Whole-Southam-thesis.pdf (11.53 MB)
Distal axon and neuromuscular junction degeneration in amyotrophic lateral sclerosis
thesisposted on 2023-05-27, 06:50 authored by Katherine SouthamKatherine Southam
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 ALS. 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.
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