The Polyadenosine RNA Binding Protein ZC3H14 is Required in Mice for Proper Working Memory, Synaptosomal Composition, and Dendritic Spine Density and Morphology Pubblico
Jones, Stephanie (Fall 2020)
Abstract
ZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), an evolutionarily conserved polyadenosine (poly(A)) RNA binding protein, is lost in a form of heritable, nonsyndromic autosomal recessive intellectual disability. This observation shows that ZC3H14 is essential for proper brain function. To investigate the role of ZC3H14 in the mammalian brain, we have generated a novel Zc3h14∆ex13/∆ex13 mutant mouse model. Utilizing these mice, we provide the first in vivo functional characterization of ZC3H14. The Zc3h14∆ex13/∆ex13 mice show defects in working memory, suggesting that loss of ZC3H14 may affect brain regions associated with memory, such as the hippocampus. Analysis of Zc3h14+/+ and Zc3h14∆ex13/∆ex13 hippocampi show loss of ZC3H14 results in elongated bulk RNA poly(A) tails, decreased dendritic spine density both in situ and in vitro, and altered steady-state proteins to include postsynaptic proteins such as CaMKIIα. CaMKIIα is critical in synaptic plasticity, which is a process highly implicated to underlie learning and memory. Loss of ZC3H14 results in increased levels of CaMKIIα in synaptosomes, as well as reduction of in vitro mushroom spine density, which is rescued upon exogenous ZC3H14 expression. In addition, ZC3H14 associates with CaMKIIα mRNA and is detected in synaptosomes, suggesting CaMKIIα may be a ZC3H14 RNA target. Together, the results generated from this novel mouse model suggest that ZC3H14 is required in higher order brain function by regulating targets critical to proper neuronal function.
Table of Contents
Chapter 1: Introduction to Dissertation ………………………………………………………...................................................................................................................................................................... 1
1.1 RNA Binding Proteins Confer Fundamental Cellular Functions and Their Dysregulation Can Result in Major Tissue-Specific Disorders …………………………............................................. 2
1.2 Neurons Rely on Complex Regulation of RNA: Long-Distance mRNP Transport/Localization and Local Translation …………………………………………......................................................... 6
1.3 Dendritic Spines are a Spatiotemporally Dynamic Morphological Feature of Neurons ………………………………………………………………………………………….............................................. 11
1.4 Dendritic Spine Function and Structure is Dependent on Components of the Post-Synaptic Density ………………………………………………………………………............................................. 14
1.5 Spine Dynamics in Neuronal Disorders ……………………………………………............................................................................................................................................................................ 19
1.6 ZC3H14 Function in Neurons ……………………………………………………….............................................................................................................................................................................. 22
1.7 Summary and Prevailing Questions ……………………………………………….............................................................................................................................................................................. 27
Chapter 2: The RNA-binding protein, ZC3H14, is required for proper polyadenylation, expression of synaptic proteins, and brain function in mice ………………………………........................ 40
2.1 Introduction ………………………………………………………………………….................................................................................................................................................................................. 41
2.2 Results ……………………………………………………………………………….................................................................................................................................................................................... 44
2.2.1 Generation and confirmation of Zc3h14Dex13/Dex13 mice ………………….............................................................................................................................................................................. 44
2.2.2 ZC3H14 is not essential but is required for normal litter and testis size ………………………………………………………………………………….......................................................................... 46
2.2.3 Zc3h14Dex13/Dex13 mice show altered ventricles in the brain ………………............................................................................................................................................................................ 47
2.2.4 Zc3h14Dex13/Dex13 mice have impaired working memory but intact learning ………………………………………………………………………………….................................................................... 48
2.2.5 Zc3h14Dex13/Dex13 mice have normal visual function and exhibit normal motor function and coordination ……………………………………………………….................................................... 50
2.2.6 Zc3h14 is required for proper poly(A) tail length control ………………................................................................................................................................................................................. 51
2.2.7 Increased expression of synaptic proteins in Zc3h14Dex13/Dex13 mice …………………………………………………………………………………............................................................................... 52
2.3 Discussion …………………………………………………………………………..................................................................................................................................................................................... 56
Chapter 3: The Polyadenosine RNA Binding Protein ZC3H14 is Required in Mice for Proper Dendritic Spine Density ……………………………………………………………….................................. 81
3.1 Introduction ………………………………………………………………………….................................................................................................................................................................................. 82
3.2 Results ……………………………………………………………………………….................................................................................................................................................................................... 85
3.2.1 Spine density in the dentate gyrus is decreased in adult mice upon loss of ZC3H14 …………………………………………………………………………................................................................. 85
3.2.2 Loss of ZC3H14 does not alter dendritic arbor development in cultured primary hippocampal neurons …………………………………………………............................................................... 86
3.2.3 Loss of ZC3H14 causes a decrease in dendritic spine density in cultured primary hippocampal neurons ………………………………………………….............................................................. 87
3.2.4 Overexpression of ZC3H14 increases dendritic spine density, specifically by increasing the number of thin spines …………………………………………...................................................... 89
3.2.5 ZC3H14 is present in synaptosomes and loss of ZC3H14 results in increased steady state levels of CaMKIIα in synaptosomes …………………….............................................................. 90
3.3 Discussion …………………………………………………………………………..................................................................................................................................................................................... 91
Chapter 4: Discussion ………………………………………………………………………........................................................................................................................................................................... 110
4.1 Introduction ………………………………………………………………………................................................................................................................................................................................... 111
4.2 Discussion of Questions and Answers to the Main Thesis Hypothesis ………….................................................................................................................................................................... 112
4.2.1 Which step(s) in RNA regulation is ZC3H14 involved in in mice? ………………………………………………………………………………….................................................................................. 112
4.2.2 Does ZC3H14 regulate all polyadenylated RNA? ……………………….................................................................................................................................................................................. 115
