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  • Here we study the impact of the common

    2018-10-24

    Here we study the impact of the common GBA-N370S mutation on the phenotype of patient-specific dopaminergic neuronal cultures differentiated from iPSC lines derived from patients with PD and identify deficits in protein homeostasis. Our results suggest that the heterozygous GBA-N370S mutation leads to a cellular gain of function, shown by misprocessing of misfolded GCase protein in the ER resulting in ER stress upregulation and autophagic/lysosomal dysfunction leading to an enlargement of the lysosomal compartment in individually identified vulnerable dopamine neurons. Together, these deficits lead to increased release of extracellular α-synuclein from GBA-N370S PD human iPSC-derived dopamine neuron cultures, which we show is not associated with exosomes. We propose that the combination of these disturbances impairs protein homeostasis in dopamine neurons contributing to their preferential vulnerability in PD. These data highlight the early pathogenic relevance of GCase function in the autophagic/lysosomal pathway in PD, and may explain the higher risk for heterozygous GBA-N370S mutation carriers to develop PD.
    Results
    Discussion Using human iPSCs derived from PD patients carrying the heterozygous GBA-N370S mutation, we have identified relevant mechanisms by which a heterozygous GBA mutation associated with PD may increase cellular susceptibility to disease. Our results highlight an important role of heterozygous mutant GCase in the disruption of protein homeostasis in dopaminergic neurons, ultimately leading to increased α-synuclein release, which may be central for the early pathogenesis of PD. Differentiation of ten independent iPSC lines from three controls and three unrelated GBA-N370S PD patients into dopaminergic neurons allowed us to robustly investigate this genetic contribution in an appropriate human model of early PD pathology. Our data suggest that the heterozygous GBA-N370S mutation results in the retention of GCase within the ER in PD iPSC-derived dopaminergic neuronal cultures. We found no evidence of a reduction of GCase protein levels in our multiple lines derived from three unrelated patients, indicating that the protein is not targeted for degradation. We have observed an overall increased order buprenorphine hydrochloride of LIMP2, a GCase-specific lysosomal receptor, in dopaminergic cultures from GBA-N370S patients. Increased LIMP2 expression is likely to be part of a cellular response to GBA mutations, as it has been previously shown that LIMP2 overexpression increases transportation of ER-retained GCase toward the lysosomes (Reczek et al., 2007). We observed the upregulation of multiple ER stress markers in GBA-N370S-derived dopaminergic neuronal cultures, which likely reflects a dysregulation of the ER homeostatic environment induced by misprocessing of misfolded GCase similar to a postmortem analysis of PD GBA patients showing UPR activation (Gegg et al., 2012). We have also identified alterations in the autophagy pathway in our PD model. Levels of autophagosomes were increased in dopamine neurons from GBA-N370S lines, and we provide multiple lines of evidence to support an impairment of general autophagic vacuole clearance. In addition, multiple lysosomal markers were upregulated in GBA-N370S dopaminergic lines consistent with an impairment of lysosomal degradation capacity. Moreover, an enlargement of the lysosomal compartment was identified, specifically within TH immunogold-identified dopaminergic neurons derived from patient\'s lines, characterized by increased number and size of lysosomal structures. Lysosomal dysfunction is a key pathological event in PD (Dehay et al., 2010), and the specificity to dopamine neurons may explain the preferential vulnerability of these neurons. Altered cellular lipid composition can be associated with ER disturbances (Basseri and Austin, 2012) and autophagic impairments (Koga et al., 2010). Although the mechanisms underlying cellular distribution of GlcCer species in neurons is not well understood, lipid contents are known to differ in membranes undergoing ER-Golgi maturation or fusion within various organelles (Halter et al., 2007). The different GlcCer species distribution observed in our neuronal cultures between control and GBA-N370S lines may be due to the different trafficking of GCase. As a result of altered trafficking, the mutant GCase enzyme may hydrolyze a species that might not have been degraded in normal cells, thereby decreasing total amounts of certain lipids (e.g., 30% reduction of C20:0 GlcCer levels in GBA-N370S neurons). On the other hand, reduced GCase concentration in particular sub-compartments may promote the accumulation of other species (e.g., ∼65% increase in C16:0 and C24:0 species) in GBA-N370S neurons.