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NMDA receptor desensitization regulated by direct binding to PDZ1-2 domains of PSD-95 Sornarajah, Lavan 2007

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NMDA RECEPTOR DESENSITIZATION REGULATED BY DIRECT BINDING PDZ1-2 DOMAINS OF PSD-95 by L A V A N SORNARAJAH B.Sc , The University of British Columbia, 2002 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENT FOR THE D E G R E E OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES Graduate Program in Neuroscience THE UNIVERSITY OF BRITISH C O L U M B I A March 2007 © Lavan Sornarajah, 2007 ABSTRACT Regulation of N M D A receptor (NMDAR) activity by desensitization is important in physiological and pathological states; N M D A R desensitization contributes in shaping synaptic responses and may be protective by limiting calcium influx during sustained glutamate insults. We previously reported that glycine-independent desensitization decreases during hippocampal neuronal development, correlating with N M D A R synaptic localization and association with post-synaptic density 95 (PSD-95). PSD-95/Discs large/zona occludens (PDZ) domains 1 and 2 of PSD-95 bind to the C-terminus of N M D A R NR2 subunits. The role of PSD-95 in anchoring signaling proteins in close proximity to NMDARs is well documented. To determine if changes in N M D A R desensitization occur because of the direct binding of PSD-95 to NMDARs or due to PSD-95 recruitment of regulatory molecules, we tested the effects of various PSD-95 constructs on N M D A R currents in human embryonic kidney 293 (HEK293) cells and neurons. In H E K cells wild-type PSD-95 markedly reduced N M D A R desensitization, and the N-terminus of PSD-95 truncated after the PDZ 1-2 domains was sufficient for this effect even when multimerization of PSD-95 was abolished. In immature neurons where PSD-95 expression is low, overexpression of either PSD-95 or PSD-95 PDZ 1-2 was sufficient to significantly decrease desensitization. In mature neurons, disruption of PSD-95/NMDAR interaction through protein kinase C (PKC) activation increased desensitization to levels found in immature neurons, and this effect was not due to direct regulation of N M D A R activity by PKC. We conclude that direct binding of PSD-95 increases stability of N M D A R responses to sustained agonist exposure in neuronal and non-neuronal cells. TABLE OF CONTENTS A B S T R A C T i i T A B L E OF CONTENTS hi LIST OF FIGURES vi A C K N O W L E D G E M E N T S vii CHAPTER I Introduction 1 1.1. Ionotropic glutamate receptor overview 1 1.2. NMDARs in synaptic plasticity : 5 1.3. N M D A R involvment in neuronal survival and excitotoxicity 6 1.4. N M D A R structural determinants of function 8 1.4.1. N M D A R subunit composition 8 1.4.2. N M D A R subunit composition and spatial-temporal distribution in the CNS 12 1.4.3. N M D A R regulation by NR1 subunits '. 13 1.4.4. N M D A R regulation by NR2 subunits 14 1.4.5. NR3 Subunits 15 1.5. N M D A R regulation by serine/threonine phosphorylation 16 1.6. N M D A R regulation by M A G U K s 18 1.6.1. SAP-102 ...18 1.6.2. PSD-95 19 1.6.3. SAP-97 and PSD-93 25 1.7. N M D A R regulation by glycine-dependent desensitization 26 1.8. N M D A R regulation by calcium-dependent inactivation 26 ii i 1.9. N M D A R regulation by glycine-independent desensitization 27 1.10. Hypothesis 28 CHAPTER II Materials and methods 29 2.1. Neuronal cell culture and transfection 29 2.1.1. Culture ..29 2.1.2. Neuronal transfection 33 2.2. HEK293 cell culture and transfection 34 2.2.1. Culture 34 2.2.2. Transfection 34 2.3. Electrophysiology 35 2.4. Co-immunoprecipatition and western blot analysis 36 2.5. Materials 37 2.6. Data analysis ...37 CHAPTER III Results 38 3.1. Age-dependent desensitization 38 3.2. P K C regulates N M D A R glycine-independent desensitization in mature neurons 38 3.2.1. PKC uncouples PSD-95 from NMDARs 38 3.2.2. Uncoupling of NMDARs/PSD-95 reduces receptor desensitization in mature hippocampal neurons 42 3.3. PSD-95 Regulates glycine-independent desensitization of NMDARs 50 3.3.1. Coexpression of PSD-95 with N M D A R subunits decreases desensitization 50 3.3.2. PDZ1-2 domains of PSD-95 are sufficient to alter N M D A R desensitization in HEK293 cells; multimeriazation not required iv for altered N M D A R desensitization 54 3.3.3. Overexpression of PDZ1-2 domains decreases desensitization in immature neurons 58 CHAPTER IV General discussion 61 4.1. Phorbol ester-induced P K C activation and regulation of neuronal N M D A R function 61 4.2. Role of PSD-95 direct binding to NMDARs on current desensitization 63 4.3. Effects of PSD-95/NMDAR interaction on N M D A R function in neurons 66 4.4. PSD-95/NMDAR association may regulate synaptic plasticity and excitotoxicity 67 4.5. Future directions 68 REFERENCES 70 v LIST OF FIGURES Figure 1-1. Membrane topology of NMDARs 3 Figure 1-2. Alternative splice variants of N M D A R NR1 subunit 10 Figure 1-3. Table of proteins that interact with PSD-95 21 Figure 1-4. Schematic representation of NMDAR-associated protein complex 23 Figure 3-1. P K C activation with 2-BP treatment does not produce additive uncoupling 40 Figure 3-2. PKC activation alters desensitization in mature neurons 44 Figure 3-3. Change in desensitization in mature neurons not due to increased peak current but altered steady state 46 Figure 3-4. P K C activation does not alter desensitization in immature neurons 48 Figure 3-5. Expression of PSD-95 regulates glycine-independent desensitization of recombinant NMDARs 52 Figure 3-6. PDZ1-2 domains are sufficient to alter N M D A R desensitization; multimerization of PSD-95 and protein targeting to the membrane are not required in regulating glycine-independent desensitization 56 . Figure 3-7. Overexpression of PSD-95 in immature neurons decreases glycine-independent desensitization 59 vi ACKNOWLEDGMENTS Foremost, I would like to thank Dr. Lynn Raymond for her supervision, patience, and insight during the course of my Masters degree. Her helpful discussions and words of encouragement provided a nurturing environment to explore my scientific curiosity. I would also like to thank the past and present members of the Raymond and Murphy laboratory for enduring hours of monotonous bar graphs during lab meetings and in return offering indispensable advice and encouragement. I would like to thank Lily Zhang for her contributions to the biochemical aspects of this project. During the course of my study, I owe a lot to Dr. Austen Milnerwood and Herman Fernandes for the intellectually stimulating conversations that occurred during our electrophysiology meetings. A special notice of appreciation goes to Cristina Vasuta for her valuable contributions in wrapping up the loose ends of this project. I would also like to thank my supervisory committee of Dr. Y u Tian Wang, Dr. Ann Marie Craig, and Dr. Brian Christie for their input and guidance. Last, but not least, I would like to thank my family for their support and Aniko Adam for being my muse. vii CHAPTER I Introduction 1.1. Ionotropic glutamate receptor overview Ionotropic glutamate receptors are ligand-gated ion channels that mediate the majority of excitatory neurotransmission in the central nervous system. Three pharmacologically distinct classes of ionotropic glutamate receptors exist named after partially selective agonists: N -methyl-D-aspartic acid r, a-amino-3-hydroxy-5-methylisoxazole-4- propionic acid (AMPA) and kainate receptors. Cloning of genes encoding individual glutamate receptor subunits identified six gene families. There is one gene family for A M P A receptors - GluR 1,2,3,4; two for kainate receptors - GluR5,6,7 and KA-1,2; and three for NMDARs - NR1, NR2A,B,C,D and NR3A,B (Hollmann and Heinemann, 1994; Dingledine et al., 1999). .Similarity in sequence suggests a common evolutionary origin for all ionotropic glutamate receptor genes; however, only subunits within the same glutamate receptor type assemble to form functional receptors (Suchanek et al., 1995; Dingledine et al., 1999). The three ionotropic glutamate receptor classes share similar structural topology. There are three transmembrane domains ( M l , M3 and M4) and a re-entrant loop on the cytoplasmic facing membrane — M2. Preceding the M l and following the M3 regions are the SI and S2 domains respectively, which are responsible for agonist binding. The N-terminus is located extracellularly while the C-terminus resides intracellularly (Dingledine et al., 1999, Figure 1-1). Early studies of conductance states suggested that N M D A receptors existed as pentamers of three NR1 and two NR2 subunits (Premkumar and Auerbach, 1997; Dingledine et al., 1999), however, more recent studies propose that glutamate receptors are tetrameric complexes of two NR1 with two NR2, or a mix of one NR2 and one NR3, subunits (Dingledine et al., 1999). 1 The three classes of receptors display distinct responses in the presence of agonist (Dingledine et al., 1999). N M D A receptors display high affinity for glutamate, which binds to the NR2 subunits, and activation requires the co-agonist glycine, which binds to NR1 subunits (Figure 1-1). These receptors exhibit relatively slow activation, deactivation and desensitization. A M P A and kainate receptors show lower affinity for glutamate, along with faster activation, deactivation and desensitization than N M D A receptors. A M P A receptors generally have low calcium permeability unless they lack the GluR2 subunit (Hollmann et al., 1991; Ozawa, 1998; Dingledine et al., 1999). Kainate receptors show moderate calcium permeability, which is higher than that observed for A M P A receptors with GluR2 subunits. N M D A receptors display high calcium permeability and voltage dependent block by extracellular magnesium. These differences allow for robust responses with the single endogenous agonist glutamate, and the differing properties of these ionotropic receptors permit specialized roles in synaptic transmission. A M P A receptors mediate the fast synaptic response while also providing the depolarization required to alleviate the magnesium block of N M D A receptors. The slow kinetics and high calcium permeability of N M D A receptors are important in regulating synaptic plasticity under physiological conditions, while overactivation can result in excitotoxity (Dingledine et al., 1999). 2 Figure 1-1. Membrane topology of NMDARs The SI and S2 domains forms the binding sites for glutamate in the NR2 subunits and for glycine in the NR1 subunits. The M2 region is a re-entrant pore loop that confers permeability to Na + , K + and Ca 2 + . Extracellular M g 2 + causes voltage-sensitive block of the receptor by residing within the pore. Intracellularly, the C-terminal tail of both the NR1 and NR2 subunits are regulated by phosphorylation sites (Y,S) as well as association with various intracellular proteins denoted in grey. Adapted from Cull-Candy and Leszkiewicz, 2004. 