amygdala (Puente et al., 2011). Anandamide negatively regulates 2-AG metabolism, of which the effect can be mimicked by the activation of TRPV1 (Maccarrone et al., 2008). There is also evidence supporting a tonic role of anandamide as an 18  endocannabinoid as chronic blockade of FAAH leads to a constant agonism of endocannabinoid system without reducing CB1R expression, which is opposite to the case of MAGL (Schlosburg et al., 2010).  Endocannabinoids are prominently involved in the suppression of synaptic transmission through multiple mechanisms, independent of synaptic nature or transmission duration (Cachope et al., 2007). CB1R-dependent self-inhibition in postsynaptic neurons has been observed in a subpopulation of neocortical interneurons and pyramidal neurons, as well as in hippocampal CA1 [reviewed in (Busquets Garcia et al., 2016)]. Accumulated evidence supports the endocannabinoid mediated communication between neurons and microglia (Navarrete and Araque, 2008, Castillo et al., 2012). Previous studies have shown that microglial cells and astrocytes are even able to produce their own 2-AG or anandamide, although it is not clear yet whether these endocannabinoids are involved in the modulation of synaptic transmission (Stella, 2009).  In contrast, although studies have shown the presence of CB2R in the brain, the role of CB2R in endocannabinoid-mediated synaptic transmission is still largely elusive [reviewed in (Atwood and Mackie, 2010)]. A recent study has reported that in medial prefrontal cortical pyramidal neurons, intracellular CB2R reduces neuronal firing through the opening of Ca2+-activated chloride channels, suggesting its involvement in the regulation of neuronal activity (den Boon et al., 2012).  1.3.4 CBR localization CB1R was first discovered in the brain. Later, by using autoradiography, in situ hybridization, and immunohistochemistry, CB1R was proven to be the most widely-expressed receptor protein from GPCR family in the brain [reviewed in (Mackie, 2005b, Kano et al., 2009)]. The brain regions 19  with high expression levels of CB1R include olfactory bulb, hippocampus, basal ganglia, and cerebellum [reviewed in (Mackie, 2005b)]. Moderate expression is found in cerebral cortex, septum, amygdala, hypothalamus, and parts of the brain stem and the dorsal horn of spinal cord, whereas regions like thalamus and ventral horn of spinal cord have low expression of CB1R [reviewed in (Mackie, 2005b)]. Several previous studies have suggested a highly concentrated expression of CB1R on presynaptic terminals, where it conducted retrograde signaling of endocannabinoids (Tsou et al., 1998, Katona et al., 1999). However, it does not exclude the existence of CB1R at postsynaptic sites, as functional studies demonstrate self-inhibition in neocortical neurons by endocannabinoids [reviewed in (Busquets Garcia et al., 2016)]. Besides neurons, CB1R is expressed, although to a much lower extent, in astrocytes, oligodendrocytes and microglia, where it has been shown to mediate synaptic transmission (Stella, 2009, Castillo et al., 2012). CB1R is as well abundantly expressed in the PNS, mostly sympathetic nerve endings, and peripheral tissues, including cardiovascular tissues, GI tract, liver, immune cells, muscle cells, reproductive system, and several types of tumor tissues (Figure 1.2)[reviewed in (Mackie, 2005b, Maccarrone et al., 2015)]. Like many other GPCRs, CB1R is primarily localized on the cell membrane, however, recent studies have also revealed the subcellular distribution of functionally active CB1R in mitochondria and endo/lysosome (Brailoiu et al., 2011, Benard et al., 2012, Busquets Garcia et al., 2016).  20   Figure 1.2 Major distribution sites and functions of CB1R function in human body.   Three years after the discovery of CB1R, another CBR, CB2R, was identified in macrophages in the spleen (Munro et al., 1993). Follow-up studies revealed a predominant expression of CB2R in immune cells and a moderate expression in other peripheral tissues [reviewed in (Howlett et al., 2002)]. In contrast, the presence of CB2R was not observed in the CNS, thus it was referred to as “the peripheral CBR” (Howlett et al., 2002). However, this concept has been challenged recently by several studies demonstrating the expression of CB2R in the brain, albeit to a much lower extent in comparison to the immune system or CB1R (Gong et al., 2006). Although the expression pattern 21  of CB2R in the CNS and PNS is comparatively limited, it is undeniable that CB2R plays an active role in neurological activities, such as nociception, drug addiction and neuroinflammation [reviewed in (Atwood and Mackie, 2010, Dhopeshwarkar and Mackie, 2014)].  1.3.5 CBR signaling Both CB1R and CB2R are members of GPCR family. They both are coupled to PTX-sensitive Gi/o protein, suppress AC and formation of cAMP upon receptor activation (Howlett et al., 2002). However, CB1R but not CB2R has also been reported to activate other G proteins in certain circumstances in a cell type- and ligand-dependent manner (Demuth and Molleman, 2006). CB1R is able to stimulate specific AC isoforms via Gβγ subunits (Rhee et al., 1998). Also, CB1R stimulates cAMP via coupling to Gs when dopamine receptor 2 (D2R) is activated simultaneously in cultured striatal neurons, and when Gi is blocked by PTX in transfected CHO-K1 cells, and in response to a relatively high concentration of WIN 55, 212-2 (WIN) in rat globus pallidus slices (Glass and Felder, 1997, Maneuf and Brotchie, 1997, Bonhaus et al., 1998). However, the same concentration of WIN but not other CB1R agonists, increases intracellular Ca2+ concentration via Gq/11 protein in transfected HEK-293 cells and cultured hippocampal neurons (Lauckner et al., 2005). Moreover, in mice hippocampal slices, astrocytic CB1R is coupled to Gq/11, increases intracellular Ca2+ concentration and triggers astrocytic release of glutamate that stimulates N-methyl-D-aspartate receptor (NMDAR) on pyramidal neurons, indirectly involved in synaptic transmission (Navarrete and Araque, 2008). Moreover, CB1R modulates the activity of several types of ion channels [reviewed in (Demuth and Molleman, 2006, Turu and Hunyady, 2010)]. CB1R has been reported to inhibit N-type Ca2+ channel in neuroblastoma cell lines, in cultured rat primary hippocampal neurons, and in mice 22  cerebellar slices (Brown et al., 2004, Turu and Hunyady, 2010). It has long been suggested, but proven only recently that CB1R regulates Ca2+ influx to inhibit GABA release in mouse hippocampal slices via modulation of the activity of presynaptic N-type Ca2+ channel (Gergely et al., 2014). Other types of Ca2+ channels, including P/Q-type, and R-type Ca2+ channels, have been shown to be negatively regulated by CB1R in various systems (Brown et al., 2004, Turu and Hunyady, 2010). On the other hand, CB1R regulates the activity of G-protein-coupled inwardly rectifying K+ channels (GIRKs) as well (Mackie et al., 1995, Robbe et al., 2001, Guo and Ikeda, 2004). CB1R activates GIRK in transfected AtT-20 cells, mouse nucleus accumbens slices, and rat sympathetic neurons injected with CB1R complementary deoxyribonucleic acid (DNA) (Mackie et al., 1995, Robbe et al., 2001, Guo and Ikeda, 2004). Previous studies have shown that in a system expressing the receptor endogenously or heterogeneously, stimulation of CB1R leads to the activation of MAPK signaling pathways, including ERK1/2, c-Jun N-terminal kinase (JNK), and p38, that are involved in the regulation of cell proliferation, cell cycle control and cell death [reviewed in (Demuth and Molleman, 2006, Howlett et al., 2010, Turu and Hunyady, 2010)]. Generally, CB1R regulates MAPK signaling in a cell type- and ligand-specific fashion (Demuth and Molleman, 2006, Howlett et al., 2010, Turu and Hunyady, 2010). For instance, CB1R-induced ERK1/2 activation can be mediated by G protein, β-arrestin, or PI3K, heavily dependent on the microenvironment and stimulus type (Bouaboula et al., 1995, Galve-Roperh et al., 2002, Turu and Hunyady, 2010). Similarly, activation of p38 has been observed upon stimulation of CB1R in human vascular endothelial cells, transfected CHO-K1 cells, and rat/mouse hippocampal slices. JNK activation has been shown in transfected CHO-K1 cells, where G proteins, PI3K and Ras were involved in the transduction [reviewed in (Demuth and Molleman, 2006)]. Moreover, JNK activation was also observed in 23  Neuro2A cells with endogenous expression of CB1R, and may be related to CB1R-mediated neurite outgrowth (He et al., 2005).  Besides the typical G proteins-dependent signaling for all GPCRs, CB1R is able to signal in a G protein-independent manner through association with other molecules such as β-arrestin (Howlett et al., 2010). β-arrestin is a key mediator for GPCR desensitization. Following receptor phosphorylation by GRK, β-arrestin binds to the receptor and initiates the internalization process, during which β-arrestin could mediate signaling pathways (McCudden et al., 2005). Desensitization of CB1R has been shown to be β-arrestin 2-dependent in various systems (Jin et al., 1999, Kouznetsova et al., 2002). And it has been reported in transfected HEK-293 cells that β-arrestin 2-mediated desensitization but not internalization of CB1R determines the time course of ERK1/2 phosphorylation upon CB1R activation (Daigle et al., 2008). Follow-up study further revealed a positive correlation between the extent of β-arrestin-mediated signaling and the duration of CB1R interaction with β-arrestin at the cell surface in a ligand-specific manner (Flores-Otero et al., 2014). Studies using β-arrestin 2 knockout mice have suggested a critical role of β-arrestin 2 in the regulation of CB1R activity (Breivogel et al., 2008, Nguyen et al., 2012). The β-arrestin 2 knockout mice displayed a comparable expression level of CB1R yet an increased sensitivity to THC, featuring an enhanced antinociception and decreased tolerance (Breivogel et al., 2008, Nguyen et al., 2012). Recent study suggested a role of β-arrestin 1 in the phosphorylation of ERK1/2, MAPK kinase 1/2 and proto-oncogene tyrosine-protein kinase Src in response to a CB1R allosteric modulator ORG27569, underscoring a biased signaling largely dependent on stimulus (Ahn et al., 2013). PI3K/Akt pathway is another key regulator of cell growth and death aside of MAPK signaling. In rat primary cultured astrocytes, human astrocyte cell line, and transfected CHO-K1 cells, CB1R 24  has been shown to activate PI3K/Akt pathway, which is responsible for the CB1R-induced protective effects on cell survival (Gomez del Pulgar et al., 2000, Galve-Roperh et al., 2002, He et al., 2005). In rat oligodendrocyte progenitors, CB1R promotes cell survival against nutrient deprivation and modulate cell differentiation via PI3K/Akt pathway (Molina-Holgado et al., 2002, Gomez et al., 2011). Similarly, in rat cortical cultured neurons, a CB1R selective agonist, HU-210, exerts neuroprotective effect against neurotoxin (S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) through activation of PI3K/Akt pathway but not MAPK pathways (Molina-Holgado et al., 2005). A previous study in mouse demonstrated that acute administration of THC activated PI3K/Akt pathway, but not ERK1/2 in several brain regions (Ozaita et al., 2007). In addition, one recent study in huntingtin knock-in striatal neuronal cells showed that CB1R protected neurons against excitotoxicity via PI3K/Akt signaling-mediated increase in brain-derived neutrophic factor (BDNF) expression (Blazquez et al., 2015).  In comparison to CB1R, CB2R has started to gain significant attention only recently. As studies accumulate, an excellent review delineating CB2R signaling became available [see review in (Dhopeshwarkar and Mackie, 2014)]. Briefly, CB2R is coupled to Gi/o protein, inhibiting the activity of AC and subsequent formation of cAMP. So far, there is no report on CB2R switching coupling to other G proteins. Instead, depending on the microenvironment, CB2R may show different efficacies in cAMP inhibition through coupling to Gi. CB2R also activates ERK1/2 via Gi protein, as shown in transfected CHO-K1 cells. Moreover, CB2R-mediated ERK1/2 activation may be linked to the role of CB2R in immune systems [reviewed in (Dhopeshwarkar and Mackie, 2014)]. CB2R has also been shown to activate p38, JNK, PI3K/Akt, and ion channels, such as Ca2+ channels and GIRK, in both endogenous and heterogeneous expression systems [reviewed in (Atwood and Mackie, 2010)]. 25   1.3.6 Physiological and pathological roles of CB1R Given the widespread distribution of CB1R in the human body, it is reasonable for one to speculate a broad spectrum of physiological roles of CB1R (Pacher et al., 2006, Kano et al., 2009, Di Marzo et al., 2015, Maccarrone et al., 2015). Indeed, CB1R and endocannabinoid systems are largely involved in various aspects of central neural activities and disorders, including appetite, learning and memory, anxiety, depression, schizophrenia, stroke, MS, neurodegeneration, epilepsy, and addiction (Pacher et al., 2006, Kano et al., 2009, Di Marzo et al., 2015). It is also affected in physiological and pathological conditions in PNS and peripheral tissues, including pain, energy metabolism, cardiovascular functions, inflammation, glaucoma, cancer, liver disorders, musculoskeletal disorders, and reproductive functions (Maccarrone et al., 2015). The expression of CB1R largely fluctuates in many pathological conditions, underscoring its critical role in a wide spectrum of biological activities (Miller and Devi, 2011). CB1R has been found to inhibit GABA and glutamate release from presynaptic terminals, which renders CB1R the ability to modulate neurotransmission (Katona et al., 1999, Gerdeman and Lovinger, 2001). This has been proposed as a plausible underlying mechanism of CB1R-mediated neuroprotection against excitotoxicity, a prominent pathological process in many neurological disorders, including neurodegenerative diseases (Marsicano et al., 2003, Katona and Freund, 2008, Chiarlone et al., 2014). Altered expression of CB1R and other elements of endocannabinoid system have been observed in various neurodegenerative diseases, such as AD, PD and HD (Pacher et al., 2006). The upregulation of CB1R and endocannabinoid system activity has been observed in basal ganglia of experimental models of PD, which could be a compensatory mechanism to the degenerated dopaminergic neurons of substantia nigra, or a pathological process 26  that contributes to the worsening of disease (Brotchie, 2003). Although changes of CB1R expression in AD patients or animal models are still controversial, the activation of CB1R has been shown to prevent amyloid β-induced neurotoxicity in several cell models [reviewed in (Aso and Ferrer, 2014)]. Moreover, activation of CB1R has been reported to be beneficial to AD animal models with memory deficits and cognitive disorders [reviewed in (Aso and Ferrer, 2014)]. On the other hand, loss of CB1R has been observed in HD and augmentation of CB1R activity through genetic or pharmacological methods ameliorates HD symptoms (discussed in details in later section) (Glass et al., 1993, Blazquez et al., 2011, Naydenov et al., 2014). These observations support a critical and possibly beneficial role of CB1R in neurodegenerative diseases.  The historical records of the anti-epileptic effect of CB1R dates back for centuries (Mechoulam, 1986). Case reports on the beneficial effect of cannabinoids on epileptic patients became available only after the identification of THC. However, studies also suggested increased seizure frequency after marijuana smoking [reviewed in (Soltesz et al., 2015)]. This kind of paradoxical effect of cannabinoids on epilepsy is not only seen in human studies but has also been reported in animal models (Wallace et al., 2002, Clement et al., 2003). Activation of CB1R by anandamide has been shown to inhibit electroshock-induced seizures in rats (Wallace et al., 2002). Conversely, CB1R activation in FAAH knockout mice displays increased susceptibility to kainic acid-induced seizures (Clement et al., 2003). The alteration of endocannabinoid system following epilepsy is cell type-specific. This concept is supported by previous animal studies showing an enhanced CB1R retrograde signaling selectively at inhibitory but not at excitatory synapses, resulting a persistent potentiation of DSI but not DSE in febrile seizures, which leads to a hyperexcitability of neurons, thus contributing to the worsening of seizures (Chen et al., 2003, Chen et al., 2007). Such alternation happens to be phase-dependent as well. The hippocampal tissues from epileptic 27  patients in acute phase display decreased CB1R density, especially in the dentate gyrus, whereas in chronic phase of epilepsy, an upregulation of CB1R has been observed (Pacher et al., 2006, Soltesz et al., 2015).  Cannabinoids are long known for their effects to stimulate appetite, prominently in a CB1R-dependent manner (Di Marzo et al., 2001, Di Marzo and Matias, 2005, Pagotto et al., 2006). The level of endocannabinoids is increased in the rat hypothalamus during fasting and return to normal after food consumption (Kirkham et al., 2002). The stimulation of appetite and feeding behavior is observed after direct injection of endocannabinoid and abolished by the administration of CB1R antagonist (Kirkham et al., 2002). Furthermore, activation of CB1R on ventrostriatal GABAergic neurons results in a hypophagic but not orexinergic effect (Kirkham et al., 2002). Olfactory process has been proposed to be involved in the positive regulation of CB1R-mediated food intake (Soria-Gomez et al., 2014). A recent study has demonstrated that CB1R-induced feeding behavior is promoted by the activation of hypothalamic proopiomelanocortin (POMC) neurons (Koch et al., 2015). Moreover, crosstalk between CB1R and important hormones involved in appetite regulation, including ghrelin, leptin, and orexin, has been extensively reported [reviewed in (Di Marzo and Matias, 2005)]. These studies suggest that CB1R-mediated regulation of appetite is through multiple mechanisms.  The regulation of pain is one of the earliest medical applications of cannabinoids (Mechoulam, 1986, Iversen, 2000). Numerous studies have documented the analgesic effect of cannabinoids in different types of pain, including chemical, mechanical, and heat pain, as well as neuropathic, inflammatory, and cancer pain [reviewed in (Fine and Rosenfeld, 2013)]. The endocannabinoid system is also involved in the regulation of nociception [reviewed in (Pacher et al., 2006, Fine and Rosenfeld, 2013)]. In addition to CB1R, there is also evidence supporting the involvement of 28  CB2R and TRPV1 in cannabinoid-mediated regulation of pain (Di Marzo and De Petrocellis, 2012, Fine and Rosenfeld, 2013). Cannabinoids used in cancer is best-known for its palliative effects, including reducing nausea and vomiting, regulating cancer pain, and stimulating appetite (Guzman, 2003). However, it is argued that cannabinoids can exert anti-tumor effect directly through the inhibition of cell proliferation and induction of apoptosis, or indirectly through the inhibition of angiogenesis, invasion and metastasis (Velasco et al., 2012). Numerous studies using synthetic/endo-/phyto-cannabinoids and endocannabinoid system regulators in various cancer cell lines support this notion (Pisanti et al., 2013). The antitumor effects of cannabinoids have also been observed in various animal tumor models (Velasco et al., 2012). In general, an enhanced endocannabinoid system is seen in tumor tissues (Sanchez et al., 2001, Guzman, 2003, Caffarel et al., 2006). However, the role of upregulated endocannabinoid system activity is still controversial as opposing results have been reported supporting a proliferative as well as anti-proliferative role of cannabinoids on cancer cells (Velasco et al., 2012, Pisanti et al., 2013). Interestingly, a bimodal effect of cannabinoids on cancer cell growth has also been observed, with low concentration being proliferative and high concentration being proapoptotic (Hart et al., 2004).  1.4 GPCR oligomerization Dimerization and/or high-order oligomerization of GPCRs is one of the fundamental mechanisms of crosstalk between individual GPCRs (Ferre et al., 2014). Initially, it was believed that GPCRs exist and function exclusively as monomers (Ferre et al., 2014). In the last two decades, significant progress has been made to dissect out the molecular and pharmacological properties of GPCR homo- and heteromerization, opposing early concept that GPCR functions 29  exclusively in monomeric entity (Gomes et al., 2016). The most striking evidence emerged from the findings on class C GPCRs (e.g. GABAB receptors, Ca2+ sensing receptors, metabotropic glutamate receptors, and taste receptors) that dimerization is obligatory for plasma membrane delivery and proper functioning of receptors (Kniazeff et al., 2011). For most class A GPCRs, monomeric entities are sufficient for receptor to function effectively [reviewed in (Ferre et al., 2014)]. However, increasing evidence over the past two decades suggests that class A GPCRs can form homomers/heteromers, with novel functional and pharmacological properties distinct from the original protomers, as first described in delta (δ)-opioid receptor (OR)/kappa-OR heterodimers in 1999 (Jordan and Devi, 1999, Ferre et al., 2014, Gomes et al., 2016). Accumulated studies support the notion that the function of GPCR also depends on receptor oligomerization [reviewed in (Ferre et al., 2014, Gomes et al., 2016)]. Although most of these observations were conducted in heterologous systems, in recent years, several studies with advanced technologies have also demonstrated the presence of GPCR heteromers in native tissues with endogenous receptor expression (Albizu et al., 2010, Bellot et al., 2015). In order to delineate protein-protein interaction in GPCR oligomerization, several techniques and methodologies have been adopted [reviewed in (Gomes et al., 2016)]. Amongst them, morphological, pharmacological, biochemical, and biophysical methods are widely used (Kaczor and Selent, 2011, Gomes et al., 2016). Morphologically, the colocalization of protomers is usually determined by immunostaining using specific antibodies against each protomer. Pharmacological aspects of receptor-receptor interaction are assessed by ligand binding assay and fragmentation complementation assay. The classical biochemical approach to determine receptor-receptor interaction is co-immunoprecipitation (Co-IP) (Gomes et al., 2016). Co-IP is usually performed in solubilized cells, using protomer-specific antibody to precipitate the target oligomer, followed by 30  immunoblotting using a different antibody against another protomer (Kaczor and Selent, 2011). On the other hand, resonance energy transfer (RET) is one of the most frequently used biophysical methods to confirm the physical interaction between GPCR protomers [reviewed in (Gandia et al., 2008)]. RET takes advantage of energy transfer between two molecules in close proximity to assess receptor interaction (Gandia et al., 2008). Depending on the type of molecule, RET is divided into fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) (Gandia et al., 2008). Advanced applications based on FRET and BRET (e.g., sequential FRET-BRET and BRET plus biomolecular fluorescence complementation) have also been used to elucidate specific aspects of GPCR oligomerization (Carriba et al., 2008, Navarro et al., 2010). Besides, proximity ligation assay is also used in the detection of GPCR oligomers (Gomes et al., 2016). Recently, the increasing development of GPCR oligomer-specific ligands and antibodies has largely facilitated the study of selective GPCR oligomers in native tissues [reviewed in (Ferre et al., 2014)]. Although controversies exist in some studies, significant amount of data has been generated by using aforesaid techniques supporting GPCR oligomerization [reviewed in (Gomes et al., 2016)]. In recent decade, advanced techniques including single-molecule total internal reflectance fluorescence microscopy and fluorescence correlation spectroscopy with photon counting histogram have been developed to understand the molecular structure and structural-functional relationship of GPCR oligomers [reviewed in (Ferre et al., 2014)]. In search of oligomer interfaces, crystallization is frequently used to obtain high-resolution structures of oligomers in various conditions [reviewed in (Ferre et al., 2014)]. Considerable amount of data has since been generated, supporting the notion that oligomer interface varies from receptor to receptor, and even from conformation to conformation [reviewed in (Ferre et al., 2014)]. 31  As a consequence of receptor allosterism brought by receptor oligomerization, novel properties are seen in ligand affinity, receptor trafficking and signaling pathways (Figure 1.3) [reviewed in (Gomes et al., 2016)]. In comparison to native monomer, the ligand affinity of one or both protomers could be different due to the protein-protein interaction (Gomes et al., 2011). Oligomerization also affects the cell surface expression of protomer and/or receptor internalization in response to ligand (Gomes et al., 2016). Moreover, in most of the proposed scenarios, oligomerization leads to biased agonism/antagonism, meaning one particular signaling pathways (conducted via same or different G proteins, or G protein-independently) of the protomers is favored by the oligomer (Lohse, 2010). There is also a report on a novel signaling pathway that is seen only in heterodimer, not shared with either protomer (Bellot et al., 2015). Importantly, the functional and pharmacological changes brought by receptor oligomerization are largely receptor- and cell type-specific (Lohse, 2010).  Figure 1.3 GPCR dimerization and property changes.  The significance of oligomerization study comes from the therapeutic implications in allosteric selectivity (Gomes et al., 2011). Certain signaling pathway may be associated with a particular 32  beneficial outcome, whereas another leads to an undesired side effect. Oligomerization favors selective signaling pathways, which is promising to enhance beneficial outcomes while reduce side effects (Ferre et al., 2014). Moreover, a recent study unraveled atypical G protein signaling generated by dual ligand-driven heterodimerization between angiotensin II receptor 1 (AT1R) and α2CAR in both heterologous expression system and native tissue (Bellot et al., 2015). This finding revealed a heterodimer-specific signaling pathway that could serve as a novel target of drug discovery. Thus, compounds selectively targeting GPCR oligomers possess the potential of better therapeutic outcomes through allosteric modulation of protomer properties. Recently, eluxadoline, a drug targeting δ-OR/mu (µ)-OR heteromers has been approved by U.S. Food and Drug Administration for the treatment of diarrhea-predominant irritable bowel syndrome, supporting the significance of GPCR oligomers in drug discovery (Fujita et al., 2014, Keating, 2017).  1.4.1 SSTR oligomerization SSTRs form homo- and heterodimers within the SSTR family (Somvanshi and Kumar, 2012). All SSTRs except SSTR1 exist as preformed homodimers in heterologous expression systems (Rocheville et al., 2000b). SSTR2 homodimers dissociate upon agonist treatment as a premise of efficient receptor internalization (Grant et al., 2004a). SSTR3 homodimers dissociate in response to agonist treatment as well (War et al., 2011). But unlike SSTR2, internalization of SSTR3 as homodimer is possible (War et al., 2011). In contrast to SSTR2 and 3, agonist treatment induces homodimerization of both SSTR4 and SSTR5, which is closely associated with receptor internalization (Rocheville et al., 2000b, Somvanshi et al., 2009). SSTR1 remains monomer regardless of agonist treatment, although it does form heterodimers with other SSTRs (Rocheville et al., 2000b). Previous studies from our lab have shown that in CHO cells, SSTR1 forms 33  heterodimer with SSTR5 in an agonist-dependent manner (Rocheville et al., 2000b). This heterodimerization modifies receptor trafficking. Upon acute agonist treatment, SSTR1 internalizes with SSTR5 and is present in the same intracellular vesicle, whereas prolonged agonist treatment leads to the upregulation of SSTR1/5 expression on the membrane. This phenomenon is not seen in the case of SSTR1 and 4 (Somvanshi et al., 2009). As we have demonstrated in a later study, regardless of agonist treatment, SSTR1 and 4 do not form heterodimer in co-transfected HEK-293 cells (Somvanshi et al., 2009). Instead, SSTR1 is resistant to agonist-induced internalization whereas SSTR4 is not, further indicating their independent presence. On the other hand, SSTR4 forms heterodimer with SSTR5 when co-expressed in HEK-293 cells, suggesting the heterodimerization between SSTR family members is specific and selective (Somvanshi et al., 2009). Besides, SSTR2 has been shown to heterodimerize with SSTR5 and SSTR3. In HEK-293 cells, SSTR2 and 5 exist as preformed heterodimers, of which expression is increased upon SSTR2 activation (Grant et al., 2008). Moreover, SSTR2 agonist displays higher efficacy at inhibiting AC, activating ERK1/2, and inhibiting cell growth in co-transfected cells. The recycling rate of SSTR2 upon activation is also increased, further enhancing SSTR2-mediated signaling, whereas no significant change is seen upon SSTR5 activation alone (Grant et al., 2008). Similar property of signaling mediated by single receptor has been reported in HEK-293 cells co-expressing SSTR2 and SSTR3 of rat origin, where these two receptors form heterodimers, with SSTR2 completely dominates the signaling while SSTR3 appears to be non-functional (Pfeiffer et al., 2001). In contrast, heterodimers composed of hSSTR2 and 3 show distinct properties that either receptor agonist is able to trigger receptor internalization and downstream signaling (War and Kumar, 2012). 