The STING pathway does not contribute to behavioural or mitochondrial phenotypes in Drosophila Pink1/parkin or mtDNA mutator models

Abstract: Mutations in PINK1 and Parkin/PRKN cause the degeneration of dopaminergic neurons in familial forms of Parkinson’s disease but the precise pathogenic mechanisms are unknown. The PINK1/Parkin pathway has been described to play a central role in mitochondrial homeostasis by signalling the targeted destruction of damaged mitochondria, however, how disrupting this process leads to neuronal death was unclear until recently. An elegant study in mice revealed that the loss of Pink1 or Prkn coupled with an additional mitochondrial stress resulted in the aberrant activation of the innate immune signalling, mediated via the cGAS/STING pathway, causing degeneration of dopaminergic neurons and motor impairment. Genetic knockout of Sting was sufficient to completely prevent neurodegeneration and accompanying motor deficits. To determine whether Sting plays a conserved role in Pink1/parkin related pathology, we tested for genetic interactions between Sting and Pink1/parkin in Drosophila. Surprisingly, we found that loss of Sting, or its downstream effector Relish, was insufficient to suppress the behavioural deficits or mitochondria disruption in the Pink1/parkin mutants. Thus, we conclude that phenotypes associated with loss of Pink1/parkin are not universally due to aberrant activation of the STING pathway

Basal mitophagy is widespread in <i>Drosophila</i> but minimally affected by loss of Pink1 or parkin.

The Parkinson's disease factors PINK1 and parkin are strongly implicated in stress-induced mitophagy in vitro, but little is known about their impact on basal mitophagy in vivo. We generated transgenic Drosophila melanogaster expressing fluorescent mitophagy reporters to evaluate the impact of Pink1/parkin mutations on basal mitophagy under physiological conditions. We find that mitophagy is readily detectable and abundant in many tissues, including Parkinson's disease-relevant dopaminergic neurons. However, we did not detect mitolysosomes in flight muscle. Surprisingly, in Pink1 or parkin null flies, we did not observe any substantial impact on basal mitophagy. Because these flies exhibit locomotor defects and dopaminergic neuron loss, our findings raise questions about current assumptions of the pathogenic mechanism associated with the PINK1/parkin pathway. Our findings provide evidence that Pink1 and parkin are not essential for bulk basal mitophagy in Drosophila They also emphasize that mechanisms underpinning basal mitophagy remain largely obscure

Mitochondrially-targeted APOBEC1 is a potent mtDNA mutator affecting mitochondrial function and organismal fitness in Drosophila

Abstract: Somatic mutations in the mitochondrial genome (mtDNA) have been linked to multiple disease conditions and to ageing itself. In Drosophila, knock-in of a proofreading deficient mtDNA polymerase (POLG) generates high levels of somatic point mutations and also small indels, but surprisingly limited impact on organismal longevity or fitness. Here we describe a new mtDNA mutator model based on a mitochondrially-targeted cytidine deaminase, APOBEC1. mito-APOBEC1 acts as a potent mutagen which exclusively induces C:G>T:A transitions with no indels or mtDNA depletion. In these flies, the presence of multiple non-synonymous substitutions, even at modest heteroplasmy, disrupts mitochondrial function and dramatically impacts organismal fitness. A detailed analysis of the mutation profile in the POLG and mito-APOBEC1 models reveals that mutation type (quality) rather than quantity is a critical factor in impacting organismal fitness. The specificity for transition mutations and the severe phenotypes make mito-APOBEC1 an excellent mtDNA mutator model for ageing research

Detection of zoonotic pathogens and characterization of novel viruses carried by commensal Rattus norvegicus in New York City

Norway rats (Rattus norvegicus) are globally distributed and concentrate in urban environments, where they live and feed in closer proximity to human populations than most other mammals. Despite the potential role of rats as reservoirs of zoonotic diseases, the microbial diversity present in urban rat populations remains unexplored. In this study, we used targeted molecular assays to detect known bacterial, viral, and protozoan human pathogens and unbiased high-throughput sequencing to identify novel viruses related to agents of human disease in commensal Norway rats in New York City. We found that these rats are infected with bacterial pathogens known to cause acute or mild gastroenteritis in people, including atypical enteropathogenic Escherichia coli, Clostridium difficile, and Salmonella enterica, as well as infectious agents that have been associated with undifferentiated febrile illnesses, including Bartonella spp., Streptobacillus moniliformis, Leptospira interrogans, and Seoul hantavirus. We also identified a wide range of known and novel viruses from groups that contain important human pathogens, including sapoviruses, cardioviruses, kobuviruses, parechoviruses, rotaviruses, and hepaciviruses. The two novel hepaciviruses discovered in this study replicate in the liver of Norway rats and may have utility in establishing a small animal model of human hepatitis C virus infection. The results of this study demonstrate the diversity of microbes carried by commensal rodent species and highlight the need for improved pathogen surveillance and disease monitoring in urban environments. Importance: The observation that most emerging infectious diseases of humans originate in animal reservoirs has led to wide-scale microbial surveillance and discovery programs in wildlife, particularly in the developing world. Strikingly, less attention has been focused on commensal animals like rats, despite their abundance in urban centers and close proximity to human populations. To begin to explore the zoonotic disease risk posed by urban rat populations, we trapped and surveyed Norway rats collected in New York City over a 1-year period. This analysis revealed a striking diversity of known pathogens and novel viruses in our study population, including multiple agents associated with acute gastroenteritis or febrile illnesses in people. Our findings indicate that urban rats are reservoirs for a vast diversity of microbes that may affect human health and indicate a need for increased surveillance and awareness of the disease risks associated with urban rodent infestation

Comprehensive Genetic Characterization of Mitochondrial Ca2+ Uniporter Components Reveals Their Different Physiological Requirements In Vivo.

