Biology subjects

Wai, T.

Publications and source records attributed to Wai, T..

11 recordsLinked to original sources

bioRxiv2026

Loss of CHCHD2 and CHCHD10 reveals differential vulnerability to bioenergetic failure in cardiac and skeletal muscle

Mutations in the mitochondrial proteins CHCHD2 and CHCHD10 cause severe neurodegenerative and neuromuscular disorders, yet their physiological functions remain poorly defined. CHCHD2 and CHCHD10 localize to the mitochondrial intermembrane space, where they assemble into a high-molecular-weight complex. Here, we generated Chchd2/Chchd10 double-knockout (DKO) mice to define the in vivo role of this complex. DKO mice developed reduced lean mass, progressive muscle weakness, and oxidative phosphorylation defects in both cardiac and skeletal muscle. Despite comparable bioenergetic impairment, CHCHD2-CHCHD10 deficiency elicited tissue-specific responses distinct from those induced by the disease-associated CHCHD10 S59L mutation. The heart accumulated enlarged mitochondria with disrupted ultrastructure and underwent adaptive proteomic remodeling that preserved myocardial contractility into adulthood. By contrast, skeletal muscle exhibited limited proteomic alterations, accompanied by profound changes in lipid composition, reduced expression of the myogenic regulators, and altered myofiber size. Mechanistically, depletion of CHCHD2 and CHCHD10 in primary satellite cells impaired proliferation and myogenic differentiation, suggesting that defective postnatal myogenesis contributes to the muscle growth defect in DKO mice. Together, these findings identify the CHCHD2-CHCHD10 complex as a critical regulator of mitochondrial integrity and demonstrate that its loss drives tissue-specific defects culminating in diverse pathological outcomes.

cell biology↗

bioRxiv2026

SARS-CoV-2 ORF9b exploits mitochondrial recruitment to TOMM70 for proteasomal protection and tunes inflammatory remodeling during lung infection

Mitochondrial outer membrane proteins are exploited by diverse intracellular pathogens, to modulate cell metabolism and innate sensing pathways. Here, we demonstrate that TOMM70-dependent mitochondrial recruitment is required to protect SARS-CoV-2 ORF9b from proteasomal degradation, a dependency conserved across ORF9b homologs from related coronaviruses. ORF9b mitochondrial recruitment requires the E477 residue of TOMM70, a surface distinct from that used by the parasite Toxoplasma gondii to engage host mitochondria. We further show that TOMM70 is not a passive scaffold: its depletion activates interferon-stimulated gene expression independently of infection and remodels host immunity distinctly from ORF9b, establishing the receptor and viral protein as mechanistically separable. Using ORF9b-deficient SARS-CoV-2, we demonstrate that ORF9b is dispensable for viral replication and pathological responses in human respiratory epithelial cells and lungs of infected golden Syrian hamsters. Omics profiling of SARS-CoV-2 infected lungs revealed an induction of pathways related to COVID-19 in the absence of ORF9b. Notably, ORF9b-deficient virus-infected lungs show elevated expression of C15ORF48, a nuclear-encoded mitochondrial protein that substitutes for Complex IV subunit NDUFA4 to attenuate inflammation. Collectively, we propose that ORF9b is a receptor-gated viral protein whose principal measurable consequence during authentic infection is a restraint on inflammatory respiratory-chain remodeling.

cell biology↗

bioRxiv2026

MTFP1 preserves β-cell cristae structure and bioenergetics to ensure insulin release and glucose homeostasis

Pancreatic {beta}-cells are uniquely dependent on mitochondrial metabolism to couple glucose sensing to insulin secretion, a process impaired in diabetes. Mitochondrial fission process 1 (MTFP1) is an inner mitochondrial membrane protein that plays pleiotropic, tissue-specific roles in mitochondrial function and dynamics. Our previous work has identified Mtfp1 mRNA as a target for miR-125b, a microRNA that negatively regulates insulin secretion from {beta}-cells. Nevertheless, the function of MTFP1 in these cells remained unexplored. Here, we show that MTFP1 is essential for normal glucose-stimulated insulin secretion (GSIS) in mouse and human cell lines and islets, and that mice with {beta}-cell-specific elimination of MTFP1 develop glucose intolerance. Whereas {beta}-cell survival and mitochondrial content were unaffected, oxidative phosphorylation and ATP production were sharply lowered. These changes were accompanied by disruption of mitochondrial cristae structure and a reduced contact surface with the endoplasmic reticulum, providing a mechanistic basis for defective stimulus-secretion coupling. Conversely, MTFP1 overexpression in mouse and human islets sufficed to improve mitochondrial respiration and GSIS. Finally, MTFP1 downregulation blocked the positive effects of miR-125b elimination in GSIS and mitochondrial respiration, unveiling MTFP1 as a downstream effector of miR-125b. Together, our findings identify MTFP1 as a critical regulator of {beta}-cell mitochondrial architecture and function, necessary for efficient insulin secretion and glucose homeostasis, and a potential therapeutic target to enhance {beta}-cell bioenergetic resilience in diabetes.

cell biology↗

bioRxiv2025

Mutant CHCHD10 disrupts cytochrome c oxidation and activates retrograde signaling in a model of cardiomyopathy.

