Inhibition of the schizophrenia-associated microRNA miR-137 disrupts Nrg1α neurodevelopmental signal transduction Public

Thomas, Kristen Therese (2017)

Permanent URL: https://etd.library.emory.edu/concern/etds/3b591947g?locale=fr
Published

Abstract

Defects in neural development play a well-substantiated but poorly understood role in schizophrenia etiology. Genomic studies have repeatedly associated variants in the gene encoding microRNA miR-137 with schizophrenia, but whether miR-137 contributes to the dysregulation of neurodevelopmental signal transduction in schizophrenia is unknown. Bioinformatic predictions suggest that the PI3K-Akt-mTOR branch of neuregulin (Nrg)/ErbB signaling may be particularly enriched with miR-137 targets. In the present study, we demonstrate that miR-137 regulates neuronal levels of p55γ, PTEN, Akt2, GSK3β, mTOR, and rictor. Inhibition of miR-137 ablates Nrg1α-induced increases in dendritic protein synthesis, phospho(Ser235/236)-S6, AMPA receptor subunits, and dendritic outgrowth, demonstrating a profound disruption of Nrg/ErbB signaling. Furthermore, inhibition of miR-137 blocks mTORC1-dependent responses to BDNF, including increased protein synthesis and dendritic outgrowth, while leaving mTORC1-independent S6 phosphorylation intact. Together, our results demonstrate that miR-137 regulates neuronal responses to Nrg1α and BDNF through convergent mechanisms and provide a novel mechanism by which miR-137 may regulate neural development and contribute to schizophrenia.

Table of Contents

Table of Contents

Chapter 1 General Introduction......................................................................................... 1

1.1 miRNAs: history and significance............................................................................ 2

1.2 miRNA biogenesis and mRNA targeting................................................................... 4

1.3 miRNA mechanisms of action.................................................................................. 8

1.4 miRNAs play diverse roles in the regulation and dysregulation of neuronal signaling 10

1.4.1 miRNAs regulate neuronal signaling.............................................................. 11

1.4.2 Neuronal signaling regulates the miRNA pathway......................................... 17

1.4.3 miRNAs regulate the neuronal circuitry......................................................... 21

1.5 The schizophrenia-associated miRNA miR-137 regulates neuronal signaling......... 25

1.5.1 miR-137 and 1p21.3 deletion syndrome........................................................ 25

1.5.2 miR-137 and schizophrenia........................................................................... 26

1.5.3 miR-137 in neuronal signaling and synaptic function.................................... 30

1.5.4 miR-137 target predictions and pathway analysis.......................................... 33

1.6 Dissertation Hypothesis and Objectives.................................................................. 34

1.7 Materials and Methods............................................................................................ 35

1.8 Figures.................................................................................................................... 36

1.9 Tables..................................................................................................................... 38

Chapter 2 miR-137 targets mRNAs encoding proteins within the

PI3K-Akt-mTOR pathway................................................................................................ 40

2.1 Introduction............................................................................................................ 41

2.1.1 The PI3K-Akt-mTOR pathway: general structure and function..................... 41

2.1.2 The PI3K-Akt-mTOR pathway in neuronal signaling..................................... 44

2.1.3 The PI3K-Akt-mTOR pathway in neurodevelopmental disease..................... 45

2.1.4 miR-137 and the PI3K-Akt-mTOR pathway.................................................. 46

2.1.5 Chapter 2 hypothesis and objectives.............................................................. 47

2.2 Results.................................................................................................................... 47

2.2.1 miR-137 negatively regulates neuronal p55γ levels....................................... 48

2.2.2 miR-137 negatively regulates neuronal PTEN levels...................................... 49

2.2.3 miR-137 negatively regulates neuronal Akt2 levels....................................... 49

2.2.4 miR-137 negatively regulates neuronal GSK3β levels................................... 49

2.2.5 miR-137 negatively regulates neuronal rictor levels....................................... 50

2.2.6 miR-137 negatively regulates neuronal mTOR levels.................................... 50

2.3 Discussion.............................................................................................................. 51

2.4 Materials and Methods............................................................................................ 54

2.5 Figures.................................................................................................................... 60

2.6 Supplemental Figures.............................................................................................. 66

Chapter 3 Inhibition of miR-137 blocks Nrg1 signaling.................................................. 67

3.1 Introduction............................................................................................................ 68

3.1.1 The Nrg/ErbB pathway: general structure and function................................. 68

3.1.2 The Nrg/ErbB pathway in neural development and function......................... 70

3.1.3 The Nrg/ErbB pathway in psychiatric disease................................................ 72

3.1.4 Chapter 3 hypothesis and objectives.............................................................. 73

3.2 Results.................................................................................................................... 74

3.2.1 Nrg1α increases dendritic phospho-S6 by an ErbB

receptor-dependent mechanism..................................................................... 74

3.2.2 Nrg1α increases dendritic phospho-S6 and mRNA translation

by a miR-137 dependent mechanism............................................................. 75

