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Distal axon and neuromuscular junction degeneration in amyotrophic lateral sclerosis


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Southam, KA 2014 , 'Distal axon and neuromuscular junction degeneration in amyotrophic lateral sclerosis', PhD thesis, University of Tasmania.

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The cause of axon degeneration in ALS remains poorly understood. In recent years it has
become clear that the onset of symptoms in ALS occurs after a potentially lengthy presymptomatic
period in which key processes become dysfunctional, notably axonal
transport, regulation of calcium and communication between motor neurons and nonneuronal
cells. This thesis aims to investigate the relationship between excitotoxicity,
non-neuronal cells and degeneration of the motor neuron axon. Substantial evidence implicates glutamate excitotoxicity in the pathogenesis of ALS.
However, the mechanism by which excitotoxicity results in axon degeneration is not well
understood. This thesis has utilised primary cell culture techniques and
immunocytochemistry to investigate the effect of targeted excitotoxin exposure to
cortical neurons. Excitotoxicity in the somatodendritic compartment resulted in
degeneration of the untreated distal axon and extensive degeneration of neuronal
structures in the treated compartment. However, targeted excitotoxicity to the distal axon
also resulted in degeneration of the axon, in the absence of degenerative changes to the
untreated somatodendritic compartment. Immunocytochemical and western blot analysis
indicated distally mediated excitotoxicity likely occurred via the AMPA receptor. In
addition, distally triggered degeneration occurred in a caspase-dependent manner. Degeneration of the neuromuscular junction occurs early in the development of ALS,
occurring in conjunction with die-back of the motor neuron distal axon. This thesis has
examined a wide range of neuromuscular junction-associated proteins in the mSOD1
G93A mouse model of ALS. Analysis of key structural proteins at the gastrocnemius
muscle indicated very early differences in the structure of the mSOD1 G93A
neuromuscular junction relative to the wild-type controls. For some structural components (rapsyn and nestin), the differences were most apparent at the onset of
physical symptoms (12 weeks), however for nerve terminals and Schwann cells,
significant differences were apparent as early as 8 weeks of age. Most previous research
has focused on loss of the presynapse from the post-synaptic acetylcholine receptors
(AChR) as a marker for loss of the neuromuscular junction, however this thesis indicates
that many key structural proteins are affected in the early stage of ALS prior to AChR
changes. Targeting such proteins may provide a novel therapeutic target for treatment in
In this thesis, primary cell culture techniques have been used to develop a novel in vitro
model for motor neurons, incorporating glial cells, motor neurons and skeletal muscle
with the spatial organisation as occurs in vivo. Spinal motor neurons co-cultured with
either glial cells or skeletal muscle under standard culture conditions developed different
morphological characteristics, with the cell feeder layer affecting the development of
neurites, axonal extension and survival during early development. Incorporation of
spatially organised cells resulted in improved survival of motor neurons, whilst
promoting robust axonal extension. The formation of rudimentary neuromuscular
junctions within the distal compartment indicated maturation of the circuit. These
cultures are the first to replicate the spatial organisation of the lower motor
neuron/neuromuscular junction circuit within an in vitro model. Additionally, such
preparations are achieved without the addition of extraneous growth factors, known to
affect later maturation of the motor neuron. Targeted excitotoxicity to the
somatodendritic compartment of mature cultures resulted in significant axon degeneration
and loss of proximal structures (soma, dendrites and axons), however targeted
excitotoxicity to the distal axon did not result in distal axon degeneration. These results indicate a number of novel findings: Firstly, the distal axon of cortical
neurons is capable of mediating excitotoxicity in a caspase-dependent manner. Secondly,
degeneration of the neuromuscular junction in vivo is preceded by changes to the
underlying structures. Thirdly, appropriate growth of motor neurons in vitro requires both
the presence of glial and muscle cells, and relevant spatial organisation of these cells.
Targeted excitotoxicity to motor neurons yielded a different response to that from
compartmented cortical cells. In conclusion, this thesis demonstrates the importance of
often under-recognised components of the lower-motor neuron - neuromuscular junction
circuit in the pathogenesis of ALS, culminating in the development of a novel cell culture
preparation for the investigation of spinal motor neurons in vitro. In addition, this thesis
has uncovered two novel sites for potential therapeutic intervention in neurodegenerative
disease; the cortical neuron axon and structural changes at the neuromuscular junction.

Item Type: Thesis - PhD
Authors/Creators:Southam, KA
Keywords: motor neuron disease, ALS, neuromuscular junction, axon degeneration, excitotoxicity, microfluidic chamber
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