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 characterization of all known uniporter components conserved in Drosophila. While loss of pore-forming MCU or EMRE abolishes fast mitochondrial Ca2+ uptake, this results in only mild phenotypes when young, despite shortened lifespans. In contrast, loss of the MICU1 gatekeeper is developmentally lethal, consistent with unregulated Ca2+ uptake. Mutants for the neuronally restricted regulator MICU3 are viable with mild neurological impairment. Genetic interaction analyses reveal that MICU1 and MICU3 are not functionally interchangeable. More surprisingly, loss of MCU or EMRE does not suppress MICU1 mutant lethality, suggesting that this results from uniporter-independent functions. Our data reveal the interplay among components of the mitochondrial Ca2+ uniporter and shed light on their physiological requirements in vivo.This work is supported by MRC core funding (MC_UU_00015/4, MC-A070-5PSB0, and MC_UU_00015/6) and an ERC starting grant (DYNAMITO; 309742) to A.J.W., as well as by the Italian Ministry of Health “Ricerca Finalizzata” (GR-2011-02351151) to E.Z. T.P.G. and J.J.L. are supported by MRC studentships awarded via the MRC MBU. V.L.H. was funded by an EMBO Long-Term Fellowship (ALTF 740-2015) co-funded by the European Commission FP7 (Marie Curie Actions, LTFCOFUND2013, GA-2013-609409)

N-terminal acetylation shields proteins from degradation and promotes age-dependent motility and longevity

Most eukaryotic proteins are N-terminally acetylated, but the functional impact on a global scale has remained obscure. Using genome-wide CRISPR knockout screens in human cells, we reveal a strong genetic dependency between a major N-terminal acetyltransferase and specific ubiquitin ligases. Biochemical analyses uncover that both the ubiquitin ligase complex UBR4-KCMF1 and the acetyltransferase NatC recognize proteins bearing an unacetylated N-terminal methionine followed by a hydrophobic residue. NatC KO-induced protein degradation and phenotypes are reversed by UBR knockdown, demonstrating the central cellular role of this interplay. We reveal that loss of Drosophila NatC is associated with male sterility, reduced longevity, and age-dependent loss of motility due to developmental muscle defects. Remarkably, muscle-specific overexpression of UbcE2M, one of the proteins targeted for NatC KO-mediated degradation, suppresses defects of NatC deletion. In conclusion, NatC-mediated N-terminal acetylation acts as a protective mechanism against protein degradation, which is relevant for increased longevity and motility. The most common protein modification in eukaryotes is N-terminal acetylation, but its functional impact has remained enigmatic. Here, the authors find that a key role for N-terminal acetylation is shielding proteins from ubiquitin ligase-mediated degradation, mediating motility and longevity.Association Francaise contre les Myopathies 261981, Canadian Institutes of Health Research (CIHR) 249843, United States Department of Health & Human Services National Institutes of Health (NIH) - USA F-12540, Portuguese national funding through Fundaco para a Ciencia e a Tecnologia (FCT) 171752-PR-2009-0222, National Funds through Fundaco para a Ciencia e a Tecnologia (FCT) G008018N, G002721N, University of Bergen MC_UU_00028/6, FDN-143264, FDN-143265, PJT-180285, PJT-463531, R01HG005853, R01HG005084, DL 57/2016/CP1361/CT0019, 2022.01782.PTDC,PTDC/BIA-BID/28441/2017,PTDC/BIA-BID/1606/2020, ALG-01-0145-FEDER-028441, PPBI-POCI-01-0145-FEDER-022122, LISBOA-01-0145-FEDER-022170info:eu-repo/semantics/publishedVersio

Le Forum, Vol. 44 #3

https://digitalcommons.library.umaine.edu/francoamericain_forum/1105/thumbnail.jp

Comprehensive Genetic Characterisation of Mitochondrial Ca2+ Uniporter Components Reveals Their Different Physiological Requirements in Vivo

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 all known uniporter components conserved in Drosophila. While loss of pore-forming MCU or EMRE abolishes fast mitochondrial Ca2+ uptake, this results in only mild phenotypes when young, despite shortened lifespans. In contrast, loss of the MICU1 gatekeeper is developmental lethal, consistent with unregulated Ca2+ uptake. Mutants for the neuronally-restricted regulator MICU3 are viable with mild neurological impairment. Genetic interaction analyses reveal that MICU1 and MICU3 are not functionally interchangeable. More surprisingly, loss of MCU or EMRE does not suppress MICU1 mutant lethality, suggesting that this results from uniporter-independent functions. Our data interrogates the interplay between components of the mitochondrial Ca2+ uniporter, and sheds light on their physiological requirements in vivo.This work is supported by MRC core funding (MC_UU_00015/4, MC-A070-5PSB0 and MC_UU_00015/6) and ERC Starting grant (DYNAMITO; 309742) to A.J.W., and the Italian Ministry of Health “Ricerca Finalizzata” [GR-2011-02351151] to E.Z. T.P.G. and J.J.L. are supported by MRC Studentships awarded via the MRC MBU. V.L.H. was funded by an EMBO Long-Term Fellowship (ALTF 740-2015) co-funded by the European Commission FP7 (Marie Curie Actions, LTFCOFUND2013, GA-2013-609409). Stocks were obtained from the Bloomington Drosophila Stock Center which is supported by grant NIH P40OD018537, and material was obtained from the Drosophila Genomics Resource Center, which is supported by NIH grant 2P40OD010949