Long-Term GABA Administration Induces Alpha Cell-Mediated Beta-like Cell Neogenesis.

The recent discovery that genetically modified α cells can regenerate and convert into ß-like cells in vivo holds great promise for diabetes research. However, to eventually translate these findings to human, it is crucial to discover compounds with similar activities. Herein, we report the identification of GABA as an inducer of α-to-ß-like cell conversion in vivo. This conversion induces α cell replacement mechanisms through the mobilization of duct-lining precursor cells that adopt an α cell identity prior to being converted into ß-like cells, solely upon sustained GABA exposure. Importantly, these neo-generated ß-like cells are functional and can repeatedly reverse chemically induced diabetes in vivo. Similarly, the treatment of transplanted human islets with GABA results in a loss of α cells and a concomitant increase in ß-like cell counts, suggestive of α-to-ß-like cell conversion processes also in humans. This newly discovered GABA-induced α cell-mediated ß-like cell neogenesis could therefore represent an unprecedented hope toward improved therapies for diabetes.

A four-step cycle driven by PI(4)P hydrolysis directs sterol/PI(4)P exchange by the ER-Golgi tether OSBP.

Several proteins at endoplasmic reticulum (ER)-Golgi membrane contact sites contain a PH domain that interacts with the Golgi phosphoinositide PI(4)P, a FFAT motif that interacts with the ER protein VAP-A, and a lipid transfer domain. This architecture suggests the ability to both tether organelles and transport lipids between them. We show that in oxysterol binding protein (OSBP) these two activities are coupled by a four-step cycle. Membrane tethering by the PH domain and the FFAT motif enables sterol transfer by the lipid transfer domain (ORD), followed by back transfer of PI(4)P by the ORD. Finally, PI(4)P is hydrolyzed in cis by the ER protein Sac1. The energy provided by PI(4)P hydrolysis drives sterol transfer and allows negative feedback when PI(4)P becomes limiting. Other lipid transfer proteins are tethered by the same mechanism. Thus, OSBP-mediated back transfer of PI(4)P might coordinate the transfer of other lipid species at the ER-Golgi interface.

Defects in AMPAR trafficking and microglia activation underlie socio-cognitive deficits associated to decreased expression of phosphodiesterase 2 a.

Phosphodiesterase 2 A (PDE2A) is an enzyme involved in the homeostasis of cAMP and cGMP and is the most highly expressed PDE in human brain regions critical for socio-cognitive behavior. In cerebral cortex and hippocampus, PDE2A expression level is upregulated in Fmr1-KO mice, a model of the Fragile X Syndrome (FXS), the most common form of inherited intellectual disability (ID) and autism spectrum disorder (ASD). Indeed, PDE2A translation is negatively modulated by FMRP, whose functional absence causes FXS. While the pharmacological inhibition of PDE2A has been associated to its pro-cognitive role in normal animals and in models of ID and ASD, homozygous PDE2A mutations have been identified in patients affected by ID, ASD and epilepsy. To clarify this apparent paradox about the role of PDE2A in brain development, we characterized here Pde2a+/- mice (homozygote animals being not viable) at the behavioral, cellular, molecular and electrophysiological levels. Pde2a+/- females display a milder form of the disorder with reduced cognitive performance in adulthood, conversely males show severe socio-cognitive deficits throughout their life. In males, these phenotypes are associated with microglia activation, elevated glutathione levels and increased externalization of Glutamate receptor (GluR1) in CA1, producing reduced mGluR-dependent Long-term Depression. Overall, our results reveal molecular targets of the PDE2A-dependent pathway underlying socio-cognitive performance. These results clarify the mechanism of action of pro-cognitive drugs based on PDE2A inactivation, which have been shown to be promising therapeutic approaches for Alzheimer's disease, schizophrenia, FXS as well as other forms of ASD.

Golgi-associated retrograde protein (GARP) complex-dependent endosomes to trans Golgi network retrograde trafficking is controlled by Rab4b.

