You may notice some images loading slow across the Open Collections website. Thank you for your patience as we rebuild the cache to make images load faster.

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The role of NMDA receptors in excitotoxicity Bakshi, Deeksha 2013

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
24-ubc_2013_spring_bakshi_deeksha.pdf [ 1.41MB ]
Metadata
JSON: 24-1.0073562.json
JSON-LD: 24-1.0073562-ld.json
RDF/XML (Pretty): 24-1.0073562-rdf.xml
RDF/JSON: 24-1.0073562-rdf.json
Turtle: 24-1.0073562-turtle.txt
N-Triples: 24-1.0073562-rdf-ntriples.txt
Original Record: 24-1.0073562-source.json
Full Text
24-1.0073562-fulltext.txt
Citation
24-1.0073562.ris

Full Text

THE ROLE OF NMDA RECEPTORS IN EXCITOTOXICITY  by  DEEKSHA BAKSHI  B.Sc. (Honours), Queen’s University, 2009  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES (Pharmaceutical Sciences)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) January 2013 © Deeksha Bakshi, 2013  ABSTRACT NMDA receptors are glutamate-gated cation channels named after their prototypical selective agonist NMDA. The channels occur as multiple subtypes, which are formed from interactions between different receptor subunits. NMDA receptor subunits are classified into three families: NR1, NR2A-D, and NR3A, B.  NMDA receptors are implicated in HD pathology. During HD, a subset of medium-sized aspiny interneurons in the striatum that co-localize SST, NPY, and the enzyme NOS are selectively spared. In contrast, medium-sized spiny cells that constitute 80 % of all striatal neurons undergo selective neurodegeneration. While it was suggested that the interneurons survive because they lack NMDA receptors, studies including from our lab have shown the presence of NR1 in SST-positive striatal neurons. The finding of NR1 expression and colocalization with SST-positive neurons indicates that NMDA receptor-induced toxicity may be regulated in a receptor-specific manner.  Therefore, the present study was conducted to  investigate whether NMDA application leads to toxicity that is receptor-specific in HEK293 cells stably transfected with NR1, NR2A, or NR2B.  The main findings of this study indicate that NMDA application causes cell death, which varies in intensity and nature, depending upon the NMDA concentration applied, and the receptor-type expressed by the cells. Cells expressing NR1 were found to undergo apoptosis but not necrosis, while cells expressing NR2A/NR2B underwent both apoptosis and necrosis in a receptor-specific manner. In cells expressing NR2A/NR2B, exposure to low concentrations of NMDA resulted in cell death that was predominantly apoptotic. In contrast, exposure to high  ii  concentrations of NMDA produced mostly necrosis.  In cells expressing NR1, NMDA  application caused apoptosis, which exhibited a gradual increase in response to greater concentrations of NMDA.  In addition, cell death through apoptosis and/or necrosis was  determined to be the greatest at all NMDA concentrations in cells expressing NR2B, followed by those expressing NR2A, and then NR1. Taken together, these results indicate that the activation of receptors formed by NR1, NR2A, or NR2B have different toxic consequences. Thus, the selective neurodegeneration observed during HD may be due to the variation in expression levels of NR1, NR2A, and NR2B between medium-sized aspiny interneurons and medium-sized spiny projection neurons.  iii  TABLE OF CONTENTS  ABSTRACT ---------------------------------------------------------------------------------------------------- ii TABLE OF CONTENTS------------------------------------------------------------------------------------ iv LIST OF FIGURES ----------------------------------------------------------------------------------------- vii LIST OF ABBREVIATIONS ------------------------------------------------------------------------------ ix ACKNOWLEDGEMENTS------------------------------------------------------------------------------- xiii CHAPTER I: Introduction ---------------------------------------------------------------------------------- 1 1.1 NMDA Receptors-------------------------------------------------------------------------------- 1 1.1.1  Nomenclature ---------------------------------------------------------------------- 1  1.1.2  Homomeric and Heteromeric Channels --------------------------------------- 1  1.1.3  Pharmacology ---------------------------------------------------------------------- 2  1.1.4  Transmembrane Topology ------------------------------------------------------- 3  1.1.5  Distribution ------------------------------------------------------------------------- 6  1.2 Cell Surface Trafficking of NMDA Receptors -------------------------------------------- 6 1.3 NMDA Receptor Internalization ---------------------------------------------------------- 7 1.3.1  Fate of NMDA Receptors after Internalization ------------------------------ 9  1.4 NMDA Receptors in Excitotoxicity and Neuronal Survival ---------------------------- 9 1.5 Excitotoxicity- An Emphasis on Huntington’s Disease -------------------------------- 14 1.5.1  NMDA Receptors and Huntington’s Disease ------------------------------ 14  1.5.2  NMDA Receptor Expression in HD ----------------------------------------- 15  1.5.3  Selective Neuronal Death in HD --------------------------------------------- 15  1.6 NMDA Receptors and ERK1/2 ------------------------------------------------------------- 16 1.6.1  MAP Kinases --------------------------------------------------------------------- 16 iv  1.6.2  ERK1/2 ---------------------------------------------------------------------------- 17  1.6.3  Regulation of ERK1/2 by NMDA Receptors ------------------------------ 18  1.7 NMDA Receptors and PI3K ----------------------------------------------------------------- 19 1.7.1  PI3K Family ---------------------------------------------------------------------- 19  1.7.2  PI3K-Akt Cascade--------------------------------------------------------------- 21  1.7.3  Regulation of PI3K by NMDA Receptors ---------------------------------- 21  1.8 NMDA Receptor Signalling Independent of Ion Flux ---------------------------------- 22 1.9 Hypothesis and Research Objectives ------------------------------------------------------ 24 1.9.1  Hypothesis ------------------------------------------------------------------------ 24  1.9.2  Research Objective 1 ----------------------------------------------------------- 26 1.9.2.1 Rationale ----------------------------------------------------------------- 26  1.9.3  Research Objective 2 ----------------------------------------------------------- 27 1.9.3.1 Rationale ----------------------------------------------------------------- 27  1.9.4  Research Objective 3 ----------------------------------------------------------- 28 1.9.4.1 Rationale ----------------------------------------------------------------- 28  CHAPTER II: Materials and Methods ------------------------------------------------------------------- 29 2.1 Transfection and Cell Culture -------------------------------------------------------------- 29 2.2 Treatment --------------------------------------------------------------------------------------- 29 2.3 Western Blot ----------------------------------------------------------------------------------- 29 2.4 MTT --------------------------------------------------------------------------------------------- 31 2.5 Determination of Apoptosis and Necrosis ----------------------------------------------- 32 2.6 Statistical Analysis ---------------------------------------------------------------------------- 33 CHAPTER III: Results ------------------------------------------------------------------------------------- 34  v  3.1 Membrane and Intracellular Localization of NR1, NR2A, or NR2B --------------- 34 3.2 NMDA-induced Death in Cells Expressing NR1, NR2A, or NR2B ---------------- 45 3.3 NMDA-induced ERK1/2 Expression in Cells Expressing NR1, NR2A or NR2B 55 3.4 NMDA-induced PI3K Expression in Cells Expressing NR1, NR2A or NR2B --- 59 CHAPTER IV: Discussion --------------------------------------------------------------------------------- 63 4.1  Discussion Overview ----------------------------------------------------------------------- 63  4.2 NMDA induces NR1, NR2A, and NR2B internalization in a concentration- and receptor-specific manner------------------------------------------------------------------------64 4.3  NMDA-induced death occurs in HEK cells expressing NR1, NR2A, or NR2B 68  4.4 NMDA-induced pERK1/2 and pPI3K expression -------------------------------------- 71 4.5 Conclusion, Physiological Relevance, and Future Studies ---------------------------- 72 REFERENCES ----------------------------------------------------------------------------------------------- 74  vi  LIST OF FIGURES Figure 1.14 Schematic representation of a NMDA receptor subunit -------------------------------- 5 Figure 1.4 Schematic representation of NMDA receptor-mediated excitotoxicity via apoptosis/necrosis -------------------------------------------------------------------------------------- -----13 Figure 3.1 Expression of NR1 in non-transfected and NR1-transfected HEK293 cells -------- 36 Figure 3.11 Expression of NR2A in non-transfected and NR2A-transfected HEK293 cells -- 37 Figure 3.12 Expression of NR2B in non-transfected and NR2B-transfected HEK293 cells--- 38 Figure 3.13 Membrane localization of NR1 in response to NMDA application ----------------- 39 Figure 3.14 Membrane localization of NR2A in response to NMDA application --------------- 40 Figure 3.15 Membrane localization of NR2B in response to NMDA application --------------- 41 Figure 3.16 Intracellular localization of NR1 in response to NMDA application---------------- 42 Figure 3.17 Intracellular localization of NR2A in response to NMDA application ------------- 43 Figure 3.18 Intracellular localization of NR2B in response to NMDA application ------------- 44 Figure 3.2 Cell viability in response to NMDA application in NR1-expressing cells----------- 47 Figure 3.21 Cell viability in response to NMDA application in NR2A-expressing cells ------- 47 Figure 3.22 Cell viability in response to NMDA application in NR2B-expressing cells ------- 48 Figure 3.23 Apoptosis in response to NMDA application in NR1-expressing cells ------------- 49 vii  Figure 3.24 Apoptosis in response to NMDA application in NR2A-expressing cells ---------- 50 Figure 3.25 Apoptosis in response to NMDA application in NR2B-expressing cells----------- 51 Figure 3.26 Necrosis in response to NMDA application in NR1-expressing cells -------------- 52 Figure 3.27 Necrosis in response to NMDA application in NR2A-expressing cells ------------ 53 Figure 3.28 Necrosis in response to NMDA application in NR2B-expressing cells ------------ 54 Figure 3.3 ERK1/2 expression in response to NMDA in NR1-expressing cells ----------------- 56 Figure 3.31 ERK1/2 expression in response to NMDA in NR2A-expressing cells ------------- 57 Figure 3.32 ERK1/2 expression in response to NMDA in NR2B-expressing cells-------------- 58 Figure 3.4 PI3K expression in response to NMDA in NR1-expressing cells --------------------- 60 Figure 3.41 PI3K expression in response to NMDA in NR2A-expressing cells ----------------- 61 Figure 3.42 PI3K expression in response to NMDA in NR2B-expressing cells ----------------- 62  viii  LIST OF ABBREVIATIONS  Akt/PKB :  Protein kinase B  ANOVA :  Analysis of variance  ASK1 :  Apoptosis signal-regulated kinase 1  ATP :  Adenosine triphosphate  BAD :  Bcl-2-associated death promoter  Bax :  Bcl-2-associated X protein  Bcl-2 :  B-cell lymphoma 2  Bcl-xL :  B-cell lymphoma-extra large  BDNF :  Brain-derived neurotrophic factor  BSA :  Bovine serum albumin  Ca2+ :  Calcium ions  c-fos :  c-FBJ murine osteosarcoma viral oncogene homolog  COS :  CV-1 in Origin and comprising the SV40 genetic material  CREB :  cAMP response element-binding protein  DHPG :  (S)-3, 5-dihydroxyphenylglycine  DMEM :  Dulbecco’s modified Eagle’s medium  DTT :  Dithiothreitol  EC50 :  Half maximal effective concentration  Elk-1 :  E-26-like protein 1  ER :  Endoplasmic reticulum  ERK :  Extracellular signal-regulated kinase  ix  ERK1/2 :  Extracellular signal-regulated kinase 1 and 2  FBS :  Fetal bovine serum  GAPs :  GTPase activating proteins  GEFs :  Guanine nucleotide exchange factors  GPCRs :  G-protein coupled receptors  Grb2 :  Growth factor receptor-bound protein 2  GDP :  Guanosine diphosphate  GFP :  Green fluorescent protein  GTP :  Guanosine triphosphate  HD :  Huntington’s disease  HEK293 :  Human embryonic kidney 293  HeLa :  Cell line from cervical cancer cells taken from patient Henrietta Lacks  htt :  Huntingtin  JNK/SAPK : c-Jun N-terminal kinase/stress-activated protein kinase MAP :  Mitogen-activated protein  MEK :  Mitogen-activated protein kinase/extracellular-signal regulated kinase kinase  MEKK :  Mitogen-activated protein kinase/extracellular-signal regulated kinase kinase kinase  MTT :  3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide  NMDA :  N-methyl-D-aspartate  NO :  Nitric oxide  NOS :  Nitric oxide synthase  NPY :  Neuropeptide Y  x  NR1 :  N-methyl-D-aspartate receptor 1  NR2A-D :  N-methyl-D-aspartate receptor 2 A-D  NR3 A, B :  N-methyl-D-aspartate receptor 3 A, B  NTC :  Non-transfected cells  PBS :  Phosphate buffered saline  PDK1 :  Phosphoinositide-dependent protein kinase 1  pERK1/2 :  phospho-Extracellular signal-regulated kinase 1 and 2  pPI3K :  phospho- Phosphoinositide-3 kinase  PI :  Phosphatidylinositol  PI (3,4)P2 :  Phosphatidylinositol 3, 4-bisphosphate  PI(3,4,5)P3 : Phosphatidylinositol 3, 4, 5-trisphosphate PI(4)P :  Phosphatidylinositol 4-monophosphate  PI(4,5)P2 :  Phosphatidylinositol 4, 5-bisphosphate  PI3K :  Phosphoinositide-3 kinase  polyQ :  Polyglutamine  RT :  Room temperature  SEM :  Standard error of the mean  SH2 :  Src homology 2  Shc :  Src homology 2 domain containing  SOS :  Son of sevenless  SST :  Somatostatin  TBS-T :  Tris-Buffered Saline with Tween 20  TC :  Transfected cells  xi  YAC :  Yeast artificial chromosome  xii  ACKNOWLEDGEMENTS  First and foremost, I would like to thank Dr. Ujendra Kumar for his time, guidance, and insight during the course of my Master’s degree. I would also like to express my gratitude to my supervisory committee and chair- Dr. Urs Hafeli, Dr. Kathleen Macleod, Dr. Wayne Riggs, and Dr. Mary Ensom for their input and assistance towards the completion of this study. Last, but not least I would like to thank my family and friends for their never-ending support.  xiii  CHAPTER I Introduction 1.1 NMDA Receptors 1.1.1 Nomenclature N-methyl-D-aspartate (NMDA) receptors are glutamate-gated cation channels named after their prototypical selective agonist NMDA (Mori and Mishina, 1995). The channels occur as multiple subtypes, which are formed from interactions between receptor subunits (Mori and Mishina, 1995). The subunits of the NMDA receptor channels are classified into three families based on sequence homology: NMDA receptor 1 (NR1), NMDA receptor 2A-D (NR2A-D), and NMDA receptor 3 A, B (NR3 A, B) (Mori and Mishina, 1995; Dingledine et al., 1999). 