Regulation of Glutamate Transporter and Receptor Function by the NHERF Scaffolding Proteins Open Access

Ritter, Stefanie Leigh (2013)

Permanent URL: https://etd.library.emory.edu/concern/etds/tt44pn013?locale=en
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Abstract

Glutamatergic neurotransmission is essential to central nervous system function and dysregulation of glutamatergic signaling is associated with multiple neurodegenerative, pathophysiological, and neuropsychiatric disease states. Levels of glutamate in the brain are controlled by glutamate transporters, and the physiological actions of glutamate are mediated by a variety of ionotropic and metabotropic glutamate receptors. Interestingly, many of these transporters and receptors possess motifs indicative of potential binding to PDZ domains, suggesting that PDZ scaffold proteins might be critical regulators of glutamate transporter and receptor function. In the studies described in this dissertation, my colleagues and I identified and characterized novel PDZ-interacting partners for the astrocytic glutamate transporter GLAST and the Group II metabotropic glutamate receptors, mGluR2 and mGluR3. Specifically, proteomic screens identified the Na+/H+ exchanger regulatory factors 1 and 2 (NHERF-1 and NHERF-2) as candidate interacting partners for GLAST and Group II mGluRs. Subsequent studies on cultured rat cortical astrocytes revealed that NHERF-2 is abundantly expressed in these cells and enhances GLAST stability and function. Moreover, interactions between Group II mGluRs and NHERF proteins were validated in a cellular context, and disruption of the C-terminal PDZ-interacting motif of mGluR2 was found to enhance receptor-mediated activation of AKT in astrocytes; however, it was unclear if this enhancement was due to disruption of associations with the NHERF proteins, since the effects of the mutation were not recapitulated by knock-out or knockdown of NHERF-1 or NHERF-2. We furthermore examined how the NHERF proteins might regulate Group II mGluRs in vivo. An electron microscopic examination of the cellular and sub-cellular distribution of Group II mGluRs in the mouse cortex in wild-type (WT), NHERF-1 KO, and NHERF-2 KO mice, revealed that loss of NHERF-2 led to a modest redistribution in the cellular targeting of Group II mGluRs without altering overall Group II mGluR expression. These studies shed light on the physiological significance of scaffold protein interactions with glutamate transporters and receptors, and thereby enhance our understanding of the molecular mechanisms underlying both normal regulation and potential dysregulation of glutamatergic neurotransmission.

Table of Contents

Table of Contents CHAPTER 1 : Introduction and Background 1.1. Glutamate and the CNS............................................................................................................................................................................................................2-6 1.1.A. Glutamatergic Neurotransmission.............................................................................................................................................................................2 1.1.B. CNS Sources of Glutamate..............................................................................................................................................................................3

1.1.C. Regulation of Extracellular Glutamate Concentrations.................................................................................................................................................3-6

1.2. Astrocytes..................................................................................................................................................................7-13 1.2.A. Overview of function.............................................................................................................................................................7-11 1.2.B. Tripartite Synapse..............................................................................................................................................................................11-12 1.2.C. Perisynaptic Astrocyte Processes...................................................................................................................................................................12-13 1.3. Excitoxicity........................................................................................................................................................................................13-14 1.4. Glutamate Transporters........................................................................................................................................................................14-17 1.4.A. Overview....................................................................................................................14-15 1.4.B. Distribution..............................................................................................................15-17 1.5. Ionotropic Glutamate Receptors................................................................................................................................................................17-19 1.5.A. Overview...............................................................................................................................................................................17 1.5.B. Distribution..............................................................................................................................................................................................18-19

1.6. G protein-coupled receptor (GPCR) Signaling............................................................................................................................................................19-21

1.7. Metabotropic Glutamate Receptors...............................................................................................................................................................................22-37 1.7.A. Overview..........................................................................................................................................................................................................22-31 1.7.B. Distribution..........................................................................................................................................................................................31-37

1.8. NAAG: A putative mGluR3-selective agonist...................................................................................................................................................................38-39

1.9. Additional excitatory amino acids: D-serine and D-aspartate.....................................................................................................................................................39

1.10. PDZ Proteins........................................................................................................................................................................................................40-41 1.11. NHERF Proteins.................................................................................................................................................................................................42-45

1.11.A. Overview.....................................................................................................................................................................................................42-44

1.11.B. Knockout mouse models.....................................................................................................................................................................................44-45

1.12. Central Hypothesis........................................................................................................................................................................................................46

CHAPTER 2 : Glutamate Transporter Regulation by the NHERF Scaffolding Proteins

2.1. Summary.........................................................................................................................................................................................................................48 2.2. Introduction........................................................................................................................................................................................................48-49 2.3. Results.............................................................................................................................................................................................................49-59 2.4. Discussion.............................................................................................................................................................................................60-62

2.5. Materials and Methods...................................................................................................................................................................................................62-65

2.6 Acknowledgments.................................................................................................................................................................................................................65

CHAPTER 3 : Group II mGluR Regulation by the NHERF Scaffolding Proteins

3.1. Summary.........................................................................................................................................................................................................................67 3.2. Introduction...........................................................................................................................................................................................................67-69 3.3. Results..................................................................................................................................................................................................................69-79 3.4. Discussion....................................................................................................................................................................................................................80-83 3.5. Materials and Methods................................................................................................................................................................................................83-88

3.6. Acknowledgments...................................................................................................................................................................................................................88

CHAPTER 4: CNS Characterization of NHERF-1 and NHERF-2 KO mice 4.1. Summary......................................................................................................................................................................................................................90-91 4.2. Introduction............................................................................................................................................................................................................91-92 4.3. Results........................................................................................................................................................................................................................92-111 4.4. Discussion.....................................................................................................................................................................................................................111-118 4.5. Materials and Methods...................................................................................................................................................................................................118-124