BACKGROUND: The trafficking of cargoes from endosomes to the trans-Golgi network requires numerous sequential and coordinated steps. Cargoes are sorted into endosomal-derived carriers that are transported, tethered, and fused to the trans-Golgi network. The tethering step requires several complexes, including the Golgi-associated retrograde protein complex, whose localization at the trans-Golgi network is determined by the activity of small GTPases of the Arl and Rab family. However, how the Golgi-associated retrograde protein complex recognizes the endosome-derived carriers that will fuse with the trans-Golgi network is still unknown. METHODS: We studied the retrograde trafficking to the trans-Golgi network by using fluorescent cargoes in cells overexpressing Rab4b or after Rab4b knocked-down by small interfering RNA in combination with the downregulation of subunits of the Golgi-associated retrograde protein complex. We used immunofluorescence and image processing (Super Resolution Radial Fluctuation and 3D reconstruction) as well as biochemical approaches to characterize the consequences of these interventions on cargo carriers trafficking. RESULTS: We reported that the VPS52 subunit of the Golgi-associated retrograde protein complex is an effector of Rab4b. We found that overexpression of wild type or active Rab4b increased early endosomal to trans-Golgi network retrograde trafficking of the cation-independent mannose-6-phosphate receptor in a Golgi-associated retrograde protein complex-dependent manner. Conversely, overexpression of an inactive Rab4b or Rab4b knockdown attenuated this trafficking. In the absence of Rab4b, the internalized cation-independent mannose 6 phosphate receptor did not have access to VPS52-labeled structures that look like endosomal subdomains and/or endosome-derived carriers, and whose subcellular distribution is Rab4b-independent. Consequently, the cation-independent mannose-6-phosphate receptor was blocked in early endosomes and no longer had access to the trans-Golgi network. CONCLUSION: Our results support that Rab4b, by controlling the sorting of the cation-independent mannose-6-phosphate receptor towards VPS52 microdomains, confers a directional specificity for cargo carriers en route to the trans-Golgi network. Given the importance of the endocytic recycling in cell homeostasis, disruption of the Rab4b/Golgi-associated retrograde protein complex-dependent step could have serious consequences in pathologies.

EFA6A, an exchange factor for Arf6, regulates early steps in ciliogenesis.

Ciliogenesis is a coordinated process initiated by the recruitment and fusion of pre-ciliary vesicles at the distal appendages of the mother centriole through mechanisms that remain unclear. Here, we report that EFA6A (also known as PSD), an exchange factor for the small G protein Arf6, is involved in early stage of ciliogenesis by promoting the fusion of distal appendage vesicles forming the ciliary vesicle. EFA6A is present in the vicinity of the mother centriole before primary cilium assembly and prior to the arrival of Arl13B-containing vesicles. During ciliogenesis, EFA6A initially accumulates at the mother centriole and later colocalizes with Arl13B along the ciliary membrane. EFA6A depletion leads to the inhibition of ciliogenesis, the absence of centrosomal Rab8-positive structures and the accumulation of Arl13B-positive vesicles around the distal appendages. Our results uncover a novel fusion machinery, comprising EFA6A, Arf6 and Arl13B, that controls the coordinated fusion of ciliary vesicles docked at the distal appendages of the mother centriole.

CHCHD10 and SLP2 control the stability of the PHB complex: a key factor for motor neuron viability.

CHCHD10 is an amyotrophic lateral sclerosis/frontotemporal dementia gene that encodes a mitochondrial protein whose precise function is unclear. Here we show that Coiled-Coil-Helix-Coiled-Coil-Helix Domain Containing protein 10 interacts with the Stomatin-Like Protein 2 and participates in the stability of the prohibitin complex in the inner mitochondrial membrane. By using patient fibroblasts and mouse models expressing the same CHCHD10 variant (p.Ser59Leu), we show that Stomatin-Like Protein 2 forms aggregates with prohibitins, found in vivo in the hippocampus and as aggresome-like inclusions in spinal motor neurons of Chchd10S59L/+ mice. Affected cells and tissues display instability of the prohibitin complex, which participates at least in part in the activation of the OMA1 cascade with OPA1 processing leading to mitochondrial fragmentation, abnormal mitochondrial cristae morphogenesis and neuronal death found in spinal cord and the hippocampus of Chchd10S59L/+ animals. Destabilization of the prohibitin complex leads to the instability of the mitochondrial contact site and cristae organizing the system complex, probably by the disruption of OPA1-mitofilin interaction. Thus, Stomatin-Like Protein 2/prohibitin aggregates and destabilization of the prohibitin complex are critical in the sequence of events leading to motor neuron death in CHCHD10S59L-related disease.