3.2.3 Nrg1α increases dendritic phospho-S6 and mRNA translation

by an mTORC1-dependent mechanism......................................................... 76

3.2.4 Nrg1α increases dendritic MAP2 by a miR-137-dependent mechanism......... 76

3.2.5 Nrg1α does not affect total or dendritic levels of miR-137............................ 77

3.2.6 Nrg1α increases dendritic outgrowth by an ErbB-dependent mechanism....... 77

3.2.7 Nrg1α increases dendritic outgrowth by a miR-137-dependent mechanism... 78

3.2.8 Nrg1α increases dendritic outgrowth by an mTOR-dependent mechanism.... 78

3.2.9 Nrg1β increases dendritic outgrowth by a miR-137-dependent mechanism... 79

3.3 Discussion.............................................................................................................. 79

3.3.1 The role of Nrg1α.......................................................................................... 79

3.3.2 Significance for schizophrenia...................................................................... 81

3.3.3 Model............................................................................................................ 82

3.3.4 Alternative mechanism.................................................................................. 83

3.4 Materials and Methods............................................................................................ 84

3.5 Figures.................................................................................................................... 89

3.6 Supplemental Figures............................................................................................ 103

Chapter 4 Regulation of AMPA receptor subunits by miR-137 and Nrg1α.................. 107

4.1 Introduction.......................................................................................................... 108

4.1.1 AMPAR subunits: general structure and function........................................ 108

4.1.2 AMPAR subunits in neurodevelopmental disease........................................ 110

4.1.3 Regulation of GluA1 by miR-137................................................................ 112

4.1.4 Regulation of AMPAR subunits by Nrg1/ErbB signaling............................. 112

4.1.5 Chapter 4 hypothesis and objectives............................................................ 113

4.2 Results.................................................................................................................. 113

4.2.1 miR-137 targets the mouse Gria1-3'UTR..................................................... 113

4.2.2 Nrg1α increases total GluA1/2 protein levels in neurons............................. 115

4.2.3 Nrg1α increases GluA1/2 protein by a miR-137-dependent mechanism...... 115

4.3 Discussion............................................................................................................ 116

4.3.1 miR-137 regulates the AMPAR subunits GluA1 and GluA2........................ 117

4.3.2 Nrg1α, miR-137, and GluA1 regulate dendritic outgrowth

and synaptic plasticity: a shared mechanism?.............................................. 118

4.3.3 Implications for neurodevelopmental disorders........................................... 119

4.4 Materials and Methods.......................................................................................... 120

4.5 Figures.................................................................................................................. 125

4.6 Supplemental Figures............................................................................................ 132

Chapter 5 miR-137 regulates mTOR-dependent responses to BDNF........................... 133

5.1 Introduction.......................................................................................................... 134

5.1.1 BDNF signaling: general structure and function........................................... 134

5.1.2 BDNF in neurodevelopmental disease......................................................... 135

5.1.3 Chapter 5 hypothesis and objectives............................................................ 137

5.2 Results.................................................................................................................. 137

5.2.1 BDNF stimulates dendritic outgrowth by an mTOR-

and miR-137-dependent mechanism........................................................... 138

5.2.2 BDNF stimulates S6 phosphorylation by an mTOR-

and miR-137-independent mechanism........................................................ 139

5.2.3 BDNF stimulates mRNA translation in dendrites by

a miR-137-dependent mechanism............................................................... 139

5.3 Discussion............................................................................................................ 140

5.3.1 Future directions.......................................................................................... 142

5.3.2 Significance for neurodevelopmental disorders........................................... 143

5.4 Materials and Methods.......................................................................................... 144

5.5 Figures.................................................................................................................. 146

5.6 Supplemental Figures............................................................................................ 151

Chapter 6 General Discussion........................................................................................ 152

6.1 Summary.............................................................................................................. 153

6.2 A novel role for miR-137 in neuronal signaling and neurodevelopment............... 153

6.3 Significance for schizophrenia and other neurodevelopmental disorders............. 154

6.3.1 miR-137 and 1p21.3 deletion syndrome...................................................... 154

6.3.2 miR-137 and schizophrenia......................................................................... 155

6.3.3 miR-137 and Fragile X Syndrome............................................................... 156

6.4 Future Directions.................................................................................................. 158

6.4.1 Examine the regulation of neuronal signaling by miR-137 in disease models 158

6.4.2 Explore the relationship between miR-137 and FMRP................................. 160

6.4.3 Identify the mechanism by which miR-137 disrupts

PI3K-Akt-mTOR signaling........................................................................... 162

6.5 Concluding Remarks............................................................................................ 164

6.6 Figures.................................................................................................................. 166

References....................................................................................................................... 167


Figures and Tables

Chapter 1 General Introduction

Figure 1.1 miR-137 targets within the Nrg/ErbB, BDNF, and LTP pathways................ 36

Figure 1.2 Proposed relationship between miR-137 targets within the

PI3K-Akt-mTOR branch of Nrg/ErbB signaling........................................... 37