Mutations in CHCHD10, a mitochondrial intermembrane space (IMS) protein implicated in proteostasis and cristae maintenance, cause multi-systemic mitochondrial disease. Heterozygous Chchd10 knock-in mice modeling the human CHCHD10S59L variant associated with Amyotrophic Lateral Sclerosis and Frontotemporal Dementia (ALS-FTD) develop a mitochondrial cardiomyopathy driven by CHCHD10 insolubility and aggregation, which is associated with chronic activation of the mitochondrial integrated stress response (mtISR). Here, we demonstrate that cardiac dysfunction in Chchd10S55L/+ mice carrying the orthologous pathogenic variant is associated with dual defects originating at the onset of disease: (1) early bioenergetic dysfunction linked to defects in the mitochondrial copper homeostasis and the oxidation of cytochrome c and (2) maladaptive mtISR signaling via the OMA1-DELE1-HRI axis. Using Oma1E324Q/E324Qknock-in mice, we show that the catalytic inactivation of the mitochondrial protease OMA1 in Chchd10S55L/+ mice delays cardiomyopathy onset without rescuing CHCHD10 insolubility, proteomic remodeling, cristae defects or OXPHOS impairment, demonstrating that mtISR can be uncoupled from the bioenergetic collapse triggered by mutant CHCHD10. Proteomic profiling of soluble and insoluble mitochondrial proteins in Chchd10S55L/+ mice reveals wide-spread disruptions of mitochondrial proteostasis, including IMS proteins involved in cytochrome c biogenesis. Defective respiration in mutant mitochondria could be rescued by the exogenous addition of cytochrome c, pinpointing IMS proteostasis disruption as a key pathogenic mechanism. Our work reveals that mutant CHCHD10 insolubility compromises metabolic resilience by impairing both mitochondrial bioenergetics and stress adaptation, offering new perspectives for the development of therapeutic targets.

cell biology↗

bioRxiv2024

Myocardial disarray drives metabolic inefficiency in human cardiomyocytes

Adult cardiomyocytes are embedded within a highly organized myocardial microenvironment that imposes critical geometric cues essential for the alignment and distribution of organelles and the shaping of their unique, rectangular cellular morphology. Despite the association of cardiomyocyte disarray with human heart disease, the functional consequences of this cellular disorganization remain poorly understood. Here, we leveraged micropatterned substrates to promote structural alignment in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), contrasting the effects of mechanical alignment on mitochondrial form and function with hiPSC-CMs cultured under standard unconstrained conditions. Cardiomyocytes cultured under unconstrained conditions exhibited misaligned sarcomeres and a perinuclear mitochondrial distribution while micropatterned hiPSC-CMs developed linear myofibrils and reconfigured sarcomere and mitochondrial organization, which increased mitochondrial respiration without augmenting mitochondrial mass. Notably, micropatterned hiPSC-CMs exhibited an increased number of mitochondrial-associated membranes, as determined by proximity ligation assays and transmission electron microscopy, suggesting enhanced interactions between the sarcoplasmic reticulum and mitochondria. Together, these findings demonstrate that mitochondrial-sarcoplasmic architecture and geometry are critical spatial features that ensure bioenergetic efficiency of cardiomyocytes. This work underscores the importance of cellular organization in cardiomyocyte metabolism and function, providing insights into the pathophysiology of cardiac diseases marked by cellular disarray.

cell biology↗

bioRxiv2023

Dynamic label-free analysis of SARS-CoV-2 infection reveals virus-induced subcellular remodeling.