3 1.2. NMDARs in synaptic plasticity Following high-frequency stimulation of the presynaptic fiber, N M D A receptor activation induces changes in the postsynaptic terminal that ultimately lead to prolonged increases in the efficiency of synaptic transmission. This modification of synaptic efficiency, termed long-term potentiation (LTP), occurs within milliseconds and persists for hours or days (Bliss and Lomo, 1973). During high frequency stimulation, presynaptic glutamate release activates A M P A receptors, which depolarize the postsynaptic membrane to alleviate the M g 2 + block of NMDARs allowing calcium to enter the neuron through the highly permeable NMDARs . Conversely, low frequency stimulation has been shown to decrease the efficiency of synaptic transmission in a process termed long-term depression (LTD). The importance of NMDARs in both LTP and LTD is supported by a variety of studies. Specific N M D A R antagonists block the induction of LTP without affecting basal synaptic transmission (Collingridge et al., 1983). The majority of synapses that undergo LTP do so in an N M D A R -dependent manner; however, rises in intracellular calcium induced by alternate sources, such as voltage-gated calcium channels, can mimic LTP (Grover and Teyler, 1990; Malenka and Nicoll, 1999). A great deal of evidence implicates that alpha -calcium-calmodulin-dependent protein kinase II (a-CaMKII) is a key component in NMDAR-dependent LTP (Malenka and Nicoll, 1999; Malenka and Bear, 2004). Postsynaptic injection of inhibitors of CaMKII or genetic deletion of a critical CaMKII subunit blocks the ability to generate LTP (Malenka et al., 1989). Paradoxically, N M D A R activation through low frequency stimulation can also lead to the induction of LTD at the same synapses that show NMDAR-dependent LTP (Malenka, 1994; Malenka and Bear, 2004). Many of the same requirements of LTP are found in LTD induction: 5 dependence on activation of AMPARs to alleviate Mg + blockade, activation of NMDARs, and rises in postsynaptic intracellular calcium levels (Dudek and Bear, 1992; Mulkey and Malenka, 1992; Malenka, 1994; Malenka and Bear, 2004). The critical components that determine whether LTP or L T D is induced are the presynaptic stimulation paradigm and the subsequent postsynaptic intracellular calcium levels. Studies suggest stimuli that induce brief, large increases in intracellular calcium (~3sec, ~10uM) activate CaMKII which leads to LTP, whereas stimulation protocols that induce prolonged, low levels of calcium (~lmin, <1|0.M) preferentially activate the serine-threonine phosphatase calcinuerin, leading to LTD (Dudek and Bear, 1992; Mulkey and Malenka, 1992; Malenka, 1994; Malenka and Bear, 2004). 1.3. NMDAR involvment in neuronal survival and excitotoxicity Numerous studies indicate that physiological activity of N M D A R activity is critical for neuronal survival. For example, in vivo blockade of N M D A receptors for only a few hours during late fetal or early neonatal life causes extensive apoptosis in many brain regions (Ikonomidou et al., 1999). Studies on cultured neurons suggest a neuroprotective role of synaptic N M D A R activity. Blockade of receptors in spinal cord cultures results in increased neuronal death caused by elimination of synaptic N M D A R activity (Brenneman et al., 1990). The mechanisms underlying synaptic NMDAR-induced neuroprotection are currently being investigated. Sub-toxic levels of agonist in vitro have been shown to mediate phosphorylation and subsequent inactivation of the pro-apoptotic protein B A D by both calcium-mediated activation of CaMKII and activation of the Ras-ERKl/2 pathway (Yano et al., 1998; Bonni et al., 1999; Brunet et al., 2001). Furthermore, synaptic activation of NMDARs in vivo results in alterations in gene expression that promote cell survival. Synaptic receptor stimulation, in both in vivo and in vitro systems, results in activation of the cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) which in turn increases the 6 transcription of several immediate early genes involved in neuronal survival (Hardingham and Bading, 2003). Although activation of NMDARs is critical for neuronal survival during development, N M D A receptors can cause cell death when intensely or chronically activated (Lipton and Rosenberg, 1994). This overactivation of NMDARs has been implicated in the etiology of many chronic neurodegenerative disorders, such as Huntington's disease, HIV-associated dementia and Alzheimer's disease ( Fan and Raymond 2007 - Progress in Neurobiol - Epub ahead of print; Lipton and Rosenberg, 1994; Lancelot and Beal, 1998; Kaul et al., 2001). Calcium entry through NMDARs activates multiple downstream pathways linked to cell death, including mitochondrial dysfunction, mitogen-activated protein kinase (MAPK) signaling, as well as calcium-activated kinases, phosphatases, and proteases (Lynch and Guttmann, 2002; Waxman and Lynch, 2005). Interestingly, NMDAR-mediated excitotoxicity appears to exhibit subunit specificity. For example, heterologous cells transfected with recombinant NMDARs show highest levels of death when expressing NR1/NR2A, followed by NR1/NR2B and finally NR1/NR2C (Waxman and Lynch, 2005). This trend is also seen developmentally in cultured neurons. As NR2B levels decrease and NR2A levels increase in neuronal development, toxicity due to N M D A R activation also increases (Waxman and Lynch, 2005). However, recent studies in acute hippocampal slices show increased NMDAR-mediated toxicity in slices from young rats compared to older animals. The authors conclude that the majority of the NMDA-mediated toxicity is caused by activation of NR2B- but not NR2A-containing N M D A receptors leading to calpain activation (Zhou and Baudry, 2006). This seemingly contradictory report may be reconciled in part by the differences in downstream signaling due to subunit dependence and subcellular localization. NR2B-containing receptors predominate at extrasynaptic sites later in development and are implicated in N M D A receptor-dependent CREB shut-off signaling pathways (Li et al., 1998; Hardingham and Bading, 2002; Hardingham et al., 2002). 7 1.4. NMDAR structural determinants of function 1.4.1. NMDAR subunit composition NMDARs are heteromeric protein complexes incorporating 3 types of subunits: NR1, NR2 and NR3. The NR1 subunit is essential in forming a functional channel and is encoded by a single gene with three alternatively spliced exons (5, 21, 22) giving rise to 8 possible isoforms (Figure 1-2). There are four possible NR2 subunits (A, B, C, and D) which are encoded by 4 different genes (Dingledine et al., 1999). NR2 subunits are important in regulating channel properties (Dingledine et al., 1999). NR3 A and B subunits are encoded by two separate genes (Chatterton et al., 2002). Expression of functional recombinant NMDARs in mammalian cells requires the co-expression of NR1 and NR2 subunits. The stoichiometry of NMDARs has not yet been established definitely, but the consensus is that NMDARs are tetramers that most often incorporate two NR1 and two of the same or different NR2 subunits (referred to as diheteromers or triheteromers, respectively) (Dingledine et al., 1999). Neurons express distinct complements of subunits, which are temporally and spatially regulated. There is evidence for expression of NMDARs that are triheteromeric combinations of NR1 with NR2A and NR2B in cerebellar granule cells and hippocampal pyramidal neurons, NR1 with NR2A and NR2C in cerebellar granule cells, and NR1 with NR2B and NR2D in cerebellar Golgi and stellate cells (Cull-Candy and Leszkiewicz, 2004). The role of NR3 subunits is still under investigation; however, recent evidence suggests NR3 may modulate receptor activity by forming triheteromeric receptors that display decreased N M D A R unitary conductance, decreased C a 2 + permeability, reduced M g 2 + sensitivity, and slightly increased mean open time compared with NR1/NR2 channels (Ciabarra et al., 1995; Das et al., 1998; Dingledine et al., 1999; Sasaki et al., 2002). 8 N M D A receptors require binding of both glutamate and glycine to activate; there is one site on the NR1 subunit that binds glycine as a full agonist and D-serine as a partial agonist, and another site on the NR2 subunit that binds glutamate as a full agonist and N M D A as a partial agonist (Dingledine et al., 1999; Gibb, 2004) (Figure 1-1). NMDARs show negative cooperativity between binding of glutamate and glycine, which manifests as glycine-depedent desensitization. However, expression of NR1 together with NR3 A or B subunits results in the formation of excitatory receptors that utilize only glycine as an agonist and are unaffected by glutamate or N M D A ; these receptors are inhibited by D-serine, a co-activator of conventional NMDARs (Chatterton et al., 2002). 9 Figure 1-2. Alternative splice variants of NMDAR NR1 subunit Alternative splicing of exons 5, 21, and 22 gives arise to 8 possible isoforms which contain different combinations of the N l , C I , C2, and C2' cassettes. From Dingledine et al., 1999 10 N1 M1 M2 M3 CZMZD=€Z> C1 C2 C2' . . asa ss domains a NR1-1 b a NR1-2 b a NR1-3 b a NR1-4 b Exon 5 Exons 21 (Dingledine, et al., 1999) 11 1.4.2. NMDAR subunit composition and spatial-temporal distribution in the CNS The eight splice variants of NR1 show regionally specific expression and do not exhibit much change during development except in the cerebellum (Laurie and Seeburg, 1994; Laurie et a l , 1995; Prybylowski and Wolfe, 2000). In the developing rat brain, NR1 mRNA is barely detectable at embryonic day 14, but increases gradually during development until the third postnatal week, after which it declines slightly to adult levels (Laurie and Seeburg, 1994). The NR1-1 and NR1-2 variants which lack the C2' cassette (see Figure 1-2), are widely and abundantly distributed throughout the brain, except for the inferior colliculus (Laurie et al., 1995). N R l - l b is highly expressed in the sensorimotor cortex, neonatal lateral caudate, thalamus, hippocampal CA3 field, and cerebellar granule cells, but is absent from adult caudate. The NR1-1 and NR1-4 splice variants display complimentary patterns; the NR1-4 splice forms are present in caudal regions such as the thalamus, colliculi and cerebullum whereas the NR1-1 variant is present in rostral structures such as cortex, caudate and hippocampus (Laurie and Seeburg, 1994). The NR1-3 mRNA variant is sparsely expressed in the central nervous system, where very low levels exist in postnatal cortex and hippocampus (Laurie and Seeburg, 1994). The four NR2 transcripts display distinct expression patterns (Cull-Candy et al., 2001; Cull-Candy and Leszkiewicz, 2004). The NR2A mRNA is distributed widely in the brain but predominates in the cerebral cortex, hippocampus, and cerebellum. In the adult brain, the NR2B subunit is selectively present in the forebrain, where it is highly expressed in the cerebral cortex, hippocampus, septum, caudate-putamen and olfactory bulb. NR2C mRNA is expressed predominantly in the granule cell layer of the cerebellum although lower levels are also detected in the olfactory bulb and thalamus. NR2D transcripts are detected at low levels in the thalamus, brain stem and olfactory bulb. In hippocampal neurons, NR2A and NR2B mRNAs are 12 prominent however NR2C and NR2D transcripts are also found in subsets of interneurons (Monyer et al., 1994; Ozawa et al., 1998). The expression of NR2 subunits is also developmentally regulated. In the developing rat brain, NR2B and NR2D mRNAs occur prenatally, whereas NR2A and NR2C mRNAs are first detected near birth. Expression of all NR2 subunit mRNA peaks around postnatal day 20 except NR2D. NR2D mRNA is present predominantly in midbrain structures and peaks around P7, then decreases to adult levels (Monyer et al., 1994). In vitro studies have shown a developmental shift in subunit expression in hippocampal neurons. Hippocampal neurons tend to express NR2B subunits early in development; these NR2B-containing receptors are present at the earliest synapses, but predominate at non-synaptic. As the neurons mature, NR2A is incorporated into synapses to form homomers of NR1/2A receptors or triheteromeric NR1/2A/2B receptors whereas homomeric NR1/2B receptors predominate at extrasynaptic sites (Williams et al., 1993; Zhong et al., 1994; L i et al., 1998; Rao et al., 1998; Stocca and Vicini, 1998; Tovar and Westbrook, 1999; L i et a l , 2002; L i et al., 2003). However, NR2A-containing receptors can still be found at non-synaptic sites and NR1/NR2B at synapses, and definite determination of the subcellular distribution of N M D A R subunits is complicated by the limitations of the available pharmacological tools (Vicini et al., 1998; Tovar and Westbrook, 1999; Thomas et al., 2006). 