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Funder: Gates Cambridge Trust; Id: http://dx.doi.org/10.13039/501100005370, Parkinson's disease-related proteins, PINK1 and Parkin, act in a common pathway to maintain mitochondrial quality control. While the PINK1-Parkin pathway can promote autophagic mitochondrial turnover (mitophagy) following mitochondrial toxification in cell culture, alternative quality control pathways are suggested. To analyse the mechanisms by which the PINK1-Parkin pathway operates in vivo, we developed methods to detect Ser65-phosphorylated ubiquitin (pS65-Ub) in Drosophila. Exposure to the oxidant paraquat led to robust, Pink1-dependent pS65-Ub production, while pS65-Ub accumulates in unstimulated parkin-null flies, consistent with blocked degradation. Additionally, we show that pS65-Ub specifically accumulates on disrupted mitochondria in vivo. Depletion of the core autophagy proteins Atg1, Atg5 and Atg8a did not cause pS65-Ub accumulation to the same extent as loss of parkin, and overexpression of parkin promoted turnover of both basal and paraquat-induced pS65-Ub in an Atg5-null background. Thus, we have established that pS65-Ub immunodetection can be used to analyse Pink1-Parkin function in vivo as an alternative to reporter constructs. Moreover, our findings suggest that the Pink1-Parkin pathway can promote mitochondrial turnover independently of canonical autophagy in vivo.

Funder: Novo Nordisk Foundation/Novo Nordisk Fonden; Id: http://dx.doi.org/10.13039/501100011747, Funder: Helsingin Yliopisto (HY); Id: http://dx.doi.org/10.13039/100007797, Funder: Sydäntutkimussäätiö (Finnish Foundation for Cardiovascular Research); Id: http://dx.doi.org/10.13039/501100005633, Funder: Jane and Aatos Erkko Foundation; Id: http://dx.doi.org/10.13039/501100004012, Mitophagy removes defective mitochondria via lysosomal elimination. Increased mitophagy coincides with metabolic reprogramming, yet it remains unknown whether mitophagy is a cause or consequence of such state changes. The signalling pathways that integrate with mitophagy to sustain cell and tissue integrity also remain poorly defined. We performed temporal metabolomics on mammalian cells treated with deferiprone, a therapeutic iron chelator that stimulates PINK1/PARKIN-independent mitophagy. Iron depletion profoundly rewired the metabolome, hallmarked by remodelling of lipid metabolism within minutes of treatment. DGAT1-dependent lipid droplet biosynthesis occurred several hours before mitochondrial clearance, with lipid droplets bordering mitochondria upon iron chelation. We demonstrate that DGAT1 inhibition restricts mitophagy in vitro, with impaired lysosomal homeostasis and cell viability. Importantly, genetic depletion of DGAT1 in vivo significantly impaired neuronal mitophagy and locomotor function in Drosophila. Our data define iron depletion as a potent signal that rapidly reshapes metabolism and establishes an unexpected synergy between lipid homeostasis and mitophagy that safeguards cell and tissue integrity.

Additional file 12 : Supplementary Figure 2. Enlargement of Fig. 3b. mRNA nuclear export analysis upon SRSF1 depletion at mRNA and FC levels.

Additional file 21 : Supplementary Figure 7. Quantification of KCNN1-4 mRNA expression levels in healthy and C9ORF72-ALS patient-derived neurons treated with control or SRSF1-RNAi lentivirus. Relative expression levels of KCNN1, KCNN2, KCNN3 and KCNN4 mRNAs were quantified using qRT-PCR in biological triplicates following normalization to U1 snRNA levels and to 100% for healthy neurons treated with Ctrl-RNAi (mean ± SEM; two-way ANOVA with Tukey’s correction for multiple comparisons; NS: not significant; ***: p

Additional file 8 : Supplementary Figure 1. Downregulation of mitosis/ cell proliferation/ cancer makers and upregulation of neuronal-related functions in healthy neurons with partial depletion of SRSF1. (A) Heatmap representing the computed fold changes for the healthy-treated neurons group. Red labels down-regulated transcripts while green depicts upregulated transcripts. (B) Heatmap representing the computed fold changes for the healthy-treated neurons group. Red labels down-regulated transcripts while green depicts upregulated transcripts. (C) Heatmap representing the computed fold changes for the healthy-treated neurons group. Red labels down-regulated transcripts while green depicts upregulated transcripts.

