AAV-mediated upregulation of VDAC1 rescues the mitochondrial respiration and sirtuins expression in a SOD1 mouse model of inherited ALS.

Mitochondrial dysfunction represents one of the most common molecular hallmarks of both sporadic and familial forms of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder caused by the selective degeneration and death of motor neurons. The accumulation of misfolded proteins on and within mitochondria, as observed for SOD1 G93A mutant, correlates with a drastic reduction of mitochondrial respiration and the inhibition of metabolites exchanges, including ADP/ATP and NAD+/NADH, across the Voltage-Dependent Anion-selective Channel 1 (VDAC1), the most abundant channel protein of the outer mitochondrial membrane. Here, we show that the AAV-mediated upregulation of VDAC1 in the spinal cord of transgenic mice expressing SOD1 G93A completely rescues the mitochondrial respiratory profile. This correlates with the increased activity and levels of key regulators of mitochondrial functions and maintenance, namely the respiratory chain Complex I and the sirtuins (Sirt), especially Sirt3. Furthermore, the selective increase of these mitochondrial proteins is associated with an increase in Tom20 levels, the receptor subunit of the TOM complex. Overall, our results indicate that the overexpression of VDAC1 has beneficial effects on ALS-affected tissue by stabilizing the Complex I-Sirt3 axis.

Special Issue "Mitochondrial Respiration in Physiology and Pathology".

Mitochondria are key organelles that regulate several functions essential for maintaining cellular homeostasis [...].

Oxygen-dependent histone lysine demethylase 4 restricts hepatitis B virus replication.

Mammalian cells have evolved strategies to regulate gene expression when oxygen is limited. Hypoxia-inducible factors (HIF) are the major transcriptional regulators of host gene expression. We previously reported that HIFs bind and activate hepatitis B virus (HBV) DNA transcription under low oxygen conditions; however, the global cellular response to low oxygen is mediated by a family of oxygenases that work in concert with HIFs. Recent studies have identified a role for chromatin modifiers in sensing cellular oxygen and orchestrating transcriptional responses, but their role in the HBV life cycle is as yet undefined. We demonstrated that histone lysine demethylase 4 (KDM4) can restrict HBV, and pharmacological or oxygen-mediated inhibition of the demethylase increases viral RNAs derived from both episomal and integrated copies of the viral genome. Sequencing studies demonstrated that KDM4 is a major regulator of the hepatic transcriptome, which defines hepatocellular permissivity to HBV infection. We propose a model where HBV exploits cellular oxygen sensors to replicate and persist in the liver. Understanding oxygen-dependent pathways that regulate HBV infection will facilitate the development of physiologically relevant cell-based models that support efficient HBV replication.

Hypoxia inducible factors inhibit respiratory syncytial virus infection by modulation of nucleolin expression.

Respiratory syncytial virus (RSV) is a global healthcare problem, causing respiratory illness in young children and elderly individuals. Our knowledge of the host pathways that define susceptibility to infection and disease severity are limited. Hypoxia inducible factors (HIFs) define metabolic responses to low oxygen and regulate inflammatory responses in the lower respiratory tract. We demonstrate a role for HIFs to suppress RSV entry and RNA replication. We show that hypoxia and HIF prolyl-hydroxylase inhibitors reduce the expression of the RSV entry receptor nucleolin and inhibit viral cell-cell fusion. We identify a HIF regulated microRNA, miR-494, that regulates nucleolin expression. In RSV-infected mice, treatment with the clinically approved HIF prolyl-hydroxylase inhibitor, Daprodustat, reduced the level of infectious virus and infiltrating monocytes and neutrophils in the lung. This study highlights a role for HIF-signalling to limit multiple aspects of RSV infection and associated inflammation and informs future therapeutic approaches for this respiratory pathogen.

The N6-methyladenosine demethylase ALKBH5 regulates the hypoxic HBV transcriptome.