4.6. Acknowledgments...........................................................................................................................................................................................................124

CHAPTER 5 : Conclusions and Implications

5.1. Overview...............................................................................................................................................................................................................126-127

5.2. GLAST interaction with NHERF-2 enhances GLAST stability.................................................................................................................................................127-129

5.3. Group II mGluR interactions with PDZ proteins, but not necessarily NHERF proteins, regulate receptor-mediated AKT signaling in cultured astrocytes......................130-133

5.4. Loss of NHERF-2 Alters Distribution of Group II mGluRs in the Cortex...........................................................................................................................................133-135

5.5. Reconciliation of cultured versus in vivo astrocytes: A case study on mGluR3....................................................................................................................135-139

5.6. NHERF Proteins and Hydrocephaly: A case for cilia dysfunction?...........................................................................................................................................140-143

5.7. Cross Talk between Astrocytic Glutamate Transporters and Glutamate Receptors:A Role for NHERF Proteins?....................................................................................143-145

5.8. The challenges of studying mGluR3 signaling in vitro and potential strategies.................................................................................................................145-148

5.9. Concluding Remarks......................................................................................................................................................................................148-149 APPENDIX...............................................................................................................................................................................................150-153 REFERENCES...............................................................................................................................................................................154-211 Figure Index

Figure 1.1. Overview of glutamate biosynthesis, storage, and release in the CNS......................................................................................................................5

Figure 1.2. Canonical mechanisms of G protein-coupled receptor signaling...................................................................................................................................21

Figure 1.3. Schematic of key structural domains of metabotropic glutamate receptors.................................................................................................................24 Figure 1.4. Overview of ultrastructural localization of mGluRs in the CNS......................................................................................................................................36

Figure 2.1. The C-terminus of GLAST binds selectively to NHERF PDZ domains............................................................................................................................53

Figure 2.2. In vitro association between GLAST and PDZ2 of NHERF-2....................................................................................................................................54

Figure 2.3. Association of NHERF-2 with GLAST in primary astrocytes and transfected cells..............................................................................................................55

Figure 2.4. Cultured astrocytes are enriched in NHERF-2 and express functional GLAST..................................................................................................................56

Figure 2.5. Knockdown of endogenous NHERF-2 in astrocytes reduces GLAST activity and protein levels..........................................................................................57

Figure 2.6. Metabotropic glutamate receptor 5 associates with GLAST in a NHERF-independent manner...................................................................................................59

Figure 3.1.The C-termini of mGluR2 and mGluR3 binds selectively to NHERF PDZ domains...................................................................................................................74

Figure 3.2. Group II mGluRs associate with NHERF-1 or NHERF-2 in a cellular context................................................................................................................75

Figure 3.3. Group II mGluR association with NHERF proteins is dependent on the last amino acid..........................................................................................................76

Figure 3.4. Examination of the effects of mutation of the Group II mGluR-PDZ interacting motif on AKT signaling........................................................................................77

Figure 3.5. Examination of mGluR2 and mGluR3 signaling in cultured astrocytes devoid of NHERF proteins.................................................................................................78

Figure 3.6. mGluR2-mediated AKT signaling is entirely mediated via Gαi in Wild-Type, NHERF-1 KO and NHERF-2 KO astrocyte cultures..........................................................79

Figure 4.1. Characterization of NHERF genotype status and protein expression in NHERF-1 and NHERF-2 mutant mice: evidence for upregulation of a NHERF-2 splice variant.....99-100

Figure 4.2. Total GLAST expression does not change in wild-type, NHERF-1 KO, or NHERF-2 KO mouse brain regions..............................................................101

Figure 4.3. Total Group II mGluR expression does not change in wild-type, NHERF-1KO, or NHERF-2 KO mouse brain regions...........................................................102

Figure 4.4. Control experiments for light microscopy reaction and evidence of ventriculomegaly in NHERF-1/-2 KO mice...........................................................................103

Figure 4.5. Representative images of Group II mGluR labeling in the cortex Wild-Type, NHERF-1 KO, and NHERF-2 KO mice...............................................................104-105

Figure 4.6. Histograms showing the relative distribution or density (counts/area) of Group II mGluR-immunoreactive elements in the cortex of wild-type, NHERF-1 KO, or NHERF-2 KO mice.................106-107

Figure 4.7. Distribution of Group II mGluR-immunoreactivy in perisynaptic astrocyte processes across WT, N1 KO and N2 KO genotypes..................................................108-109

Figure 4.8. Three-dimensional reconstruction of labeled perisynaptic glial processes: Validation of criteria used in perisynaptic glial classification...............................110

Figure 4.9. Comparison of the percentage of mice that died prematurely from hydrocephaly..........................................................................................................111

Figure A.1. Treatment of cultured rat astrocytes with NHERF-2 siRNA results in the specific decrease in NHERF-2 expression and the resulting decrease in total GLAST expression.................151

Figure A.2. Group II mGluRs are not detectable in cultured astrocytes via Western blot analysis........................................................................................................152

Figure A.3. Preliminary data to ascertain most robust Group II mGluR signaling output in cultured astrocytes expressing mGluR2 or mGluR3................................................153

Tables

Table 1.1. Summary of potencies (EC50/IC50 values in µM) of several compounds acting on metabotropic glutamate receptors (mGluRs) 1-8.................................................27

Table 1.2. Classification of PDZ Consensus Motifs on Glutamate Transporters and Metabotropic Glutamate Receptors........................................................................................41

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