Defective mitochondria‐lysosomal axis enhances the release of extracellular vesicles containing mitochondrial DNA and proteins in Huntington's disease

Abstract

Mitochondrial and autophagy dysfunction are mechanisms proposed to be involved in the pathogenesis of several neurodegenerative diseases. Huntington's disease (HD) is a progressive neurodegenerative disorder associated with mutant Huntingtininduced abnormalities in neuronal mitochondrial dynamics and quality control. Former studies suggest that the removal of defective mitochondria may be compromised in HD. Mitochondrial quality control (MQC) is a complex, well-orchestrated pathway that can be compromised through mitophagy dysregulation or impairment in the mitochondria-lysosomal axis. Another mitochondrial stress response is the generation of mitochondrial-derived vesicles that fuse with the endolysosomal system and form multivesicular bodies that are extruded from cells as extracellular vesicles (EVs). In this work, we aimed to study the presence of mitochondrial components in human EVs and the relation to the dysfunction of both mitochondria and the autophagy pathway. We comprehensively characterized the mitochondrial and autophagy alterations in premanifest and manifest HD carriers and performed a proteomic and genomic EVs profile. We observed that manifest HD patients exhibit mitochondrial and autophagy impairment associated with enhanced EVs release. Furthermore, we detected mitochondrial DNA and proteins in EVs released by HD cells and in neuronal-derived EVs including VDAC-1 and alpha and beta subunits of ATP synthase F1. HD-extracellular vesicles transport higher levels of mitochondrial genetic material in manifest HD patients, suggesting an alternative pathway for the secretion of reactive mitochondrial components. This study provides a novel framework connecting EVs enhanced release of mitochondrial components to mitochondrial and lysosomal dysfunction in HD.

Extracellular vesicles improve GABAergic transmission in Huntington’s disease iPSC-derived neurons

“…In this study, we used a previously published protocol (Beatriz et al ., 2022; Delli Carri et al ., 2013; Lopes et al, 2020) to promote neural induction in pluripotent cell colonies and obtain mature neurons (Figure S1A). Pluripotency (Nanog and OCT4) and neural induction (Nestin) marker were assessed in iPSCs and NSCs, respectively (Figure 1A).…”

“…Fibroblasts were grown in DMEM medium (#D5648, Gibco) supplemented with 9 mM sodium bicarbonate, 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Gibco). Induced pluripotent stem cells (iPSC) were reprogrammed from human dermal fibroblasts (from two HD carriers derived lines with 43 (fHD3) and 46 (fHD1) CAG repeats and two CTRs (fCTR1, fCTR3; Figure 1D) (Beatriz et al ., 2022). iPSCs were maintained in Geltrex®-coated plates with StemFlex medium (Gibco).…”

“…iPSCs were maintained in Geltrex®-coated plates with StemFlex medium (Gibco). Differentiation into a striatal-specific neuronal phenotype was performed as previously described (Beatriz et al ., 2022). Briefly, neuronal induction media was supplemented with 5 μM Dorsomorphin (Sigma) and 10 μM SB431542 (Peprotech) until day five and later 1 μM XAV 939 (Peprotech) and 200 ng/ml Sonic Hedgehog (SHH C-25II; R&D) were added until day 12 (Chambers et al ., 2009; Delli Carri et al ., 2013; Nicoleau et al ., 2013).…”

“…Isolation of EVs was performed as previously described (Beatriz et al ., 2022). Briefly, fibroblasts were cultured in in medium depleted of EVs by centrifuging the FBS at 100 000 x g for 18 hours at 4°C.…”

“…As previously described (Beatriz et al ., 2022; Lopes et al, 2020), cells were lysed in a lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% TritonX-100) supplemented with protease inhibitor cocktail (Sigma), and protein was quantified using the Bio-Rad Protein Assay (Bio-Rad). For protein denaturation, SDS sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 5% glycerol, 600 mM DTT, 0.01% bromophenol blue) was added to protein lysates (30 μg for fibroblasts) and heated for five minutes at 95°C.…”