1.1.2 Homomeric and Heteromeric Channels When expressed in Xenopus oocytes, the NR1 subunit forms homomeric channels that conduct current in response to rapid treatment with 10 M glutamate or 100 M NMDA (Mori and Mishina, 1995). However, this conductance is small compared to that observed when NR1 is expressed in conjunction with one of the four NR2 subunits, indicating that heteromeric interactions likely result between NR1 and NR2A-D (Mori and Mishina, 1995). In mammalian cells, the expression of NR1 or one of the four NR2 subunits alone does not result in the production of channels that conduct current in response to the rapid application of 100 M NMDA (Mori and Mishina, 1995). However, current-conducting channels are formed when NR1 is co-expressed with one of the NR2 subunits (Mori and Mishina, 1995). Such heteromeric interactions have also been determined in vivo via co-immunoprecipitation. Previous studies  1  have shown that immunoprecipitate prepared from the rat cerebral cortex with NR1 antibody exhibits NR2A and NR2B expression (Sheng et al., 1994).  Hence, NMDA receptors are  believed to be heteromeric channels. Interestingly, a study performed by Wang and Thukral demonstrated that pre-synaptic NMDA receptors on noradrenergic terminals share many characteristics with homomeric NR1 channels (Wang and Thukral, 1996). It was thus concluded that some endogenous NMDA receptors may be present as homomeric entities and composed of only NR1 (Wang and Thukral, 1996). However, as mentioned earlier, the functionality of homomeric NMDA receptor channels has been largely negated by earlier studies (Mori and Mishina, 1995). The stoichiometry of heteromeric NMDA receptor channels is not yet established (Dingledine et al., 1999). Although, the general consensus is that the channels are tetramers, which most often contain two NR1 and two NR2 subunits of the same or different type (Dingledine et al., 1999). Studies have shown that NR1 also interacts with NR3 subunits to generate a glycine receptor that is unaffected by glutamate (Dingledine et al., 1999). In addition, NR3 subunits can modulate channel function by associating with NR1/NR2 complexes to form triheteromeric channels that exhibit decreased activity (Dingledine et al., 1999). 1.1.3 Pharmacology The agonist affinities of NMDA receptor channels are dependent upon subunit composition (Waxman and Lynch, 2005). The EC50 (half maximal effective concentration) values of glutamate as determined in Xenopus oocytes are 0.45, 1.0, 1.8, and 2.9 M for NR1/NR2D, NR1/NR2C, NR1/NR2B and NR1/NR2A channels, respectively (Risgaard et al., 2010). In comparison, the EC50 values of NMDA as determined in Xenopus oocytes are 8.3, 22,  2  23, and 75 M for NR1/NR2D, NR1/NR2C, NR1/NR2B and NR1/NR2A channels, respectively (Risgaard et al., 2010). These values thus indicate that NR1/NR2A channels have the lowest agonist affinity, while NR1/NR2B and NR1/NR2C channels have intermediate agonist affinities, and NR1/NR2D channels have the highest agonist affinity (Waxman and Lynch, 2005). Variation in the agonist affinities of NMDA receptor channels corresponds to the differences in their deactivation kinetics. Channels containing NR2A produce currents that cease rapidly (t~120 msec) in comparison to those containing NR2B/NR2C (t~400 msec), and NR2D (t~5 sec) (Bloodgood and Sabatini, 2009). Channel conductance is also subunit-dependent (Bloodgood and Sabatini, 2009). NR2A and NR2B-containing channels conduct nearly twice as much current as those containing NR2C and NR2D (Bloodgood and Sabatini, 2009). As a result, stimulation of NR2A-containing channels results in large and fast currents, while conductance through NR2B-containing channels is also large but longer-lasting (Bloodgood and Sabatini, 2009). NR2C and NR2B-containing channels induce the smallest and longest-lasting currents (Bloodgood and Sabatini, 2009). 1.1.4 Transmembrane Topology The NMDA receptor subunits share a common transmembrane topology (Paoletti and Neyton, 2007). Each subunit contains three transmembrane domains (M1, 3 and 4), a cytoplasmfacing pore loop (M2), and an extracellular loop between M3 and M4 (Paoletti and Neyton, 2007). The pre-M1 region and the M3-M4 extracellular loop are the S1 and S2 domains respectively, which together form the agonist-binding domain (Paoletti and Neyton, 2007). The subunits also have an extracellular N-terminus, and an intracellular C-terminus that varies in size  3  depending upon the subunit, and contains sites for associating with intracellular proteins (Paoletti and Neyton, 2007).  4  Figure 1.14 Schematic representation of a NMDA receptor subunit. (Figure was reproduced from: Johnson JW 2003. Acid tests of NMDA receptor gating basics. Mol Pharmacol 63: 11991201.)  5  1.1.5 Distribution In the adult rat brain, the NR1 subunit is distributed ubiquitously (Petralia et al., 1994). NR2A expression is also found throughout the brain, while NR2B expression is limited to the forebrain (Wenzel et al., 1995). In comparison, expression of NR2C is largely restricted to the cerebellum, and that of NR2D is found in diencephalic, mesencephalic, and brain stem structures (Wenzel et al., 1995). NR3A is expressed in the subcortical regions, amygdala, hippocampus, thalamus, hypothalamus, brainstem and spinal cord (Wong et al., 2002). In the case of NR3B, expression is predominant in the spinal cord and brain stem (Chatterton et al., 2002). 1.2 Cell Surface Trafficking of NMDA Receptors The export of NMDA receptor channels from the endoplasmic reticulum (ER) is an important determinant of their trafficking to the cell surface. Human NR1 and NR2A subunits are retained in the ER when expressed alone in COS or human embryonic kidney (HEK) 293 cells (McIlhinney et al., 1998). Rodent NR1 and NR2B subunits also remain in the ER when expressed individually in COS (CV-1 in Origin and comprising the SV40 genetic material) cells (Horak et al., 2008). However, co-expression of NR1 with NR2A or NR2B in the adorementioned studies, results in the assembly of both subunits in the ER and their appearance on the cell surface (McIlhinney et al., 1998; Horak et al., 2008). The processes responsible for the ER retention of NR1 and NR2 subunits have been examined using chimeras of a transmembrane protein Tac (internleukin-2 receptor subunit) and NR1 and NR2B C-termini (Stephenson et al., 2008). The NR1 C-terminal chimera was found to be retained in the ER due to a three aminoacid residue motif RRR in the NR1 C-terminal (Stephenson et al., 2008). The NR2B C-terminal chimera is also retained in the ER, indicating the presence of an ER retention signal (Stephenson  6  et al., 2008). However, the precise motif of this signal is not yet determined (Stephenson et al., 2008). Horak et al. (2008) showed that NR1 and NR2B subunits truncated immediately after the fourth transmembrane domain (M4) do not reach the cell surface when expressed individually in transfected cells.  In contrast, when the two subunits are co-expressed, robust cell surface  expression takes place (Horak et al., 2008). This finding indicates the presence of at least one other retention signal in the remaining portion of NR1 and NR2B (Horak et al., 2008). Further truncations revealed the presence of additional ER retention signals in the third transmembrane domains (M3) of the two subunits (Horak et al., 2008). Moreover, the M3 region of NR2B was identified as being responsible for negating the NR1 M3 retention signal, while the M3 and M4 regions of NR1 are responsible for negating the NR2B M3 retention signal (Horak et al., 2008). The precise mechanism by which the ER retention signals in NR1 and NR2 subunits are masked after receptor assembly remains unclear (Horak et al., 2008). After exiting the ER, NMDA receptor complexes are processed in the Golgi apparatus followed by insertion into synaptic or extrasynaptic membranes (Stephenson et al., 2008). 1.3 NMDA Receptor Internalization The membrane expression of NMDA receptors has been reported as being relatively stable in comparison to that of other receptors (Nong et al., 2004).  NMDA receptor  internalization in 15-21 day old rat cortical cultures has been reported as being 5 % or less when measured at the end of a 30 min period (Ehlers, 2000). However, it was later discovered that the level of NMDA receptor internalization varies depending upon the developmental stage of neurons (Roche et al., 2001).  In rat cortical cultures, about 22 % of NMDA receptors  internalization in 30 min at 7 days in vitro, while the internalization decreases to about 5 % at 18  7  days in vitro (Roche et al., 2001). Hence, the extent of NMDA receptor internalization declines as neurons mature. During the early development stages of rat cortical cultures, the predominantly expressed NR2 subunit is NR2B (Nong et al., 2004). This is also the case for developing neurons in vivo (Nong et al., 2004).  The NR2B C-terminal carries the amino acid sequence YEKL, an  internalization motif of the type YXX, which signals clathrin-mediated endocytosis in many proteins (Roche et al., 2001).  Roche et al. examined the role of the NR2B C-terminal in  regulating receptor internalization by expressing chimeras of Tac and wild type or mutant NR2B C-termini in HeLa (cell line from cervical cancer cells taken from patient Henrietta Lacks) cells and hippocampal neurons (Roche et al., 2001). They found that the wild type NR2B C-terminal chimera internalizes, whereas exclusion of the YEKL motif via an eleven amino acid truncation of the NR2B C-terminal prevents internalization of the mutant NR2B C-terminal chimera (Roche et al., 2001). The internalization induced by the NR2B C-terminal is clathrin-dependent, as coexpression of a dominant negative form of dynamin (K44A dynamin), a GTPase involved in clathrin-mediated endocytosis, inhibits the process (Roche et al., 2001). Further investigation was carried out using the yeast two-hybrid assay, which showed that a direct interaction occurs between the NR2B C-terminus and the 2 subunit of the AP-2 adaptor protein, a protein complex that connects cargo proteins to clathrin-coated pits (Lavezzari et al., 2003). This interaction ceases when point mutations are made to the NR2B YEKL motif, which also impairs the internalization of the NR2B C-terminus chimera (Lavezzari et al., 2003). Hence, these results indicate that the YEKL motif in the NR2B C-terminus induces receptor internalization that takes place through clathrin-mediated endocytosis.  8  The decline in NMDA receptor internalization during neuronal maturation may be due to the concurrent decrease in NR2B expression, and increase in NR2A expression (Nong et al., 2004). The chimera of Tac and NR2A C-terminal has been shown to internalize at a rate that is lower than that of Tac-NR2B C-terminal chimera (Nong et al., 2004). 1.3.1 Fate of NMDA Receptors after Internalization Recent work has investigated the fate of NMDA receptors after internalization. Roche et al. (2001) found that internalized NR2A and NR2B subunits tagged with FLAG epitope differ in their subsequent intracellular trafficking. Following internalization, both subunits are present in the early endosomal compartment (Roche et al., 2001). However, the subunits are differently localized afterwards (Roche et al., 2001). Namely, FLAG-NR2B largely co-localizes with green fluorescent protein (GFP)-Rab11, a marker for recycling endosomes. In comparison, FLAGNR2A exhibits little co-localization with GFP-Rab11 (Roche et al., 2001).  Instead, a  progressive loss of FLAG-NR2A signal occurs, indicating FLAG-NR2A degradation (Roche et al., 2001). It is not known whether the differences in NMDA receptor subunit trafficking after internalization are due to molecular determinants within the subunits (Nong et al., 2004). 1.4 NMDA Receptors in Excitotoxicity and Neuronal Survival Glutamate is a major excitatory neurotransmitter in the brain (Wang and Qin, 2010). The interactions of glutamate with specific membrane receptors mediate important neurologic functions including synaptic plasticity (Wang and Qin, 2010). However, abnormal glutamate levels and glutamate receptor activity can result in neuronal injury or death through excitotoxicity (Lucas and Newhouse, 1957; Olney, 1969; Wang and Qin, 2010). Excitotoxicity refers to toxicity resulting from the over-activation of glutamate receptors (Olney, 1969; Wang 9  and Qin, 2010). Excitotoxicity has been a subject of great interest due to its implication in neurodegenerative diseases such as ischemia, amyotrophic lateral sclerosis, Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease (Doble, 1999; Wang and Qin, 2010).  The  molecular pathways involved in excitotoxicity are not fully elucidated (Wang and Qin, 2010). However, studies have found the excessive activation of glutamate-gated NMDA receptor channels to have several adverse consequences including impairment of intracellular calcium homeostasis, dysfunction of the mitochondria, increase in nitric oxide (NO) production and free radicals, irregular kinase activity, persistent activation of proteases, and increases in expression of pro-death transcription factors and immediate early genes (Wang and Qin, 2010). The toxic consequences of NMDA receptor activation are believed to largely depend upon the receptor’s high permeability to calcium ions (Ca 2+) (Sattler and Tymianski, 2000). For instance, Hartley et al. (1993) reported a positive correlation between Ca 2+ influx and death in mice cortical neurons exposed to glutamate. Moreover, NMDA receptor antagonists that attenuate glutamate-induced neuronal death were found to do so by reducing intracellular Ca2+ accumulation in neurons (Hartley et al., 1993). Hence, Ca2+ entry through NMDA receptors is implicated in the activation of downstream processes linked to cell death (Sattler and Tymianski, 2000; Waxman and Lynch, 2005).  Ca2+-independent NMDA receptor- mediated excitotoxicity has also been reported,  although the mechanism by which this toxicity occurs has not yet been investigated (Chen et al., 1998). The mode of excitotoxic cell death is not uniform (Wang and Qin, 2010). In rat cortical cultures, mild insult to the cells by exposure to low NMDA concentrations leads to death that is predominantly apoptotic (Bonfoco et al., 1995). In contrast, intense insult by treatment with high NMDA concentrations produces mostly necrosis (Bonfoco et al., 1995).  