A new mechanism for mtDNA pathogenesis: impairment of post-transcriptional maturation leads to severe depletion of mitochondrial tRNA(Ser(UCN)) caused by T7512C and G7497A point mutations

We have studied the consequences of two homoplasmic, pathogenic point mutations (T7512C and G7497A) in the tRNA(Ser(UCN)) gene of mitochondrial (mt) DNA using osteosarcoma cybrids. We identified a severe reduction of tRNA(Ser(UCN)) to levels below 10% of controls for both mutations, resulting in a 40% reduction in mitochondrial protein synthesis rate and in a respiratory chain deficiency resembling that in the patients muscle. Aminoacylation was apparently unaffected. On non-denaturating northern blots we detected an altered electrophoretic mobility for G7497A containing tRNA molecules suggesting a structural impact of this mutation, which was confirmed by structural probing. By comparing in vitro transcribed molecules with native RNA in such gels, we also identified tRNA(Ser(UCN)) being present in two isoforms in vivo, probably corresponding to the nascent, unmodified transcripts co-migrating with the in vitro transcripts and a second, faster moving isoform corresponding to the mature tRNA. In cybrids containing either mutations the unmodified isoforms were severely reduced. We hypothesize that both mutations lead to an impairment of post-transcriptional modification processes, ultimately leading to a preponderance of degradation by nucleases over maturation by modifying enzymes, resulting in severely reduced tRNA(Ser(UCN)) steady state levels. We infer that an increased degradation rate, caused by disturbance of tRNA maturation and, in the case of the G7497A mutant, alteration of tRNA structure, is a new pathogenic mechanism of mt tRNA point mutations

Molecular dysfunction associated with the human mitochondrial 3302A>G mutation in the MTTL1 (mt-tRNA(Leu(UUR))) gene

The gene encoding mt-tRNA(Leu(UUR)), MT-TL1, is a hotspot for pathogenic mtDNA mutations. Amongst the first to be described was the 3302A>G transition which resulted in a substantial accumulation in patient muscle of RNA19, an unprocessed RNA intermediate including mt-16S rRNA, mt-tRNA(Leu(UUR)) and MTND1. We have now been able to further assess the molecular aetiology associated with 3302A>G in transmitochondrial cybrids. Increased steady-state levels of RNA19 was confirmed, although not to the levels previously reported in muscle. This data was consistent with an increase in RNA19 stability. The mutation resulted in decreased mt-tRNA(Leu(UUR)) levels, but its stability was unchanged, consistent with a defect in RNA19 processing responsible for low tRNA levels. A partial defect in aminoacylation was also identified, potentially caused by an alteration in tRNA structure. These deficiencies lead to a severe defect in respiration in the transmitochondrial cybrids, consistent with the profound mitochondrial disorder originally associated with this mutation

The Ras related GTPase Miro is not required for mitochondrial transport in Dictyostelium discoideum

Ras-related GTPases of the Miro family have been implicated in mitochondrial homeostasis and microtubule-dependent transport. They consist of two GTP-binding domains separated by calcium-binding motifs and of a C-terminal transmembrane domain that targets the protein to the outer mitochondrial membrane. We disrupted the single Miro-encoding gene in Dictyostelium discoideum and observed a substantial growth defect that we attribute to a decreased mitochondrial mass and cellular ATP content. However, mutant cells even showed an increased rate of oxygen consumption, while glucose consumption, mitochondrial transmembrane potential and production of reactive oxygen species were unaltered. Processes characteristic of the multicellular stage of the D. discoideum life cycle were also unaltered. Although mitochondria occasionally use microtubules for transport in D. discoideum, their size and distribution were not visibly affected. We found Miro in all branches of the eukaryotic tree with the exception of a few protist lineages (mainly those lacking typical mitochondria). Trypanosomatids and ciliates possess structurally unique homologs lacking the N-terminal or the C-terminal GTPase domain, respectively. We propose that in D. discoideum, as in yeasts and plants, Miro plays roles in mitochondrial homeostasis, but the ability to build a complex that regulates its association to kinesin for microtubule-dependent transport probably arose in metazoans

Alice in the digital wonderland : pediatric teaching during the COVID-19 pandemic. A survey and comment of the Teaching Working Group of the German Society of Pediatrics and Adolescent Medicine (DGKJ)