Assessing the impact of SARS-CoV-2 on organelle dynamics allows a better understanding of the mechanisms of viral replication. We combine label-free holo-tomographic microscopy (HTM) with Artificial Intelligence (AI) to visualize and quantify the subcellular changes triggered by SARS-CoV-2 infection. We study the dynamics of shape, position and dry mass of nucleoli, nuclei, lipid droplets (LD) and mitochondria within hundreds of single cells from early infection to syncytia formation and death. SARS-CoV-2 infection enlarges nucleoli, perturbs LD, changes mitochondrial shape and dry mass, and separates LD from mitochondria. We then used Bayesian statistics on organelle dry mass states to define organelle cross-regulation (OCR) networks and report modifications of OCR that are triggered by infection and syncytia formation. Our work highlights the subcellular remodeling induced by SARS-CoV-2 infection and provides a new AI-enhanced, label-free methodology to study in real-time the dynamics of cell populations and their content.

cell biology↗

bioRxiv2023

Mtfp1 ablation enhances mitochondrial respiration and protects against hepatic steatosis

Hepatic steatosis is the result of an imbalance between nutrient delivery and metabolism in the liver. It is the first hallmark of Non-alcoholic fatty liver disease (NAFLD) and is characterized by the accumulation of excess lipids in the liver that can drive liver failure, inflammation, and cancer. Mitochondria control the fate and function of cells and compelling evidence implicates these multifunctional organelles in the appearance and progression of liver dysfunction, although it remains to be elucidated which specific mitochondrial functions are actually causally linked to NAFLD. Here, we identified Mitochondrial Fission Process 1 protein (MTFP1) as a key regulator of mitochondrial and metabolic activity in the liver. Deletion of Mtfp1 in hepatocytes is physiologically benign in mice yet leads to the upregulation of oxidative phosphorylation (OXPHOS) activity and mitochondrial respiration, independently of mitochondrial biogenesis. Consequently, hepatocyte-specific knockout mice are protected against high fat diet-induced hepatic steatosis and metabolic dysregulation. Additionally, we find that deletion of Mtfp1 in liver mitochondria inhibits mitochondrial permeability transition pore opening in hepatocytes, conferring protection against apoptotic liver damage in vivo and ex vivo. Our work uncovers novel functions of MTFP1 in the liver, positioning this gene as an unexpected regulator of OXPHOS and a therapeutic candidate for NAFLD.

physiology↗

bioRxiv2021

Mitochondrial fission process 1 (MTFP1) controls bioenergetic efficiency and prevents inflammatory cardiomyopathy and heart failure in mice

Mitochondria are paramount to the metabolism and survival of cardiomyocytes. Here we show that Mitochondrial Fission Process 1 (MTFP1) is essential for cardiac structure and function. Constitutive knockout of cardiomyocyte MTFP1 in mice resulted in adult-onset dilated cardiomyopathy (DCM) characterized by sterile inflammation and cardiac fibrosis that progressed to heart failure and middle-aged death. Failing hearts from cardiomyocyte-restricted knockout mice displayed a general decline in mitochondrial gene expression and oxidative phosphorylation (OXPHOS) activity. Pre-DCM, we observed no defects in mitochondrial morphology, content, gene expression, OXPHOS assembly nor phosphorylation dependent respiration. However, knockout cardiac mitochondria displayed reduced membrane potential and increased non-phosphorylation dependent respiration, which could be rescued by pharmacological inhibition of the adenine nucleotide translocase ANT. Primary cardiomyocytes from pre-symptomatic knockout mice exhibited normal excitation-contraction coupling but increased sensitivity to programmed cell death (PCD), which was accompanied by an opening of the mitochondrial permeability transition pore (mPTP). Intriguingly, mouse embryonic fibroblasts deleted for Mtfp1 recapitulated PCD sensitivity and mPTP opening, both of which could be rescued by pharmacological or genetic inhibition of the mPTP regulator Cyclophilin D. Collectively, our data demonstrate that contrary to previous in vitro studies, the loss of the MTFP1 promotes mitochondrial uncoupling and increases cell death sensitivity, causally mediating pathogenic cardiac remodeling.

physiology↗

bioRxiv2021

Enhanced cGAS-STING-dependent interferon signaling associated with mutations in ATAD3A

Mitochondrial DNA (mtDNA) has been suggested to drive immune system activation, but the induction of interferon signaling by mtDNA has not been demonstrated in a Mendelian mitochondrial disease. We initially ascertained two patients, one with a purely neurological phenotype, and one with features suggestive of systemic sclerosis in a syndromic context, and found them both to demonstrate enhanced interferon-stimulated gene (ISG) expression in blood. We determined each to harbor a previously described de novo dominant-negative heterozygous mutation in ATAD3A, encoding ATPase family AAA domain-containing protein 3A (ATAD3A). We identified five further patients with mutations in ATAD3A, and recorded up-regulated ISG expression and interferon alpha protein in four of them. Knockdown of ATAD3A in THP-1 cells resulted in increased interferon signaling, mediated by cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). Enhanced interferon signaling was abrogated in THP-1 cells and patient fibroblasts depleted of mtDNA. Thus, mutations in the mitochondrial membrane protein ATAD3A define a novel type I interferonopathy. SummaryDominant-negative mutations in ATAD3A, a ubiquitously expressed mitochondrial protein, cause mitochondrial DNA-dependent up-regulation of type I interferon signaling in the context of neurological disease and autoimmunity, thereby defining a novel type I interferonopathy.