Hence, the 10  intensity of the insult is suggested to dictate whether excitotoxicity occurs through apoptosis or necrosis (Bonfoco et al., 1995). Apoptosis and necrosis are two forms of cell death with distinct morphological and biochemical features (Bonfoco et al., 1995). Apoptosis is characterized by changes in nuclear morphology, including chromatin condensation and fragmentation, as well as cell shrinkage, plasma membrane blebbing, and division of the cell into apoptotic bodies that contain nuclear/cytoplasmic substances (Golstein and Kroemer, 2007). Necrosis, on the other hand, is defined by the dilation and dysfunction of cell organelles, in particular the mitochondria, and plasma membrane lysis (Golstein and Kroemer, 2007). Another distinguishing factor is energy levels.  Intracellular energy levels are dissipated in necrosis, but not in apoptosis  (Ankarcrona et al., 1995). The above-mentioned toxic effects of NMDA receptors are contrary to the observations that NMDA receptor activity is important for neuronal survival. The inhibition of NMDA receptors in vivo by receptor antagonists during the late fetal or early neonatal stage produces apoptosis in many regions of the rat brain (Ikonomidou et al., 1999). Furthermore, in the adult rat brain, in vivo administration of NMDA receptor antagonists leads to increase in neuronal loss when applied after traumatic brain injury or during ongoing neurodegeneration (Ikonomidou et al., 2000). Hence, NMDA receptors are involved in both neuronal survival and death. NMDA receptors are expressed at synaptic and extrasyaptic sites (Hardingham et al., 2002). In hippocampal neurons, extrasynaptic NMDA receptors contain mostly NR1 and NR2B subunits, while synaptic NMDA receptors are also composed of NR2A (Hardingham et al., 2002). Hardingham et al. demonstrated that the location of the NMDA receptor in an important determinant of whether its activation leads to pro-survival or pro-death events (Hardingham et al., 2002). The study determined that in hippocampal neurons, synaptic NMDA receptor activity 11  is neuroprotective, while extrasynaptic NMDA receptor activity leads to cell death (Hardingham et al., 2002). The method by which synaptic and NMDA receptors mediate their opposing effects were also investigated. It was found that Ca2+ influx through synaptic NMDA receptors activates the transcription factor cAMP response element-binding (CREB), causing the expression of the CREB-regulated pro-survival gene that encodes brain derived neurotrophic factor (BDNF) (Hardingham et al., 2002). In contrast, Ca2+ influx through extrasynaptic NMDA receptors activates a dominant CREB shut-off pathway that prevents the BDNF expression (Hardingham et al., 2002).  12  Figure 1.4 Schematic representation of NMDA receptor-mediated excitotoxicity via apoptosis/necrosis. (Figure was reproduced from: Beal MF 2000. Energetics in the pathogenesis of neurodegenerative diseases. Trends Neurosci 23 (7): 298-304.)  13  1.5 Excitotoxicity- An Emphasis on Huntington’s disease 1.5.1 NMDA Receptors and Huntington’s disease Huntington’s disease (HD) is an inherited neurodegenerative disorder characterized by cognitive deficits, motor decline, and mood disturbances (Fan and Raymond, 2007). The genetic disorder is due to an > 36 expansion in the polyglutamine (polyQ) region of the huntingtin (htt) protein, with longer repeat length linked to an earlier age of onset (Fan and Raymond, 2007). HD mainly affects the striatum, and causes the selective death of medium-sized Golgi type I spiny cells, which constitute 80 % of all striate neurons (Fan and Raymond, 2007). In contrast, a subclass of medium-sized aspiny interneurons that colocalize somatostatin (SST), neuropeptide Y (NPY), and nitric oxide synthase (NOS), survive the degenerative process (Dawbarn et al., 1985; Ferrante et al., 1985; Kumar, 2004).  NMDA receptors are implicated in HD  neuropathology (Fan and Raymond, 2007). The post-mortem brains of HD patients exhibit a disproportionate loss of NMDA receptors in the striatum, indicating that NMDA receptor expression increases susceptibility of the neurons to cell death (Fan and Raymond, 2007). In addition, the administration of NMDA receptor agonists to rodents or nonhuman primates in vivo, or the application of these agents to striatal cultures in vitro replicates the selective pattern of death in striate neurons during HD (Beal et al., 1986; Fan and Raymond, 2007). Interestingly, recent work from our laboratory has demonstrated comparable neurochemical changes including NMDA receptor expression in striatal neurons from HD transgenic mice and SST receptor 1/5 double knockout mice (Rajput et al., 2011).  14  1.5.2 NMDA Receptor Expression in HD Transgenic mice expressing full-length poly Q-expanded human htt, such as the yeast artificial chromosome (YAC) 46, YAC72, and YAC128 mice, have been developed to study the mechanisms underlying HD (Fan and Raymond, 2007). These mice contain 46, 72, and 128 polyQ repeats, respectively, and exhibit the pattern of selective degeneration of striate neurons during HD (Fan and Raymond, 2007). Previous work revealed that medium-sized spiny neurons of the YAC 72 mice exhibit an increase in NMDA receptor-mediated current and toxicity in comparison to wild-type (Fan and Raymond, 2007). Fan et al. demonstrated that the overexpression of NR1 and NR2B subunits at the cell membrane of medium-sized spiny neurons is responsible for the enhanced activity and toxicity of NMDA receptors in YAC72 mice (Fan et al., 2007). Altered NMDA receptor expression has also been examined in striate neurons of the R6/2 HD transgenic mice (Rajput et al., 2011), which express truncated human htt with 150 polyQ repeats (Fan and Raymond, 2007). It was found that NR1 and NR2A expression at the cell membrane is increased in the R6/2 mice when compared to wild-type (Rajput et al., 2011). In contrast, expression of NR2B is decreased at the cell membrane relative to wild-type (Rajput et al., 2011). 1.5.3 Selective Neuronal Death in HD The processes underlying the selective sparing of medium-sized aspiny interneurons and susceptibility of medium-sized spiny neurons remain elusive. Koh et al. suggested that mediumsized aspiny interneurons survive because they have a paucity of NMDA receptors (Koh et al., 1986). However, more recently, many studies including from our laboratory have used cultured 15  cells and tissue sections to demonstrate the presence of NR1 in SST-positive striate neurons (Williams et al., 1991; Augood et al., 1994; Kumar et al., 1997). The finding of NR1 expression and co-localization with SST-positive striatal neurons indicates that NMDA receptor-mediated excitotoxicity during HD may be regulated in a receptor subtype-selective manner. 1.6 NMDA Receptors and ERK1/2 1.6.1 MAP Kinases Mitogen-activated protein (MAP) kinases are a family of enzymes that regulate a wide range of cellular processes by phosphorylating serines and threonines of target proteins (Pearson et al., 2001). These include cell growth and differentiation, as well as cell survival and death (Pearson et al., 2001). The MAP kinase family is comprised of three subfamilies (Pearson et al., 2001). The first subfamily is the extracellular signal-regulated kinase (ERK) which contains several isoforms (Pearson et al., 2001; Bogoyevitch and Court, 2004). Of these, only ERK 1 and 2 have been thoroughly characterized (Pearson et al., 2001). The two additional MAP kinase families are known as c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 (Pearson et al., 2001). The JNK subfamily has at least ten isoforms that are encoded by three genes (JNK1-3), while the p38 subfamily contains four isoforms () (Pearson et al., 2001). MAP kinases are regulated by protein kinase cascades (Pearson et al., 2001). Each MAP kinase cascade is a three-tiered cascade: a MAP kinase is activated by an upstream MAPK/ERK kinase (MEK), which in turn is activated by an upstream MEK kinase (MEKK) (Pearson et al., 2001). MEKs are a family of dual specificity enzymes that phosphorylate serine/threonine and tyrosine residues of their MAP kinase substrates (Pearson et al., 2001). MEKs display very 16  narrow substrate specificity; most activate only one or two MAP kinases (Pearson et al., 2001). In contrast, MEKKs, which are a family of serine/threonine protein kinases, are capable of activating multiple MAP kinase cascades (Pearson et al., 2001). The ERK1 and 2 cascades include the Ras-Raf-MEK1/2 pathway, while cascades of JNKs and p38s include the RacMEKK-MEK4/7 and the Rac-MEKK-MEK3/6 pathways, respectively (Pearson et al., 2001). 1.6.2 ERK1/2 ERK1 and 2 (ERK1/2) are serine/threonine protein kinases with molecular masses of 44 and 42 kDa, respectively (Rubinfeld and Seger, 2005). The activation of these enzymes is mediated by cell-surface receptors such as receptor tyrosine kinases, heterotrimeric G-protein coupled receptors, and ligand-gated ion channels (Rubinfeld and Seger, 2005). The signalling pathway leading to the activation of ERK1/2 by receptor tyrosine kinases has been the most well-defined, and is often used as a prototype for other MAP kinase cascades (Rubinfeld and Seger, 2005). This mode of ERK1/2 activation involves the recruitment of adaptor proteins such as Src homology 2 domain-containing (Shc) and Growth factor receptor-bound protein 2 (Grb2), which bind phosphotyrosine residues of the activated receptor via their SH2 (Src homology 2) domains (Rubinfeld and Seger, 2005). The guanine nucleotide exchange factor son of sevenless (SOS) then interacts with the receptor-adaptor protein complex and induces the small guanosine triphosphate (GTP) -binding protein Ras to exchange its guanosine diphosphate (GDP) for GTP (Rubinfeld and Seger, 2005). GTP-bound Ras binds MEKK (Raf-1, B-Raf, A-Raf), and causes it to become activated (Rubinfeld and Seger, 2005). Subsequently, MEKK phosphorylates and activates MEK1/2 (Ser218 and 222 in MEK1), which in turn activates ERK1/2 (Rubinfeld and Seger, 2005). The activation of ERK1/2 is executed by phosphorylation of both its tyrosine and threonine residues (Thr202 and Tyr204 in ERK1; Thr185 and Tyr187 in ERK2) (Rubinfeld and 17  Seger, 2005).  Phosphorylated ERK1/2 translocate to the nucleus to regulate target gene  expression (Rubinfeld and Seger, 2005). 1.6.3 Regulation of ERK1/2 by NMDA Receptors The ERK1/2 signalling pathway is activated by Ca2+ entry through NMDA receptors (Bading and Greenberg, 1991; Thomas and Huganir, 2004). NMDA receptor activity has been shown to stimulate Ras, which is activated by specific guanine nucleotide exchange factors (GEFs), and inhibited by GTPase activating proteins (GAPs) (Thomas and Huganir, 2004). The calcium-calmodulin regulated GEF RasGEF1 has also been found to bind directly to NR2B (Krapivinsky et al., 2003).  However, NMDA receptors do not always promote ERK1/2  signalling. Studies have demonstrated that the location of NMDA receptors affects the outcome of receptor activation (Kim et al., 2005; Ivano et al., 2006). The stimulation of extrasynaptic NMDA receptors does not lead to ERK1/2 activation, while stimulation of both synaptic and extrasynaptic NMDA receptors does (Ivano et al., 2006). Extrasynaptic NMDA receptors have also been reported to inactivate Ras (Kim et al., 2005). Thus, these findings indicate that synaptic and extrasynaptic NMDA receptors have an opposite effect on Ras-ERK signalling. The interaction between NR2B and RasGEF1 has been found to cause ERK1/2 activation (Krapivinsky et al., 2003). Surprisingly, NR2A, which is the predominant NR2 subunit in cortical/hippocampal synapses, does not interact with RasGEF1, and does not activate ERK1/2 (Krapivinsky et al., 2003).  Since NR2B-containing NMDA receptors are predominant at  extrasynaptic sites, it has been suggested differences in the regulation of ERK1/2 between synaptic and extrasynaptic NMDA receptor is not due to the variations in receptor composition (Krapivinsky et al., 2003). Rather, NR2B-containing NMDA receptors have opposing effects on  18  ERK1/2 activity based on their location (Krapivinsky et al., 2003). Differences in the signalling pathways of synaptic and extrasynaptic NMDA receptors that cause their opposing effect on ERK1/2 activity remain undetermined. Since the activation of extrasynaptic NMDA receptors is believed have toxic consequences (Hardingham et al., 2002), ERK1/2 inactivation may contribute to the toxicity mediated by these receptors. 1.7 NMDA Receptors and PI3K 1.7.1 PI3K Family In response to extracellular stimuli, phosphoinositide-3 kinases (PI3Ks) phosphorylate the 3-position of the inositol ring of phosphoinositides (Koyasu, 2003).  The resultant  phosphoinositides regulate important cellular processes such as cell growth and survival (Koyasu, 2003). The PI3Ks are divided into three classes (IA/IB, II, and III) on the basis of structural characteristics and substrate specificity (Koyasu, 2003). All PI3Ks contain a C2 domain and a catalytic domain (Koyasu, 2003).  The C2 domain of PI3Ks interacts with  phospholipid in a calcium-dependent manner (Koyasu, 2003). Class I and Class II PI3Ks have a Ras-binding domain that is located at their N-terminus (Koyasu, 2003). Class I PI3Ks interact with phosphatidylinositol (PI), phosphatidylinositol 4monophosphate (PI (4) P) and phosphatidylinositol 4, 5-bisphosphate (PI(4,5)P2) to produce PI 3-phosphate (PI3P), PI 3, 4-bisphosphate (PI (3,4) P2) and PI3, 4, 5-trisphosphate (PI (3,4,5) P3) (Koyasu, 2003). They are further section into IA and IB subclasses on the basis of differences in activation methods (Koyasu, 2003). Class IA PI3Ks are heterodimeric molecules that are composed of a 110-kDa (p110) catalytic subunit that associates with a 50-85 kDa regulatory subunit (Koyasu, 2003). The 19  activation of these enzymes is mediated by stimulation of receptor tyrosine kinases (Koyasu, 2003). Class IA PI3Ks have three types of p110 catalytic subunit: p110and p110which are widely expressed in many tissues and organs, and p110which is predominantly expressed in leukocytes (Koyasu, 2003). In addition, class IA PI3Ks have five different regulatory subunits: p85, p55, p50 are derived from alternative splicing of a single gene, while p85 and p55 are encoded by separate genes (Koyasu, 2003). Of these, p85 is the most abundantly expressed regulatory subunit (Koyasu, 2003). The p85 and p85 subunits have two Src homology domains 2 (SH2) that interact with phosphotyrosine residues of adaptor proteins (Koyasu, 2003). This activates the p110 kinase activity, which is inhibited in the p85-110 complex (Koyasu, 2003). The p110 subunit also contains a Ras-binding domain that can associate with GTP-bound Ras, and may serve as an alternative activation pathway for the subunit (Koyasu, 2003). The class IB PI3K enzyme p110is expressed mainly in leukocytes (Koyasu, 2003). This enzyme is structurally similar to the p110 subunits of class 1A PI3Ks, but lacks domain necessary for binding p85 (Koyasu, 2003). Instead, p110binds a different regulatory subunit termed p101, and is activated via G-protein coupled receptors (Koyasu, 2003). There are three Class II PI3Ks: PI3K-C2 and PI3K-C2 which are present ubiquitously, and PI3K-C2  that is expressed predominantly in hepatocytes (Koyasu, 2003). Class II PI3Ks phosphorylate PI and PI(4)P to form PI(3)P and PI (3, 4) P2 (Koyasu, 2003). Class III PI3K contains only one member (Vps34p), which interacts with PI to produce PI3P. Unlike class I PI3Ks, the activation mechanisms of class II and class II PI3Ks are largely unknown (Koyasu, 2003).  