Wild-type C9orf72 drives proteasomal dysfunction and mutant aggregates via a Stat1-Isg15 axis in Huntington's disease.

USP28 Deficiency is Linked to Impaired Ubiquitin-dependent Proteostasis in Huntington’s Disease

Parkinson's disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35.

Mutations in several genes are known to cause familial forms of Parkinson's disease (PD), including mutations in the vacuolar protein sorting 35 ortholog (VPS35) gene linked to late-onset, autosomal dominant PD. VPS35 encodes a core subunit of the retromer complex which functions in endosomal sorting and recycling. It remains unclear how the pathogenic D620N mutation in VPS35 disrupts retromer function to induce neurodegeneration in PD. Using cell- and rodent-based models expressing D620N VPS35, we performed interactome proteomics to identify alterations underlying the pathogenic effects of D620N VPS35 in PD. Using overexpression of VPS35 variants in HEK-293 T cells, we conducted tandem affinity purification (TAP) or co-immunoprecipitation (co-IP) with protein chemical crosslinking to determine the native and non-native protein interactomes of wild-type (WT) and D620N VPS35, respectively. Notably, we can confirm the reduced interaction of D620N VPS35 with components of the WASH complex. Additionally, using a viral-mediated gene transfer model of human D620N VPS35 overexpression in adult rat brain, we identify the first brain-specific protein interactome of VPS35. These overexpression models reveal remarkably similar interaction profiles of WT and D620N VPS35, suggesting that the D620N mutation has a subtle effect on the overall VPS35 protein interactome. We also conducted proteomic analysis of brain tissue from a D620N VPS35 knockin (KI) mouse model that expresses VPS35 at endogenous levels. Using co-IP from hemibrain or striatal extracts of WT and D620N VPS35 KI mice, we reveal a high degree of similarity between the brain interactomes of WT and D620N VPS35, further suggesting a subtle effect of the D620N mutation on VPS35 protein interactions. Notably, in both hemibrain and striatum, we find a selective decrease in the interaction of two known interactors, TBC1D5 and VPS29, with D620N VPS35. We also performed global proteomic analysis of striatal tissue from D620N VPS35 KI mice and reveal a high degree of similarity between WT and D620N, further suggesting a subtle effect of this mutation. Together, our study provides a comprehensive evaluation of the VPS35 protein interactome and reveals a selective effect of the PD-linked D620N mutation in mammalian cells and brain. Our study provides key insight into the mechanisms of retromer dysfunction in VPS35-linked PD.

Structure-function dissection of huntingtin exon 1 identifies a PRD-driven modifier of neuronal toxicity in Huntington’s disease

The Huntingtin gene (HTT) contains a conserved, yet expandable CAG repeat within exon 1. While the pathogenic expansion in Huntington’s Disease (HD) is well studied, the role of surrounding domains remains unclear. Using genome-edited mini-organoids and neurons, we dissected HTT exon 1 and found species-specific toxicity: the human variant caused more severe deficits than the mouse. Swapping the proline-rich domain (PRD) - the most divergent region - revealed its key role: the mouse PRD mitigated, while the human PRD worsened neuronal phenotypes. Omics profiling showed that pathogenic human exon 1 induced broad protein dysregulation, largely reversed by mouse PRD replacement. Bioinformatics implicated the actin cytoskeleton and transcriptional coactivator MKL2/MRTFB. We validated MKL2/MRTFB dysregulation in HD models and showed that restoring its expression rescued neuronal abnormalities. These findings highlight the PRD’s contribution to HD toxicity and point to MKL2/MRTFB and the cytoskeleton as candidate mediators.

C9orf72-associated and sporadic FTD patient iPSC-microglia show differences in phagocytosis and gene expression.

C9orf72 hexanucleotide repeat expansion (C9-HRE) is a major genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (FTD). However, approximately half of the FTD patients are sporadic without a clear genetic background. To compare characteristics of microglia from different FTD subtypes, we generated induced pluripotent stem cell-derived microglia (iMG) from sporadic and C9-HRE-carrying behavioral variant FTD (bvFTD) patients and healthy controls. C9-HRE iMG displayed C9-HRE-associated RNA foci and dipeptide repeat proteins. All bvFTD iMG had fewer LAMP2-A-positive vesicles compared to control iMG. Additionally, C9-HRE iMG showed significantly increased LC3BII/I conversion after bafilomycin A1 treatment and altered phagocytic activity. The gene expression profile of C9-HRE iMG only modestly differed from the control iMG, but was greatly different from the sporadic bvFTD patient iMG. Our data show alterations in phagocytic and autophagosomal/lysosomal pathways and gene expression profiles between C9-HRE and sporadic bvFTD iMG for the first time.

Effects of homozygous PARK7 gene mutations L166P and M26I on BNIP3/BNIP3L interactions and ER-mitochondria proximity

Mutations in PARK7, which encodes DJ-1, cause autosomal recessive early-onset Parkinson’s disease. DJ-1 contributes to mitochondrial homeostasis and ER-mitochondria communication, but how the pathogenic L166P and M26I variants affect BNIP3/BNIP3L-associated phenotypes remains incompletely understood. Here, we examined DJ-1 variant-dependent changes in BNIP3/BNIP3L interactions, mitochondrial morphology, ER-mitochondria proximity-associated readouts, and global intracellular Ca 2+ responses in PC-12 and SH-SY5Y cell models. AlphaFold3-based interface prediction, co-immunoprecipitation, and GST pull-down assays supported interactions involving DJ-1, BNIP3, and BNIP3L, including binding to selected BNIP3- and BNIP3L-derived peptide regions. DJ-1 WT and M26I showed detectable binding to BNIP3-derived regions, whereas the unstable L166P variant showed reduced BNIP3 binding even after partial restoration of L166P abundance with MG132. Reciprocal co-immunoprecipitation and knockdown experiments further supported an association between BNIP3 and BNIP3L in these cells. In L166P expressing cells with GRP75 knockdown, BNIP3 depletion increased the ER-mitochondria distance and reduced the length of the ER-mitochondria proximity region. Colocalization analyses showed DJ-1 variant-dependent changes in proximity-associated imaging readouts, while immunoblotting identified reduced VDAC1 and MFN1 levels after BNIP3 depletion in mutant expressing cells. BNIP3 depletion reduced the relative 2-APB evoked Fluo-4 response in WT, L166P and M26I expressing cells by 40.7%, 76.7%, and 42.5%, respectively. Overall, these findings may reflect altered DJ-1/BNIP3/BNIP3L interaction profiles and BNIP3 sensitive changes in ER-mitochondria proximity associated and global intracellular Ca 2+ readouts, particularly in L166P-expressing cells, although direct validation is still required.

Dissecting the transcriptome of Parkin (PRKN)-linked Parkinson’s disease using 2D and 3D hiPSC-neuronal models

This work generates human induced pluripotent stem cell-derived midbrain dopaminergic neurons using both a 2D differentiation protocol and a recently developed 3D method based on the microencapsulation of hiPSCs in small alginate/fibronectin beads.