Summary Microglia have recently been established as key regulators of brain development. However, their role in neuronal subtype specification remains largely unknown. Using three different co-culture setups, we show that microglia-secreted factors enhance dopaminergic differentiation of somatic and induced pluripotent stem cell-derived human neural stem cells (NSCs). The effect was consistent across different NSC and microglial cell lines and was independent of prior microglial activation, although restricted to microglia of embryonic origin. We provide evidence that the effect is mediated through reduced cell proliferation and decreased apoptosis and necrosis orchestrated in a sequential manner during the differentiation process. tumor necrosis factor alpha, interleukin-1β, and insulinlike growth factor 1 are identified as key mediators of the effect and shown to directly increase dopaminergic differentiation of human NSCs. These findings demonstrate a positive effect of microglia on dopaminergic neurogenesis and may provide new insights into inductive and protective factors that can stimulate in vitro derivation of dopaminergic neurons.

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Mutations in the PARK2 gene encoding parkin, an E3 ubiquitin ligase, are associated with autosomal recessive early-onset Parkinson's disease (PD). While parkin has been implicated in the regulation of mitophagy and proteasomal degradation, the precise mechanism leading to neurodegeneration in both sporadic and familial PD upon parkin loss-of-function mutations remains unknown. Cultures of isogenic induced pluripotent stem cell (iPSC) lines with and without PARK2 knockout (KO) enable mechanistic studies of the effect of parkin deficiency in human dopaminergic neurons. In the present study, we used such cells to investigate the impact of PARK2 KO on the lysosomal compartment combining different approaches, such as mass spectrometry-based proteomics, electron microscopy (TEM) analysis and functional assays. We discovered a clear link between parkin deficiency and lysosomal alterations. PARK2 KO neurons exhibited a perturbed lysosomal morphology, displaying significantly enlarged and electron-lucent lysosomes as well as an increased total lysosomal content, which was exacerbated by mitochondrial stress. In addition, we found perturbed autophagic flux and decreased lysosomal enzyme activity suggesting an impairment of the autophagylysosomal pathway in parkin-deficient cells. Interestingly, activity of the GBA-encoded enzyme, b-glucocerebrosidase, was significantly increased suggesting the existence of a compensatory mechanism. In conclusion, our data provide a unique characterization of the morphology, content, and function of lysosomes in PARK2 KO neurons, thus revealing a new important connection between mitochondrial dysfunction and lysosomal dysregulation in PD pathogenesis.