Post-transcriptional regulation of BDNF and the Cdk5 pathway by the neuronal RNA binding protein HuD Open Access

Allen, Megan (2016)

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Establishment of the neuronal network is essential for brain function. This multifaceted process requires intricate regulation of gene expression through both transcriptional and post-transcriptional mechanisms. In particular, post-transcriptional regulation of mRNAs offers the advantage of rapid and local control of the cellular proteome. Such sophisticated regulation depends on both cis-regulatory elements and trans-acting factors, such as RNA-binding proteins (RBPs) and miRNAs. One key factor for neuronal development and function is the RBP, HuD, which binds target mRNAs to regulate stability and translation. The list of HuD ligands is rapidly expanding. However, how HuD regulates mRNA targets in vivo to coordinate sophisticated biological paradigms is unknown. In this dissertation we uncover HuD-dependent post-transcriptional regulation of two key pathways for neuronal development and function. We show that HuD selectively binds and stabilizes an mRNA isoform of the brain derived neurotrophic factor (BDNF) and therefore promotes expression of BDNF protein to be transported to hippocampal mossy fiber axonal boutons. We also discovered that post-transcriptional regulation by a HuD-miRNA molecular loop selectively up-regulates a Cyclin-dependent kinase 5 (Cdk5) activator called p39. This selective regulation occurs concurrently with histone-acetylation-dependent transcription of p39, which enhances Cdk5 activity in the postnatal brain. We reveal essential functions of the Cdk5 activator p39 in neuronal network formation. Moreover, HuD-regulated Cdk5 signaling governs the projection of hippocampal mossy fiber axons. Given the functional interplay between BDNF and Cdk5 signaling, the cooperative regulation of both pathways by HuD revealed by our studies provides an intriguing example of higher-order coordination of gene networks. Thus, these studies advance our understanding of how post-transcriptional regulation is employed to govern multifaceted aspects of neuronal function in the brain.

Table of Contents

Chapter 1: Introduction to Dissertation 1

1.1 Post-transcriptional regulation of cellular mRNAs is essential for the development and function of brain neurons 2

1.1.1 Molecular mechanisms that govern cellular mRNA behavior in neurons 2

1.1.2Trans-acting factors governing neuronal mRNA abundance81.1.3 Coordination between mRNA binding proteins and miRNAs 11

1.1.4 RBPs and miRNAs in neurological disease 12

1.2 HuD is a specific RNA-binding protein that governs neuronal development and function 15

1.2.1 Conservation and identification of the ELAV family of proteins151.2.2 HuD function in neuronal development and in the adult brain 17

1.2.3 Molecular mechanisms and mRNA targets of HuD regulation 19

1.2.4 HuD and neurological disorders 22

1.3 Cdk5 signaling, which is critical for brain development and is dysregulated in neurological disease, may be regulated by HuD 23

1.3.1 The discovery of Cdk5 and its activators, p35 and p39 24

1.3.2 Cdk5 activity regulates normal brain function 271.3.3 Cdk5 activity is dysregulated in neurological disease 301.3.4 The available amount of Cdk5 activators determines Cdk5 activity 32

1.3.5 Emerging evidence suggests post-transcriptional regulation of the Cdk5 activator, p35, by HuD 34

1.4 Summary and prevailing questions 35

Chapter 2: HuD promotes BDNF expression in brain neurons via selective stabilization of the BDNF long 3'UTR mRNA 52

2.1 Introduction 53

2.2 Results 54

2.2.1 HuD selectively enhances expression of the BDNF long 3'UTR reporter through a highly conserved ARE 54

2.2.2 HuD directly binds and stabilizes RNA that carries the ARE segment in the BDNF long 3'UTR 56

2.2.3 HuD expression levels selectively regulate the abundance of the endogenous BDNF long 3'UTR 58

2.2.4 A HuD transgene selectively up-regulates BDNF long 3'UTR mRNA and BDNF protein in the hippocampal mossy fiber pathway 59

2.3 Discussion 60

Chapter 3: HuD differentially regulates Cdk5 activator expression post-transcriptionally to control neuronal Cdk5 function 79

3.1 Introduction 80

3.2 Results 82

3.2.1 HuD stabilizes Cdk5 activator mRNAs in brain neurons through conserved 3'UTR AREs 82

3.2.2 HuD binds and increases the abundance of both p35 and p39 mRNAs but selectively up-regulates p39 protein in the hippocampus 84

3.2.3 miR-101a is up-regulated by HuD and selectively targets the p35 3'UTR to inhibit p35 protein expression 85

3.2.4 HuD-dependent upregulation of p39 enhances phosphorylation of Cdk5 targets and mossy fiber axonal projection in the hippocampus 86

3.3 Discussion 88

Chapter 4: p39 drives Cdk5 activity increase in postnatal neurons and governs neuronal network formation and epileptic response 116

4.1 Introduction 117

4.2 Results 118

4.2.1 Histone acetylation-mediated transcription selectively up-regulates p39 during neuronal differentiation 118

4.2.2 Loss of p39 attenuates overall Cdk5 activity but differentially affects phosphorylation of Cdk5 targets in brain neurons 119

4.2.3 Essential function of p39 in advancing axonal development, dendritic spine formation, and synaptogenesis 120

4.2.4 Loss of p39 results in dysregulation of Cdk5 targets at synapses and ameliorated response to pharmacologically induced seizure 122

4.3 Discussion 123

Chapter 5: Discussion: Concluding remarks, unresolved questions, and future directions 151

Chapter 6:Material and Methods 168

6.1 Chapter 2169

6.2 Chapter 3173

6.3 Chapter 4178

Chapter 7: References 184

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