1.4.3. NMDAR regulation by NR1 subunits The NR1 splice variants modulate N M D A R properties. The pH sensitivity of NMDARs is determined by the presence of exon 5; at physiological pH NR1 subunits that contain exon 5 are fully active while NR1 subunits lacking exon 5 are partially blocked. NR1 subunits containing exon 5 also display no potentiation by polyamines or voltage-dependent block by zinc (Traynelis et al., 1995). However these effects are also dependent on the identity of the NR2 subunits present in the N M D A R complex. Assembly of NR1 splice variants that lack exon 5 13 with NR2C or NR2D subunits produces NMDARs that are insensitive to zinc blockade or proton inhibition (Paoletti et al., 1997; Traynelis et al., 1998). Exon 5 of NR1 can also regulate the deactivation of NMDARs containing NR2B subunits. The presence of exon 5 increases the deactivation rate of recombinant NR1/2B receptors, whereas exon 5 has no effect on NR1/2A receptors (Vicini et al., 1998; Rumbaugh et al., 2000). The NR1 subunit also plays a critical role in the trafficking of NMDARs from the ER to the surface membrane. NR2A and NR2B subunits are unable to reach the cell surface unless assembled with NR1, yet most splice variants of NR1 can reach the cell surface without NR2 subunits present (Fukaya et al., 2003). The trafficking of NR1 subunits from the ER is regulated by splice variants. ER export is accelerated by NR1 subunits containing the C2' domain and slowed by NR1 subunits expressing the C2 cassette (Mu et al., 2003). Moreover, the process of forward trafficking from the ER to the cell surface is tightly regulated by coordinated P K A and P K C phosphorylation of the sites flanking the R X R motif within the CI cassette of NR1 subunits (Scott et al., 2001; Scott et al., 2003). Additionally, NR1 splice variants display selective targeting to specific cell regions (Mu et al., 2003). 1.4.4. NMDAR regulation by NR2 subunits Many of the subunit-dependent properties of NMDARs are controlled by the NR2 subunit. NR2 subunits have been shown to modulate agonist affinity, N M D A R single-channel conductance, channel open probability, sensitivity to zinc inhibition, block by extracellular Mg2+, and channel kinetics (Dingledine et a l , 1999; Cull-Candy et al., 2001; Cull-Candy and Leszkiewicz, 2004). Diheteromeric NR1/NR2A receptors show lower sensitivity for glutamate compared to NR1/NR2B receptors but display a higher open probability. NR2A- or NR2B-containing NMDARs generate 'high-conductance' channel openings with a high sensitivity to 2+ block by Mg , whereas NR2C- or NR2D-containing receptors give rise to 'low-conductance' 14 openings with a lower sensitivity to extracellular Mg + (Dingledine et al., 1999; Cull-Candy et al., 2001; Cull-Candy and Leszkiewicz, 2004). Further differences in NR2 subunits with regard to agonist affinity and various forms of desensitization are discussed later. There is much evidence suggesting populations of NMDARs may exist as two NR1 subunits and two different NR2 subunits to produce a functionally distinct triheteromeric receptor complex (Vicini et al., 1998; Tovar and Westbrook, 1999). Transfection of NR1, NR2A and NR2B subunits into recombinant systems results in receptors that display reduced ifenprodil sensitivity, distinguishing them from homomeric NR1/NR2B receptors that are inhibited by low micromolar concentrations of ifenprodil and infenprodil-insensitive NR1/NR2A receptors (Tovar and Westbrook, 1999). The presence of triheteromeric receptors that exhibit distinct functional and pharamacologial properties increases the diversity of signaling available in the central nervous system. 1.4.5. NR3 Subunits The recently identified NR3 subunits (A and B) co-assemble with NR1 to form diheteromeric functional excitatory glycine receptors that are unaffected by glutamate or N M D A , inhibited by D-serine, relatively calcium impermeable, and also insensitive to magnesium block, the pore blocker MK-801 or competitive antagonist memantine (Chatterton et al., 2002). Triheteromeric NR1/NR2/NR3 receptors exist early in development and regulate NMDARs by decreasing unitary conductance. Knockout mice lacking the NR3A gene display enhanced N M D A responses and increased dendritic spines in early postnatal cerebrocortical neurons (Das et al., 1998). 15 1.5. NMDAR regulation by serine/threonine phosphorylation Most phosphorylation sites on glutamate receptors have been shown to be on the intracellular C-terminal domain (Swope et al., 1999). NMDARs can be phosphorylated by P K A , PKC, casein kinase-2 (CK2) and CaMKII. The balance of phosphorylated and unphosphorylated states is maintained by the aforementioned kinases' activity and the calcium/calmodulin-dependent phosphatase calcineurin. Twelve isoforms of P K C have been discovered and are categorized into three structurally and functionally distinct groups. Conventional cPKC isoforms (PKCa, PKCp and PKCy) are diacylglycerol sensitive and responsive to calcium. The novel nPKC isoforms (PKC5, PKCe, PKCr | and PKC0) are sensitive to diacylglycerol but calcium insensitive. The atypical aPKC isoforms (PKC^ and PKCt/X) are insensitive to both calcium and diacylglcerol, and are regulated by the PB1 domain of their N-terminus (Battaini, 2001; Parker and Murray-Rust, 2004). P K C activity has varied and profound effects on N M D A R function. P K C phosphorylation has been shown to increase open probability, decrease the external magnesium block of NMDARs , inhibit clustering of receptors, regulate release of receptors from the endoplasmic reticulum and promote surface expression (Chen and Huang, 1992; Kelso et al., 1992; Ehlers et al., 1995; Tingley et al., 1997; Dingledine et al., 1999; Lu et al., 1999; Zheng et al., 1999; Lan et al., 2001; Scott et al., 2001; Scott et al., 2003). Various metabotropic receptors have been shown to potentiate N M D A R current through P K C activation (Markram and Segal, 1990; Aniksztejn et al., 1992; Chen and Huang, 1992; Dildy-Mayfield and Harris, 1994). However, P K C augmentation of N M D A R current does not completely depend on phosphorylation of the receptor itself as experiments involving NMDARs assembled from subunits lacking all known sites of P K C phosphorylation can still exhibit P K C potentiation (Zheng et al., 1999). These data suggest that PKC-induced potentiation of N M D A R activity does not occur by direct phosphorylation of the receptor protein but rather through downstream 16 signaling. In support of this, PKC potentiation of N M D A R current is absent in neurons from knockout mice lacking src kinase, suggesting that the effect of PKC is through tyrosine phosphorylation (Lu et al., 1999). Trafficking and clustering of NMDARs subunits are heavily regulated by P K C activity. P K C activation and subsequent phosphorylation of Ser890 of the C I cassette of NR1 can inhibit clustering of NR1 (Ehlers et al., 1995; Tingley et al., 1997). NR1 subunit phosphorylation also regulates forward trafficking of NMDARs from the ER; PKC phosphorylation sites flanking the R X R motif of NR1 subunits expressing the CI cassette suppress ER retention (Scott et al., 2001; Scott et al., 2003). As well, activation of PKC allows for the rapid delivery of NMDARs from vesicular, plasma membrane-proximal pools in a SNARE-dependent, exocytotic manner resulting in increased surface expression (Lan et al., 2001). Compared to the work on PKC modulation of NMDARs, little is known about P K A , CK2, and C A M K I I phosphorylation of NMDARs . The NR1 CI cassette contains a P K A phosphorylation site that is involved in forward trafficking from the ER. Coordinate phosphorylation of sites flanking the R X R motif of NR1 promotes release of receptors from the ER for trafficking to the plasma membrane. In hippocampal neurons P K A activation by 0-adrenergic receptors potentiated N M D A R activity indirectly through calcineurin inhibition (Raman et al., 1996). CK2 activity can potentiate N M D A R responses by increasing N M D A R channel open probability, although whether these effects are through direct phosphorylation of NMDARs has yet to be determined (Lieberman and Mody, 1999). On the other hand, CK2 phosphorylation of site 1480 in NR2B reduces interaction with PSD-95 and other PDZ domain-containing proteins (see next section), decreasing N M D A R surface expression (Chung et al., 2004). Along with kinase activity, the level of serine/threonine phosphorylation is also controlled by activity of phosphatases PP1, PP2A or PP2B. Inhibition of PP1 and/or PP2A with 17 the use of the specific inhibitor calyculin A occluded the PKA-mediated potentiation of striatal N M D A R current (Blank et al., 1997). Calcineurin activity has been shown to down-regulate N M D A R activity through desensitization at the C-terminus of the NR2A subunit in a dephosphorylation dependent manner (Tong et al., 1995; Krupp et al., 2002). 1.6. NMDAR regulation by MAGUKs The NR2 subunits of NMDARs contain intracellular C-terminal domains that are critical for signaling and anchoring of NMDARs. One crucial feature of the NR2 C-terminus in regulating N M D A R function is the terminal, conserved sequence ESDV of NR2A and NR2B, and ESEV for NR2C and NR2D. These sequences are known ligands for PSD-95/Dlg/ZO-l (PDZ) domains which are modular protein interaction domains specialized for binding to short peptide motifs in over 400 proteins in mice and humans (Kim and Sheng, 2004). Typically, the PDZ scaffold and its associated multiprotein complex are targeted to a specific subcellular site to perform a specialized local function. The PDZ domain was originally recognized in three proteins, postsynaptic density 95kDa (PSD-95), ZO-1 and Dig that are all members of a superfamily of membrane associated guanylate kinases (MAGUKs). The PSD-95 family of M A G U K s includes PSD-93/Chapsyn-110, synapse associated protein 102 kDa (SAP-102), and synapse associated protein 102 kDa (SAP-97). These proteins are roughly 70% homologous to each other, and all contain three N-terminal PDZ domains, an SH3 domain and a C-terminal guanylate kinase-like domain (Kim and Sheng, 2004). 1.6.1. SAP-102 Studies from intact brains early in development suggest expression of PSD-95 and PSD-93 are low in the hippocampus whereas SAP-102 is highly expressed at synapses (Sans et al., 2000). The high level of SAP-102 at immature synapses coincides with the early developmental 18 expression of the NR2B subunit, whereas the later expression of NR2A coincides with the prevalence of PSD-93 and PSD-95 at synaptic sites. Furthermore, co-immunoprecipitation studies suggest a preference for NR2A-containing receptors in complexes with PSD-95 or PSD-93 whereas SAP-102 is largely associated with NR2B-containing receptors (Sans et al., 2000). In support of SAP-102's role in developing synapses, acute knockdown in immature hippocampal slices resulted in a 50% reduction of A M P A R EPSCs while acute knockdown in mature slices caused no noticeable alterations in synaptic transmission (Elias et al., 2006). SAP-102 may also play a role in trafficking NMDARs from the endoplasmic reticulum and golgi apparatus to the synapse in a PDZ dependent manner as a part of an exocyst complex (Sans et al., 2003). 1.6.2. PSD-95 PSD-95 is the best characterized of the M A G U K family of proteins and has been shown to have varied effects in synaptic regulation. Deletion mutagenesis studies established that the N-terminal region of PSD-95, containing palmitoylated Cys-3 and Cys-5, regulates both membrane targeting and multimerization (Craven et al., 1999; Hsueh and Sheng, 1999; Christopherson et a l , 2003). Expression of PSD-95 in non-neuronal cell lines has been shown to cluster Shaker type K + channels and reduces the single-channel conductance of the Kir2.3 inward rectifier K + channnel (Kim et al., 1995; Kim and Sheng, 1996; Nehring et al., 2000). PSD-95 regulates A M P A receptor clustering in neurons although it is not required for proper N M D A receptor trafficking and clustering (El-Husseini et al., 2000). Evidence suggests PSD-95 is involved in surface delivery of NMDARs, expression of NMDARs, and stabilization of NR2B-containing receptors at synaptic sites (Lin et al., 2004; Prybylowski et al., 2005). Additionally, PSD-95 has been implicated in regulating glycine-independent desensitization; overexpression of PSD-95 in immature neurons results in a decrease in desensitization, and 19 conditions that shift either PSD-95 or N M D A receptors away from synaptic sites in mature neurons result in increased N M D A R desensitization (Li et al., 2003). Despite the large body of evidence suggesting a role for PSD-95 in regulating N M D A R receptor function, overexpression of PSD-95 selectively enhances A M P A R mediated EPSCs while NMDAR-mediated EPSCs remain unaltered (Schnell et al., 2002; Rumbaugh et al., 2003; Elias et al., 2006). Furthermore, PSD-95 knockout mice show no alterations in synaptic transmission (Elias et al., 2006). These surprising results may partly be explained by functional redundancy and compensation by other members of the M A G U K family of proteins; acute knockdown of PSD-95 results in a reduction of A M P A R mEPSC frequency similar to the PSD-95/PSD-93 double knockout mice (Elias et al., 2006). Problems arise when isolating whether the effects of PSD-95 are through a direct interaction or are secondary to its role as a scaffolding molecule. Figure 1-3 displays a non-exhaustive list of PSD-95 binding partners and to which domains they interact with. The complexity that arises in the neuronal post-synaptic density from the wide network of molecules PSD-95 can recruit and interact with causes difficulty in directly assessing the role of PSD-95 in a neuronal preparation (Figure 1-4). 