Table 1.1 miR-137 targets are enriched in pathways relevant to neuronal signaling...... 38

Table 1.2 Predicted miR-137 targets within the PI3K-Akt-mTOR branch

of Nrg/ErbB signaling.................................................................................... 39

Chapter 2 miR-137 targets mRNAs encoding proteins within the PI3K-Akt-mTOR pathway

Figure 2.1 miR-137 targets the PIK3R3-3'UTR and regulates neuronal p55γ protein.... 60

Figure 2.2 miR-137 targets the PTEN-3'UTR and regulates neuronal PTEN protein..... 61

Figure 2.3 miR-137 regulates neuronal Akt2 protein.................................................... 62

Figure 2.4 miR-137 targets the GSK3B-3'UTR and regulates neuronal GSK3β

protein levels without affecting phosphorylation at Ser9............................... 63

Figure 2.5 miR-137 targets the RICTOR-3'UTR and regulates neuronal rictor protein.. 64

Figure 2.6 miR-137 regulates neuronal mTOR levels but not phosphorylation at Ser2448........... 65

Supplemental Figure 2.1 Validation of pre-miR-137 overexpression plasmid used

in neuronal experiments................................................................................ 66

Chapter 3 Inhibition of miR-137 blocks Nrg1 signaling

Figure 3.1 Timeline for experiments using acute Nrg1α stimulation.............................. 89

Figure 3.2 Nrg1α increases dendritic phospho(Ser235/236)-S6 by an ErbB

receptor-dependent mechanism..................................................................... 90

Figure 3.3 Inhibition of miR-137 blocks Nrg1α-induced increases in dendritic

phospho(Ser235/236)-S6 and mRNA translation........................................... 91

Figure 3.4 Inhibition of mTORC1 blocks Nrg1α-induced increases in dendritic

phospho(Ser235/236)-S6 and mRNA translation........................................... 93

Figure 3.5 Nrg1α increases dendritic MAP2 by a miR-137-dependent mechanism....... 95

Figure 3.6 Experimental timeline and Sholl methodology for Nrg1α morphology

experiments................................................................................................... 96

Figure 3.7 Nrg1α increases dendritic outgrowth in DIV4 primary cortical neurons

by an ErbB receptor-dependent mechanism.................................................. 97

Figure 3.8 Nrg1α increases dendritic outgrowth by a miR-137-dependent mechanism. 98

Figure 3.9 Inhibition of mTOR blocks Nrg1α-induced dendritic outgrowth................ 100

Figure 3.10 Inhibition of miR-137 blocks Nrg1β-induced dendritic outgrowth........... 101

Figure 3.11 Model for miR-137 regulating Nrg1α signal transduction......................... 102

Supplemental Figure 3.1 Inhibition of miR-137 increases dendritic GSK3β without

blocking Nrg1α-induced dephosphorylation at Ser9.................................... 103

Supplemental Figure 3.2 Model for Nrg1α regulating miR-137 levels or activity........ 104

Supplemental Figure 3.3 Acute Nrg1α treatment does not affect total or dendritic

levels of miR-137........................................................................................ 105

Supplemental Figure 3.4 Two-day Nrg1α treatment does not affect miR-137 levels... 106

Chapter 4 Regulation of AMPA receptor subunits by miR-137 and Nrg1α

Figure 4.1 miR-137 inhibits Gria1-3'UTR luciferase reporter activity........................ 125

Figure 4.2 miR-137 associates with Gria1 mRNA in vitro in a site and

sequence-specific manner........................................................................... 126

Figure 4.3 miR-137 inhibits GluA1 and GluA2 protein levels in cortical neurons...... 127

Figure 4.4 Nrg1α increases GluA1 and GluA2 protein levels without affecting

mRNA levels............................................................................................... 128

Figure 4.5 Inhibition of miR-137 blocks Nrg1α-induced increases in dendritic

GluA1 and GluA2....................................................................................... 129

Figure 4.6 Inhibition of miR-137 blocks Nrg1α induced GluA1 synthesis.................. 130

Figure 4.7 Model for miR-137 regulating Nrg1α-induced changes in GluA1 levels.... 131

Supplemental Figure 4.1 Nrg1α does not affect miR-137's ability to assemble

on Gria1 mRNA in vitro.............................................................................. 132

Chapter 5 miR-137 regulates mTOR-dependent responses to BDNF

Figure 5.1 BDNF stimulates dendritic outgrowth by an mTOR- and miR-137-

dependent mechanism................................................................................. 146

Figure 5.2 BDNF stimulates S6 phosphorylation by an mTOR- and miR-137-

independent mechanism.............................................................................. 148

Figure 5.3 BDNF stimulates mRNA translation in dendrites by a miR-137-

dependent mechanism................................................................................. 150

Supplemental Figure 5.1 30 min treatment with BDNF does not affect miR-137

levels in cortical neurons............................................................................. 151

Chapter 6 General Discussion

Figure 6.1 Proposed roles for miR-137 at the glutamatergic synapse 166

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