Objective: In this study, we aim to generate and characterise patient-derived midbrain dopaminergic neurons (mDANs) of the A30P Parkinson’s disease (PD) familial case. Our goal is to established A30P-associated phenotypes, which could be addressed on a comprehensive imaging-based HCS/HTS on our automated system.

Background: Parkinson’s disease (PD) is the second most common neurodegenerative disease. PD is characterised by the four cardinal motor symptoms, bradykinesia being the key symptom necessary to confirm the diagnosis of the disease. Approximately 20% of PD cases are known to be familial. Amongst these cases, mutations in SNCA, the gene encoding alpha synuclein was found to give an autosomal dominant inheritance of the disease. In 1998, our group discovered one of these point mutations of SNCA, an Alanine to Proline mutation at position 30 (A30P). This was confirmed upon autopsy with the presence of Lewy Bodies, Lewy Neurites and abundant alpha synuclein aggregates.

Methods: We generated a patient-derived cellular model of SNCA with the A30P point mutation, by obtaining fibroblasts from the index patient, an age-matched gender-matched non-PD control, and the unaffected brother of the patient. We reprogrammed these fibroblasts using retroviruses, and generated induced pluripotent stem cells (iPSCs). From these iPSCs, we used small molecules to differentiate these iPSCs into neural precursor cells (smNPCs), and then further differentiated these cells into mDANs.

Results: We successfully obtained enriched cultures of ≥85% neurons (CD200+/CD49f-) when analysed using FACS. From these, approximately 20% were confirmed by immunocytochemistry as being midbrain (FoxA2+) dopaminergic (TH+), and demonstrate the capability to actively fire and release dopamine, all characteristics of a good model for PD. Further characterisation by PCR and protein immunoblotting additionally showed the presence of mDANs markers on our model d50 cultures. We now aim to investigate A30P specific phenotypes, and analyse the interplay between neuronal and non-neuronal populations in the same microenvironment.

Conclusions: We were able to generate a robust, repeatable, standardised model for the A30P familial case of PD. Phenotypic differences were identified and consistently obtained, and can now be integrated on our automated image-based HCS/HTS system.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/characterisation-of-the-a30p-mutation-in-alpha-synuclein-gene-in-patient-derived-cellular-model-of-parkinsons-disease/