Abstract
Amyotrophic lateral sclerosis (ALS) is the most common form of Motor Neuron Disease (MND) and has a devastatingly short prognosis of only 3-5 years post diagnosis. Around 5000 people are affected by MND every year in the UK and a person’s chances of developing MND is around 1 in 300. There are currently no effective treatments and in order to develop treatments we need to understand the disease better.Synaptopathies and synaptic loss are becoming increasingly recognised as central factors in neuropathies. Synaptic dysfunction and loss are thought to be one of the earliest pathological events in many neurodegenerative diseases, including ALS. Further, increased synapse loss has been shown in the prefrontal cortex of ALS patients who present with cognitive impairment, which around 50% of ALS patients experience in addition to impaired motor function. This cognitive impairment can be so severe that ~15% of ALS patients are diagnosed with co- morbid Frontotemporal Dementia (ALS-FTD).
The overarching aim of this project was to identify changes in the cortical synaptic proteome of ALS, whilst also identifying synaptic changes specific to the cognitive impaired phenotype of ALS and the presence of the most common genetic link to ALS and FTD, a C9ORF72 hexanucleotide repeat expansion (C9ORF72-HRE) . The rationale behind these aims was to identify molecular mechanisms that could be underlying this synaptopathy and synapse loss, as despite the profound synaptic pathology in both ALS and FTD, the underlying molecular mechanisms are still unknown. To address this, we generated synaptoneurosome (SNS) fractions from human primary motor cortex (BA4) and dorsolateral prefrontal cortex (BA9) from nonneurological controls (n=11) and ALS cases (n=18). ALS samples were stratified by the presence of cognitive impairment based on the Edinburgh Cognitive and Behavioural ALS Screen (ECAS) score and C9ORF72- HRE. Through pooling these samples and conducting TMT-LC MS/MS proteomics we identified >6000 proteins in our SNS samples. Enrichment analysis of our data showed high enrichment for synapses and synaptic processes, and we validated our approach with western blotting and array tomography. Further, we identified protein changes specific to stratifications of cognitive impairment and C9ORF72-HRE status. Utilisation of robust unbiased bioinformatic analyses identified protein expression clusters for group comparisons of interest, which were taken forward for further enrichment analysis. Subsynaptic localisation of dysregulated proteins were also identified in each comparison, revealing many postsynaptic protein changes in C9ORF72- HRE. Developing the field’s understanding of the molecular processes behind this synaptic pathological phenomenon, across multiple different ALS stratified cohorts, could assist in the development of therapeutics to target ALS pathology at its earliest stage to prevent or slow down disease progression.
In summary, this project provides the first ever characterisation of the human cortical ALS synaptic proteome. I have identified synaptic proteome changes specific to ALS, ALS with cognitive impairment (ALSci) and C9+ve ALS in both the primary motor cortex and the dorsolateral prefrontal cortex. Through robust bioinformatic analyses of these thresholded changed proteins I have identified molecular mechanisms specific to each ALS stratification, which shines a light on potential pathogenic mechanisms leading to the synaptopathy and synapse loss seen in ALS.
| Date of Award | 2024 |
|---|---|
| Original language | English |
| Supervisor | Christopher Henstridge (Supervisor) |