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

<jats:title>Abstract</jats:title><jats:p>Parkinson’s 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<jats:italic>SNCA</jats:italic>, as well as duplication and triplication of the<jats:italic>SNCA</jats:italic>locus. In our cellular model, aggregates of alpha-synuclein occurred only within the GFAP<jats:sup>+</jats:sup>astrocytes carrying the pathogenic<jats:italic>SNCA</jats:italic>mutations. Assessment of spontaneous cytosolic calcium (Ca<jats:sup>2+</jats:sup>) release using Fluo4 revealed that<jats:italic>SNCA</jats:italic>mutant astrocytes released excess Ca<jats:sup>2+</jats:sup>compared to controls. Unbiased evaluation of 3D mitochondrial morphometric parameters showed that these<jats:italic>SNCA</jats:italic>mutant astrocytes had increased mitochondrial fragmentation and decreased mitochondrial connectivity compared to controls, and reduced mitochondrial bioenergetic function. This comprehensive assessment of different pathogenic<jats:italic>SNCA</jats:italic>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<jats:italic>SNCA</jats:italic>are dysfunctional, suggesting a contributory role for dysfunctional astrocytes in the disease mechanism and pathogenesis of PD.</jats:p><jats:sec><jats:title>Table of Contents Image</jats:title><jats:fig id="ufig1" position="float" fig-type="figure" orientation="portrait"><jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="053470v1_ufig1" position="float" orientation="portrait"/></jats:fig></jats:sec><jats:sec><jats:title>Main Points</jats:title><jats:list list-type="bullet"><jats:list-item><jats:p>We used a novel serum-free protocol to generate midbrain-specific functional astrocytes from Parkinson’s disease patients carrying pathological mutations in<jats:italic>SNCA</jats:italic></jats:p></jats:list-item><jats:list-item><jats:p>Patient-derived astrocytes show morphological and functional impairments</jats:p></jats:list-item></jats:list></jats:sec>