34  SSTRs have been reported to form heterodimers with various GPCRs, including D2R, βARs, ORs [reviewed in (Somvanshi and Kumar, 2012)]. Among these receptors, the most thoroughly studied is the heterodimerization with D2R. First report came out in the year of 2000, when Rocheville et al. described SSTR5 and D2R heterodimerization in co-transfected CHO cells and the enhanced cAMP inhibition upon simultaneous receptor activation (Rocheville et al., 2000a). Later it was reported that in heterologous expression systems, SSTR2 and D2R forms heterodimer, but in an agonist-dependent manner (Baragli et al., 2007). Similar to SSTR5/D2R, heterodimerization of SSTR2 and D2R is accompanied by an enhanced D2R signaling. However, in cultured striatal neurons, SSTR2/D2R heterodimers exist constitutively and dissociates upon D2R antagonist treatment (Baragli et al., 2007). Since these findings on interaction between SSTRs and D2R, efforts have been made to apply this new concept to finding new therapies for particular pathological conditions, especially neuroendocrine tumors and pituitary adenomas (Gatto and Hofland, 2011). Combined treatment of SST analogs and D2R agonists has been proved effective clinically (Pivonello et al., 2005, Colao et al., 2007). Chimeric molecules specifically targeting SSTR2/D2R or SSTR5/D2R have been introduced to enhance the beneficial effects of SSTR/D2R heterodimers in cancer therapy [reviewed in (Culler, 2011)].  1.4.2 CB1R oligomerization Numerous studies have shown that CB1R forms oligomers in various types of cells. The first study claiming the evidence of CB1R homomers was emerged in 2000 by using Co-IP, displaying a band of high molecular weight from rat brain membranes preparations, suggesting the presence of a constitutive multimeric complex of CB1R (Mukhopadhyay et al., 2000). This observation was confirmed by using several antibodies developed against different epitopes of CB1R and an 35  antibody preferentially recognizing CB1R homodimer in immunoprecipitation, immunoblotting and immunostaining studies (Hajos et al., 2000, Katona et al., 2001, Wager-Miller et al., 2002, Mackie, 2005a). CB1R has also been reported to form heteromers with its own family member, CB2R, in endogenous and recombinant expression systems (Callen et al., 2012). The authors also found that in transfected neuroblastoma cells, ligand (agonist or antagonist) binding to either receptor diminished CBR–induced Akt phosphorylation and neuritogenesis, which was termed as bidirectional cross-antagonism (Callen et al., 2012). Similar cross-antagonism in ERK1/2 phosphorylation was observed in CB1R/G protein-coupled receptor 55 heteromers in a heterologous expression system (Martinez-Pinilla et al., 2014). Besides its own family member, CB1R has been shown to dimerize with several other members of GPCR family (Wager-Miller et al., 2002, Kearn et al., 2005, Ellis et al., 2006, Rios et al., 2006, Carriba et al., 2007, Hudson et al., 2010a). The most thoroughly studied combination is CB1R/D2R (Hudson et al., 2010a). As early as 1997, Glass and Felder noticed that concurrent stimulation of CB1R and D2R in cultured rat primary striatal neuron lead to an accumulation of cAMP, despite the fact that activation of either CB1R or D2R inhibits cAMP formation via coupling to Gi (Glass and Felder, 1997). Later, Kearn et al. demonstrated the constitutive yet agonist-enhanced expression of CB1R/D2R heterodimer in heterologous system and confirmed the switch of G protein coupling from Gi to Gs in response to concurrent receptor stimulation (Kearn et al., 2005). The blockade of CB1R-dependent Gi signaling has also been described in the case of adenosine A2A receptor (A2AR)-CB1R heteromerization (Carriba et al., 2007). When co-expressed, CB1R is able to conduct an effective Gi signaling only when A2AR is co-activated (Carriba et al., 2007). Later, the same group detected the presence of A2AR/D2R/CB1R heteromer using sequential BRET-FRET in transfected HEK-293 cells and the potential regulatory role of 36  the oligomer in ERK1/2 phosphorylation in mice striatal slices, bringing this cross-talk to a more complicated level (Carriba et al., 2008, Navarro et al., 2010). Moreover, levodopa, the most common medication for PD, has been shown to disrupt A2AR/D2R/CB1R heteromer in rats and primates, implying a potential therapeutic mechanism and novel pharmaceutical target (Bonaventura et al., 2014, Pinna et al., 2014). CB1R/µ-OR heteromer has also been observed in transfected cells and is characterized by a reciprocal antagonism that activation of one receptor leads to the attenuation of the other receptor-mediated signaling (Rios et al., 2006). Later this effect was argued to be attributed to CB1R constitutive activity (Canals and Milligan, 2008). In the case of CB1R/orexin receptor 1, treatment of either antagonist not only results in translocation of both receptors together from intracellular vesicles to the cell surface, but also decreases the potency of the other receptor agonist to activate ERK1/2 (Ellis et al., 2006). CB1R and AT1R form heteromer in transfected cells, resulting in the potentiation of AT1R signaling. The heteromer is as well found in hepatic stellate cells, where it is upregulated under pathological conditions (Rozenfeld et al., 2011). Moreover, the potentiated AT1R-mediated signaling and subsequent mitogenic and profibrogenic effect can be blocked by CB1R antagonist (Rozenfeld et al., 2011). Other GPCRs have been shown to heteromerize with CB1R include β-2AR and δ-OR (Hudson et al., 2010b, Rozenfeld et al., 2012).  1.5 Excitotoxicity Excitotoxicity refers to a pathological situation where over-excitation of neuronal cells by excitatory neurotransmitters induces neurotoxicity, or even cell death. This phenomenon was first described in 1957 and formally termed in 1978. In the CNS, excessive glutamate release and/or insufficient glutamate clearance results in prolonged opening of ionotropic glutamate receptors, 37  especially NMDARs, allowing continuous influx of Ca2+. Intracellular Ca2+ overload triggers a series of signaling cascades, including activation of Ca2+-dependent enzymes, generation of oxidative stress, dysfunction of mitochondria and endoplasmic reticulum (ER), finally leading to cell death [reviewed in (Hara and Snyder, 2007)]. Recent studies on excitotoxicity have revealed a more complex picture than the classical one described above. Excessive release of glutamate from the presynaptic terminals activate both synaptic and extrasynaptic NMDARs. The subsequent effect is opposite, as synaptic NMDARs activate multiple neuroprotective pathways including the stimulation of cAMP response element-binding protein (CREB) function through nuclear Ca2+ signaling, whereas extrasynaptic NMDARs induce CREB dephosphorylation and several other pro-apoptotic signaling cascades [reviewed in (Lewerenz and Maher, 2015)]. On the other hand, glutamate released from astrocytes is also able to activate extrasynaptic NMDARs (Angulo et al., 2004). In addition to glutamate, other excitotoxins, including quinolinic acid (QUIN), an endogenous metabolite of tryptophan metabolism, have also been demonstrated to induce excitotoxicity (Lewerenz and Maher, 2015). Excitotoxicity is a prominent pathogenic process in many neurodegenerative diseases, both acute and chronic (Doble, 1999). It is well documented that excitotoxicity induced by abrupt insult plays a critical role in acute neurodegenerative diseases including stroke, traumatic brain injury, and status epilepticus (Doble, 1999, Lewerenz and Maher, 2015). These diseases all feature sudden excessive glutamate release from neurons due to membrane depolarization or mechanical damage that leads to neuronal cell death (Doble, 1999, Lewerenz and Maher, 2015). However, in the case of chronic neurodegenerative diseases, excitotoxicity is more like a secondary pathogenic factor and the extent of its contribution to the gradual progression of disease pathogenesis is not clear (Lewerenz and Maher, 2015). Nevertheless, a significant amount of data has been generated 38  supporting the involvement of excitotoxicity in chronic neurodegenerative diseases including amyotrophic lateral sclerosis, AD, PD and HD (Lewerenz and Maher, 2015).  1.5.1 Huntington’s disease HD is an inherited disease caused by an expansion of CAG repeats on exon 1 of huntingtin gene located on chromosome 4, with a threshold of HD being 35 repeats or more. The length of CAG repeats is related to the age of onset of disease in an inverse way that individual carrying longer CAG repeats tend to develop HD in an earlier stage of life (Zuccato et al., 2010, Ross and Tabrizi, 2011). The mean age of HD onset is 40 years-old, and usually will lead to death after 15-20 years. The prevalence of HD in Western European population is 4-10 per 100,000 (Zuccato et al., 2010, Ross and Tabrizi, 2011). However, due to the unstable and expanding nature of CAG repeats, individuals having 27-35 repeats are at risk of developing HD or passing HD to their offspring. Typical symptoms of HD include progressive emotional, motor and cognitive disorders that eventually lead to severe dementia, rendering the patient extremely vulnerable (Zuccato et al., 2010, Ross and Tabrizi, 2011). The key pathological change in HD is the prominent atrophy and neuronal cell loss in striatum, particularly the preferential degeneration of medium-sized spiny neurons (MSNs) and selective sparing of medium-sized aspiny interneurons positive to SST, neuropeptide Y (NPY), and neuronal nitric oxide synthase (nNOS) (Ferrante et al., 1985, Reiner et al., 1988, Ferrante et al., 1997). In advanced stages of HD, both spiny and aspiny neurons are markedly degenerated and other parts of the brain are greatly damaged as well (Zuccato et al., 2010). Encoded by huntingtin gene, Huntingtin (Htt) is a 347 kilodalton (kDa) protein expressed ubiquitously in human and rodents, with CNS being the most concentrated region. The 39  polyglutamine (polyQ) tract starts at 18th amino acid near the N-terminus, and forms a polar zipper structure that binds to transcription factors that contain a polyQ region to modulate gene transcription [reviewed in (Zuccato et al., 2010)]. Later researchers found that Htt interacts with a wide range of proteins, underscoring its critical role in embryonic development, anti-apoptosis, axonal and vesicle transportation, and synaptic activity [reviewed in (Zuccato et al., 2010)]. Inactivation of huntingtin gene leads to embryonic death. Mutant Htt (mHtt) with an elongated polyQ tract has a tendency to form cytoplasmic aggregates and nuclear inclusion, which is one of the hallmarks of HD [reviewed in (Zuccato et al., 2010)]. Studies have suggested that aggregates and inclusions could be pathogenic, although disputes exist as there is evidence supporting a neuroprotective role against the toxic soluble fragments of mHtt (Zuccato et al., 2010, Miller et al., 2011). The mutation of Htt leads to a situation where the normal Htt function is lost, along with increased toxicity in the neurons, resulting in massive neurodegeneration (Zuccato et al., 2010). Several other molecular pathways have been proposed to contribute to the pathogenesis of HD, however, the exact underlying mechanism is not fully elucidated yet [reviewed in (Zuccato et al., 2010)]. Excitotoxicity is proposed to be a pathogenic mechanism of HD. The first evidence supporting this hypothesis came from the replication of the histological, biochemical and behavioral changes of HD by intrastriatal injection of QUIN, an endogenous NMDAR agonist in rodents and primates (Beal et al., 1986, Beal et al., 1991, Ferrante et al., 1993). Later it was found that QUIN levels in cortex and striatum were increased in HD patients and animal models (Guidetti et al., 2004). Moreover, decreased glutamate uptake and enhanced NMDAR sensitivity were also reported, supporting the role of excitotoxicity in HD (Fan and Raymond, 2007).  40  mHtt has been shown to participate in several aspects of downstream signaling pathways in excitotoxicity. First, normal Htt is associated with NMDAR via postsynaptic density protein 95 (PSD95) (Sun et al., 2001). Such association is weakened by expanded polyQ tract in mHtt, leading to NMDAR sensitization (Sun et al., 2001). mHtt also induces mis-localization of PSD95-NMDAR subtype 2B to extrasynaptic site, thus enhancing NMDAR-mediated toxicity (Fan et al., 2012); second, mHtt sensitizes inositol 1,4,5-triphosphate receptors on ER, causing increased Ca2+ release from ER to cytoplasm (Tang et al., 2003, Tang et al., 2005); third, mHtt directly decreases the Ca2+ threshold required to open mitochondrial permeability transition pore, that will dissipate mitochondrial membrane potential, leading to mitochondrial damage, excessive Ca2+ release, increased oxidative stress and initiation of apoptosis [reviewed in (Zuccato et al., 2010)].  1.5.2 SST in excitotoxicity and HD The role of SST as neurotransmitter and neuromodulator has been shown in regulation of several key neurotransmitters, including dopamine, GABA, and glutamate (Patel, 1999). Previous studies have shown that glutamate induces SST release via the activation of NMDAR, whereas SST in turn inhibits excitatory synaptic transmission (Grilli et al., 2004). SST-mediated activation of SSTR2 at the presynaptic terminals of mouse cerebrocortical neurons inhibits the release of glutamate, implicating its role in excitotoxicity (Grilli et al., 2004). It has been shown later in rat Schaffer collateral-CA1 synapses, that SSTR2 activation prevents status epilepticus via inhibition of glutamate release (Kozhemyakin et al., 2013). The neuroprotective role of SST has been observed in various neurodegenerative disorders including AD, PD, HD (Epelbaum et al., 1983, Burgos-Ramos et al., 2008, Tallent and Qiu, 2008). Unlike several neurotransmitters and neuropeptides that have been reported to decrease in HD, 41  two independent studies by Aronin et al. and Nemeroff et al. described respectively that SST levels were increased in HD striatum and globus pallidus (Aronin et al., 1983, Nemeroff et al., 1983). Later, two studies not only confirmed the increase of SST levels in human HD basal ganglia, but also reported the selective sparing of a subpopulation of striatal medium-sized aspiny interneurons co-expressing SST, NPY and nNOS in HD striatum, unveiling another key character of HD besides the preferential degeneration of MSN (Vincent and Johansson, 1983, Dawbarn et al., 1985, Ferrante et al., 1985). One study from our lab described that, QUIN, a substance used to resemble HD, increases SST mRNA expression and SST release in medium and cellular content (Beal et al., 1986, Patel et al., 1991). Further study using cultured rat primary striatal neurons revealed the neuroprotective effect of SST against N-methyl-D-aspartate (NMDA) - and/or QUIN-induced cell death (Kumar, 2008). This effect was blocked by PTX, suggesting a role of SSTRs (Kumar, 2008). Accordingly, our lab also reported that SSTR1 and 5 double knockout mice resembles the neurochemical changes as seen in HD transgenic R6/2 mice, emphasizing the role of SSTRs in HD pathology (Rajput et al., 2011). On the other hand, others have shown that SSTR2 is expressed in MSN and SSTR2 knockout mice display impaired motor coordination, further implicating the involvement of SSTRs in motor disorders (Allen et al., 2003).  1.5.3 CB1R in excitotoxicity and HD As stated earlier, numerous studies have shown that CB1R plays a neuroprotective role against excitotoxicity induced by various stimulus (Marsicano et al., 2003, Kim et al., 2006a, Blazquez et al., 2011, Zoppi et al., 2011). It has been demonstrated recently that in mouse brain, the neuroprotective effect exerted by CB1R against excitotoxicity is restricted to the CB1R population located on glutamatergic terminals (Chiarlone et al., 2014). In addition to the prominent inhibitory 42  effects on Ca2+ influx and glutamate release, CB1R-mediated neuroprotection also involves inhibition of nitric oxide (NO) production, reduction of zinc mobilization, and increase of BDNF expression (Khaspekov et al., 2004, Kim et al., 2006a, Sanchez-Blazquez et al., 2013). Recent studies even implicated a direct physical interaction between CB1R and NMDAR in the aid of histidine triad nucleotide-binding protein 1, which allows CB1R to negatively regulate NMDAR activity and protects neural cells from excitotoxicity (Sanchez-Blazquez et al., 2013, Vicente-Sanchez et al., 2013). The neuroprotective effects of cannabinoids in both acute and chronic neurodegenerative diseases are well documented (Pacher et al., 2006). As CB1R is highly expressed in MSNs, which account for the majority of striatal neurons and preferentially degenerate in HD, it is reasonable to predict that CB1R is largely affected in HD (Graveland et al., 1985, Herkenham et al., 1990, Marsicano and Lutz, 1999). Decreased expression of CB1R has been first reported in 1993 in substantia nigra of HD patients using autoradiography (Glass et al., 1993). Further studies revealed the loss of CB1R as an early sign of HD before the onset of actual neuronal cell loss, and is progressive along the worsening of HD (Glass et al., 2000). This observation is confirmed at mRNA level as well as CB1R immunoreactivity in several transgenic HD mice models [reviewed in (Pacher et al., 2006)]. A recent study described downregulation of CB1R not only in MSNs but also in a subpopulation of interneurons positive to NPY/nNOS/SST in both transgenic HD mice and HD patients (Horne et al., 2013). A delayed loss of CB1R in HD transgenic mice R6/1 was seen in enriched environment, accompanied by delays onset of motor disorders and slows disease progression (Glass et al., 2004). Moreover, knockout of CB1R leads to the worsening of motor performances, increased susceptibility to 3-nitropropionic acid (3-NP), and exacerbated striatal atrophy and Htt aggregates in HD transgenic mice R6/2 (Blazquez et al., 2011, Mievis et al., 2011). 43  Selective increase of CB1R expression in MSNs improves the survival of excitatory projection neurons, but not motor performances of HD transgenic R6/2 mice (Naydenov et al., 2014). Administration of THC has been reported to ameliorate motor disorders, striatal atrophy and Htt aggregates in transgenic mice, although controversies exist (Dowie et al., 2010, Blazquez et al., 2011). Activation of CB1R inhibits glutamate release but increases BDNF release from presynaptic terminals in mice (Marsicano et al., 2003). Further investigation in HD cell models discovered that CB1R activation can protect striatal cells against excitotoxicity through increase of BDNF expression via PI3K/Akt pathway (Blazquez et al., 2011).  1.6 STHdh cell lines To elucidate the complex molecular mechanism associated with neuronal cell death in HD, striatal neuronal cultures treated with either QUIN, 3-NP, or mHtt were widely studied in the past (Zuccato et al., 2010). In 2000, Dr. MacDonald’s group established STHdh cells, a striatal cell line expressing normal and mHtt (Trettel et al., 2000). STHdh cells (STHdhQ111/Q111 and STHdhQ7/Q7) are striatal progenitor cells from E14 mutant HdhQ111/Q111 knock-in mouse and wild-type (wt) littermate embryos, conditionally immortalized by the temperature-sensitive mutant of SV40 Large T Antigen (Cattaneo and Conti, 1998, Wheeler et al., 2000). At 33 Celsius degree (°C), homozygous mutant cells have longer doubling time than wt cells, while both cease proliferation at nonpermissive temperature (39 °C) (Trettel et al., 2000). STHdh cells express nestin, and can be differentiated in growth medium supplemented with a “dopamine cocktail” into a microtubule-associated protein 2-positive, neuronal-like morphology with multiple processes (Trettel et al., 2000). It has been reported that after overnight serum deprivation, STHdh cells exit cell cycle, increase the expression of dopamine and cAMP-regulated phosphoprotein of 32 kDa 44  and D2R, and enhance neurite outgrowth, making the cells a model of MSN (Trettel et al., 2000). With these unique neuronal-like features, STHdh cells serve as a suitable model to delineate the molecular mechanism involved in neuronal cell death in HD. Htt with 7 (wt) or 111 (mutant) glutamine tract is well-expressed in variants of STHdh cells, in alternate forms in both cytoplasm and nucleus (Trettel et al., 2000). STHdhQ111/Q111 cells display an increased p53 levels, an enlarged ER, and a higher basal activity of iron pathway, representing a stressed phenotype induced by mHtt (Trettel et al., 2000). Gines et al. reported an enhanced basal Akt activity in mutant cells, which is PI3K-, NMDAR-, and Ca2+-dependent, representing an early pro-survival response to abnormal NMDAR activity and mitochondrial deficits caused by mHtt (Gines et al., 2003a). Tropomyosin receptor kinase B (TrkB) expression is reported to be decreased in mutant cells (Gines et al., 2010). As a consequence, TrkB-mediated activation of MAPK/ERK1/2 but not PI3K/Akt or PLCγ is impaired, which leads to an enhanced susceptibility to oxidative stress in mutant cells (Gines et al., 2010). Further characterization revealed a reduced basal cAMP synthesis and an impaired mitochondrial respiratory chain, implicating energy deficits in mutant cells (Gines et al., 2003b). Follow-up study discovered that mHtt on the outer membrane of mitochondria largely decreased the Ca2+ threshold for the opening of mitochondrial permeability transition pore, rendering mutant cells more susceptible to 3-NP (Choo et al., 2004, Ruan et al., 2004). Later studies showed that mitochondrial ATP production is decreased in mutant cells, leading to a poorer ability to handling Ca2+ flux in comparison to wt cells (Ruan et al., 2004, Milakovic and Johnson, 2005, Seong et al., 2005, Oliveira et al., 2006). A gene expression analysis discovered that mHtt did not significantly affect mitochondrial pathways in STHdhQ111/Q111 cells, except decrease in peroxisome proliferator-activated receptor γ coactivator 1-α expression (Cui et al., 2006, Lee et al., 2007). Instead, the impaired mitochondrial 45  energy metabolism may be an indirect result of abnormal Htt functions induced by expanded polyQ tract.  1.7 Summary of background SST was first identified in hypothalamus as a GH-releasing inhibitory peptide and later found to be widely expressed throughout the CNS and many other peripheral tissues. Along with its wide distribution, SST plays a critical role not only in many physiological processes, including hormone secretion, appetite regulation, neurotransmission, and neuromodulation, but also in pathological conditions such as epilepsy and neurodegeneration. Such diverse and fundamental effects of SST are mediated through the binding to five specific receptors, namely SSTR1-5, all belonging to the GPCR family. Cannabinoids have long been used in medical practice for seizure alleviation, pain regulation, and appetite control. However, prolonged usage of cannabinoids have been largely limited due to psychoactive effect and drug addiction. Cannabinoids bind to CBRs, which are members of the GPCR family. CB1R is the predominant subtype in CNS and the major subtype associated with cannabinoids-mediated neurotransmission and neuromodulation. CB1R also plays a crucial role in physio-/pathological conditions including pain management, appetite control, epilepsy and neurodegeneration. Studies have shown significant overlapping properties between these two receptor subtypes: (i) SSTR and CB1R have been described respectively to be expressed in the same brain regions. (ii) Both SSTR and CB1R have been shown to form oligomers with other GPCRs. (iii) SSTR and CB1R are deeply involved in neurotransmission and neuromodulation. Moreover, both have been implicated to exert neuroprotective effects in excitotoxicity. 46  Given this compelling evidence, we propose possible interactions between SSTR2 or SSTR5 and CB1R which might serve as a new therapeutic target in neurological disorders.  1.8 Hypothesis From the preceding discussion, the emerging hypothesis is “SSTR2/CB1R and SSTR5/CB1R may function in heteromeric complex as novel receptor entities with distinct pharmacological and signaling properties that might play an important role in excitotoxicity via modulation of signaling pathways responsible for neuronal cell death and survival.”  1.9 Specific Aims Specific Aim 1: To determine the colocalization and interaction of SST/CB1R in rat brain. Specific Aim 2: To determine the functional consequences of receptor heterodimerization in stable mono- and cotransfected HEK-293 cell. Specific Aim 3: To determine whether SSTR2/CB1R and SSTR5/CB1R interact and modulate excitotoxicity in in vitro HD model.  47  Chapter 2: Colocalization of CB1R with SST and nNOS in rat brain hypothalamus 2.1 Background The hypothalamus exhibits a complex array of functions in response to various stimuli and is known to be a prominent physiological regulator of the CNS. Several neurotransmitters, neuropeptides and receptor proteins are well expressed in hypothalamus. Amongst them, endocannabinoids play a crucial role in various biological activities, including appetite stimulation, energy balance, pain relief, cognition, memory, anxiety, motor behavior, and autonomic and neuroendocrine responses (Marsicano et al., 2003, Pacher et al., 2006, Castillo et al., 2012, Di Marzo et al., 2015, Maccarrone et al., 2015). The cannabinoids are also linked to MS, schizophrenia and depression (Pacher et al., 2006). Such different arrays of biological effects in the CNS and PNS are mediated by two CBRs, namely CB1R and CB2R, which belong to the GPCR superfamily. CB1R is highly expressed in CNS in a region-specific manner in different neuronal population, while CB2R is predominantly present in peripheral tissues. Despite low expression in the hypothalamus, CB1R is crucially involved in regulation of food intake and energy balance through hypothalamic circuits (Di Marzo and Matias, 2005, Bellocchio et al., 2008). The levels of endocannabinoids increase in rat hypothalamus during fasting and return to normal after food intake (Hanus et al., 2003). The stimulation of appetite is observed after direct injection of endocannabinoid into rat ventromedial hypothalamic nucleus (VMH) and abolished by the administration of CB1R antagonists (Jamshidi and Taylor, 2001). Also, in target tissues, the biological effects of endocannabinoids are independent of CBRs (De Petrocellis and Di Marzo, 48  2010). These observations indicate the possibility of direct and indirect role of other hormones in association with CB1R in regulation of appetite. SST, also known as somatotropin release-inhibiting factor, was first isolated from hypothalamus as GH inhibitory peptide (Brazeau et al., 1973). SST positive neurons are present throughout the brain with enriched SST like immunoreactivity in hypothalamus, specifically in the periventricular nucleus (PeVN) constituting 80% of SST immunoreactivity in CNS (Finley et al., 1981). Relatively low expression of SST occurs in the paraventricular nucleus (PVN), suprachiasmatic nucleus, arcuate nucleus (AN), and VMH (Johansson et al., 1984). SST via five different receptor subtypes play various crucial roles in mammalian brain including neurodegenerative and neuropsychological disorders, pain relief, inflammation and nociception (Kumar and Grant, 2010). In the hypothalamus, SST not only serves hypophysiotropic function, but also influences several other neuronal functions through local circuits. For instance, SST regulates the release of GHRH, corticotropin releasing factor (CRF), norepinephrine and SST itself (Patel, 1999). Recent studies have described potential role of SST in inhibition of ghrelin, one of the major hormones affecting food intake and energy balance, indicating the role of SST in regulation of appetite (Silva et al., 2005, Seoane et al., 2007). Also, the presence of SSTRs in 35% POMC mRNA-containing neurons in AN might support this notion (Fodor et al., 1998). NO is the most prominent second messenger in CNS and PNS, where it is produced mainly by nNOS. Previous studies have shown that nNOS positive neurons are well expressed in hypothalamus, the brain region displaying highest concentration other than cerebellum (Rodrigo et al., 1994). In hypothalamus, nNOS is associated with several physiological functions, including energy balance and regulation of appetite (Calabrese et al., 2007). Inhibition of nNOS leads to decreased food intake and weight loss while food deprivation results in enhanced nNOS activity 49  (Ueta et al., 1995, O'Shea and Gundlach, 1996). In addition, nNOS has also been implicated in modulation of several appetite-related hormones, including ghrelin, NPY and leptin. Ghrelin and NPY have been shown to increase nNOS in hypothalamus while leptin does the opposite (Morley et al., 1999, Gaskin et al., 2003). Furthermore, ghrelin-induced feeding is blocked by nNOS inhibitor, and leptin-regulated body energy control requires hypothalamic nNOS-positive neurons (Gaskin et al., 2003, Leshan et al., 2012). The comparable distributional pattern and overlapping functional roles suggest that CB1R in hypothalamus might colocalize with SST and nNOS. However, to date, no study has shown whether SST and nNOS are coexpressed in CB1R positive neurons in hypothalamus. On the other hand, SST and nNOS have been shown to express in the same neuron in cortical and striatal regions. However, whether they colocalize in hypothalamus is not known. Accordingly, in the present study, we investigated CB1R colocalization with SST and nNOS in hypothalamus. Taken together, our results showed region specific colocalization of CB1R with SST and nNOS, as well as colocalization of SST and nNOS in rat brain hypothalamus.  2.2 Materials and Methods 2.2.1 Animals Male Sprague-Dawley rats (body weight 200-250 g) were obtained from the University of British Columbia animal care unit. The protocols regarding animal care were followed in compliance with the principles of the Canadian Council on Animal Care and were approved by the University of British Columbia Animal Care Committee. 2.2.2 Materials SST mouse monoclonal antibody (Cat. No. sc-74556) and CB1R goat polyclonal antibody (Cat. No. sc-10066) were purchased from Santa Cruz (Dallas, TX). nNOS rabbit polyclonal antibody 50  (Cat. No. AB5380) was purchased from Millipore (Billerica, MA). Normal goat serum (NGS) was purchased from Vector Laboratories (Burlingame, CA). Fluorescein isothiocyanate (FITC) and cyanine 3 (Cy3)-conjugated goat anti-mouse, goat anti-rabbit and donkey anti-goat secondary antibodies were obtained from Jackson Laboratories (West Grove, PA). All other reagents and chemicals were of analytical grade and obtained from various commercial sources. 