20 Figure 1-3. Table of proteins that interact with PSD-95 By binding polypeptides through its various domains, PSD-95 can assemble large macromolecular complexes. From Kim and Sheng, 2004. Table 1 Proteins that interact with PSD-95 family scaffolds Interacting protein Comments on the interacting proteins References PDZ domains NR2A-D Subunits of NMDA receptors 131 GluR6 Subunit of kainate receptors 132 82GluR Subunit of 6-tonotropic glutamate receptors 133 |31 -adrenergic receptor G-protein-coupled receptor 25 nAChRc Subunit of neuronal nicothic acetylcholine receptor 76,77 5-HT2Aand 5-HT2C Rc Subunits of 5-HT (serotonin) receptors 134 ErbB4 A receptor tyrosine kinase for neuregulin 135,136 Kv1 Voltage-gated potassium channel 14 Kir2, Kir3, K M and Kir5 Inward-rectifying potassium channels 32,124 Neurol igin A postsynaptic membrane protein that binds to |3-neurexins and regulates synaptic adhesion and development 26,28.30 Stargazin family proteins Tetra-spanning transmembrane proteins required for surface and synaptic expression of AMPA receptors 64 nNOS Neuronal nitric oxide synthase 3,33 SynGAP An abundant RasGAP of the PSD that regulates synaptic plasticity 35,36.39,40 Kalirin-7 A guanine nucleotide exchange factor for Rac1 that regulates spine morphogenesis 53 Fyn, Lyn, Src and Yes Src family non-receptor protein tyrosine knases; might also interact /vith the SH3 domain of PSD-95 48,49 Cypin A cytosolic protein that regulates dendrite patterning by promoting microtubule assembly 137 CRIPT A microtubule-binding protein 18 Sec8 A subunit of the exocyst complex involved in protein and vesicle trafficking 115 KIFIBa A motor of the kinesin superfamily 111 SH3 domain Pyk2 A non-receptor tyrosine kinase regulated by calcium and PKC and required for LTP induction 50.138 GK domain GKAP/SAPAP An abundant multi-domain scaffold of PSD that links PSD-95 with Shank 139 SPAR A postsynaptic RapGAP that regulates spine morphogenesis 57 SH3 and GK domains KA2 GluR Subunit of kainate receptors 132 AKAP79/150 An anchoring protein that binds to protein kinase A and protein phosphatase 1 41 121domain CASK Mammalian homologue of LIN2 140.141 Myosin VI A minus-end-directed actin-based motor 113 Only proteins that interact directly vjith PSD-95 family scaffolds are listed. These interactions might not apply to all members of the PSD-95 family. Owing to space limitations, this list is not comprehensive and not all relevant references are cited AMPA, u-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; GK. guanylate kinase-like domain; LTP, long-term potentiation: NMDA, W-rnethyl-L-aspartate; PKC. protein kinase C; PSD-95, postsynaptic density protein 95; Rac. Rap and Ras, small monomelic G-proteins; RapGAP, Rap GTPase-actwating protein; RasGAP, Ras GTPase-actK'ating protein; SH3 domain, Src homology 3 domain; Shank, SH3 and ankyrin repeat-containing protein. (Kim and Sheng, 2004) Figure 1-4. Schematic representation of NMDAR-associated protein complex Schematic representation of NMDAR-associated protein complex. The cytoplasmic tails of NMDARs also bind directly to cytoskeletal and signaling proteins, including oc-actinin, calmodulin and PSD-95. PSD-95 interacts with a multitude of cytoplasmic signaling molecules thereby connecting NMDARs to multiple signal transduction pathways. From Sheng and Kim, 2002. 23 24 1.6.3. SAP-97 and PSD-93 Much less is known about SAP-97 or PSD-93 in the regulation of receptor function. SAP-97 has been found to be present in both axons and postsynaptically at excitatory synapses (Muller et al., 1995; Valtschanoff et al., 2000; Aoki et al., 2001; Rumbaugh et al., 2003). The differential targeting of SAP-97 is accomplished through splice variations; SAP-97 expressing the 13 splice insert target preferentially to postsynaptic spines whereas splice forms lacking this motif are localized diffusely (Rumbaugh et al., 2003). Overexpression of synaptically targeted SAP-97 in neurons results in altered spine morphology, increased surface A M P A receptors, and increased A M P A R mEPSC frequency (Rumbaugh et al., 2003). SAP-97 is unique in that it is the only M A G U K to bind directly to A M P A R through a PDZ interaction with the GluRl subunit (Leonard et al., 1998). PSD-93 is homologous to the previously described M A G U K s but displays some unique characterisitics. PSD-93 is largely localized to post-synaptic densities although it has been detected utilizing electron microscopy diffusely on pre-synaptic membranes, in the cytoplasm of axons and in dendritic shafts (Sans et al., 2000; Aoki et al., 2001). Although PSD-93 is N -terminally palmitoylated, it does not require palmitoylation for membrane targeting (Firestein et al., 2000). Overexpression of PSD-93 results in an increase in AMPAR-mediated evoked EPSC amplitude while leaving NMDAR-mediated EPSCs unaffected in cultured hippocampal neurons (Schnell et al., 2002; Elias et al., 2006). However, PSD-93 knockout mice show decreased surface expression of NR2A and NR2B subunits, reduced NMDAR-mediated mEPSCs and attenuated evoked NMDAR-dependent EPSCs without altering A M P A R surface expression or synaptic function in neurons from the spinal dorsal horn and forebrain (Tao et al., 2003). 25 1.7. NMDAR regulation by glycine-dependent desensitization In the continuous presence of glutamate (or N M D A ) and low concentrations of co-agonist glycine, N M D A R current responses diminish over time. This form of desensitization is indicative of the negative allosteric coupling between the glutamate binding sites of the NR2 subunits and the glycine binding sites on the NR1 subunits, and manifests as a decrease in glycine affinity. This form of desensitization can be overcome by high glycine concentrations, on the order of 50 uM (Mayer et al., 1989; McBain and Mayer, 1994; Dingledine et al., 1999; Gibb, 2004). 1.8. NMDAR regulation by calcium-dependent inactivation Intracellular calcium can reversibly inhibit N M D A R receptor activity through the process of calcium-dependent inactivation (CDI) (Rosenmund and Westbrook, 1993). CDI is sensitive to influx of calcium through the N M D A R and it can be abolished by high intracellular calcium buffering (Rosenmund and Westbrook, 1993). Recombinant N M D A receptor studies have shown CDI to be NR2 subunit dependent; inactivation of recombinant N M D A receptors requires either the NR2A or NR2D subunits, whereas inactivation of diheteromeric NR1/NR2B or NR1/NR2C receptors does not occur (Krupp et al., 1996). The mechanism for CDI relies on the calcium binding protein calmodulin, which interacts with the NR1 subunit near the membrane-proximal region (also called "CO") when bound to calcium (Wyszynski et al., 1997; Zhang et al., 1998). The inactivating effect of calmodulin is prevented by overexpressing a region of the cytoskeletal protein a-actinin known to interact with the CO region of NR1 (Zhang et al., 1998). a-actinin anchors NMDARs to F-actin, which when depolymerized, has been shown to inhibit N M D A R channel activity in a calcium- and ATP-dependent manner (Rosenmund and Westbrook, 1993; Zhang et al., 1998). Furthermore, calcium can bind directly to a-actinin and decrease its affinity for NR1 binding (Krupp et al., 1999). Together, these data suggests that the 26 displacement of a-actinin by calcium/calmodulin leads to CDI by disrupting N M D A R anchoring to the actin cytoskeleton. This process can be modulated by second messenger systems, P K C activity, and calcineurin activity, and is thought to be important in regulating synaptic transmission (Tong et al., 1995; Dingledine et al., 1999; Lu et al., 2000). 1.9. NMDAR regulation by glycine-independent desensitization Glycine-independent desensitization is characterized by the decay in N M D A R current during sustained, saturating concentrations of both agonists. Glycine-independent desensitization is NR2 subtype-dependent; NR2A and NR2B subtypes exhibit this form of desensitization whereas NR2C and NR2D subtypes do not (Villarroel et al., 1998). The structural determinants of glycine-independent desensitization were proposed to be located on the N terminus of the NR2 subunits. It has been suggested that two regions in particular of the NR2 subunits are critical for two distinct components of glycine-independent desensitization. Mutations of the pre-Ml region of NR2A selectively eliminated the slow component of desensitization while substitution of the amino terminal domain (ATD) of NR2A with the corresponding region of NR2C eliminated the fast component of glycine-independent desensitization (Krupp et al., 1998; Villarroel et al., 1998). However, recent studies suggest these two domains, are involved in zinc-dependent desensitization and not glycine-independent desensitization. The fast component of desensitization of NMDARs, previously ascribed to glycine-independent desensitization, is caused by a negative allosteric interaction between the zinc binding site in the ATD and the glutamate binding site in the S1/S2 domain rather than true glycine-independent desensitization (Zheng et al., 2001). In a follow-up study, alterations in either the pre-Ml or A T D sites were found to be critical for the component of desensitization that is caused by ambient zinc acting at the extracellular, high affinity site of the NR2A subunit. However, mutation of the lurcher motif, located near the extracellular end of the M3 region, of 27 either NR1 or NR2 subunits reduces glycine-independent desensitization as well as proton sensitivity and the deactivation rate (Hu and Zheng, 2005). The role of the lurcher motif in desensitization may be a common feature of all ionotropic glutamate receptors; recent studies have shown mutations in analogous regions of the GluRl A M P A receptor subunit alter the desensitization properties as well (Klein and Howe, 2004). 1.10. Hypothesis The extent of glycine-independent N M D A R desensitization significantly decreases over the course of neuronal development when comparing N M D A R current recorded from cultured immature neurons (4-7 days in vitro) to that in mature neurons (14-23 days in vitro) (Li et al 2003). Several lines of evidence indicate that the decrease in desensitization with development is correlated with the localization of NMDARs to the synapse and their association with PSD-95 (Li et al 2003). Over-expression of PSD-95 in immature neurons decreased the amount of N M D A R desensitization. Chronic treatment of mature neurons with picrotoxin, which causes chronic N M D A R activation by decreasing inhibitory input, leads to a decreased number of synaptic NMDARs while PSD-95 distribution remains unaffected (Crump et al., 2001). Treatment of mature neurons with 2-bromopalmitate results in PSD-95 dispersion from the synapse by inhibiting palmitoylation while N M D A receptor clustering remains intact. In both conditions, N M D A R desensitization in mature neurons increased to levels found in immature neurons (Li et al., 2003). However, it is not clear whether the effect of PSD-95 is secondary to its role as a scaffolding protein, anchoring protein kinases and phosphatases in close proximity to NMDARs , or if PSD-95 binding to NMDARs could directly alter receptor desensitization. In this thesis, I test the hypothesis that direct binding of NMDARs to PSD-95 and not synaptic localization is important in regulating glycine-independent desensitization. 