Bidirectional control of dendritic mRNA translation, glutamate receptor expression, and synapse structure by the CPEB-associated polyadenylation machinery Open Access

Swanger, Sharon (2012)

Permanent URL: https://etd.library.emory.edu/concern/etds/s1784m43w?locale=en%5D
Published

Abstract

Bidirectional control of dendritic mRNA translation, glutamate receptor expression, and synapse structure by the CPEB-associated polyadenylation machinery


Neurons are highly polarized cells that extend elaborate dendritic arbors and have
thousands of synaptic inputs. The post-transcriptional control of gene expression through
dendritic mRNA localization and local protein synthesis is an important means for
regulating postsynaptic protein expression. Moreover, translational control of dendritic
mRNAs is essential for certain forms of synaptic plasticity, learning, and memory. CPEB
(cytoplasmic polyadenylation element binding protein) is one RNA binding protein that
regulates local translation in dendrites as well as synaptic structure and function.
However, the mechanism by which it regulates these processes is unknown. Herein, we
identify a poly(A) polymerase, a deadenylase, and the translation inhibitory factor
neuroguidin as components of a dendritic CPEB-associated polyadenylation complex.
Synaptic stimulation induces phosphorylation of CPEB, expulsion of the deadenylase
from the ribonucleoprotein complex, and mRNA polyadenylation in dendrites.
Furthermore, these CPEB-associated translation factors bidirectionally regulate dendritic
spine morphology as well as AMPA receptor surface expression in cultured hippocampal
neurons. One CPEB target mRNA is that encoding GluN2A, which is an NMDA receptor
subunit and a critical regulator of synapse function and plasticity. We found that GluN2A
mRNA is localized to dendrites and associates with CPEB. The dendritic transport and
local translation of GluN2A mRNA is regulated target sequence within
GluN2A mRNA. The CPEB-associated poly(A) polymerase promotes dendritic GluN2A
protein expression and surface expression of GluN2A-containing NMDA receptors;
whereas, the negative translation factor neuroguidin inhibits GluN2A expression in
dendrites and at the cell surface. Moreover, protein synthesis and this poly(A) polymerase
are required for activity-induced translation of GluN2A and membrane insertion of
GluN2A-containing NMDA receptors. These results identify a pivotal role for dendritic
mRNA polyadenylation and the opposing effects of CPEB-associated translation factors
in regulating receptor expression and synapse structure at glutamatergic synapses as well
as activity-induced membrane insertion of NMDA receptors during synaptic plasticity.

Table of Contents

TABLE OF CONTENTS

Page
CHAPTER ONE: General Introduction........................................................................ 1
1.1 The significance of protein synthesis in synaptic plasticity, learning, and
memory........................................................................................................................ 4
1.2 Activity-induced local protein synthesis in dendrites................................................. 5
1.3 mRNA localization to dendrites.................................................................................. 8
1.3.1 Activity-induced dendritic mRNA transport....................................................... 9
1.3.2 Cis-acting elements mediate mRNA-specific dendritic transport....................... 11
1.4 Specific mRNAs are translated within dendrites........................................................ 13
1.5 General translational control mechanisms.................................................................. 15
1.5.1 The translation process........................................................................................ 15
1.5.2 General mechanisms controlling translation initiation........................................ 17
1.6 mRNA-specific mechanisms of translational control................................................. 18
1.6.1 RNA binding proteins and ribosome recruitment................................................ 19
1.6.2 Cytoplasmic polyadenylation............................................................................... 21
1.7 Bidirectional control of dendritic mRNA translation by mRNA binding
proteins........................................................................................................................ 22
1.7.1 RNA binding proteins mediate dendritic mRNA transport................................. 23
1.7.2 Translational repression and synaptic activation of local protein synthesis........ 24
1.8 Cytoplasmic polyadenylation element binding protein............................................... 26
1.8.1 The CPEB family of RNA binding proteins........................................................ 26
1.8.2 The role of CPEB in synaptic plasticity, learning, and memory......................... 27

1.8.3 Synaptic activity regulates CPEB phosphorylation............................................. 28
1.8.4 CPEB-mediated regulation of mRNA transport and translation.......................... 29
1.8.5 Putative CPEB-associated translational regulators in the brain........................... 31
1.9 Thesis hypothesis and objectives................................................................................ 31
CHAPTER TWO: The CPEB-associated polyadenylation complex is regulated by
activity and controls dendritic mRNA polyadenylation................................................ 38
2.1 Introduction..............

