Axonal transport of spinal muscular atrophy (SMA) disease protein survival of motor neuron (SMN) and rescue of axonal defects by the mRNA-binding proteins IMP1 and HuD Público

Abstract

Spinal muscular atrophy (SMA) is a fatal autosomal recessive genetic disorder that causes motor neuron degeneration and muscle atrophy. It is caused by a mutation or deletion in the survival of motor neuron (SMN1) gene and the consequent reduced levels of SMN protein. SMN is ubiquitously expressed, and its best-characterized function is in the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). Since SMN is essential for the survival of all cells, it is still unknown why motor neurons are selectively affected in SMA. SMN has been shown to localize to motor neuron axons, and to associate with mRNA-binding proteins, such as IMP1 and HuD, which are decreased in SMA motor neuron axons. This suggests an alternative role for SMN in the regulation of axonal mRNA localization, which is impaired in SMA. In this study, I explored this non-canonical role for SMN by overexpressing IMP1 and HuD in primary cultured spinal motor neurons from a severe SMA mouse model. I observed a rescue of axon length and axonal growth cone protein levels of GAP-43, a locally-translated protein important for axon outgrowth and cytoskeletal maintenance, providing further support for a role of SMN in axonal mRNA metabolism. I further investigated this pathway by adapting the recently-developed split kinesin assay for use in fixed primary spinal motor neurons. This is a promising new assay to potentially identify the motor proteins involved in SMN axonal transport. Finally, I conducted pilot experiments for the future use of γ-secretase modulators to increase axonal transport in murine SMA spinal motor neurons.

Table of Contents

INTRODUCTION ..........................................................................................................1

The roles of SMN .........................................................................................................1

Axonal defects in SMA animal models ...............................................................................3

IMP1 and HuD associate with SMN ...................................................................................4

Active transport of SMN in the axon ................................................................................5

Goals .........................................................................................................................6

MATERIALS AND METHODS ..........................................................................................7

SMA mouse model.........................................................................................................7

Primary motor neuron cell culture and transfection .............................................................7

Immunocytochemistry ...................................................................................................8

Image acquisition and analysis ........................................................................................8

Split kinesin assay.........................................................................................................9

Immunoprecipitation (IP) and western blot analysis ............................................................11

Drug treatment ...........................................................................................................11

Axon length analysis ....................................................................................................11

Statistical analysis .......................................................................................................12

RESULTS ....................................................................................................................12

1) Rescue of axonal defects in SMA motor neurons via IMP1 and HuD overexpression ................12

IMP1 and HuD overexpression rescues axon length defect in SMA motor neurons ......................12

Overexpression of IMP1 and HuD rescues GAP-43 protein levels in SMA motor neuron

growth cones ..............................................................................................................13

2) Identification of kinesin motor proteins that mediate the axonal transport of SMN .................16

Split kinesin assay ........................................................................................................16

The KIF1A tail domain is transported to axon terminals upon addition of the linker drug ..............16

Normally dendritically-localized Transferrin receptor is transported to the axonal growth cone

via dimerization of myc-KIF13B tail and KIF5C-tdTM motor domains ........................................18

SMN may associate with the KIF21B tail ............................................................................20

Modification to the split kinesin assay is required to test KIF5C association with SMN .................21

3) γ-secretase modulators as modifiers of axonal transport in primary motor neurons .................24

DISCUSSION ...............................................................................................................28

Overexpression of IMP1 and HuD rescues axonal defects in SMA motor neurons .........................28

The split kinesin assay is a promising new approach to study motor-dependent transport in

primary spinal motor neurons ............................................................................................29

KIF21B may be involved in the axonal and/or dendritic transport of SMN ...................................31

Modified assays are required to validate KIF5C as a motor protein for SMN axonal transport .........31

Pilot experiments show that Sfida compounds affect axonal transport in motor neurons ...............33

Conclusions ...................................................................................................................34

REFERENCES.................................................................................................................36

About this Honors Thesis

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School	Emory College
Department	Neuroscience and Behavioral Biology
Degree	BS
Submission	Honors Thesis
Language	English
Research Field	Biology, Neuroscience Biology, Cell
Palabra Clave	mRNA binding proteins kinesin SMN SMA local translation axonal transport mRNA localization
Committee Chair / Thesis Advisor	Rossoll, Wilfried O, Emory University Bassell, Gary, Emory University
Committee Members	Liu, Robert C, Emory University

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