28 CHAPTER II Materials and Methods 2.1. Neuronal cell culture and transfection 2.1.1. Culture Embryonic hippocampal culture was prepared based on a protocol modified from that described previously (Brewer et al., 1993; Brewer, 1995). The detailed protocol is described at below. Solutions 1. Poly-D-lysine (PDL, Sigma P-6407): 50ug/ml. 2. Eagle's balanced salt solution (EBSS) (1 L): EBSS salts (Sigma E-6132) 8.7 g, glucose (Sigma G-5767) 2.01g, NaHC03 (Sigma S-5761) 2.2 g. 3. EBSS+ (13.3 ml): EBSS solution 13.3 ml, L-cysteine HC1 2.34 mg, EDTA (50 mM) 0.133 ml. 4. Papain (Sigma P-3125) solution: 20 U/ml (diluted with EBSS+). Papain solution was kept in 37°C until clear, and oxygenated for 3 min just before use. 5. 10/10 solution (20 ml): EBSS 20 ml, BSA (Sigma A-3350) 200 mg, ovomucoid (trypsin inhibitor, Sigma T-9253) 200 mg. The solution was made just before use and kept 37 °C for 1 hr. 6. DNAase stock solution (1 ml): DNAase (Sigma DN25) 5 mg, EBSS 1 ml. The stock solution was stored at -20 °C. 7. DNAase working solution in (5 ml): EBSS 4.5 ml, 10/10 solution 0.5 ml, DNAase stock solution 100 ul. This solution was made immediately before use. 8. 2-mercapto-ethanol (BME, Sigma, M-7522) stock (10 ml): B M E 7.2 ul, Neurobasal mediumlO ml. 29 9. Plating and feed medium (500 ml): B27 (Gibco 17504-044) 10 ml, glutamine 0.5 mM, penicillin/streptomycin (10 U/ml), B M E 1.25 ml, Neurobasal Medium (NBM, Gibco 21103-049) 500 ml. Procedure 1. The following solutions were prepared (all sterile) before making culture. a. poly-D-lysine (PDL): 50ug/ml b. sterile Mil l i -Q H20 c. ethylenediaminetetraacetic acid (EDTA) stock (50 mM) d. Phosphate buffered solution (PBS e. EBSS f. 10/10 g. DNAase (5mg/ml in EBSS, stored at -20 °C) h. Plating medium (keep no longer than 14 days) 2. The plates (24-well-plate, with coverslips in it) were coated with 50|xg/ml PDL overnight. The PDL working solution was diluted from the stock solution with sterile Mil l i -Q H20. 3. PDL was aspirated from the plate. The plates were air-dried for a couple of hours. 4. Plating medium was prepared (if not made the day before). 5. The following materials were prepared in the dissection hood: a. petri dishes (100, 65 & 35mm) b. conical tubes (50 & 15 ml) c. syringes (30 & 10 ml) & syringe filters (0.22 um Millex-GV low-binding) d. dissection tools (in 70% ethanol for sterilization) e. under-pad for initial dissection f. cold PBS 30 6. Papain solution (15 ml) was made at a concentration of 20 U/ml in EBSS+. The solution was kept in 37 °C water bath until clear (~30min), and was then oxygenated for 3 min. The solution was filtered (with 0.22 um filter) to sterilize before use. 7. While the papain solution was heating, the anesthetized 17-18-day gestation rat was decapitated. The dissection was immediately performed in the dissection hood. 8. Dissection: a. The rat was put on a fresh under-pad in the hood, with ventral side up. The abdomen was sprayed well with 70% ethanol to sterilize. b. Cold PBS was prepared in two 10-cm plates, with each about half full. c. The skin of the abdomen was lifted using heavy forceps, and was cut open using heavy scissors. d. The uterus was lifted using heavy forceps and was cut free at the base and from the mesenteries. The uterus was then placed in a dish of cold PBS. e. The uterus was cut open, and fetuses were removed (without placenta) to another dish of cold PBS. f. Cold PBS was prepared in two 65-mm dishes. g. Fetuses were decapitated, and the heads were placed in one65-mm dish with fresh PBS, rinsed and transferred to another dish of PBS. h. Additional cold PBS was prepared in several 35-mm dishes, which were placed on ice. i . Brains were removed using two pairs of fine forceps. To do this, the head was oriented with the crown up and nose to left (if you are right-handed). The points the left hand forceps were placed into the eye sockets to hold the head steady. One prong of the right hand forceps was used to slit the skull along centerline in the nose-to-nape direction. Flaps of the skull were peeled back, and the brain was scooped out using the closed tips of the right hand forceps. The brains were placed in one of the 35-mm dishes with cold PBS (on ice). 31 j . The hippocampi were removed under a dissection microscope, and were placed in another 35-mm dish of cold PBS on ice.. 9. Digestion: a. Hippocampi were added to the sterile, warm papain solution, and were triturated for 2-3 times with 10 ml pipette to break up the tissue. b. The hippocampi were then incubated for 5-10 min at 37 °C water bath with mixing every 3 min. c. The tissue was gently centrifuged for 2-3 min by (1000 rpm) in one 15 ml tube. d. DNAase working solution was prepared while spinning. The stock DNAase aliquots were used directly from the -20 °C freezer. 200 ul of DNAase stock solution was diluted into 5 ml of 1/1 solution, which was diluted 10 times from the 10/10 solution. The solution was filtered to sterilize. e. The hippocampi pellet was resuspend in the DNAase solution. A 10-ml pipette was used to triturate the tissue for 4-5 times, and then a fire-polished, cotton-plugged Pasteur pipette was used to triturate for about ten times until tissue was broken up (do not over-triturate or make a lot of air bubbles). f. 3-5 ml of 10/10 solution was added to the tube to create gradient (drip 10/10 solution slowly down along the side of a slanted tube). g. Cells were gently centrifuged to form pellet (1000 rpm, 30 seconds). 10. The supernatant solution was removed from the tube without the pelleted cells intact. The pellet was washed once gently with 5 ml of plating medium. 11. The pellet was resuspended in 6 ml of plating medium and cells were counted using a hemacytometer. A l l living cells in the 16 squares (4X4 bounded by triple lines) were counted. The number of cells/ml = the number of cells in the 16 squares X 25 X 104. 32 12. The cells were plated on the dry, PDL-coated coverslips in the 24-well-plate at a desired density. Excellent long-term viability can be achieved after 4 weeks in culture with greater than 90% viability for cells plated at 640 cells/mm2 (2.26 X 105 cells/ml if you plate 0.5 ml of cells into one well of 24-well-plate) and greater than 50% viability for cells plated at 160 cells/. mm2 (0.57 X 105 cells/ml) (Brewer et al., 1993; Brewer, 1995). For my experiments, the cells were plated at a density of about 300-400 cells / mm2. 13. One-half of the medium was replaced with fresh medium at 4 days in vitro (DIV). One-half of the medium was changed twice a week thereafter. 2.1.2. Neuronal transfection Neurons were transfected with 1.2ug of D N A per 24 well of either PSD-95-green fluorescent protein (GFP), PSD-95 PDZ1-2 GFP or GFP alone, using a calcium phosphate kit (Clontech, Mountain View, CA). Briefly, neurons were transfected at 4 DIV and were used for patch-clamp recording 2-3 d after transfection. The detailed procedure follows for transfection of 4 wells in a 24 well plate: Solutions: 1. 2 M calcium solution (Clontech) 2. 2X Hepes-buffered saline (HBS, Clontech) Procedure 1. Add 8ug of D N A along with 12.4 uL of 2M calcium solution and add sterile water to make lOOuL of final volume 2. Add D N A solution to lOOuL 2X HBS while vortexing gently 3. Leave solutions at room temperature for 10 minutes 4. Add 50uL of solution drop-wise into each well while rocking gently into 500uL of conditioned media 33 5. 6. Incubate at 37°, 5% C02 for 10 minutes Wash with and replace wells with fresh media 2.2. HEK293 cell culture and transfection 2.2.1. Culture Human embryonic kidney (HEK) 293 cells (CRL 1573; American Type Culture Collection, Rockville, MD) were maintained at 37° and 5% C02 in minimum essential medium containing Earle's salts and supplemented with L-glutamine (2 mM), sodium pyruvate ( ImM), penicillin/streptomycin (100 units/ml), and 10% fetal bovine serum. The cells were passaged every 3-4 days and plated at a density of ~1 x 106/ml at 10-24 hr before transfection. 2.2.2. Transfection Cells were transfected according to the method of calcium phosphate precipitation, as previously described (Chen and Okayama, 1988; L i et al., 2003). Briefly, a total of 12 ug of plasmid D N A was used for each 10-cm plate. Cells were transfected with a 1:1:2 ratio of cDNAs encoding N R l - l a , NR2 (A or B), and either GFP, PSD-95-GFP, PSD-95 PDZ1-2-GFP, GAP 14 PDZ1-2-GFP, Prenylated PSD-95-GFP, or GAP14. After precipitation with 3 M sodium acetate and 100% ethanol, the plasmid DNAs were resuspended in 450 ul 0.1 X TE, and mixed with CaC12 (250 M , 50 ul) and N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (Aarts et al.) (2 X , 500 (al). The mixture was incubated for 20 min at room temperature. During this time, cells (in 10-cm plates) were in the 3% C02, 37 °C incubator for 20 min. The mixture containing DNAs was then added to each plate. After incubation for 8-10 hrs in the 3% C02, 37 °C incubator, the cell medium containing the transfection reagents was replaced by fresh medium. N M D A R antagonist memantine (50 uM) was included in the medium to protect the cells from excitotoxicity (Raymond et al., 1996). 34 Transfected cells were maintained on glass coverslips and used at 20-36 hr after the start of transfection. 2.3. Electrophysiology Cultured neurons were used for recording at 4-7 DIV ("immature"), or >13 DIV ("mature"). Recordings from HEK293 cells were made 12-24 hours following the end of transfection. Conventional whole-cell patch clamp recording was conducted as previously described (Hamill et al., 1981). Electrodes were fabricated from borosilicate glass (Warner Instruments, Hamden, CT) using a Narashige (Tokyo, Japan) PP-83 electrode puller. Open tip resistance was 5-6 M Q for electrodes containing (in mM) 115 Cs-methanesulfonate, 10 HEPES, 20 K2-creatine phosphate, 4 MgATP, 10 B A P T A , as well as 50 U/ml creatine phosphokinase, pH 7.26 , 310 mOsm. Both neurons and HEK293 cells were superperfused with external recording solution, containing (in mM): 167 NaCl, 2.4 KC1, 10 HEPES, 10 glucose, 0.2 CaC12, pH 7.3 (325 mOsm). Tetrodotoxin (TTX) (300 nM) and glycine (50 uM) were added just before use. Agonist (1 m M N M D A ) was dissolved in the same solution used to bathe the cells and gravity fed to the cells through one side of a theta-tube (Chen et al., 1997). A l l other drugs, dissolved in the external bathing solution, were included in both the control and agonist side of the theta-tube. Computer-controlled solenoid-driven valves were used to rapidly switch between the two solutions. Agonist was applied for 10 sec at 1-min intervals. For experiments in cultured hippocampal neurons, large, pyramidal-shaped neurons were selected for recording. A l l recordings were made in voltage-clamp mode at a holding potential of -70 mV. Data were acquired using the Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA). Currents were filtered at 1 kHz and digitized at 10 kHz. pClamp 8.1 software (Axon 35 Instruments) was used for data acquisition and analysis. Access resistance and cell capacitance were regularly monitored and recordings were abandoned if access resistance exceeded 20 M Q . 