Chronic hepatitis B is a global health problem and current treatments only suppress hepatitis B virus (HBV) infection, highlighting the need for new curative treatments. Oxygen levels influence HBV replication and we previously reported that hypoxia inducible factors (HIFs) activate the basal core promoter (BCP). Here we show that the hypoxic-dependent increase in BCP-derived transcripts is dependent on N6-methyladenosine (m6A) modifications in the 5' stem loop that regulate RNA half-life. Application of a probe-enriched long-read sequencing method to accurately map the HBV transcriptome showed an increased abundance of pre-genomic RNA under hypoxic conditions. Mapping the transcription start sites of BCP-RNAs identified a role for hypoxia to regulate pre-genomic RNA splicing that is dependent on m6A modification. Bioinformatic analysis of published single cell RNA-seq of murine liver showed an increased expression of the RNA demethylase ALKBH5 in the peri-central low oxygen region. In vitro studies with a human hepatocyte derived HepG2-NTCP cell line showed increased ALKBH5 gene expression under hypoxic conditions and a concomitant reduction in m6A-modified HBV BCP-RNA and host RNAs. Silencing the demethylase reduced the level of BCP-RNAs and host gene (CA9, NDRG1, VEGFA, BNIP3, FUT11, GAP and P4HA1) transcripts and this was mediated via reduced HIFα expression. In summary, our study highlights a previously unrecognized role for ALKBH5 in orchestrating viral and cellular transcriptional responses to low oxygen.

CTCF regulates hepatitis B virus cccDNA chromatin topology.

Hepatitis B Virus (HBV) is a small DNA virus that replicates via an episomal covalently closed circular DNA (cccDNA) that serves as the transcriptional template for viral mRNAs. The host protein, CCCTC-binding factor (CTCF), is a key regulator of cellular transcription by maintaining epigenetic boundaries, nucleosome phasing, stabilisation of long-range chromatin loops and directing alternative exon splicing. We previously reported that CTCF binds two conserved motifs within Enhancer I of the HBV genome and represses viral transcription, however, the underlying mechanisms were not identified. We show that CTCF depletion in cells harbouring cccDNA-like HBV molecules and in de novo infected cells resulted in an increase in spliced transcripts, which was most notable in the abundant SP1 spliced transcript. In contrast, depletion of CTCF in cell lines with integrated HBV DNA had no effect on the abundance of viral transcripts and in line with this observation there was limited evidence for CTCF binding to viral integrants, suggesting that CTCF-regulation of HBV transcription is specific to episomal cccDNA. Analysis of HBV chromatin topology by Assay for Transposase Accessible Chromatin Sequencing (ATAC-Seq) revealed an accessible region spanning Enhancers I and II and the basal core promoter (BCP). Mutating the CTCF binding sites within Enhancer I resulted in a dramatic rearrangement of chromatin accessibility where the open chromatin region was no longer detected, indicating loss of the phased nucleosome up- and down-stream of the HBV enhancer/BCP. These data demonstrate that CTCF functions to regulate HBV chromatin conformation and nucleosomal positioning in episomal maintained cccDNA, which has important consequences for HBV transcription regulation.

The N6-methyladenosine demethylase ALKBH5 regulates the hypoxic HBV transcriptome.

Chronic hepatitis B is a global health problem and current treatments only suppress hepatitis B virus (HBV) infection, highlighting the need for new curative treatments. Oxygen levels influence HBV replication and we previously reported that hypoxia inducible factors (HIFs) activate the basal core promoter to transcribe pre-genomic RNA. Application of a probe-enriched long-read sequencing method to map the HBV transcriptome showed an increased abundance of all viral RNAs under low oxygen or hypoxic conditions. Importantly, the hypoxic-associated increase in HBV transcripts was dependent on N6-methyladenosine (m6A) modifications and an m6A DRACH motif in the 5' stem loop of pre-genomic RNA defined transcript half-life under hypoxic conditions. Given the essential role of m6A modifications in the viral transcriptome we assessed the oxygen-dependent expression of RNA demethylases and bioinformatic analysis of published single cell RNA-seq of murine liver showed an increased expression of the RNA demethylase ALKBH5 in the peri-central low oxygen region. In vitro studies with a human hepatocyte derived HepG2 cell line showed increased ALKBH5 gene expression under hypoxic conditions. Silencing the demethylase reduced the levels of HBV pre-genomic RNA and host gene (CA9, NDRG1, VEGFA, BNIP3, FUT11, GAP and P4HA1) transcripts and this was mediated via reduced HIFα expression. In summary, our study highlights a previously unrecognized role for ALKBH5 in orchestrating viral and cellular transcriptional responses to low oxygen.