Sterol transfer, PI4P consumption, and control of membrane lipid order by endogenous OSBP.

The network of proteins that orchestrate the distribution of cholesterol among cellular organelles is not fully characterized. We previously proposed that oxysterol-binding protein (OSBP) drives cholesterol/PI4P exchange at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi network (TGN). Using the inhibitor OSW-1, we report here that the sole activity of endogenous OSBP makes a major contribution to cholesterol distribution, lipid order, and PI4P turnover in living cells. Blocking OSBP causes accumulation of sterols at ER/lipid droplets at the expense of TGN, thereby reducing the gradient of lipid order along the secretory pathway. OSBP consumes about half of the total cellular pool of PI4P, a consumption that depends on the amount of cholesterol to be transported. Inhibiting the spatially restricted PI4-kinase PI4KIIIß triggers large periodic traveling waves of PI4P across the TGN These waves are cadenced by long-range PI4P production by PI4KIIα and PI4P consumption by OSBP Collectively, these data indicate a massive spatiotemporal coupling between cholesterol transport and PI4P turnover via OSBP and PI4-kinases to control the lipid composition of subcellular membranes.

Aminopeptidase A contributes to biochemical, anatomical and cognitive defects in Alzheimer's disease (AD) mouse model and is increased at early stage in sporadic AD brain.

One of the main components of senile plaques in Alzheimer's disease (AD)-affected brain is the Aß peptide species harboring a pyroglutamate at position three pE3-Aß. Several studies indicated that pE3-Aß is toxic, prone to aggregation and serves as a seed of Aß aggregation. The cyclisation of the glutamate residue is produced by glutaminyl cyclase, the pharmacological and genetic reductions of which significantly alleviate AD-related anatomical lesions and cognitive defects in mice models. The cyclisation of the glutamate in position 3 requires prior removal of the Aß N-terminal aspartyl residue to allow subsequent biotransformation. The enzyme responsible for this rate-limiting catalytic step and its relevance as a putative trigger of AD pathology remained yet to be established. Here, we identify aminopeptidase A as the main exopeptidase involved in the N-terminal truncation of Aß and document its key contribution to AD-related anatomical and behavioral defects. First, we show by mass spectrometry that human recombinant aminopeptidase A (APA) truncates synthetic Aß1-40 to yield Aß2-40. We demonstrate that the pharmacological blockade of APA with its selective inhibitor RB150 restores the density of mature spines and significantly reduced filopodia-like processes in hippocampal organotypic slices cultures virally transduced with the Swedish mutated Aß-precursor protein (ßAPP). Pharmacological reduction of APA activity and lowering of its expression by shRNA affect pE3-42Aß- and Aß1-42-positive plaques and expressions in 3xTg-AD mice brains. Further, we show that both APA inhibitors and shRNA partly alleviate learning and memory deficits observed in 3xTg-AD mice. Importantly, we demonstrate that, concomitantly to the occurrence of pE3-42Aß-positive plaques, APA activity is augmented at early Braak stages in sporadic AD brains. Overall, our data indicate that APA is a key enzyme involved in Aß N-terminal truncation and suggest the potential benefit of targeting this proteolytic activity to interfere with AD pathology.

Accumulation of amyloid precursor protein C-terminal fragments triggers mitochondrial structure, function, and mitophagy defects in Alzheimer's disease models and human brains.