2.4. Co-immunoprecipatition and western blot analysis Batches of 14-17 DIV hippocampal neuronal cultures in 10 cm dishes were treated with drugs or vehicle as described in Results, then each dish was collected in 1 ml Harvest buffer containing 1 mM EGTA, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 ug/ml aprotinin, 20 ug/ml pepstatin A, and 20 ug/ml leupeptin in PBS. Cell suspensions were processed as described previously (Li et al., 2003). Briefly, after centrifugation, pellets were lysed and solubilized in Harvest buffer containing 0.1% Sodium dodecyl sulfate (SDS) and 0.8% Triton X -100 (0.5 ml final volume). One-tenth of the lysate was reserved for input loading, and the remainder was incubated with protein-A and protein-G beads then briefly centrifuged to remove nonspecifically bound proteins. The supernatant was incubated with 10 ug of rabbit polyclonal anti-NR2A antibody (Upstate Biotechnology, Chicago, IL) for 1 hr at 4°C, then Protein-A and protein-G beads were added for another 1 hr incubation period. Beads were washed three times with 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 1% Triton X-100. Proteins were eluted from the beads and denatured by boiling in loading buffer for 5 min, then loaded to SDS-PAGE. Proteins from each group of different treatment conditions (vehicle, 2-bromopalmitate (2-BP), 12-O-tetradecanoylphorbol-13-acetate (TP A), and TP A with 2-BP; or vehicle, RO-320432 (RO), and TPA) were loaded to the same gel. After transfer, membranes were probed with antibodies against NR2A (the same as used for immunoprecipitation, at 1 (xg/ml) and PSD-95 (mouse monocolonal; 5 ug/ml; Chemicon). Bands were visualized using Enhanced Chemiluminescence (Amersham, Piscataway, NJ) and densities were quantified by densitometric analysis (Li et al., 2003). The PSD-95 to NR2A band-density ratio was calculated to determine the amount of PSD-95 co-immunoprecipated with NR2A. 36 2.5. Materials A l l chemicals were purchased from Sigma (St. Louis, MO). A l l PSD-95 constructs were described previously (Craven et al., 1999; El-Husseini et al., 2000; Schnell et al., 2002; Christopherson et al., 2003; Prange et al., 2004). N R l - l a (Dingledine et al., 1999), NR2B and NR2A (el) cDNAs were described previously (Chen et al., 1997). Tissue culture reagents were obtained from Invitrogen (Carlsbad, CA). Stock solutions of 1 m M N M D A and 100 uM glycine were each stored in individual aliquots for up to 6 weeks at -200C. TPA was dissolved in dimethyl sulfoxide (DMSO) at a concentration of ImM and diluted to a final concentration of lOOnM. A 200mM stock solution of 2-BP in ethanol was diluted to a final concentration of lOOuM. RO was dissolved in DMSO for a stock concentration of 2mM and used at l u M . 2.6. Data analysis Results are presented as mean ± SE. Sets of different results were compared using one-way A N O V A followed by Bonferroni post-test or Student's t-test as appropriate, and significant differences were determined at the 95% confidence intervals unless otherwise indicated. Three to 10 responses of each cell were averaged for estimation of steady-state to peak current (Iss/Ip). 37 CHAPTER III Results 3.1. Age-dependent desensitization N M D A R desensitization attenuates receptor activation during sustained exposure to agonists (Tong et al., 1995). Glycine-independent desensitization can be isolated in cultured hippocampal neurons by recording N M D A R currents in response to 10-sec applications of saturating concentrations of agonist — 1 mM N M D A with 50 u M glycine — in low external calcium and a high intracellular concentration of B A P T A (Mayer et al., 1989; Tong and Jahr, 1994). Previously, we demonstrated a developmental decrease in this form of N M D A R desensitization recorded from cultured hippocampal neurons, which could be explained by receptor localization to the synapse; however, the reduction in desensitization also correlated with co-association of NMDARs with PSD-95 (Li et al., 2003). 3.2. PKC regulates NMDAR glycine-independent desensitization in mature neurons 3.2.1. PKC uncouples PSD-95 from NMDARs Previously we have shown that treatments that disrupt NMDAR/PSD-95 interactions result in increased desensitization in mature neurons (Li et al 2003). Treatment of mature hippocampal neurons with phorbol esters to activate P K C increases diffuse staining of N M D A R clusters throughout dendrites, indicating a shift from synaptic to non-synaptic localization; under these conditions, PSD-95 staining remained punctate and clustered at the synapse suggesting that NMDARs and PSD-95 become uncoupled after PKC activation (Fong et al., 2002). To confirm that treatment with phorbol esters results in dissociation of NMDARs from PSD-95, we treated neurons with 100 nM TPA for 10, 30 or 60 minutes and examined the interaction between NMDARs and PSD-95 by co-immunoprecipitation with an anti-NR2A 38 antibody. TPA significantly decreased the amount of PSD-95 co-immunoprecipitated with NR2A compared with control after a 60-min treatment (PSD-95/NR2A ratio scaled to control was 77 ± 13%, 67 ± 15%, and 51 ± 13% after 10-, 30- and 60-min TPA treatments, respectively; n=5 independent experiments with all four conditions done in parallel; P <0.01 for control vs. 60-min TPA). Although phorbol esters activate P K C over the course of minutes, prolonged treatment can reduce P K C activity in many cell types (Wagey et al., 2001). To confirm that the effect of a 60-min TPA treatment was not a result of down-regulation of P K C activity, we treated neurons with 1 uM RO, which inhibits P K C activity by binding to the catalytic region ATP binding cassette (Birchall et al., 1994). Consistent with our hypothesis that NR2A and PSD-95 co-association was reduced because of P K C activation by TPA, treatment with RO for 10 minutes had no effect on the co-immunoprecipitation of NR2A and PSD-95 (PSD-95/NR2A ratio scaled to control was 98 ± 19% and 64 ± 12% after 10-min RO and 60-min TPA treatments, respectively; n=5 independent experiments with all three conditions done in parallel; P<0.05 for control vs. 60-min TPA). Previous studies have shown that inhibition of PSD-95 palmitoylation by treatment with 2-BP shifts PSD-95 away from synapses and reduces co-localization with NMDARs (Craven et al., 1999). Indeed, 6-hr treatment with 100 uM 2-BP caused a significant reduction in co-immunoprecipitation of PSD-95 with NR2A (Figure 3-1), similar to previously published results (Li et al., 2003). Co-treatment of neurons with 2-BP (6 hours) and TPA (60 min) resulted in a reduction in the association of PSD-95 with NR2A that was not significantly different than the decrease found for either treatment alone (Figure 3-1). Together, these results indicate that P K C activation by TPA results in a partial uncoupling of NR2A and PSD-95 that is similar, and not additive, to that produced by 2-BP. 39 Figure 3-1. PKC activation with 2-BP treatment does not produce additive uncoupling (a) Representative blot of co-immunoprecipitation. PSD-95 band density was significantly reduced in IP lanes with TPA alone or TPA plus 2-BP treatments compared to control. Combined 1-hour TPA and 6-hour 2-BP treatment did not produce additive reduction in NR2A/PSD-95 association, (b) Pooled data showing mean ± SE of PSD-95/NR2A band density normalized to control (n = 8 independent experiments for all conditions). **P <0.01 for control vs. 60-min TPA. *P <0.05 for control vs. 2-BP and 2-BP + 60-min TPA. Figure contributed by Lily Zhang. 40 3.2.2. Uncoupling of NMDARs/PSD-95 reduces receptor desensitization in mature hippocampal neurons To determine whether PKC-induced dissociation of PSD-95 from NMDARs could alter desensitization, we recorded NMDA-evoked current from cultured mature hippocampal neurons after a 60-min treatment with 100 n M TPA or control solution. TPA treatment caused a marked increase in desensitization to levels similar to those found in immature neurons (Figure 3-2), whereas shorter treatments with TPA (10 min) or inhibition of P K C with RO - both treatments that did not affect coupling between PSD-95 and NR2A - did not alter N M D A R desensitization (Figure 3-2). This is another argument in favor of PSD-95 uncoupling being responsible for the TPA effect on N M D A R desensitization; if the PKC -mediated increase in N M D A R desensitization was due to receptor phosphorylation alone, should see it after 10 min. As a control, incubation with an inactive phorbol ester, 4a-TPA (100 nM), for 1 hour did not alter N M D A R current desensitization (Figure 3-2). Consistent with the similar reduction in co-association of NR2A and PSD-95 produced by treatment with TPA or 2-BP or the two together, these treatments also produced a similar, non-additive, reduction in N M D A R current Iss/Ip (Figure 3-2). Both treatments result in a dissociation of NMDARs from PSD-95: TPA treatment results in extrasynaptic localization of NMDARs while PSD-95 distribution remains unaltered at synaptic sites (Fong et al., 2002); 2-BP treatment results in removal of PSD-95 from synaptic sites while N M D A R distribution is unaltered at the synapse (Craven et al., 1999). The increase in desensitization that results from either treatment suggests binding of PSD-95 to NMDARs is critical in regulating this process. Previous work indicates that P K C activation increases N M D A R peak current and surface expression in Xenopus oocytes (Lan et al., 2001). However, we found no change in peak N M D A R current density after a 60-min treatment with lOOnM TPA (Figure 3-3), consistent with 42 a previous study using a biochemical approach to show that N M D A R surface expression in cultured hippocampal neurons was unchanged following an identical TPA treatment (Fong et al., 2002). TPA has also been shown to enhance N M D A R currents in hippocampal slices (Chen and Huang, 1992) and cultures (Xiong et al., 1998). To further test whether TPA directly alters channel gating under our experimental conditions, we determined whether TPA treatment had any effect on N M D A R current in immature hippocampal neurons that express low levels of PSD-95. TPA did not significantly change the N M D A R Iss/Ip, peak current or current density compared with the control treatment (control vs. 60-min TPA showed: Iss/Ip of 34 ± 9% vs. 29 ± 11% and peak current density of 31 ± 10 pA/pF vs. 37 ± 13 pA/pF; n=15 for control n=7 for 60-min TPA; recordings were made from neurons at 4-5 DIV, Figure 3-4). These results indicate that the effect of TPA on N M D A R desensitization is not a result of direct P K C modulation of N M D A R current or surface expression. Instead, they suggest that association of PSD-95 with NMDARs, disrupted by P K C activation, underlies the reduced N M D A R desensitization found in mature compared with immature neurons. 43 Figure 3-2. PKC activation alters desensitization in mature neurons. (a) Representative traces of mature neurons under control conditions, treated with TPA for 1 hour, and treated with TPA for one hour and 6-hour 2-BP. (b) 1-hour TPA treatment reduced Iss/Ip of mature (>14 DIV) neurons. Shorter 10-minute TPA treatments, 10-minute incubation with a P K C inhibitor (RO), or 1-hour treatment with an inactive phorbol (4ccTPA) did not alter desensitization of mature neurons. Combined TPA and 2-BP treatments did not further increase the extent of desensitization. Control, n=23; 60-min TPA, n=9; 10-min TPA, n=7; 10-min RO, n=9; 1-hr aTPA, n=8; 2-BP + TPA, n=9. **P<0.01 by One way A N O V A followed by Bonferroni post-test. 44 45 Figure 3-3. Change in desensitization in mature neurons not due to increased peak current but altered steady state. Mature neurons displayed no changes in current density with any treatment. Control, n=12; 60-min TPA, n=9; 10-min TPA, n=7; 10-min RO, n=9; 1-hr 4ocTPA, n=8; 2-BP + TPA, n=9. F>0.05 by One way A N O V A . 46 Figure 3-4. PKC activation does not alter desensitization in immature neurons. (a) Immature neurons displayed no changes in Iss/Ip with 60-min TPA treatment. Control, n=15; 60-min TPA, n=7; .P>0.05 by student's t-test. (b) TPA does not alter peak current density in immature neurons. Immature neurons displayed no changes in current density with any treatment. Control, n=15; 60-min TPA, n=7. P>0.05 by student's t-test. 48 Peak Current Density (pA/pF) - t - t r o r o M W £ > . c > u i 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 3.3. PSD-95 Regulates glycine-independent desensitization of NMDARs 3.3.1. Coexpression of PSD-95 with NMDAR subunits decreases desensitization PSD-95 recruits many scaffolding and signaling molecules to the post-synaptic density directly through its various domains or indirectly through macromolecular M A G U K - A K A P complexes (Colledge et al., 2000; Sheng and Pak, 2000; Lim et al., 2002; Kim and Sheng, 2004). To assess whether the previously reported effect of PSD-95 on N M D A R desensitization (Li et al., 2003) occurred as a result of neuronal-specific mechanisms, we compared N M D A R currents from HEK293 cells transfected with either GFP (control) or PSD-95, in combination with different subtypes of NMDARs. HEK293 cells expressing GFP and NR1/NR2A showed significantly greater desensitization compared to cells co-expressing GFP and NR1/NR2B (Figure 3-5), as previously reported (Krupp et al., 1996; Dingledine et al., 1999). Strikingly, immature neurons that predominantly express NR1 and NR2B subunits exhibit more extensive N M D A R desensitization than recombinant NR1/NR2B in HEK293 cells (compare Figure 3-2 with 3-5). Furthermore mature hippocampal neurons that express largely NR2A-containing receptors (Li et al., 2002) show significantly less desensitization compared with either NMDARs in immature neurons or NR1/NR2A expressed in non-neuronal cells (compare Figure 3-2 with 3-5). The co-expression of PSD-95 significantly reduced the extent of NMDA-evoked current desensitization in both NR1/NR2A- and NR1/NR2B-expressing cells (Figure 3-5). Moreover, the steady-state to peak ratio for N M D A R current in PSD-95-expressing cells was similar for NR1/NR2A and NR1/NR2B, and also resembled the ratio found in mature cultured hippocampal neurons (for N M D A R current in PSD-95-expressing HEK293 cells was 64 ± 7%, n=l 1 for NR1/NR2A and 71 ± 7%, n=12 for NR1/NR2B, whereas this ratio was 75 ± 9%, n=23 in mature hippocampal neurons; P>0.05, by one-way A N O V A , Figure 3-2,3-5). These data demonstrate that the role of PSD-95 in regulating N M D A R desensitization is independent of other neuronal-50 and synapse-specific proteins and suggest that direct binding of PSD-95 to NMDARs is sufficient to alter glycine-independent desensitization. 