ASPP2 binds to hepatitis C virus NS5A protein via an SH3 domain/PxxP motif-mediated interaction and potentiates infection.

Hepatitis C virus (HCV) infects millions of people worldwide and is a leading cause of liver disease. Despite recent advances in antiviral therapies, viral resistance can limit drug efficacy and understanding the mechanisms that confer viral escape is important. We employ an unbiased interactome analysis to discover host binding partners of the HCV non-structural protein 5A (NS5A), a key player in viral replication and assembly. We identify ASPP2, apoptosis-stimulating protein of p53, as a new host co-factor that binds NS5A via its SH3 domain. Importantly, silencing ASPP2 reduces viral replication and spread. Our study uncovers a previously unknown role for ASPP2 to potentiate HCV RNA replication.

Molecular components of the circadian clock regulate HIV-1 replication.

Human immunodeficiency virus 1 (HIV-1) causes major health burdens worldwide and still lacks curative therapies and vaccines. Circadian rhythms are endogenous daily oscillations that coordinate an organism's response to its environment and invading pathogens. Peripheral viral loads of HIV-1 infected patients show diurnal variation; however, the underlying mechanisms remain unknown. Here, we demonstrate a role for the cell-intrinsic clock to regulate rhythmic HIV-1 replication in circadian-synchronized systems. Silencing the circadian activator Bmal1 abolishes this phenotype, and we observe BMAL1 binding to the HIV-1 promoter. Importantly, we show differential binding of the nuclear receptors REV-ERB and ROR to the HIV-long terminal repeat at different circadian times, demonstrating a dynamic interplay in time-of-day regulation of HIV-1 transcription. Bioinformatic analysis shows circadian regulation of host factors that control HIV-1 replication, providing an additional mechanism for rhythmic viral replication. This study increases our understanding of the circadian regulation of HIV-1, which can ultimately inform new therapies.

ERK1/2-dependent TSPO overactivation associates with the loss of mitophagy and mitochondrial respiration in ALS.

Mitochondrial dysfunction and the loss of mitophagy, aimed at recycling irreversibly damaged organelles, contribute to the onset of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease affecting spinal cord motor neurons. In this work, we showed that the reduction of mitochondrial respiration, exactly oxygen flows linked to ATP production and maximal capacity, correlates with the appearance of the most common ALS motor symptoms in a transgenic mouse model expressing SOD1 G93A mutant. This is the result of the equal inhibition in the respiration linked to complex I and II of the electron transport chain, but not their protein levels. Since the overall mitochondrial mass was unvaried, we investigated the expression of the Translocator Protein (TSPO), a small mitochondrial protein whose overexpression was recently linked to the loss of mitophagy in a model of Parkinson's disease. Here we clearly showed that levels of TSPO are significantly increased in ALS mice. Mechanistically, this increase is linked to the overactivation of ERK1/2 pathway and correlates with a decrease in the expression of the mitophagy-related marker Atg12, indicating the occurrence of impairments in the activation of mitophagy. Overall, our work sets out TSPO as a key regulator of mitochondrial homeostasis in ALS.

VDAC1 Knockout Affects Mitochondrial Oxygen Consumption Triggering a Rearrangement of ETC by Impacting on Complex I Activity.