Several lines of recent evidence indicate that the amyloid precursor protein-derived C-terminal fragments (APP-CTFs) could correspond to an etiological trigger of Alzheimer's disease (AD) pathology. Altered mitochondrial homeostasis is considered an early event in AD development. However, the specific contribution of APP-CTFs to mitochondrial structure, function, and mitophagy defects remains to be established. Here, we demonstrate in neuroblastoma SH-SY5Y cells expressing either APP Swedish mutations, or the ß-secretase-derived APP-CTF fragment (C99) combined with ß- and γ-secretase inhibition, that APP-CTFs accumulation independently of Aß triggers excessive mitochondrial morphology alteration (i.e., size alteration and cristae disorganization) associated with enhanced mitochondrial reactive oxygen species production. APP-CTFs accumulation also elicit basal mitophagy failure illustrated by enhanced conversion of LC3, accumulation of LC3-I and/or LC3-II, non-degradation of SQSTM1/p62, inconsistent Parkin and PINK1 recruitment to mitochondria, enhanced levels of membrane and matrix mitochondrial proteins, and deficient fusion of mitochondria with lysosomes. We confirm the contribution of APP-CTFs accumulation to morphological mitochondria alteration and impaired basal mitophagy in vivo in young 3xTgAD transgenic mice treated with γ-secretase inhibitor as well as in adeno-associated-virus-C99 injected mice. Comparison of aged 2xTgAD and 3xTgAD mice indicates that, besides APP-CTFs, an additional contribution of Aß to late-stage mitophagy activation occurs. Importantly, we report on mitochondrial accumulation of APP-CTFs in human post-mortem sporadic AD brains correlating with mitophagy failure molecular signature. Since defective mitochondria homeostasis plays a pivotal role in AD pathogenesis, targeting mitochondrial dysfunctions and/or mitophagy by counteracting early APP-CTFs accumulation may represent relevant therapeutic interventions in AD.

REDD1 deficiency protects against nonalcoholic hepatic steatosis induced by high-fat diet.

Nonalcoholic fatty liver disease is a chronic liver disease which is associated with obesity and insulin resistance. We investigated the implication of REDD1 (Regulated in development and DNA damage response-1), a stress-induced protein in the development of hepatic steatosis. REDD1 expression was increased in the liver of obese mice and morbidly obese patients, and its expression correlated with hepatic steatosis and insulin resistance in obese patients. REDD1 deficiency protected mice from the development of hepatic steatosis induced by high-fat diet (HFD) without affecting body weight gain and glucose intolerance. This protection was associated with a decrease in the expression of lipogenic genes, SREBP1c, FASN, and SCD-1 in liver of HFD-fed REDD1-KO mice. Healthy mitochondria are crucial for the adequate control of lipid metabolism and failure to remove damaged mitochondria is correlated with liver steatosis. Expression of markers of autophagy and mitophagy, Beclin, LC3-II, Parkin, BNIP3L, was enhanced in liver of HFD-fed REDD1-KO mice. The number of mitochondria showing colocalization between LAMP2 and AIF was increased in liver of HFD-fed REDD1-KO mice. Moreover, mitochondria in liver of REDD1-KO mice were smaller than in WT. These results are correlated with an increase in PGC-1α and CPT-1 expression, involved in fatty acid oxidation. In conclusion, loss of REDD1 protects mice from the development of hepatic steatosis.

On-site secretory vesicle delivery drives filamentous growth in the fungal pathogen Candida albicans.

Candida albicans is an opportunistic fungal pathogen that colonises the skin as well as genital and intestinal mucosa of most healthy individuals. The ability of C. albicans to switch between different morphological states, for example, from an ellipsoid yeast form to a highly polarised, hyphal form, contributes to its success as a pathogen. In highly polarised tip-growing cells such as neurons, pollen tubes, and filamentous fungi, delivery of membrane and cargo to the filament apex is achieved by long-range delivery of secretory vesicles tethered to motors moving along cytoskeletal cables that extend towards the growing tip. To investigate whether such a mechanism is also critical for C. albicans filamentous growth, we studied the dynamics and organisation of the C. albicans secretory pathway using live cell imaging and three-dimensional electron microscopy. We demonstrate that the secretory pathway is organised in distinct domains, including endoplasmic reticulum membrane sheets that extend along the length of the hyphal filament, a sub-apical zone exhibiting distinct membrane structures and dynamics and a Spitzenkörper comprised of uniformly sized secretory vesicles. Our results indicate that the organisation of the secretory pathway in C. albicans likely facilitates short-range "on-site" secretory vesicle delivery, in contrast to filamentous fungi and many highly polarised cells.