Mitochondrial Ca2+ uptake is an important mediator of metabolism and cell death. Identification of components of the highly conserved mitochondrial Ca2+ uniporter has opened it up to genetic analysis in model organisms. Here we report a comprehensive genetic characterisation of the known uniporter components conserved in Drosophila. While loss of MCU or EMRE abolishes fast mitochondrial Ca2+ uptake, this results in surprisingly mild phenotypes. In contrast, loss of the regulatory gatekeeper component MICU1 has a much more severe phenotype, being developmental lethal, consistent with unregulated Ca2+ uptake. Mutants for MICU3 are viable with mild neurological phenotypes. Genetic interaction studies reveal that MICU1 and MICU3 are not functionally interchangeable. More surprisingly, loss of MCU or EMRE does not suppress MICU1 mutant lethality, suggesting that the lethality results from MCU-independent functions. This study helps shed light on the physiological requirements of the mitochondrial Ca2+ uniporter, and provides a suite of tools to interrogate their interplay in homeostasis and disease conditions.

Parkinson disease is a debilitating and incurable neurodegenerative disorder affecting ∼1-2% of people over 65 years of age. Oxidative damage is considered to play a central role in the progression of Parkinson disease and strong evidence links chronic exposure to the pesticide paraquat with the incidence of the disease, most probably through the generation of oxidative damage. In this work, we demonstrated in human SH-SY5Y neuroblastoma cells the beneficial role of superoxide dismutase (SOD) enzymes against paraquat-induced toxicity, as well as the therapeutic potential of the SOD-mimetic compound M40403. Having verified the beneficial effects of superoxide dismutation in cells, we then evaluated the effects using Drosophila melanogaster as an in vivo model. Besides protecting against the oxidative damage induced by paraquat treatment, our data demonstrated that in Drosophila M40403 was able to compensate for the loss of endogenous SOD enzymes, acting both at a cytosolic and mitochondrial level. Because previous clinical trials have indicated that the M40403 molecule is well tolerated in humans, this study may have important implication for the treatment of Parkinson disease.

Compelling evidence indicates that two autosomal recessive Parkinson's disease genes, PINK1 (PARK6) and Parkin (PARK2), cooperate to mediate the autophagic clearance of damaged mitochondria (mitophagy). Mutations in the F-box domain-containing protein Fbxo7 (encoded by PARK15) also cause early-onset autosomal recessive Parkinson's disease, by an unknown mechanism. Here we show that Fbxo7 participates in mitochondrial maintenance through direct interaction with PINK1 and Parkin and acts in Parkin-mediated mitophagy. Cells with reduced Fbxo7 expression showed deficiencies in translocation of Parkin to mitochondria, ubiquitination of mitofusin 1 and mitophagy. In Drosophila, ectopic overexpression of Fbxo7 rescued loss of Parkin, supporting a functional relationship between the two proteins. Parkinson's disease-causing mutations in Fbxo7 interfered with this process, emphasizing the importance of mitochondrial dysfunction in Parkinson's disease pathogenesis.

Hexanucleotide repeat expansions in the C9ORF72 gene are the commonest known genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Expression of repeat transcripts and dipeptide repeat proteins trigger multiple mechanisms of neurotoxicity. How repeat transcripts get exported from the nucleus is unknown. Here, we show that depletion of the nuclear export adaptor SRSF1 prevents neurodegeneration and locomotor deficits in a Drosophila model of C9ORF72-related disease. This intervention suppresses cell death of patient-derived motor neuron and astrocytic-mediated neurotoxicity in co-culture assays. We further demonstrate that either depleting SRSF1 or preventing its interaction with NXF1 specifically inhibits the nuclear export of pathological C9ORF72 transcripts, the production of dipeptide-repeat proteins and alleviates neurotoxicity in Drosophila, patient-derived neurons and neuronal cell models. Taken together, we show that repeat RNA-sequestration of SRSF1 triggers the NXF1-dependent nuclear export of C9ORF72 transcripts retaining expanded hexanucleotide repeats and reveal a novel promising therapeutic target for neuroprotection.