Regulation of translation in neuronal function and development Open Access

Lau, Anthony G. (2010)

Permanent URL: https://etd.library.emory.edu/concern/etds/rj430484j?locale=en
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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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