2.2.3 Immunocytochemistry Rats were perfused with 4% paraformaldehyde freshly prepared in 0.1M phosphate-buffered saline. Brains were taken out and post-fixed in same perfusion solution for 2 hours (h). 40 micrometer (μm) thick free-floating brain sections passing through hypothalamus were processed for indirect double label immunofluorescence colocalization as described previously (Kumar, 2007). Briefly, rat brain sections were incubated in 5% NGS and 0.2% Triton X100 in Tris-buffered saline (TBS) for 1 h at room temperature. Sections were then incubated overnight at 4o C in a humid atmosphere with anti-goat CB1R (1:200) and anti-mouse SST (1:200) or anti-rabbit nNOS (1:200) antibodies in TBS containing 1% NGS. Brain sections were then washed in TBS for 3 times and incubated for 2 h in presence of FITC and Cy3-conjugated secondary antibodies for the final colour development. Following three subsequent washes in TBS, brain sections were mounted onto the slide and photographed using Carl Zeiss confocal microscope (LSM 700, AxioObserver). All the photographs composites were made using Adobe Photoshop (San Jose, CA) and merged images showing colocalization were generated in ZEN Lite blue edition software (Jena, Germany). 2.2.4 Western Blot Analysis CB1R immunoreactivity in hypothalamic tissue lysate was determined using Western blotting analysis as previously described (Somvanshi and Kumar, 2014). Briefly, 15 microgram (µg) of 51  tissue protein were solubilized in Laemmli sample buffer and fractionated on 10% sodium dodecyl sulfate (SDS) - polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membrane. Membrane was probed with goat anti-CB1R antibody (1:500) overnight. Following three subsequent washes membrane was incubated in donkey anti-goat secondary antibody and signals were developed with chemiluminescence and photographed using FluorChem software (Alpha Innotech). 2.2.5 Quantitative Analysis The relative percentage distribution of neurons displaying colocalization between SST, CB1R and nNOS was determined as previously described (Kumar, 2007). 40 µm coronal rat brain sections passing through hypothalamus were double-labelled for SST, CB1R and nNOS and processed for quantitative analysis using Carl Zeiss confocal microscope (LSM 700, AxioObserver) at 20X magnification. Neurons were considered immunoreactive if the staining of soma was distinctively stronger than background. Neurons showing immunoreactivity in both green and red channels, represented by orange to yellow in merged images, were considered showing colocalization. Five to ten sections per brain obtained from five different rat brains were subjected for quantification. 8~10 areas in interested regions were randomly selected for neuron-counting in each section. Only neuronal cell bodies showing intact morphology and immunoreactivity were counted. No dot or nerve fibre-like structures were included in this quantitative analysis. A total of 250~300 neurons positive to given proteins were analysed for colocalization in each combination. The mean percent of neurons showing colocalization was calculated from total neurons and results are presented as mean ± standard error (SE) (n=5).  52  2.3 Results 2.3.1 Specificity of immunoreactivity and colocalization In the present study, to determine the specificity of CB1R antibody, wt and CB1R transfected HEK-293 cells were used. As shown in Figure 2.1A, CB1R like immunoreactivity was not seen in wt HEK-293 cells whereas CB1R-transfected cells showed intensive receptor like immunoreactivity at cell surface (Figure 2.1B). On the other hand, CB1R transfected cells in absence of primary antibody were devoid of receptor-like immunoreactivity (Figure 2.1C). To further characterize the possibility of any bleed through, rat brain sections were processed for SST primary antibody and then exposed to both FITC and Cy3-conjugated secondary antibodies. Specific staining was detected in green channel but not in red channel (Figure 1D-F). We also characterized the specificity of CB1R antibody in rat hypothalamic tissue lysate using Western blot. As shown in Figure 1G left panel, two strong bands were observed at 64 and 53 kDa respectively, representing the glycosylated and non-glycosylated form of CB1R, whereas no such band was detected in the absence of primary antibody. Furthermore, the specificity of CB1R antibody used in this study has been demonstrated by Sanford et al. by immunohistochemistry using antibody preadsorbed with blocking peptide (Sanford et al., 2008). Several studies using this antibody revealed a similar distributional pattern of CB1R immunoreactivity which mainly confined in soma and cytoplasm (Kalifa et al., 2011). Taken these results together, the immunoreactivity seen in presence of selective antibodies was considered specific. 53   Figure 2.1 Photomicrographs illustrating the specificity of CB1R antibody and immunofluorescence in HEK-293 cells, hypothalamus section and tissue lysate. Wild-type and CB1R transfected HEK-293 cells were incubated with CB1R antibody at a dilution of 1:200 overnight at 4 °C, followed by Cy3-conjugated secondary antibody (dilution 1:800) for 2 h at room temperature. In wild-type cells (panel A), no receptor like immunoreactivity was detected, whereas transfected cells exhibited strong cell surface immunoreactivity for CB1R (arrows, panel B). CB1R transfected cells probed only with secondary antibody in absence of primary antibody were devoid of specific fluorescence (panel C). In the lower panel, hypothalamic section stained for SST showed only green fluorescence (panel D) but not red (panel E). No bleed through was detected. In panel G, representative Western blots analysis showed CB1R expression in rat hypothalamus tissue lysate. Rat hypothalamus tissue lysate was processed for Western blot analysis as described in Material and Methods section. In the presence of CB1R primary antibody, two strong bands were detected at the expected molecular sizes of 64 and 53 kDa respectively, representing the expression of CB1R in hypothalamus. No band was observed in the absence of receptor specific antibody (right panel). Scale bar = 20 µm (panels A-C) and 40 µm (panels D-F) respectively.  54  Indirect immunofluorescence analysis of CB1R like immunoreactivity revealed that CB1R was well expressed in all major nuclei of hypothalamus. The intensity of receptor like immunoreactivity varied from mild to strong, and was observed on the membrane as well as inside the cells. In PVN, CB1R positive neurons showed strong colocalization with SST, whereas some neurons expressing SST were devoid of CB1R immunoreactivity (Figure 2.2A-C). SST-positive neurons were expressed in high density in PeVN when compared to other regions. As shown in Figure 2.2D-F, in PeVN, large percentage of SST-positive neurons exhibited colocalization with CB1R. In neuronal cells, SST like immunoreactivity was primarily confined in apical ending of neuronal cell bodies with strong colocalization with CB1R (Figure 2.2F). Furthermore, neuronal processes displayed variable degree of colocalization and ependymal cells of third ventricle exhibited weak SST and CB1R like immunoreactivity with poor colocalization (Figure 2.2F). In addition, neuronal populations either expressing CB1R or SST like immunoreactivity were also present in PeVN (Figure 2.2F). In contrast, sparsely distributed SST positive neurons showing colocalization with CB1R or neuronal population displaying only CB1R like immunoreactivity were seen in VMH (Figure 2.2G-I). In AN, neurons identified exhibit strong to moderate colocalization between SST and CB1R (Figure 2.2J-L). In median eminence (ME), SST and CB1R like immunoreactivity was mostly seen in nerve fibers (Figure 2.2M-O). Dorsomedial hypothalamic nucleus was devoid of SST and CB1R like immunoreactivity (data not shown).  Quantitative analysis of SST and CB1R colocalization revealed significantly distinct pattern of coexpression in different hypothalamic nuclei. As shown in Fig 2.3, in PVN, 59.38% neurons exhibited colocalization, whereas in PeVN and VMH, 66% and 25% neurons displayed colocalization between SST and CB1R respectively. 55   56  Figure 2.2 Representative confocal photomicrographs illustrating the colocalization of SST and CB1R in hypothalamus.  Rat brain sections were incubated overnight with SST monoclonal (1:200) and anti-goat CB1R antibodies (1:200). Following three washes in TBS, sections were incubated with goat anti mouse FITC and donkey anti goat Cy3-conjugated secondary antibodies as described in Materials and Methods section. Note three distinct neuronal populations displaying either colocalization or individual staining for SST and CB1R in different hypothalamic regions. Neurons positive to SST (arrowheads), CB1R (tilted arrows) and colocalization (arrows) in merged images are indicated in respective panel. Neuronal processes and nerve fibers showing colocalization are indicated by asterisks in individual merged panel. In PVN, strong colocalization immunoreactivity was observed (arrows, in panel C). In PeVN, neurons expressing either SST (arrowheads) or CB1R (tilted arrows) and colocalization (arrows) were seen (panel D-F). The majority of neurons in VMH were either positive to only CB1R (tilted arrows) or displaying colocalization (arrows, panel G-I). Neuronal cell bodies in AN (panel J-L) and innervated nerve fibers in ME (panel M-O) exhibited strong colocalization between CB1R and SST. In addition, neuronal processes and nerve fibers were seen positive to CB1R throughout the hypothalamus (asterisks, panel C, F). Scale bar = 40 µm.  Figure 2.3 Quantitative analysis of neurons coexpressing SST and CB1R in three hypothalamic nuclei.  Neurons positive to SST and/or CB1R were counted from PeVN, PVN, and VMH and data were presented as the percentage of neurons coexpressing SST and CB1R in neurons positive to either SST or CB1R. Data were collected from 5 rat brains and a total number of 250~300 neurons were analyzed. Bars present the mean ± SE (n=5). 57  2.3.2 CB1R and nNOS are coexpressed in hypothalamus Using double labeled immunofluorescence immunocytochemistry, three different populations either exhibiting nNOS or CB1R like immunoreactivity and colocalization between nNOS and CB1R were observed. As shown in Figure 2.4, mild to strong colocalization between nNOS and CB1R were present in most nuclei of hypothalamus, including PVN (A-C), PeVN (D-F), VMH (G-I) and AN (J-L). Neuronal populations devoid of colocalization but positive to either nNOS or CB1R were frequently seen in most regions except AN (Figure 2.4L). Intensely innervated nerve fibers positive to nNOS and CB1R in ME were devoid of colocalization (Figure 2.4M-O). In addition to neuronal cells, processes as well as interconnected nerve fibers displaying variable patterns of immunoreactivity, mostly positive to CB1R, were seen throughout the hypothalamus. The ependymal cells of third ventricle exhibited weak nNOS and CB1R like immunoreactivity (Figure 2.4D-F). We next determined the percentage distribution of neurons displaying colocalization between nNOS and CB1R. As shown in Figure 2.5, in PVN, PeVN and VMH, 60%, 85% and 30% of total neurons exhibited colocalization respectively, whereas remaining neurons displayed either CB1R or nNOS like immunoreactivity. 58   59  Figure 2.4 Confocal photomicrographs depicting colocalization of CB1R with nNOS positive neurons in rat brain hypothalamus.  nNOS like immunoreactivity was identified using FITC-conjugated secondary antibodies (green) whereas CB1R positive neurons were recognized in presence of Cy3-conjugated (red) secondary antibodies. In merged images, neurons expressing either nNOS or CB1R or colocalization are indicated by arrowheads, tilted arrows and arrows respectively, while neuronal processes and nerve fibers showing colocalization are indicated by asterisks. As shown in PVN (panel A-C), PeVN (panel D-F) and VMH (panel G-I), not all neurons positive to nNOS or CB1R displayed colocalization. Three different neuronal populations either positive to nNOS or CB1R or both were frequently seen in most hypothalamic regions except AN (panel J-L). Nerve fibers in ME displaying nNOS or CB1R like immunoreactivity were devoid of colocalization (panel M-O). Scale bar = 40 µm.  Figure 2.5 Representative histograms showing quantitative analysis of neurons coexpressing CB1R and nNOS in PeVN, PVN and VMH.  Neurons positive to CB1R and/or nNOS were counted from selective hypothalamic regions and results were presented as the percentage of neurons coexpressing CB1R and nNOS. Data were collected from 5 rat brains and a total number of 250~300 neurons were analyzed. Bars present the mean ± SE (n=5).  2.3.3 Colocalization of SST and nNOS in hypothalamus, cortex and striatum Having seen the region specific expression of nNOS and SST with CB1R, we next determined whether nNOS and SST also exhibited distinct and region specific colocalization in hypothalamus. As shown in Figure 2.6, SST and nNOS positive neurons exhibited variable intensity of 60  immunoreactivity in PVN (A-C), PeVN (D-F) and VMH (G-I) respectively. In PVN, nNOS positive neurons were relatively more in numbers than SST positive neurons and displayed weak colocalization (Figure 2.6A-C). Conversely, PeVN was enriched with SST positive neurons (Figure 2.6D-F). Large population of SST positive neurons in PeVN was lacking colocalization, whereas majority of nNOS positive neurons in PeVN displayed mild to strong colocalization (Figure 2.6D-F). In contrast to PVN and PeVN, SST and nNOS positive neurons were comparable and exhibited strong colocalization in VMH (Figure 2.6G-I). All SST positive neurons showed colocalization with nNOS in addition to neurons stained only for nNOS (Figure 2.6G-I). Interestingly, in agreement with several previous studies, all nNOS positive neurons in cortex (Figure 2.6J-L) and striatum (Figure 2.6M-O) exhibited colocalization with SST. However, in cortical brain regions, we also noticed some neurons expressing only SST but not nNOS (Figure 2.6L). Quantitative analysis of SST and nNOS in hypothalamic regions is displayed in Figure 2.7. In PVN, 21% neurons exhibited strong colocalization. SST and nNOS were colocalized in 55% of total neurons positive to SST or nNOS in PeVN, whereas in VMH, 53% neurons displayed colocalization. 61   62  Figure 2.6 Representative confocal photomicrographs showing colocalization between SST and nNOS in rat brain hypothalamus, cortex and striatum.  SST immunoreactivity was identified by FITC fluorescence (green) while nNOS was localized by Cy3 fluorescence (red). Colocalization of SST with nNOS in merged image is indicated by arrows. Neurons positive to only SST or nNOS are indicated by arrowheads and titled arrows respectively. Neuronal processes and nerve fibers showing colocalization are indicated by asterisks. PVN was enriched with nNOS positive neurons which displayed weak colocalization with SST (arrows, panel C). Large population of nNOS positive neurons in PVN was devoid of colocalization (tilted arrows, panel C). In contrast, SST positive neurons in PeVN displayed selective and specific colocalization (arrows, panel F). Note that not all SST positive neurons in PeVN exhibited colocalization (arrowheads, panel F). In contrast, comparatively strong colocalization was seen between SST and nNOS in VMH (arrows, panel I). In both of cortex (panel L) and striatum (panel O), all nNOS positive neurons displayed colocalization with SST, whereas some cortical neurons showed only SST like immunoreactivity (arrowheads, panel L). In cortex and striatum, strong immunoreactivity was also seen in neuronal processes and nerve fibers. Scale bar = 40 µm.  Figure 2.7 Quantitative analysis of neurons coexpressing SST and nNOS in three hypothalamic nuclei.  Neurons positive to SST and/or nNOS were counted from PeVN, PVN, and VMH and data were presented as the percentage of neurons coexpressing SST and nNOS over neurons positive to either SST or nNOS. Data were collected from 5 rat brains and a total number of 250~300 neurons were analyzed. Bars present the mean ± SE (n=5).  63  2.4 Discussion In the present study, we describe the colocalization of CB1R with SST and nNOS to establish a possible relationship in hypothalamic functions, specifically in the regulation of appetite. SST and nNOS are coexpressed with CB1R in hypothalamus in region specific manner. Comparable colocalization of SST and nNOS with CB1R might be associated with cannabinoids induced stimulation of appetite as well as other functions linked to CB1R in hypothalamus. To our knowledge, this is the first attempt to establish the physical association between CB1R and neurons positive to SST and nNOS in hypothalamus. Furthermore, almost 50% SST positive neurons in hypothalamus are devoid of nNOS like immunoreactivity, distinct from cortical and striatal regions. The functional significance of our observations are twofold: first, we uncovered that SST and nNOS colocalized with CB1R; second, the colocalization of SST with nNOS, specifically in PeVN, was different in cortex and striatum. Colocalization of CB1R with SST does not necessarily mean that SST is a physiological regulator of CB1R in hypothalamus, however, it does strengthen our speculation that CB1R and SST might function in concert. SST positive neurons are rich in PeVN and colocalize with CB1R, suggesting that the release of SST may indirectly stimulate CB1R positive neurons in VMH or directly activate CB1R positive neurons in AN to stimulate appetite. It has been described previously that through intracerebroventricular administration, low dose of SST in rats increases food intake whereas high dose results in loss of appetite (Aponte et al., 1984, Danguir, 1988). Importantly, such effects were reversed in presence of SSTR2 antagonist, suggesting that SST-induced appetite might be linked to SSTR2 activation. Notably, intraperitoneal injection had no similar effect, suggesting the central effect of SST (Stengel et al., 2010). Additionally, enhanced SST release in pituitary during food restriction may well account for the activated food drive in 64  such condition (Mounier et al., 1989). Furthermore, studies have also suggested that CB1R is required for α-melanocyte stimulating hormone, the cleavage product of POMC, to modulate appetite (Verty et al., 2004). Activation of POMC neurons leads to an anorexigenic effect while antagonism causes obesity (Elias et al., 1999). Considering the presence of SST in hypothalamic POMC neurons, it is also highly possible that SST may indirectly exert inhibitory effect on POMC positive neurons and stimulate appetite.  In hypothalamus, PVN and VMH are known to be the centers of satiety and inhibition of PVN neural activity stimulates appetite and weight gain (Grandison and Guidotti, 1977). The administration of GABA in PVN and VMH leads to increased food intake (Kelly et al., 1977). Consistent with previous studies, GABAergic neurons express CB1R in VMH (Reguero et al., 2011). Moreover, SST and CB1R are known to inhibit GABA release (Leresche et al., 2000, Wilson and Nicoll, 2002). However, studies have also revealed the inhibitory effect of SST and CB1R on glutamatergic neurons, whereas the activation of excitatory neurons in lateral hypothalamus stimulates appetite (Stanley et al., 2011). In this direction, we predict that the role of SST and CB1R in food intake is differentially regulated by different nuclei in hypothalamus by modulating GABAergic and glutamatergic neurotransmission.  In the CNS and PNS, NO is recognized as a neurotransmitter. NO has been shown to exert a profound role in mediating the release of hypothalamic peptides through neurons containing nNOS. Neuroendocrine function of nNOS is supported by the fact that these neurons in the hypothalamus contain oxytocin, vasopressin (VP), and CRF (Yamada et al., 1996). In addition, NOergic neurons actively participate in controlling the release of CRF, prolactin, VP, GHRH, and SST (Karanth et al., 1993, Ota et al., 1993, Aguila, 1994, Rettori et al., 1994). Similar interactions between CB1R and several important hypothalamic hormones, such as leptin, CRF, and NPY, 65  have been well documented (Bellocchio et al., 2008). Di, S. and colleagues described that the release of glucocorticoids in PVN leads to activation of presynaptic CB1R in glutamatergic neurons and postsynaptic nNOS in GABAergic neurons, consequently leading to the reduction of glucocorticoids-induced hormone release, such as VP and oxytocin (Di et al., 2003, Di et al., 2009). These results clearly indicate the existence of two distinct populations of neurons expressing CB1R and nNOS in PVN and their differential regulation in energy control. Taken into consideration that glucocorticoids increase food intake by activating presynaptic CB1R in PVN, whether nNOS exert any plausible role in concert with CB1R in the regulation of appetite is not known and future studies are needed (Malcher-Lopes et al., 2006). Moreover, CB1R has been shown to interact with nNOS in several in vivo and in vitro models. CB1R-mediated nNOS inhibition has been observed in rat neurogenesis zones as well as cultured rat cerebellar granule cells, supporting their interaction in other areas (Hillard et al., 1999, Kim et al., 2006b). Having seen the colocalization of CB1R with SST and nNOS respectively, it will be interesting to know whether SST and nNOS modulate CB1R activity together to affect appetite and energy control. Ghrelin-induced hyperphagic effect is abolished by CB1R antagonist and in CB1R knockout mice (Kola et al., 2008). The central administration of ghrelin and NPY has been shown to increase nNOS activity (Morley et al., 1999, Gaskin et al., 2003). Intracerebroventricular injection of ghrelin increases NPY mRNA expression in AN in both fed and fasted rats (Seoane et al., 2003). NPY, on the other hand, is also increased by CB1R agonists while decreased by CB1R antagonists (Gamber et al., 2005). We have previously shown that SST, nNOS and NPY are coexpressed in striatum and cortex (Dawson et al., 1991a). In contrast, in PeVN, two populations of SST positive neurons either expressing SST alone or exhibiting colocalization with nNOS were seen in present study. Whether these SST positive neurons express NPY needs to be determined. 66  In hypothalamus, SST and nNOS are coexpressed, suggesting a possible regulatory role of NPY in the function of SST (Hisano et al., 1990). Together, these observations support the model that CB1R, SST and nNOS might regulate appetite and energy control through direct interaction or indirect crosstalk with other hormones in the hypothalamus. In conclusion, we anticipate that SST and nNOS in combination with cannabinoids may participate in the regulation of physiological activities of hypothalamus. Whether ablation of SST and nNOS in general or conditional knocking down in PeVN and VMH in particular will perturb the role of CB1R is not known and needs to be determined to derive any direct pharmacological significance. Most importantly, our results emphasize that SST/nNOS positive neurons, which are selectively preserved in excitotoxicity and afford neuroprotection in the cortex and striatum, may also function as a physiological regulator of appetite in hypothalamus. Moreover, the quantitative analysis of SST and CB1R colocalization in a region-specific manner is an indication of selective preference of SSTR subtypes. It would be interesting to determine whether SSTR subtypes colocalize with CB1R in rat brain regions and further studies are in progress in this direction. Taken together, results presented here uncovered a potential therapeutic avenue for the development of new drugs taking advantage of crosstalk between SST, nNOS and cannabinoid systems, in order to reduce the psychoactive side effects of currently available cannabinoid-based drugs and benefit patients suffering from anorexia with Human immunodeficiency virus infection and acquired immune deficiency syndrome, as well as emesis with chemotherapy.   67  Chapter 3: Colocalization of CB1R with SST and nNOS in rat brain hippocampus 3.1 Background Cannabinoid receptors are prominent members of the GPCR family. They are expressed in different parts of the brain at distinct densities. Two different isoforms of CBRs have been cloned and characterized pharmacologically, namely CB1R and CB2R. The widespread distribution of CBRs in central and peripheral tissues is associated with various functions including learning, memory, cognition, as well as regulation of pain and behavior. In the CNS, CB1R function as neuromodulator, regulating neurotransmitter release in a retrograde route (Marsicano et al., 2003). The localization of CB1R has been observed on both excitatory and inhibitory synapses in many brain regions including the hippocampus (Katona et al., 1999, Kawamura et al., 2006). Notably, despite GABAergic interneurons carry the majority of central CB1R, evidence indicates that only the CB1R subpopulation expressed on glutamatergic neurons is responsible for many if not all cannabinoid-related physiological effects, including neuroprotection against excitotoxicity (Monory et al., 2006, Chiarlone et al., 2014). The hippocampus is a critical component of the limbic system and is directly associated with regulation of learning and memory as well as several other endocrine functions (Bliss and Collingridge, 1993). A dysfunctional hippocampus not only results in impaired memory but is also associated with several prominent neurological disorders. The hippocampal tissue from epileptic patients displays decreased CB1R density, especially in the dentate gyrus (Ludanyi et al., 2008). In the hippocampus, neurogenesis triggered by high chronic doses of cannabinoids is mediated via CB1R (Jiang et al., 2005).  68  Like cannabinoid, SST functions as a neurotransmitter and neuromodulator and has been linked to seizures, memory and cognitive function (Liang and Wu, 1990). SST is expressed in glutamic acid decarboxylase-containing neurons and colocalizes with a subpopulation of GABAergic interneurons in rat dentate gyrus (Tallent, 2007). The loss of SST-positive interneurons in the dentate gyrus has been recognized as a hallmark of epilepsy and in neuropsychological disorders such as schizophrenia and bipolar disorder (Tallent and Qiu, 2008, Konradi et al., 2011a, Konradi et al., 2011b). Furthermore, SST-mediated suppression of long-term potentiation (LTP) further attests its role in both epileptogenesis and memory formation in the hippocampus (Baratta et al., 2002).  NO is a gaseous retrograde messenger synthesized by three different NOS. nNOS, the predominant form in neurons, is abundantly expressed in the hippocampus (Vincent and Kimura, 1992, Harooni et al., 2009). Studies in hippocampal pyramidal cells have shown that nNOS at postsynapse produces NO to conduct retrograde signaling to GABAergic interneurons (Szabadits et al., 2007). nNOS participates in LTP in cultured rat hippocampal neurons while nNOS inhibitor depresses LTP in rats (Bohme et al., 1993, Arancio et al., 1996).  There is increasing evidence suggesting that cannabinoids play a neuroprotective role in neurodegenerative diseases and function as both neurotrophic and anti-inflammatory factors (Tanasescu et al., 2013). SST is well recognized as a neurotrophic factor, exerting neuroprotective effects against excitotoxicity, and as a classical histopathological hallmark of various neurological diseases (Tallent and Qiu, 2008). We have recently shown anti-inflammatory effect of SST in cytokines- and lipopolysaccharide-induced toxicity in human brain endothelial cells (Basivireddy et al., 2013). Interestingly, the common molecular mechanisms attributed to the neuroprotective role of CB1R, SST and nNOS are the regulation of Ca2+ influx and glutamate release (Dawson et 69  al., 1991b, Boehm and Betz, 1997, Marsicano et al., 2003). Furthermore, nNOS positive neurons which colocalize with SST are selectively preserved in neurodegenerative diseases and NMDA-induced excitotoxicity in cultured striatal neurons (Aronin et al., 1983, Beal et al., 1986, Kumar, 2004). Whether it be in the hippocampus or elsewhere, CB1R is similar to SST and nNOS in their coexpression in glutamatergic, cholinergic and GABAergic neurons and their playing of a crucial role in modulation of LTP and LTD. However, it is currently unknown whether SST and nNOS positive neurons coexpress CB1R in the hippocampus. Accordingly, in the present study, double-labeled immunofluorescence was performed to determine the functional association between SST/nNOS and CB1R in adult rat brain hippocampus. Our results revealed the comparable pattern of colocalization between CB1R and SST or nNOS in rat brain hippocampus.  3.2 Materials and Methods 3.2.1 Animals Male Sprague-Dawley rats (body weight 200-250 g) were obtained from UBC animal care unit. The protocols regarding animal care were followed in compliance with the principles of the Canadian Council on Animal Care and were approved by the University of British Columbia Animal Care Committee. 3.2.2 Materials Mouse anti-SST monoclonal (Cat. No. sc-74556) and goat anti-CB1R polyclonal antibody (Cat. No. sc-10066) were purchased from Santa Cruz (Dallas, TX). Rabbit anti-nNOS polyclonal antibody (Cat. No. AB5380) was purchased from Millipore (Billerica, MA). FITC and Cy3-conjugated goat anti-mouse, goat anti-rabbit and donkey anti-goat secondary antibodies were obtained from Jackson Laboratories (West Grove, PA). NGS was purchased from Vector 70  Laboratories (Burlingame, CA). All other reagents and chemicals were of analytical grade and obtained from various commercial sources. 