Demarest, T. G. | bioRxiv Science

bioRxiv2026

Restoring Parkin Function: An AAV Gene Therapy Approach for Early-Onset Parkinson's Disease

BackgroundBiallelic loss-of-function mutations in PRKN gene (encoding Parkin protein) cause early-onset Parkinsons disease (EOPD). Parkin is a crucial component of PINK1-Parkin pathway, which marks damaged mitochondria for degradation via mitophagy. Without functional Parkin, damaged mitochondria accumulate, causing oxidative stress and neurodegeneration. ObjectiveInvestigate Parkin gene replacement via AAV gene therapy as a potential treatment for Parkin-dependent EOPD. MethodsWe initially validated phosphorylated ubiquitin Ser65 (pUbSer65) as an indicator of Parkin-mediated mitophagy initiation. We evaluated AAV-mediated PRKN replacement (hereafter, AAV-Parkin) in a Parkin knockout neuroblastoma cell line (SH-SY5Y cells) and feasibility of delivery in mouse and rat models. ResultsOur research showed pUbSer65 signal was reduced in Parkin-KO SH-SY5Y cells when compared to wild-type cells after mitochondrial stress, indicating deficiency in initiation of mitophagy. AAV-mediated human PRKN gene replacement successfully restored these pUbSer65 levels in knockout cells. We saw restoration in patient-derived fibroblasts following AAV-Parkin overexpression. We developed a translatable gene therapy approach using rodents. We demonstrated the feasibility of delivering AAV-Parkin directly into the substantia nigra (SN) of wild-type rats. Using an AAV1 capsid with Ef1a promoter, we achieved dose-dependent Parkin expression and identified a well-tolerated dose. We also evaluated multiple promoters in a proprietary Spark100 capsid, finding Ef1a and Synapsin1 (Syn1) were most effective for transducing dopaminergic neurons in the SN of mice without causing adverse effects. These findings established a well-tolerated vector dose and an optimal capsid-promoter combination. ConclusionsOur results support the potential of AAV-Parkin gene therapy as a disease-modifying approach for Parkin-deficient EOPD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/737487v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@16dd13corg.highwire.dtl.DTLVardef@c3dfcdorg.highwire.dtl.DTLVardef@19a310dorg.highwire.dtl.DTLVardef@a66f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2019

Self-assembly of multi-component mitochondrial nucleoids via phase separation

Mitochondria contain an autonomous and spatially segregated genome. The organizational unit of their genome is the nucleoid, which consists of mitochondrial DNA (mtDNA) and associated architectural proteins. Here, we show that phase separation is the primary physical mechanism for assembly and size-control of the mitochondrial nucleoid. The major mtDNA-binding protein TFAM spontaneously phase separates in vitro via weak, multivalent interactions into viscoelastic droplets with slow internal dynamics. In combination, TFAM and mtDNA form multiphase, gel-like structures in vitro, which recapitulate the in vivo dynamic behavior of mt-nucleoids. Enlarged, phase-separated, yet transcriptionally active, nucleoids are present in mitochondria from patients with the premature aging disorder Hutchinson-Gilford Progeria Syndrome (HGPS) and are associated with mitochondrial dysfunction. These results point to phase separation as an evolutionarily conserved mechanism of genome organization. HighlightsO_LIMitochondrial genomes are organized by phase separation. C_LIO_LIThe main packaging protein TFAM and mtDNA combine to form viscoelastic, multiphase droplets in vitro. C_LIO_LIMitochondrial nucleoids exhibit phase behavior in vivo, including dynamic rearrangements and heterogenous organization. C_LIO_LICoalescence and enlargement of mt-nucleoids occur upon loss of mitochondrial homeostasis as well as in prematurely aged cells and are associated with mitochondrial dysfunction. C_LI

cell biology↗