SummaryHeterozygous mutations in the glucocerebrosidase gene (GBA) represent the strongest common genetic risk factor for Parkinson's disease (PD), the second most common neurodegenerative disorder. However, the molecular mechanisms underlying this association are still poorly understood. Here, we have analyzed ten independent induced pluripotent stem cell (iPSC) lines from three controls and three unrelated PD patients heterozygous for the GBA-N370S mutation, and identified relevant disease mechanisms. After differentiation into dopaminergic neurons, we observed misprocessing of mutant glucocerebrosidase protein in the ER, associated with activation of ER stress and abnormal cellular lipid profiles. Furthermore, we observed autophagic perturbations and an enlargement of the lysosomal compartment specifically in dopamine neurons. Finally, we found increased extracellular α-synuclein in patient-derived neuronal culture medium, which was not associated with exosomes. Overall, ER stress, autophagic/lysosomal perturbations, and elevated extracellular α-synuclein likely represent critical early cellular phenotypes of PD, which might offer multiple therapeutic targets.

Mutations in the glucocerebrosidase gene (GBA), which cause Gaucher disease, are also potent risk factors for Parkinson's disease. We examined whether a genetic burden of variants in other lysosomal storage disorder genes is more broadly associated with Parkinson's disease susceptibility. The sequence kernel association test was used to interrogate variant burden among 54 lysosomal storage disorder genes, leveraging whole exome sequencing data from 1156 Parkinson's disease cases and 1679 control subjects. We discovered a significant burden of rare, likely damaging lysosomal storage disorder gene variants in association with Parkinson's disease risk. The association signal was robust to the exclusion of GBA, and consistent results were obtained in two independent replication cohorts, including 436 cases and 169 controls with whole exome sequencing and an additional 6713 cases and 5964 controls with exome-wide genotyping. In secondary analyses designed to highlight the specific genes driving the aggregate signal, we confirmed associations at the GBA and SMPD1 loci and newly implicate CTSD, SLC17A5, and ASAH1 as candidate Parkinson's disease susceptibility genes. In our discovery cohort, the majority of Parkinson's disease cases (56%) have at least one putative damaging variant in a lysosomal storage disorder gene, and 21% carry multiple alleles. Our results highlight several promising new susceptibility loci and reinforce the importance of lysosomal mechanisms in Parkinson's disease pathogenesis. We suggest that multiple genetic hits may act in combination to degrade lysosomal function, enhancing Parkinson's disease susceptibility.

Autosomal-recessive early-onset parkinsonism is clinically and genetically heterogeneous. The genetic causes of approximately 50% of autosomal-recessive early-onset forms of Parkinson disease (PD) remain to be elucidated. Homozygozity mapping and exome sequencing in 62 isolated individuals with early-onset parkinsonism and confirmed consanguinity followed by data mining in the exomes of 1,348 PD-affected individuals identified, in three isolated subjects, homozygous or compound heterozygous truncating mutations in vacuolar protein sorting 13C (VPS13C). VPS13C mutations are associated with a distinct form of early-onset parkinsonism characterized by rapid and severe disease progression and early cognitive decline; the pathological features were striking and reminiscent of diffuse Lewy body disease. In cell models, VPS13C partly localized to the outer membrane of mitochondria. Silencing of VPS13C was associated with lower mitochondrial membrane potential, mitochondrial fragmentation, increased respiration rates, exacerbated PINK1/Parkin-dependent mitophagy, and transcriptional upregulation of PARK2 in response to mitochondrial damage. This work suggests that loss of function of VPS13C is a cause of autosomal-recessive early-onset parkinsonism with a distinctive phenotype of rapid and severe progression.

ObjectiveParkinson’s disease is characterised neuropathologically by α-synuclein aggregation. Currently, there is no blood test to predict the underlying pathology or distinguish Parkinson’s from atypical parkinsonian syndromes. We assessed the clinical utility of serum neuronal exosomes as biomarkers across the spectrum of Parkinson’s disease, multiple system atrophy and other proteinopathies.MethodsWe performed a cross-sectional study of 664 serum samples from the Oxford, Kiel and Brescia cohorts consisting of individuals with rapid eye movement sleep behavioural disorder, Parkinson’s disease, dementia with Lewy bodies, multiple system atrophy, frontotemporal dementia, progressive supranuclear palsy, corticobasal syndrome and controls. Longitudinal samples were analysed from Parkinson’s and control individuals. We developed poly(carboxybetaine-methacrylate) coated beads to isolate L1 cell adhesion molecule (L1CAM)-positive extracellular vesicles with characteristics of exosomes and used mass spectrometry or multiplexed electrochemiluminescence to measure exosomal proteins.ResultsMean neuron-derived exosomal α-synuclein was increased by twofold in prodromal and clinical Parkinson’s disease when compared with multiple system atrophy, controls or other neurodegenerative diseases. With 314 subjects in the training group and 105 in the validation group, exosomal α-synuclein exhibited a consistent performance (AUC=0.86) in separating clinical Parkinson’s disease from controls across populations. Exosomal clusterin was elevated in subjects with non-α-synuclein proteinopathies. Combined neuron-derived exosomal α-synuclein and clusterin measurement predicted Parkinson’s disease from other proteinopathies with AUC=0.98 and from multiple system atrophy with AUC=0.94. Longitudinal sample analysis showed that exosomal α-synuclein remains stably elevated with Parkinson’s disease progression.ConclusionsIncreased α-synuclein egress in serum neuronal exosomes precedes the diagnosis of Parkinson’s disease, persists with disease progression and in combination with clusterin predicts and differentiates Parkinson’s disease from atypical parkinsonism.