Functional characterisation of SV2C and its variants as a Parkinson’s Disease-associated gene

• Main Authors: Chang, Chu-Hua, Chew, Elaine Guo Yan, Tandiono, Moses, Zhang, Tian, Gong, Ximing, Xia, Yun, Foo, Jia Nee
• Other Authors: Interdisciplinary Graduate School (IGS)
• Format: Conference or Workshop Item
• Language: English
• Published: 2023
• Subjects: Science::Medicine; Parkinson’s Disease; Gene
• Online Access: https://hdl.handle.net/10356/170356; https://www.sfn.org/meetings/neuroscience-2023/sessions-and-events
• Institution: Nanyang Technological University

Parkinson’s Disease (PD) is the second most common neurodegenerative disease characterised by the loss of dopaminergic neurons in the substantia nigra pars compacta in the midbrain. Extensive effort has been devoted to identifying genetic risk factors for PD to allow for early intervention and management of PD patients. In a genome-wide association study conducted in the East Asian population (Foo et al., 2020), SV2C was identified as a novel risk locus for PD where its lead SNP (rs246814) tagged a missense variant (rs31244) p.Asp543Asn (D543N) in the SV2C gene that could potentially introduce a new N-glycosylation site (Asn-X-Ser/Thr) in the luminal domain of the encoded synaptic vesicle glycoprotein. SV2C was reported to be highly expressed in the midbrain and the loss of SV2C in mice showed reduced dopamine release in the striatum. However, the exact function of SV2C in dopaminergic neurons of the midbrain is still unclear. This study aims to characterise the functional effect of SV2C and its variants in human stem cell-derived midbrain dopaminergic (mDA) neurons. SV2C gene was first knocked out in H9 human embryonic stem cells and the differentiated SV2C-KO mDA neurons formed neuronal-like projections and were positive for mature midbrain markers TH and NURR1, suggesting that the loss of SV2C did not affect the direct differentiation of mDA neurons. Due to the heterogeneity of cells upon differentiation, a midbrain reporter H9-PITX3-mCherry knock-in cell line was generated to enrich the population of mDA neurons for functional characterisation. CRISPR/Cas9-mediated site-specific mutagenesis will also be performed to generate SV2C D543N variant in H9 cells and the N-glycosylation of SV2C will be investigated. Ongoing functional assessment of SV2C-KO and SV2C D543N mDA neurons will include measuring the dopamine release and synaptic vesicle (SV) trafficking using a fluorescent SV marker synapto-pHlourin. These data would give insights into the role of SV2C as a PD risk gene and its contribution to PD pathogenesis.