20  1.7.2 PI3K-Akt Cascade The PI3K-Akt cascade is a signalling pathway that has an important role in cell survival (Cantley, 2002).  PI3K, which can be activated in a calcium-dependent manner by  calcium/calmodulin, catalyzes the phosphorylation of PI(4,5)P2 to PI(3,4,5)P3 in the membrane (Cantley, 2002).  The kinase phosphoinositides-dependent protein kinase (PDK1) and its  substrate protein kinase B (Akt/PKB) are recruited to the membrane via association with PIP3 through their pleckstrin homology domains (Cantley, 2002).  Subsequently, PDK1  phosphorylates and activates Akt, which promotes cell survival and growth through phosphorylation-mediated activation/inactivation of its substrates (Cantley, 2002). For instance, Akt phosphorylates and inactivates the pro-apoptotic B-cell lymphoma 2 (Bcl-2) family member Bcl-2-associated death promoter (BAD), which inhibits its interaction with pro-survival Bcl-2 family members Bcl-2 and B-cell lymphoma-extra large (Bcl-xL) (Cantley, 2002). The JNK/p38 activator apoptosis signal-regulated kinase 1 (ASK1) is also inactivated by Akt phosphorylation, and p53 activity is inhibited by Akt, resulting in the decrease of Bcl-2-associated X protein (Bax) expression and neuronal death (Cantley, 2002). 1.7.3 Regulation of PI3K by NMDA Receptors The PI3K/Akt cascade is involved in the pro-survival effects of NMDA receptors (Hetman and Kharebava, 2006). In cultured rat cerebellar granule neurons, PI3K inhibition with the antagonists LY294002 or wortmann, has been demonstrated to decrease the protective effect of NMDA receptors against apoptosis (Zhang et al., 1998; Hetman and Kharebava, 2006). It was also found that NMDA activates Akt in a PI3K-dependent manner, and the expression of a  21  dominant negative mutant form of Akt inhibits the protective effects of NMDA (Hetman and Kharebava, 2006; Lafon-Cazal et al., 2002; Bhave et al., 1999). The role of PI3K in NMDA receptor-mediated survival of neurons other than cerebellar granule cells is unclear (Hetman and Kharebava, 2006). NMDA receptor activity has been shown to increase PI3K/Akt signalling in primary cultures of rodent striatal or cortical neurons (Perkinton et al., 2002). PI3K may also be involved in NMDA receptor-mediated protection of forebrain neurons (Hetman and Kharebava, 2006). Akt activation was demonstrated in striatal neurons from a genetic mouse model of HD (Gines et al., 2003). The Akt activation, which was believed to be a compensatory process, was inhibited by NMDA receptor antagonists (Gines et al., 2003). While there are several targets the mediate the anti-apoptotic signalling by PI3K/Akt, it is not known which ones are associated in the protective signalling downstream of NMDA receptors (Hetman and Kharebava, 2006). 1.8 NMDA Receptor-mediated Signalling Independent of Ion Flux The functionality of ligand-gated ion channels has traditionally been based on their ability to mediate ion flux (Vissel et al., 2001). However, this classification of ion channel functionality has been challenged. Studies have suggested that agonist-binding to ligand-gated ion channels such as NMDA receptors can also activate intracellular signalling cascades independently of ion flux (Vissel et al., 2001). In HEK293 cells expressing NR1 and NR2A, repeated agonist application was found to cause a reduction in the peak amplitude of NR1/NR2A currents, and a loss in the number of functional NMDA receptor channels (Vissel et al., 2001). Subsequently, it was determined that the current decline occurs independently of ion flux (Vissel et al., 2001). This was demonstrated by repeatedly exposing the cells to agonist in the presence  22  of Mg2+, which hinders the NMDA receptor channel pore (Vissel et al., 2001). The current was subsequently measured in Mg2+-free solution, to determine whether the current decline occurs in the absence of ion flux (Vissel et al., 2001). It was thus found that inhibition of NMDA receptor-mediated ion flux with Mg2+ does not prevent the reduction in NR1/NR2A currents (Vissel et al., 2001). By comparison, exposing cells to the non-receptor tyrosine kinase Src, or the tyrosine phosphatase inhibitor bpV, was shown to prevent current decline, indicating that tyrosine dephosphorylation is involved in the process (Vissel et al., 2001).  Furthermore,  truncations of NR1 and NR2A revealed that the current decline is dependent upon tyrosine residues in NR1 (Y837) and NR2A (Y842), both of which are present near the M4 region of the subunits (Vissel et al., 2001). The Y842 residue in NR2A is a putative YXX consensus-binding motif for the 2 subunit of the adaptor protein AP-2, a protein complex that connects cargo proteins to clathrin-coated pits (Vissel et al, 2001). The co-expression of a dominant negative 2 (W421A) was found to inhibit the decline of NR1/NR2A currents, suggesting that agonistmediated tyrosine dephosphorylation regulates the association of this protein with the NMDA receptor channel (Vissel et al., 2001). Hence, these results indicate that agonist-binding to NMDA receptors is relayed to intracellular or membrane spanning regions of the receptor subunits.  It was thus concluded that agonist-binding may be a method by which NMDA  receptor-associated proteins can induce intracellular cascades independently of ion flux. The co-application of NMDA and the mGluR5 agonist (S)-3, 5-dihydroxyphenylglycine (DHPG) to striatal neuronal cultures results in the synergistic increase of ERK1/2 phosphorylation (Yang, 2004). The increase in ERK1/2 phosphorylation is independent of NMDA receptor-mediated ion flux, as the non-competitive open-channel blocker MK801, and extracellular Mg2+ do not significantly block NMDA and DHPG induced ERK1/2 23  phosphorylation (Yang, 2004). Similarly, the addition of the cell permeable Ca 2+ chelators BAPTA-AM and Calcium Green-1/AM to neurons does not inhibit the ERK1/2 phosphorylation (Yang, 2004). Furthermore, NMDA and DHPG are able to synergistically increase ERK1/2 phosphorylation in Ca2+/Na+-free medium, indicating that NMDA receptor-mediated ion flux is not required for the process (Yang, 2004). ERK1/2 activation through this pathway is able to phosphorylate the transcription factors E-26-like protein 1 (Elk-1) and CREB, and cause c-FBJ murine osteosarcoma viral oncogene homolog (c-Fos) expression (Yang, 2004). Hence, these results suggest that ERK1/2 phosphorylation can occur independently of ion flux through NMDA receptors, and underlies a membrane to nucleus communication that is important for transcriptional regulation. 1.9 Hypothesis and Research Objectives 1.9.1 Hypothesis In the striatum, a subset of medium-sized aspiny interneurons that co-localize SST, NPY, and the enzyme NOS are selectively spared during HD (Dawbarn et al., 1985; Ferrante et al., 1985). In contrast, medium-sized spiny cells that constitute 80 % of all striatal neurons undergo selective neurodegeneration (Fan and Raymond, 2007). Although it was initially suggested that the interneurons survive because they lack NMDA receptors, studies including those from our lab have shown the presence of NR1 in SST-positive striatal neurons (Koh et al., 1986; William et al., 1991; Augood et al., 1994; Kumar et al., 1997). The finding of NR1 expression and colocalization with SST-positive neurons indicates that NMDA receptor-induced toxicity may be regulated in a receptor-specific manner. That is, differences in the expression levels of NMDA  24  receptor subunits, leading to the prevalence of different NMDA receptor subtypes in these neurons may account for their opposing fates during HD. Mammalian cells expressing NR1 and NR2 subunits alone have been reported to form channels that do not elicit current responses to glutamate/NMDA (Mori and Mishina, 1995). However, these studies did not account for time-dependent effects, and measured cell response to rapid agonist exposure only, which may differ from when agonists are applied for longer periods of time. Furthermore, agonist-binding to NMDA receptor channels has been found to induce the activation of intracellular signalling cascades independently of ion flux (Vissel et al., 2001; Yang, 2004). Since NMDA receptor-mediated excitotoxicity has been found to occur in the absence of Ca2+ (Chen et al., 1998), it is possible that agonist-binding alone can induce intracellular cascades leading to cell death.  From the aforementioned findings, it can be  hypothesized that NMDA-induced toxicity may occur in cells expressing NR1, NR2A, or NR2B, in a receptor-type dependent manner. Since neurons express multiple NMDA receptor subunits, this makes it difficult to characterize the effects of activating a single NMDA receptor subtype. Therefore, this study was accomplished by using HEK293 cells, which do not express NMDA receptor subunits, as vehicles for the selective expression of NMDA receptors. Of the NMDA receptor subunits, NR1, NR2A, and NR2B were selected, as they are the predominant subunits expressed in the adult forebrain (Fan and Raymond, 2007). To address the aforementioned hypothesis, the following three research objectives were constructed:  25  1. To determine the membrane and intracellular expressions of NR1, NR2A, and NR2B in response to NMDA. 2. To investigate the effect of NMDA application on cell viability, and subsequently characterize the nature of death in cells expressing NR1, NR2A, or NR2B. 3. To determine the molecular mechanisms underlying the NMDA-induced death of cells expressing NR1, NR2A, or NR2B. 1.9.2 Research Objective 1 Determine the membrane and intracellular expressions of NR1, NR2A, and NR2B in response to NMDA. 1.9.2.1 Rationale The localization of NMDA receptors can contribute to toxicity. Medium-sized spiny neurons of the YAC mouse model of HD display increased NMDA receptor-mediated ion flux and toxicity, when compared to neurons from the wild-type (Fan and Raymond, 2007). Fan et al. demonstrated that this enhanced NMDA receptor activity and toxicity are due to an increase in NR1 and NR2B expression at the membrane of medium-sized spiny neurons (Fan et al., 2007). Hence, NMDA receptor over-expression at the membrane can have toxic consequences. NMDA receptor internalization may also play a role in NMDA-induced toxicity. Earlier studies have found that receptor internalization can affect signal transduction (Sorkin and Zastrow, 2009). For instance, proteins known as -arrestins, which target G-protein coupled receptors (GPCRs) to clathrin-coated pits for endocytosis, can act as adaptor proteins that interact with both the agonist-bound internalized receptor and proteins involved in signal transduction to activate MAP kinase cascades (Nong et al., 2004). Therefore, although the effect of NMDA  26  receptor internalization on signal transduction is unknown, it is possible that the process can regulate intracellular signalling pathway(s) linked to cell death through a similar mechanism. The above-mentioned findings indicate that the membrane expression and internalization of NMDA receptors may have toxic effects that contribute to NMDA-induced cell death. Therefore, the first objective of this study is to determine the membrane and intracellular expression of NR1, NR2A, and NR2B in response to toxic levels of NMDA via western blot analysis. 1.9.3 Research Objective 2  Investigate the effect of NMDA application on cell viability, and subsequently characterize the nature of death in cells expressing NR1, NR2A, or NR2B. 1.9.3.1 Rationale The application of NMDA to striatal cultures results in cell death that progressively increases with greater concentrations of NMDA (Kumar, 2004). NMDA-induced cell death is believed to be primarily mediated by the mitochondria, which have the ability to sequester large amounts of intracellular Ca2+ (Waxman and Lynch, 2005; Wang and Qin, 2010). Excessive Ca 2+ uptake by the mitochondria due to NMDA receptor over-activation results in the depolarization of the mitochondrial membrane, and the consequent inhibition of the electron transport chain and ATP formation (Waxman and Lynch, 2005; Wang and Qin, 2010). These outcomes make the cell more vulnerable to death insults, and studies have found that the degree of mitochondrial membrane depolarization is analogous to the level of cell death (Waxman and Lynch, 2005; Wang and Qin, 2010). These findings thus suggest that the application of NMDA leads to cell death that occurs largely via mitochondrial dysfunction. Hence, the first part of the second 27  objective is to investigate whether NMDA can have such toxic effects in cells expressing NR1, NR2A, and NR2B individually.  This experiment will also determine whether there is an  association between the NMDA receptor expressions mentioned in objective 1, and NMDAinduced cell death measured in terms of mitochondrial dysfunction. The mode of excitotoxic cell death is not uniform (Wang and Qin, 2010). In rat cortical cultures, mild insult to the cells by treatment with low NMDA concentrations results in cell death that is mostly apoptotic (Bonfoco et al., 1995). In contrast, intense insult by the application of greater NMDA concentrations leads to predominantly necrotic cell death (Bonfoco et al., 1995). Hence, the intensity of the insult can also dictate the nature of NMDA-induced cell death. Therefore, since NMDA can produce both apoptosis and necrosis, after examining the level of NMDA-induced cell death, we will also determine the nature of the death. 1.9.4 Research Objective 3 Determine the molecular mechanisms underlying the NMDA-induced death of cells expressing NR1, NR2A, or NR2B. 1.9.4.1 Rationale After assessing the level and nature of NMDA-induced death, we will determine the underlying molecular mechanisms. Since the downstream signalling pathways following the activation of NR1, NR2A, and NR2B alone have not been previously studied, we arbitrarily chose to examine the signalling molecules ERK1/2 and PI3K. Given the anti- and pro-survival functions of ERK1/2 (Hardingham et al., 2002; Lu and Xu, 2006), it is anticipated that ERK1/2 will be either activated or inactivated during NMDA-induced toxicity. Since PI3K is known to have pro-survival functions (Hetman and Kharebava, 2006), it is anticipated that PI3K will be inactivated during NMDA-induced toxicity. 28  CHAPTER II Materials and Methods 2.1 Transfection and Cell Culture Stable transfections of HEK293 cells expressing NR1, NR2A or NR2B were developed. The rat NR1 cDNA was inserted into the pcDNA3.1+hygro plasmid (hygromycin resistance), while the cDNA of NR2A and NR2B was inserted into the pcDNA3.1+neo plasmid (neomycin resistance). Cells were transfected with 5g of DNA with Lipofectamine reagent. Stable transfections were selected and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10 % FBS (fetal bovine serum), and 400 g/ml of hygromycin or 700 g/ml of neomycin at 37oC and 5 % CO2. 