Regulation of translation in neuronal function and development Público
Lau, Anthony G. (2010)
Abstract
Regulation of translation in neuronal function and
development
By Anthony G. Lau
Brain development and function requires sophisticated regulation of
gene
expression achieved by both transcriptional and
post-transcriptional mechanisms. While
transcriptional regulation can have long-term effects on gene
expression, rapid and local
changes in protein levels, far beyond the capabilities of
transcription, are due to intricate
mechanisms of post-transcriptional regulation and essential for
brain function. However,
key mechanisms underlying post-transcriptional regulation in
neurons remain poorly
understood. In this dissertation, the roles of both cis- and
trans-acting factors controlling
translation and subcellular localization of mRNA were explored
during brain
development and function. First we examined the ability of distinct
3' untranslated
regions (3'UTRs), long or short, in differential regulation of
translation and subcellular
localization of transcripts encoding the brain-derived neurotrophic
factor (BDNF) upon
neuronal stimulation. We showed that the short 3'UTR restricts BDNF
mRNA in
neuronal soma and supports active BDNF translation while the long
3'UTR promotes
dendritic localization and represses translation of BDNF mRNA at
rest. We further
demonstrated that, neuronal activation releases translational
repression of the long 3'UTR
BDNF mRNA, which is accompanied by a rapid activation of the
tropomyosin kinase
receptor B in the hippocampus.
We next explored the role of the fragile X mental retardation
protein (FMRP), a
selective RNA-binding protein, in hippocampal development. Loss of
FMRP resulted in
dysregulated translation of the microtubule associated protein 1B,
which is associated
with abnormal projection of mossy fiber axons in the neonatal
hippocampus, which offers
a comprehensive model for the functional consequence of
translational dysregulation in
abnormal neuronal network development due to FMRP deficiency.
Lastly, in an effort to
elucidate trans-acting factors mediating translational regulation
of the long 3'UTR BDNF
mRNA, we found that the long 3'UTR BDNF mRNA is associated with
FMRP as well as
a specific microRNA, miR-128. In addition, miR-128 suppresses
translation of luciferase
reporter in a BDNF long 3'UTR -dependent manner, and neuronal
activation causes
significant down-regulation of miR-128. Taken together, these data
provide novel
insights regarding cis- and trans-acting factors that control
translation to accommodate
brain development and function.
Table of Contents
Table of Contents
CHAPTER 1: Introduction to
Dissertation.................................................................1
1.1 Introduction: regulation of mRNA translation and
localization governs neuronal
development and
plasticity...........................................................................................2
1.1.1 Activity-dependent protein synthesis and local mRNA
translation in synaptic
plasticity
..................................................................................................................3
1.1.2 Synaptic circuitry and function of hippocampal neurons
.................................6
1.1.3 Cis- and trans-acting factors that control mRNA translation
and localization ..7
1.2 Brain-Derived Neurotrophic Factor (BDNF) expression and the
functional link to
translational regulation
..............................................................................................11
1.2.1 BDNF gene expression and post-translational processing
.............................11
1.2.2 BDNF signaling and physiological functions
................................................15
1.2.3 Role of BDNF in the
synapse........................................................................16
1.2.4 Regulation of BDNF expression
...................................................................18
1.2.4.1 Transcriptional
regulation..........................................................................18
1.2.4.2 Post-transcriptional regulation of BDNF
mRNA........................................20
1.2.5 BDNF in
disease...........................................................................................23
1.3 The fragile X mental retardation protein (FMRP) governs
neuronal translation and
brain function
............................................................................................................24
1.3.1 Fragile X syndrome and the role of FMRP in translation
regulation..............24
1.3.2 FMRP in synaptic development and
function................................................28
1.4 MicroRNA in
neurons.........................................................................................31
1.4.1 MicroRNA, a novel class of small non-coding RNA that controls
brain
development and function
......................................................................................31
1.4.2 MicroRNA in synaptic development and
function.........................................33
1.5 Goals for Dissertation
.........................................................................................34
CHAPTER 2: The distinct 3'UTRs differentially regulate
subcellular localization
and translation of BDNF mRNA in the
hippocampus..........................................38
2.1 Introduction
........................................................................................................39
2.2
Results................................................................................................................41
2.2.1 Long 3'UTR BDNF mRNA is preferentially localized to dendrites
in primary
cultured
neurons.....................................................................................................41
2.2.2 The long 3' UTR of BDNF mRNA is a bona fide cis-acting
translation
repressor at rest
......................................................................................................45
2.2.3 Neuronal activity causes translation derepression of the long
3' UTR BDNF
mRNA.
..................................................................................................................51
2.2.4 The BDNF long 3'UTR is essential for seizure-induced rapid
TrkB activation
in hippocampal mossy fibers
..................................................................................61
2.3 Discussion
..........................................................................................................70
2.4 Materials and
Methods........................................................................................77
Chapter 3: Deficiency of the fragile X mental retardation
protein results in
dysregulated MAP1B translation and aberrant mossy fiber
projection during
hippocampal
development.....................................................................................87
3.1 Introduction
.........................................................................................................88
3.2
Results................................................................................................................90
3.2.1 FMRP represses translation of MAP1B mRNA
............................................90
3.2.2 FMRP siRNA promotes neurite extension and resistance to
neurite retraction
in response to microtubule disruption in cultured
cells............................................91
3.2.3 Knocking down MAP1B expression results in aberrant neurite
protrusion in
culture....................................................................................................................99
3.2.4 Loss of FMRP leads to aberrantly elevated MAP1B protein
levels in
hippocampal mossy fiber axons during neonatal
development..............................102
3.2.5 Colocalization of FMRP and MAP1B mRNA in the mossy fiber
terminals.105
3.2.6 Abnormal projection of MFs and increased ZnT3 in the mossy
fibers of Fmr1
KO mice during hippocampal
development..........................................................110
3.3 Discussion
........................................................................................................114
3.4 Materials and
Methods......................................................................................117
Chapter 4: Trans-acting factors potentially involved in
regulating the translation
of the long 3'UTR BDNF mRNA
........................................................................123
4.1 Introduction
......................................................................................................124
4.2
Results..............................................................................................................126
4.2.1 FMRP preferentially associates with the long 3' UTR BDNF
mRNA..........126
4.2.2 miR-128 specifically represses translation of luciferase
reporter that harbors
the BDNF long 3'
UTR.........................................................................................127
4.2.3 Expression of miR-128 in the
brain.............................................................133
4.2.4 Down-regulation of miR-128 level upon neuronal stimulation
....................136
4.3 Discussion
........................................................................................................139
4.4 Materials and
Methods......................................................................................144
Chapter 5: Conclusion and Future Directions
........................................................151
Chapter 6: References
..............................................................................................157
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