No abstract

No abstract

No abstract

CLN3 disease is a lysosomal storage disorder associated with fatal neurodegeneration that is caused by mutations in CLN3, with most affected individuals carrying at least one allele with a 966 bp deletion. Using CRISPR/Cas9, we corrected the 966 bp deletion mutation in human induced pluripotent stem cells (iPSCs) of a compound heterozygous patient (CLN3 Δ 966 bp and E295K). We differentiated these isogenic iPSCs, and iPSCs from an unrelated healthy control donor to neurons and identified disease-related changes relating to protein synthesis, trafficking and degradation, and in neuronal activity which were not apparent in CLN3-corrected or healthy control neurons. CLN3 neurons showed numerous membrane-bound vacuoles containing diverse storage material, and hyperglycosylation of the lysosomal LAMP1 protein. Proteomic analysis showed increase in lysosomal-related proteins and many ribosomal subunit proteins in CLN3 neurons that was accompanied by downregulation of proteins related to axon guidance and endocytosis. CLN3 neurons also had lower electrophysical activity as recorded using microelectrode arrays. These data implicate interrelated pathways in protein homeostasis and neurite arborization as contributing to CLN3 disease, and which could be potential targets for therapy.

Short-chain quinones (SCQs) have been investigated as potential therapeutic candidates against mitochondrial dysfunction, which was largely thought to be associated with the reversible redox characteristics of their active quinone core. We recently reported a library of SCQs, some of which showed potent cytoprotective activity against the mitochondrial complex I inhibitor rotenone in the human hepatocarcinoma cell line HepG2. To better characterize the cytoprotection of SCQs at a molecular level, a bioactivity profile for 103 SCQs with different compound chemistries was generated that included metabolism related markers, redox activity, expression of cytoprotective proteins and oxidative damage. Of all the tested endpoints, a positive correlation with cytoprotection by SCQs in the presence of rotenone was only observed for the NAD(P)H:quinone oxidoreductase 1 (NQO1)-dependent reduction of SCQs, which also correlated with an acute rescue of ATP levels. The results of this study suggest an unexpected mode of action for SCQs that appears to involve a modification of NQO1-dependent signaling rather than a protective effect by the reduced quinone itself. This finding presents a new selection strategy to identify and develop the most promising compounds towards their clinical use.