Dysregulation of astrocytic DNAJC6 contributes to sporadic Parkinson’s disease pathogenesis
Wahyu Handoko Wibowo Darsono, Yeongran Hwang, Erica Valencia, Leonardo Tejo Gunawan, Seung Jae Hyeon, Hoon Ryu, Thor D. Stein, Mi-Yoon Chang, Noviana Wulansari, Sang-Hun Lee
Wahyu Handoko Wibowo Darsono, Yeongran Hwang, Erica Valencia, Leonardo Tejo Gunawan, Seung Jae Hyeon, Hoon Ryu, Thor D. Stein, Mi-Yoon Chang, Noviana Wulansari, Sang-Hun Lee
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Research Article Clinical Research Neuroscience

Dysregulation of astrocytic DNAJC6 contributes to sporadic Parkinson’s disease pathogenesis

Abstract

Loss-of-function mutations in DNAJC6, encoding the cochaperone auxilin (HSP40 family), cause familial juvenile-onset Parkinson’s disease (PD). Given the chaperone role of DNAJC6 in cellular homeostasis in adult neurons, we hypothesized that DNAJC6 dysfunction may not be limited to juvenile-onset disorders but could also be associated with adult-onset brain diseases. Here, we show that DNAJC6 expression is significantly downregulated in postmortem substantia nigra tissues and transcriptomic datasets from patients with late-onset sporadic PD. Consistently, human pluripotent stem cell–derived midbrain cultures exhibited reduced DNAJC6 expression under multiple PD-associated conditions. Mechanistically, DNAJC6 loss resulted from impaired transcription mediated by the midbrain-specific factors NURR1/FOXA2 and reduced protein stability regulated by LRRK2. Beyond neurons, DNAJC6 was robustly expressed in astrocytes and similarly downregulated in sporadic PD contexts. Astrocytic DNAJC6 deficiency impaired phagocytic, autolysosomal, and mitochondrial functions while promoting a proinflammatory phenotype, thereby exacerbating neurodegenerative pathology. Importantly, epigenetic restoration of DNAJC6 in neurons and astrocytes using a CRISPRa-AAV9 system in the substantia nigra of an α-synuclein–induced PD mouse model alleviated behavioral deficits and neuropathology. These findings provide evidence that DNAJC6 dysregulation is associated with pathogenic processes in sporadic PD and suggest that targeting neuronal and astrocytic DNAJC6 could represent a potential disease-modifying strategy.

Authors

Wahyu Handoko Wibowo Darsono, Yeongran Hwang, Erica Valencia, Leonardo Tejo Gunawan, Seung Jae Hyeon, Hoon Ryu, Thor D. Stein, Mi-Yoon Chang, Noviana Wulansari, Sang-Hun Lee

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Figure 2

Mechanisms underlying the loss of DNAJC6 in PD environments: impaired Nurr1- and Foxa2-mediated transcription and LRRK2-induced protein instability.

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Mechanisms underlying the loss of DNAJC6 in PD environments: impaired Nu...
(A) DNAJC6 expression levels in mouse NSC (GSE54086) and glia cultures (GSE145489) transduced with Nurr1 (N) and Foxa2 (F) compared with mock controls (C). Data are presented as fold changes of N + F/C (indicated within bars) with corresponding bar color intensity. (B) Downregulation of DNAJC6 mRNA levels by shRNA-mediated KD of Nurr1 and Foxa2 in mouse primary midbrain neuron + astrocyte cultures, as measured by qPCR. (C) qPCR-based determination of DNAJC6, Nurr1, and Foxa2 mRNA expression levels in hESC-derived midbrain cultures treated with α-syn PFF. (D) Rescue of DNAJC6 downregulation in α-syn PFF-treated human midbrain cultures by forced expression of Nurr1 and Foxa2, as measured by qPCR. (E) Schematic representation of predicted Nurr1 and Foxa2 binding sites on the human DNAJC6 promoter. TSS, transcription start site. (F) ChIP-qPCR analysis showing Nurr1 and Foxa2 protein binding at DNAJC6 promoter regions identified in E. (G and H) ChIP-qPCR analysis demonstrating reduced Nurr1 and Foxa2 recruitment to the DNAJC6 promoter in human midbrain cultures treated with α-syn PFF. (I) DNAJC6 protein stability assay in α-syn PFF-treated and untreated human midbrain cultures. Representative WB images and quantification of DNAJC6 levels normalized to β-actin over 48 h following cycloheximide (CHX) treatment (50 μg/mL). (J) Effects of autophagy (Bafilomycin A1) and proteasome (MG132) inhibitors on DNAJC6 protein stability in α-syn PFF-treated human midbrain cultures. (K and L) Physical interaction between DNAJC6 and LRRK2 proteins demonstrated by co-IP (K) and proximity ligation assay (PLA; L) in α-syn PFF-treated and untreated human midbrain cultures. (K) Co-IP analysis showing LRRK2-mediated serine phosphorylation of DNAJC6. Proteins from human midbrain cultures, either treated or untreated with α-syn PFF, were immunoprecipitated using an anti-LRRK2 antibody, followed by immunoblotting with an anti-DNAJC6 antibody. Left blot: 1% input of whole-cell lysate. Right blot: immunoblot analysis of co-IP proteins probed for DNAJC6 and Pan-phosphoserine (P-SERINE). The DNAJC6 and phosphorylated DNAJC6 bands are indicated by arrows. Normal IgG was used as a negative control to confirm binding specificity. The LRRK2 IP blot demonstrates the successful enrichment of the protein. Scale bars: 15 μm. (M) Increased LRRK2 kinase activity in response to α-syn PFF treatment, estimated by LRRK2 autophosphorylation at Ser935. (N and O) Rescue of DNAJC6 protein decline by LRRK2 inhibition (PFE-360) in α-syn PFF-treated human midbrain cultures (N) and in midbrain cultures derived from LRRK2 G2019S-hiPSCs (O). n = 3 independent reactions (B–H), n = 3 independent experiments (I–O); *P < 0.05, **P < 0.01, ***P < 0.0001, ****P < 0.0001; unpaired 2-tailed t test (B, C, F–H), 1-way ANOVA followed by Tukey’s post hoc analysis (D, J, and N), or paired 2-tailed t test (I, K–M, and O).