Voltage-Dependent Anion-selective Channel isoform 1 (VDAC1) is the most abundant isoform of the outer mitochondrial membrane (OMM) porins and the principal gate for ions and metabolites to and from the organelle. VDAC1 is also involved in a number of additional functions, such as the regulation of apoptosis. Although the protein is not directly involved in mitochondrial respiration, its deletion in yeast triggers a complete rewiring of the whole cell metabolism, with the inactivation of the main mitochondrial functions. In this work, we analyzed in detail the impact of VDAC1 knockout on mitochondrial respiration in the near-haploid human cell line HAP1. Results indicate that, despite the presence of other VDAC isoforms in the cell, the inactivation of VDAC1 correlates with a dramatic impairment in oxygen consumption and a re-organization of the relative contributions of the electron transport chain (ETC) enzymes. Precisely, in VDAC1 knockout HAP1 cells, the complex I-linked respiration (N-pathway) is increased by drawing resources from respiratory reserves. Overall, the data reported here strengthen the key role of VDAC1 as a general regulator of mitochondrial metabolism.

17ß-Oestradiol Protects from Hepatitis C Virus Infection through Induction of Type I Interferon.

BACKGROUND AND AIMS: Sex hormones are widely recognised to act as protective factors against several viral infections. Specifically, females infected by the hepatitis C virus display higher clearance rates and reduced disease progression than those found in males. Through modulation of particle release and spread, 17ß-oestradiol controls HCV's life cycle. We investigated the mechanism(s) behind oestrogen's antiviral effect. METHODS: We used cell culture-derived hepatitis C virus in in vitro assays to evaluate the effect of 17ß-oestradiol on the innate immune response. Host immune responses were evaluated by enumerating gene transcripts via RT-qPCR in cells exposed to oestrogen in the presence or absence of viral infection. Antiviral effects were determined by focus-forming unit assay or HCV RNA quantification. RESULTS: Stimulation of 17ß-oestradiol triggers a pre-activated antiviral state in hepatocytes, which can be maintained for several hours after the hormone is removed. This induction results in the elevation of several innate immune genes, such as interferon alpha and beta, tumour necrosis factor, toll-like receptor 3 and interferon regulatory factor 5. We demonstrated that this pre-activation of immune response signalling is not affected by a viral presence, and the antiviral state can be ablated using an interferon-alpha/beta receptor alpha inhibitor. Finally, we proved that the oestrogen-induced stimulation is essential to generate an antiviral microenvironment mediated by activation of type I interferons. CONCLUSION: Resulting in viral control and suppression, 17ß-oestradiol induces an interferon-mediated antiviral state in hepatocytes. Oestrogen-stimulated cells modulate the immune response through secretion of type I interferon, which can be countered by blocking interferon-alpha/beta receptor alpha signalling.

Small Extracellular Vesicles Secreted by Nigrostriatal Astrocytes Rescue Cell Death and Preserve Mitochondrial Function in Parkinson's Disease.

Extracellular vesicles (EVs) are emerging as powerful players in cell-to-cell communication both in healthy and diseased brain. In Parkinson's disease (PD)-characterized by selective dopaminergic neuron death in ventral midbrain (VMB) and degeneration of their terminals in striatum (STR)-astrocytes exert dual harmful/protective functions, with mechanisms not fully elucidated. Here, this study shows that astrocytes from the VMB-, STR-, and VMB/STR-depleted brains release a population of small EVs  in a region-specific manner. Interestingly, VMB-astrocytes secreted the highest rate of EVs, which is further exclusively increased in response to CCL3, a chemokine that promotes robust dopaminergic neuroprotection in different PD models. The neuroprotective potential of nigrostriatal astrocyte-EVs is investigated in differentiated versus undifferentiated SH-SY5Y cells exposed to oxidative stress and mitochondrial toxicity. EVs from both VMB- and STR-astrocytes counteract H2 O2 -induced caspase-3 activation specifically in differentiated cells, with EVs from CCL3-treated astrocytes showing a higher protective effect. High resolution respirometry further reveals that nigrostriatal astrocyte-EVs rescue neuronal mitochondrial complex I function impaired by the neurotoxin MPP+ . Notably, only EVs from VMB-astrocyte fully restore ATP production, again specifically in differentiated SH-SY5Y. These results highlight a regional diversity in the nigrostriatal system for the secretion and activities of astrocyte-EVs, with neuroprotective implications for PD.

