The complex I subunit NDUFA10 selectively rescues Drosophila pink1 mutants through a mechanism independent of mitophagy.

Mutations in PINK1, a mitochondrially targeted serine/threonine kinase, cause autosomal recessive Parkinson's disease (PD). Substantial evidence indicates that PINK1 acts with another PD gene, parkin, to regulate mitochondrial morphology and mitophagy. However, loss of PINK1 also causes complex I (CI) deficiency, and has recently been suggested to regulate CI through phosphorylation of NDUFA10/ND42 subunit. To further explore the mechanisms by which PINK1 and Parkin influence mitochondrial integrity, we conducted a screen in Drosophila cells for genes that either phenocopy or suppress mitochondrial hyperfusion caused by pink1 RNAi. Among the genes recovered from this screen was ND42. In Drosophila pink1 mutants, transgenic overexpression of ND42 or its co-chaperone sicily was sufficient to restore CI activity and partially rescue several phenotypes including flight and climbing deficits and mitochondrial disruption in flight muscles. Here, the restoration of CI activity and partial rescue of locomotion does not appear to have a specific requirement for phosphorylation of ND42 at Ser-250. In contrast to pink1 mutants, overexpression of ND42 or sicily failed to rescue any Drosophila parkin mutant phenotypes. We also find that knockdown of the human homologue, NDUFA10, only minimally affecting CCCP-induced mitophagy, and overexpression of NDUFA10 fails to restore Parkin mitochondrial-translocation upon PINK1 loss. These results indicate that the in vivo rescue is due to restoring CI activity rather than promoting mitophagy. Our findings support the emerging view that PINK1 plays a role in regulating CI activity separate from its role with Parkin in mitophagy.

The Drosophila inner-membrane protein PMI controls crista biogenesis and mitochondrial diameter.

Cristae are mitochondrial inner-membrane structures that concentrate respiratory chain complexes and hence regulate ATP production. Mechanisms controlling crista morphogenesis are poorly understood and few crista determinants have been identified. Among them are the Mitofilins that are required to establish crista junctions and ATP-synthase subunits that bend the membrane at the tips of the cristae. We report here the phenotypic consequences associated with the in vivo inactivation of the inner-membrane protein Pantagruelian Mitochondrion I (PMI) both at the scale of the whole organism, and at the level of mitochondrial ultrastructure and function. We show that flies in which PMI is genetically inactivated experience synaptic defects and have a reduced life span. Electron microscopy analysis of the inner-membrane morphology demonstrates that loss of PMI function increases the average length of mitochondrial cristae in embryonic cells. This phenotype is exacerbated in adult neurons in which cristae form a dense tangle of elongated membranes. Conversely, we show that PMI overexpression is sufficient to reduce crista length in vivo. Finally, these crista defects are associated with impaired respiratory chain activity and increases in the level of reactive oxygen species. Since PMI and its human orthologue TMEM11 are regulators of mitochondrial morphology, our data suggest that, by controlling crista length, PMI influences mitochondrial diameter and tubular shape.

Partial loss of MCU mitigates pathology in vivo across a diverse range of neurodegenerative disease models.

Mitochondrial calcium (Ca2+) uptake augments metabolic processes and buffers cytosolic Ca2+ levels; however, excessive mitochondrial Ca2+ can cause cell death. Disrupted mitochondrial function and Ca2+ homeostasis are linked to numerous neurodegenerative diseases (NDs), but the impact of mitochondrial Ca2+ disruption is not well understood. Here, we show that Drosophila models of multiple NDs (Parkinson's, Huntington's, Alzheimer's, and frontotemporal dementia) reveal a consistent increase in neuronal mitochondrial Ca2+ levels, as well as reduced mitochondrial Ca2+ buffering capacity, associated with increased mitochondria-endoplasmic reticulum contact sites (MERCs). Importantly, loss of the mitochondrial Ca2+ uptake channel MCU or overexpression of the efflux channel NCLX robustly suppresses key pathological phenotypes across these ND models. Thus, mitochondrial Ca2+ imbalance is a common feature of diverse NDs in vivo and is an important contributor to the disease pathogenesis. The broad beneficial effects from partial loss of MCU across these models presents a common, druggable target for therapeutic intervention.

High-content phenotypic screen to identify small molecule enhancers of Parkin-dependent ubiquitination and mitophagy.

Mitochondrial dysfunction and aberrant mitochondrial homeostasis are key aspects of Parkinson's disease (PD) pathophysiology. Mutations in PINK1 and Parkin proteins lead to autosomal recessive PD, suggesting that defective mitochondrial clearance via mitophagy is key in PD etiology. Accelerating the identification and/or removal of dysfunctional mitochondria could therefore provide a disease-modifying approach to treatment. To that end, we performed a high-content phenotypic screen (HCS) of ∼125,000 small molecules to identify compounds that positively modulate mitochondrial accumulation of the PINK1-Parkin-dependent mitophagy initiation marker p-Ser65-Ub in Parkin haploinsufficiency (Parkin +/R275W) human fibroblasts. Following confirmatory counter-screening and orthogonal assays, we selected compounds of interest that enhance mitophagy-related biochemical and functional endpoints in patient-derived fibroblasts. Identification of inhibitors of the ubiquitin-specific peptidase and negative regulator of mitophagy USP30 within our hits further validated our approach. The compounds identified in this work provide a novel starting point for further investigation and optimization.

