Investigating the role of SOD1 in sporadic amyotrophic lateral sclerosis patients using structural proteomics

Background

Amyotrophic lateral sclerosis (ALS) is a complex neurological disorder characterised by the progressive degeneration of motor neurons. SOD1 (superoxide dismutase 1) was the first familial genetic risk factor identified for ALS and accounts for 20% of familial ALS cases. Neurodegeneration in these patients is thought to be caused by a toxic gain of function of misfolded SOD1, mediated by mutations that disrupt metal ion binding, formation of disulphide bonds and dimerisation.

Recent phase 1/2/3 trials of an antisense oligonucleotide (Tofersen) targeting SOD1 in patients with mutations in the SOD1 gene showed an improvement in motor function as well as biomarkers of target engagement and therapeutic efficacy. However, most patients have sporadic (sALS), so a genetically targeted therapeutic strategy is not possible.

SOD1 is abundant in the CNS (approximately 1 percent of brain protein) and is a protein particularly susceptible to oxidative post-translational modification and misfolding. Once misfolded SOD1 can propagate in a prion like fashion between CNS cells. 

There is evidence that SOD1 function in sALS is important. Secretomes from SOD1 and sporadic ALS patient-derived astrocytes, when co-cultured with healthy control motor neurons, cause toxicity. Knockdown of SOD1 in these astrocytes reduces this secretome-mediated toxicity. Furthermore, we have shown that astrocytes from sALS patients exhibit aggregates of misfolded SOD1. We, therefore, hypothesise that SOD1 dysfunction in sALS is important and that Tofersen may have the potential to be used in the treatment of at least some cases of sALS where the genetic cause is unknown.

Objectives

• Investigate structural changes of SOD1 protein in stem cell-derived astrocytes from SOD1-ALS and sALS patients versus healthy controls.

• Identify which structural changes are also present in CSF from the same patients used to generate astrocytes.

• Assess whether structural differences in SOD1 CSF can be used to stratify which sALS cases may be suitable for therapeutic targeting of SOD1.

Experimental Approach

This project will exploit a unique resource of CSF samples and reprogrammed astrocytes from the same cohort of ALS patients. To assess structural changes in SOD1 in these samples, we will use immunopurification of SOD1 coupled with covalent protein painting and quantitative proteomics. In this strategy, the conformation of a protein is inferred by the relative accessibility of protein residues to chemical labelling. The abundance of labelled residues is then determined by quantitative mass spectrometry of digested proteins. In CSF samples, we will also use a global strategy to assess structural changes in all proteins (including SOD1) to provide an unbiased assay of protein conformation changes in sALS. The project will also use immunofluorescence microscopy to assess SOD1 localisation/aggregation, assays to measure the binding of SOD1 to metal ions, as well as SOD1 activity and glutathione oxidation assays in astrocytes.

Specialist training and career development

The supervisors bring together expertise from clinical neurology, stem cells and disease modelling and proteomics. Hence the project will require specialist training to develop a variety of skills. This interdisciplinary project combines molecular & cell biology with protein biochemistry and state-of-the-art quantitative analytical methods (i.e., mass spectrometry-based proteomics). In-depth training and access to the mass spectrometry facility will be provided by the Co-Supervisor (MC). Given the nature of the data produced using these approaches, you will also be trained in statistical analysis and computational biology.

You will attend weekly lab meetings and monthly meetings with the supervisory team. You will have the opportunity to present your work at local symposia and national/international conferences (e.g., the MNDA International Symposium on ALS/MND), where you will have opportunities to grow your scientific network and identify career opportunities post-PhD.

Entry Requirements:

Candidates must have a first or upper second-class honours degree or significant research experience.

How to apply:

Please complete a University Postgraduate Research Application form available here: www.shef.ac.uk/postgraduate/research/apply

Please clearly state the prospective main supervisor in the respective box and select Neuroscience as the department.

Enquiries:

Interested candidates should in the first instance contact Professor Shaw ([Email Address Removed])

Proposed start date: 01/10/2023