Inhibition of the schizophrenia-associated microRNA miR-137 disrupts Nrg1α neurodevelopmental signal transduction Pubblico
Thomas, Kristen Therese (2017)
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.................................................................................. 81.4 miRNAs play diverse roles in the regulation and dysregulation of neuronal signaling 10
1.4.1 miRNAs regulate neuronal signaling.............................................................. 111.4.2 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.................................................................................................................... 361.9 Tables..................................................................................................................... 38
Chapter 2 miR-137 targets mRNAs encoding proteins within the
PI3K-Akt-mTOR pathway................................................................................................ 402.1 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.................................................. 673.1 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........................... 1335.1 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........................................................................................ 1526.1 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
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................................................................................................... 96Figure 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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