Pileggi, C. | bioRxiv Science

bioRxiv2020

Cardiomyocyte-specific Srsf3 deletion reveals a mitochondrial regulatory role

Srsf3 was recently reported as being necessary to preserve RNA stability via an mTOR mechanism in a cardiac mouse model in adulthood. Here, we demonstrate the link between Srsf3 and mitochondrial integrity in an embryonic cardiomyocyte-specific Srsf3 conditional knockout (cKO) mouse model. Fifteen-day-old Srsf3 cKO mice showed dramatically reduced (below 50%) survival and reduced left ventricular systolic performance, and histological analysis of these hearts revealed a significant increase in cardiomyocyte size, confirming the severe remodelling induced by Srsf3 deletion. RNA-seq analysis of the hearts of 5-day-old Srsf3 cKO mice revealed early changes in expression levels and alternative splicing of several transcripts related to mitochondrial integrity and oxidative phosphorylation. Likewise, the levels of several protein complexes of the electron transport chain decreased, and mitochondrial complex I-driven respiration of permeabilized cardiac muscle fibres from the left ventricle was impaired. Furthermore, transmission electron microscopy analysis showed disordered mitochondrial length and cristae structure. Together with its indispensable role in the physiological maintenance of mouse hearts, these results highlight the previously unrecognized function of Srsf3 in regulating mitochondrial integrity.

physiology↗

bioRxiv2020

A mouse model for spinal muscular atrophy provides insights into non-alcoholic fatty liver disease pathogenesis

Background & aims Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder leading to paralysis and death in children. SMA patients are more susceptible to dyslipidemia as well as liver steatosis, features reproduced in SMA mouse models. As current pre-clinical models of NAFLD are invariably imperfect and generally take a long time to develop, the rapid development of liver steatosis in SMA mice provides a means to identify molecular markers of non-alcoholic fatty liver disease (NAFLD). Here, we investigated whether Smn2B/- mice, a model of severe SMA, display typical features of NAFLD/non-alcoholic steatohepatitis (NASH).Methods Biochemical, histological, electron microscopy, proteomic, and high-resolution respirometry were used.Results The Smn2B/- mice develop steatohepatitis early in life. The consequent liver damage arises from mitochondrial reactive oxygen species production and results in impaired hepatic function including alterations in protein output, complement, coagulation, iron homeostasis, and IGF-1 metabolism. The steatohepatitis is reversible by AAV9-SMN gene therapy. The NAFLD phenotype is likely due to non-esterified fatty acid (NEFA) overload from peripheral lipolysis, subsequent to hyperglucagonemia compounded by reduced muscle use. Mitochondrial β-oxidation contributed to hepatic damage as we observed enhanced hepatic mitochondrial β-oxidation and reactive oxygen species production. Hepatic mitochondrial content, however, was decreased. In contrast to typical NAFLD/NASH, the Smn2B/- mice lose weight due to their neurological condition, develop hypoglycemia and do not develop hepatic fibrosis.Conclusion The Smn2B/- mice represent a good model of microvesicular steatohepatitis. Like other models, it is not representative of the complete NAFLD/NASH spectrum. Nevertheless, it offers a reliable, low-cost, early onset model that is not dependent on diet to identify molecular players in NAFLD pathogenesis and can serve as one of the very few models of microvesicular steatohepatitis for both adult and pediatric populations.Competing Interest StatementMarc-Olivier Deguise received honoraria and travel accommodations from Biogen for speaking engagements at the SMA Summit 2018 held in Montreal, Canada and SMA Academy 2019 held in Toronto, Canada. Rashmi Kothary received honoraria and travel accommodations from Roche as an invited speaker at their global and national board meetings in 2019. RK and the Ottawa Hospital Research Institute have a licensing agreement with Biogen for the Smn2B/- mouse model. Thomas H. Gillingwater has served on global and UK advisory boards for Roche. These COI are outside the scope of this study. All other authors have no competing interests to declare.List of abbreviationsAGCautomatic gain controlALPalkaline phosphataseALTalanine aminotransferaseASTaspartate aminotransferaseBaxBCL2 associated X proteinDAVIDThe Database for Annotation, Visualization and Integrated DiscoveryESEnrichment ScoreFasRFas receptorH&EHematoxylin & eosinHFDhigh fat dietIGF-1insulin-like growth factor 1IGFbp1insulin like growth factor binding protein 1IGF1Rinsulin like growth factor 1 receptorigfalsinsulin like growth factor binding protein acid labile subunitIPAingenuity pathway analysisMCDmethionine and choline deficient dietMCLMarkov Clustering AlgorithmNAFLDnon-alcoholic fatty liver diseaseNASHnon-alcoholic steatohepatitisNEFAnon-esterified fatty acidPpostnatal dayp21cyclin dependent kinase inhibitor 1Ap53tumor protein p53PASPeriodic acid-SchiffSMAspinal muscular atrophySMN1Survival motor neuron 1TMTTandem Mass TaggingTNFR1TNF receptor superfamily member 1AView Full Text

pathology↗

bioRxiv2020

Parkinson Disease-Linked Parkin Mediates Redox Reactions That Lower Oxidative Stress In Mammalian Brain

We recently hypothesized that parkin plays a role in redox homeostasis and provided evidence that it directly reduces hydrogen peroxide (H2O2) in vitro. Here, we examined this anti-oxidant activity in vivo. Informed by findings in human brain, we demonstrate that elevated oxidative stress promotes parkin insolubility in mice. In normal mouse brain parkin was partially oxidized, e.g., at cysteines 195 and 252, which was augmented by oxidative stress. Although under basal conditions H2O2 levels were unchanged in adult prkn-/- brain, a parkin-dependent reduction of cytosolic H2O2 was observed when mitochondria were impaired, either due to neurotoxicant exposure (MPTP) or Sod2 haploinsufficiency. In accordance, markers of oxidative stress, e.g., protein carbonylation and nitrotyrosination, were elevated in the cytosol but not in mitochondria from prkn-/- mice. Nevertheless, this rise in oxidative stress led to changes in mitochondrial enzyme activities and the metabolism of glutathione in cells and mammalian brain. In parkins absence reduced glutathione concentrations were increased including in human cortex. This compensation was not due to new glutathione synthesis but attributed to elevated oxidized glutathione (GSSG)-reductase activity. Moreover, we discovered that parkin also recycled GSSG to its reduced form. With this reaction, parkin became S-glutathionylated, e.g., at cysteines 59 and human-specific 95. This oxidative modification was reversed by glutaredoxin. Our results demonstrate that cytosolic parkin mediates anti-oxidant reactions including H2O2 reduction and glutathione regeneration. These reducing activities lead to a range of oxidative modifications in parkin itself. In parkin-deficient brain oxidative stress rises despite changes to maintain redox balance.

neuroscience↗