51 Figure 3-5. Expression of PSD-95 regulates glycine-independent desensitization of recombinant NMDARs. (a) Representitive traces of N M D A R currents in HEK293 cells expressing N M D A R subunits with GFP or PSD-95-GFP. Currents were normalized for comparison of desensitization. (b) Expression of PSD-95 significantly reduced the extent of NMDA-evoked current desensitization in both NR1/NR2A- and NRl/NR2B-expressing cells. NR1/2A + GFP, n=13; NR1/2A + PSD-95-GFP, n=l l ; NR1/2B + GFP, n= l l ; NR1/2B + PSD-95; *P<0.05, **P<0.01; One way A N O V A followed by Bonferroni post-test. 52 las mm 4 mm NISI/SI + PSO-95 3.3.2. PDZ1-2 domains of PSD-95 are sufficient to alter NMDAR desensitization in HEK293 cells; multimeriazation not required for altered NMDAR desensitization PSD-95 contains three N-terminal PDZ domains, a central Src homology 3 (SH3) domain, and a C-terminal guanylate kinase-like (GK) domain, which can each recruit various proteins to the membrane (Cho et al., 1992; Kim et al., 1995; Kornau et al., 1995). NMDARs directly interact with PDZ1 and PDZ2 of PSD-95 through the C-terminal PDZ-binding motif (ESDV) of the NR2 subunit (Kornau et al., 1995). Deletion mutagenesis studies established that the N-terminal region of PSD-95, containing palmitoylated Cys-3 and Cys-5, regulates both membrane targeting and multimerization (Craven et al., 1999; Hsueh and Sheng, 1999; Christopherson et al., 2003). In order to isolate which domains of PSD-95 are required for regulating N M D A R desensitization, we utilized PSD-95 constructs with deletions of specific domains and/or with mutations that eliminated PSD-95 multimerization. In HEK293 cells expressing NR1/NR2A, co-transfection of PSD-95 truncated after domain PDZ2 (PSD-95 PDZ1-2-GFP) resulted in significantly reduced NMDA-evoked current desensitization compared to cells co-expressing only GFP, and an Iss/Ip ratio similar to that found with full-length wild-type PSD-95 (Figure 3-6). Although the PSD-95 PDZ1-2 construct lacks many of the domains involved in recruiting kinases and phosphatases to the membrane, it can multimerize, allowing proteins such as GluRl to co-cluster via free PDZ domains (Schnell et al., 2002). To assess the role of PSD-95 multimerization in the regulation of N M D A R desensitization we used two different PSD-95 mutants that targeted to the membrane but remained in monomeric form: a variant of the truncated PSD-95 PDZ1-2-GFP construct, in which the N-terminal 13 amino acids were replaced with the first 12 amino acids of growth associated protein-43 (GAP12 PDZ1-2-GFP); and a variant of the C3,5S mutant of full-length PSD-95, in which the prenylation motif of paralemmin 54 was added to the C terminus (Prenyl-PSD-95-GFP). The Iss/Ip of NMDA-evoked currents recorded from HEK293 cells expressing these multimerization-deficient constructs was not significantly different from that observed in cells expressing full-length wildtype PSD-95-GFP, indicating that multimerization does not play a role in regulating glycine-independent N M D A R desensitization (Figure 3-6). Although mutating the N-terminus of PSD-95 should block multimerization, these constructs were GFP-tagged and GFP may form oligomers (Jain et al., 2001). However, we transfected the same constructs without the GFP tag and found no significant difference in the Iss/Ip (data not shown). As a control, we recorded N M D A R current from cells expressing the first 12 amino acids of GAP43 fused to GFP only (GAP12-GFP) together with NR1/NR2A. This construct had no effect on N M D A R desensitization (Figure 3-6; Iss/Ip was not significantly different from that found in cells co-expressing GFP and NR1/NR2A), confirming that it is the PDZ domains of the GAP 12 PDZ1-2-GFP protein that are responsible for altering N M D A R current desensitization. This evidence strongly supports the idea that PSD-95 regulation of N M D A R desensitization occurs through direct binding of PDZ 1-2 domains to the N M D A R and does not require domains involved in scaffolding other proteins in proximity to NMDARs. 55 Figure 3-6. PDZ1-2 domains are sufficient to alter NMDAR desensitization; multimerization of PSD-95 and protein targeting to the membrane are not required in regulating glycine-independent desensitization. NR1/2A + PSD-95-GFP, n=l l ; NR1/2A + PSD PDZ1-2-GFP, n=l l ; NR1/2A + GAP 12 PDZ1-2, n=7; NR1/2A + Prenyl PSD-95, n=8; NR1/2A + GAP12, n=8; **P<0.01. One way A N O V A followed by Bonferroni post-test. 56 Iss/Ip —J 3.3.3. Overexpression of PDZ1-2 domains decreases desensitization in immature neurons To determine whether expression of PSD-95 PDZ1-2-GFP, including only the N-terminal region through PDZ1 and PDZ2, in immature cultured hippocampal neurons is sufficient to reduce NMDA-evoked current desensitization to levels found in mature neurons, we transfected GFP, PSD-95-GFP, or PSD-95 PDZ1-2-GFP plasmids into 4 DIV neurons and compared NMDA-evoked currents recorded at 6 DIV. Expression of PSD-95-GFP in immature neurons resulted in a decrease in NMDA-evoked current desensitization compared to GFP-transfected controls and an Iss/Ip similar to that observed in mature neurons (compare Figures 3-3 with 3-7). Notably, there was no significant difference in peak current between neurons transfected with GFP and PSD-95 (1093 ± 205 pA, n=7 and 1264 ± 148 pA, n=12, respectively). Transfection of PSD-95 PDZ1-2-GFP also significantly reduced N M D A R desensitization compared to GFP-transfected neurons, although the effect was smaller than that observed for full-length PSD-95-GFP (Figure 3-7). From these data, our observations in HEK293 cells, and our previously published results (Li et al., 2003), we conclude that binding of PSD-95 PDZ1-2 domains to N M D A R s plays a critical role in reducing receptor desensitization over the course of neuronal development. 58 Figure 3-7. Overepression of PSD-95 in Immature Neurons Decreases Glycine-independent Desensitization. (a) Representitive traces of N M D A R responses in immature neurons overexpressing GFP, PSD-95 PDZ1-2-GFP or PSD-95-GFP. (b) Overexpression of PSD-95 PDZ1-2-GFP or PSD-95-GFP in immature hippocampal neurons results in decreased desensitization compared to GFP transfected controls. GFP h=7; PSD-95 PDZ1-2-GFP, n=6; PSD-95-GFP, n=12; *P<0.05, **/><0.01. One way A N O V A followed by Bonferroni post-test. 59 a 60 CHAPTER IV General Discussion The major findings of this study indicate that direct binding of PSD-95 to NMDARs decreases the extent of glycine-independent desensitization in HEK293 cells and neurons. The first two PDZ domains of PSD-95 were sufficient to mediate this effect, which did not require PSD-95 multimerization. Moreover, desensitization can be regulated by P K C activity by altering the interaction between PSD-95 and NMDARs: mature neurons expressing high levels of PSD-95 show little desensitization, however P K C activation uncouples PSD-95 from NMDARs and increases desensitization to levels observed in immature neurons. Interestingly, binding of PSD-95 to the C-terminus of NR2 subunits modulates N M D A R desensitization gating that is largely determined by amino acid residues in the lurcher motif, part of the channel vestibule (Hu and Zheng, 2005). However, there is precedence for changes in C-terminal domains of N M D A R subunits affecting function of distant domains. For example, src-mediated phosphorylation of NR2A C-terminal residues relieves N M D A R zinc inhibition, which is determined by the amino terminal domain (Zheng et al., 1998). 4.1. Phorbol ester-induced PKC activation and regulation of neuronal NMDAR function Our data demonstrate that one-hour TPA treatment of mature neurons results in increased N M D A R desensitization, without altering peak current, through P K C activation and subsequent uncoupling of PSD-95 from NMDARs; as well, TPA did not alter N M D A R peak current in immature neurons expressing only low levels of PSD-95. A previous study using immunocytochemical techniques also showed dissociation of PSD-95 and NMDARs , without alterations in N M D A R surface expression, by one-hour TPA treatment in mature hippocampal neurons (Fong et al., 2002). 61 In contrast to results with chronic (1-hour) phorbol ester treatments, acute phorbol ester treatments have been shown to alter N M D A R peak current by increasing surface number, with a trend towards increasing channel opening rate (Xiong et al., 1998; Zheng et al., 1999; Lan et al., 2001); however, it may be that these effects of phorbol esters are observed under basal conditions only for NMDARs containing NRl-4b splice variants, which exhibit the highest surface expression and undergo the largest P K C potentiation of all NR1 splice variants when expressed in in Xenopus oocytes (Kelso et al., 1992; Okabe et al., 1999; Lan et al., 2001). Interestingly, in HEK293 cells expressing N R l - l a and NR2A or NR2B, P K C activation enhanced glycine-independent desensitization without altering the peak current (Jackson et al., 2006), consistent with the idea that the acute effects of phorbol esters on N M D A R function are specific to NMDARs composed of certain NR1 splice variants and/or that these effects differ between heterologous systems. In the latter study, the effects of phorbol esters on N M D A R desensitization were still evident despite mutagenesis of the major P K C sites on the NR1 subunit, and the removal of the C-terminal tail of NR1 or NR2A (Jackson et al., 2006). In contrast, our data indicates that TPA enhances N M D A R desensitization by disrupting PSD-95 binding to the C-terminus of NR2; differences between our result and that found by (Jackson et al.) may be, in part, accounted for by differences in P K C isoform expression and activity between HEK293 cells and hippocampal neurons. In other studies in neurons, an increase in the N M D A R surface expression occurred after acute TPA treatment in conjunction with the phosphatase inhibitor okadaic acid (Lan et al., 2001), which has been shown to substantially augment the peak current potentiation produced by phorbol ester treatment (Lu et al., 1999; Lan et al., 2001). Loading neurons with the constitutively active form of P K C also resulted in N M D A R peak current potentiation (Xiong et al., 1998). Our stimulation conditions activated only endogenous PKC without inhibition of phosphatases, which may not be sufficient to mediate increases in neuronal N M D A R peak current density and surface expression. 62 A recent study has shown that P K C potentiation of N M D A R currents in Xenopus oocytes can be occluded by co-expression of PSD-95, and is dependent on phosphorylation of Serine-1462, which lies within the PDZ-binding motif of NR2A (Liao et al., 2000; Lin et al., 2006). These results suggest that the lack of N M D A R current potentiation in mature neurons following phorbol ester treatment may be explained by the high levels of co-association of PSD-95 with NMDARs, which are largely localized to synapses (Li et al., 2003). However, when we uncoupled PSD-95 from NMDARs with 2-bromopalmitate and subsequently activated PKC, we still found no potentiation of N M D A R peak current amplitude or density. Moreover, the lack of potentiation of N M D A R current density by TPA in immature neurons, which express low levels of PSD-95, also indicates that regulation of N M D A R function by phosphorylation is different in heterologous compared with neuronal cells. 