2.2 Treatment Concentration-dependent effects of NMDA were examined by treating cells with 50 M, 100 M, 300 M, 1 mM, or 5 mM of NMDA in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10 % FBS, and 400 g/ml of hygromycin or 700 g/ml of neomycin for 24 hours at 37oC and 5 % CO2. 2.3 Western Blot Cell lysate from untreated and NMDA-treated cells were analyzed for the expression of NR1, NR2A, or NR2B, as well as for phosphorylated and total ERK1/2 and PI3K expression. Membrane and cytosolic extracts were prepared by lysing cells in lysis buffer (20mM Tris-HCl, 2.5 mM dithiothreitol (DTT), 1:100 protease and phosphatase inhibitors; pH 7.5), followed by centrifugation at 2000 rpm for 10min at 4oC, and then a second centrifugation at 13000 rpm for 29  70min at 4oC; the pellets (membrane fractions) were re-suspended in Tris-HCl.  Protein  concentrations were determined by the Bradford assay, using known concentrations of bovine serum albumin (BSA) as standards.  25 g of protein was taken from each sample and  solubilised in Laemmli sample buffer (Sigma-Aldrich) with 5 % 2-merceptoethanol and denatured at 99 for 5 min before fractionation by electrophoresis on 8 % (for NR1, NR2A, NR2B, and PI3K) or 10 % (for ERK1/2) polyacrylamide gel, depending upon the size of the protein of interest. Thereafter, proteins were transferred onto 0.2 m nitrocellulose membranes in transfer buffer (20 mM Tris, 192 mM glycine, and 20 % methanol), and then blocked with 5 % bovine serum albumin (BSA) in Tris-buffer saline with 0.2 % tween (TBS-T) at room temperature (RT) for 1 hour. The membranes were subsequently probed with specific primary antibodies diluted in 5 % BSA at 4oC for 16 hours. The primary antibodies against NR1 (monoclonal-Abcam), NR2A (rabbit polyclonal-Sigma-Aldrich), and NR2B (monoclonalChemicon) were diluted to 1:500, while those against phospho (Thr 202 and Tyr 204 of ERK1; Thr 185 and Tyr 187 of ERK2)-ERK1/2 (rabbit polyclonal-Cell Signalling), total ERK1/2 (rabbit polyclonal-Cell Signalling), phospho (Tyr 458) - PI3K (rabbit polyclonal-Cell Signalling), and total PI3K (rabbit polyclonal-Cell Signalling) were diluted to 1:1000. The primary antibodies against -actin (monoclonal-Sigma-Aldrich), which was used as a house-keeping protein, were diluted to 1:2000. After incubation with primary antibodies, membranes were washed three times with TBS-T and incubated with goat anti-mouse or goat anti-rabbit secondary antibodies at a dilution of 1:2000 in 5 % BSA for 2 hours at RT. The membranes were again washed three times with TBS-T, and then incubated with chemiluminescence reagent for 1 minute at RT. Proteins of interest were detected via Alpha Innotech FluorChem 8800 gel box imager.  30  Relative protein expression was derived from western blot analysis. The intensity of the protein bands was measured by the Alpha Innotech FluorChem software. The expression of each protein of interest (NR1, NR2A, NR2B, pERK1/2, and pPI3K) was determined by taking the ratio of the protein band intensity to that of the corresponding -actin (for NR1, NR2A, and NR2B), or total ERK1/2 and PI3K (for pERK1/2 and pPI3K). This value was then compared to the value of the control group to obtain protein expression relative to control. Results were expressed as the mean of the relative protein expression from three independent experiments. 2.4 MTT The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay is based on a colorimetric reaction, in which the mitochondrial succinate dehydrogenase enzyme reduces the yellow tetrazolium salt MTT into a blue formazan product, which can be detected and quantified spectrophotometrically. The amount of formazan product is considered to be proportional to the number of viable cells.  Hence, cell viability is measured in terms of  mitochondrial function. The effect of NMDA application on cell viability of HEK293 cells expressing NR1, NR2A, or NR2B was determined by the MTT assay. The experiment was performed by seeding 10,000 cells per well onto a 96-well plate containing DMEM supplemented with 10% FBS and 400 g/ml of hygromycin or 700 g/ml of neomycin; cells were maintained at 37oC and 5 % CO2. At 80 % confluency, culture medium was removed and cells were incubated for 24 hours with new medium containing 50 M-5 mM of NMDA. Medium was also placed in empty wells to form blank solutions. After the 24-hour treatments, 1.5 mg/ml MTT (Sigma-Aldrich) in phosphate buffer saline (PBS) was added to each of the wells. The 96-well plate was then 31  incubated at 37oC and 5 % CO2 for 3-4 hours to allow for the MTT to be metabolized. Subsequently, the MTT solution was removed and formazan crystals were solubilised by incubation in DMSO for 15 min.  Absorbance at 575 nm was measured using a  spectrophotometer. The absorbance of each sample was subtracted from that of the blank, and then compared to control to yield % cell viability. 2.5 Determination of Apoptosis and Necrosis Apoptosis and necrosis were determined by Hoechst 3352 and propidium iodide, respectively. Cells expressing NR1, NR2A, or NR2B were seeded onto poly-D-lysine coated coverslips containing DMEM supplemented with 10% FBS and 400 g/ml of hygromycin or 700 g/ml of neomycin in 24-well plates. At 80 % confluency, culture medium was removed and cells were incubated for 24 hours with new medium containing 50 M-5 mM of NMDA. Posttreatment, cells were washed three times with PBS, and then fixed with 4 % paraformaldehyde in PBS for 20min on ice. Following three subsequent washes in PBS, cells were incubated with 1g/ml Hoechst 3352 in PBS for 10min at RT. Cells were then washed with PBS twice, and incubated with 25g/ml propidium iodide in PBS for 5 min at RT. After three washes with PBS, the coverslips were mounted onto slides. The slides were observed under Leica microscope attached to Retiga 2002R camera. For each of the control and experimental groups, a total of 300-350 cells were counted from 8 randomly selected areas of the coverslip and a ratio of apoptotic/necrotic to total number of cells was calculated from every area. These 8 values were then used to obtain the mean % apoptosis/necrosis.  32  2.6 Statistical Analyses Results were expressed as mean + standard error of the mean (SEM). Sets of different results were compared via one-way analysis of variance (ANOVA) followed by Dunnett’s test. The mean values were considered as statistically significant at P<0.05.  33  CHAPTER III Results 3.1  Membrane and Intracellular Localization of NR1, NR2A, and NR2B NMDA receptor expression in HEK293 cells stably transfected with NR1, NR2A, or  NR2B was first verified via western blot analysis (Figures 3.1-3.12). It was found that all three receptors were expressed in the membrane and intracellular fractions from transfected cells, but not from non-transfected cells. In addition, the level of localization for each receptor was found to be similar in the membrane, as well as the intracellular fractions. Previous studies have suggested that the localization of NMDA receptors may affect NMDA-induced cell death (Nong et al., 2004; Fan et al., 2007). To investigate whether this is the case in HEK293 cells expressing NR1, NR2A, or NR2B, the membrane and intracellular localization of the receptors were determined through western blot analysis (Figures 3.13-3.18). Cells were treated with a range of NMDA concentrations that were previously shown to cause neuronal death in striatal cultures (Kumar, 2004). Thereafter, it was found that when compared to control, the application of 1 mM and 5 mM NMDA significantly decreased the membrane localization of NR1  (34 + 15 % and 35 + 8% in 1 mM and 5 mM NMDA-treated cells,  respectively; n=3; P<0.05 for control vs. treatments) and NR2A (29 + 9 % and 29 + 8% in 1 mM and 5 mM NMDA-treated cells, respectively; n=3; P<0.05 for control vs. treatments), indicating that receptor internalization took place at these concentrations (Figures 3.13 and 3.14). The intracellular localization of NR2A was also significantly decreased relative to control after treatment with 5 mM NMDA (42 + 9% in 5 mM NMDA-treated cells; n=3; P<0.05 for control vs. treatments) (Figure 3.17). In the case of NR2B, membrane localization was significantly 34  decreased in comparison to control following the application of 100 M and 300 M NMDA (17 + 2.6 % and 15 + 4% in 100 M and 300 M NMDA-treated cells, respectively; n=3; P<0.05 for control vs. treatments) (Figure 3.15). Interestingly, however, the membrane localization of NR2B in response to 1 mM and 5 mM of NMDA was not significantly different from control, which suggests that although NR2B internalizes at intermediate NMDA concentrations, it is stabilized at the membrane at high NMDA concentrations. Since the decreases in NR1, NR2A, and NR2B membrane localizations were not accompanied by increases in the intracellular localizations of these receptors, it is likely that the receptors were degraded following endocytosis (Figures 3.16-3.18). Thus, the trafficking of NMDA receptors was found to be dependent upon the agonist concentration, as well as receptor-type. Subsequently, we examined whether cell death occurred under the same treatment conditions.  35  Figure 3.1: (A) Representative western blot of the membrane and intracellular localization of NR1 in non-transfected cells (NTC) and transfected cells (TC). Protein extracts (25 g) from NTC and TC were separated by SDS-PAGE and probed with affinity-purified antibodies (primary: monoclonal anti-NR1; secondary: goat anti-mouse). Blots were stripped and reprobed for -actin. It was found that NR1 was expressed in the membrane and intracellular fractions from transfected cells, but not from non-transfected cells. (B) The densitometric quantification of NR1 expression in TC relative to NTC. The values shown in the bar graph were calculated as follows. The protein band intensity of NR1 expression was normalized to that of the corresponding -actin. This value was then divided by that of the normalized background detection in NTC to yield receptor expression relative to NTC. Values represent mean + SEM; n=3  36  Figure 3.11: (A) Representative western blot of the membrane and intracellular localization of NR2A in non-transfected cells (NTC) and transfected cells (TC). Protein extracts (25 g) from NTC and TC were separated by SDS-PAGE and probed with affinity-purified antibodies (primary: rabbit polyclonal anti-NR2A; secondary: goat anti-rabbit). Blots were stripped and reprobed for -actin. It was found that NR2A was expressed in the membrane and intracellular fractions from transfected cells, but not from non-transfected cells. (B) The densitometric quantification of NR2A expression in TC relative to NTC. Values represent mean + SEM; n=3. Please refer to legend of Figure 3.1 for more details.  37  Figure 3.12: (A) Representative western blot of the membrane and intracellular localization of NR2B in non-transfected cells (NTC) and transfected cells (TC). Protein extracts (25 g) from NTC and TC were separated by SDS-PAGE and probed with affinity-purified antibodies (primary: rabbit polyclonal anti-NR2B; secondary: goat anti-rabbit). Blots were stripped and reprobed for -actin. It was found that NR2B was expressed in the membrane and intracellular fractions from transfected cells, but not from non-transfected cells. (B) The densitometric quantification of NR2B expression in TC relative to NTC. Values represent mean + SEM; n=3. Please refer to legend of Figure 3.1 for more details.  38  Figure 3.13: Representative western blot and densitometric quantification of NR1 membrane localization in control and NMDA-treated cells. Protein extracts (25 g) were fractioned by SDS-PAGE and probed with affinity-purified antibodies (primary: monoclonal anti-NR1; secondary: goat anti-mouse). Blots were stripped and reprobed for -actin. Application of 1 mM and 5 mM NMDA significantly decreased the localization of NR1 Values are expressed as mean + SEM, n=3; *P<0.05 for control vs. treatment by one-way ANOVA followed with Dunnett’s test.  39  Figure 3.14: Representative western blot and densitometric quantification of NR2A membrane localization in control and NMDA-treated cells. Protein extracts (25 g) were fractioned by SDS-PAGE and probed with affinity-purified antibodies (primary: rabbit polyclonal anti-NR2A; secondary: goat anti-rabbit). Blots were stripped and reprobed for -actin. NR2A localization was significantly decreased in response to the application of 1 mM and 5 mM NMDA. Values are expressed as mean + SEM, n=3; *P<0.05 for control vs. treatment by one-way ANOVA followed with Dunnett’s test.  40  Figure 3.15: Representative western blot and densitometric quantification of NR2B membrane localization in control and NMDA-treated cells. Protein extracts (25 g) were fractioned by SDS-PAGE and probed with affinity-purified antibodies (primary: rabbit polyclonal anti-NR2B; secondary: goat anti-rabbit). Blots were stripped and reprobed for -actin. NR2B localization was significantly decreased in response to treatment with 100 M and 300 M NMDA. Values are expressed as mean + SEM, n=3; *P<0.05 for control vs. treatment by one-way ANOVA followed with Dunnett’s test.  41  Figure 3.16: Representative western blot and densitometric quantification of NR1 intracellular localization in control and NMDA-treated cells. NR1 localization did not significantly differ from control in response to any of the treatment conditions. Values are expressed as mean + SEM, n=3; *P<0.05 for control vs. treatment by one-way ANOVA followed with Dunnett’s test. Please refer to legend of Figure 3.13 for more details of experiment.  42  Figure 3.17: Representative western blot and densitometric quantification of NR2A intracellular localization in control and NMDA-treated cells. Treatment with 5 mM NMDA significantly decreased NR2A localization. Values are expressed as mean + SEM, n=3; *P<0.05 for control vs. treatment by one-way ANOVA followed with Dunnett’s test. Please refer to legend of Figure 3.14 for more details of experiment.  43  Figure 3.18: Representative western blot and densitometric quantification of NR2B intracellular localization in control and NMDA-treated cells. NR2B localization did not significantly different from control in response to any of the treatment conditions. Values are expressed as mean + SEM, n=3; *P<0.05 for control vs. treatment by one-way ANOVA followed with Dunnett’s test. Please refer to legend of Figure 3.15 for more details of experiment.  