Objective: To generate mechanistic insight into how the SYNJ1 p.Arg258Gln mutation leads to neurodegeneration in juvenile Parkinsonism.

Background: In humans, the SYNJ1 homozygous p.Arg258Gln missense mutation leads to juvenile Parkinsonism (PARK20). Strikingly, patients carrying mutations in the SYNJ1 gene that result in complete loss of SYNJ1 expression display more severe phenotypes, including early onset refractory seizures and juvenile lethality. These studies suggest a role for SYNJ1 mutations in neurodegenerative disease and indicate a phenotype-genotype correlation. The synaptojanin 1 protein (SYNJ1) plays an important role in synaptic vesicle cycling, and regulation of autophagic flux. A potential role for SYNJ1 in mitochondria has not been investigated.

Methods: Patient derived SYNJ1 homozygous p.Arg258Gln and control induced pluripotent stem cells (IPSCs) were exposed to small molecules to generate neural progenitor cells (NPCs) and subsequently differentiated for 3 weeks to induced dopaminergic (iDA) neurons. To investigate the role of SYNJ1 in autophagy, mitochondria and vesicle recycling, NPCs and iDA neurons were exposed to conditions that induce autophagy (starvation), mitophagy (CCCP and valinomycin treatment) and control conditions, and probed for markers of autophagy (i.e. WIPI2), mitophagy (i.e. Parkin) and synaptic vesicle recycling pathways (i.e. clathrin) by immunocytochemistry and Western blotting techniques. To analyze the functional impact of a SYNJ1 mutation on mitochondria in NPCs, the oxygen consumption rate was measured by extracellular flux analysis (Seahorse) including a mitochondrial stress test.

Results: Our data indicate that the SYNJ1 p.Arg258Gln mutation affects the autophagy pathway as indicated by increased levels of WIPI2 expression in patient derived NPCs and iDA neurons. Furthermore, increased number of Clathrin clusters and reduction of Clathrin cluster size, indicate that the SYNJ1 p.Arg258Gln mutation also disturbs proteins of the synaptic vesicle recycling pathway in iDA neurons. Finally, Parkin protein levels were increased in SYNJ1 p.Arg258Gln patient derived NPCs, and down regulated during mitophagy. Strikingly, patient derived NPCs showed elevated mitochondrial activity in terms of basal respiration, ATP production, maximal respiration and energy-coupling efficiency.

Conclusions: Although further research is needed to clarify how the SYNJ1 p.Arg258Gln mutation leads to neurodegeneration, our data suggest a role for SYNJ1 in several cellular pathways, including autophagy, vesicle recycling and mitophagy.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/increased-parkin-expression-in-a-park20-synj1-mutation-ipscs-based-model/