4.2. Role of PSD-95 direct binding to NMDARs on current desensitization In a previous report (Li et al., 2003), several lines of evidence indirectly implicated the interaction of PSD-95 with NMDARs in regulating glycine-independent desensitization; conditions where PSD-95 targeting to the synapse was disrupted by inhibition of palmitoylation, or where NMDARs were removed from the synapse by chronic treatment with picrotoxin, both resulted in an increase of N M D A R desensitization (Li et al., 2003). The complexity of interactions that occur between PSD-95 and NMDARs makes it difficult to isolate which mechanisms may be involved in regulating N M D A R desensitization in neurons. In HEK293 cells, expression of either wild-type PSD-95 or the N-terminal region of PSD-95 truncated after the PDZ 1-2 domains reduced N M D A R desensitization, to levels similar to those found in mature hippocampal neurons, indicating other neuronal-specific proteins and signaling pathways are not required for the effect of PSD-95 on N M D A R desensitization. However, neuronal-specific mechanisms could still regulate N M D A R desensitization. For example, immature hippocampal 63 neurons that express predominantly NR2B-containing receptors and have lower levels of PSD-95 in early development show more N M D A R desensitization than HEK293 cells expressing NR1/NR2B. The extent of neuronal NR1/NR2B current desensitization in the absence of PSD-95 expression may be regulated by cell-specific receptor phosphorylation, since desensitization is enhanced by phosphorylation of NR2B Serl303 (Sessoms-Sikes et al., 2005) and reduced by tyrosine phosphatase inhibition in NR2B-containing immature neurons (Li et al., 2003). Conversely, mature neurons exhibit low levels of desensitization while expressing high levels of NR2A-containing receptors, which desensitize extensively in HEK293 cells. This may be explained by a direct interaction with PSD-95: NMDARs in mature neurons are largely synaptic and colocalize and presumably interact with PSD-95, while PSD-95 coexpression with NR1/NR2A in HEK293 cells was sufficient to decrease desensitization. Co-expression of PSD-95 with NR1/NR2A in HEK293 cells was previously reported to increase the number of functional channels at the cell surface (Lin et al., 2004). We did not directly address this question in our electrophysiological recordings from transfected HEK293 cells, due to the high cell-to-cell variability in N M D A R expression. Notably, the Iss/Ip did not correlate with the peak current amplitude in HEK293 cells. In neurons, however, we did not observe any change in peak current after overexpression of PSD-95 or with dissociation of NMDARs and PSD-95, suggesting other neuronal-specific mechanisms may contribute to regulating N M D A R surface expression and occlude the effect of PSD-95. Interestingly, PDZ interactions have also been shown to stabilize NMDARs at synaptic sites. Previously, it has been shown that overexpression of PSD-95 in immature cerebellar granule cells resulted in a decrease in current density (Losi et al., 2003). PSD-95 cotransfection can retard the internalization rate of chimeras containing the C-terminus of the NR2B subunit (Roche et al., 2001). PSD-95 also has been shown to stabilize NR2B-containaing receptors at the synapse while trafficking of NR2A- or NR2B-containing receptors to the surface occurred in a PSD-95 64 independent manner (Prybylowski et al., 2005). This evidence suggests the larger role of PSD-95 regulation of NMDARs in neurons may be in maintenance of surface receptors rather than through forward trafficking and surface delivery. With overexpression of full-length PSD-95 in immature neurons, we observed a significant decrease in N M D A R glycine-independent desensitization compared to GFP-transfected controls, an effect that was attenuated though still significant with expression of the truncated mutant, PSD-95 PDZ1-2-GFP. PSD-95 PDZ1-2-GFP has been shown to cluster at synaptic sites, although less efficiently than full-length PSD-95 (Craven et al., 1999). Although the PSD-95 PDZ1-2-GFP construct contains the N-terminal palmitoylation sites involved in membrane targeting, a second signal found in the last 13-25 amino acids of PSD-95 is also required for proper synaptic targeting (Craven et al., 1999); absence of this region in the truncated mutant results in a "semi-clustered" phenotype. Furthermore, the PDZ3 domain binds the postsynaptic protein CRIPT, linking PSD-95 to the microtubule cytoskeleton. Disruption of the CRIPT/PSD-95 interaction prevented the association of PSD-95 with microtubules and inhibited synaptic targeting in hippocampal neurons (Passafaro et al., 1999). In HEK293 cells, expression of truncated mutants did not show a significant difference in N M D A R current desensitization compared to full-length PSD-95. As targeting or clustering of NMDARs is independent of PSD-95 in neurons (El-Husseini et al., 2000), an inefficient synaptic targeting or clustering of PSD-95 PDZ1-2-GFP suggests that NMDARs and PSD-95 PDZ1-2-GFP could be in non-overlapping subcellular compartments, resulting in less effective interaction. This might, at least in part, explain the discrepancy between the overexpression in neurons versus HEK293 cells, where targeting to the plasma membrane is sufficient to colocalize NMDARs and PSD-95. PSD-95 exists predominantly in an a isoform containing two cysteine residues that can be palmitoylated to regulate membrane targeting as well as multimerization, however a rarer (3 isoform containing an L27 domain instead of a palmitoylation motif is also present in neurons. 65 The P isoform has been shown to regulate PSD-95 clustering as well as A M P A receptor function in an activity-dependent manner through the CaMKII pathway (Chetkovich et al., 2002; Schluter et al., 2006). In our overexpression studies we have not utilized any constructs containing the N -terminal domain of the |3 isoform, and in mature neurons expressing endogenous PSD-95, the a isoform has been shown to account for more than 80% of the total PSD-95 expression. In support of this, mature neurons exhibit high sensitivity to treatments that interfere with palmitoylation of PSD-95 (Li et al., 2003; Schluter et al., 2006). 4.3. Effects of PSD-95/NMDAR interaction on NMDAR function in neurons Previous studies investigating the role of PSD-95 in modulating neuronal N M D A R currents yielded apparently conflicting results. In mice expressing the truncated C-terminus NR2A subunit ( N R 2 A A C / A C ) , which is unable to bind PSD-95, hippocampal neurons in brain slice have reduced N M D A R spontaneous and evoked excitatory postsynaptic currents (EPSCs) and dramatic reductions in hippocampal CA1 long-term potentiation (LTP) (Sprengel et al., 1998; Steigerwald et al., 2000); consistent with our findings, N M D A R current desensitization was increased in whole-cell patches from these neurons (Steigerwald et al., 2000). Also consistent with our results, in mice carrying a targeted mutation in the PSD-95 gene that leaves the first two PDZ domains intact by introducing a stop codon in the PDZ3 domain, there were no alterations of whole-cell N M D A R current in cultured neurons or gross differences in synaptic transmission compared to wild-type (Migaud et al., 1998). On the other hand, hippocampal slices from PSD-95 knock-out mice (PSD-95 7), or after acute knock-down of PSD-95 using short hairpin RNAs (shRNA), showed no difference in NMDAR-mediated EPSCs (Nakagawa et al., 2004; Elias et al., 2006). Furthermore, acute disruption of PSD-95/NMDAR interactions utilizing peptides that competitively bind to the first two PDZ domains of PSD-95 in hippocampal slices showed no effect on N M D A receptor-66 mediated EPSC amplitude (Lim et al., 2003). However, these studies did not directly examine N M D A R desensitization, and glycine-independent desensitization may be difficult to assess in slice preparations, since single evoked responses reflect transient agonist exposure. Desensitization has been shown to shape synaptic responses, but this was observed during repetitive stimulation (Tong et al., 1995). We postulate that disruption of the NMDAR/PSD-95 interaction shifts the relationship between N M D A R activation and activity-dependent plasticity to the right, such that higher N M D A R activity levels are required to induce long-term potentiation. In this case, acute interference with the NMDAR/PSD-95 interaction may result in no apparent change in LTP at stimulation frequencies that were formerly in the saturating range for inducing LTP (Migaud et al., 1998; Lim et al., 2003; Malenka and Bear, 2004). 4.4. PSD-95/NMDAR association may regulate synaptic plasticity and excitotoxicity N M D A R desensitization limits calcium influx during periods of repeated synaptic stimulation or prolonged neuronal exposure to glutamate as may occur during ischemia. PSD-95 binding to NR2 subunits to reduce N M D A R desensitization may contribute to the shift in stimulation frequencies required to induce NMDAR-dependent synaptic plasticity that occur during brain development (Philpot et al., 2003), with consequences for new learning. As well, the effect of NMDAR/PSD-95 binding on the integrated calcium current in response to sustained glutamate insults may contribute to neuronal sensitivity to excitotoxicity. It has been shown that N M D A R antagonists prevent toxicity in in vitro models of ischemia however clinical trials utilizing N M D A R antagonists have largely been disappointing due to troubling, unwanted side effects (Lee et al., 1999; Brauner-Osborne et al., 2000). Regulating desensitization by disrupting NMDAR/PSD-95 interactions may provide a novel approach for protection from excitotoxicity. 67 4.5. Future directions We have shown here that direct binding of PSD-95 to NMDARs regulates glycine-independent desensitization. Although this finding is interesting there remains much to be studied. Members of the PSD-95 family of M A G U K s share approximately 70% homology, and a large body of evidence suggests a degree of functional redundancy exists within the family (Kim and Sheng, 2004). PSD-95 and PSD-93 knock-out mice show no alterations in synaptic transmission; however, acute knockdown of either protein or creation of a double knockout results in severely altered neurotransmission (Elias et al., 2006). It would be important to determine whether other members of the M A G U K family of proteins such as PSD-93 or SAP-102 confer altered desensitization profiles to NMDARs through direct binding to the C-terminus of the NR2 subunit. Although we suggest alterations in desensitization may be relevant in both learning/memory and excitotoxicity, we do not address these processes directly in this thesis. It has been previously published that disruption of binding between NR2B-containing NMDARs and PSD-95 resulted in no alterations in calcium entry or whole-cell responses while decreasing NMDA-induced toxicity through an uncoupling of NMDARs from downstream toxic signaling pathways (Sattler et al., 1999; Aarts et al., 2002). One possible explanation for the unaltered whole-cell currents and calcium signal may be the small differences between the integrated current in cells expressing NR2B-containing NMDARs with and without PSD-95. Although PSD-95 significantly decreased the extent of desensitization of NR2B-containing NMDARs in HEK293 cells in our study, the differences are much less robust compared to cells expressing NR2A-containing NMDARs with and without PSD-95. This compounded with the possibility that uncoupling caused by the peptide may be incomplete could easily result in little alteration in whole cell currents or calcium signals. 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