3.2.3 Immunocytochemistry Rats were deeply anaesthetized with halothane and perfused with heparinized cold normal saline followed by freshly prepared 4% paraformaldehyde in 0.1M phosphate-buffered saline. Brains were then dissected out and post-fixed in the same solution for 2 h. 40 μm thick free-floating brain sections were processed for colocalization using double labelled immunofluorescence immunocytochemistry as described previously (Kumar, 2007). Briefly, coronal brain sections were incubated in TBS containing 5% NGS for 1 h at room temperature, followed by incubation with anti-goat CB1R (1:200) and anti-mouse SST (1:200) or anti-rabbit nNOS (1:200) antibodies in 1% NGS prepared in TBS overnight at 4o C in a humid atmosphere. Following three washes in TBS, brain sections were subsequently incubated with FITC and Cy3-conjugated secondary antibodies for 2 h at room temperature for final colour development. Sections were washed in TBS, mounted onto the slide and photographed using Carl Zeiss confocal microscope (LSM 700, AxioObserver). Adobe Photoshop (San Jose, CA) was used to make composites and merged images showing colocalization were generated using ZEN Lite blue edition software (Jena, Germany). 3.2.4 Quantitative analysis The percentage distribution of neurons displaying colocalization between SST, CB1R and nNOS was determined as previously described (Kumar, 2007). 40 µm coronal rat brain sections passing through hippocampus were double-labelled for SST, CB1R and/or nNOS and photographed using Carl Zeiss confocal microscope (LSM 700, AxioObserver) at 20X magnification. Five to ten sections obtained from five different rat brains were subjected for 71  quantification. The total neuronal population displaying either protein like immunoreactivity were counted from 8~10 randomly selected areas. Only neuronal cell bodies showing intact morphology and immunoreactivity were counted. No dot or nerve fiber-like structures were included in quantitative analysis. A total of ~250-300 neurons positive to given proteins were analysed. The mean percent of neurons showing colocalization was calculated from total neurons and presented as mean ± SE (n=5).  3.3 Results The specificity of immunoreactivity was confirmed by omission of primary antibodies as described previously (Zou et al., 2015). In absence of primary antibodies, no specific immunoreactivity was observed. Accordingly, the expression seen in the presence of selective antibodies was taken into account as specific staining. The specificity of CB1R antibodies was also confirmed in HEK-293 cells stably transfected with CB1R. As shown in Figure 3.1A-C, CB1R transfected cells exhibited strong cell surface expression and such staining was not seen in wt cells (Figure 3.1A) nor in the absence of primary antibodies (Figure 3.1C). In addition, we also confirmed whether any bleed through of secondary antibodies account for nonspecific staining. As shown in Figure 3.1D-F, no bleed through from green channel to red channel was detected in absence of Cy3 secondary antibody. Furthermore, CB1R immunoreactivity shown here was consistent with several previous studies using the same antibody (Sanford et al., 2008, Kalifa et al., 2011). 72   Figure 3.1 Photomicrographs illustrating the specificity of CB1R antibody.  Wild-type and CB1R transfected HEK-293 cells were incubated with CB1R antibody at a dilution of 1:500 overnight at 4 ºC, followed by Cy3-conjugated secondary antibody (dilution 1:800) for 2 h at room temperature. In wild-type cells (A) no receptor like immunoreactivity was detected whereas transfected cells exhibited strong cell surface immunoreactivity for CB1R (B, arrows). CB1R transfected cells probed only with secondary antibody in absence of primary antibody were devoid of specific fluorescence (C). Note that no bleed through was detected when hippocampal section stained for SST using FITC conjugated secondary antibody showed only green fluorescence. Scale bar = 20 µm (A-C) and 40 µm (D-F) respectively.  3.3.1 SST in hippocampus is expressed in selective CB1R positive neurons SST and CB1R positive neuronal cells were well expressed in different layers of the hippocampus and dentate gyrus. Neuronal population either displaying colocalization or single staining for SST and CB1R-like immunoreactivity exhibited variable degrees of intensity. SST 73  and CB1R immunoreactivity in interneurons which appeared in soma were relatively strong in the pyramidal layer. As shown in Figure 3.2B-D, in the CA1, the majority of interneurons expressing SST and CB1R were confined to the pyramidal layer, whereas interneurons in the stratum oriens and radiatum displayed colocalization with strong SST and CB1R-like immunoreactivity in neuronal processes. Neuronal cell bodies with dendrites were also occasionally observed in pyramidal layer with strong colocalization at apical dendrites (Figure 3.2D, J). CA2 (Figure 3.2E-G) and CA3 (Figure 3.2H-J) regions also displayed comparable distribution and colocalization between SST and CB1R as seen in the CA1 region. In the granule cell layer of the dentate gyrus, most neuronal cells displayed variable degrees of colocalization. Also, at the border of hilus, neuronal cells with processes stretching into the granule cell layer often displayed colocalization (Figure 3.3). Dense communicating nerve fibers positive to only SST were observed in the hilus (Figure 3.3A, C). 74   Figure 3.2 Representative confocal photomicrographs illustrating the colocalization of SST and CB1R in CA1-3 areas of hippocampus.  75  Rat brain sections were incubated with SST monoclonal (1:200) and anti-goat CB1R antibodies (1:200) overnight at 4 ºC in humid atmosphere. Following three washes in TBS, sections were incubated with goat anti mouse FITC and donkey anti-goat Cy3-conjugated secondary antibodies for final color development as described in Experimental Procedures section. SST (green) and CB1R (red) are well expressed in all three CA regions of hippocampus (A-J). Colocalization of SST with CB1R in merged image (yellow) is indicated by arrows (D, G, and J). Neuronal processes and nerve fibers showing colocalization are indicated by thin arrows in merged images. The interneurons confined to pyramidal layers displayed strong colocalization in CA1 (B-D), CA2 (E-G) and CA3 (H-J) regions and indicated by arrows in respective panels. Neuronal processes positive to both CB1R and SST were occasionally observed in stratum oriens and radiatum (D, G, and J). Scale bar = 200 µm (A) and 40 µm (B-J). SO, stratum oriens; SP, stratum pyramidal layer; SR, stratum radiatum; ML, molecular layer; GL, granular layer; PML, polymorphic layer.  Figure 3.3 Representative confocal photomicrographs illustrating the colocalization of SST and CB1R in dentate gyrus. Rat brain sections were processed as described in legend to Figure 3.2. SST expression was visualized by FITC (green) in panels A and D. CB1R immunoreactivity was identified by red immunofluorescence in the same areas of dentate gyrus (B, E). Colocalization of SST with CB1R in merged image is shown in yellow and indicated by arrows (C, F). 76  Neurons positive to only SST are indicated by arrowheads (C). Neuronal processes and nerve fibers showing colocalization are indicated by thin arrows in merged images. Interneurons showing strong to mild colocalization were frequently observed in granular layer adjacent to polymorphic layer (A-C) and in hilus (D-F). Note neurons in granular layer (C) and hilus (F) with long processes showing strong colocalization. Scale bar = 40 µm.  3.3.2 Colocalization of CB1R and nNOS in rat brain hippocampus  In hippocampal formation, a comparable pattern of colocalization between CB1R and nNOS was observed. As illustrated in Figure 3.4, nNOS positive interneurons confined to the pyramidal layer displayed moderate level of colocalization with CB1R. In addition to neuronal cell bodies, strong colocalization was also seen in neuronal processes and nerve fibers (Figure 3.4D). As with in CA1 region, CB1R and nNOS in CA2 showed comparable immunoreactivity and colocalization (Figure 3.4E-G). In the stratum oriens and lacunosum-moleculare, we also observed a few of interneurons displayed mild colocalization or showed single staining for nNOS and/or CB1R (data not shown). In CA3, the pattern of colocalization between nNOS and CB1R was relatively different from CA1 and CA2, with interneurons showing colocalization observed mainly in stratum oriens (Figure 3.4H-J). Interestingly, as shown in Figure 3.5, a distinct pattern of colocalization was observed in the dentate gyrus. Neuronal cells either showing moderate colocalization or immunoreactivity only for CB1R were frequently seen. In the hilus, we also noticed neuronal cells with axons and arborizing dendrites displaying strong colocalization (Figure 3.5G-I). 77   78  Figure 3.4 Confocal photomicrographs depicting subcellular distribution and colocalization of CB1R with nNOS positive neurons in CA1-3 areas of hippocampus. nNOS like immunoreactivity was identified using FITC-conjugated secondary antibodies (green) and shown in panels B, E, H whereas CB1R positive neurons were visualized in presence of Cy3-conjugated secondary antibodies (red) and shown in panels C, F, I. In merged images (D, G, J) neurons expressing either nNOS or CB1R, or colocalization between CB1R and nNOS are indicated by arrowheads, tilted arrows and arrows respectively. Colocalization in neuronal processes and nerve fibers are indicated by thin arrows. In CA1, neurons positive to CB1R exhibited colocalization with nNOS in pyramidal layer (B-D). Mild to strong colocalization was well displayed in interneurons in both CA2 (E-G) and CA3 (H-J). Interneurons showing only nNOS immunoreactivity were seen in CA3 (J) and indicated by arrowheads. Scale bar = 200 µm (A) and 40 µm (B-J).  79  Figure 3.5 Double labelled immunofluorescence confocal photomicrographs depicting colocalization of CB1R with nNOS positive neurons in dentate gyrus. nNOS and CB1R like immunoreactivity was identified as described in legend to Figure 3.4, using FITC and Cy3-conjugated secondary antibodies. Neuronal cells displaying only CB1R (red) immunoreactivity or colocalization (yellow), in merged images are indicated by tilted arrows and arrows respectively. The neuronal processes and nerve fibers showing colocalization are indicated by thin arrows in respective panels. Note a distinct population of interneurons in dentate gyrus either exhibiting colocalization or only CB1R like immunoreactivity (F). Receptor colocalization with nNOS was also seen in axons and dendrites in hilus is indicated by thin arrows in merged images (C, F, I). Scale bar = 40 µm.  3.3.3 In hippocampus not all SST positive neurons express nNOS  We recently demonstrated that SST positive neurons displaying colocalization with nNOS is not only restricted in the cortex and striatum but also exist in the hypothalamus (Zou et al., 2015). In extension to these observations, here we determined whether SST positive neurons colocalize with nNOS in the hippocampus. As shown in Figure 3.6 and 3.7, SST and nNOS positive neurons displayed comparable colocalization in CA1-3 regions as well as in the dentate gyrus. In the pyramidal layer and hilus, nNOS-like immunoreactivity was predominantly confined to interneurons and displayed strong colocalization with SST (Figure 3.6 and 3.7). Note the significant changes in colocalization in neuronal processes and dendrites. In CA1-3, interneurons dispersed in the stratum radiatum displayed strong to mild colocalization (Figure 3.6). Consistent with one previous study (Dun et al., 1994a), interneurons merged in the pyramidal layer not only displayed strong colocalization but also expressed SST-like immunoreactivity only (Figure 3.6B-M). As displayed in Figure 3.6K-M, few interneurons confined to CA3 with multiple dendrites or neuronal process strongly positive to SST were devoid of nNOS expression. In CA1-3, all nNOS 80  positive neurons colocalized with SST (Figure 3.6B-M). Furthermore, nerve fibers exhibiting either only SST or nNOS expression or mild colocalization were observed (Figure 3.6D, G, J, M). However, the pattern of colocalization was different in the dentate gyrus. As shown in Figure 3.7, interneurons restricted to either the granule cell layer or the hilus exhibited colocalization or only SST-like immunoreactivity. Interestingly, large interneurons in hilus with multiple neuronal processes were only positive to SST (Figure 3.7D-F). These results clearly indicate two SST positive neuronal subpopulations, either displaying or devoid of colocalization. At the distal end of the hilus, neurons positive to either SST or nNOS were devoid of colocalization (Figure 3.7G-I). 81   82  Figure 3.6 Representative confocal photomicrographs of hippocampus showing colocalization between SST and nNOS in CA1-3 regions. SST immunoreactivity was identified by FITC fluorescence (green) whereas nNOS like immunoreactivity is identified by Cy3 fluorescence (red). Coexpression of SST with nNOS is shown by yellow in merged image and indicated by arrows (D, G, and J). Neurons positive to only SST are indicated by arrowheads. Extensive communicating nerve fibers and neuronal processes showing colocalization are indicated by thin arrows. Note a discrete population of interneurons in CA1-3 regions, either displaying colocalization or SST like immunoreactivity alone (D, G, J). Neurons positive to both SST and nNOS were mostly confined in pyramidal layer (D, G, and J). Large neurons with long dendrites only positive to SST were also seen in pyramidal layer (K-M). Scale bar = 200 µm (A) and 40 µm (B-M).  83  Figure 3.7 Representative double label immunofluorescence confocal photomicrographs illustrating colocalization between SST and nNOS in dentate gyrus. SST and nNOS immunoreactivity was identified as described in legend to Figure 3.6. Colocalization of SST and nNOS in merged image is indicated by arrows. Neurons positive to only SST or nNOS are indicated by arrowheads and titled arrows in merged images respectively. Neuronal processes and nerve fibers showing colocalization are indicated by thin arrows. Interneurons positive to either SST only or both SST and nNOS were well expressed in granular layer and hilus (A-C, D-F). Note that neuronal population showing only SST or nNOS at the distal end of hilus (G-I). Scale bar = 40 µm. 3.3.4 Quantitative analysis To further support our morphological characterization, quantitative analysis was performed to determine the percentage of colocalization between CB1R and SST/nNOS as well as SST and nNOS in the hippocampus including CA1-3 and the dentate gyrus. As shown in Figure 3.8, 95± 4% CB1R positive neurons exhibited colocalization with SST whereas 80 ± 11% of SST positive neurons displayed colocalization with CB1R. On the other hand, 69 ± 6% CB1R positive neurons colocalized with nNOS. Conversely, only 64 ±4% nNOS positive neurons exhibited colocalization with CB1R (Figure 3.8). Although, SST and nNOS are known to be coexpressed in many brain regions, quantitative analysis revealed that in the hippocampus 57 ±11% of SST positive neurons coexpress nNOS whereas a large percentage (88 ± 4%) of nNOS positive neurons colocalized with SST. This analysis revealed the presence of three different neuronal populations in the hippocampus. The distinct distributional pattern of colocalization between SST and nNOS was significantly evident in the dentate gyrus and CA regions of the hippocampus. 84   Figure 3.8 Quantitative analysis of the expression of CB1R in SST and nNOS positive neurons and SST in nNOS positive neurons in hippocampus. In randomly selected areas of hippocampus, neurons positive to CB1R were analyzed for the colocalization with SST and nNOS. To determine the colocalization between SST and nNOS, neurons positive to SST were counted in hippocampal formation and analyzed for the expression of nNOS as described in Experimental Procedures. Data were collected from 5 rat brains and a total number of 250~300 neurons were analyzed. Bars represent the mean ± SE (n=5).  3.4 Discussion Several previous studies have shown overlapping functions of SST, nNOS and CB1R in the CNS, including excitotoxicity, regulation of appetite and memory and cognitive function. We recently demonstrated the colocalization of CB1R with SST and nNOS in rat brain hypothalamus (Zou et al., 2015). However, it is currently unknown whether neurons expressing CB1R in the hippocampus are also positive to SST or nNOS and attest their overlapping function. In the present study, we described the colocalization of CB1R with SST and nNOS in rat brain hippocampus. Results showed that CB1R, SST and nNOS are expressed at various densities in different layers of hippocampus and dentate gyrus. In addition to neuronal populations displaying colocalization, single staining of CB1R, SST and nNOS in different layers were seen all over the hippocampus. 85  The specific distribution and selective pattern of colocalization described here not only correlate with the role of CB1R, SST and nNOS in the hippocampus, but also suggest the reciprocal contributions to their respective regulation of hippocampus activities. However, the cellular and molecular mechanisms and functional consequences of interaction between CB1R/SST/nNOS are not well understood. The colocalization of CB1R with SST and nNOS presented here supports the supposition that CB1R and SSTR subtypes might functionally interact in a heteromeric complex. Given the abundance of CB1R in GABAergic system and its association with behavioral complexity, the results of this study provide further insight on the role of SST and nNOS in regulation of GABAergic functions. These findings establish a crucial link between CB1R, SST and nNOS in regulating pharmacological and physiological activities in the hippocampus. The subcellular distribution of CB1R described here is consistent with several previous studies (Pettit et al., 1998, Moldrich and Wenger, 2000, Kalifa et al., 2011). CB1R-like immunoreactivity is mainly confined to neuronal cell bodies with occasional expression in axonal processes. Although the lack of receptor immunoreactivity in neuronal processes was surprising, several previous studies by using antibodies against N-terminus have shown similar pattern of CB1R distribution in hippocampus (Sanford et al., 2008, Zarate et al., 2008, Kalifa et al., 2011). In addition, studies using electron microscopy suggest that CB1R immunoreactivity in soma may represent functional receptors before translocation or phosphorylation, which may not be recognized by antibodies raised against certain epitopes on C-terminus (Katona et al., 1999, Bodor et al., 2005). SST and CB1R positive neurons are well expressed in the hippocampus and are associated with several pathological conditions (Tsou et al., 1998, Wilson and Nicoll, 2001, Tallent, 2007, Tallent and Qiu, 2008). The inhibition of neurotransmitter release such as glutamate and GABA in the brain by SST and CB1R supports their role in regulation of synaptic function (Katona et al., 1999, 86  Patel, 1999, Marsicano et al., 2003). It has been shown that 95% of CB1R positive neurons in rat hippocampus are positive to GABA; however, GABAergic interneurons clearly possess a large subpopulation displaying no CB1R immunoreactivity (Tsou et al., 1999). Similar observations as described here have also been reported in mouse forebrain (Marsicano and Lutz, 1999). Upon activation, SST-positive neurons co-expressing GABA simultaneously release SST and GABA (Gamse et al., 1980, Kosaka et al., 1988). SST and CB1R are known to modulate GABA release (Katona et al., 1999). Although the role of cannabinoids in modulation of GABAergic system is undisputed, the precise molecular mechanisms involved are not well understood. Taking this into consideration, the possible interaction between CB1R and SST along with the consequent effect on GABA release cannot be excluded from discussion. Furthermore, as hippocampus is the prominent brain region responsible for memory and cognition, involvement of cannabinoids in memory through GABAergic system might suggest the role of SST in cognitive function and memory process in the brain with anticipated crosstalk amongst these three systems (Ranganathan and D'Souza, 2006). From a biochemical perspective, similarities in cellular signaling between SST and CB1R might shed light on the possible mechanism. SST, in a Gi dependent manner, exerts a crucial role in regulation of hippocampal activities with important implication in seizure through inhibition of excitatory transmission and enhancement of K+ currents in CA1 region (Tallent and Siggins, 1999, Baratta et al., 2002). Similarly, CB1R coupled to Gi, inhibit cAMP and thus stimulate inwardly rectifying K+ channels (IRKs) in the CNS (Tanasescu et al., 2013). CB1R-modulated cAMP-independent of K+ channel activation has also been characterized (Demuth and Molleman, 2006). These shared properties further strengthen the hypothesis of a synergistic role of SST and CB1R in regulation of some key aspects of brain physiology. Of note, in the striatum, CB1R in association with D2R has been reported to enhance cAMP formation, supporting its Gs 87  coupling (Giuffrida et al., 1999). With existing information, it can be argued that CB1R lacking colocalization with SST may be involved in differential G protein coupling. Most importantly, interneurons showing colocalization and SST alone in the dentate gyrus might exert distinct effects in regulation of Ca2+ influx in excitotoxicity. CB1R and nNOS are associated with several neurological disorders. Activated CB1R are known to decrease glutamate release, and thus attenuate NMDAR-mediated activation of nNOS and production of NO (Hillard et al., 1999). Studies have shown the neuroprotective effect of CB1R agonists in NMDA-induced toxicity by reducing NO production in rat retina and murine cerebral cortex (El-Remessy et al., 2003, Kim et al., 2006a). In cultured murine cortical neurons, CB1R and nNOS are colocalized, further suggesting their interaction (Kim et al., 2006a). Moreover, similar observations have also been made in mouse dentate gyrus demonstrating that CB1R stimulate adult neurogenesis, at least partly by inhibiting nNOS (Kim et al., 2006b). Consistent with previous studies, the pattern of colocalization presented here between CB1R and nNOS indicates their crosstalk in physiological processes of hippocampus such as neurogenesis, or under pathological conditions, including excitotoxicity and seizures. All SST positive interneurons show colocalization with nNOS in the striatum (Figueredo-Cardenas et al., 1996). In the cortex, two different types of SST positive neurons, either expressing nNOS or lacking colocalization, are frequently seen (Dawson et al., 1991b, Zou et al., 2015). Conversely, in the hypothalamus, we found three different populations either displaying colocalization or expressing single staining (Zou et al., 2015). Interestingly, in the hippocampus, we noticed comparable distribution pattern as seen in cortical brain regions displaying either colocalization or only SST expression. These observations raised the question whether nNOS positive neurons lacking SST expression is due to the presence of specific proteins or 88  morphological distinction. Several previous studies have shown that the cortex and hippocampus are involved in memory and cognitive function as well as associated with seizure and excitotoxicity (Goldman-Rakic, 1987, Bliss and Collingridge, 1993). Consistent with these studies, the pattern of colocalization seen between SST and nNOS supports the notion that SST and nNOS might function in a concert in hippocampal activities, i.e. memory, excitotoxicity and seizures. The expression of CB1R with SST in CA1 and the dentate gyrus of the hippocampus is important specifically in excitotoxicity and seizures. While the biological effects of SST are mediated by five different receptor subtypes, the subcellular distribution of SSTR subtypes in the hippocampus is not well understood (Patel, 1999). Therefore, the identification and characterization of SSTR subtypes with CB1R would be essential to attest any possible interactions, and future studies are in progress in this direction. It is unknown whether SST stimulates the release of endogenous endocannabinoid and activates CBR directly via binding to its five different receptor subtypes or indirectly through endocrine function. In addition, several studies have demonstrated that SSTR subtypes functionally interact with many other GPCRs in a heteromeric complex (reviewed in Somvanshi and Kumar, 2012). However, it needs to be determined whether SSTR subtypes exist in the same cell and function in a concert with CB1R. Moreover, future studies on characterization of SSTR subtypes and CB1R will enable us to delineate the exact role of SST in combination with CB1R in projection neurons and interneurons in different parts of the brain. Most importantly, it needs to be determined whether activation of CB1R has any effect on SSTR expression and trafficking in neuronal cells prepared from hippocampus or other brain regions. Furthermore, with the existing notion that modulation of behavioral responses of cannabinoid is linked to GABAergic system, overlapping properties 89  associated with the functions of SST and nNOS might contribute to delineate the molecular mechanism at the cellular levels, at least in regulation of endogenous endocannabinoids. Taken together, immunohistochemical characterization of CB1R colocalization with SST and nNOS adds new insight on the role of CB1R in combination with SST and nNOS in excitotoxicity and seizure.   90  Chapter 4: SSTR5 is a prominent regulator of signaling pathways in cells with coexpression of CB1R 4.1 Background CB1R belongs to the GPCR family and is the major CBRs that is well-expressed in both CNS and PNS. In CNS, most of the physiological effects of THC, the main psychoactive component in cannabis, are mediated by CB1R (Matsuda et al., 1990). CB1R is widely expressed in the CNS, and plays a crucial role in neurotransmission, neuromodulation, and synaptic plasticity upon activation by its endogenous ligand, endocannabinoids (Howlett et al., 2002). Endocannabinoids bind to CB1R and inhibit the release of several neurotransmitters from presynaptic terminals and exert neuroprotective effects against excitotoxicity (Marsicano et al., 2003). Given that endocannabinoids exert such predominant role in synaptic communication, any interference in this system could initiate severe pathophysiological conditions. Previous studies have reported decreased expression of CB1R in AD, PD and HD (Bisogno and Di Marzo, 2010). CB1R knockout mice display an increased susceptibility to excitotoxin along with sustained neurodegeneration, indicating the pivotal role of CB1R in neuroprotection (Mievis et al., 2011). The neuroprotective role of CB1R has been reported both in vivo and in vitro (Ramirez et al., 2005, Liu et al., 2009a). In peripheral tissues, CB1R is associated with several pathophysiological functions, including cardiac functions, energy metabolism and bone formation (Pacher et al., 2006). Despite these promising therapeutic benefits, the use of cannabinoids in clinical practice is limited, due to undesired psychoactive effects and increased incidence of schizophrenia (Mackie, 2006). Like cannabinoids, SST plays a critical role in many pathological conditions through binding to five different receptor subtypes, namely SSTR1-5. SSTR subtypes are well-expressed in the 91  CNS in a region- and receptor-specific manner (Schindler et al., 1996, Patel, 1999, Ramirez et al., 2004, Kumar, 2007). Although the expression of SSTR5 in the CNS is relatively low in comparison to other SSTR subtypes, its pathophysiological significance in neurological disorders has been supported by several studies (Craft et al., 1999, Stroh et al., 1999, Kumar, 2005, Watson et al., 2009). In AD patients, decreased expression of SSTR5 like immunoreactivity has been observed in cortical brain regions (Kumar, 2005). SSTR5 is suggested to play a role in SST analog-mediated memory facilitation in both AD patients and healthy adults (Craft et al., 1999, Watson et al., 2009). Furthermore, our recent study has demonstrated comparable neurochemical changes in the striatum of HD transgenic R6/2 mice and mice deficient in SSTR1/5 (Rajput et al., 2011). In addition, SSTR5 is involved in protection of rat retina against AMPA-induced excitotoxicity (Kiagiadaki et al., 2010, Chen et al., 2014). Taken together, these observations suggest a potential beneficial role of SSTR5 in neurological disorders, including excitotoxicity. At present, it is well established that GPCRs function as dimers or even higher-order oligomers in a manner distinct from the native receptor (Kumar and Grant, 2010, Ferre et al., 2014, Gomes et al., 2016). Numerous studies have shown that SSTRs, as well as CBRs, form homo- and/or heterodimers either within the family or with other GPCRs including dopamine, opioid, orexin and adenosine receptors and members of receptor tyrosine kinase family (Kearn et al., 2005, Ellis et al., 2006, Rios et al., 2006, Carriba et al., 2007, Hudson et al., 2010a, Somvanshi and Kumar, 2012). There is growing evidence that GPCR heterodimerization not only modulate pharmacological properties of receptors, but also brings novel signaling to the interacting protomers in a receptor-specific fashion. For instance, interaction between D2R and CB1R leads to a switch of preferential G-protein coupling from Gi to Gs, whereas heterodimerization of D2R with either SSTR2 or SSTR5 increases dopamine affinity and augments D2R-mediated signaling 92  with significant clinical implication in pituitary tumor treatment (Glass and Felder, 1997, Rocheville et al., 2000a, Kearn et al., 2005, Tichomirowa et al., 2005, Baragli et al., 2007, Saveanu and Jaquet, 2009).  