An In Vivo Model to Study the Regulation of FMRP Restricted; Files Only

Santoro, Michael Robert (2016)

Permanent URL: https://etd.library.emory.edu/concern/etds/st74cq65s?locale=en
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Abstract

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability. The syndrome results from the lack of fragile X mental retardation protein (FMRP), an mRNA-binding protein which plays a key role in learning and memory. FMRP's activity seems to be regulated by the phosphorylation of a key serine (Ser499 in the mouse). Previous work in cell culture and fruit flies suggests that when FMRP is phosphorylated at this residue it represses the translation of its target mRNAs and that dephosphorylation of the protein releases this repression, allowing the synthesis of new proteins. In this project I created a line of phosphomutant mice to further study the regulation of FMRP. Specifically, I mutated Ser499 to alanine, creating a constitutively unphoshporylated form of the protein. Surprisingly, these mice did not resemble either WT or KO mice in an assay of new protein synthesis. They were indistinguishable from WT mice in terms of susceptibility to audiogenic seizures, macroorchidism, and behavioral assays. These results suggest that the current view of FMRP regulation may be incomplete. The protein may be phosphorylated differently in vivo or alternative isoforms of FMRP may play a more important role than has previously been appreciated.

Table of Contents

Chapter 1 Introduction........................................................................................ 1

Identification of FXS as a Distinct Syndrome......................................................................... 1

Identification of the FMR1 Gene.......................................................................................... 2

Animal Models of FXS........................................................................................................ 4

FMRP Expression and Functional Protein Domains................................................................. 5

FMRP is an mRNA-binding Protein...................................................................................... 8

FMRP is a Translational Repressor...................................................................................... 10

Mechanisms of Translational Repression............................................................................... 11

mGluR Theory of Fragile X Syndrome................................................................................ 13

Phosphorylation Regulates FMRP-Mediated Translation Inhibition.......................................... 15

Chapter 2 Phosphomutant FMRP..................................................................... 18

Creation of Mice............................................................................................................... 20

Phenotype of Mice............................................................................................................. 33

Discussion........................................................................................................................ 41

Chapter 3 Polyribosome Profiles................................................................. 47

Background...................................................................................................................... 47

Polyribosome Profiles on Brain Lysate................................................................................. 51

Polyribosome Profiles in Cortical Neurons........................................................................... 54

Polyribosome Profiles in MEFs........................................................................................... 55

In Vitro Run-Off Assays.................................................................................................... 56

Discussion........................................................................................................................ 57

Chapter 4 Western Blots.................................................................................... 61

Loading Controls............................................................................................................... 61

DARPP32 and SHANK1 Levels in Crude SNS..................................................................... 70

MAP1B Levels in Hippocampi............................................................................................ 74

Discussion........................................................................................................................ 75

Chapter 5 Perspectives........................................................................................ 77

Measuring FXS Phenotypes................................................................................................ 77

Alternative Isoforms of FMR1............................................................................................ 78

Further Uses of Ser499Ala Mice......................................................................................... 79

Conclusions...................................................................................................................... 80

Chapter 6 Materials and Methods................................................................ 82

Mouse Husbandry.............................................................................................................. 82

Cloning Fmr1 Gene........................................................................................................... 82

PCR of pStart-K................................................................................................................ 84

Site Directed Mutagenesis................................................................................................... 84

Insertion of Neomycin Resistance Cassette............................................................................ 86

Creation of Probes for Southerns......................................................................................... 87

Digestion of ES Cell DNA for Southern Blots...................................................................... 89

Digestion of Tail-Snip DNA for Southern Blots.................................................................... 89

DNA Gel for Southerns...................................................................................................... 89

Probe Labeling by Nick Translation..................................................................................... 90

Southern Blot.................................................................................................................... 90

Mouse Genotyping by PCR/Digest of Exon 15...................................................................... 91

Genotyping Phosphomutant Mice by PCR............................................................................ 92

Western Blot of pFMRP/FMRP........................................................................................... 92

Reverse Transcription of Fmr1 mRNA................................................................................. 94

PCR of Fmr1 cDNA.......................................................................................................... 95

qPCR of Fmr1 Isoforms..................................................................................................... 95

Audiogenic Seizures.......................................................................................................... 96

SNS Protein Synthesis....................................................................................................... 96

Marble Burying................................................................................................................. 97

Nestlet Shredding.............................................................................................................. 97

Testis Mass....................................................................................................................... 98

Statistics........................................................................................................................... 98

Polyribosome Profiles with Tris.......................................................................................... 98

Polyribosome Profiles with HEPES..................................................................................... 99

Polyribosome Profiles Using IVTEBP Method...................................................................... 100

Polyribosome Profiles on MEFs........................................................................................ 101

Polyribosome Profiles on Neurons..................................................................................... 102

Western Blot of Sucrose Fractions..................................................................................... 103

cDNA Synthesis from Polyribosome Profile Fractions......................................................... 104

qPCR of Polyribosome Profile Fractions............................................................................ 104

Standard Curves on Film.................................................................................................. 105

Total Protein Stain Using Ponceau S.................................................................................. 106

Standard Curve Using Fluorescent Westerns....................................................................... 106

Western Blots of Crude SNS............................................................................................. 107

Western Blots of MAP1B in Hippocampi........................................................................... 109

References................................................................................................................ 110

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