Regulation of mGluR3 by the Proteasome and Cellular Stress: Implications for the Treatment of Schizophrenia 公开

Reiff, Rachel Elizabeth (2012)

Permanent URL: https://etd.library.emory.edu/concern/etds/2227mq53v?locale=zh
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Abstract

Schizophrenia is a serious psychiatric disease that has a profound impact on sufferers, caretakers, and society as a whole. Although many medications that treat symptoms of schizophrenia exist, they have limited efficacy and often produce severe adverse effects. The metabotropic glutamate receptor three (mGluR3) has recently been identified as a potential therapeutic target for the treatment of schizophrenia. Past studies have shown that mGluR3 protein levels may be abnormally low in the brains of schizophrenic patients, and that an agonist for mGluR3 and the related receptor mGluR2 might be useful in alleviating symptoms of schizophrenia. However, much is still unknown about the activity and regulation of mGluR3, and in order to make progress toward developing drugs that target mGluR3, it is first necessary to gain a better understanding of the receptor both in normal states and in disease.

The purpose of this project was to study the ways that mGluR3 is trafficked, degraded, and regulated in a cellular context. Particular focus was given to degradation pathways of mGluR3, as the manipulation of degradation may be a useful therapeutic tool for increasing receptor expression and activity. We found that mGluR3 and mGluR2 are degraded robustly by the proteasome in both primary cells and a heterologous overexpression system. Additionally, we found that the proteasomal degradation of mGluR3 is ubiquitin-independent. Our data also indicated that mGluR2 and mGluR3 are upregulated by cellular stress induced by dimethyl sulfoxide treatment. Lastly, we found that mGluR2 and mGluR3 signal through the AKT survival pathway, thereby supporting a role for the receptors in neuroprotection.

These findings provide novel insight into the activity and regulation of mGluR3 and will contribute important information to the field of schizophrenia research. Through an enhanced understanding of mGluR3 trafficking, signaling, and degradation, it will be possible to assess the role of the receptor in schizophrenia more closely and to design greatly needed therapeutic drugs.

Table of Contents

I. Introduction......1
- Schizophrenia......1
- The Dopamine Hypothesis of Schizophrenia......2
- The Glutamate Hypothesis of Schizophrenia......3
II. Background......5
- Metabotropic Glutamate Receptor Three......5
- Connections between mGluR3 and Schizophrenia......7
III. Materials and Methods......10

- Degradation Pathway Assessment......10
- Western Blot......11
- Site-Directed Mutagenesis......12
- Degradation of mGluR3-ZIK versus mGluR3......12
- Co-Immunoprecipitation of mGluR3 and mGluR3-ZIK with HA-Ubiquitin......12
- Co-Immunoprecipitation of mGluR3 with β-arrestins......13
- Comparison of Degradation Pathways of mGluR2 and mGluR3......14
- Assessment of Group II mGluR Degradation in Primary Cortical Rat Neurons......15
- Assessment of Group II mGluR Signaling in Primary Neurons......15
- Evaluation of mGluR3 Surface Expression via Biotinylation......17
- Evaluation of Cycloheximide Effects on Group II mGluR Detection......18
IV. Results and Discussion......19
- Degradation of mGluR3 Occurs through the Proteasome in HEK-293T Cells......19
- Proteasomal Degradation of mGluR3 is Ubiquitin-Independent......19
- β-arrestins 1 and 2 Physically Interact with mGluR3 in a Cellular Context......21
- Group II mGluRs are Degraded by the Proteasome in Primary Neurons......22
- Expression of Group II mGluRs is Upregulated by DMSO Treatment in Neurons......23
- Activation of Group II mGluRs Leads to AKT Signaling in Primary Rat Neurons......23
- A Higher-Order mGluR3 Species is Expressed on the Cell Surface, but Surface Biotinylation Cannot be Used to Evaluate the Effects of MG132......25
- Cycloheximide Has Unexpected Effects on Group II mGluR Detection in Neurons......25
V. Conclusions......26
VI. Figures......30
- Figure 1......30
- Figure 2......31
- Figure 3......32
- Figure 4......33
- Figure 5......35
- Figure 6......36
- Figure 7......37
- Figure 8......38
- Figure 9......39
- Figure 10......40
- Figure 11......41
- Figure 12......42
VII. References......43

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