"Establishing a Medium Spiny Neuron Differentiation Protocol for ADCY5 Disease Modeling"
Host Mentor: Jeanne Loring
Center for Regenerative Medicine, Scripps Research Institute
Familial Dyskinesia with Facial Myokymia (FDFM) is a rare autosomal dominant movement disorder characterized by involuntary tremors in the limbs and facial muscles with a wide range in severity of symptoms. Mutations in a gene highly expressed in the striatum, the adenylate cyclase 5 gene (ADCY5), have been linked to FDFM with the most severe phenotype associated with a gain of function mutation linked to elevated cAMP levels at amino acid 418 (arginine to tryptophan). For this reason, iPSCs generated from patient and relative control cells will be differentiated into the principal projection neuron of the striatum, the medium spiny neuron (MSN). The dose and duration of purmorphamine for patterning was optimized with existing iPSCs with a dual SMAD inhibition plus Wnt inhibitor neural induction. Immunocytchemistry revealed the highest co-expression of DARPP32 and CTIP2 in 0.5 uM purmorphamine producing 6.48% MSNs in comparison to 2.69% and 0% in 0.4 uM and 0.6 uM, respectively. Further optimization is needed as the highest co-expression occurred in cell clumps which were not able to be accurately quantified. An alternative protocol will be used in the future utilizing Activin A instead of purmorphamine having produced over 50% MSNs. These MSNs along with reciprocal CRISPR gene editing could be used to evaluate the validity of cAMP as a parameter for disease modelling for screening of compounds for potential treatments since few exist for a disease with a young age of onset where one would be impactful.