CB1R and SSTR5 share a number of overlapping properties at molecular level; both receptors are coupled to Gi/o to inhibit AC, activate MAPK pathways, inhibit voltage-dependent Ca2+ channels and activate IRKs, thus playing critical roles in physiological responses of neuronal cells (Patel, 1999, Howlett et al., 2002). Neuroprotective role of SST and cannabinoids in excitotoxicity, oxidative stress, and traumatic and ischemic brain injury are well-established and associated with modulation of signaling pathways including ERK1/2 and PI3K (Molina-Holgado et al., 2005, Hu et al., 2010, Kumar and Grant, 2010). However, nothing is currently known whether CB1R interacts with any SSTR subtypes and if such interaction exists, what the functional consequences are. We recently have reviewed that SSTR5 is one of the most dynamic receptors that displays significant diversity upon heterodimerization with other members of GPCR family (Somvanshi and Kumar, 2012). CB1R heterodimerizes with D2R and tend to switch its coupling from Gi to Gs (Glass and Felder, 1997). Our previous study demonstrated D2R heterodimerization with SSTR5 (Rocheville et al., 2000a). It is tempting to determine whether this phenomenon is unique for CB1R in the combination with D2R or a common feature of CB1R. Accordingly, in the present study, we employed multiple techniques to determine the possible interaction between SSTR5 and CB1R in rat brain with endogenous expression and HEK-293 cells stably transfected with hSSTR5 and/or hCB1R. Our results show that SSTR5 and CB1R are coexpressed in rat brain cortex, striatum and hippocampus and CB1R is expressed in SSTR5 immunoprecipitate. In cotransfected HEK-293 cells, SSTR5 and CB1R constituted a functional complex and displayed novel properties in modulation of downstream signaling. At present, no molecular mechanism is in place that could 93  help to minimize undesired side effect of cannabinoids while enhancing its potential medical use. Results described here showing complex formation between CB1R and SSTR5 provides possibility of developing a new therapeutic in drugs of abuse.  4.2 Materials and Methods 4.2.1 Animals Male Sprague-Dawley rats (body weight 200-250 g) were obtained from the University of British Columbia animal care unit. Protocols regarding animal care were followed in compliance with principles of the Canadian Council on Animal Care and were approved by the University Animal Care Committee (Protocol #A06-0419). 4.2.2 Materials SST-14 was procured from Bachem (Torrance, CA) and non-peptide SSTR5 agonist L-817818 was kindly provided by Dr. S. P. Rohrer from Merck & Co. WIN was purchased from the Tocris Cookson Inc., Ellisville, MO (Authorization 31251.09.13). NGS was purchased from Vector Laboratories, Burlingame, CA. SSTR5 antibody was produced in our laboratory and has been well characterized as described previously (Kumar et al., 1999). CB1R anti-goat polyclonal antibody was purchased from Santa Cruz, CA. Antibodies against hemagglutinin (HA) and cMyc were purchased from Sigma-Aldrich, Inc., St. Louis, MO. FITC- and Cy3-conjugated secondary antibodies were obtained from Jackson ImmunoResearch, ON. Rabbit polyclonal antibodies for phospho (p)- and total (t)-ERK1/2, and t-PI3K were purchased from Cell Signaling Technology, Danvers, MA. P-PKA, t-PKA, and p-PI3K antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, CA. The cAMP assay kit was purchased from BioVision, Inc., CA, USA. Other reagents were of analytical reagent (AR) grade and obtained from various sources. 94  4.2.3 Cell culture and transfection cMyc-hSSTR5 in pcDNA-3.1/Hygro and HA-hCB1R in pcDNA-3.1/Neo were used to prepare stable mono- and cotransfected HEK-293 cell lines by using Lipofectamine transfection reagent. Clones surviving antibiotic selection were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 700 µg/milliliter (mL) neomycin and/or 400 µg/mL hygromycin. Receptor expression is comparable to physiological level, as determined previously by radioligand binding assay (Grant et al., 2008). 4.2.4 Immunofluorescence immunohistochemistry Rats were deeply anesthetized with halothane and perfused with heparinized cold normal saline followed by freshly prepared 4% paraformaldehyde in 0.1M TBS. Brains were then dissected out and post-fixed in the same solution for 2 h. 40 µm thick brain sections passing through cortex, striatum and hippocampus were incubated in 5% NGS in TBS for 1 h at room temperature to block the unspecific binding sites. Sections were then incubated in a mixture of specific primary rabbit anti-SSTR5 (1:200 dilution) and goat anti-CB1R (1:100 dilution) antibodies in 1% NGS for overnight at 4 °C. Following three subsequent washes in TBS, brain sections were incubated in the mixtures of FITC- and Cy3-conjugated secondary antibodies for 2 h at room temperature. The sections were mounted and observed under Leica Confocal microscope. Specificity of antibodies and immunoreactivity was validated in the presence of pre-immune serum and/or antigen pre-adsorbed antibody as described previously (Kumar, 2007, Zou and Kumar, 2015, Zou et al., 2015). 4.2.5 Co-IP Cortex and striatum were dissected and homogenized using tissue homogenization buffer. Protein concentration was determined by Bradford assay. 200 µg of tissue protein was solubilized in 1 mL binding buffer (50 millimolar (mM) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 95  (HEPES), 2 mM CaCl2, 5 mM MgCl2, pH 7.5) followed by treatment with receptor agonists alone or in combination for 30 minutes (min) at 37 °C. Protein samples were then incubated with primary anti-SSTR5 antibody (1: 400 dilution) at 4 °C overnight to immunoprecipitate SSTR5. In case of cotransfected cells, 250 µg of membrane protein extract from control and agonist-treated cells were solubilized in 1 mL of radioimmune precipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris–HCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, pH 8.0) for 1 h at 4 °C. CB1R in membrane extracts was immunoprecipitated by incubation with anti-HA specific antibody (1:500 dilution) overnight at 4 °C. Samples were further incubated with protein A/G agarose beads for 2 h at 4 °C. Agarose beads were subsequently washed three times with phosphate buffered saline (PBS) and solubilized in Laemmli sample buffer (Bio-Rad) containing 5% β-mercaptoethanol. Samples were fractionated by SDS-PAGE. The fractionated proteins were transferred onto 0.2 millimeter nitrocellulose membrane and blocked with 5% non-fat milk for 1 h at room temperature. Membrane was incubated overnight at 4 °C with anti-CB1R primary antibody (1:500 dilution) for tissue lysates and anti-cMyc primary antibody (1:500 dilution) for cell membrane fractions. After three washes, membrane was incubated with secondary antibody for 2 h at room temperature. Bands were detected by chemiluminescence reagent and images were taken using Alpha Innotech Fluorchem 800 (Alpha Innotech Co., San Leandro, CA) gel box imager. Specificity of immunoprecipitation was validated using blank beads and wt and CB1R or SSTR5 mono-transfected HEK-293 cells. 4.2.6 Microscopic photobleaching FRET (pbFRET) analysis Cotransfected HEK-293 cells with stable expression of hSSTR5 and hCB1R were treated with L-817818 (50 nM) and WIN [1 micromolar (µM)] alone or in combination for 15 min at 37 °C and processed for immunocytochemistry as previously described (Grant et al., 2004a). Briefly, 96  cells were grown to 70% confluency, fixed with 4% paraformaldehyde on ice for 20 min and followed by incubation with 5% NGS in PBS at room temperature for 1 h. Cells were then incubated with monoclonal anti-cMyc (1:500 dilution) and/or polyclonal anti-HA (1:500 dilution) primary antibodies at 4 °C overnight, followed by FITC- (1:400 dilution) and Cy3- (1:800 dilution) conjugated secondary antibodies respectively to create donor-acceptor pair. Multiple regions on plasma membrane were analyzed for photobleaching decay on a pixel-by-pixel basis. Relative FRET efficiency was calculated as described previously (Rocheville et al., 2000b). 4.2.7 Receptor internalization HEK-293 cells stably cotransfected with cMyc-hSSTR5/HA-hCB1R were grown to 60-70% confluency on poly-D-lysine coated coverslips. Cells were treated with SSTR5 specific agonist L-817818 (50 nM) and CB1R specific agonist WIN (1 µM) alone or in combination for 15 min at 37 °C and processed for immunofluorescence immunocytochemistry as described earlier (Grant et al., 2004b). Posttreatment cells were fixed with 4% paraformaldehyde and incubated with 5% NGS in PBS for 1 h at room temperature. Cells were then first incubated with primary antibodies against HA (1:500 dilution) and cMyc (1:500 dilution) at 4 °C overnight, followed by incubation with secondary Cy3 (1:800 dilution) and FITC (1:400 dilution) conjugated antibodies for 2 h at room temperature. Cells were viewed and photographed using Leica confocal microscope. Figure composites were constructed using Adobe Photoshop and merged images displaying colocalization were generated by Image J software, NIH. 4.2.8 Coupling to AC Mono- and cotransfected cells were incubated with receptor specific agonists alone or in combination for 30 min at 37 °C in the presence of 20 µM FSK and 0.5 mM 3-isobutyl-1-methylxanthine. Control and agonist-treated cells were then lysed in 0.1 M HCl and cAMP level 97  was determined by enzyme-linked immunosorbent assay using a cAMP Kit from BioVision, Inc. CA, USA as per manufacturer’s instruction and as described earlier (Somvanshi et al., 2011a). 4.2.9 Western blot analysis Cotransfected HEK-293 cells were treated with SST-14 (50 nM), L-817818 (50 nM) and WIN (1 µM) alone or in combination as well as in a concentration-dependent manner for 15 min at 37 °C. Cells were washed with cold PBS and lysed in RIPA buffer. 15 μg of protein were solubilized in Laemmli sample buffer containing 5% β-mercaptoethanol, fractionated by SDS-PAGE and transferred onto nitrocellulose membrane. Membranes were processed for p- and t-PKA (1:500 dilution), p- and t-ERK1/2 (1:1000 dilution), and p- and t-PI3K (1:500 dilution). Images were captured using Alpha Innotech FluorChem 8800 gel box imager. β-actin was used as loading control. Bands were quantified by densitometry analysis using FluorChem software (Alpha Innotech). 4.2.10 Statistical analysis Results were presented as mean ± SE. Statistical analysis was carried out using Graph Pad Prism 5.0 and statistical differences were taken at p values < 0.05. Results represent at least three independent experiments.  4.3 Results 4.3.1 SSTR5 and CB1R are coexpressed in rat brain regions To determine the expression and colocalization of SSTR5 and CB1R in CNS, double labeled immunofluorescence immunocytochemistry was performed in 40 µm thick free-floating coronal rat brain sections. We focused on three different brain regions, including cortex, striatum and hippocampus that are affected most in several neuropathological conditions. As shown in Figure 98  4.1, SSTR5 and CB1R were expressed in all three brain regions and displayed region-specific colocalization. In cortex, strong SSTR5 expression was seen in pyramidal neurons in deep layers along with receptor like immunoreactivity in neuronal processes and nerve fibers (Figure 4.1A). On the contrary, CB1R like immunoreactivity was largely confined to fiber-like structures while expressing moderately in sparsely distributed neuronal cell body (Figure 4.1B). Colocalization of SSTR5 and CB1R was mostly detected in neuronal processes and soma. Moderate colocalization was also present on the cell surface of SSTR5-positive pyramidal neurons, surrounded by CB1R-positive interneuron terminals (Figure 4.1C). In the striatum, CB1R-positive neurons were relatively higher in number than SSTR5 positive neurons (Figure 4.1D and E). Three different neuronal populations displaying either colocalization or single staining were frequently observed in the striatum (Figure 4.1F). In the hippocampal CA1 and CA2 regions, the majority of neurons positive to both SSTR5 and CB1R were confined to stratum pyramidal layer with scattered SSTR5 positive interneurons (Figure 4.1I and L). Besides neuronal cell bodies, nerve fibers and neuronal processes expressing colocalization were also present in hippocampus, especially in CA1 region (Figure 4.1I). In comparison to CA1, receptor colocalization in CA2 was mild with fewer SSTR5 positive neurons (Figure 4.1L). Neuronal cells displaying only SSTR5 expression were also seen in stratum oriens and stratum pyramidal layer (Figure 4.1L). The presence of SSTR5 and CB1R coexpression in rat brain postulates the possibility of receptor interaction in the CNS. 99   Figure 4.1 Representative confocal photomicrographs showing the colocalization of SSTR5 and CB1R in rat brain.  40 µm free-floating rat brain sections were probed with SSTR5 and CB1R antibodies, followed by incubation with FITC and Cy3 conjugated secondary antibodies to visualize SSTR5 and CB1R as described in experimental procedures. Neuronal populations expressing SSTR5, CB1R and colocalization are shown in green, red and yellow (merged images) respectively. In the cortex (panels A-C), SSTR5/CB1R colocalization was mostly seen in neuronal bodies and nerve fibers. Strong SSTR5-like immunoreactivity was expressed in pyramidal neurons, whereas CB1R was expressed widely in nerve fibers and sparsely distributed neuronal cell bodies. In the striatum (panels D-F), SSTR5 and CB1R showed moderate to strong colocalization (arrows, panel F), along with neuronal cells showing single staining (open and solid arrowheads, panel F). In the hippocampus CA1 (panels G-I) and CA2 (panels J-L) regions, 100  interneurons only positive to SSTR5 were seen in SP (open arrowheads, panels I and L) with neuronal population displaying moderate colocalization (arrows, panels I and L). SSTR5-experssing neurons were also observed in the peripheral zone of stratum oriens of CA2 region (asterisk, panel L). In contrast, CB1R was well-distributed in nerve fibers and neuronal processes (thin arrow, panel I and L). SO, stratum oriens; SP, stratum pyramidal layer; SR, stratum radiatum. Scale bar = 40 µm.  4.3.2 CB1R is expressed in SSTR5 immunoprecipitates from rat brain tissue In support of our colocalization studies and to gain additional biochemical evidence, we next determined whether CB1R is expressed in SSTR5 immunoprecipitate prepared from cortical and striatal tissue lysates using Co-IP as described in Section 4.2. Tissue lysate preparation was treated with L-817818 (50 nM) and WIN (1 µM) alone or in combination for 30 min at 37 °C. Results depicted in Figure 4.2 demonstrate expression of CB1R in SSTR5 immunoprecipitate at the expected molecular size of ~110 kDa without any significant changes upon treatment with receptor specific agonist alone or in combination. Also, an extra band at relatively smaller molecular size was seen only in the cortical tissue lysate in control and upon treatment with L-817818 but not in the striatum. We next confirmed input by determining the expression of SSTR5 using receptor specific antibody. As shown in Figure 4.2 (lower panel), SSTR5 was detected as monomer as well as in homodimer entity at the expected molecular size of ~58 and ~110 kDa in both cortex and striatum respectively. These results indicate that CB1R and SSTR5 in cortex and striatum exist in a stable constitutive complex, independent of receptor activation. 101   Figure 4.2 Co-IP analysis illustrating CB1R expression in SSTR5 immunoprecipitates. Cortical and striatal SSTR5 immunoprecipitates were blotted for CB1R. Note a single band representing SSTR5/CB1R complex at the expected molecular size of ~110 kDa in cortical and striatal extracts without any significant changes upon agonist treatments (Upper panel). The input was confirmed in SSTR5 immunoprecipitates blotted with SSTR5 specific antibody. Note bands representing SSTR5 homodimer (~110 kDa) and monomer (~58 kDa) (Lower panel).  102  Figure 4.3 Co-IP analysis displaying constitutive SSTR5/CB1R heterodimerization in cotransfected cells. Cotransfected cells expressing SSTR5/CB1R were treated with receptor specific agonists alone or in combination for 30 min and processed for co-IP. Membrane fractions prepared from control and treated cells were immunoprecipitated with HA antibody for CB1R and immunoblotted with cMyc antibody for SSTR5. (A) Note strong band at expected molecular size of ~110 kDa representing constitutive complex formation between CB1R and SSTR5 with no significant effect upon agonist treatment. (B) CB1R membrane immunoprecipitates from wild type (regular) and CB1R or SSTR5 mono-transfected HEK293 cells were blotted for SSTR5. Only a nonspecific band at ~77 kDa is observed in membrane lysate of SSTR5 mono-transfected cells, indicating the specificity of immunoprecipitation.  4.3.3 SSTR5 and CB1Rs exist as constitutive heterodimers in HEK-293 cells Having seen the expression of SSTR5/CB1R complex in rat brain cortex and striatum, we next examined interaction between SSTR5 and CB1R in stably-cotransfected HEK-293 cells using Co-IP. Briefly, cotransfected HEK-293 cells were grown to 70% confluency and treated with 50 nM SST, 50 nM L-817818 and 1 µM WIN alone or in combination for 15 min at 37ºC. Posttreatment membrane fractions prepared from control and treated cells were immunoprecipitated for CB1R with HA antibody and probed for SSTR5 with cMyc antibody. In untreated cotransfected cells, strong band at the expected molecular size of ~110 kDa was detected in CB1R immunoprecipitate, representing SSTR5/CB1R complex. Expression of SSTR5 in CB1R immunoprecipitate was without any discernible changes upon receptor activation except following treatment with WIN alone (Figure 4.3A). Specificity of heterodimerization was also confirmed in SSTR5 immunoprecipitate blotted for CB1R (data not shown). In addition, specificity of antibody and receptor heterodimerization was confirmed in non-transfected HEK-293 cells (regular) and in cells mono-transfected with CB1R or SSTR5. In such experimental conditions, no specific band was visible except a nonspecific band at ~77 kDa in SSTR5 mono-transfected cells (Figure 4.3B). 103  These results demonstrate a physical association between SSTR5 and CB1R, yet insufficient to justify direct protein-protein interaction due to distance limitation of Co-IP techniques. Accordingly, cells were processed for microscopic pbFRET analysis at cell surface. 4.3.4 Agonist-dependent suppression of SSTR5 and CB1Rs heterodimerization To obtain biophysical evidences in support of physical interaction between SSTR5/CB1R at cell membrane, microscopic pbFRET analysis was accomplished in cotransfected cells. As shown in Figure 4.4A, in control cells high effective FRET efficiency of 18.48±0.67% is an indication of constitutive SSTR5/CB1R heterodimers. Treatments with receptor specific agonists L-817818 (50 nM) and WIN (1 µM) alone or in combination exhibited a significant decrease in relative FRET efficiency to 12.36±1.18%, 16.57±1.47%, 9.05±0.55% respectively, suggesting an agonist-induced dissociation of receptor heterodimerization. Previous studies have shown that CB1R exists as dimer or tetramer both in vitro and in vivo, whereas SSTR5 exists as monomer under basal condition and dimerizes upon agonist treatment (Rocheville et al., 2000b, Wager-Miller et al., 2002). To elucidate the molecular mechanism responsible for the agonist-induced loss in FRET efficiency, cotransfectants were processed for homodimerization of SSTR5 and CB1R using pbFRET analysis. As shown in Figure 4.4B, SSTR5 exhibited a relative FRET efficiency of 5.45±1.76% under basal condition, which was increased to 16.90±1.57% and 13.62±1.43% following treatment with L-817818 in the absence or presence of WIN respectively, suggesting agonist induced homodimerization of SSTR5. In contrast, CB1R exists as preformed dimer in basal condition with relative FRET efficiency of 10.92±2.07%. Treatment with WIN alone or in combination with L-817818 resulted in significant loss in FRET efficiency to 5.18±0.95% and 4.24±0.76%, suggesting the disruption of preformed CB1R homodimer (Figure 4.4C). Taken together, these results demonstrate that CB1R and SSTR5 exist 104  in a heteromeric complex at cell surface of cotransfected cells. Simultaneous receptor activation leads to the loss of heterodimerization between SSTR5 and CB1R, which could potentially be associated with the preferential SSTR5 homodimer formation and loss of CB1R homodimers.  Figure 4.4 Representative histograms displaying relative FRET efficiency of homo- and heterodimerization in cotransfected cells. HEK293 cells stably expressing SSTR5/CB1R were subjected to microscopic pbFRET analysis as described in experimental procedures. Upon treatment with receptor specific agonist L-817818 (50 nM) and WIN (1 µM) alone or in combination for 15 min at 37 °C, cells were incubated with anti-HA (CB1R) and/or anti-cMyc (SSTR5) primary antibodies overnight. FITC and Cy3 conjugated secondary antibodies were used to generate donor-acceptor pair. (A) SSTR5/CB1R heterodimerization in response to agonist treatments. Note a significant decrease in FRET efficiency upon treatment with SSTR5 agonist alone or in combination with CB1R agonist, but not with CB1R agonist alone. (B) Cotransfected cells exhibited SSTR5 homodimer formation upon SSTR5 agonist treatment with or without CB1R agonist. (C) Dissociation of constitutive CB1R homodimer to monomer upon treatment with CB1R agonist with or without SSTR5 agonist. Results are presented as mean ± S.E.M. of three independent experiments. Data were analyzed using one-way ANOVA and post hoc Dunnett’s was applied to compare with control. *, p<0.05. 4.3.5 Agonist-dependent internalization of CB1R and SSTR5 in cotransfected HEK-293 cells Cell surface expression and agonist-induced internalization of receptors are critical determinants of protein-protein interaction. Having seen the loss in FRET efficiency upon 105  concurrent receptor activation, we next investigated whether receptor expression and internalization could be associated. Cotransfected HEK-293 cells were treated with L-817818 (50 nM) and WIN (1 µM) alone and/or in combination for 15 min at 37ºC and processed for immunofluorescence immunocytochemistry. As shown in Figure 4.5, in control cells, CB1R and SSTR5 were expressed on cell membrane and intracellularly (panel C and D). Activation of SSTR5 or CB1R in the presence of receptor specific agonist induced internalization of only corresponding receptor, leaving alternate receptor on cell surface (panel H and L, thin arrows). Furthermore, receptor colocalization at cell membrane was significantly diminished upon concurrent treatment of receptor agonists (panel O, arrowheads). Interestingly, scattered SSTR5 like immunoreactivity was observed on cell membrane following co-activation of both receptors (panel P, thin arrows). Results described here indicate internalization of SSTR5 and CB1R in a complex upon simultaneous co-activation of both receptors in cotransfected HEK-293 cells, which might be potentially associated with agonist-induced loss in FRET efficiency of heterodimer. 106   Figure 4.5 Agonist-induced internalization of SSTR5 and CB1R in cotransfected HEK-293 cells. HEK-293 cells with stable SSTR5/CB1R expression were treated with receptor specific agonists alone or in combination for 15 min at 37 °C as described in the legend to Figure 4 and processed for immunofluorescence immunocytochemistry. Boxed cells in merged images were enlarged to show membrane receptor distribution in panel D, H, L and P. Under basal condition, CB1R (red) and SSTR5 (green) displayed colocalization (yellow) in merged image on cell membrane and intracellularly (arrows, panels C and D). Note decreased cell membrane expression of corresponding receptor upon single agonist treatment while leaving alternate protomer on the cell membrane (thin arrows, panels G, H, K, and L). Simultaneous activation of both receptors enhanced intracellular distribution 107  (arrowheads, panel O) and loss of cell membrane expression. Note the cell membrane expression of SSTR5 even in the presence of both agonists (thin arrows, panel P), indicating two different pool of SSTR5. Images represent at least three independent experiments. In panels A-C, E-G, I-K, and M-O, scale bar = 25 µm; in panels D, H, L, and P, scale bar = 5 µm.  4.3.6 Inhibition of cAMP in cells cotransfected with SSTR5/CB1R is prominently regulated by SSTR5 Here we intended to compare the inhibition of FSK-stimulated cAMP in mono- and cotransfected HEK-293 cells. As shown in Figure 4.6A, SSTR5 monotransfected cells displayed concentration-dependent inhibition of FSK-stimulated cAMP to maximum inhibition of 42.23±3.44% in the presence of SST. Treatment of CB1R mono-transfected cells with WIN induced four folds less inhibitory effect on cAMP in comparison to SST (Figure 4.6B). In comparison, cotransfected cells treated with L-817818 (50 nM) or WIN (1 µM) alone inhibited cAMP level by 23.15±3.30% and 5.67±1.07% respectively, comparable to results seen in mono-transfected cells (Figure 4.6C). Concomitant application of both agonists L-817818 (50 nM) and WIN (1 µM) exhibited 21.46±1.17% inhibition of FSK-stimulated cAMP, comparable to inhibition induced by activation of SSTR5 alone (Figure 4.6D). We further extended our study and determined the concentration dependent effect of receptor agonists on cAMP inhibition. As shown in Figure 6D, in the presence of L-817818 (50 nM), comparable inhibitory effect on cAMP was maintained despite the increasing concentration of WIN (100 nM to 2 µM). On the contrary, increasing concentration of SSTR5 agonist L-817818 (10 nM to 500 nM) in the presence of WIN (1 µM) displayed enhanced inhibition of FSK-stimulated cAMP (8.72±0.44% to 59.23±1.01%) in a concentration-dependent manner. These results indicate that SSTR5 exerts a predominant inhibitory role on FSK-stimulated cAMP 108  formation in cotransfected cells. Furthermore, changes in cAMP in response to SSTR5 and CB1R agonist were sensitive to PTX treatment, indicating predominant Gi coupling of SSTR5/CB1R complex in regulation of cAMP signaling (Figure 4.6E).  Figure 4.6 cAMP is predominantly regulated by SSTR5 in cotransfected cells. Receptor coupling to adenylyl cyclase was determined by inhibition of FSK-stimulated cAMP in mono- and cotransfected HEK293 cells. Briefly, cells were grown to 70~80% confluency and treated in a concentration-dependent manner with receptor specific agonists alone or in combination for 30 min in the presence of FSK and IBMX. Posttreatment cells were lysed and cAMP level was measured using a cAMP Kit as described in experimental procedures. (A and B) In SSTR5 or CB1R mono-transfected cells, activation of corresponding receptor reduced cAMP formation in a concentration-dependent fashion. (C) In cotransfected cells, activation of either receptor exhibited inhibition of cAMP comparable to mono-transfected cells. (D) Upon concurrent treatment, inhibition of cAMP was 109  enhanced in response to increasing concentration of L-817818 but not WIN. (E) Receptor-induced cAMP inhibition was significantly attenuated in cells pretreated with PTX. Results are presented as mean ± S.E. of three independent experiments performed in triplicates. Data were analyzed using one-way ANOVA and post hoc Dunnett’s was applied to compare with FSK-treated cells. *, p<0.05.  Figure 4.7 The phosphorylation of PKA in cotransfected cells is in parallel to cAMP. Cell lysates prepared from control and treated cells as indicated were subjected to Western blot analysis for PKA phosphorylation. (A) Activation of SSTR5 with receptor specific agonist alone or in combination inhibited PKA phosphorylation significantly, whereas in response to WIN, PKA phosphorylation was enhanced in comparison to control. Note significant inhibition of CB1R-induced phosphorylation in the presence of SST and SSTR5 agonist. (B) Upon treatment with increasing concentration of WIN in the presence of L-817818 (50 nM), the status of PKA phosphorylation was comparable to control. Note the inhibition of WIN-induced PKA phosphorylation in the presence of increasing concentration of L-817818. β-actin was used as loading control. Data are representative of three independent experiments. Data analysis was performed by using one-way ANOVA and post hoc Dunnett's to compare against basal level. *, p<0.05. 110  4.3.7 PKA is regulated in cAMP-dependent manner To assess whether agonist-induced changes in PKA phosphorylation are in agreement with the changes in cAMP, PKA phosphorylation level was determined using Western blotting analysis in cotransfected cells. As shown in Figure 4.7A, in comparison to control, the status of PKA phosphorylation was decreased significantly upon treatment with SST (50 nM) or L-817818 (50 nM). Conversely, WIN (1 µM) enhanced PKA phosphorylation, which was abrogated in cells treated with SST or L-817818 in combination (Figure 4.7A). As shown in Figure 4.7B, increasing concentration of WIN (50 nM to 1 µM) with SSTR5 agonist L-817818 (50nM) had no discernible effect on PKA phosphorylation. In comparison, increasing concentration of SSTR5 agonist L-817818 (10 to 500 nM) with WIN (1 µM) exhibited dual effects on PKA phosphorylation. As shown in Figure 4.7B, at lower concentration of SSTR5 agonist with WIN (1 µM), the increased PKA activation was probably due to CB1R activation, whereas inhibition with increasing concentration of SSTR5 agonist was associated with the activation of SSTR5. Taken together, these results indicate that changes in PKA phosphorylation are comparable to cAMP, but in receptor specific manner. 4.3.8 Receptor-mediated ERK1/2 signaling is altered in cotransfected cells SST-mediated inhibition of cell proliferation via SSTR subtypes is a unique property which involves ERK1/2 activation. CB1R and SSTRs have been reported to activate ERK1/2 in a PKA-dependent manner as well as independent of Gi protein (Daigle et al., 2008, Grant et al., 2008). To derive direct physiological significance of SSTR5 and CB1R heterodimerization, we next determined the status of ERK1/2 phosphorylation. As shown in Figure 4.8A, HEK-293 cells with stable coexpression of SSTR5 and CB1R exhibited upregulation of ERK1/2 phosphorylation upon treatment with receptor specific agonist alone or in combination. Moreover, concomitant activation 111  of SSTR5 and CB1R uncovered the predominant role of SSTR5 in regulation of ERK1/2 phosphorylation in cotransfected cells. As shown in Figure 4.8B, increasing concentration of L-817818, but not of WIN, revealed a gradual enhanced phosphorylation of ERK1/2. We have previously shown Gi dependency on SSTR5-induced ERK1/2 phosphorylation (Grant et al., 2008). Accordingly, to determine whether CB1R mediated activation of ERK1/2 is also dependent on Gi, cells were treated with PTX and processed for ERK1/2 phosphorylation. As shown in Figure 4.8C, cells pre-exposed to PTX displayed partial blockade of CB1R induced ERK1/2 phosphorylation. Taken together, these results indicate that CB1R modulate ERK1/2 phosphorylation in Gi dependent manner and activation of ERK1/2 in cotransfected cells is primarily regulated by SSTR5.  