α-Synuclein A53T Promotes Mitochondrial Proton Gradient Dissipation and Depletion of the Organelle Respiratory Reserve in a Neuroblastoma Cell Line.

α-synuclein (αSyn) is a small neuronal protein whose accumulation correlates with Parkinson's disease. αSyn A53T mutant impairs mitochondrial functions by affecting substrate import within the organelle, activity of complex I and the maximal respiratory capacity. However, the precise mechanism initiating the bioenergetic dysfunction is not clearly understood yet. By overexpressing αSyn A53T in SH-SY5Y cells, we investigated the specific changes in the mitochondrial respiratory profile using High-Resolution Respirometry. We found that αSyn A53T increases dissipative fluxes across the intermembrane mitochondrial space: this does not compromise the oxygen flows devoted to ATP production while it reduces the bioenergetic excess capacity of mitochondria, providing a possible explanation of the increased cell susceptibility observed in the presence of further stress stimuli.

Inflammatory Gene Expression Associates with Hepatitis B Virus cccDNA- but Not Integrant-Derived Transcripts in HBeAg Negative Disease.

Chronic hepatitis B virus (HBV) infection is a global health problem that presents as a spectrum of liver disease, reflecting an interplay between the virus and the host immune system. HBV genomes exist as episomal covalently closed circular DNA (cccDNA) or chromosomal integrants. The relative contribution of these genomes to the viral transcriptome in chronic hepatitis B (CHB) is not well-understood. We developed a qPCR method to estimate the abundance of HBV cccDNA- and integrant-derived viral transcripts and applied this to a cohort of patients diagnosed with CHB in the HBe antigen negative phase of disease. We noted a variable pattern of HBV transcripts from both DNA templates, with preS1/S2 mRNAs predominating and a significant association between increasing age and the expression of integrant-derived mRNAs, but not with inflammatory status. In contrast, cccDNA-derived transcripts were associated with markers of liver inflammation. Analysis of the inflammatory hepatic transcriptome identified 24 genes significantly associated with cccDNA transcriptional activity. Our study uncovers an immune gene signature that associates with HBV cccDNA transcription and increases our understanding of viral persistence.

Voltage Dependent Anion Channel 3 (VDAC3) protects mitochondria from oxidative stress.

Unraveling the role of VDAC3 within living cells is challenging and still requires a definitive answer. Unlike VDAC1 and VDAC2, the outer mitochondrial membrane porin 3 exhibits unique biophysical features that suggest unknown cellular functions. Electrophysiological studies on VDAC3 carrying selective cysteine mutations and mass spectrometry data about the redox state of such sulfur containing amino acids are consistent with a putative involvement of isoform 3 in mitochondrial ROS homeostasis. Here, we thoroughly examined this issue and provided for the first time direct evidence of the role of VDAC3 in cellular response to oxidative stress. Depletion of isoform 3 but not isoform 1 significantly exacerbated the cytotoxicity of redox cyclers such as menadione and paraquat, and respiratory complex I inhibitors like rotenone, promoting uncontrolled accumulation of mitochondrial free radicals. High-resolution respirometry of transiently transfected HAP1-ΔVDAC3 cells expressing the wild type or the cysteine-null mutant VDAC3 protein, unequivocally confirmed that VDAC3 cysteines are indispensable for protein ability to counteract ROS-induced oxidative stress.

Viral genome wide association study identifies novel hepatitis C virus polymorphisms associated with sofosbuvir treatment failure.

