Regulation of protein synthesis and stress granule dynamics in neural cells by Cdh1-APC Open Access

Abstract

Maintaining a homeostatic balance between protein degradation and protein synthesis at synapses is necessary for learning and memory. Perturbation of protein homeostasis can compromise synaptic function, and several neurodevelopmental brain disorders are characterized by dysregulated protein homeostasis, including Angelman Syndrome, Fragile X Syndrome, and Autism Spectrum Disorders. Ubiquitination is a post-translational modification that is a primary mechanism of targeting proteins for degradation. While the E3 Ubiquitin Ligase Anaphase Promoting Complex and its regulatory subunit Cdh1 (Cdh1-APC) has been previously shown to regulate learning and memory, the underlying molecular mechanisms are unclear. In this study, we have identified a novel role of Cdh1-APC as a regulator of protein synthesis in neurons. Inhibition of Cdh1-APC activity leads to a decrease in protein synthesis in postmitotic cortical neurons. Proteomic profiling revealed that Cdh1-APC interacts with known regulators of translation, including mRNA binding proteins, initiation factors, ribosomal proteins, and stress granule proteins. Inhibition and knockdown of Cdh1-APC activity caused an increase in stress granule formation, suggesting a novel mechanism by which Cdh1-APC may regulate the dynamic balance between translational repression within stress granules and protein synthesis. Furthermore, the interaction between Cdh1-APC and the Fragile X Mental Retardation Protein (FMRP), an RNA binding protein necessary for proper neurodevelopment, was shown to regulate stress granule formation and downstream changes in protein synthesis. We propose a model in which Cdh1-APC targets key stress granule proteins, such as FMRP, and inhibits the formation of stress granules, leading to increases in protein synthesis. As Cdh1-APC has been primarily studied in the context of mitotic cells and progressing cell cycle, these findings from neurons demonstrate a novel role for Cdh1-APC independent of its well-characterized targeting of mitotic proteins for degradation. Elucidation of an interdependent role for Cdh1-APC in regulation of stress granules and protein synthesis in cortical neurons has implications for how Cdh1-APC can regulate protein synthesis-dependent synaptic plasticity underlying learning and memory.

Table of Contents

Chapter 1 : General Introduction................................................................................................ 1

1.1. Protein Synthesis.................................................................................................................. 3

1.1.1. Local Protein Synthesis in Neurons.............................................................................. 4

1.1.2. Stress Granules.............................................................................................................. 6

1.2. Ubiquitination....................................................................................................................... 8

1.2.1. Addition of Ubiquitin onto Substrates.......................................................................... 9

1.2.2. Ubiquitination is Dysregulated in Neurodevelopmental Disorders............................ 11

1.3. Fragile X Syndrome........................................................................................................... 13

1.3.1. Dysregulation of Dendritic Spines in Fragile X Syndrome........................................ 14

1.3.2. Aberrant mGluR-LTD in Fragile X Syndrome........................................................... 15

1.3.3. Dysregulated Protein Synthesis in Fragile X Syndrome............................................. 16

1.4. FMRP Functions as a Translational Repressor.................................................................. 17

1.4.1. FMRP Directly Binds RNA........................................................................................ 18

1.4.2. FMRP Interacts with the RNA-Induced Silencing Complex...................................... 20

1.4.3. FMRP Binds Polyribosomes....................................................................................... 20

1.4.4. Regulation of FMRP and FMR1 mRNA by Association with other RNA-binding Proteins      22

1.5. Reversal of FMRP-mediated Repression........................................................................... 23

1.5.1. FMRP Modification as a Molecular Switch................................................................ 24

1.5.2. Ubiquitination of FMRP.............................................................................................. 26

1.6. The Anaphase Promoting Complex (APC)........................................................................ 27

1.6.1. Role of APC in Mitotic Cells...................................................................................... 29

1.6.2. Role of APC in Postmitotic Neurons.......................................................................... 31

1.7. Critical Questions in the Field............................................................................................ 35

1.8. Dissertation Hypothesis and Objectives............................................................................. 35

1.9. Figures................................................................................................................................ 37

Chapter 2 : Materials & Methods.............................................................................................. 42

Cell Culture............................................................................................................................... 43

Pharmacology............................................................................................................................ 44

Plasmids..................................................................................................................................... 44