Figure 4.8 Changes in the phosphorylation status of ERK1/2 upon agonist treatment in cotransfected cells. Cells expressing SSTR5 and CB1R were treated with SST, L-817818, and WIN alone or in combination for 15 min at 37 °C. Protein extracts from each treatment group were analyzed for ERK1/2 phosphorylation level. (A) Both of single and combined agonist treatment strongly activated ERK1/2 without any significant difference among treatments. (B) In the presence of L-817818 (50 nM) with increasing concentration of WIN, p-ERK1/2 level was not altered in comparison to control. However, ERK1/2 phosphorylation was enhanced in a concentration-dependent manner with 112  L-817818 in the presence of WIN (1 µM). (C) Pretreatment of cells with PTX abrogated WIN-induced ERK1/2 phosphorylation. β-actin was used as loading control. Data are representative of three independent experiments. Data analysis was done by using one-way ANOVA and post hoc Dunnett's to compare against basal level. *, p<0.05.  4.3.9 PI3K Phosphorylation is regulated in time and receptor dependent manner To determine if heterodimerization between SSTR5 and CB1R is involved in the modulation of PI3K, a signaling pathway associated with tumor progression and neuroprotection, cotransfected HEK-293 cells were treated with receptor specific agonist as indicated for 15 min at 37 ºC and processed for the expression and phosphorylation of PI3K. As shown in Figure 4.9A, PI3K phosphorylation was not changed significantly in comparison to control, whether cells were treated with SSTR5 and CB1R agonists alone or in combination. Also, status of PI3K phosphorylation was indistinguishable from control upon treatment with different concentrations of either receptor agonist in combination as indicated (Figure 4.9B). However, interesting observations emerged from time dependent experiments. As shown in Figure 4.9C, upon treatment with L-817818 (50 nM), p-PI3K level was lowest at 5 min and increased at extended time points (10-30 min), albeit still lower than control. In contrast, there was a time-dependent bi-phasic effect of CB1R activation exhibiting decreased PI3K phosphorylation following 10 and 30 min treatment, but comparable to control at 5 and 15 min of treatment. Interestingly, prolonged (10-30 min) activation of both receptors suppressed PI3K phosphorylation whereas upon 5 min treatment, the status of PI3K phosphorylation remained comparable to control. These results not only suggest time-dependent changes in PI3K phosphorylation but also uncovered the predominant role of CB1R at early point and SSTR5 following extended treatment (Figure 4.9C). 113   Figure 4.9 Time-dependent modulation of PI3K phosphorylation upon SSTR5 and CB1R activation. Following treatment as indicated in legend to Figure 8, cell lysate was processed for PI3K phosphorylation. The status of PI3K phosphorylation was indistinguishable from cells either treated with agonist alone or in combination (A) or with different concentrations of agonist as indicated (B). Note time-dependent changes in PI3K phosphorylation in the presence of receptor specific agonists (C). β-actin was used as loading control. Data are representative of three independent experiments. Data analysis was done by using two-way ANOVA and Bonferroni posttest to compare against basal level. *, p<0.05.  4.4 Discussion GPCRs function as homo- and heterodimers with efficient functional translation distinct from monomers and homodimers entity (Ferre et al., 2014, Gomes et al., 2016). Although the 114  pathophysiological significance of such interaction is still awaited, studies have addressed such issue in conditions including tumor biology, neurodegeneration and immunological diseases (Mellado et al., 2001, George et al., 2002, Ferre et al., 2014). SSTR2 and SSTR5 functionally interact with D2R, regulating receptor binding properties and coupling to AC (Rocheville et al., 2000a, Baragli et al., 2007). To extend these observations and elucidate the role of SSTR5 in a broader sense, present study was undertaken to describe interaction between CB1R and SSTR5 in rat brain with endogenous expression and also in stably cotransfected HEK-293 cells. SSTR5 and CB1R colocalize in different rat brain regions as well as at the cell membrane of transfected cells, supporting the possibility of receptor heterodimerization. Receptor heterodimerization is further strengthened by the expression of CB1R in SSTR5 immunoprecipitates prepared from rat brain cortex and striatum. In cotransfected HEK-293 cells, the expression of SSTR5 was observed in CB1R immunoprecipitates prepared from membrane extracts, in addition to the high relative FRET efficiency of heterodimer at cell surface. Furthermore, the modulation of signal transduction pathways confirmed functional and positive crosstalk between SSTR5 and CB1R. To our knowledge, this is the first comprehensive description of SSTR5 and CB1R heterodimerization in rat brain and cotransfected HEK-293 cells, with morphological, biochemical and physiological significance. Previous studies using transfected cells either expressing CB1R or SSTR5 have described receptor internalization in response to agonists (Hukovic et al., 1996, Hsieh et al., 1999, Cescato et al., 2006). SSTR5 in response to SST exhibits time-dependent internalization in transfected CHO-K1 cells that shows no upregulation upon prolonged agonist treatment (Hukovic et al., 1996). On the other hand, CB1R displayed constitutive and agonist-induced internalization and recycling (Leterrier et al., 2004). In mono-transfected HEK-293 cells, CB1R locates 115  predominantly in intracellular compartments and translocates to membrane when forming heteromeric complex with other GPCRs (Canals and Milligan, 2008, Rozenfeld et al., 2011). Consistent with these observations, we show CB1R expression and colocalization with SSTR5 at cell membrane and intracellularly. SSTR5 and CB1R internalization was receptor-specific in response to selective agonists, whereas receptor complex internalizes when both receptors are activated simultaneously. These observations are in contrast to previous studies describing SSTR2/SSTR5 and SSTR2/SSTR3 suggesting that receptor internalization is restricted to the activated protomer only (Pfeiffer et al., 2001, Grant et al., 2008). Internalization of SSTR5/CB1R in complex upon concurrent activation is an indication of stable complex formation, opposing the concept that GPCRs are not in static complex. Interestingly, we found sparsely distributed SSTR5 like immunoreactivity at cell surface following treatment with both agonists simultaneously. We believe that remaining SSTR5 at cell surface uncover two important features: first, SSTR5 exists at membrane independently as well as in complex with CB1R; second, remaining receptors at cell surface are responsible for the preferential homodimerization upon concomitant agonist treatment (Figure 4.4B). Arguably, lack of CB1R at cell surface favors dissociation of preformed CB1R homodimers due to receptor internalization (Figure 4.4C).  Changes in FRET analysis in parallel to our colocalization results are consistent with the notion that the receptor expression at cell surface is crucial in receptor homo- and heterodimerization, as we described previously (Rocheville et al., 2000b). In HEK-293 cells stably expressing receptors at physiological level, SSTR5 and CB1R exist as constitutive heteromers at cell surface. FRET efficiency of SSTR5/CB1R heterodimer decreases significantly in the presence of SSTR5 agonist, but not with CB1R agonist. Loss in FRET efficiency upon co-activation of SSTR5/CB1R possibly attributes to receptor co-internalization. Despite significant loss in FRET efficiency upon 116  concurrent stimulation of both receptors, we find enhanced inhibition of FSK-stimulated cAMP which is predominantly regulated by SSTR5. How might the activation of SSTR5 and CB1R enhance cAMP inhibition despite losing relative FRET efficiency in cotransfected cells? One possible explanation is that agonist-induced changes in receptor organization and conformation at cell surface lead to the loss of relative FRET efficiency. The other plausible mechanism could be preferential homodimerization of SSTR5 and dissociation of CB1R dimers which is supported by our in vitro observations. We have previously shown that SSTR5 exists as monomer in basal condition and displays homodimerization in the presence of agonist, whereas CB1R exists as homodimers, shown by others (Rocheville et al., 2000b, Wager-Miller et al., 2002, Mackie, 2005a). Consistently, increased inhibition of cAMP formation upon SSTR5 agonist treatment underscores the role of preferential homodimerization of SSTR5 in cotransfected cells. Whereas the masking of WIN induced concentration-dependent inhibition of cAMP formation upon concomitant treatment with SSTR5 agonist clearly indicates the predominant role of SSTR5 in the heterodimeric complex.  The effector signaling molecule cAMP has been studied most for GPCRs whereas other signal transduction pathways are still elusive. Previous studies have shown rapid increase in ERK1/2 phosphorylation in response to short-term activation of CB1R, whereas sustained activation leads to diminished ERK1/2 phosphorylation (Daigle et al., 2008). Consistent with these observations, we found increased ERK1/2 phosphorylation upon activation of single or both receptors simultaneously. Inhibition of PKA phosphorylation may also account for increased ERK1/2 with CB1R agonist. This positive crosstalk is Gi-dependent since such changes were sensitive to PTX. On the other hand, PKA-independent phosphorylation of ERK1/2, which is not blocked by PTX, should be attributed to CB1R. Our previous study has shown that SSTR5 is unable to initiate the 117  recruitment of β-arrestin, another important mediator of ERK1/2 signaling besides G protein (Grant et al., 2008). CB1R has been shown to recruit β-arrestin 2 rapidly to cell membrane and regulate ERK1/2 in response to receptor activation (Daigle et al., 2008). It is also possible that CB1R regulates ERK1/2 through another G protein other than Gi, as it is suggested that WIN-stimulated CB1R may switch coupling from Gi to other G proteins when Gi is unavailable (Lauckner et al., 2005). Our findings are in line with the above studies, as SSTR5 is coupled to Gi and associated with regulation of downstream signaling in complex with CB1R. Based on this hypothesis, we argue that SSTR5 in heteromeric complex with CB1R plays a major role in Gi-dependent signaling while CB1R is responsible for Gi-independent signaling, revealing a biased-signaling regulation. In addition to modulation of ERK1/2 phosphorylation, studies using mono-transfected cells either expressing SSTR5 or CB1R have shown their effect on PI3K activation (Gomez del Pulgar et al., 2000, Somvanshi et al., 2011b). Both negative and positive functional interactions between ERK1/2 and PI3K have been observed in a cell type-dependent manner. In the presence of PI3K inhibitor, CB1R-induced phosphorylation of ERK1/2 is reversed in prostate PC-3 and U373MG human astrocytoma cells, but transiently activated in Neuro2A cells (Galve-Roperh et al., 2002, Sanchez et al., 2003, Graham et al., 2006). Furthermore, functional independence between ERK1/2 and PI3K has also been reported. HU-210-mediated activation of CB1R results in PI3K phosphorylation in STHdh mouse striatal neuroblast and retinal neurons, whereas in Neuro2A cells, HU-210 activates ERK1/2 but not PI3K (Graham et al., 2006, Blazquez et al., 2015, Kokona and Thermos, 2015). Our findings are in agreement with the latter studies, as receptor activation leads to decreased PI3K phosphorylation albeit non-significant. Considering ERK1/2 activation upon receptor stimulation, it is possible that SSTR5/CB1R complex favors cAMP/PKA/ERK1/2 118  pathway over PI3K signaling, implying a receptor complex-specific regulation of downstream signal transductions. Interesting observations emerged from time-dependent experiments showing efficient inhibition of PI3K upon co-activation of both receptors and attesting positive crosstalk between SSTR5 and CB1R with implication in cell proliferation. Also, it should be noted that activation of PI3K signaling pathways with sustained activation of either SSTR5 or CB1R might be involved in neuroprotection. Whether time-dependent gain and loss of PI3K activity in the presence of CB1R agonist is associated with CB1R internalization and translocation back to membrane warrants future studies.  Consistent with previous studies showing that receptor heterodimerization is associated with receptor trafficking and functions, here we show that co-activation of both receptors leads to receptor internalization in a complex, whereas activation of one protomer is without any effect on the other. In cotransfactants, despite the disruption of heterodimers, enhanced inhibition of cAMP can be obtained because of preferential homodimerization of one receptor without any influence from the status of the other interacting protomer. In addition, we observed heterodimerization-dependent and independent regulation of signaling transduction pathways. In conclusion, the results we describe here provide first direct evidence for the role of CB1R and SSTR5 in rat brain and transfected cells, which are relevant to several physiological functions including cell proliferation, neuroprotection and perception of pain. These results in a broader sense explored the possible interaction between SSTR5 and CB1R and described functional consequences that can serve as a potential therapeutic approach in designing new drug targeting CB1R in combination with SSTR5 that might play an important role in abrogating cannabinoid associated unexpected side effect. Future studies are warranted to identify the molecular mechanisms associated with 119  chronic and acute activation of CB1R and SSTR5 that might contribute to distinguishing beneficial effects from unwanted side effects with the use of cannabinoids.   120  Chapter 5: Role of SSTRs and CB1R in protection of striatal neurons and regulation of signaling pathways  5.1 Background HD is an inherited neurodegenerative disorder caused by an abnormal expansion of the CAG repeat in exon 1 of huntingtin gene. This gene encodes for a ubiquitously expressed cytoplasmic protein, Htt, with highest expression in neurons of CNS. The threshold of disease onset is above 35 repeats, with an inverse correlation between the length of repeats and the age of HD onset. The clinical symptoms of HD include chorea, dystonia, and impaired cognition. The neuropathological hallmark of HD is the severe atrophy of neostriatum with marked neuronal loss and gliosis (Zuccato et al., 2010). Striatal neuronal loss happens preferentially in the MSNs, which make up over 90% of all striatal neurons. A subclass of medium aspiny interneurons that coproduce SST, NPY, and nNOS, however, survive the early neurodegenerative process (Ferrante et al., 1985). The mechanism underlying the selective neuronal loss and survival is unclear. Excitotoxicity, particularly through overactivation of NMDAR, is proposed to be one of the pathogenic mechanisms of neuronal loss in HD (Doble, 1999, Zuccato et al., 2010). This notion is supported by the observation that the concentration of QUIN, an endogenous NMDA agonist, is elevated in the brain of HD patients and NMDAR is overactivated in HD models (Guidetti et al., 2004, Fan and Raymond, 2007). Local high NMDAR expression in MSNs have been linked to their selective susceptibility in HD (Landwehrmeyer et al., 1995). Moreover, intrastriatal injections of NMDAR agonists such as QUIN, glutamate or NMDA to rodents in vivo, or the application of these agents to neuronal cultures in vitro replicates the pattern of selective neurodegeneration as normally seen in HD (Beal et al., 1986, Beal et al., 1991, Kumar, 2004). Nevertheless, excessive excitatory 121  neurotransmission is not the only cause for the progression of HD (Zuccato et al., 2010). Perturbed expression of dopamine, GABA, SST and CB1R have also been associated with HD pathogenesis, although the detailed mechanisms associated with these prominent molecules are not well elucidated yet (Zuccato et al., 2010). CB1R is one of the most widespread members of GPCR family in CNS, with high density seen in different brain regions, including hippocampus, cortex, cerebellum and basal ganglia (Mackie, 2005b, Kano et al., 2009). Upon activation by endocannabinoids, it is involved in a wide range of physio- and pathological activities, including learning, memory, motor behavior, appetite control, pain regulation, and neurodegenerative diseases (Pacher et al., 2006). In CNS, CB1R expressed at GABAergic and glutamatergic terminals has been shown to negatively regulate the release of GABA and glutamate through endocannabinoid-mediated retrograde signaling, contributing to the prevention of synaptic hyperactivation and attesting its neuroprotective role in several neurodegenerative diseases (Katona et al., 1999, Gerdeman and Lovinger, 2001, Marsicano et al., 2003, Katona and Freund, 2008, Chiarlone et al., 2014). This concept is further strengthened by the fact that downregulation of CB1R at the levels of mRNA and protein has been observed in many neurological disorders including HD, whereas genetic rescue of CB1R improves striatal neuronal survival in HD transgenic R6/2 mice (Miller and Devi, 2011, Naydenov et al., 2014). SST is an inhibitory peptide that suppresses hormone release and cell growth. In addition, SST in CNS serves as a neurotransmitter and neuromodulator and is involved in the regulation of a variety of neurological activities, including cognition, locomotor, sensory and autonomic functions (Schindler et al., 1996, Patel, 1999, Kumar and Grant, 2010). There is a considerable amount of evidence suggesting that SST plays a critical role in various neurodegenerative diseases including AD, HD, PD, MS as well as neuropsychological disorders such as major depression and 122  schizophrenia (Aronin et al., 1983, Epelbaum et al., 1983, Sorensen, 1987, Beal et al., 1988, Burgos-Ramos et al., 2008, Tallent and Qiu, 2008, Lin and Sibille, 2013). SST exerts such profound effects via binding to five SSTRs, expressed at various densities throughout the brain (Bruno et al., 1993). All five SSTRs inhibit cAMP formation via coupling to PTX-sensitive Gi proteins. SSTR subtypes have also been shown to regulate several downstream signal transduction pathways including MAPKs and ion channels (Patel, 1999, Kumar and Grant, 2010). The overlapping morphological, biochemical and pharmacological properties have signified the importance of both SST and cannabinoid in HD. CB1R is highly expressed in MSNs, thus largely downregulated during the progression of HD (Herkenham et al., 1990, Glass et al., 1993, Glass et al., 2000). The loss of striatal CB1R contributes to the pathogenesis of HD whereas genetic rescue of CB1R is beneficial in improving neuronal survival in R6/2 mice (Blazquez et al., 2011, Naydenov et al., 2014). In addition, CB1R-mediated neuroprotective effect has been observed in various excitotoxicity models (Kim et al., 2006a, Pacher et al., 2006, Zoppi et al., 2011). In comparison, medium-sized aspiny interneurons positive to SST are selectively preserved at the early stage of HD and expression level of SST is increased in the basal ganglia in HD (Aronin et al., 1983, Dawbarn et al., 1985, Ferrante et al., 1985). The most profound connection between HD and SST was established using SSTR1 and 5 double knock-out mice, which reproduced a neurochemical phenotype in the striatum comparable as seen in R6/2 mice (Rajput et al., 2011). Moreover, we have shown that SST protects striatal neurons against NMDA and QUIN-induced excitotoxicity in a PTX-sensitive manner, indicating the involvement of SSTR subtypes in neuroprotective effect of SST (Kumar, 2008). These results imply the crucial role that SSTR and CB1R might play in the pathogenesis of HD. However, it is not known whether these two receptor subtypes functionally interact with each other in HD. We have recently shown the colocalization 123  of CB1R and SST in rat brain hypothalamus and hippocampus (Zou and Kumar, 2015, Zou et al., 2015). In extension to the colocalization studies using rat brain, we recently described that these two receptors functionally interact with each other and exist as constitutive heterodimers that dissociate upon receptor activation in HEK-293 cells transfected with SSTR5 and CB1R (Zou et al., 2017). Concurrent activation of both SSTR5 and CB1R in cotransfected cells lead to a SSTR5-dominant role in the modulation of signaling pathways as seen in inhibition of cAMP and activation of ERK1/2 (Zou et al., 2017). However, the physiological significance and therapeutic implication of such crosstalk between SSTRs and CB1R is unclear. A number of HD models developed throughout these years have provided a better approach to study the process of cell death than in HD patients (Zuccato et al., 2010). Neurochemical models of HD utilizing neurotoxins such as 3-NP and QUIN, mimic key pathogenic events of HD, including mitochondrial dysfunction, DNA damage as well as apoptotic and necrotic cell death (Beal et al., 1986, Beal et al., 1991). Genetic models such as transgenic R6/2 mice provide the opportunity to understand the correlation between the toxicity of mHtt and the pathogenesis of HD (Zuccato et al., 2010). In addition, recently a conditionally immortalized striatal cell line developed from mutant (STHdhQ111/111) and wt (STHdhQ7/7) Htt knock-in mice have been used frequently (Trettel et al., 2000). These cells have been well characterized for biochemical and neurochemical features of HD, including impaired mitochondrial function, increased sensitivity to neurotoxins, and abnormal signal transduction pathways (Trettel et al., 2000, Ruan et al., 2004, Gines et al., 2010). However, despite the significant role of SST in HD, SSTRs have not been characterized in these striatal neuronal cells which represent a model of striatal projection neurons. Accordingly, in the present study, we investigated the expression, colocalization, and possible functional interactions between SSTR2/CB1R and SSTR5/CB1R in both STHdhQ111/Q111 and 124  STHdhQ7/Q7 cells as well as R6/2 mice brain. Our results presented here show that both SSTRs and CB1R exert neuroprotective effects through modulating the cell survival pathways, specifically ERK1/2, against QUIN-induced excitotoxicity, whereas concurrent activation of SSTR and CB1R diminish such pro-survival effect, suggesting receptor crosstalk in STHdh cells.  5.2 Materials and Methods 5.2.1 Animals Post-fixed brains from wt and HD transgenic R6/2 mice (7 and 11-week-old) were purchased from Jackson Laboratory.  5.2.2 Materials SST was procured from Bachem (Torrance, CA) and non-peptide SSTR2 agonist L-779976 and SSTR5 agonist L-817818 were kindly provided by Dr. S. P. Rohrer from Merck & Co. WIN and ACEA were purchased from the Tocris Cookson Inc., Ellisville, MO (Authorization 31250.09.13 and 31251.09.13). All drugs were constituted according to suppliers’ instructions, in either dimethyl sulfoxide or ethanol at a final concentration <0.1% (v/v). NGS was purchased from Vector Laboratories (Burlingame, CA). SSTR2 and 5 antibodies were produced in our laboratory and has been well characterized as described previously. CB1R anti-goat polyclonal antibody was purchased from Santa Cruz, CA. CB1R anti-guinea pig polyclonal antibody was purchased from Frontier Institute, Japan. FITC- and Cy3-conjugated secondary antibodies were obtained from Jackson ImmunoResearch, ON. Rabbit polyclonal antibodies for p- and t-ERK1/2, p- and t-Akt, and t-PI3K were purchased from Cell Signaling Technology, Danvers, MA. p-PI3K antibody was purchased from Santa Cruz Biotechnology, Santa Cruz, CA. Other chemicals and reagents were of AR grade and obtained from various sources. 125  5.2.3 Cell cultures Conditionally immortalized striatal cells from E14 mutant knock-in HdhQ111/Q111 (STHdhQ111/Q111) and their wt littermates (STHdhQ7/Q7), were originally developed by Dr. M. E. McDonald and purchased from Coriell Institute (Camden, NJ)(Trettel et al., 2000). Cells were maintained at permissive temperature of 33 °C in DMEM medium supplemented with 10% FBS, 1% streptomycin-penicillin, 2 mM L-glutamine and 400 µg/mL Geneticin in a humidified atmosphere containing 5% CO2. 5.2.4 Double-labeled fluorescence Immunohistochemistry 40 µm thick free floating coronal brain sections from wt and R6/2 (7 and 11 weeks) old mice were collected in TBS and incubated with 5% NGS containing 0.2% Triton-X in TBS. Sections were then incubated with primary anti-goat CB1R (1:500) antibody in combination with anti-rabbit SSTR2 (1:500) or SSTR5 (1:500) primary antibodies overnight at 4°C. After three washes in TBS, sections were incubated with corresponding FITC- and Cy3-conjugated secondary antibodies for 2 h at room temperature for final color development. Brain section were mounted on slides and photographed under Leica confocal microscope. Wildtype (STHdhQ7/Q7) and mutant (STHdhQ111/Q111) striatal neuronal cells were grown on poly-D-lysine coated coverslips in 24-well plate to 50-70% confluency. After fixation with 4% paraformaldehyde on ice for 20 min, Cells were permeabilized (P) or not (NP) with 0.2% Triton-X at room temperature for 15 min before proceeding to blocking with 5% NGS for 1 h. Cells were probed with primary antibodies against CB1R (1:500) in combination with anti-SSTR2 (1:500) or anti-SSTR5 (1:500) primary antibodies at 4 °C overnight. After three washes in PBS, sections were incubated with corresponding FITC- and Cy3-conjugated secondary antibodies for 2 h at room temperature. Cells were washed with PBS once and then incubated with 5 µg/mL Hoechst 126  33258 for 30 min for nuclear staining. After washing with PBS, cells were mounted and observed under Leica confocal microscope. Specificity of antibodies and immunoreactivity was validated as previously described and supported by other studies (Kumar et al., 1999, Kumar, 2007, Kano et al., 2009, Zou et al., 2017). 5.2.5 MTT assay Wildtype (STHdhQ7/Q7) and mutant (STHdhQ111/Q111) cells were seeded at a density of 5,000 cells/well in 96-well plate and allowed to grow overnight. Cells were starved in serum-free medium for 24 h, followed by incubation with Locke’s solution (154 Mm NaCl, 5.6 mM KCl, 2.3 mM CaCl2, 3.6 mM NaHCO3, 5 mM HEPES, 20 mM glucose and 10 µM glycine) supplemented with 1 µM SST, 1 µM SSTR2 specific agonist L-779976, 1 µM SSTR5 specific agonist L-817818, 1 µM WIN, and 1 µM ACEA, or respective vehicles in the presence or absence of 3 mM QUIN for 5 h (unless otherwise indicated). The Locke’s solution was replaced with serum-free DMEM supplemented with the corresponding drugs as indicated earlier. After 24 h, cells were washed twice with PBS and then incubated with 20 microliter (µL) of 5 mg/mL MTT solution for 4 h. The medium was then aspirated and replaced by 200 µL isopropanol. The plate was then read on spectrophotometer at 570 nanometer. 5.2.6 Co-IP Cells were grown in 6-well plate and allowed to reach 80% confluency. To assess the effect of agonist on receptor interaction, cells were treated with L-779976 (1 µM), L-817818 (1 µM), ACEA (1 µM) alone or in combination for 30 min at 33 °C. After one wash with cold PBS, cells were lysed in RIPA buffer. 250 µg of protein from whole cell lysates was immunoprecipitated with primary anti-SSTR2 or anti-SSTR5 antibody (1:500) overnight at 4 °C. Samples were then incubated with protein A/G agarose beads for 2 h at 4 °C to allow sufficient binding. The beads 127  were then washed with cold PBS and solubilized in Laemmli sample buffer containing 5% β-mercaptoethanol. Samples were fractionated by SDS-PAGE and transferred to nitrocellulose membrane. Membranes were then incubated with primary anti-CB1R antibody (1:500) overnight at 4 °C and probed with HRP-conjugated secondary antibody for 2 h at room temperature. Chemiluminescence reagents were used for developing and images were captured using Alpha Innotech Fluorchem 800 gel box imager. Specificity of immunoprecipitation was validated as previously described (Zou et al., 2017). 5.2.7 Western Blot Wildtype (STHdhQ7/Q7) and mutant (STHdhQ111/Q111) cells were grown in 6-well plate to 80% confluency. After treatment as indicated, cells were washed with cold PBS and lysed with RIPA buffer. Membrane and cytosolic portion were separated by centrifugation at 12,000 g at 4 °C for 1 h. 15 µg of protein were solubilized in Laemmli sample buffer containing 5% β-mercaptoethanol and was then fractionated by SDS-PAGE. Samples were transferred to nitrocellulose membrane, followed by blocking with 5% non-fat milk for 1 h at room temperature. Primary antibodies used to probe target proteins including CB1R (1:500), SSTR2 (1:500), and SSTR5 (1:500). For signaling changes, 15 µg of protein from whole cell lysates were fractioned using SDS-PAGE. Primary antibodies raised against p- and t-ERK1/2 (1:1000), p- and t-PI3K (1:500), phosphatase and tensin homologue (PTEN) (1:1000) were used. After overnight incubation with primary antibodies, membranes were further incubated with secondary antibodies for 2 h at room temperature. Membranes were developed using chemiluminescence reagents and images were captured using Alpha Innotech Fluorchem 800 gel box imager. β-actin was used as loading control for whole cell lysates and cytosolic portion. 128  5.2.8 Statistical analysis Results were presented as mean ± SE. Statistical analysis was carried out using Graph Pad Prism 5.0 and statistical differences were taken at p values < 0.05. Results represent at least three independent experiments.  