4.2.3 What functional impact does loss of ZC3H14 have on higher order brain function? ……………………………………………………………………….................................................................. 118
4.2.4 Is loss of ZC3H14 accompanied by structural changes in the brain? ………………………………………………………………………………….............................................................................. 121
4.2.5 Does ZC3H14 affect neuron morphology? ……………………………...................................................................................................................................................................................... 125
4.2.6 Does ZC3H14 affect the proteomic composition of dendritic spines? …………………………………………………………………………………............................................................................ 131
4.3 Concluding remarks ………………………………………………………………................................................................................................................................................................................. 136
Chapter 5: Materials and Methods …………………………………………………………....................................................................................................................................................................... 140
5.1 Chapter 2 …………………………………………………………………………..................................................................................................................................................................................... 141
5.2 Chapter 3 …………………………………………………………………………..................................................................................................................................................................................... 154
Chapter 6: References ……………………………………………………………………….......................................................................................................................................................................... 160
List of Figures
Chapter 1:
Figure 1-1: Steps of post-transcriptional RNA processing, with examples of dysregulated RNA binding proteins resulting in tissue specific disorders ……………………………........................ 29
Figure 1-2: Dendritic spines serve as major postsynaptic sites of local translation …………………………………………………………………………………………......................................................... 31
Figure 1-3: General schematic of different dendritic spine morphology classifications ………………………………………………………………………………………….................................................. 32
Figure 1-4: Schematic of the brain-wide phases that regulate dendritic spine density over the course of life in mammals …………………………………………………………................................. 33
Figure 1-5: The role of CaMKII in cytoskeletal stabilization within dendritic spines …………………………………………………………………………………………...................................................... 34
Figure 1-6: Changes in dendritic spine density, morphology, and proportion of spine types associated with various neurological disorders over the course of neuronal development …………………………………………………………………………….......................................................................................................................................................................................................... 35
Figure 1-7: Evolutionarily conserved domain structure of ZC3H14 protein and its orthologues …………………………………………………………………………….................................................... 36
Figure 1-8: ZC3H14 domain structure and isoform alternative splicing ………………................................................................................................................................................................... 38
Figure 1-9: Pedigree of Family-1 and Family-2 of independent ZC3H14 mutation inheritance ………………………………………………………………………………................................................... 39
Chapter 2:
Figure 2-1: Generation of Zc3h14∆ex13/∆ex13 mice ………………………………………........................................................................................................................................................................ 62
Figure 2-2: Zc3h14∆ex13/∆ex13 mice are viable …………………………………………............................................................................................................................................................................ 64
Figure 2-3: Analysis of brain and hippocampal morphology in Zc3h14∆ex13/∆ex13 mice …………………………………………………………………………………………................................................... 65
Figure 2-4: Analysis of Zc3h14∆ex13/∆ex13 mice in behavioral paradigms ……………….................................................................................................................................................................. 67
Figure 2-5: ZC3H14 is required for proper poly(A) tail length control in mice …………………………………………………………………………………………................................................................ 69
Figure 2-6: Proteomic analysis of Zc3h14+/+ versus Zc3h14∆ex13/∆ex13 hippocampi …………………………………………………………………………………………......................................................... 71
Figure 2-7: Validation of proteomic changes in Zc3h14+/+ compared to Zc3h14∆ex13/∆ex13 hippocampi …………………………………………………………………………….......................................... 73
Figure 2-S1: Mass spectrometry analysis of Zc3h14∆ex13/∆ex13 truncation products …………………………………………………………………………………………........................................................ 74
Figure 2-S2: Statistical values for body weight ………………………………………............................................................................................................................................................................ 75
Figure 2-S3: Visual function and motor coordination assessments ……………………................................................................................................................................................................... 76
Figure 2-S4: Heatmap with hierarchical clustering of significantly different proteins ………………………………………………………………………………………….................................................... 78
Table 2-S1: General behavioral tests …………………………………………………............................................................................................................................................................................... 80
Chapter 3:
Figure 3-1: Zc3h14∆ex13/∆ex13 mice show decreased neuronal protrusions compared to control …………………………………………………………………………………............................................... 97
Figure 3-2: Primary hippocampal neurons from Zc3h14∆ex13/∆ex13 mice exhibit no significant difference in dendritic arborization at DIV12 …………………………........................................... 99
Figure 3-3: Primary hippocampal neurons from Zc3h14∆ex13/∆ex13 mice cultured in vitro (DIV19) shows a statistically significant decrease in total dendritic spine density …………………………………………………………………………………………........................................................................................................................................................................................ 101
Figure 3-4: Primary hippocampal neurons from Zc3h14∆ex13/∆ex13 mice cultured in vitro (DIV19) show a statistically significant decrease in mushroom-shaped spines that can be rescued by expressing ZC3H14 isoform 1 …………………………………………….............................................................................................................................................................................................. 102
Figure 3-5: Overexpression of ZC3H14 in cultured primary hippocampal neurons (DIV19) causes increased dendritic spine density ……………………………………........................................ 104
Figure 3-6: The increase in spine density detected in DIV19 primary hippocampal neurons that overexpress ZC3H14 is primarily due to an increase in the number of thin dendritic spines ……………………………………………………………………….............................................................................................................................................................................................................. 106
Figure 3-7: CaMKIIα is enriched in synaptosomal fraction from Zc3h14∆ex13/∆ex13 mice compared to control ………………………………………………………………….......................................... 108
Chapter 4:
Figure 4-1: Model of RNA Regulation by ZC3H14 in Neurons …………………….......................................................................................................................................................................... 138
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