Antibodies................................................................................................................................. 45

FMRP Immunoprecipitation..................................................................................................... 46

FLAG Immunoprecipitation...................................................................................................... 47

Puromycylation......................................................................................................................... 48

FMRP Expression...................................................................................................................... 48

Western Blotting........................................................................................................................ 48

Silver Staining........................................................................................................................... 49

Proteomics Analysis.................................................................................................................. 49

Bioinformatic Analysis.............................................................................................................. 50

Ubiquitin Immunoprecipitation................................................................................................. 50

Immunofluorescence................................................................................................................. 51

Quantification of Stress Granules............................................................................................. 51

Statistical analysis..................................................................................................................... 52

Chapter 3 : Cdh1-APC Regulates FMRP-Mediated Protein Synthesis................................. 53

3.1. Introduction........................................................................................................................ 54

3.2. Results................................................................................................................................ 55

3.2.1. Cdh1-APC Ubiquitinates FMRP Downstream of mGluR5 Signaling in Cortical Neurons       55

3.2.2. Cdh1-APC Reverses FMRP-mediated Repression of Protein Synthesis.................... 56

3.2.3. Cdh1-APC Interaction with FMRP Affects FMRP Expression.................................. 57

3.2.4. Cdh1-APC Interaction with FMRP Regulates Protein Synthesis............................... 58

3.3. Discussion.......................................................................................................................... 59

3.4. Figures................................................................................................................................ 63

3.5. Supplemental Figures......................................................................................................... 70

Chapter 4 : Cdh1-APC Associates with Translational Machinery and is a Novel Regulator of Protein Synthesis  71

4.1. Introduction........................................................................................................................ 72

4.2. Results................................................................................................................................ 73

4.2.1. Cdh1-APC Regulates Protein Synthesis Independent of Cell Cycle.......................... 73

4.2.2. Cdh1-APC interactome is enriched with regulators of protein synthesis................... 73

4.3. Discussion.......................................................................................................................... 75

4.4. Figures................................................................................................................................ 79

4.5. Tables................................................................................................................................. 83

4.6. Supplemental Figures....................................................................................................... 104

Chapter 5 : Cdh1-APC Regulates Stress Granule Dynamics............................................... 105

5.1. Introduction...................................................................................................................... 106

5.2. Results.............................................................................................................................. 107

5.2.1. Cdh1-APC Interacts with Stress Granule Proteins.................................................... 107

5.2.2. Cdh1-APC Antagonizes Stress Granule Assembly................................................... 107

5.2.3. Stress granule formation reduces protein synthesis.................................................. 109

5.2.4. Cdh1-APC regulates stress granules in a FMRP-dependent mechanism.................. 109

5.3. Discussion........................................................................................................................ 111

5.4. Figures.............................................................................................................................. 114

5.5. Tables............................................................................................................................... 120

5.6. Supplemental Figures....................................................................................................... 139

Chapter 6 : General Discussion................................................................................................ 144

6.1. Summary.......................................................................................................................... 145

6.2. Cdh1-APC activity across the lifespan............................................................................. 145

6.2.1. Cdh1-APC and neurodevelopment............................................................................ 146

6.2.2. Cdh1-APC and neurodegeneration............................................................................ 147

6.2.3. Relationship between neurodevelopment and neurodegeneration............................ 148

6.3. Insight into mRNP granules............................................................................................. 149

6.4. Targeting of E3 ligases as potential therapeutic options.................................................. 150

6.5. Future Directions.............................................................................................................. 152

6.5.1. Consequences of Cdh1-APC interaction with FMRP and other proteins................. 152

6.5.2. Determine if Cdh1 localization affects protein synthesis.......................................... 155

6.5.3 Identify if Cdh1-APC solely regulates protein synthesis via FMRP......................... 157

6.5.4. Elucidate if Cdh1 regulates spine density downstream of FMRP............................. 158

6.6. Concluding Remarks........................................................................................................ 159

6.7. Figures.............................................................................................................................. 161

References.................................................................................................................................. 165

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	Ph.D.
Submission	Dissertation
Language	English
Keyword	FMRP
Committee Chair / Thesis Advisor	Gary J. Bassell, Emory University
Committee Members	Victor Faundez, Emory University Yue Feng, Emory University Nicholas T. Seyfried, Emory University Chad McKinley Hales, Emory University

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