The pathophysiological role of TDP43 in amyotrophic lateral sclerosis due to C9orf72 mutations

Amyotrophic lateral sclerosis (ALS), the commonest type of motor neuron disease, is a devastating, form of specific neurodegeneration leading to progressive weakness and death from respiratory failure a median of 30 months from symptom onset. It affects one in 400 people during their lifetime. There are currently no effective treatments. The cause for the disease is complex and poorly understood, but recently there have been major breakthroughs which open up significant new possibilities for developing more effective treatments. The most important discovery is the finding of an error in a gene called C9orf72, which affects up to 10% of all patients with ALS. As with many newly discovered mutations, its effects on cells and the specific reason why motor neurons are vulnerable are still poorly understood.

At the Oxford University Nuffield Department of Clinical Neuroscience we will study this mutation and its effects on human nerve cells by taking skin samples form patients and transforming them into stem cells. We will then grow the stem cells into "motor" nerve cells, the special type of cell affected in ALS. This approach has the advantage of allowing us to study the mutation in the specific cell type that is affected in this condition. The other advantage is that we will be studying human tissue that has come from patients who have developed the disease. Using the cells we have grown, we will describe the mutation in detail. Then we will use gene therapy techniques to reverse the effects of the mutation to provide the initial steps towards exploring whether gene therapy has a role to play in treating ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative neurological disorder that predominantly affects the motor system and results in death within 1-5 years of onset. There are no effective treatments. Its pathophysiology is poorly understood and multiple hypotheses exist. There is abundant evidence that RNA splicing is defective in ALS. This is supported by the fact that the signature protein found in the inclusions in ALS, TDP-43, is an RNA binding protein. The most common mutation in ALS patients is a hexanucleotide repeat in the intron of C9orf72, found in 40% of familial and 7% of sporadic cases. Patients with C9orf72 mutations have a characteristic clinical picture and have TDP-43 positive inclusions, such as those found in the majority of ALS patients, but also have atypical TDP43 negative inclusions. The function of C9orf72 as well as its relationship with TDP-43 are still unknown. We will investigate this using the two following hypotheses:

Hypothesis 1: Motor neurons with the C9orf72 mutation have different physiology and pathology from sporadic ALS cells and controls. This will be tested using motor neurons derived from iPS cells derived from relevant patients and controls from the ALS clinic. I will analyse survival, TDP43 pathology, neurophysiology and response to cellular stress. Results will be validated using the TDP43 mouse model available in the host laboratory, a C9orf72 mouse model in development and human autopsy findings.

Hypothesis 2: The C9orf72 mutation interferes with splicing of mRNA species normally targeted by TDP-43. I will extract RNA from the above cell lines and look for splicing abnormalities using RNA sequencing. I will then create antisense oligonucleotides to silence the mutation and analyse for reversal of the effects identified.

We propose basic research into the molecular pathophysiology of amyotrophic lateral sclerosis (ALS). Its outcome will not have a direct commercial or therapeutic exploitation. If successful, this project will provide the first evidence of the use of gene therapy and antisense oligonucleotide to treat ALS in vitro.
Future studies will then be needed to establish their benefit in multicellular organisms and disease models, before eventually therapeutic applications can be considered in the longer term. In the long term this research could potentially benefit 10-20% of future patients with ALS after 10 to 20 years.
This research is of particular importance to patient organisations such as the motor neuron disease association, as it offers hope for a potentially disease modifying drug in the field and as such is of interest to patients, families and donors.