A novel CISD2 mutation associated with a classical Wolfram syndrome phenotype alters Ca2+ homeostasis and ER-mitochondria interactions.

Wolfram syndrome (WS) is a progressive neurodegenerative disease characterized by early-onset optic atrophy and diabetes mellitus, which can be associated with more extensive central nervous system and endocrine complications. The majority of patients harbour pathogenic WFS1 mutations, but recessive mutations in a second gene, CISD2, have been described in a small number of families with Wolfram syndrome type 2 (WFS2). The defining diagnostic criteria for WFS2 also consist of optic atrophy and diabetes mellitus, but unlike WFS1, this phenotypic subgroup has been associated with peptic ulcer disease and an increased bleeding tendency. Here, we report on a novel homozygous CISD2 mutation (c.215A > G; p.Asn72Ser) in a Moroccan patient with an overlapping phenotype suggesting that Wolfram syndrome type 1 and type 2 form a continuous clinical spectrum with genetic heterogeneity. The present study provides strong evidence that this particular CISD2 mutation disturbs cellular Ca2+ homeostasis with enhanced Ca2+ flux from the ER to mitochondria and cytosolic Ca2+ abnormalities in patient-derived fibroblasts. This Ca2+ dysregulation was associated with increased ER-mitochondria contact, a swollen ER lumen and a hyperfused mitochondrial network in the absence of overt ER stress. Although there was no marked alteration in mitochondrial bioenergetics under basal conditions, culture of patient-derived fibroblasts in glucose-free galactose medium revealed a respiratory chain defect in complexes I and II, and a trend towards decreased ATP levels. Our results provide important novel insight into the potential disease mechanisms underlying the neurodegenerative consequences of CISD2 mutations and the subsequent development of multisystemic disease.

Essential and selective role of SNX12 in transport of endocytic and retrograde cargo.

The endosomal protein-sorting machineries play vital roles in diverse physiologically important cellular processes. Much of the core membrane-sorting apparatus is conserved in evolution, such as retromer, which is involved in the recycling of a diverse set of cargoes via the retrograde trafficking route. Here, in an RNAi-based loss-of-function study, we identified that suppression of SNX12 leads to a severe blockage in CIM6PR (also known as IGF2R) transport and alters the morphology of the endocytic compartments. We demonstrate that SNX12 is involved in the early phase of CIM6PR transport, and mediates receptor recycling upstream of the other well-established SNX components of retromer. Ultra-structural analysis revealed that SNX12 resides on tubulo-vesicular structures, despite it lacking a BAR domain. Furthermore, we illustrate that SNX12 plays a key role in intraluminal vesicle formation and in the maturation of a subpopulation of early endosomes into late endosomes, thereby regulating selective endocytic transport of cargo for degradation. This study therefore provides evidence for the existence of early endosomal subpopulations that have differential roles in the sorting of the cargoes along endocytic degradative pathways.

Bax inhibitor-1 protects from nonalcoholic steatohepatitis by limiting inositol-requiring enzyme 1 alpha signaling in mice.

