Characterizing Motor Neuron-specific mRNA Processing Defects in an Intermediate Mouse Model of Spinal Muscular Atrophy Öffentlichkeit

Abstract

Spinal Muscular Atrophy (SMA) is a neuromuscular disease characterized by a progressive loss of spinal motor neurons and consequently, a loss of locomotor abilities. SMA is directly caused by reduced levels of the ubiquitously expressed survival of motor neuron (SMN) protein, yet the molecular mechanisms by which reduced levels of SMN cause the dysfunction and degeneration of motor neurons remain elusive. By combining motor neuron–specific tagging of ribosomes with affinity purification of translating ribosomes, we have performed a comprehensive RNA-seq study to establish the profile of ribosome-bound mRNAs, or “translatome”, in spinal motor neurons at pivotal time points in an intermediate mouse model of SMA. At postnatal day 9 (P9), before the onset of any disease-associated symptoms, we observed an early and persistent upregulation of transcripts involved in p53-mediated signaling pathway. We also observed a reduction of several markers of motor neuron subpopulations, including Matrix Metalloproteinase-9 (MMP9) at P9 and P19, suggesting that fast fatigable motor neurons may be more vulnerable to SMN depletion. Further analysis of splicing alterations present in our SMA translatome profiles identify novel SMN-dependent splicing perturbations that may influence SMA pathology in motor neurons. Finally, we adopted a powerful method to evaluate changes to the proteome specifically in SMA motor neuron in vivo. Taken together, data from these studies provide a comprehensive assessment of motor neuron-specific changes in mRNA processing at early and severe time-points in disease in vivo, revealing novel targets that may contribute to motor neuron degeneration in SMA.

Table of Contents

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

Abstract ............................................................................................................................................ 2

1.1 SMA clinical background ......................................................................................................... 3

1.1.2 SMA is caused by reduced SMN protein levels ....................................................... 3

1.2 Animals Models Recapitulate SMA Phenotypes ...................................................................... 4

1.3 SMA Pathology is Not Limited to Motor Neurons .................................................................... 8

1.4 SMA is caused by reduced RNP assembly ................................................................................ 9

1.5 The SMN complex is an assembly machine for spliceosomal snRNPs ................................... 11

1.5.1 SMA deficiency causes widespread splicing defects ............................................... 13

1.6 RNA localization is integral to neuronal developmental and function .................................... 15

1.6.1 Assembly and transport of neuronal mRNPs .......................................................... 18

1.6.2 Mechanisms of mRNA trafficking and local translation in axons ........................... 21

1.6.3 SMN and Motor Neuron Susceptibility .................................................................. 22

1.6.4 SMN acts as a molecular chaperone for the assembly of mRNPs .......................... 24

1.7 Other mRNA-processing functions of SMN ............................................................................ 29

1.8 Rationale, Hypothesis, and Objectives ................................................................................... 31

1.8.1 Rationale ................................................................................................................. 31

1.8.2 Hypothesis ............................................................................................................... 32

1.8.3 Objectives ................................................................................................................ 32

1.9 Figures ..................................................................................................................................... 33

Chapter 2: Characterization of Motor Neuron-specific mRNA processing and localization defects in a mouse model of Spinal Muscular Atrophy .......................................................................................... 40

Abstract .......................................................................................................................................... 41

2.1 Introduction .............................................................................................................................. 42

2.2 Results ..................................................................................................................................... 46

2.2.1 Translatome profiling of SMA motor neurons at early symptomatic and severe time points ................................................................................................................................. 46

2.2.2 Upregulation of p53-mediated signaling precedes the onset of NMJ denervation and motor neuron loss .............................................................................................................. 48

2.2.3 Down-regulated transcripts suggests SMN-deficiency selectively affects specific motor neuron subtypes ...................................................................................................... 49

2.2.4 Translatome Findings Reflect Changes in Protein Expression ............................... 50

2.2.5 Aberrant Splicing Can be Detected in the Translatome of Early Symptomatic and Late Symptomatic Motor Neurons .................................................................................... 51

2.3 Discussion ............................................................................................................................... 53

2.4 Tables and Figures .................................................................................................................. 61

2.5 Characterizing the Axonal Translatome .................................................................................. 73

2.5.1 Results ...................................................................................................................... 73

2.5.2 Discussion ................................................................................................................ 76

2.5.3 Tables and Figures ................................................................................................... 79

2.6 Methods .................................................................................................................................. 85

Chapter 3: Examining the Motor Neuron-specific Proteome in a mouse model of SMA ................. 90

3.1 Characterization of the motor neuron-specific proteome in SMA .......................................... 90

3.2 Conclusions and Future Directions .......................................................................................... 91

3.3 Methods ................................................................................................................................... 92

3.4 Figures ..................................................................................................................................... 95

Chapter 4: Future Directions and Concluding Remarks ...................................................................... 99

4.1 Comprehensive characterization of spinal motor neuron subtypes in SMA .......................... 100

4.2 Explore translatome differences between cholinergic neurons in the brain of SMA mice .... 101

4.3 AAV-mediated Rescue of Translatome Candidates in SMA Mice ....................................... 102

4.4 Concluding Remarks ..............................................................................................................103

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Cell Biology, Neuroscience Biology, Molecular
Stichwort	MetRS Translatome Survival of Motor Neuron SMA SMN RiboTag Motor neurons Spinal muscular atrophy
Committee Chair / Thesis Advisor	Bassell, Gary , Emory University Rossoll, Wilfried, Mayo Clinic
Committee Members	English, Arthur , Emory University Feng, Yue, Emory University Alvarez, Francisco, Emory University

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