IPSC-derived midbrain astrocytes from Parkinson's disease patients carrying pathogenic SNCA mutations exhibit alpha-synuclein aggregation, mitochondrial fragmentation and excess calcium release

Abstract

Parkinsons disease (PD) is characterized by the loss of A9 midbrain dopaminergic neurons and the accumulation of alpha-synuclein aggregates in remaining neurons. Many studies of the molecular and cellular basis of neurodegeneration in PD have made use of iPSC-derived neurons from patients with familial PD mutations. However, approximately half of the cells in the brain are glia, and their role facilitating neurodegeneration is unclear. We developed a novel serum-free protocol to generate midbrain astrocytes from patient-derived iPSCs harbouring the pathogenic p.A30P, p.A53T mutations in SNCA, as well as duplication and triplication of the SNCA locus. In our cellular model, aggregates of alpha-synuclein occurred only within the GFAP+ astrocytes carrying the pathogenic SNCA mutations. Assessment of spontaneous cytosolic calcium (Ca2+) release using Fluo4 revealed that SNCA mutant astrocytes released excess Ca2+ compared to controls. Unbiased evaluation of 3D mitochondrial morphometric parameters showed that these SNCA mutant astrocytes had increased mitochondrial fragmentation and decreased mitochondrial connectivity compared to controls, and reduced mitochondrial bioenergetic function. This comprehensive assessment of different pathogenic SNCA mutations derived from PD patients using the same cellular model enabled assessment of the mutation effect, showing that p.A53T and triplication astrocytes were the most severely affected. Together, our results indicate that astrocytes harbouring the familial PD mutations in SNCA are dysfunctional, suggesting a contributory role for dysfunctional astrocytes in the disease mechanism and pathogenesis of PD. Table of Contents Image O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/053470v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@974d72org.highwire.dtl.DTLVardef@15d9da0org.highwire.dtl.DTLVardef@1177d4eorg.highwire.dtl.DTLVardef@123c0c2_HPS_FORMAT_FIGEXP M_FIG C_FIG Main PointsO_LIWe used a novel serum-free protocol to generate midbrain-specific functional astrocytes from Parkinsons disease patients carrying pathological mutations in SNCA C_LIO_LIPatient-derived astrocytes show morphological and functional impairments C_LI