Neuroligin-mediated neurodevelopmental defects are induced by mitochondrial dysfunction and prevented by lutein in C. elegans

Complex-I-deficiency represents the most frequent pathogenetic cause of human mitochondriopathies. Therapeutic options for these neurodevelopmental life-threating disorders do not exist, partly due to the scarcity of appropriate model systems to study them. Caenorhabditis elegans is a genetically tractable model organism widely used to investigate neuronal pathologies. Here, we generate C. elegans models for mitochondriopathies and show that depletion of complex I subunits recapitulates biochemical, cellular and neurodevelopmental aspects of the human diseases. We exploit two models, nuo-5/NDUFS1- and lpd-5/NDUFS4-depleted animals, for a suppressor screening that identifies lutein for its ability to rescue animals' neurodevelopmental deficits. We uncover overexpression of synaptic neuroligin as an evolutionarily conserved consequence of mitochondrial dysfunction, which we find to mediate an early cholinergic defect in C. elegans. We show lutein exerts its beneficial effects by restoring neuroligin expression independently from its antioxidant activity, thus pointing to a possible novel pathogenetic target for the human disease.We would like to thank Professor Tom Johnson at the University of Colorado Boulder and Professor Shane Rea (now at University of Washington) for making possible theinitial customized HMAD RNAi screening. We also would like to thank Alison Kell, Jenny Cho, and Alessandro Torgovnick for technical help with RNAi screen and lifespan; Professor Proksch at the Heinrich Heine University of Duesseldorf for the compound library used in the suppressor screen; Núria Benseny-Cases at MIRAS beamline in ALBA Synchrotron Light Source, Cerdanyola del Vallès in Barcelona, and Genis Rabost for technical assistance with FTIR measurements and analysis; Anthony Luz and Ian Ryde at Duke University for assistance with Seahorse XFe24 analysis and DNA damage assays respectively; Dirk Schwitters for qPCR on brains from WT and NDUFS4−/− mice. Finally, we thank the Caenorhabditis Genetics Center (funded by the National Institutes of Health Office of Research Infrastructure Programs: P40OD010440) as well as the National Bioresource Project (NBRP) for C. elegans strains and Professors Sieburth, Kaplan and Calahorro for providing additional mutants used in this work. The Wood-Whelan fellowship covered Silvia Maglioni costs to visit Joel Meyer Laboratory. This work was possible thanks to financial support from the German Research Foundation (DFG grants VE663-3/1 and VE663/8-1), the Federal Ministry of Education and Research (JPI-HDHL, Grant no. 01EA1602), and the Heinrich Heine University of Duesseldorf (Strategic Research Funding 2014) to NV; the National Institutes of Health (P42ES010356) to J.N.M., a fellowship from the China Scholarship Council (CSC201607030005) to Z.L.; the Spanish Ministry of Science, Innovation and Universities (RTI2018-096273-B-I00) and the ‘Severo Ochoa’ Programme for Centers of Excellence in R&D (SEV-2015-0496) to A.L.; the ERC Stg 337327 MitoPexLyso to N.R.With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).Peer reviewe

Modulation of oxidative phosphorylation and redox homeostasis in mitochondrial NDUFS4 deficiency via mesenchymal stem cells

Abstract Background Disorders of the oxidative phosphorylation (OXPHOS) system represent a large group among the inborn errors of metabolism. The most frequently observed biochemical defect is isolated deficiency of mitochondrial complex I (CI). No effective treatment strategies for CI deficiency are so far available. The purpose of this study was to investigate whether and how mesenchymal stem cells (MSCs) are able to modulate metabolic function in fibroblast cell models of CI deficiency. Methods We used human and murine fibroblasts with a defect in the nuclear DNA encoded NDUFS4 subunit of CI. Fibroblasts were co-cultured with MSCs under different stress conditions and intercellular mitochondrial transfer was assessed by flow cytometry and fluorescence microscopy. Reactive oxygen species (ROS) levels were measured using MitoSOX-Red. Protein levels of CI were analysed by blue native polyacrylamide gel electrophoresis (BN-PAGE). Results Direct cellular interactions and mitochondrial transfer between MSCs and human as well as mouse fibroblast cell lines were demonstrated. Mitochondrial transfer was visible in 13.2% and 6% of fibroblasts (e.g. fibroblasts containing MSC mitochondria) for human and mouse cell lines, respectively. The transfer rate could be further stimulated via treatment of cells with TNF-α. MSCs effectively lowered cellular ROS production in NDUFS4-deficient fibroblast cell lines (either directly via co-culture or indirectly via incubation of cell lines with cell-free MSC supernatant). However, CI protein expression and activity were not rescued by MSC treatment. Conclusion This study demonstrates the interplay between MSCs and fibroblast cell models of isolated CI deficiency including transfer of mitochondria as well as modulation of cellular ROS levels. Further exploration of these cellular interactions might help to develop MSC-based treatment strategies for human CI deficiency

The long non-coding RNA HOTAIRM1 promotes tumor aggressiveness and radiotherapy resistance in glioblastoma

Glioblastoma is the most common malignant primary brain tumor. To date, clinically relevant biomarkers are restricted to isocitrate dehydrogenase (IDH) gene 1 or 2 mutations and O6-methylguanine DNA methyltransferase (MGMT) promoter methylation. Long non-coding RNAs (lncRNAs) have been shown to contribute to glioblastoma pathogenesis and could potentially serve as novel biomarkers. The clinical significance of HOXA Transcript Antisense RNA, Myeloid-Specific 1 (HOTAIRM1) was determined by analyzing HOTAIRM1 in multiple glioblastoma gene expression data sets for associations with prognosis, as well as, IDH mutation and MGMT promoter methylation status. Finally, the role of HOTAIRM1 in glioblastoma biology and radiotherapy resistance was characterized in vitro and in vivo. We identified HOTAIRM1 as a candidate lncRNA whose up-regulation is significantly associated with shorter survival of glioblastoma patients, independent from IDH mutation and MGMT promoter methylation. Glioblastoma cell line models uniformly showed reduced cell viability, decreased invasive growth and diminished colony formation capacity upon HOTAIRM1 down-regulation. Integrated proteogenomic analyses revealed impaired mitochondrial function and determination of reactive oxygen species (ROS) levels confirmed increased ROS levels upon HOTAIRM1 knock-down. HOTAIRM1 knock-down decreased expression of transglutaminase 2 (TGM2), a candidate protein implicated in mitochondrial function, and knock-down of TGM2 mimicked the phenotype of HOTAIRM1 down-regulation in glioblastoma cells. Moreover, HOTAIRM1 modulates radiosensitivity of glioblastoma cells both in vitro and in vivo. Our data support a role for HOTAIRM1 as a driver of biological aggressiveness, radioresistance and poor outcome in glioblastoma. Targeting HOTAIRM1 may be a promising new therapeutic approach