Bidirectional control of dendritic mRNA translation, glutamate receptor expression, and synapse structure by the CPEB-associated polyadenylation machinery Pubblico
Swanger, Sharon (2012)
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
About this Dissertation
School | |
---|---|
Department | |
Subfield / Discipline | |
Degree | |
Submission | |
Language |
|
Research Field | |
Parola chiave | |
Committee Chair / Thesis Advisor | |
Committee Members |
Primary PDF
Thumbnail | Title | Date Uploaded | Actions |
---|---|---|---|
|
Bidirectional control of dendritic mRNA translation, glutamate receptor expression, and synapse structure by the CPEB-associated polyadenylation machinery () | 2018-08-28 14:40:59 -0400 |
|
Supplemental Files
Thumbnail | Title | Date Uploaded | Actions |
---|