Endoplasmic reticulum (ER) stress is activated in nonalcoholic fatty liver disease (NAFLD), raising the possibility that ER stress-dependent metabolic dysfunction, inflammation, and cell death underlie the transition from steatosis to steatohepatitis (nonalcoholic steatohepatitis; NASH). B-cell lymphoma 2 (BCL2)-associated X protein (Bax) inhibitor-1 (BI-1), a negative regulator of the ER stress sensor, inositol-requiring enzyme 1 alpha (IRE1α), has yet to be explored in NAFLD as a hepatoprotective agent. We hypothesized that the genetic ablation of BI-1 would render the liver vulnerable to NASH because of unrestrained IRE1α signaling. ER stress was induced in wild-type and BI-1-/- mice acutely by tunicamycin (TM) injection (1 mg/kg) or chronically by high-fat diet (HFD) feeding to determine NAFLD phenotype. Livers of TM-treated BI-1-/- mice showed IRE1α-dependent NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation, hepatocyte death, fibrosis, and dysregulated lipid homeostasis that led to liver failure within a week. The analysis of human NAFLD liver biopsies revealed BI-1 down-regulation parallel to the up-regulation of IRE1α endoribonuclease (RNase) signaling. In HFD-fed BI-1-/- mice that presented NASH and type 2 diabetes, exaggerated hepatic IRE1α, X-box binding protein 1 (XBP1), and C/EBP homologous protein (CHOP) expression was linked to activated NLRP3 inflammasome and caspase-1/-11. Rises in interleukin (IL)-1ß, IL-6, monocyte chemoattractant protein 1 (MCP1), chemokine (C-X-C motif) ligand 1 (CXCL1), and alanine transaminase (ALT)/aspartate transaminase (AST) levels revealed significant inflammation and injury, respectively. Pharmacological inhibition of IRE1α RNase activity with the small molecules, STF-083010 or 4µ8c, was evaluated in HFD-induced NAFLD. In BI-1-/- mice, either treatment effectively counteracted IRE1α RNase activity, improving glucose tolerance and rescuing from NASH. The hepatocyte-specific role of IRE1α RNase activity in mediating NLRP3 inflammasome activation and cell death was confirmed in primary mouse hepatocytes by IRE1α axis knockdown or its inhibition with STF-083010 or 4µ8c. CONCLUSION: Targeting IRE1α-dependent NLRP3 inflammasome signaling with pharmacological agents or by BI-1 may represent a tangible therapeutic strategy for NASH. (Hepatology 2018).

Mitochondrial defect in muscle precedes neuromuscular junction degeneration and motor neuron death in CHCHD10S59L/+ mouse.

Recently, we provided genetic basis showing that mitochondrial dysfunction can trigger motor neuron degeneration, through identification of CHCHD10 encoding a mitochondrial protein. We reported patients, carrying the p.Ser59Leu heterozygous mutation in CHCHD10, from a large family with a mitochondrial myopathy associated with motor neuron disease (MND). Rapidly, our group and others reported CHCHD10 mutations in amyotrophic lateral sclerosis (ALS), frontotemporal dementia-ALS and other neurodegenerative diseases. Here, we generated knock-in (KI) mice, carrying the p.Ser59Leu mutation, that mimic the mitochondrial myopathy with mtDNA instability displayed by the patients from our original family. Before 14 months of age, all KI mice developed a fatal mitochondrial cardiomyopathy associated with enhanced mitophagy. CHCHD10S59L/+ mice also displayed neuromuscular junction (NMJ) and motor neuron degeneration with hyper-fragmentation of the motor end plate and moderate but significant motor neuron loss in lumbar spinal cord at the end stage of the disease. At this stage, we observed TDP-43 cytoplasmic aggregates in spinal neurons. We also showed that motor neurons differentiated from human iPSC carrying the p.Ser59Leu mutation were much more sensitive to Staurosporine or glutamate-induced caspase activation than control cells. These data confirm that mitochondrial deficiency associated with CHCHD10 mutations can be at the origin of MND. CHCHD10 is highly expressed in the NMJ post-synaptic part. Importantly, the fragmentation of the motor end plate was associated with abnormal CHCHD10 expression that was also observed closed to NMJs which were morphologically normal. Furthermore, we found OXPHOS deficiency in muscle of CHCHD10S59L/+ mice at 3 months of age in the absence of neuron loss in spinal cord. Our data show that the pathological effects of the p.Ser59Leu mutation target muscle prior to NMJ and motor neurons. They likely lead to OXPHOS deficiency, loss of cristae junctions and destabilization of internal membrane structure within mitochondria at motor end plate of NMJ, impairing neurotransmission. These data are in favor with a key role for muscle in MND associated with CHCHD10 mutations.

