title: Investigation of the pathological mechanisms in canine degenerative myelopathy and the potential involvement of extracellular vesicles in disease progression
Creators: name: Black, Lauren Elaine
Publisher: name: University of Glasgow
Year: 2024

description: Canine degenerative myelopathy (DM) is a progressive and lethal adult-onset neurodegenerative disease with nonspecific clinical signs that can end in tetraplegia and respiratory dysfunction. It is frequently identified in German shepherd dogs (GSD) but has been described in other breeds. A definitive diagnosis is reached after histopathological examination of the spinal cord where axon degeneration and demyelination are characterised. Mutations in the gene encoding superoxide dismutase 1 (SOD1) are thought to have a pathological role in the disease and genotyping of the Sod1 gene can be used with clinical signs and histology to diagnose DM. The genetics, clinical signs and histology of DM suggests it may be a good naturally occurring large animal model for some forms of the human motor neurone disease, amyotrophic lateral sclerosis (ALS). This study aimed to 1) establish if defective cellular clearance pathways play a role in the aggregation of SOD1 and 2) if these defects contribute to the secretion of SOD1 positive extracellular vesicles (EVs) that 3) can spread mutant protein in a prion-like manner. Finally, 4) was to validate these findings with spinal cord tissue from DM cases using proteomics and biochemistry.

In vitro studies using a neuroblastoma derived cell line (SK-N-SH) were conducted to assess the effect of disrupting various cell clearance and toxicity pathways on wildtype (WT) and mutant SOD1 aggregation and EV production. The reducing agent dithiothreitol increased the propensity of WT- and DM-SOD1 to aggregate (p ≤ 0.01) but did not have a statistically significant impact on the production of SOD1 positive EVs from cells. The autophagy inhibitor chloroquine increased the percentage of cells with DM-SOD1 aggregates (p ≤ 0.01), but not WT-SOD1 aggregates. EV secretion was not statistically significantly affected by chloroquine treatment in cells with DM-SOD1, but there was a significant increase in the EV marker flotillin-1 from cells containing WT-SOD1 (p ≤ 0.01). The proteasome inhibitor MG312 significantly increased the number of cells with WT- and DM-SOD1 aggregates (p &lt; 0.0001), but they were higher in DM-SOD1 transfected cells (p ≤ 0.05). Flotillin-1 showed a downward trend from treated cells however this was only statistically supported with EVs from WT-SOD1 containing cells (p ≤ 0.01). WT- and DM-SOD1 showed an upwards trend in the EV fraction, but only reached significance in EVs from the cells containing DM-SOD1 (p ≤ 0.01). Overall, disruption to the main protein processing pathways caused the induction of nontypical clearance pathways and some of these appear to be less effective in the presence of the DM associated Sod1 mutation. Further, mutant SOD1 may have an impact on the stabilisation of the cell membrane as indicated by changes to associated proteins and this could have subsequent effects on protein clearance, particularly at the level of the endosome pathway and EVs. Further studies indicated there is a potential for EVs to spread WT- and DM-SOD1 to other cells in culture which suggests EVs could be recruited in DM for the spread of mutant SOD1 to other cells and may contribute to the progression of DM throughout the thoracic spinal cord and to the cervical and lumbar regions.

Biochemical and proteomics analysis of spinal cords from control and DM dogs suggested axon and myelin integrity was disrupted and astrocytes were activated at early stages of DM. Evidence suggested these changes were the consequence of altered cellular metabolism, intracellular structure and protein processing. The Sod1 mutation caused an apparent reduction in SOD1 enzyme activity further suggesting the mutation is a contributor to the pathogenesis and progression of DM. Changes to plasma membrane organisation were also highlighted in the ex vivo study and may indicate perturbations to protein and lipid turnover.

Ultimately the findings presented in this thesis contribute to the understanding of DM pathogenesis and will aid the search for DM biomarkers to enable earlier diagnosis, monitor disease progression and identify treatment targets.