Investigating the Role of mitophagy in Amyotrophic Lateral Sclerosis - Biomedicine, PhD (GW4 BioMed MRC DTP)

About
Supervisory Team: Dr Akshay Bhinge
Location: Exeter, Streatham Campus

Project
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that is characterized by the relentless loss of motor neurons i.e. nerves that carry signals form the brain and spinal cord to the muscles. Patients suffer progressive muscle paralysis and usually die within 3 years of diagnosis due to respiratory failure. There are currently no effective treatments available for ALS. Understanding the molecular changes accompanying motor neuron death will be paramount in delivering drugs to halt or reverse the loss of neurons. Approximately 10% of ALS cases are familial with mutations identified in genes with diverse functions such as SOD1 and FUS, while the remaining 90% are termed sporadic i.e. these patients have no clear family history of the disease.

However, mitochondrial dysfunction accompanying neuronal hyperactivity has been observed in both familial and sporadic cases. In this project, the student will use a variety of cutting-edge techniques to identify the interplay of mitochondrial maintenance mechanisms and neuronal activity in causing motor neuron death in ALS. Recently, loss-of-function mutations causing ALS have been found in the genes TBK1 and OPTN that are involved in the mitophagy pathway. Mitophagy is a process via which the cell removes defective mitochondria. However, the mechanism through which these mutations cause disease is unknown. The project will first focus on creating induced pluripotent stem cells (iPSC) bearing loss-of-function mutations in TBK1 and OPTN via the CRISPR-Cas9 genome editing technology. Mutant iPSC will then be differentiated into spinal motor neurons and will be characterized for defects in the mitophagy pathway by measuring the mitochondrial membrane potential in live neurons as well as co-localization of mitochondria with autophagosomes and autolysosomes (part of the mitophagy machinery) using fluorescent microscopy.

Since mitochondrial health is intricately linked to neuronal activity and survival, mutant motor neurons will be investigated for aberrations in neuronal activity by electrophysiology and neuronal survival by immunofluorescence. Next, genome wide molecular changes due to the ALS causing mutations will be identified using RNA-sequencing. Network analysis based on the genomic data will be used to guide CRISPR screens to rescue mitophagy defects in ALS motor neurons. Top hits from the screen will be further explored to assay effects on neuronal firing and survival in vitro. To answer whether these defects are observed in other ALS mutations, the identified genes will be validated using motor neurons differentiated from mutant SOD1 and FUS iPSC that are already available.

This project adopts a multi-disciplinary approach to understanding the role of mitophagy in ALS and takes advantage of the diverse expertise of all three supervisors. The student will be trained in multiple state-of-the-art methods in the areas of disease modelling using iPSC, CRISPR-Cas9 mediated genome editing and screening, high-content imaging, mitochondrial biology, electrophysiology and bioinformatics. The results of this project are expected to deliver crucial insights linking mechanistic defects in mitophagy to neuronal activity that lead to death in ALS neurons. In addition, we are pursing a collaboration with a leading pharmaceutical company that will allow us to pursue candidate genes identified in this project into future translational studies.

Start date: October 2018

Most studentships will be 3.5 years full time or up to 7 years part-time, and can be longer where additional training is undertaken.



How to apply
APPLICATIONS OPEN ON 25 SEPTEMBER AND CLOSE AT 17:00 ON 24 NOVEMBER 2017.

IMPORTANT: In order to apply for this project, you should apply using the DTP’s online application form. More information on the application process may be found here: http://www.gw4biomed.ac.uk/projects-2/for-students/

You do NOT need to apply to the University of Exeter at this stage – only those applicants who are successful in obtaining an offer of funding from the DTP will be required to submit an application to study at Exeter.