Loss of MICOS complex integrity and mitochondrial damage, but not TDP-43 mitochondrial localisation, are likely associated with severity of CHCHD10-related diseases.

Following the involvement of CHCHD10 in FrontoTemporal-Dementia-Amyotrophic Lateral Sclerosis (FTD-ALS) clinical spectrum, a founder mutation (p.Gly66Val) in the same gene was identified in Finnish families with late-onset spinal motor neuronopathy (SMAJ). SMAJ is a slowly progressive form of spinal muscular atrophy with a life expectancy within normal range. In order to understand why the p.Ser59Leu mutation, responsible for severe FTD-ALS, and the p.Gly66Val mutation could lead to different levels of severity, we compared their effects in patient cells. Unlike affected individuals bearing the p.Ser59Leu mutation, patients presenting with SMAJ phenotype have neither mitochondrial myopathy nor mtDNA instability. The expression of CHCHD10S59L mutant allele leads to disassembly of mitochondrial contact site and cristae organizing system (MICOS) with mitochondrial dysfunction and loss of cristae in patient fibroblasts. We also show that G66V fibroblasts do not display the loss of MICOS complex integrity and mitochondrial damage found in S59L cells. However, S59L and G66V fibroblasts show comparable accumulation of phosphorylated mitochondrial TDP-43 suggesting that the severity of phenotype and mitochondrial damage do not depend on mitochondrial TDP-43 localization. The expression of the CHCHD10G66V allele is responsible for mitochondrial network fragmentation and decreased sensitivity towards apoptotic stimuli, but with a less severe effect than that found in cells expressing the CHCHD10S59L allele. Taken together, our data show that cellular phenotypes associated with p.Ser59Leu and p.Gly66Val mutations in CHCHD10 are different; loss of MICOS complex integrity and mitochondrial dysfunction, but not TDP-43 mitochondrial localization, being likely essential to develop a severe motor neuron disease.

Expression patterns of sterol transporters NPC1 and NPC2 in the cnidarian-dinoflagellate symbiosis.

The symbiotic interaction between cnidarians (e.g., corals and sea anemones) and photosynthetic dinoflagellates of the genus Symbiodinium is triggered by both host-symbiont recognition processes and metabolic exchange between the 2 partners. The molecular communication is crucial for homeostatic regulation of the symbiosis, both under normal conditions and during stresses that further lead to symbiosis collapse. It is therefore important to identify and fully characterise the key players of this intimate interaction at the symbiotic interface. In this study, we determined the cellular and subcellular localization and expression of the sterol-trafficking Niemann-Pick type C proteins (NPC1 and NPC2) in the symbiotic sea anemones Anemonia viridis and Aiptasia sp. We first established that NPC1 is localised within vesicles in host tissues and to the symbiosome membranes in several anthozoan species. We demonstrated that the canonical NPC2-a protein is mainly expressed in the epidermis, whereas the NPC2-d protein is closely associated with symbiosome membranes. Furthermore, we showed that the expression of the NPC2-d protein is correlated with symbiont presence in healthy symbiotic specimens. As npc2-d is a cnidarian-specific duplicated gene, we hypothesised that it probably arose from a subfunctionalisation process that might result in a gain of function and symbiosis adaptation in anthozoans. Niemann-Pick type C proteins may be key players in a functional symbiosis and be useful tools to study host-symbiont interactions in the anthozoan-dinoflagellate association.

FATE1 antagonizes calcium- and drug-induced apoptosis by uncoupling ER and mitochondria.

