Loss-of-function mutations of DNAJC6, encoding HSP40 auxilin, have recently been identified in patients with early-onset Parkinson’s disease (PD). To study the roles of DNAJC6 in PD pathogenesis, we used human embryonic stem cells with CRISPR-Cas9–mediated gene editing. Here, we show that DNAJC6 mutations cause key PD pathologic features, i.e., midbrain-type dopamine (mDA) neuron degeneration, pathologic α-synuclein aggregation, increase of intrinsic neuronal firing frequency, and mitochondrial and lysosomal dysfunctions in human midbrain-like organoids (hMLOs). In addition, neurodevelopmental defects were also manifested in hMLOs carrying the mutations. Transcriptomic analyses followed by experimental validation revealed that defects in DNAJC6-mediated endocytosis impair the WNT-LMX1A signal during the mDA neuron development. Furthermore, reduced LMX1A expression during development caused the generation of vulnerable mDA neurons with the pathologic manifestations. These results suggest that the human model of DNAJC6-PD recapitulates disease phenotypes and reveals mechanisms underlying disease pathology, providing a platform for assessing therapeutic interventions.

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Intraneuronal inclusions of misfolded α-synuclein (α-syn) and prion-like spread of the pathologic α-syn contribute to progressive neuronal death in Parkinson’s disease (PD). Despite the pathologic significance, no efficient therapeutic intervention targeting α-synucleinopathy has been developed. In this study, we provide evidence that astrocytes, especially those cultured from the ventral midbrain (VM), show therapeutic potential to alleviate α-syn pathology in multiple in vitro and in vivo α-synucleinopathic models. Regulation of neuronal α-syn proteostasis underlies the therapeutic function of astrocytes. Specifically, VM-derived astrocytes inhibited neuronal α-syn aggregation and transmission in a paracrine manner by correcting not only intraneuronal oxidative and mitochondrial stresses but also extracellular inflammatory environments, in which α-syn proteins are prone to pathologic misfolding. The astrocyte-derived paracrine factors also promoted disassembly of extracellular α-syn aggregates. In addition to the aggregated form of α-syn, VM astrocytes reduced total α-syn protein loads both by actively scavenging extracellular α-syn fibrils and by a paracrine stimulation of neuronal autophagic clearance of α-syn. Transplantation of VM astrocytes into the midbrain of PD model mice alleviated α-syn pathology and protected the midbrain dopamine neurons from neurodegeneration. We further showed that cografting of VM astrocytes could be exploited in stem cell–based therapy for PD, in which host-to-graft transmission of α-syn pathology remains a critical concern for long-term cell therapeutic effects.

The LIF-JAK2-STAT3 pathway is the central signal transducer that maintains undifferentiated mouse embryonic stem cells (mESCs), which is achieved by the recruitment of activated STAT3 to the master pluripotency genes and activation of the gene transcriptions. It remains unclear, however, how the epigenetic status required for the master gene transcriptions is built into LIF-treated mESC cultures. In this study, Jak2, but not Stat3, in the LIF canonical pathway, establishes an open epigenetic status in the pluripotency gene promoter regions. Upon LIF activation, cytosolic JAK2 was translocalized into the nucleus of mESCs, and reduced DNA methylation (5mC levels) along with increasing DNA hydroxymethylation (5hmC) in the pluripotent gene (Nanog/Pou5f1) promoter regions. In addition, the repressive histone codes H3K9m3/H3K27m3 were reduced by JAK2. Activated JAK2 directly interacted with the core epigenetic enzymes TET1 and JMJD2, modulating its activity and promotes the DNA and histone demethylation, respectively. The JAK2 effects were attained by tyrosine phosphorylation on the epigenetic enzymes. The effects of JAK2 phosphorylation on the enzymes were diverse, but all were merged to the epigenetic signatures associated with open DNA/chromatin structures. Taken together, these results reveal a previously unrecognized epigenetic regulatory role of JAK2 as an important mediator of mESC maintenance.