Die Umstellung der pädiatrischen Lehre im Medizinstudium von Präsenzveranstaltungen auf digitale Formate war und ist für Lehrende und Studierende eine besondere Herausforderung. Basierend auf einer Umfrage unter pädiatrischen Universitätskliniken fasst die AG Lehre der DGKJ im folgenden Beitrag bisherige Erfahrungen zur digitalen Lehre im Sommersemester 2020 zusammen. Die Umfrageteilnehmer von 17 pädiatrischen Universkliniken berichteten über ein umfangreiches Spektrum oft kurzfristig entwickelter digitaler Lehrformate. Dabei waren Vorlesungen und Seminare gut, Unterrichtsformate mit direktem Patientenbezug und Fertigkeiten-/Kommunikationstrainings deutlich weniger gut durch digitale Formate ersetzbar. Die Implementierung digitaler Unterrichtsformate führte zu einem deutlich erhöhten Zeitaufwand für die Lehrenden. Digitale Lehrangebote benötigen eine gute IT-Infrastruktur, sie sollten in ein Curriculum eingebunden sein und durchweg direkte Austauschmöglichkeiten zwischen Studierenden und Lehrenden vorsehen. Lehrenden sollten Schulungen in Didaktik zur digitalen Lehre und zu IT-Kenntnissen angeboten werden. Diese Ergebnisse decken sich mit der Literatur zu digitalen Lehrformaten allgemein sowie zur Lehre im Rahmen der COVID-19-Pandemie im Besonderen. Die hier vorgestellten Erfahrungen sollen nicht nur die Entwicklung digitaler pädiatrischer Lehrformate während der bestehenden Pandemie erleichtern, sondern auch darüber hinaus die Konzeption neuer digitaler Lehrangebote für die Pädiatrie im Medizinstudium anregen. Insbesondere müssen neue digitale Ersatzformate für den Unterricht am Patienten entwickelt werden. Die Stellungnahme wurde im Konsens von der AG Lehre der DGKJ erarbeitet und vom DGKJVorstand verabschiedet.The COVID-19 pandemic led to a rapid switch from undergraduate classroom teaching to online-teaching; a challenging process for teachers and students. Based on a recent online survey among German pediatric university hospitals the “AG Lehre der DGKJ” (teaching working group of the German Society of Pediatrics and Adolescent Medicine) summarizes latest experiences with e-learning during the summer term of 2020. The survey participants from 17 pediatric university hospitals report that the large spectrum of e-learning formats could sufficiently replace classical lectures and seminars but could not fully replace teaching involving direct contact to patients. The introduction of new digital teaching formats is time-consuming, needs high-quality IT infrastructure, should be embedded in a continuous curriculum and provide the possibility of regular exchange between students and teachers. Teachers should be provided with the opportunity for training in didactic methods and IT skills. These results correspond to the literature on e-learning in general and undergraduate medical education during the COVID-19 pandemic in particular. The experiences summarized here should not only facilitate the development of e-learning tools during the ongoing pandemic but also stimulate to establish e-learning as a valuable component of future pediatric medical education. New digital substitutes for teaching involving pediatric patients need to be developed. The statement was drafted by consensus by the German Society of Pediatrics and Adolescent Medicine Working Group on Teaching and approved by the DGKJ board

Prohibitins control cell proliferation and apoptosis by regulating OPA1-dependent cristae morphogenesis in mitochondria

Prohibitins comprise an evolutionarily conserved and ubiquitously expressed family of membrane proteins with poorly described functions. Large assemblies of PHB1 and PHB2 subunits are localized in the inner membrane of mitochondria, but various roles in other cellular compartments have also been proposed for both proteins. Here, we used conditional gene targeting of murine Phb2 to define cellular activities of prohibitins. Our experiments restrict the function of prohibitins to mitochondria and identify the processing of the dynamin-like GTPase OPA1, an essential component of the mitochondrial fusion machinery, as the central cellular process controlled by prohibitins. Deletion of Phb2 leads to the selective loss of long isoforms of OPA1. This results in an aberrant cristae morphogenesis and an impaired cellular proliferation and resistance toward apoptosis. Expression of a long OPA1 isoform in PHB2-deficient cells suppresses these defects, identifying impaired OPA1 processing as the primary cellular defect in the absence of prohibitins. Our results therefore assign an essential function for the formation of mitochondrial cristae to prohibitins and suggest a coupling of cell proliferation to mitochondrial morphogenesis