Cu,Zn-Superoxide Dismutase (SOD1) and Oxidative Stress in the Pathogenesis of Axonal Degeneration Open Access

Fischer, Lindsey R. (2009)

Permanent URL: https://etd.library.emory.edu/concern/etds/9g54xj24w?locale=en
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Abstract

Axonal degeneration is a common pathologic feature in peripheral neuropathy and neurodegenerative disease. Despite progress in understanding downstream pathways of axon death, the initial trigger(s) for axonal degeneration in neurodegenerative diseases, and motor neuron disease specifically, are unknown. Growing evidence supports the idea that axons are vulnerable to oxidative stress-mediated injury, but models of peripheral neuropathy due to compromised antioxidant defenses are lacking.

Here, we demonstrate that genetic deletion of Cu,Zn-superoxide dismutase (SOD1) causes axonal degeneration both in vitro and in vivo. Sod1-/- dorsal root ganglia showed spontaneous axon degeneration in culture that was mimicked by siRNA knockdown of SOD1 and exposure to superoxide-generating herbicides. Sod1-/- primary motor neurons also showed poor axon outgrowth that was attenuated by antioxidant treatment. In vivo, denervation and morphologic abnormalities at hind limb neuromuscular junctions were seen by 4 months and progressed out to 18 months. Denervation correlated with oxidative stress in nerve, but not muscle, as determined by measurement of the GSH redox potential. Sensory fibers were spared in vivo. These data provide proof of principle that chronic oxidative stress is sufficient to cause a distal motor axonopathy in vivo.

We also investigated two strategies to rescue Sod1-/- axons. First, Sod1-/- mice were crossed with ‘slow Wallerian degeneration' (WldS) mice, an approach that conferred axonal protection in other models, but did not prevent axonal degeneration in the SOD1 KO. Next, a targeted approach was used to test the hypothesis that mitochondria are a source of superoxide leading to axonal degeneration in the Sod1-/- model. Sod1-/- mice were crossed with transgenic mice expressing wild-type SOD1 exclusively in the mitochondrial intermembrane space (IMS). Robust, long-term rescue of axons was observed. Thus, while SOD1 is normally found throughout the nucleus, cytoplasm, and IMS, restricted expression of SOD1 in the IMS is sufficient for the survival of motor axons. Future studies are needed to determine how IMS-SOD1 influences mitochondrial trafficking and function in axons, and to investigate whether oxidative damage in mitochondria contributes to axonal degeneration in other animal models and in human motor neuropathies.

Table of Contents

TABLE OF CONTENTS
Chapter I: Introduction ................................................................................1

Axonal degeneration..................................................................................1
Types of axonal degeneration......................................................................1
Programmed axon death ............................................................................3
Axonal degeneration in neurodegenerative disease.........................................7

Mechanisms of axonal degeneration .........................................................18
Summary.............................................................................................. 22
Oxidative Stress ......................................................................................23
Reactive oxygen species ............................................................................23
Superoxide dismutases...............................................................................27
SOD1 and motor neuron disease ...............................................................31
Neuromuscular phenotype of SOD1 KO mice ..........................................35
Summary....................................................................................................38
Research Overview................................................................................................39
Chapter II: Materials and Methods...............................................................................42
Animals..................................................................................................................42
Neuropathology .....................................................................................................44
Tissue analysis .......................................................................................................47
Primary neuronal cultures......................................................................................48
Statistical analysis..................................................................................................52
Chapter III: Loss of Cu,Zn-superoxide dismutase (SOD1) and superoxide-
generating herbicides cause axonal degeneration in mouse DRG cultures................53

Introduction………………………………………………………………………53
Results……………………………………………………………………………55
Discussion………………………………………………………………………..67
Chapter IV: Genetic deletion of SOD1 causes a distal motor axonopathy ................72
Introduction………………………………………………………………………72
Results……………………………………………………………………………74
Discussion………………………………………………………………………..91
Chapter V: WldS does not protect against oxidative stress-mediated axonal
degeneration in the SOD1 knockout model...................................................................97
Introduction………………………………………………………………………97
Results……………………………………………………………………………99
Discussion………………………………………………………………………108
Chapter VI: Targeted expression of SOD1 in the mitochondrial intermembrane
space is sufficient to prevent motor axonopathy in the SOD1 knockout mouse......113
Introduction..........................................................................................................113
Results .................................................................................................................114
Discussion............................................................................................................123
Chapter VII: Summary and future directions............................................................127
REFERENCES ..............................................................................................................130


LIST OF FIGURES

1.1. Early neuropathology from a human ALS case..........................................................10
1.2. NMJ immunocytochemistry from SOD1G93A gastrocnemius muscle.........................14
1.3. Light microscope images of L4 ventral roots from SOD1G93A mice..........................15
1.4. Nissl-stained motor neurons in the ventral horn of SOD1G93A lumbar spinal cord....16
1.5. Mitochondrial ROS release and compartmentalization of SOD enzymes..................29
1.6. SOD1 activity studies in red blood cells from FALS and SALS patients ..................33
3.1. SOD1 is required for DRG axon outgrowth and survival ..........................................56
3.2. Axonal degeneration due to loss of SOD1 precedes cell body degeneration .............58
3.3 Antioxidant protection in Sod1-/- DRGs .....................................................................60
3.4 Degeneration of mature DRG axons is triggered by siRNA knockdown of SOD1 ....62
3.5. Superoxide-generating herbicides paraquat (PQ) and diquat (DQ) are toxic to DRGs

in vitro....................................................................................................................65
3.6. DRG axons are more sensitive to PQ and DQ toxicity than their parent cell bodies .66
4.1. Systemic deletion of SOD1 causes a neuromuscular phenotype................................76
4.2. Sod1-/- mice display progressive denervation of hind limb muscles .........................77
4.3. Loss of SOD1 causes morphologic abnormalities at the NMJ ...................................79
4.4. Sod1-/- mice lack pathologic involvement of lumbar spinal cord and proximal
axons ...................................................................................................................83
4.5. Distal sensory fibers do not degenerate in Sod1-/- mice ............................................85
4.6. Loss of SOD1 leads to a more oxidized GSH redox state in peripheral nerve, but not
muscle, at 4 months ...............................................................................................88
4.7. Poor axon outgrowth in Sod1-/- primary motor neurons is rescued by antioxidant

treatment ................................................................................................................89
5.1. WldS does not prevent the onset of denervation in Sod1-/- mice..............................101
5.2. WldS does not prevent development of morphologic abnormalities in distal motor
axons of Sod1-/- mice by four months of age......................................................102
5.3. Sod1-/-, WldS mice retain the 'slow Wallerian degeneration' phenotype.................103
5.4. WldS does not alter the redox potential ( Eh) in Sod1-/- tissue, calculated from HPLC
measurement of GSH and GSSG.........................................................................105
5.5. WldS fails to protect Sod1-/- DRG axons..................................................................106
5.6. WldS fails to protect DRG axons against oxidative stress due to treatment with the
redox-cycling herbicide, paraquat (PQ)...............................................................107
6.1. Generation of IMS-SOD1 transgenic mice ..............................................................116
6.2. Breeding of IMS-SOD1 and Sod1-/- mice ...............................................................117
6.3 IMS-SOD1 rescues axon outgrowth in Sod1-/- primary motor neurons ...................120
6.4. IMS-SOD1 normalizes mitochondrial density in motor axons ...............................121
6.5. IMS-SOD1 is sufficient for long-term maintenance of NMJ innervation and
morphology in vivo ..............................................................................................122





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