Mitochondrial quality control and neurological disease: an emerging connection.

The human brain is a highly complex organ with remarkable energy demands. Although it represents only 2% of the total body weight, it accounts for 20% of all oxygen consumption, reflecting its high rate of metabolic activity. Mitochondria have a crucial role in the supply of energy to the brain. Consequently, their deterioration can have important detrimental consequences on the function and plasticity of neurons, and is thought to have a pivotal role in ageing and in the pathogenesis of several neurological disorders. Owing to their inherent physiological functions, mitochondria are subjected to particularly high levels of stress and have evolved specific molecular quality-control mechanisms to maintain the mitochondrial components. Here, we review some of the most recent advances in the understanding of mitochondrial stress-control pathways, with a particular focus on how defects in such pathways might contribute to neurodegenerative disease.

Leveraging the immune system during chemotherapy: moving calreticulin to the cell surface converts apoptotic death from "silent" to immunogenic.

In contrast to prior belief, tumor cell apoptosis is not necessarily silent but can be immunogenic. By tracing how anthracyclines and gamma-irradiation trigger immunogenic cell deaths, we found that they were causally connected to the exposure of calreticulin on the tumor cell surface, before apoptosis in the tumor cell itself occurred. Furthermore, we showed that calreticulin exposure was necessary and sufficient to increase proimmunogenic killing by other chemotherapies. Our findings suggest that calreticulin could serve as a biomarker to predict therapy-associated immune responses, and that tactics to expose calreticulin might improve the clinical efficacy of many cancer therapies.

Lipid Transfer Proteins and Membrane Contact Sites in Human Cancer.

Lipid-transfer proteins (LTPs) were initially discovered as cytosolic factors that facilitate lipid transport between membrane bilayers in vitro. Since then, many LTPs have been isolated from bacteria, plants, yeast, and mammals, and extensively studied in cell-free systems and intact cells. A major advance in the LTP field was associated with the discovery of intracellular membrane contact sites (MCSs), small cytosolic gaps between the endoplasmic reticulum (ER) and other cellular membranes, which accelerate lipid transfer by LTPs. As LTPs modulate the distribution of lipids within cellular membranes, and many lipid species function as second messengers in key signaling pathways that control cell survival, proliferation, and migration, LTPs have been implicated in cancer-associated signal transduction cascades. Increasing evidence suggests that LTPs play an important role in cancer progression and metastasis. This review describes how different LTPs as well as MCSs can contribute to cell transformation and malignant phenotype, and discusses how "aberrant" MCSs are associated with tumorigenesis in human.

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.

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

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.

Enhancing nucleotide metabolism protects against mitochondrial dysfunction and neurodegeneration in a PINK1 model of Parkinson's disease.

Mutations in PINK1 cause early-onset Parkinson's disease (PD). Studies in Drosophila melanogaster have highlighted mitochondrial dysfunction on loss of Pink1 as a central mechanism of PD pathogenesis. Here we show that global analysis of transcriptional changes in Drosophila pink1 mutants reveals an upregulation of genes involved in nucleotide metabolism, critical for neuronal mitochondrial DNA synthesis. These key transcriptional changes were also detected in brains of PD patients harbouring PINK1 mutations. We demonstrate that genetic enhancement of the nucleotide salvage pathway in neurons of pink1 mutant flies rescues mitochondrial impairment. In addition, pharmacological approaches enhancing nucleotide pools reduce mitochondrial dysfunction caused by Pink1 deficiency. We conclude that loss of Pink1 evokes the activation of a previously unidentified metabolic reprogramming pathway to increase nucleotide pools and promote mitochondrial biogenesis. We propose that targeting strategies enhancing nucleotide synthesis pathways may reverse mitochondrial dysfunction and rescue neurodegeneration in PD and, potentially, other diseases linked to mitochondrial impairment.

Reticulon-1C acts as a molecular switch between endoplasmic reticulum stress and genotoxic cell death pathway in human neuroblastoma cells.

