Investigating the role of adenosine deaminase in motor neuron disease

In a recent study published in Brain (https://doi.org/10.1093/brain/awy353) and highlighted in a number of media articles (www.sheffield.ac.uk/news/nr/disrupted-pathway-motorneurone-disease-1.830534) we have identifed a novel disruption in an energy generating pathway in astrocytes (neuronal support cells) derived from motor neurone disease (MND) patients. The pathway involves the conversion of the nucleoside adenosine to inosine, which is controlled by the enzyme adenosine deaminase (ADA). We have found that ADA is reduced in astrocytes derived from patients with a mutation that causes MND and in patients with no identified mutation. Loss of ADA would reduce inosine production and cause a build-up of adenosine that can be toxic in the central nervous system. The aim of this project is to further investigate this exciting novel discovery by ascertaining whether increasing ADA levels by gene therapy in MND patient derived astrocytes;
1. Reduces adenosine mediated astrocyte toxicity.
2. Increases astrocyte bioenergetic capacity.
3. Increases astrocyte antioxidant capacity.
4. Reduced DNA damage by increasing DNA repair mechanisms in astrocytes.
5. Increases astrocyte mediated support to motor neurones in co-culture.

The project will involve extensive tissue culture of induced neuronal progenitor derived astrocytes including lentiviral delivery of ADA targeted vectors. The project will include the use of cutting edge technologies to analyse cell function including an OmniLog™ phenotypic analyser, an XF24 metabolic flux analyser, an IN Cell automated microscope and 96 well plate fluorescent readers. Furthermore, the project will involve basic biochemical techniques such as SDS-PAGE, western blot and immunofluorescence. All protocols are in place (https://doi.org/10.1093/brain/awy353), as well as the expertise to modulate the level of ADA by gene therapy.

The project will be performed at the Sheffield Institute for Translational Neuroscience (SITraN), a world class neurodegenerative disease research institute, which is part of the Sheffield Biomedical Research Centre and the newly formed Neuroscience research
institute. https://www.sheffield.ac.uk/neuroscience-institute/home