44  3.2 NMDA-induced Death in Cells Expressing NR1, NR2A, or NR2B The application of NMDA to striatal cultures leads to cell death that progressively increases with greater concentrations of NMDA (Kumar, 2004). Cell death from NMDA is considered to occur largely via mitochondrial dysfunction (Waxman and Lynch, 2005; Wang and Qin, 2010). Therefore, we examined whether NMDA also has such a toxic effect on cells expressing NR1, NR2A, or NR2B by using the MTT assay to measure cell viability in terms of mitochondrial function. Accordingly, it was determined that cells expressing NR1 did not exhibit a significant decrease in viability when compared to control after treatment with any of the NMDA concentrations (Figure 3.2). In comparison, 5 mM NMDA led to a significant reduction in the viability of cells expressing NR2A (69% + 8%; n=3; P<0.05 for control vs. treatment) and NR2B (54% + 18%; n=3; P<0.05 for control vs. treatment) (Figures 3.21 and 3.22). Hence, these results show that cell viability decreases in response to 5 mM NMDA in NR2A and NR2B expressing cells. Subsequently, the nature of the death was determined by exposing cells to Hoechst 3352 and propidium iodide, which are dyes that stain the nucleus of apoptotic and necrotic cells, respectively. It was found that the application of NMDA to NR1-expressing cells caused a significant increase in apoptosis from control, in a concentration-dependent manner (% average apoptosis was 2.3-,3.7-,5.3-, 6.2-,7.2-,and 8.1 % in control, 50 M, 100 M, 300 M, 1 mM , and 5 mM NMDA-treated cells, respectively; n=3; P<0.05 for control vs. treatment) (Figure 3.23). In contrast, apoptosis significantly increased at low NMDA concentrations, and then decreased at higher NMDA concentrations in cells expressing NR2A (% average apoptosis was 2.4-,7.8-,7.2-, 6.0-,4.9-,and 4.5 % in control, 50 M, 100 M, 300 M, 1 mM , and 5 mM NMDA-treated cells, respectively; n=3; P<0.05 for control vs. treatment) and NR2B (% average 45  apoptosis was 2.7-,6.3-,8.1-, 7.1-,5.3-,and 4.5 % in control, 50 M, 100 M, 300 M, 1 mM , and 5 mM NMDA-treated cells, respectively; n=3; P<0.05 for control vs. treatment) (Figures 3.24 and 3.25). Interestingly, necrosis was detected in NR2A and NR2B, but not in NR1 expressing cells (Figures 3.26-3.28). A significant increase in necrosis that was dependent upon the applied NMDA concentration was observed in cells expressing NR2A (% average necrosis was 0.7-,2.9-,4.1-, 5.9-,8.0-,and 11.3 % in control, 50 M, 100 M, 300 M, 1 mM , and 5 mM NMDA-treated cells, respectively; n=3; P<0.05 for control vs. treatment) and NR2B (% average necrosis was 1.4-,5.0-,6.8-, 12.7-,15.0-,and 17.0 % in control, 50 M, 100 M, 300 M, 1 mM , and 5 mM NMDA-treated cells, respectively; n=3; P<0.05 for control vs. treatment) (Figures 3.27 and 3.28). Therefore, taken together, these results suggest that the form of cell death and the degree of apoptosis/necrosis varies depending upon the type of receptor expressed, and the concentration of NMDA treatment. The occurrence of apoptosis in cells expressing NR1, which did not exhibit decreased cell viability as determined by the MTT assay, indicates that the mitochondria of these cells are functional during apoptosis. Subsequently, we determined the possible molecular mechanisms mediating these effects.  46  Figure 3.2: Effect of NMDA application on the viability of cells expressing NR1 as determined by the MTT assay. The viability of cells expressing NR1 did not significant differ from control in response to any of the NMDA concentrations. Values are shown as mean + SEM, n=3; *P<0.05 for control vs. treatment by one-way ANOVA followed with Dunnett’s test.  Figure 3.21: Effect of NMDA application on the viability of cells expressing NR2A as determined by the MTT assay. Treatment with 5 mM NMDA caused a significant decrease in the % cell viability of NR2A-expressing cells. Values are shown as mean + SEM, n=3; *P<0.05 for control vs. treatment by one-way ANOVA followed with Dunnett’s test. 47  Figure 3.22: Effect of NMDA application on the viability of cells expressing NR2B as determined by the MTT assay. Treatment with 5 mM NMDA caused a significant decrease in the % cell viability of NR2B-expressing cells. Values are shown as mean + SEM, n=3; *P<0.05 for control vs. treatment by one-way ANOVA followed with Dunnett’s test.  48  Figure 3.23: Representative photomicrographs and quantification of NMDA-induced apoptosis, as determined by Hoechst 3352 staining, in cells expressing NR1. For each treatment group, 300-350 cells were counted from 8 randomly selected areas of the coverslip. It was found that NMDA application significantly increased apoptosis at all concentrations (shown in photomicrographs by the arrows). Values are shown as overall % apoptosis + SEM, calculated by taking the mean of the % apoptosis from three independent experiments (n=3); *P<0.05 for control vs. treatment by one-way ANOVA followed with Dunnett’s test.  49  Figure 3.24: Representative photomicrographs and quantification of NMDA-induced apoptosis, as determined by Hoechst 3352 staining, in cells expressing NR2A. It was found that NMDA application significantly increased apoptosis at all concentrations (shown in photomicrographs by the arrows). Values are shown as overall % apoptosis + SEM, calculated by taking the mean of the % apoptosis from three independent experiments (n=3); *P<0.05 for control vs. treatment by one-way ANOVA followed with Dunnett’s test. Please refer to legend of Figure 3.23 for more details of experiment.  50  Figure 3.25: Representative photomicrographs and quantification of NMDA-induced apoptosis, as determined by Hoechst 3352 staining, in cells expressing NR2B. It was found that NMDA application significantly increased apoptosis at all concentrations (shown in photomicrographs by the arrows). Values are shown as overall % apoptosis + SEM, calculated by taking the mean of the % apoptosis from three independent experiments (n=3); *P<0.05 for control vs. treatment by one-way ANOVA followed with Dunnett’s test. Please refer to legend of Figure 3.23 for more details of experiment.  51  Figure 3.26: Representative photomicrographs and quantification of NMDA-induced necrosis, as determined by propidium iodide staining, in cells expressing NR1. For each treatment group, 300-350 cells were counted from 8 randomly selected areas of the coverslip. Necrosis was not significantly different from control in response to any of the NMDA treatments in NR1expressing cells. Values are expressed as overall % necrosis + SEM, calculated by taking the mean of the % apoptosis from three independent experiments (n=3); *P<0.05 for control vs. treatment by one-way ANOVA followed with Dunnett’s test.  52  Figure 3.27: Representative photomicrographs and quantification of NMDA-induced necrosis, as determined by propidium iodide staining, in cells expressing NR2A. NMDA application significantly increased necrosis at all concentrations in cells expressing NR2A. The arrows in the photomicrographs are pointing to a representative necrotic nucleus. Values are expressed as overall % necrosis + SEM, calculated by taking the mean of the % apoptosis from three independent experiments (n=3); *P<0.05 for control vs. treatment by one-way ANOVA followed with Dunnett’s test. Please refer to legend of Figure 3.26 for more details of experiment.  53  Figure 3.28: Representative photomicrographs and quantification of NMDA-induced necrosis, as determined by propidium iodide staining, in cells expressing NR2B. NMDA application significantly increased necrosis at all concentrations in cells expressing NR2B. The arrows in the photomicrographs are pointing to a representative necrotic nucleus. Values are expressed as overall % necrosis + SEM, calculated by taking the mean of the % apoptosis from three independent experiments (n=3); *P<0.05 for control vs. treatment by one-way ANOVA followed with Dunnett’s test. Please refer to legend of Figure 3.26 for more details of experiment.  54  3.3 NMDA-induced ERK1/2 Expression in Cells Expressing NR1, NR2A or NR2B Having measured the level and nature of NMDA-induced cell death, we then investigated the potential molecular mechanisms responsible for the observed effects. As the downstream signalling pathways after the activation of NR1, NR2A, or NR2B are unknown, we arbitrarily decided to examine the signalling molecule ERK1/2. ERK1/2 is believed to have pro-survival functions, and thought to be inactivated after the stimulation of death-promoting extrasynaptic NMDA receptors (Hardingham et al., 2002). It was determined that on the whole, expression of phosphorylated (active) ERK1/2 did not deviate considerably from control in NR1 and NR2A expressing cells (Figures 3.3 and 3.31). By comparison, ERK1/2 expression was decreased relative to control in cells expressing NR2B following the application of NMDA at all concentrations (ERK1: 47 + 9 %, 60 + 15 %, 39 + 9 %, 22 + 3.6 %, 23 + 2.5 % and ERK2: 64 + 0.4 %, 75 + 0.1%, 44 + 2.3 %, 32 + 5.2 %, 32 + 5.3 % after 50 M, 100 M, 300 M, 1 mM and 5 mM treatments, respectively; n=2) (Figure 3.32). Due to the small sample size, statistical analysis was not conducted for these experiments.  55  Figure 3.3: Representative western blot and densitometric quantification of phosphorylated ERK1/2 (pERK1/2) expression in NR1 expressing control and NMDA-treated cells. Protein extracts (25 g) were fractioned by SDS-PAGE and probed with affinity-purified antibodies (primary: rabbit polyclonal anti-pERK1/2; secondary: goat anti-rabbit). Blots were stripped and reprobed for ERK1/2 and -actin. Values are expressed as mean + SEM, n=2.  56  Figure 3.31: Representative western blot and densitometric quantification of phosphorylated ERK1/2 (pERK1/2) expression in NR2A expressing control and NMDA-treated cells. Values are expressed as mean + SEM, n=2. Please refer to legend of Figure 3.3 for more details of experiment.  57  Figure 3.32: Representative western blot and densitometric quantification of phosphorylated ERK1/2 (pERK1/2) expression in NR2B expressing control and NMDA-treated cells. Values are expressed as mean + SEM, n=2. Please refer to legend of Figure 3.3 for more details of experiment.  58  3.4 NMDA-induced PI3K Expression in Cells Expressing NR1, NR2A or NR2B The signaling molecule PI3K was also examined to investigate the potential molecular mechanism responsible for the observed effects of NMDA. PI3K was arbitrarily selected as the downstream signalling pathways are the individual activation of NR1, NR2A, and NR2B are unknown. Given the known pro-survival functions of PI3K (Hetman and Kharebava, 2006), it is believed that the signalling molecule is inactivated during NMDA-induced toxicity. It was found that phosphorylated (active) PI3K expression was not significantly different from control in cells expressing NR1, NR2A or NR2B (Figures 3.4-3.42).  59  Figure 3.4: Representative western blot and densitometric quantification of phosphorylated PI3K (pPI3K) expression in NR1 expressing control and NMDA-treated cells. Protein extracts (25 g) were fractioned by SDS-PAGE and probed with affinity-purified antibodies (primary: rabbit polyclonal anti-pPI3K; secondary: goat anti-rabbit). Blots were stripped and reprobed for PI3K and -actin. The expression of pPI3K did not significantly differ from control in response to any of the NMDA concentrations. Values are shown as mean + SEM, n=3; *P<0.05 for control vs. treatment by one-way ANOVA followed with Dunnett’s test.  60  Figure 3.41: Representative western blot and densitometric quantification of phosphorylated PI3K (pPI3K) expression in NR2A expressing control and NMDA-treated cells. It was found pPI3K expression did not significantly differ from control in response to any of the NMDA concentrations. Values are shown as mean + SEM, n=3; *P<0.05 for control vs. treatment by one-way ANOVA followed with Dunnett’s test. Please refer to legend of Figure 3.4 for more details of experiment.  61  Figure 3.42: Representative western blot and densitometric quantification of phosphorylated PI3K (pPI3K) expression in NR2B expressing control and NMDA-treated cells. It was found that pPI3K expression did not significantly differ from control in response to any of the NMDA concentrations. Values are shown as mean + SEM, n=3; *P<0.05 for control vs. treatment by one-way ANOVA followed with Dunnett’s test. Please refer to legend of Figure 3.4 for more details of experiment.  62  CHAPTER IV DISCUSSION 4.1 Discussion Overview During HD, medium-sized spiny projection neurons of the striatum undergo selective degeneration (Fan and Raymond, 2007).  In contrast, a subset of medium-sized aspiny  interneurons of the striatum that co-localize SST, NPY, and the enzyme NOS are selectively spared (Dawbarn et al., 1985; Ferrante et al., 1985). The process underlying the selective sparing of interneurons and the susceptibility of projection neurons is undetermined. While it was initially suggested that the interneurons were spared as they lacked NMDA receptors, studies have shown the presence of NR1 in SST-positive striatal neurons (Koh et al., 1986; William et al., 1991; Augood et al., 1994; Kumar et al., 1997). These findings suggest that differences in the expression levels of NMDA receptor subunits, leading to the prevalence of different NMDA receptor subtypes in these neurons may underlie their opposite fates during HD. Accordingly, the present study investigated whether NMDA-induced toxicity is regulated in a receptor-specific manner, by determining the toxic effects of NMDA application on HEK293 cells expressing NR1, NR2A, or NR2B. The main findings of this study indicate that NMDA application causes cell death, which varies in intensity and nature, as determined by apoptosis and necrosis, depending upon the concentration of NMDA applied, and the receptor-type expressed by the cells.  Cells  expressing NR1 were found to undergo apoptosis but not necrosis, while cells expressing NR2A or NR2B underwent both apoptosis and necrosis in a receptor-specific manner.  In cells  expressing NR2A or NR2B, exposure to low concentrations of NMDA resulted in cell death that 63  was predominantly apoptotic. In contrast, exposure to high concentrations of NMDA produced mostly necrosis. In cells expressing NR1, NMDA application caused apoptosis, which exhibited a gradual increase in response to greater concentrations of NMDA. In addition, cell death through apoptosis and/or necrosis was determined to be the greatest at all NMDA concentrations in cells expressing NR2B, followed by those expressing NR2A, and then NR1. Taken together, these results indicate that the activation of receptors formed by NR1, NR2A, or NR2B have different toxic consequences. Thus, the selective neurodegeneration observed during HD may be due to the variation in expression levels of NR1, NR2A, and NR2B between medium-sized aspiny interneurons and medium-sized spiny projection neurons. 