Damage or stress in many organelles may trigger apoptosis by several not yet fully elucidated mechanisms. A cell death pathway is induced by endoplasmic reticulum (ER) stress elicited by the unfolded protein response and/or by aberrant Ca(2+) signalling. Reticulon-1C (RTN-1C) belongs to the reticulon family, neuroendocrine-specific proteins localized primarily on the ER membrane. In the present study, we demonstrate that RTN-1C is able to modulate, in a mutually exclusive way, the cellular sensitivity to different apoptosis pathways in human neuroblastoma cells. In fact, the increase of RTN-1C protein levels per se results in ER stress-induced cell death, mediated by an increase of cytosolic Ca(2+), and significantly sensitizes cells to different ER stress inducers. In line with these findings, the reduction of RTN-1C, by antisense DNA expression, reduced the sensitivity to ER-stressors. In the presence of high RTN-1C levels, genotoxic drugs become ineffective as a consequence of the cytoplasm translocation of p53 protein, while the silencing of endogenous RTN-1C results in the potentiation of the genotoxic drugs action. These data indicate that RTN-1C is able to modulate the cellular sensitivity to different apoptotic pathways representing a promising molecular target for new drug development.

Ecto-calreticulin in immunogenic chemotherapy.

The conventional treatment of cancer relies upon radiotherapy and chemotherapy. Such treatments supposedly mediate their effects via the direct elimination of tumor cells. Nonetheless, there are circumstances in which conventional anti-cancer therapy can induce a modality of cellular demise that elicits innate and cognate immune responses, which in turn mediate part of the anti-tumor effect. Although different chemotherapeutic agents may kill tumor cells through an apparently homogeneous apoptotic pathway, they differ in their capacity to stimulate immunogenic cell death. We discovered that the pre-apoptotic translocation of intracellular calreticulin (endo-CRT) to the plasma membrane surface (ecto-CRT) is critical for the recognition and engulfment of dying tumor cells by dendritic cells. Thus, anthracyclines and gamma-irradiation that induce ecto-CRT cause immunogenic cell death, while other pro-apoptotic agents (such as mitomycin C and etoposide) induce neither ecto-CRT nor immunogenic cell death. Depletion of CRT abolishes the immunogenicity of cell death elicited by anthracyclines, while exogenous supply of CRT or enforcement of CRT exposure by pharmacological agents that favor CRT translocation can enhance the immunogenicity of cell death. For optimal anti-tumor vaccination and immunogenic chemotherapy, the same cells have to expose ecto-CRT and to succumb to apoptosis; if these events affect different cells, no anti-tumor immune response is elicited. These results may have far reaching implications for tumor immunology because (i) ecto-CRT exposure by tumor cells allows for the prediction of therapeutic outcome and because (ii) the re-establishment of ecto-CRT may ameliorate the efficacy of chemotherapy.

Endoplasmic reticulum stress induces apoptosis by an apoptosome-dependent but caspase 12-independent mechanism.

The endoplasmic reticulum (ER) is the cellular site of polypeptide folding and modification. When these processes are hampered, an unfolded protein response (UPR) is activated. If the damage is too broad, the mammalian UPR launches the apoptotic program. As a consequence, mobilization of ER calcium stores sensitizes mitochondria to direct proapoptotic stimuli. We make use of a mouse Apaf1-deficient cell system of proneural origin to understand the roles played in this context by the apoptosome, the most studied apoptotic machinery along the mitochondrial pathway of death. We show here that in the absence of the apoptosome ER stress induces cytochrome c release from the mitochondria but that apoptosis cannot occur. Under these circumstances, Grp78/BiP and GADD153/CHOP, both hallmarks of UPR, are canonically up-regulated, and calcium is properly released from ER stores. We also demonstrate that caspase 12, a protease until now believed to play a central role in the initiation of ER stress-induced cell death in the mouse system, is dispensable for the mitochondrial pathway of death to take place.

Role of the autophagic-lysosomal system on low potassium-induced apoptosis in cultured cerebellar granule cells.

Apoptotic and autophagic cell death have been implicated, on the basis of morphological and biochemical criteria, in neuronal loss occurring in neurodegenerative diseases and it has been shown that they may overlap. We have studied the relationship between apoptosis and autophagic cell death in cerebellar granule cells (CGCs) undergoing apoptosis following serum and potassium deprivation. We found that apoptosis is accompanied by an early and marked proliferation of autophagosomal-lysosomal compartments as detected by electron microscopy and immunofluorescence analysis. Autophagy is blocked by hrIGF-1 and forskolin, two well-known inhibitors of CGC apoptosis, as well as by adenovirus-mediated overexpression of Bcl-2. 3-Methyladenine (3-MA) an inhibitor of autophagy, not only arrests this event but it also blocks apoptosis. The neuroprotective effect of 3-MA is accompanied by block of cytochrome c (cyt c) release in the cytosol and by inhibition of caspase-3 activation which, in turn, appears to be mediated by cathepsin B, as CA074-Me, a selective inhibitor of this enzyme, fully blocks the processing of pro-caspase-3. Immunofluorescence analysis demonstrated that cathepsin B, normally confined inside the lysosomal-endosomal compartment, is released during apoptosis into the cytosol where this enzyme may act as an execution protease. Collectively, these observations indicate that autophagy precedes and is causally connected with the subsequent onset of programmed death.