Regulation of A-type potassium channel Kv4.2 expression by FMRP and miR-324-5p Público

Yao, Xiaodi (2012)

Permanent URL: https://etd.library.emory.edu/concern/etds/3197xm16b?locale=es
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Abstract

Regulation of A-type potassium channel Kv4.2 expression

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability
that is caused by loss of the Fragile X mental retardation protein (FMRP), a RNA binding
protein involved in various aspects of mRNA regulation. One prominent characteristic of
FXS is high susceptibility to seizure resulting from neuronal hyperexcitability; yet the
underlying molecular mechanisms remain elusive. Potassium channels play critical roles
in controlling neuronal excitability. Recent findings suggest that FMRP might directly
regulate voltage-gated potassium channels. Voltage-gated Kv4.2 potassium channel is the
major component mediating the A-type K+ outward current thereby controlling neuronal
excitability in the hippocampus and has been implicated in epilepsy. Thus we
investigated the possible involvement of FMRP to regulate Kv4.2 that might explain the
high susceptibility to epilepsy in FXS. In this dissertation we uncovered two novel
posttranscriptional regulation mechanisms to modulate the expression of Kv4.2. We show
that FMRP associates with Kv4.2 mRNA and positively regulates Kv4.2 mRNA
translation and protein expression in neurons. Moreover, both total and cell surface
protein levels of Kv4.2 are reduced in a mouse model of FXS. Our results suggest that
loss of FMRP results in Kv4.2 downregulation which might contribute to excess neuronal
excitability in Fmr1 KO mice and thus imply a potential mechanism underlying FXS-
associated epilepsy. In contrast to FMRP, the microRNA (miRNA) miR-324-5p was
shown to be an inhibitory regulator for Kv4.2 expression in neurons. Our results show
that miR-324-5p targets to the 3'UTR of Kv4.2 mRNA and inhibits Kv4.2 protein
expression. Furthermore, miRNA-324-5p
overexpression reduced surface expression of Kv4.2 in neurons. Taken together, our
results suggest that miR-324-5p may regulate Kv4.2 expression and functions during
neuronal development and plasticity. Collectively, my thesis work provides new insights
into the molecular mechanisms in regulating neuronal excitability and eplepitogenesis.
Dysregulation of Kv4.2 expression through two mechanisms may be involved in epilepsy.
Our findings therefore provide a molecular basis for future studies toward a better
understanding the interplay of these mechanisms in the pathogenesis of FXS and other
forms of epilepsy.

Table of Contents


Table of Contents
Chapter I. General Introduction………………………………………………………..1

Section 1.1 Fragile X syndrome and FMR1 gene …………………….………..…2
Section 1.2 The FMRP Protein……………….......……………………………….4
Section 1.3 FMRP is an RNA binding protein………………………….………...5
Section 1.4 Functions of FMRP in mRNA metabolism and dynamics……...…..8
Section 1.4.1 FMRP represses translation………………………………9
Section 1.4.2 Possible mechanism of FMRP-mediated translational
inhibition………………………..………………………………………..13
Section 1.4.3 FMRP positively regulates translation……………….…...16
Section 1.4.4 FMRP regulates mRNA stability……………….…….…...17
Section 1.4.5 FMRP regulates mRNA transport…..………………….….18
Section 1.4.6 FMRP is involved in mRNA editing…………...…………21
Section 1.4.7 FMRP functions in protein-protein interaction…………....21
Section 1.5 Roles for FMRP in neuronal and synaptic function………..…….…23
Section 1.6 FMRP and epilepsy…………………………….……………….…..27
Section 1.7 Kv4.2 potassium channel, a possible link between FMRP and
epilepsy…………...…………….………………………………………………..28
Section 1.7.1 The role of Kv4.2 for synaptic function…………….…….29


Section 1.7.2 The role of Kv4.2 in epilepsy…………………….……....30
Section 1.8 Thesis objectives………………………………………………...….32
Section 1.9 Figures and legends…………………………………………………34
Chapter II. Fragile X Mental Retardation Protein Regulates Protein Expression and
mRNA Translation of the Potassium Channel Kv4.2...………………………………38

Section 2.1 Abstract………………………………………………………….…..39
Section 2.2 Introduction……………………..……………………………….…..40
Section 2.3 Materials and Methods……………………………………………....42
Section 2.4 Results…………………………………………………..……….…..45
Section 2.5 Discussion……………………………………………….......………49
Section 2.6 Acknowledges……………………………..…………………….…..52
Section 2.7 Figures and legends……………………………………………..…..53
Chapter III. miR-324-5p regulates the expression of the potassium channel
Kv4.2……………………………………………………………………………………62
Section 3.1 Introduction…………………………………………………….…..63
Section 3.2 Materials and Methods……………………………………………..67
Section 3.3 Results………………………………………………………….…..70
Section 3.4 Discussion…………………………………………….…...………..75
Section 3.5 Figures and legends………………………………………...……….79
Chapter IV. Summary and Future Directions………………………………………..87
Chapter V. References……………………………………………………..…….…….97


Table of Figures

Figure 1-1 A schematic view of the FMR1 gene……………………………………...…34
Figure 1-2 A schematic of FMRP protein …….……………………………………...….36
Figure 2-1 Kv4.2 protein levels are reduced in Fmr1 KO hippocampal dendrites ……...53
Figure 2-2 Reduced levels of Kv4.2 at neuronal cell surfaces in Fmr1 KO mice can be
partially rescued by antagonizing mGlu5. ……………………………………..……..…55
Figure 2-3 Kv4.2 mRNA translation is reduced in synaptic fractions from Fmr1 KO
cortices.………………………………………………………………………………..…57
Figure 2-4 FMRP regulation of Kv4.2 mRNA is mediated by both 5′- and 3′-UTRs of
Kv4.2 mRNA …………………………………………………………………………....59
Figure 3-1 Kv4.2 mRNA is a putative target of miR-324-5p. ………………………….79
Figure 3-2 Point mutation in the seed region of Kv4.2 mRNA abolishes the function of
miR-324-5p Kv4.2 mRNA. ………………………………………………..……………81
Figure 3-3 miR-324-5p is expressed in the hippocampus ………...………………….....83
Figure 3-4 miR-324-5p regulates the total and cell surface levels of Kv4.2. …………...85
Figure 4-1 Proposed models for the cooperation of FMRP and miR-324-5p in the
regulation of Kv4.2 expression.….………………………………………………….…...95

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