4.2 NMDA induces NR1, NR2A, and NR2B internalization in a concentration- and receptor-specific manner The membrane and intracellular localizations of NR1, NR2A, and NR2B were determined in response to NMDA via western blot analyses. Previously, human NR1 and NR2A were demonstrated to be retained in the ER when expressed alone in COS/HEK293 cells (McIlhinney et al., 1998). The ER retention of rodent NR1 and NR2B was also reported in COS cells expressing these receptors individually (Horak et al., 2008). In contrast to the findings of these studies, our results indicate that NR1, NR2A, and NR2B of rat origin exhibit membrane localization when expressed alone in HEK293 cells.  Previously, a relationship between  increased NMDA receptor activity and cell-surface expression was demonstrated (Fan et al., 2007). Thus, the membrane localization of rat NR1, NR2A, and NR2B suggests that the receptors may be forming functional channels when expressed individually in HEK293 cells. The discrepancy between our results and those of previous studies in terms of receptor localization may be due to differences between the trafficking of rat and human NMDA 64  receptors, as well as the cell-type in which the receptors are expressed. The overall sequence identities between rat and human NR1, NR2A, and NR2B are 99.3 %, 95.3 %, and 98.5 %, respectively (Hedegaard et al., 2011). Hence, the ER retention of human NR1 and NR2A in HEK293 cells, as reported by McIlhinney et al. may be due to interspecies variation between rat and human NMDA receptors, leading to differences in their trafficking (McIlhinney et al., 1998). Furthermore, earlier work has shown that NMDA receptors respond differently depending upon the cell-type in which they are expressed. Rat NR1 forms homomeric channels that conduct current responses to rapid treatment with 10 M glutamate or 100 M NMDA in Xenopus oocytes (Mori and Mishina, 1995). However, when expressed alone in mammalian cells, rat NR1 expression does not result in the production of channels that conduct current in response to the rapid application of agonists (Mori and Mishina, 1995). Hence, the ER retention of rodent NR1 and NR2B, as reported by Horak et al. may be due to the receptors being expressed in COS cells instead of HEK293 cells (Horak et al., 2008). In addition to the results mentioned above, it was found that NMDA affects the membrane and/or intracellular localization of NR1, NR2A, and NR2B in a concentration- and receptor-specific manner. More specifically, the membrane localization of NR1 and NR2A were determined to be significantly decreased in comparison to control after the application of 1 mM and 5 mM NMDA. In contrast, NR2B membrane localization was significantly decreased at 100 M and 300 M NMDA.  The intracellular localization of NR2A was also significantly  decreased at 5 mM NMDA. The observed decreases in the membrane localization of NR1, NR2A, and NR2B suggest the occurrence of concentration-dependent receptor internalization. Previously, 27 % of NMDA receptors were reported to be internalized in neuronal cultures after treatment with 50 M or 1 mM NMDA in the presence of 100 M glycine (Nong et al., 2003). 65  This finding is not comparable with the results obtained in the present study, as the NMDA concentrations at which receptor internalization occurred, and/or the level of internalization at the respective NMDA concentrations, are different from those observed by Nong et al. (Nong et al., 2003). The discrepancy may be due to the multiple NMDA receptor subunits expressed in neurons, which can interact to generate various ion channels with properties that differ from those produced by NR1, NR2A, and NR2B alone in HEK293 cells. It is also important to note that since the intracellular localization of these receptors did not increase at the corresponding NMDA concentrations, receptor degradation likely occurred following internalization. In the case of NR2A, the significant decrease in its intracellular localization suggests that the level of NR2A degradation was greater as compared to that of NR1 and NR2B. Previous studies have found that receptor internalization can affect signal transduction (Sorkin and Zastrow, 2009). For example, proteins known as -arrestins, which target G-protein coupled receptors (GPCRs) to clathrin-coated pits for endocytosis, can function as adaptor proteins that interact with both the agonist-bound internalized receptor and proteins involved in signal transduction to activate MAP kinase cascades (Nong et al., 2004). Although the effect of NMDA receptor internalization on signal transduction has not been determined, it is possible that the process can regulate intracellular signalling pathway(s) through a similar mechanism. NR1, NR2A, and NR2B internalization was found to occur at NMDA concentrations that induced cell death as determined by apoptosis and necrosis (Figures 3.21 and 3.22). Thus, it is possible that the internalization process of these receptors led to cell death by modulating intracellular signalling pathway(s). However, as previously mentioned, the involvement of NMDA receptor internalization on signal transduction regulation is unknown. Therefore, further studies are required to investigate whether and how such a phenomenon takes place. It is also important to 66  note that the level of internalization of the three receptors did not differ amongst the NMDA concentrations at which internalization was detected.  However, the level of cell death as  determined by apoptosis and/or necrosis, increased with greater NMDA at these concentrations. Therefore, if receptor internalization were to have a toxic effect, an additional toxic process must be occurring to account for the increased cell death. Receptor internalization is also a mode of desensitization (Ferguson, 2001). Desensitization refers to the dampening of receptor response to agonist with time to defend against receptor overstimulation. Thus, is it also possible that the observed internalization of NR1, NR2A, and NR2B are the result of desensitization, which has been initiated to defend against toxicity from NMDA receptor over-stimulation.  Further studies are required to  investigate whether the internalization of NR1, NR2A, and NR2B has a role in mediating NMDA-induced toxicity, or defending against NMDA-induced toxicity via desensitization. Taken together, the aforementioned findings of this study show that NMDA induces the internalization of NR1, NR2A, and NR2B in a concentration and receptor-specific manner. Moreover, since receptor internalization was found to occur at NMDA concentrations that produced cell death, a relationship may exist between NMDA receptor internalization and toxicity. 4.3 NMDA-induced death occurs in HEK-293 cells expressing NR1, NR2A, or NR2B Cell death after NMDA application was analyzed in HEK293 cells expressing NR1, NR2A, or NR2B. Previously, it was reported that mammalian cells expressing NR1 or members of NR2 alone do not form functional channels that conduct current after treatment with glutamate or NMDA (Mori and Mishina, 1995). However, our results show that NMDA induces death in 67  cells expressing NR1, NR2A, or NR2B, which is indicative of NMDA receptor function in these cells. Furthermore, data from cell death analyses revealed that NMDA induces cell death which varies in intensity and nature depending upon the NMDA concentration and receptor-type expressed by the cells.  Cell death as determined by apoptosis and/or necrosis gradually  increased with the application of greater NMDA concentrations in each of the cell-types. In addition, cell viability, as determined by the MTT assay, was significantly decreased from control at the highest NMDA concentration (5 mM) in NR2A- and NR2B-expressing cells. Since the activation of NMDA receptors by NMDA has been shown to have toxic consequences (Wang and Qin, 2010), the observed increase in cell death with greater NMDA concentrations suggests a gradual rise in the activation of NR1, NR2A, and NR2B in these cells. In contrast to previous studies, the above-mentioned results of this study indicate that NR1, NR2A, and NR2B may be forming functional channels in HEK293 cells expressing these receptors individually.  Although it was previously demonstrated that mammalian cells  expressing NR1 or members of NR2 alone do not form functional channels that conduct current after agonist treatment (Mori and Mishina, 1995), these studies did not account for timedependent effects, and measured cell response to rapid agonist exposure only. Such a response is not sufficient to determine receptor functionality, as it may differ from when agonists are applied for longer periods of time, as is the case in the present study. Furthermore, agonist-induced NMDA receptor-mediated Ca2+-influx has been shown to be time-dependent, and to increase with agonist exposure-time (Nonaka et al., 1998). Hence, cells expressing NR1, NR2A, and NR2B may elicit current responses when treated for longer time-periods. In addition, although ligand-gated ion channels have traditionally been defined as functional based on their ability to mediate ion flux, this classification of ion channel functionality has been challenged (Vissel et 68  al., 2001). Previous studies have shown that agonist-binding to ligand-gated ion channels such as NMDA receptors can also lead to signal transduction, and the activation of downstream signaling cascades independently of ion flux (Vissel et al., 2001; Yang, 2004). These findings collectively suggest that the observed cell death can be attributed to the interaction between NMDA and NR1, NR2A, or NR2B, resulting in the activation of downstream signalling pathways linked to cell death, in an ion flux dependent or independent manner. Apoptosis and necrosis are two forms of cell death with distinct morphological and biochemical features (Bonfoco et al., 1995). In rat cortical cultures, mild insult to the cells by exposure to low NMDA concentrations leads to cell death that is predominantly apoptotic (Bonfoco et al., 1995). In contrast, intense insult by treatment with high NMDA concentrations produces mostly necrosis (Bonfoco et al., 1995). This effect of NMDA is consistent with that seen in cells expressing NR2A or NR2B, indicating that the channels produced by these subunits respond similarly to NMDA receptor channels in neurons. In contrast, cells expressing NR1 did not exhibit such a pattern of cell death. In NR1-expressing cells, NMDA application led to apoptosis only. The level of apoptosis was significantly greater when compared to control, and increased in response to greater concentrations of NMDA. Heteromeric interactions between NMDA receptor subunits in vivo have been demonstrated by co-immunoprecipitation studies (Mori and Mishina, 1995). Hence, the interaction between NR1 and another NMDA receptor subunit in the rat cortical cultures may account for this discrepancy, and mediate the pattern of cell death observed by Bonfoco et al. (Bonfoco et al., 1995). Furthermore, neurons express multiple NMDA receptor subunits, and the combined effect of their activation may deviate from that observed by the activation of NR1 alone in HEK293 cells.  69  Intracellular ATP levels are an important determinant of the form of NMDA receptormediated cell death (Tsujimoto, 1997).  It was reported that immediately after glutamate  application, neuronal death occurs via necrosis (Lipton and Nicotera, 1998). These neurons exhibited characteristics of necrosis including mitochondrial dysfunction, declined ATP levels, swollen nuclei, and scattered intracellular debris in the incubation medium (Lipton and Nicotera, 1998). Neurons surviving this necrotic phase were shown to regain their mitochondrial activity and ATP levels, and subsequently undergo apoptosis (Lipton and Nicotera, 1998). Thus, these findings indicate that necrosis is an ATP-independent process, while apoptosis is largely ATPdependent. Since mitochondrial dysfunction is known to occur during apoptosis, ATP necessary for the remaining apoptotic process is believed to be supplied by glycolysis (Tsujimoto, 1997). We found that NMDA-induced death by apoptosis and/or necrosis occurs at all concentrations in each of the cell-types. However, cell viability was significantly decreased at the highest NMDA concentration of 5 mM only, in NR2A- and NR2B-, but not in NR1-expressing cells. Since cell viability was measured in terms of mitochondria function, our data suggests that the mitochondria were functional at the NMDA concentrations in which cell viability did not significantly differ from control. In this study, apoptosis was determined in terms of nuclear fragmentation, which has been found to occur at an early stage of apoptosis that precedes mitochondrial damage (Martin et al., 1998). Therefore, the observed apoptosis in NR1, NR2A, and/or NR2B-expressing cells may have been at a stage in which the mitochondria were functional.  In contrary to apoptosis, mitochondrial damage has been reported to occur  throughout necrosis (Martin et al., 1998). It is thus surprising that a progressive decrease in cell viability was not observed, as necrosis increased in cells expressing NR2A/NR2B. A study conducted by McKeague et al. demonstrated that although cells exposed to the toxin H2O2  70  displayed necrosis as determined by propidium iodide staining after four hours of treatment, the MTT assay did not detect a decrease in cell viability until 24 hours post-treatment (McKeague et al., 2003). It was concluded that the discrepancy existed because the MTT assay does not consider the possible rise in cell number in a cycling cell population (McKeague et al., 2003). Therefore, it is possible that the MTT assay was unable to measure decreased cell viability due to relatively lower levels of necrosis at the respective NMDA concentrations. In such cases, the cell division from cells unaffected by NMDA, which ultimately attributes to a much high cell number, may have decreased the usefulness of the MTT assay. 4.4 NMDA-induced pERK1/2 and pPI3K expression Having measured the level and nature of NMDA-induced cell death, we then investigated the potential molecular mechanisms underlying the observed effects.  As the downstream  signalling pathways following the activation of channels formed by NR1, NR2A, or NR2B are unknown, we arbitrarily selected to examine the signalling molecules ERK1/2 and PI3K. The data from western blot analyses of pERK1/2 expression shows that on the whole, NMDA treatment did not lead to a considerable deviation from control in pERK1/2 expression of NR1and NR2A-expressing cells. By comparison, pERK1/2 expression was decreased from control in response to NMDA treatment in NR2B-expressing cells. Although statistical analyses were not conducted on these results, the observed decrease in pERK1/2 expression of NR2B-expressing cells is quite interesting. Ivano et al. had demonstrated that the activation of synaptic NMDA receptors enhances pERK1/2 expression, while the activation of extrasynaptic NMDA receptors reduces pERK1/2 expression (Ivano et al., 2006). It was also reported that NR2B-containing NMDA receptors predominate at these extrasynaptic sites (Krapivinsky et al., 2003). Therefore, if the decreased pERK1/2 expression in NR2B-expressing cells is found to be significantly 71  different from control, this would indicate that channels formed by NR2B alone are sufficient to cause the effect on ERK1/2 observed in neurons. Western blot analyses of pPI3K expression revealed that pPI3K expression did not significantly differ from control at any of the NMDA concentrations in the three cell-types. Thus, it was found that PI3K did not contribute to the observed toxicity in HEK293 cells expressing NR1, NR2A, or NR2B. 4.5 Conclusion, Physiological Relevance, and Future Studies The major findings of this study revealed that NMDA application leads to cell death, which varies in intensity and nature depending upon the NMDA concentration applied, and NMDA receptor-type expressed by the cells.  