................................................................................................... 39
2.2 Results......................................................................................................................... 41
2.2.1 The cytoplasmic polyadenylation machinery is found in dendrites and at
synapses............................................................................................................... 41
2.2.2 Interaction and co-localization of CPEB complex proteins in neurons............... 43
2.2.3 PARN is expelled from the polyadenylation complex following NMDA-
induced CPEB phosphorylation........................................................................... 45
2.2.4 Dendritic polyadenylation is induced by NMDAR activation and
bidirectionally regulated by PARN and Gld2...................................................... 46
2.2.5 NMDA-induced polyadenylation is dependent upon CPEB phosphorylation.... 48
2.3 Discussion................................................................................................................... 78
2.4 Experimental Procedures............................................................................................. 82
CHAPTER THREE: Gld2 and Ngd bidirectionally regulate dendritic spine
morphology and AMPA receptor surface expression in hippocampal neurons......... 88
3.1 Introduction................................................................................................................. 89
3.2 Results......................................................................................................................... 91
3.2.1 CPEB-associated translation factors regulate dendritic spine morphology......... 91

3.2.2 Gld2 and Ngd bidirectionally regulate GluA1 surface expression...................... 93
3.3 Discussion................................................................................................................... 99
3.4 Experimental Procedures............................................................................................. 103
CHAPTER FOUR: CPEB and associated translation factors regulate activity-
induced dendritic GluN2A mRNA translation and NMDA receptor membrane
insertion............................................................................................................................... 106
4.1 Introduction................................................................................................................. 107
4.2 Results......................................................................................................................... 113
4.2.1 CPEB interacts with GluN2A mRNA.................................................................. 113
4.2.2 GluN2A mRNA is localized to dendrites............................................................ 114
4.2.3 CPEB and the CPE sequence regulate dendritic localization of GluN2A
mRNA.................................................................................................................. 115
4.2.4 Gld2 and Ngd bidirectionally regulate dendritic GluN2A protein expression.... 116
4.2.5 Gld2 and Ngd bidirectionally regulate the surface expression of GluN2A-
containing NMDA receptors................................................................................ 116
4.2.6 Chemical LTP induces a protein synthesis-dependent increase in GluN2A-
containing NMDA receptor surface expression................................................... 117
4.2.7 Chemical LTP induces CPEB phosphorylation and dendritic mRNA
polyadenylation....................................................................................................119
4.2.8
Gld2 depletion occludes and Ngd depletion potentiates the chemical LTP-
induced synthesis and surface expression of GluN2A......................................... 119
4.2.9 Gld2 is required for increased dendritic GluN2A expression during chemical
LTP...................................................................................................................... 120

4.2.10 GluN2A mRNA is translated in dendrites in a CPE-dependent manner........... 120
4.3 Discussion................................................................................................................... 151
4.4 Experimental Procedures............................................................................................. 156
CHAPTER FIVE: General Discussion............................................................................. 164
5.1 RNA binding proteins mediate mRNA transport and bidirectional translational
control in dendrites...................................................................................................... 166
5.2 Stimulus-specific control of local protein synthesis by RNA binding proteins.......... 169
5.3 Synaptic regulation by mRNA binding proteins in health and disease....................... 170
5.4 Input-specific local protein synthesis.......................................................................... 175
5.5 Concluding remarks..................................................................................................... 177
REFERENCES................................................................................................................... 184
APPENDIX 1: Automated 4D analysis of dendritic spine morphology........................ 219
A1.1 Introduction.............................................................................................................. 220
A1.2 Results and Discussion............................................................................................. 222
A1.2.1 Automated detection and 3D measurement of dendritic spines......................... 222
A1.2.2 Automated tracking of dendritic spines in live neurons..................................... 226
A1.2.3 Acute BDNF treatment induces synapse maturation through spine
remodeling.......................................................................................................... 228
A1.2.4 Inhibiting PI3 kinase activity rescues dendritic spine defects in neurons from
Fmr1 KO mice.................................................................................................... 234
A1.3 Conclusions.............................................................................................................. 237
A1.4 Experimental Procedures.......................................................................................... 250
A1.5 References................................................................................................................ 255

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