5.3 Results 5.3.1 Colocalization of SSTR2, SSTR5 and CB1R in R6/2 mice Colocalization pattern of SSTR2 and CB1R in R6/2 mice brain We first investigated the expression and colocalization of SSTR2/CB1R and SSTR5/CB1R in cortex, striatum and hippocampus of wt and R6/2 mice brain sections by using double labeled immunofluorescence immunohistochemistry. In three different brain regions, SSTR subtypes and CB1R displayed region- and receptor-specific colocalization in an age-dependent manner in R6/2 mice brain when compared to wt. Receptor-like immunoreactivity was mainly expressed in pyramidal neurons in deep layers of cortex (Figure 5.1A). As shown in Figure 5.1A, cortical brain region from wt displayed three subpopulations of neuronal cells expressing either only SSTR2 (green) or CB1R (red) or colocalization (yellow). Colocalization between SSTR2 and CB1R was mostly confined to neuronal cell bodies. This pattern of colocalization was also observed in 7- and 11-week-old R6/2 mouse brain, albeit with a reduced intensity of immunoreactivity (Figure 5.1B and C). Similarly, in striatum, receptor-like immunoreactivity and colocalization was mainly confined to neuronal cell bodies, with much lower intensity in R6/2 mouse brain (Figure 5.1E and F) than wt (Figure 5.1D). In hippocampal CA1 region (Figure 5.1G-I), the majority of neurons positive to both SSTR2 and CB1R were seen in stratum pyramidal layer, with a few interneurons showing only SSTR2-like immunoreactivity expressed in or close to stratum pyramidal layer. In 129  comparison to wt, the intensity of immunofluorescence was greatly decreased in 11-week-old R6/2 mouse brain (Figure 5.1I), whereas relative expression and colocalization was not significantly changed in 7-week-old one (Figure 5.1H).  Figure 5.1 Representative confocal photomicrographs showing colocalization of CB1R and SSTR2 in regions of wt and HD transgenic R6/2 mice (7- and 11-week-old) mice brain. 40 µm brain sections from wt and R6/2 mice were incubated with primary antibodies against CB1R and/or SSTR2 overnight, followed by incubation with corresponding secondary antibodies. Neuronal cells displaying only CB1R (red) are indicated by arrowheads. Neurons showing only SSTR2-like immunoreactivity (green) are indicated by open arrows. Colocalization is shown in yellow and indicated by solid arrows in respective panels. Neuronal processes are indicated by thin arrows. Higher magnification view of neurons is shown in Inset. In wt mouse brain, CB1R and SSTR2 are well-expressed in cortex (A), striatum (D) and hippocampal CA1 region (G). In comparison to wt and 7-130  week-old R6/2 mice, the intensity of receptor-like immunoreactivity in 11-weeks-old mouse brain was decreased (panels C, F, and I). SO, stratum oriens; SP, stratum pyramidal layer; SR, stratum radiatum. Scale bar = 40 µm.  Figure 5.2 Double labelled immunofluorescence confocal photomicrographs depicting expression and colocalization of CB1R and SSTR5 in brain regions of wt and R6/2 mice. 40 µm brain sections from wt and R6/2 mouse were stained for the expression of CB1R and SSTR5 as described in legend to Figure 5.1. SSTR5-like immunoreactivity is shown in green and indicated by open arrows, and CB1R is shown in red and indicated by arrowheads. Colocalization is indicated by solid arrows and shown in yellow. Neuronal processes are indicated by thin arrows. Inset shows a higher magnification of part of the image. Both SSTR5 and CB1R are well-expressed in cortex (A-C), striatum (D-F) and hippocampus (G-I). Strong to moderate colocalization of SSTR5/CB1R is observed in all three regions in wt mouse. The intensity of colocalization is largely decreased in 11-week-old R6/2 mouse. Neurons positive to only SSTR5 or CB1R are also seen frequently. SO, stratum oriens; SP, stratum pyramidal layer. Scale bar = 40 µm. 131  Colocalization pattern of SSTR5 and CB1R in R6/2 mice brain As shown in Figure 5.2, neuronal cells displaying SSTR5 (green) or CB1R (red) or colocalization (yellow) were well-expressed in all three brain regions, with strong to moderate intensity. In cortex of wt mouse (Figure 5.2A), pyramidal neurons displaying SSTR5 or CB1R or both were frequently seen in deep layers. The expression of either receptor was largely decreased in R6/2 mouse brain (Figure 5.2B and C). In striatum, colocalization between SSTR5/CB1R was also observed in wt section (Figure 5.2D). Comparative analysis of CB1R-like immunoreactivity revealed relatively less expression in R6/2 mice brain of 11-week-old (Figure 5.2F) than 7-week-old (Figure 5.2E). In hippocampus, the majority of CB1R-like immunoreactivity and colocalization was present in pyramidal neurons (Figure 5.2G). Interneurons positive to only SSTR5 were scattered in stratum pyramidal layers and stratum oriens (Figure 5.2G). The intensity of receptor-like immunoreactivity for both receptors was decreased in 11-week-old R6/2 mouse brain, with a greater reduction in CB1R than SSTR5 (Figure 5.2I). 5.3.2 Expression of SSTR2, SSTR5 and CB1R in STHdh cells changes upon receptor activation Expression of SSTR2, SSTR5, and CB1R in STHdh cells In order to assess the role of SSTRs and CB1R in pathogenesis in HD, here we used STHdh cells, a well-developed striatal neuron-like cell line with knock-in wt (STHdhQ7/Q7) and mHtt (STHdhQ111/Q111). Previous studies have shown the endogenous expression of CB1R in STHdhQ7/7 and STHdhQ111/111 cells, however, whether these cells also express SSTRs endogenously is not known (Blazquez et al., 2011, Laprairie et al., 2013). Accordingly, we first determined the expression and distributional pattern of SSTR2/5 and CB1R using immunostaining and immunoblot analysis. As shown in Figure 5.3 and 5.4, all three receptors were well expressed 132  in both cell lines with distinct sites of expression. Unlike the well-known membrane localization of GPCRs in homologous or heterologous expression system, the relative expression of CB1R-like immunoreactivity (red) was prominently seen intracellularly rather than at cell surface in both STHdhQ7/7 and STHdhQ111/111 cells. In comparison to CB1R, SSTR2 (green) displayed higher membrane expression and comparable intracellular expression in both STHdh cells (Figure 5.3). As shown in Figure 5.4, like CB1R, the expression of SSTR5 (green) was predominantly in cytoplasm. Importantly, no notable difference was seen in intensity of immunoreactivity between STHdhQ7/7 and STHdhQ111/111 cells. Furthermore, strong colocalization between SSTR2/CB1R and SSTR5/CB1R was observed in both cell lines intracellularly, as well as occasionally spotted punctated receptor staining in the processes (Figure 5.3 and 5.4). In order to support results in immunostaining, receptor expression was also determined in whole cell lysates, cytosolic and membrane fractions using Western blot analysis. As shown in Figure 5.5, SSTR2, SSTR5 and CB1R were well expressed in whole cell lysates and cytosolic fraction at the expected molecular size of 57, 58, and 53 kDa. The receptor expression in membrane fractions was significantly lower than cytosolic fraction. We also noted relatively higher expression of CB1R in membrane fraction prepared from STHdhQ111/111 in comparison to STHdhQ7/7 cells. In contrast, SSTR subtypes expression was comparable in both cells. 133   Figure 5.3 Representative photomicrographs showing colocalization of SSTR2 and CB1R in STHdhQ7/7 and STHdhQ111/111 cells. Cells were grown as described in Section 5.2. Receptor colocalization was determine in permeabilized (P) and non-permeabilized (NP) cells to distinguish membrane and intracellular expression SSTR2 (green), CB1R (red), nucleus (blue) with Hoechst, and colocalization of SSTR2/CB1R (yellow) are shown in individual and merged images. SSTR2 displayed higher membrane expression in STHdhQ111/111 cells than in STHdhQ7/7 cells, whereas cytosolic expression was comparable. In both wt (STHdhQ7/7) and mutant (STHdhQ111/111) cells, the majority of CB1R-like immunoreactivity was detected predominantly in intracellular compartments. Colocalization was mainly detected intracellularly. Scale bar = 40 µm. 134   Figure 5.4 Representative confocal images showing colocalization of SSTR5 and CB1R in STHdhQ7/7 and STHdhQ111/111 cells. Cells were grown and processed as described in legend to Figure 5.3. Cells were incubated with primary antibodies raised against SSTR5 and CB1R, followed by corresponding secondary antibodies for final color development. SSTR5 is shown in green, CB1R is shown in red. Nucleus was stained with Hoechst 33258, shown in blue. Receptor colocalization is presented as yellow in merged image. Both SSTR5 and CB1R displayed an intracellular-predominant distributional pattern in STHdhQ7/7 and STHdhQ111/111 cells. Colocalization was mostly expressed as punctated staining in cytoplasm. Scale bar = 40 µm. 135   Figure 5.5 Representative immunoblots showing receptor expression in both STHdhQ7/7 and STHdhQ111/111 cells. 15 µg of protein from whole cell lysate, membrane and cytosolic fraction was analyzed using Western blotting. CB1R showed limited membrane expression in STHdhQ7/7 cells and comparatively higher expression in STHdhQ111/111 cells, whereas the cytosolic expression was comparable between STHdhQ7/7 cells and STHdhQ111/111 cells. In contrast, SSTR2 showed higher membrane expression in STHdhQ7/7 cells. Membrane expression of SSTR5 was low in both STHdh cells. Images represent at least three independent experiments.  Agonist-induced changes in receptor expression in STHdhQ7/7 and STHdhQ111/111 cells Agonist induced internalization and membrane expression of most receptor proteins are crucial to the regulation of signal transduction pathways. Accordingly, we investigated the changes in receptor expression in cytosolic and membrane fractions prepared from STHdhQ7/7 and STHdhQ111/111 cells in response to agonist treatments. As shown in Figure 5.6A, in STHdhQ7/7 cells, intracellular expression of CB1R was increased in response to SSTR2 and 5 specific agonist as well as CB1R agonist WIN and ACEA treatments, but not upon treatment of SST. The membrane expression of CB1R, was decreased upon agonist treatment relatively to the greater extent in presence of SST, which had no effect on CB1R expression in cytoplasmic fraction. The cytosolic 136  expression of SSTR2 and 5 was comparable to control in response to receptor specific agonist. Unlike CB1R, the expression level of SSTR2 and 5 in membrane fraction was weak, without any significant changes following agonist treatment. In contrast to STHdhQ7/7 cells, in striatal cells with mHtt (STHdhQ111/111), the cytosolic expression of CB1R was unaltered whereas SSTR2 was decreased upon all agonist treatment and SSTR5 was increased when cells were treated with SST and SSTR5 specific agonist (Figure 5.6B). The expression of CB1R and SSTR subtypes in membrane fractions prepared from STHdhQ111/111 cells was changed in a receptor agonist-specific manner. CB1R expression in membrane fraction was decreased upon treatment with CB1R specific agonists and SST but not with SSTR specific agonists. SSTR2 expression in membrane fractions was changed in response to SSTR2 and 5 selective agonists but not CB1R agonists, whereas SSTR5 expression level was increased in most cases when compared to control (Figure 5.6B).  Figure 5.6 Agonist-induced changes of receptor expression in STHdhQ7/7 and STHdhQ111/111 cells. 137  15 µg of protein of membrane and cytosolic fraction prepared from control and treated cells as indicated in Section 5.2 were subjected to Western Blot analysis. (A) Receptor expression changes upon agonist treatment in SThdhQ7/7 cells. Note that CB1R expression was increased in cytosol in response to all agonist treatment except SST, whereas its expression on membrane was decreased upon all agonist treatment especially SST and WIN. The expression of either SSTR2 or 5 in cytosol was comparable to control upon agonist treatment. Membrane expression of SSTR2 was decreased upon all agonist treatment, whereas SSTR5 was increased in response to SSTR2 agonist treatment. (B) Agonist-induced changes in receptor expression in STHdhQ111/111 cells. Expression of CB1R in cytosol was unaltered upon agonist treatment, whereas the membrane expression of CB1R was decreased. Cytosolic expression of SSTR2 was decreased, while membrane expression was increased upon treatment of SSTR2 specific agonist. Expression of SSTR5 was increased in cytosol upon receptor activation by SST or specific agonist. Whereas on membrane, treatment of different agonists lead to SSTR5 expression increased to various extents. Results are representative of three independent experiments.  5.3.3 Receptor agonist-induced changes in signaling pathways in STHdh cells Having seen receptor specific changes in receptor-like immunoreactivity and distribution, we next investigated the effect of such changes on cell proliferative signaling molecules including ERK1/2, PI3K and PTEN. ERK1/2 Phosphorylation is more responsive in the presence of QUIN First, we determined the time-dependent changes on ERK1/2 phosphorylation upon receptor-specific agonist treatment. As shown in Figure 5.7, in STHdhQ7/7 cells, the phosphorylation of ERK1/2 in response to L-779976, L-817818 or ACEA was increased significantly at 15 min. At prolonged treatment of 6 h, elevated ERK1/2 phosphorylation was observed upon treatment of L-817818 and ACEA. In STHdhQ111/111 cells, the level of pERK1/2 was significantly increased at 5 min upon treatment of SSTR2 or CB1R specific agonist but not with the activation of SSTR5. The level of pERK1/2 then decreased or remained stable at 15 min. At prolonged treatment, however, 138  pERK1/2 was not significantly altered in response to any receptor agonist albeit suppressed in comparison to early time point of 5 mins. Accordingly, 15 min incubation time was taken into consideration for the rest of the experiments on signaling pathways.  Figure 5.7 Time- and agonist-dependent changes in ERK1/2 phosphorylation. Cells were treated with 1 µM L-779976, 1 µM L-817818 or 1 µM ACEA for indicated time. Cell lysates were subjected to Western Blot analysis for the ERK1/2 phosphorylation. In STHdhQ7/7 cells, maximum ERK1/2 phosphorylation was observed at 15 min upon treatment of any of the three selected agonists. Note the increased tERK1/2 expression in response to prolonged ACEA treatment. In STHdhQ111/111 cells, pERK1/2 level was significantly increased at 5 min in response to L-779976 and ACEA. β-actin was used as an internal loading control. Data are presented as mean ± SEM of three independent experiments. Statistical analysis was performed by using two-way ANOVA and Bonferroni posttest to compare against control. *, p<0.05.  QUIN is an endogenous NMDAR agonist and has been regularly used to induce excitotoxicity and mimic HD in neuronal models (Beal et al., 1986, Beal et al., 1991). Therefore, we determined the changes in pERK1/2 in STHdhQ7/7 and STHdhQ111/111 cells upon treatment with receptor agonist 139  in the presence or absence of QUIN. As shown in Figure 5.8A, receptor agonist induced insignificant changes in ERK1/2 phosphorylation in STHdhQ7/7. Conversely, activation of SSTR2 and CB1R but not SSTR5 in STHdhQ111/111 cells significantly increased ERK1/2 phosphorylation. As shown in Figure 5.8B, in STHdhQ7/7 and STHdhQ111/111 cells, QUIN inhibited basal pERK1/2 level in comparison to control. However, in the presence of receptor specific agonists, QUIN-mediated inhibition of pERK1/2 was abolished and the phosphorylation ratio of ERK1/2 was largely increased upon most agonist treatments in both STHdhQ7/7 and STHdhQ111/111 cells, with the exception of SSTR5 specific agonist-treated STHdhQ7/7 cells. These results indicate a protective effect of SSTRs and CB1R agonists against QUIN (Figure 5.8B). 140   Figure 5.8 Agonist and/or QUIN-induced changes in ERK1/2 phosphorylation in STHdhQ7/7 and STHdhQ111/111 cells. Cells were treated with 1 µm SST, 1 µm L-779976, 1 µm L-817818, 1 µm WIN-55212, or 1 µm ACEA in the absence or presence of 3 mM QUIN for 15 min. Then cell lysates were collected and 15 µg of protein from each treatment group were subjected to Western Blot analysis. (A) Changes in ERK1/2 phosphorylation in response to receptor agonist alone. In STHdhQ7/7 cells, ERK1/2 phosphorylation was increased albeit insignificantly upon agonist treatment. In STHdhQ111/111 cells, increase in ERK1/2 phosphorylation was observed in agonist-treated cells, especially 141  upon treatment of SSTR2 agonist, WIN and ACEA. (B) Agonist-induced changes in ERK1/2 phosphorylation in the presence of QUIN. In STHdhQ7/7 cells, all agonist treatments except SSTR5 agonist induced over 1.5 folds increase of ERK1/2 phosphorylation over control. In STHdhQ111/111 cells, significant increase of ERK1/2 phosphorylation was observed in all agonist treatment. β-actin was used as an internal loading control. Data are presented as mean ± SE of three independent experiments. Statistical analysis was performed by using one-way ANOVA and post hoc Dunnett’s test to compare against control. *, p<0.05.  Expression of cell survival PI3K pathway is suppressed in STHdhQ111/111 cells Considering that PI3K signaling pathway is important in cell survival, we investigated the status of PI3K phosphorylation in response to agonist treatment in the presence or absence of QUIN (Franke et al., 1997). As shown in Figure 5.9A, upon agonist treatment, pPI3K levels in STHdhQ7/7 cells were comparable to control, whereas decreased in STHdhQ111/111 cells albeit insignificantly. In the presence of QUIN, pPI3K in STHdhQ7/7 cells remained comparable to control in presence of SST and SSTR2 agonist but was increased significantly in response to SSTR5 agonist and CB1R agonists (Figure 5.9B). Comparatively, no significant changes in PI3K phosphorylation were seen in STHdhQ111/111 cells regardless of treatment with SSTRs or CB1R agonists (Figure 5.9B). Taken together these results suggest that PI3K pathways is less responsive and intrinsically downregulated in cells with mHtt. 142   Figure 5.9 PI3K activity was increased upon agonist treatment in the presence of QUIN in STHdhQ7/7 cells. Cells were processed as described in legend to Figure 5.8 and probed for phosphorylation of PI3K. (A) Agonist-induced changes in PI3K phosphorylation status were insignificant in both STHdhQ7/7 and STHdhQ111/111 cells, except significant decrease of pPI3K upon WIN treatment in STHdhQ111/111 cells. (B) In the presence of QUIN, agonist treatment markedly increased the activity of PI3K in STHdhQ7/7 cells upon treatment of SSTR5 agonist and ACEA but not in STHdhQ111/111 cells. β-actin was used as an internal loading control. Data are presented as mean ± SE of 143  three independent experiments. Statistical analysis was performed by using one-way ANOVA and post hoc Dunnett’s test to compare against control. *, p<0.05.  PTEN activity is intrinsically suppressed in mutant knock-in striatal neuronal cells During embryogenesis, the role of PTEN in cell proliferation is well defined (Di Cristofano et al., 1998). Furthermore, PTEN is essential to NMDAR-dependent LTD, thus directly associated with synaptic and cognitive function, as seen in AD models (Jurado et al., 2010, Knafo et al., 2016). In addition to the changes in cell survival signaling pathways, we next determined PTEN expression in STHdhQ7/7 and STHdhQ111/111 cells in response to SSTR and CB1R specific agonist with and without QUIN. As shown in Figure 5.10A, STHdhQ7/7 cell exhibited decreased PTEN expression upon treatment of agonists especially SSTR5 agonist and WIN which decreased PTEN significantly. In comparison, PTEN expression was not significantly changed upon treatment of SSTR agonists but decreased in response to CB1R agonists in STHdhQ111/111 cells. However, in the presence of QUIN, no significant increase in PTEN expression was observed in STHdhQ7/7 cells upon treatment with agonist (Figure 5.10B). As shown in Figure 5.10B, in STHdhQ111/111 cells, QUIN-mediated decrease in PTEN expression was abrogated in response to receptor agonists especially ACEA. 144   Figure 5.10 Changes in PTEN expression upon agonist and/or QUIN treatment in both STHdhQ7/7 and STHdhQ111/111 cells. Cells were treated and processed as described in legend to Figure 5.8. Membrane was probed for PTEN expression. (A) In STHdhQ7/7 cells, the level of PTEN was comparable to control upon treatment with all agonists except SSTR5 agonist and WIN, whereas treatment of CB1R agonists reduced PTEN level in STHdhQ111/111 cells. (B) In QUIN-treated STHdhQ7/7 cells, PTEN expression was upregulated in response to SSTR agonist while downregulated when treated with CB1R agonists. In STHdhQ111/111 cells, however, the expression of PTEN was increased upon treatment with ACEA. β-actin was used as an internal loading control. Data are presented as mean ± SE of three independent experiments. Statistical analysis was performed by using one-way ANOVA and post hoc Dunnett’s test to compare against control. *, p<0.05. 145   5.3.4 Role of SSTRs and CB1R in protection against QUIN induced toxicity in STHdh cells Having seen receptor-induced changes in survival signaling pathways, it is of our interest to find out whether these changes affect cell survival in QUIN-induced toxicity. Therefore, we aimed to determine QUIN-induced cell death in presence or absence of receptor agonists using MTT assay. As shown in Figure 5.11, STHdhQ7/7 cells displayed 17.87±1.18% cell death in presence of QUIN (3 mM) whereas STHdhQ111/111 cells exhibited increased cell death of 29.21±1.51%. To determine whether activation for SSTR subtypes and CB1R protect cells from QUIN induced toxicity, cells were treated with specific agonist as indicated. As shown in Figure 5.11, all agonists blocked QUIN-induced cell death significantly in both STHdhQ7/7 cells and STHdhQ111/111 cells. These results suggest that activation of either SSTR or CB1R is beneficial to cell survival against excitotoxicity.  Figure 5.11 Striatal cells with expression of mHtt are more susceptible to QUIN. Cells were grown in 96-well plate and serum-starved for 24 h and followed by incubation with Locke’s solution containing 1 µM SST, 1 µM SSTR2 specific agonist L-779976, 1 µM SSTR5 specific agonist L-817818, 1 µM WIN 146  and 1 µM ACEA for 5 h in the presence of 3 mM QUIN. Posttreatment medium was replaced with QUIN-free medium containing receptor agonist and incubated for additional 24 h. Cells were then processed for cell viability using MTT assay. In STHdhQ7/7 cells, QUIN induced 17.87±1.18% cell death which was decreased to 3.73±1.68%~7.53±1.75% upon agonist treatment. In STHdhQ111/111 cells, QUIN-induced cell death was relatively higher as 29.21±1.51%, which was suppressed to 14.08±3.08%~20.83±1.37% with receptor agonist significantly. Data are presented as percentage of cell death in comparison to control and are as mean ± SE of three independent experiments performed in triplicates. Statistical analysis was performed by using one-way ANOVA, followed by Bonferroni’s multiple comparison test. *, p<0.05 versus control; #, p<0.05 versus QUIN-treated cells.  5.3.5 Effects of combined treatment on QUIN-induced cell death, receptor interaction and ERK1/2 signaling Combined agonist treatment diminished the protective effect of single agonist As we have seen the neuroprotective effect of single receptor activation in both STHdh cells, we next determined whether combined agonist treatment exerts an enhanced protective effect on cell survival. Accordingly, STHdhQ7/7 and STHdhQ111/111 cells were treated with receptor specific agonists in combination as indicated in the presence of QUIN and processed for MTT assay. As shown in Figure 5.12, to our surprise, the neuroprotective effect induced by single receptor agonist treatment was abolished in the presence of combined agonists. The only exception is STHdhQ7/7 cells upon treatment in combination of SSTR2 agonist and ACEA, exhibiting 6.60±1.20% cell death, comparable to single agonist treatment. These results were intriguing, at the same time raised questions regarding the status of receptor expression, dimerization and signaling pathway following combined agonist treatment. 147   Figure 5.12 Combined treatment failed to protect cells from QUIN-induced toxicity. STHdhQ7/7 and STHdhQ111/111 cells were grown in 96-well plate and serum-starved for 24 h. Cells were then incubated with Locke’s solution containing 1 µM L-779976 or 1 µM L-817818 along with 1 µM ACEA in the presence of 3 mM QUIN for 5 h. The treatment was replaced with QUIN-free medium containing corresponding drugs. After 24 h incubation, cell viability was determined using MTT assay. In STHdhQ7/7 cells, QUIN-induced cell death was rescued by combined treatment of ACEA with L-779976 but not with L-817818. In STHdhQ111/111 cells, combined treatment failed to rescue the cells from the toxicity induced by QUIN. Data are presented as percentage of cell death in comparison to control and are as mean ± SE of three independent experiments performed in triplicates. Statistical analysis was performed by using one-way ANOVA, followed by Bonferroni’s multiple comparison test. *, p<0.05 versus control; #, p<0.05 versus QUIN-treated cells.  Combined agonist treatment delayed ERK1/2 phosphorylation in STHdhQ111/111 cells As we have shown earlier in Figure 5.7, cell survival signaling pathways were affected upon receptor activation. We then examined whether ERK1/2 activity was affected upon concurrent activation of SSTRs and CB1R in a time-dependent manner. As shown in Figure 5.13, in STHdhQ7/7 cells, the time-dependent changes in ERK1/2 phosphorylation upon combined treatments were comparable to treatment of ACEA alone, but not SSTR2 or 5 agonist. 148  Interestingly, in STHdhQ111/111 cells, treatment of either combination of agonists delayed the activation of ERK1/2 from 5 min to 15 min, when compared to single agonist treatment. The phosphorylation ratio and expression level of ERK1/2 were not significantly altered at prolonged treatment in comparison to single agonist treatment.  Figure 5.13 Loss of pERK1/2 in STHdhQ111/111 cells upon prolonged receptor activation. Cells were treated with 1 µM SSTR2 and SSTR5 specific agonist in combination with 1 µM ACEA for different time points as indicated in Section 5.2. Note comparable pattern of increased p-ERK1/2 at 15 and 60 mins in STHdhQ7/7 cells. In STHdhQ111/111 cells, significant increase in ERK1/2 phosphorylation was observed at 15 min and then suppressed upon extended receptor activation. β-actin was used as an internal loading control. Data are presented as mean ± SE of three independent experiments. Statistical analysis was performed by using two-way ANOVA and Bonferroni posttest to compare against control. *, p<0.05.   149  Expression of receptor complex changed upon combined agonist treatment Our previous study on SSTR5 and CB1R heterodimerization and the results presented here showing receptor colocalization (Figure 5.3 and 5.4) suggest the possibility of interaction between SSTRs and CB1R in STHdhQ7/7 and STHdhQ111/111 cells (Zou et al., 2017). Accordingly, we assessed receptor-receptor interaction between SSTR2/CB1R and SSTR5/CB1R using Co-IP in STHdhQ7/7 and STHdhQ111/111 cells following treatment with single and combined agonists. Whole cell lysates prepared from control and treated cells were subjected to Co-IP. SSTR2 and SSTR5 immunoprecipitates were probed for CB1R expression respectively. As shown in Figure 5.14, in control condition, a band at approximately 110 kDa was detected in both STHdhQ7/7 and STHdhQ111/111 cells representing SSTR2/CB1R (left panel) and SSTR5/CB1R (right panel) respectively. In SSTR2 immunoprecipitates prepared from STHdhQ7/7 cells, the intensity of the band showing SSTR2/CB1R complex was increased upon treatment with SSTR2 agonist and ACEA alone but comparable to control upon combined treatment. In the case of SSTR5/CB1R, the expression of CB1R in SSTR5 immunoprecipitates from STHdhQ7/7 cells was increased to the greater extent upon combine treatment in comparison to treatment with single receptor agonist. In STHdhQ111/111 cells, combined treatment increased the expression of SSTR2/CB1R complex. However, the expression of CB1R in SSTR5 immunoprecipitates was decreased in STHdhQ111/111 cells following treatment with receptor agonist alone or in combination.  150   Figure 5.14 CB1R is expressed in SSTR2 and SSTR5 immunoprecipitates. Co-IP analysis displaying changes in interaction of SSTR2/CB1R and SSTR5/CB1R upon agonist treatment in STHdhQ7/7 and STHdhQ111/111 cells. Cells were treated with 1 µM SSTR2 specific agonist L-779976, 1 µM SSTR5 specific agonist L-817818, 1 µM CB1R agonist ACEA alone or in combination for 30 min. 250 µg of total protein from control and treated cells was immunoprecipitated for SSTR2 or SSTR5 and immunoblotted for CB1R as described in Section 5.2. In SSTR2 immunoprecipitates, a strong band at expected molecular weight of ~110 kDa was observed in untreated STHdhQ7/7 and STHdhQ111/111 cells. In STHdhQ7/7 but not in STHdhQ111/111 cells, CB1R expression was changed in response to agonist. The expression of CB1R in SSTR5 immunoprecipitate was relatively higher in response to agonist when compared to control in STHdhQ7/7, whereas in STHdhQ111/111 cells, CB1R expression decreased upon agonist treatment. Images are representative of at least three independent experiments.  Taken together, these results indicate that receptor crosstalk upon concurrent activation differs from single receptor activation and may account for the loss of protective effect as well as delayed 151  ERK1/2 phosphorylation in cells treated with two agonists in combination in comparison to single receptor activation.  