It was found that cells expressing NR1 underwent  apoptosis but not necrosis, while cells expressing NR2A or NR2B underwent both apoptosis and necrosis in a receptor-specific manner. In cells expressing NR2A or NR2B, application of low NMDA concentrations resulted in cell death that was mostly apoptotic. In contrast, application of greater NMDA concentrations produced mostly necrosis. In cells expressing NR1, NMDA application led to apoptosis, which displayed a gradual increase in response to greater concentrations of NMDA. Moreover, cell death via apoptosis and/or necrosis was found to be the greatest at all NMDA concentrations in cells expressing NR2B, followed by those expressing NR2A, and then NR1. These results collectively suggest that the activation of receptors formed by NR1, NR2A, or NR2B have distinct toxic consequences.  Therefore, the selective  neurodegeneration observed during HD may be due to the variation in expression levels of NR1, NR2A, and NR2B between medium-sized aspiny interneurons and medium-sized spiny projection neurons.  72  While the above-mentioned results are interesting, there remains more to be elucidated. Heteromeric interactions between NMDA receptor subunits have been demonstrated in vivo (Mori and Mishina, 1995). This indicates that NMDA-induced toxicity may also be regulated by interactions between NR1 and NR2A/NR2B. Thus, a similar study using HEK293 cells coexpressing NMDA receptor subunits should be performed to investigate the outcome of receptor interaction on toxicity. Moreover, to determine whether the observed phenomenon occurs in neurons, a replicate study should be conducted in striatal cultures by using subunit-specific antagonists.  Additionally, since NMDA-induced toxicity resulting from the activation of  channels formed by NR1, NR2A, or NR2B has not been previously examined,  whether the  process takes place via an ion flux dependent or independent manner should be investigated.  73  REFERENCES  Ankarcrona M, Dypbukt JM, Bonfoco E, Zhivotovsky B, Orrenius S, Lipton SA, Nicotera P 1995. Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function Neuron 15:961-973. Augood SJ, McGowan EM, Emson PC 1994. Expression of N-methyl-D-aspartate receptor subunit NR1 messenger RNA by identified striatal somatostatin cells. Neuroscience 59:7-12. Bading H, Greenberg M 1991. Stimulation of protein tyrosine phosphorylation by NMDA receptor activation Science 253:912-914. Beal MF 2000. Energetics in the pathogenesis of neurodegenerative diseases. Trends Neurosci 23: 298-304 Beal MF, Kowall NW, Ellison DW, Mazurek MF, Swartz KJ, Martin JB 1986. Replication of the neurochemical characteristics of Huntington's disease by quinolinic acid Nature 321:168-171. Bhave SV, Ghoda L, Hoffman PL 1999. Brain-derived neurotrophic factor mediates the antiapoptotic effect of NMDA in cerebellar granule neurons: signal transduction cascades and site of ethanol action. J Neurosci 19:3277-3286. Bloodgood BL, Sabatini BL. 2009. NMDA Receptor-Mediated Calcium Transients in Dendritic Spines. In Van Dongen AM, editor. Biology of the NMDA Receptor, Boca Raton (FL): Taylor & Francis Group, LLC. Bogoyevitch MA, Court NW 2004. Counting on mitogen-activated protein kinases--ERKs 3, 4, 5, 6, 7 and 8 Cell Signal 16:1345-1354. Bonfoco E, Krainc D, Ankarcrona M, Nicotera P, Lipton SA 1995. Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-D-aspartate or nitric oxide/superoxide in cortical cell cultures Proc Natl Acad Sci U S A 92:7162-7166. Cantley LC 2002. The phosphoinositide 3-kinase pathway Science 296:1655-1657. Chatterton JE, Awobuluyi M, Premkumar LS, Takahashi H, Talantova M, Shin Y, Cui J, Tu S, Sevarino KA, Nakanishi N, Tong G, Lipton SA, Zhang D 2002. Excitatory glycine receptors containing the NR3 family of NMDA receptor subunits. Nature 415:793-798. Chen Q, Olney JW, Lukasiewicz PD, Almli T, Romano C 1998. Ca2+-independent excitotoxic neurodegeneration in isolated retina, an intact neural net: a role for Cl- and inhibitory transmitters Mol Pharmacol 53:564-572. Dawbarn D, De Quidt ME, Emson PC 1985. Survival of basal ganglia neuropeptide Ysomatostatin neurones in Huntington's disease Brain Res 340:251-260. 74  Dingledine R, Borges K, Bowie D, Traynelis SF 1999. The glutamate receptor ion channels Pharmacol Rev 51:7-61. Doble A 1999. The role of excitotoxicity in neurodegenerative disease: implications for therapy. Pharmacol Ther 81:163-221. Ehlers MD 2000. Reinsertion or Degradation of AMPA Receptors Determined by ActivityDependent Endocytic Sorting Neuron 28:511-525. Fan MM, Fernandes HB, Zhang LY, Hayden MR, Raymond LA 2007. Altered NMDA receptor trafficking in a yeast artificial chromosome transgenic mouse model of Huntington's disease J Neurosci 27:3768-3779. Fan MM, Raymond LA 2007. N-methyl-D-aspartate (NMDA) receptor function and excitotoxicity in Huntington's disease Prog Neurobiol 81:272-293. Ferguson SS 2001. Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol Rev 53:1-24. Ferrante R, Kowall N, Beal M, Richardson E, Bird E, Martin J 1985. Selective sparing of a class of striatal neurons in Huntington's disease Science 230:561-563. Gines S, Ivanova E, Seong IS, Saura CA, MacDonald ME 2003. Enhanced Akt signaling is an early pro-survival response that reflects N-methyl-D-aspartate receptor activation in Huntington's disease knock-in striatal cells. J Biol Chem 278:50514-50522. Golstein P, Kroemer G 2007. Cell death by necrosis: towards a molecular definition Trends Biochem Sci 32:37-43. Hardingham GE, Fukunaga Y, Bading H 2002. Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways Nat Neurosci 5:405-414. Hartley DM, Kurth MC, Bjerkness L, Weiss JH, Choi DW 1993. Glutamate receptor-induced 45Ca2+ accumulation in cortical cell culture correlates with subsequent neuronal degeneration. J Neurosci 13:1993-2000. Hedegaard MK, Hansen KB, Andersen KT, Brauner-Osborne H, Traynelis SF 2011. Molecular pharmacology of human NMDA receptors Neurochem Int . Hetman M, Kharebava G 2006. Survival Signaling Pathways Activated by NMDA Receptors Current Topics in Medicinal Chemistry 6:787 -799. Horak M, Chang K, Wenthold RJ 2008. Masking of the endoplasmic reticulum retention signals during assembly of the NMDA receptor J Neurosci 28:3500-3509.  75  Ikonomidou C, Bosch F, Miksa M, Bittigau P, Vockler J, Dikranian K, Tenkova TI, Stefovska V, Turski L, Olney JW 1999. Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain. Science 283:70-74. Ikonomidou C, Stefovska V, Turski L 2000. Neuronal death enhanced by N-methyl-D-aspartate antagonists Proc Natl Acad Sci U S A 97:12885-12890. Ivanov A, Pellegrino C, Rama S, Dumalska I, Salyha Y, Ben-Ari Y, Medina I 2006. Opposing role of synaptic and extrasynaptic NMDA receptors in regulation of the extracellular signalregulated kinases (ERK) activity in cultured rat hippocampal neurons. J Physiol 572:789-798. Johnson JW 2003. Acid tests of NMDA receptor gating basics. Mol Pharmacol 63: 1199-1201. Kim MJ, Dunah AW, Wang YT, Sheng M 2005. Differential roles of NR2A- and NR2Bcontaining NMDA receptors in Ras-ERK signaling and AMPA receptor trafficking Neuron 46:745-760. Koh J, Peters S, Choi D 1986. Neurons containing NADPH-diaphorase are selectively resistant to quinolinate toxicity Science 234:73 -76. Koyasu S 2003. The role of PI3K in immune cells Nat Immunol 4:313-319. Krapivinsky G, Krapivinsky L, Manasian Y, Ivanov A, Tyzio R, Pellegrino C, Ben-Ari Y, Clapham DE, Medina I 2003. The NMDA Receptor Is Coupled to the ERK Pathway by a Direct Interaction between NR2B and RasGRF1 Neuron 40:775 -784. Kumar U 2004. Characterization of striatal cultures with the effect of QUIN and NMDA Neurosci Res 49:29-38. Kumar U, Asotra K, Patel SC, Patel YC 1997. Expression of NMDA receptor-1 (NR1) and huntingtin in striatal neurons which colocalize somatostatin, neuropeptide Y, and NADPH diaphorase: a double-label histochemical and immunohistochemical study. Exp Neurol 145:412424. Lafon-Cazal M, Perez V, Bockaert J, Marin P 2002. Akt mediates the anti-apoptotic effect of NMDA but not that induced by potassium depolarization in cultured cerebellar granule cells Eur J Neurosci 16:575-583. Lavezzari G, McCallum J, Lee R, Roche KW 2003. Differential binding of the AP-2 adaptor complex and PSD-95 to the C-terminus of the NMDA receptor subunit NR2B regulates surface expression. Neuropharmacology 45:729-737. Lipton SA, Nicotera P 1998. Calcium, free radicals and excitotoxins in neuronal apoptosis Cell Calcium 23:165-171.  76  Lu Z, Xu S 2006. ERK1/2 MAP kinases in cell survival and apoptosis IUBMB Life (International Union of Biochemistry and Molecular Biology: Life) 58:621-631. Lucas DR, Newhouse JP 1957. The toxic effect of sodium L-glutamate on the inner layers of the retina. AMA Arch Ophthalmol 58:193-201. Martin LJ, Al-Abdulla NA, Brambrink AM, Kirsch JR, Sieber FE, Portera-Cailliau C 1998. Neurodegeneration in excitotoxicity, global cerebral ischemia, and target deprivation: A perspective on the contributions of apoptosis and necrosis. Brain Res Bull 46:281-309. McIlhinney RAJ, Le Bourdellès B, Molnár E, Tricaud N, Streit P, Whiting PJ 1998. Assembly intracellular targeting and cell surface expression of the human N-methyl-d-aspartate receptor subunits NR1a and NR2A in transfected cells Neuropharmacology 37:1355 -1367. McKeague AL, Wilson DJ, Nelson J 2003. Staurosporine-induced apoptosis and hydrogen peroxide-induced necrosis in two human breast cell lines. Br J Cancer 88:125-131. Mori H, Mishina M 1995. Structure and function of the NMDA receptor channel Neuropharmacology 34:1219 -1237. Nonaka S, Hough CJ, Chuang DM 1998. Chronic lithium treatment robustly protects neurons in the central nervous system against excitotoxicity by inhibiting N-methyl-D-aspartate receptormediated calcium influx Proc Natl Acad Sci U S A 95:2642-2647. Nong Y, Huang YQ, Ju W, Kalia LV, Ahmadian G, Wang YT, Salter MW 2003. Glycine binding primes NMDA receptor internalization. Nature 422:302-307. Nong Y, Huang YQ, Salter MW 2004. NMDA receptors are movin' in Curr Opin Neurobiol 14:353-361. Olney JW 1969. Brain lesions, obesity, and other disturbances in mice treated with monosodium glutamate. Science 164:719-721. Paoletti P, Neyton J 2007. NMDA receptor subunits: function and pharmacology Curr Opin Pharmacol 7:39-47. Pearson G, Robinson F, Beers Gibson T, Xu BE, Karandikar M, Berman K, Cobb MH 2001. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions Endocr Rev 22:153-183. Perkinton MS, Ip JK, Wood GL, Crossthwaite AJ, Williams RJ 2002. Phosphatidylinositol 3kinase is a central mediator of NMDA receptor signalling to MAP kinase (Erk1/2), Akt/PKB and CREB in striatal neurones. J Neurochem 80:239-254.  77  Petralia RS, Wang YX, Wenthold RJ 1994. The NMDA receptor subunits NR2A and NR2B show histological and ultrastructural localization patterns similar to those of NR1. J Neurosci 14:6102-6120. Rajput PS, Kharmate G, Norman M, Liu SH, Sastry BR, Brunicardi CF, Kumar U 2011. Somatostatin receptor 1 and 5 double knockout mice mimic neurochemical changes of Huntington's disease transgenic mice. PLoS One 6:e24467. Risgaard R, Hansen KB, Clausen RP 2010. Partial agonists and subunit selectivity at NMDA receptors. Chemistry 16:13910-13918. Roche KW, Standley S, McCallum J, Dune Ly C, Ehlers MD, Wenthold RJ 2001. Molecular determinants of NMDA receptor internalization. Nat Neurosci 4:794-802. Rubinfeld H, Seger R 2005. The ERK Cascade: A Prototype of MAPK Signaling Mol Biotechnol 31:151-174. Sattler R, Tymianski M 2000. Molecular mechanisms of calcium-dependent excitotoxicity. J Mol Med (Berl) 78:3-13. Sheng M, Cummings J, Roldan LA, Jan YN, Jan LY 1994. Changing subunit composition of heteromeric NMDA receptors during development of rat cortex. Nature 368:144-147. Sorkin A, von Zastrow M 2009. Endocytosis and signalling: intertwining molecular networks. Nat Rev Mol Cell Biol 10:609-622. Stephenson FA, Cousins SL, Kenny AV 2008. Assembly and forward trafficking of NMDA receptors (Review) Mol Membr Biol 25:311-320. Thomas GM, Huganir RL 2004. MAPK cascade signalling and synaptic plasticity Nat Rev Neurosci 5:173-183. Tsujimoto Y 1997. Apoptosis and necrosis: intracellular ATP level as a determinant for cell death modes Cell Death Differ 4:429-434. Vissel B, Krupp JJ, Heinemann SF, Westbrook GL 2001. A use-dependent tyrosine dephosphorylation of NMDA receptors is independent of ion flux Nat Neurosci 4:587-596. Wang JK, Thukral V 1996. Presynaptic NMDA receptors display physiological characteristics of homomeric complexes of NR1 subunits that contain the exon 5 insert in the N-terminal domain. J Neurochem 66:865-868. Wang Y, Qin ZH 2010. Molecular and cellular mechanisms of excitotoxic neuronal death. Apoptosis 15:1382-1402.  78  Waxman EA, Lynch DR 2005. N-methyl-D-aspartate receptor subtypes: multiple roles in excitotoxicity and neurological disease. Neuroscientist 11:37-49. Wenzel A, Scheurer L, Kunzi R, Fritschy JM, Mohler H, Benke D 1995. Distribution of NMDA receptor subunit proteins NR2A, 2B, 2C and 2D in rat brain. Neuroreport 7:45-48. Williams JS, Berbekar I, Weiss S 1991. N-methyl-D-aspartate evokes the release of somatostatin from striatal interneurons in primary culture. Neuroscience 43:437-444. Wong HK, Liu XB, Matos MF, Chan SF, Perez-Otano I, Boysen M, Cui J, Nakanishi N, Trimmer JS, Jones EG, Lipton SA, Sucher NJ 2002. Temporal and regional expression of NMDA receptor subunit NR3A in the mammalian brain. J Comp Neurol 450:303-317. Yang L 2004. A Novel Ca2+-Independent Signaling Pathway to Extracellular Signal-Regulated Protein Kinase by Coactivation of NMDA Receptors and Metabotropic Glutamate Receptor 5 in Neurons Journal of Neuroscience 24:10846 -10857. Zhang FX, Rubin R, Rooney TA 1998. N-Methyl-D-aspartate inhibits apoptosis through activation of phosphatidylinositol 3-kinase in cerebellar granule neurons. A role for insulin receptor substrate-1 in the neurotrophic action of n-methyl-D-aspartate and its inhibition by ethanol. J Biol Chem 273:26596-26602.  79  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.24.1-0073562/manifest

Comment

Related Items