Persistent hepatitis C virus (HCV) infection is a major cause of chronic liver disease, worldwide. With the development of direct-acting antivirals, treatment of chronically infected patients has become highly effective, although a subset of patients responds less well to therapy. Sofosbuvir is a common component of current de novo or salvage combination therapies, that targets the HCV NS5B polymerase. We use pre-treatment whole-genome sequences of HCV from 507 patients infected with HCV subtype 3a and treated with sofosbuvir containing regimens to detect viral polymorphisms associated with response to treatment. We find three common polymorphisms in non-targeted HCV NS2 and NS3 proteins are associated with reduced treatment response. These polymorphisms are enriched in post-treatment HCV sequences of patients unresponsive to treatment. They are also associated with lower reductions in viral load in the first week of therapy. Using in vitro short-term dose-response assays, these polymorphisms do not cause any reduction in sofosbuvir potency, suggesting an indirect mechanism of action in decreasing sofosbuvir efficacy. The identification of polymorphisms in NS2 and NS3 proteins associated with poor treatment outcomes emphasises the value of systematic genome-wide analyses of viruses in uncovering clinically relevant polymorphisms that impact treatment.

Small Hexokinase 1 Peptide against Toxic SOD1 G93A Mitochondrial Accumulation in ALS Rescues the ATP-Related Respiration.

Mutations in Cu/Zn Superoxide Dismutase (SOD1) gene represent one of the most common causes of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder that specifically affects motor neurons (MNs). The dismutase-active SOD1 G93A mutant is responsible for the formation of toxic aggregates onto the mitochondrial surface, using the Voltage-Dependent Anion Channel 1 (VDAC1) as an anchor point to the organelle. VDAC1 is the master regulator of cellular bioenergetics and by binding to hexokinases (HKs) it controls apoptosis. In ALS, however, SOD1 G93A impairs VDAC1 activity and displaces HK1 from mitochondria, promoting organelle dysfunction, and cell death. Using an ALS cell model, we demonstrate that a small synthetic peptide derived from the HK1 sequence (NHK1) recovers the cell viability in a dose-response manner and the defective mitochondrial respiration profile relative to the ADP phosphorylation. This correlates with an unexpected increase of VDAC1 expression and a reduction of SOD1 mutant accumulation at the mitochondrial level. Overall, our findings provide important new insights into the development of therapeutic molecules to fight ALS and help to better define the link between altered mitochondrial metabolism and MNs death in the disease.

Voltage-Dependent Anion Selective Channel Isoforms in Yeast: Expression, Structure, and Functions.

Mitochondrial porins, also known as voltage-dependent anion selective channels (VDACs), are pore-forming molecules of the outer mitochondrial membranes, involved in the regulation of metabolic flux between cytosol and mitochondria. Playing such an essential role, VDAC proteins are evolutionary conserved and isoforms are present in numerous species. The quest for specific function(s) related to the raise of multiple isoforms is an intriguing theme. The yeast Saccharomyces cerevisiae genome is endowed with two different VDAC genes encoding for two distinct porin isoforms, definitely less characterized in comparison to mammalian counterpart. While yVDAC1 has been extensively studied, the second isoform, yVDAC2, is much less expressed, and has a still misunderstood function. This review will recapitulate the known and poorly known information in the literature, in the light of the growing interest about the features of VDAC isoforms in the cell.

Alpha-Synuclein and Mitochondrial Dysfunction in Parkinson's Disease: The Emerging Role of VDAC.

Alpha-Synuclein (αSyn) is a protein whose function is still debated, as well as its role in modulation of mitochondrial function in both physiological and pathological conditions. Mitochondrial porins or Voltage-Dependent Anion Channel (VDAC) proteins are the main gates for ADP/ATP and various substrates towards the organelle. Furthermore, they act as a mitochondrial hub for many cytosolic proteins, including αSyn. This review analyzes the main aspects of αSyn-mitochondria interaction, focusing on the role of VDAC and its emerging involvement in the pathological processes.