Several stimuli induce programmed cell death by increasing Ca(2+) transfer from the endoplasmic reticulum (ER) to mitochondria. Perturbation of this process has a special relevance in pathologies as cancer and neurodegenerative disorders. Mitochondrial Ca(2+) uptake mainly takes place in correspondence of mitochondria-associated ER membranes (MAM), specialized contact sites between the two organelles. Here, we show the important role of FATE1, a cancer-testis antigen, in the regulation of ER-mitochondria distance and Ca(2+) uptake by mitochondria. FATE1 is localized at the interface between ER and mitochondria, fractionating into MAM FATE1 expression in adrenocortical carcinoma (ACC) cells under the control of the transcription factor SF-1 decreases ER-mitochondria contact and mitochondrial Ca(2+) uptake, while its knockdown has an opposite effect. FATE1 also decreases sensitivity to mitochondrial Ca(2+)-dependent pro-apoptotic stimuli and to the chemotherapeutic drug mitotane. In patients with ACC, FATE1 expression in their tumor is inversely correlated with their overall survival. These results show that the ER-mitochondria uncoupling activity of FATE1 is harnessed by cancer cells to escape apoptotic death and resist the action of chemotherapeutic drugs.

Targeting eIF5A Hypusination Prevents Anoxic Cell Death through Mitochondrial Silencing and Improves Kidney Transplant Outcome.

The eukaryotic initiation factor 5A (eIF5A), which is highly conserved throughout evolution, has the unique characteristic of post-translational activation through hypusination. This modification is catalyzed by two enzymatic steps involving deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). Notably, eIF5A may be involved in regulating the lifespan of Drosophila during long-term hypoxia. Therefore, we investigated the possibility of a link between eIF5A hypusination and cellular resistance to hypoxia/anoxia. Pharmacologic targeting of DHPS by N1-guanyl-1,7-diaminoheptane (GC7) or RNA interference-mediated inhibition of DHPS or DOHH induced tolerance to anoxia in immortalized mouse renal proximal cells. Furthermore, GC7 treatment of cells reversibly induced a metabolic shift toward glycolysis as well as mitochondrial remodeling and led to downregulated expression and activity of respiratory chain complexes, features characteristic of mitochondrial silencing. GC7 treatment also attenuated anoxia-induced generation of reactive oxygen species in these cells and in normoxic conditions, decreased the mitochondrial oxygen consumption rate of cultured cells and mice. In rats, intraperitoneal injection of GC7 substantially reduced renal levels of hypusinated eIF5A and protected against ischemia-reperfusion-induced renal injury. Finally, in the preclinical pig kidney transplant model, intravenous injection of GC7 before kidney removal significantly improved graft function recovery and late graft function and reduced interstitial fibrosis after transplant. This unconventional signaling pathway offers an innovative therapeutic target for treating hypoxic-ischemic human diseases and organ transplantation.

Arf6 exchange factor EFA6 and endophilin directly interact at the plasma membrane to control clathrin-mediated endocytosis.

Members of the Arf family of small G proteins are involved in membrane traffic and organelle structure. They control the recruitment of coat proteins, and modulate the structure of actin filaments and the lipid composition of membranes. The ADP-ribosylation factor 6 (Arf6) isoform and the exchange factor for Arf6 (EFA6) are known to regulate the endocytic pathway of many different receptors. To determine the molecular mechanism of the EFA6/Arf6 function in vesicular transport, we searched for new EFA6 partners. In a two-hybrid screening using the catalytic Sec7 domain as a bait, we identified endophilin as a new partner of EFA6. Endophilin contains a Bin/Amphiphysin/Rvs (BAR) domain responsible for membrane bending, and an SH3 domain responsible for the recruitment of dynamin and synaptojanin, two proteins involved, respectively, in the fission and uncoating of clathrin-coated vesicles. By using purified proteins, we confirmed the direct interaction, and identified the N-BAR domain as the binding motif to EFA6A. We showed that endophilin stimulates the catalytic activity of EFA6A on Arf6. In addition, we observed that the Sec7 domain competes with flat but not with highly curved lipid membranes to bind the N-BAR. In cells, expression of EFA6A recruits endophilin to EFA6A-positive plasma membrane ruffles, whereas expression of endophilin rescues the EFA6A-mediated inhibition of transferrin internalization. Overall, our results support a model whereby EFA6 recruits endophilin on flat areas of the plasma membrane to control Arf6 activation and clathrin-mediated endocytosis.