5.4 Discussion The role of SST and cannabinoid in excitotoxicity and pathogenesis of HD is well established (Dawbarn et al., 1985, Kumar, 2008, Blazquez et al., 2011). However, with significant overlapping functional and structural properties as well as colocalization in different brain regions, the crosstalk between SSTR and CBR subtypes is rarely studied in pathological conditions. We recently described that SSTR5 and CB1R form constitutive heterodimers in heterologous expressing system, displaying novel and distinct properties in the regulation of downstream signaling pathways in comparison to native receptor (Zou et al., 2017). Whether SSTRs and CB1R interact and exert similar effects as seen in HEK-293 cells in pathological condition is not known. Accordingly, in the present study, we first determined the colocalization between SSTR2/CB1R and SSTR5/CB1R in brain sections from wt and R6/2 mice. We then took the advantage of conditionally immortalized striatal neuronal wt (STHdhQ7/7) and mutant (STHdhQ111/111) cells. Here, for the first time, we characterized the expression of SSTR2 and 5 in STHdh cell lines and explored their role in both STHdhQ7/7 and STHdhQ111/111 cells under normal as well as excitotoxic conditions. We also investigated the expression and functionality of CB1R in both STHdh cells. Our results showed that activation of either SSTRs or CB1R exerted protective effect against QUIN-induced excitotoxicity in both STHdh cells. We observed selective changes in ERK1/2 phosphorylation in response to receptor agonist treatment. However, such protection was diminished upon concurrent activation of receptors, which might be associated with delayed ERK1/2 phosphorylation and altered receptor heterodimer composition. To our knowledge, this is 152  the first comprehensive description of characterization of SSTR subtypes in association with CB1R in in vivo and in vitro models of HD. The selective sparing of SST-positive neurons and the loss of CB1R expression in HD are well established (Dawbarn et al., 1985, Ferrante et al., 1985, Blazquez et al., 2011). Importantly, the loss of SSTR2 may be associated with disrupted motor behavior whereas decreased SSTR5 expression may account for impaired memory and cognitive function (Craft et al., 1999, Stroh et al., 1999, Allen et al., 2003, Kumar, 2005, Watson et al., 2009). However, nothing is known about the status of SSTR subtypes in HD. In the present study, we showed for the first time that SSTR2/CB1R and SSTR5/CB1R colocalized in three brain regions in wt mice. Whereas in R6/2 mouse brains, not only the intensity of receptor-like immunoreactivity but also the number of neuronal cells displaying colocalization were decreased in a region- and age-dependent manner. In comparison to wt and 7-week-old R6/2 mice, SSTR/CB1R like immunoreactivity was severely perturbed at the age of 11 weeks. These results are consistent with several previous studies, supporting the possible interaction between SSTR2/CB1R and SSTR5/CB1R in pathological conditions (McCaw et al., 2004, Blazquez et al., 2011, Rajput et al., 2011). These neurochemical changes along with the well-defined loss of CB1R may together contribute to the pathogenesis of HD. In wt or mutant STHdh cells, CB1R-like immunoreactivity was prominently confined intracellularly with weak expression at cell surface. This pattern of distribution is supported by previous study showing that 4% of total CB1R was expressed on the plasma membrane of STHdh 7/7 cells (Laprairie et al., 2013). The intracellular localization of CB1R is not restricted to STHdh cells only but also has been seen in other neuronal cell lines and cultured primary neurons, where it is mostly found in endo/lysosomes and actively participates in the regulation of intracellular Ca2+ 153  storage (Rozenfeld, 2011). Recently, the presence of functional CB1R has also been observed in mitochondria and associated with cellular respiration and energy metabolism (Benard et al., 2012). These studies support the intracellular-predominant yet fully functional expression of CB1R as presented here. However, the exact location of intracellular CB1R in STHdh cells remains unknown and warrants future investigation. Previous studies have shown that ACEA induces total CB1R expression while reduces its membrane expression as a result of agonist-induce receptor endocytosis in STHdhQ7/7 cells, supporting our agonist-induced changes in CB1R expression in STHdhQ7/7 cells (Laprairie et al., 2013). On the other hand, in mutant cells, the intracellular expression of CB1R was not altered upon agonist treatment whereas membrane expression of CB1R was decreased, comparable to STHdhQ7/7 cells. Our results indicate that in STHdhQ111/111 cells, the presence of mHtt might play a role in agonist-induced expression of CB1R, but not agonist-induced endocytosis. Consistent with previous studies, it is highly possible that mHtt may interact with several transcriptional factors including CREB to interfere with the transcription of CB1R (Blazquez et al., 2011, Laprairie et al., 2013, Blazquez et al., 2015). In addition to CB1R, present study for the first time demonstrated the expression of SSTR2 and 5 in STHdh cells. Although in comparison to CB1R, SSTR2 and 5 displayed relatively higher membrane expression, with intracellular expression comparable to CB1R. Such intracellular-predominant expression pattern has been observed in breast cancer cells overexpressing SSTR3 with anti-proliferative effect (War et al., 2015). Whether SSTR2 and 5 located intracellularly exert any specific effects in STHdh cells is not known. SSTR2 and 5 expressions is not only influenced by SSTR agonists, but also modulated by CB1R agonists, further supporting an interaction between SSTR and CB1R, which is consistent with our Co-IP result (Figure 14). 154  Mutant cells exhibit relatively slow growth rate, increased susceptibility to stress and toxicity as well as impaired mitochondrial function in comparison to STHdhQ7/7 cells, indicating the crucial role of mHtt in modulating intrinsic cell survival pathways (Trettel et al., 2000, Ruan et al., 2004). These observations are consistent with our results showing increased neuronal cell death in STHdhQ111/111 cells upon QUIN-induced toxicity than STHdhQ7/7 cells. Activation of SSTR and CB1R increased the cell viability in both STHdhQ7/7 and STHdhQ111/111 cells, which is in line with previous studies showing that CB1R exerts neuroprotective effect against NMDA toxicity in STHdhQ7/7 cells (Blazquez et al., 2011, Blazquez et al., 2015). Moreover, our results on cell survival signaling pathways such as ERK1/2 and PI3K revealed a lower basal level of total proteins in STHdhQ111/111, supporting its impaired ability to encounter stress. We also observed agonist-induced activation of ERK1/2 and PI3K, which is supported by previous studies showing activation of ERK1/2 and PI3K in response to CB1R activation in STHdh cells, as well as SSTR activation in heterologous expression system (Patel, 1999, Kumar and Grant, 2010, Laprairie et al., 2013, Blazquez et al., 2015). ERK1/2 has been associated with protection against oxidative stress, whereas PI3K has been suggested to mediate BDNF expression (Gines et al., 2010, Blazquez et al., 2015). Our results presented here are in line with these previous studies. Moreover, in comparison to STHdhQ7/7 cells, we observed an impaired PI3K phosphorylation in STHdhQ111/111 cells upon receptor activation and in the presence of QUIN, suggesting a suppressed survival response in presence of mHtt. Considering the neuroprotective effect seen in both STHdh cells, we believe that ERK1/2 is the signaling pathway mediating such effect induced by SSTR and CB1R activation. In MTT assay using combined agonists, we observed results in contrast to the existing notion that simultaneous activation of two receptor account for enhanced signaling pathways. The 155  activation of both receptors showed insignificant effect on cell survival against QUIN-induced excitotoxicity. It is possible that concurrent activation leads to changes in receptor expression and functionality that attenuate the cell survival signaling, as seen in the delayed ERK1/2 phosphorylation upon combined agonist treatment in STHdhQ111/111 cells. Another possibility is that concurrent activation of receptors modifies the composition of heterodimers present on the cell membrane. As previously reported, D2R is endogenously expressed in STHdhQ7/7 cells and forms heterodimer with CB1R (Bagher et al., 2016). Co-activation of D2R influences CB1R coupling to G protein and β-arrestin-mediated ERK1/2 signaling (Glass and Felder, 1997, Jarrahian et al., 2004, Kearn et al., 2005, Bagher et al., 2016). In SH-SY5Y cells, mutual antagonism has been reported in heterodimer of CB1R and CB2R that co-activation of both receptors abolished neuritogenesis induced by either receptor agonist (Callen et al., 2012). Similarly, we have reported earlier in recombinant system that CB1R forms heterodimer with SSTR5 which lead to a SSTR5-predominant signaling in cAMP/PKA/ERK pathway (Zou et al., 2017). SSTR2 and 5 have been shown to heterodimerize with each other as well as with D2R (Rocheville et al., 2000a, Rocheville et al., 2000b, Grant et al., 2008). These results suggest that the activation of two receptors at a time tends to mask the effect of one receptor due to the endogenous presence of the other receptor. In present experimental condition, the potential role of D2R in the regulation of SSTR subtypes and CB1R via heterodimerization cannot be ruled out from discussion. This is further supported by the fact that D2R-mediated switch of CB1R coupling to G protein results in opposing effect to the single receptor activation (Bagher et al., 2016). Such interaction bears clinical significance, as evidence suggests that D2R antagonists might be beneficial in treating early HD symptoms [reviewed in (Pidgeon and Rickards, 2013)]. Furthermore, whether altered expression of receptor complex upon combined agonist treatment is 156  associated with the diminished protective effects is not known. To address this complex issue of how the combined treatment-induced changes in receptor-receptor interaction and signaling pathways are involved in loss of neuroprotective, further studies are in progress in this direction. Taken together, in the present study, we have shown that expression and colocalization of SSTR2/CB1R and SSTR5/CB1R was decreased along with the progression of HD in R6/2 mice brain. Activation of receptors results in an upregulation of cell survival and related signaling pathways against excitotoxicity. Results in STHdhQ7/7 and STHdhQ111/111 cells also demonstrate that SSTR2/CB1R and SSTR5/CB1R physically interact with each other. This interaction is subjected to the regulation of receptor activity by agonist. Combined agonist treatment altered the time-course of ERK1/2 phosphorylation and receptor complex composition, which may be associated with the diminished neuroprotective effects. The results presented here explored the role of SSTR/CB1R interaction in striatal neurons with expression of mHtt, providing novel insight for the role SSTR and CB1R in excitotoxicity.  157  Chapter 6: Concluding Remarks 6.1 Overall Discussion The anti-proliferative and hormone-inhibiting effects of SST have drawn great attention and been thoroughly studied for decades, fostering its clinical applications with beneficial outcomes, particularly in the treatment of neuroendocrine tumors (Patel, 1999). In contrast, the role of SST as a neurotransmitter/neuromodulator remains elusive. Despite its widespread distribution in CNS and frequent usage as a marker for GABAergic interneurons, its functionality in CNS has not been thoroughly explored (Kumar and Grant, 2010). Yet early studies have demonstrated the significance of SST in CNS, serving as a background for further exploration of the promising potential of SST in the treatment of various neurological disorders (discussed in Chapter 1). On the other hand, cannabinoids and endocannabinoid system have gained tremendous attention in recent years. Since the discovery of THC, the major psychoactive component in cannabis, the role of cannabinoids has been thoroughly investigated in various fields, including appetite control, pain regulation, cancer therapy, and most importantly, neurodegeneration (Pacher et al., 2006). It has been well-documented that cannabinoids, mainly through the regulation of CB1R activity, modulates the major excitatory and inhibitory neurotransmission in CNS. Crosstalk between endocannabinoid system and glutamatergic, GABAergic, dopaminergic systems has been intensively examined (Kano et al., 2009). However, no such studies have been accomplished on the interaction between the endocannabinoid and the SST system, despite their overlapping distribution in CNS. Moreover, considering that both cannabinoids and SST are involved in the regulation of neurotransmission, the possible interaction between these two systems may hold significant clinical implication in excitotoxicity and associated neurological disorders. As the main central effectors of cannabinoids and SST, CB1R and SSTRs all belong to GPCR family, which 158  serves as the major target of more than 50% of therapeutic drugs in the market (Foord et al., 2005, Millar and Newton, 2010). In recent years, the interaction between GPCRs as oligomeric complex has been observed and characterized in homologous and heterologous expression systems (Ferre et al., 2014). The clinical significance and therapeutic potential of such interaction are well supported by studies in various disease models (Gomes et al., 2016). Accordingly, this thesis attempted to explore the possible crosstalk between SST and endocannabinoid systems in CNS, mainly focusing on characterizing the interaction of SSTR and CB1R as heteromeric complex, and its role in pathological conditions. To address the crosstalk between the endocannabinoid and SST system in the CNS, we first examined the colocalization between CB1R and SST in rat brain with endogenous expression. Previous studies have shown that SST synthesized in hypothalamus accounts for most of SST expression in the brain, actively participating in the regulation of hypothalamic hormones secretion (Finley et al., 1981). CB1R, despite its low expression in hypothalamus, is also actively involved in the central regulation of appetite (Di Marzo and Matias, 2005). Both SST and cannabinoids have been reported to affect food intake behavior and several key appetite-related hormones including leptin and ghrelin (Silva et al., 2005, Bellocchio et al., 2008). The colocalization between SST and CB1R presented here suggests a crosstalk between these two systems in the regulation of appetite. Recently, POMC neurons have been shown to promote CB1R-induced acute feeding during satiety (Koch et al., 2015). Lower doses of ACEA depolarize POMC neuron, whereas higher doses hyperpolarize it (Koch et al., 2015). POMC neurons in AN project to SST-positive neurons in PeVN, whereas a subpopulation of POMC neurons in AN is also SST-positive (Fodor et al., 1998). These observations suggest a direct regulation of SST-positive neurons from CB1R through local circuit or an indirect effect via POMC neurons. Taken these observations and the colocalization 159  seen in the present study into account, CB1R may play a role in modulating the bimodal effect of SST on food intake reported previously (Aponte et al., 1984, Danguir, 1988). This effect is reversed in the blockade of SSTR2, the SSTR subtype involved in the regulation of Ca2+ influx and neurotransmitter release (Danguir, 1988). These observations bring up the possible interaction between SSTR and CB1R, with the regulatory effect in food intake. In contrast to hypothalamus, CB1R is highly-expressed in hippocampus, with well-characterized differential distribution and distinct roles in glutamatergic and GABAergic neurons (Tsou et al., 1998, Katona et al., 1999, Kawamura et al., 2006, Monory et al., 2006, Chiarlone et al., 2014). SST in hippocampus is expressed on GABAergic interneurons and exerts inhibitory effect on neurotransmitter release (Baratta et al., 2002, Tallent, 2007). Impairment of both short-term and long-term memory has been associated with hippocampal CB1R activity (Ranganathan and D'Souza, 2006). On the other hand, several lines of evidence suggest that SST facilitates learning and memory [reviewed in (Liguz-Lecznar et al., 2016)]. Considering these opposite roles in memory formation, colocalization between hippocampal SST and CB1R described in the present study may indicate a negative regulation among these two systems in the control of memory formation. In addition, SST has been described as an endogenous anti-epileptic with evidence supporting its critical role in seizure control (Tallent and Qiu, 2008). In contrast, effect of cannabinoid on epilepsy is paradoxical, which may be associated with distinct effects of CB1R expressed on glutamatergic and GABAergic neurons (Wallace et al., 2002, Clement et al., 2003). The colocalization between SST and CB1R described here provide a possible method to selectively regulate the effect of CB1R in epilepsy. Previous studies have also reported that cannabinoids and SST inhibit glutamate release in hippocampus through presynaptic receptors respectively (Boehm and Betz, 1997, Misner and Sullivan, 1999). Our results showing the colocalization between CB1R 160  and SST in hippocampal regions are in line with these observations. Also, it brings up the possibility of CB1R and SSTR interaction at presynaptic site, as well as its potential role in the regulation of neuronal hyperactivity as seen in seizures. In addition, we determined the colocalization between nNOS and SST or CB1R. As a retrograde gaseous neurotransmitter and second messenger, NO actively participates in a wide spectrum of biological activities, including neurotransmission, vascular tone, gene transcription and protein modification (Moncada et al., 1991). It is also responsible for causing nitrosative stress that could potentially damage DNA and harm the cells (Ridnour et al., 2004). The association of nNOS with both CB1R and SST have been reported previously (Ferrante et al., 1985, Dun et al., 1994b, Howlett et al., 2010). Here we demonstrated the colocalization between nNOS and SST or CB1R in hypothalamus and hippocampus, providing first-hand evidence supporting the regulatory role of SST and CB1R on NO production. The colocalization between SST and CB1R described in Chapter 2 and 3 supports the possibility of SSTR/CB1R interaction in CNS. Moreover, as a retrograde neurotransmitter producer and nitrosative stress inducer, the presence of nNOS on SST/CB1R-expressing neurons underscores the physiological significance of such interaction in modulating neurotransmission and neuroprotection. Based on these observations, we aimed to investigate whether SSTR and CB1R interact with each other as heterodimer complex in Chapter 4. It is well-documented that GPCR function in concert as oligomeric complex. Both SSTRs and CB1R have been reported to form heterodimers with other GPCRs, including D2R and µ-OR (Howlett et al., 2010, Hudson et al., 2010a, Somvanshi and Kumar, 2012). Moreover, changes in pharmacological properties of such interaction have been characterized in various physiological and pathological conditions (Ferre et al., 2014). Accordingly, having seen the colocalization between SST and CB1R, our main object 161  in Chapter 4 was to ascertain the physical and functional interaction of SSTR5 and CB1R of human origin. First we investigated the colocalization of CB1R and SSTR5 in rat brain, and then studied receptor internalization, interaction and signal transduction pathways in HEK-293 cells stably cotransfected with hCB1R and hSSTR5. Our results showed that in rat brain cortex, striatum, and hippocampus, CB1R and SSTR5 colocalized and interacted with each other in an agonist-insensitive manner. In cotransfected HEK-293 cells, SSTR5 and CB1R existed in a constitutive heteromeric complex under basal condition, which was disrupted upon agonist treatments. These different receptor activities could result from the different microenvironments as well as receptors origins, as previous studies have demonstrated that SSTR of rat and human origin may behave differently (Hukovic et al., 1996, Nouel et al., 1997, Roosterman et al., 1997, Roth et al., 1997). Furthermore, concurrent receptor activation led to preferential formation of SSTR5 homodimer and dissociation of CB1R homodimer. Agonist-induced dissociation of heterodimers have been observed in both SSTR2 and SSTR3 homodimers (Grant et al., 2004a, War et al., 2011). The co-existence of multiple subpopulations of receptor monomers and dimers on the cell membrane has been visualized for M1 muscarinic receptors (Hern et al., 2010). The observation that SSTR5 forms homodimer upon agonist treatment is consistent with our previous study on SSTR5 homodimerization in mono-transfected CHO cells (Rocheville et al., 2000b). Others have reported that CB1R exists as constitutive homodimer or higher-order oligomer, and we showed the dissociation of pre-formed CB1R homodimer in response to agonist treatment (Wager-Miller et al., 2002, Mackie, 2005a). Taken together, concurrent receptor activation lead to the dissociation of CB1R/SSTR5 heterodimer and CB1R homodimer, and preferential formation of SSTR5 homodimer, as well as receptor internalization alone or in a complex, leaving relatively more SSTR5 on the cell membrane, as seen in receptor internalization assay. These results supported 162  enhanced inhibition of cAMP formation observed in response to increasing concentration of SSTR5 specific agonist in the presence of CB1R agonist. Another possible contributor to this could be the enhanced SSTR5 homodimerization with increased functionality. On the other hand, lack of changes in CB1R-mediated cAMP inhibition in the presence of increased CB1R agonist could result from SSTR5-dominated Gi signaling in CB1R/SSTR5 heterodimer which remained comparatively stable in the presence of CB1R agonist. Our speculation is supported by the insignificant change of cAMP level upon PTX pretreatment, as CB1R but not SSTR5 has been reported to switch its coupling to Gs when Gi is unavailable (Glass and Felder, 1997, Maneuf and Brotchie, 1997, Bonhaus et al., 1998). Furthermore, we showed that the changes in ERK1/2 activity was in parallel to the fluctuation of cAMP inhibition upon combined treatment, suggesting a correlation between ERK1/2 and cAMP that ERK1/2 activity was Gi-dependent thus regulated mainly by SSTR5. It has been reported previously that CB1R-induced ERK1/2 activation could be dependent on cAMP/PKA pathway in several heterologous expression systems, whereas in CB1R-transfected HEK-293 cells, ERK1/2 phosphorylation was Gi-independent and β-arrestin-dependent (Bouaboula et al., 1995, Kearn et al., 2005, Daigle et al., 2008). The partial blockade of ERK1/2 activity by PTX shown in the present study supported the involvement of Gi in ERK1/2 phosphorylation, and also revealed a Gi-independent signaling of ERK1/2 phosphorylation, which should be attributed to CB1R. As shown by previous studies, CB1R was able to recruit β-arrestin and initiate ERK1/2 phosphorylation upon receptor desensitization, whereas SSTR5 was unable to do so (Daigle et al., 2008, Grant et al., 2008). In addition, we investigated the changes of PI3K phosphorylation and found that in comparison to cAMP/PKA/ERK1/2 pathway, PI3K activity was not significantly affected, albeit the time-course of PI3K phosphorylation was altered in response to agonist treatment. Taken together, results presented here demonstrated that with predominant 163  role of SSTR5, the functional consequences of crosstalk between SSTR5 and CB1R resulting in the regulation of receptor trafficking and signal transduction pathways, opening new therapeutic avenue in cancer biology and excitotoxicity. To further elucidate the significance of the interaction and crosstalk between these two systems, we next investigated the receptor-receptor interaction in pathological conditions described in Chapter 5. First, we characterized the colocalization of SSTR2/CB1R and SSTR5/CB1R in brain sections obtained from wt and R6/2 mice. R6/2 mouse is a well-stablished and widely-used transgenic model of HD, closely resembling the neuroanatomical abnormality of HD (Zuccato et al., 2010). Our observations on the decreased expression and colocalization between SSTRs and CB1R are consistent with previous studies displaying a downregulation of receptor expression in HD in an age-dependent manner (McCaw et al., 2004, Blazquez et al., 2011). Next, we took the advantage of a previously-established immortalized mouse striatal neuronal cell lines with wt (SThdhQ7/7) or mHtt (STHdhQ111/111) knock-in, in which we induced excitotoxicity using QUIN. These striatal cells have been characterized previously, featuring an enhanced susceptibility to stress and toxins in mutant cells (Trettel et al., 2000). Activation of either SSTR or CB1R protected cells from QUIN-induced toxicity, along with enhanced ERK1/2 phosphorylation. The observations on CB1R described here are consistent with several previous studies, and the characterization of SSTRs in these neuronal cells is reported for the first time (Blazquez et al., 2011, Blazquez et al., 2015). Most importantly, we showed a distinct effect on cell survival upon combined treatment with receptor specific agonist when compared to the activation of either SSTR subtype or CB1R. Although the detailed molecular mechanisms are awaited, in the present experimental condition, combined agonist treatment-induced delay of ERK1/2 phosphorylation and changes in receptor complex composition might be in support of these results (Figure 6.1). 164  Moreover, previous studies have demonstrated the presence of D2R/CB1R heterodimer in STHdhQ7/7 cells, with a preferential coupling to Gs instead of Gi and subsequent reversed effect on downstream signaling, as seen in other recombinant system (Jarrahian et al., 2004, Bagher et al., 2016). On the other hand, we have reported previously that SSTR2 or 5 heterodimerize with D2R, resulting in an enhanced D2R signaling, which in turn, may facilitate the switch of G protein coupling of CB1R (Rocheville et al., 2000a, Baragli et al., 2007). Thus, a more complicated aspect involving D2R should be taken into consideration to elucidate receptor interaction and specific effects upon combined agonist treatment.  Figure 6.1 Schematic illustration summarizing the crosstalk between SSTRs and CB1R. Left panel, interaction between SSTR5 and CB1R. It has been shown that activation of SSTR5 or CB1R respectively leads to the inhibition of cAMP/PKA and the activation of ERK1/2. In co-transfected HEK-293 cells, we observed that SSTR5 and CB1R form heterodimers, displaying a SSTR5-predominant effect in the regulation of cAMP/PKA and ERK1/2 signalings. In STHdh cells, single receptor activation leads to ERK1/2 activation and a protective effect against excitotoxicity, but such effect is diminished upon combined agonist treatment. Right panel, crosstalk between 165  SSTR2 and CB1R. Activation of either SSTR2 or CB1R increases ERK1/2 activity and also exerts neuroprotective effect against excitotoxicity in STHdh cells. This neuroprotective effect is maintained upon concomitant rececptor activation, but is suppressed in cells expressing mHtt. Whether SSTR2 and CB1R functionally interact with each other as heterodimer is not known. Arrows of different colors indicate signaling or effect from receptor of corresponding color. Green arrows indicate the effect upon concomitant activation of both receptors.  Moreover, the diminished protective effects upon combined treatment implicate physiological significance. As both cannabinoids and SST exert inhibitory effects on glutamate and GABA release, the colocalization seen in Chapter 2 and 3 may represent a local feedback circuit, that CB1R and SSTR may regulate the activity of each other via heterodimerization, to maintain a balance between excitatory and inhibitory neurotransmission. Recent studies have uncovered the presence of mitochondrial CB1R in both hypothalamic POMC neurons and hippocampal CA1 neurons, emphasizing its role in the regulation of neuronal energy metabolism (Benard et al., 2012, Koch et al., 2015). Mitochondrial CB1R is involved in ACEA-induced protection against cell death after cerebral ischemia/reperfusion injury (Ma et al., 2015). In addition, CB1R located in endo/lysosomes has been associated with regulation of internal Ca2+ storage (Brailoiu et al., 2011). Activation of endo/lysosome located CB1R increases intracellular Ca2+, opposite to the effect of membrane-bound CB1R (Brailoiu et al., 2011). In the present study, CB1R confined in intracellular compartments of STHdh cells may play similar roles in the regulation of energy and Ca2+ concentration, although it is not clear how the agonist-induced protective effects against QUIN-induced toxicity are assigned among membrane- and intracellular-expressed CB1R. Also, different membrane/cytosol expression ratio of CB1R in STHdhQ7/7 and STHdhQ111/111 cells may attribute to their distinct responses to excitotoxic stress and ability to survive. In addition, SSTR2 and 5 have both displayed intracellular colocalization with CB1R. It 166  is likely that SSTR2 and 5 co-exist with CB1R in the endo/lysosomes as a result of heterodimer endocytosis. However, the possible presence of SSTR2/CB1R and SSTR5/CB1R complex in mitochondria cannot be ruled out from discussion.  6.2 Overall Conclusion 1. CB1R and SST colocalize in rat brain hypothalamus and hippocampus in a region-specific manner.  2. In co-transfected HEK-293 cells, CB1R and SSTR5 form constitutive heterodimer that dissociates upon agonist treatment. Concurrent receptor activation results in a SSTR5-predominant regulation of cAMP/PKA/ERK signaling. 3. Expression and colocalization of SSTR2/CB1R and SSTR5/CB1R decrease in R6/2 mice in an age-dependent manner. In STHdh cells, expression of SSTR2, SSTR5 and CB1R changes upon agonist treatment. Activation of either receptor protects cells from QUIN-induced toxicity, mainly through the activation of ERK1/2. Combined agonist treatment results in a diminished protective effect, along with a delayed ERK1/2 phosphorylation and altered receptor complex composition.  6.3 Future Study 1. The exact mechanism of the diminished protective effect upon combined treatment in STHdh cells remains elusive. Characterization of receptor interaction upon QUIN treatment could provide some clues. 2. 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