Identification of Aberrantly Expressed Synaptic Proteins in the Mouse Model of Fragile X Syndrome Público

Abstract

Fragile X syndrome (FXS) is the most commoly inherited form of intellectual disability. A
mutation in the FMR1 gene of the X chromosome results in the loss of fragile X mental
retardation protein (FMRP), an RNA-binding protein. The mechanism by which disability
results, however, is unclear. Mouse Models of FXS exhibit altered synaptic plasticity that
contributes to enhanced long-term depression (LTD) induced by mGluR activation inthw
hippocampus. This mGluR-dependent LTD (mGluR-LTD) requires new protein synthesis to be
induced and maintained. The mGluR theory of FXS proposes that FMRP is a regulatory agent
that suppresses protein synthesis at the synapse. Activation of mGluR in the synapse initiates
local protein synthesis, the products of which induce LTD via endocytosis of AMPA receptors.
Consequently, in the pathological state, the loss of translation inhibition from FMRP results in
increased synthesis of LTD-inducing proteins and enhanced LTD. However, the LTD-inducing
proteins that are regulated by FMRP are not yet identified. Using high throughput proteomic
analysis and Western blotting on synaptoneurosome (SNS) preparation of hippocampi, our study
identified 3 synaptic proteins differentially expressed between wild-type (WT) and Fmr1
knockout (KO) mice: Eukaryotic elongation factor 1a (eEF1A), Neuronal axonal protein 22
(NAP22) and cyclin G associated kinase (GAK), suggesting these are regulated by FMRP and
may contribute to the FXS pathophysiology.

Table of Contents

Table of Contents
Abstract …………………………………………………………………………………………1
Introduction ……………………………………………………………………………………………..2
Materials and Methods ………………………………………………………………………………..5
Results ………………………………………………………………………………………………….9
Figures and Tables ……………………………………………………………………………………14
Figure 1. mGluR Theory of FXS
Figure 2. Synaptoneurosome Preparation Scheme
Figure 3. Electron microscopy images show intact synapses
Figure 4. Western blotting demonstrates enrichment of SNS
Table 1. Densitometry Data for PSD-95 Enrichment in SNS
Figure 5. SNS sample aggregation after SDS and Iodoacetamide treatment
Figure 6. Optimal conditions for preparation of soluble samples
Figure 7. Coomassie staining used to determine protein concentration of MS sample 1
Table 2. Mass, Volume and Protein Concentration of MS Sample 1
Figure 8. Commassie staining used to determine protein concentration of MS sample 2
Table 3. Mass, Volume and Protein Concentration of MS Sample 2
Table 4. MS-Identified Candidate Proteins
Figure 9. Western blotting verifies overexpression of eEF1A
Table 5. Densitometry Data of eEF1A Western Blot
Figure 10. Western blotting verifies overexpression of NAP22
Table 6. Densitometry Data of NAP22 Western Blot
Figure 11. Western blotting verifies overexpression of GAK
Table 7. Densitometry Data of GAK Western Blot

Discussion …………………………………………………………………………………………….. 30
Sources …………………………………………………………………………………………………34

About this Honors Thesis

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School	Emory College
Department	Neuroscience and Behavioral Biology
Degree	BS
Submission	Honors Thesis
Language	English
Research Field	Biology, Neuroscience
Palabra Clave	FXS NBB FMRP
Committee Chair / Thesis Advisor	Warren, Stephen, Emory University
Committee Members	Frenzel, Kristen E, Emory University Crutcher, Michael D, Emory University

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