immunology↗

bioRxiv2021

High-throughput phenotypic screen for genetic modifiers in patient-derived OPA1 mutant fibroblasts identifies PGS1 as a functional suppressor of mitochondrial fragmentation

Mutations affecting the mitochondrial fusion protein Optic Atrophy 1 (OPA1) cause autosomal dominant optic atrophy (DOA) - one of the most common form of mitochondrial disease. The majority of patients develop isolated optic atrophy, but about 20% of OPA1 mutation carriers manifest more severe neurological deficits as part of a "DOA+" phenotype. OPA1 deficiency causes mitochondrial fragmentation and also disrupts cristae organization, oxidative phosphorylation, mitochondrial DNA (mtDNA) maintenance, and cell viability. It has not yet been established whether phenotypic severity can be modulated by genetic modifiers of OPA1. To better understand the genetic regulation of mitochondrial dynamics, we established a high-throughput imaging pipeline using supervised machine learning (ML) to perform unbiased, quantitative mitochondrial morphology analysis that was coupled with a bespoke siRNA library targeting the entire known mitochondrial proteome (1531 genes), providing a detailed phenotypic screening of human fibroblasts. In control fibroblasts, we identified known and novel genes whose depletion promoted elongation or fragmentation of the mitochondrial network. In DOA+ patient fibroblasts, we identified 91 candidate genes whose depletion prevents mitochondrial fragmentation, including the mitochondrial fission genes DNM1L, MIEF1, and SLC25A46, but also genes not previously linked to mitochondrial dynamics such as Phosphatidyl Glycerophosphate Synthase (PGS1), which belongs to the cardiolipin (CL) synthesis pathway. PGS1 depletion reduces CL content in mitochondria and rebalances mitochondrial dynamics in OPA1-deficient fibroblasts by inhibiting mitochondrial fission, which improves defective respiration, but does not rescue mtDNA depletion, cristae dysmorphology or apoptotic sensitivity. Our data reveal that the multifaceted roles of OPA1 in mitochondria can be functionally uncoupled by modulating mitochondrial lipid metabolism, providing novel insights into the cellular relevance of mitochondrial fragmentation. This study illustrates the power of a first-in-kind objective automated imaging approach to uncover genetic modifiers of mitochondrial disease through high-throughput phenotypic screening of patient fibroblasts.

cell biology↗

bioRxiv2020

Distinct molecular signatures of fission predict mitochondrial degradation or proliferation

Mitochondrial fission is a highly regulated process which, when disrupted, can alter metabolism, proliferation and apoptosis1-3. The downstream effects have implications for many diseases, from neurodegeneration4-6 to cardiovascular disease7,8 and cancer9,10. Key components of the fission machinery have been identified: constriction sites are initiated by the endoplasmic reticulum (ER)11 and actin12 before dynamin-related protein 1 (Drp1)13 is recruited to the outer mitochondrial membrane via adaptor proteins14-17, where it drives constriction and scission of the membrane18. In the life cycle of mitochondria, fission is important for the biogenesis of new mitochondria as well as the clearance of dysfunctional mitochondria via mitophagy3,19. Global regulation of fission on the cellular level is insufficient to explain how fate decisions are made at the single organelle level, so it is unknown how those dual functions arise, blocking progress in developing therapies that target mitochondrial activity. However, systematically studying mitochondrial division to uncover fate determinants is challenging, since fission is unpredictable, and mitochondrial morphology is extremely heterogeneous. Furthermore, because their ultrastructure lies below the diffraction limit, the dynamic organization of mitochondria and their interaction partners are hard to study at the single organelle level. We used live-cell structured illumination microscopy (SIM) and instant SIM20 for fast multi-colour acquisition of mitochondrial dynamics in Cos-7 cells and mouse cardiomyocytes. We analysed hundreds of fission events, and discovered two functionally and mechanistically distinct types of fission. Mitochondria divide peripherally to shed damaged material into smaller daughter mitochondria that subsequently undergo mitophagy, whereas healthy mitochondria proliferate via midzone division. Both types are Drp1-mediated, but they rely on different membrane adaptors to recruit Drp1, and ER and actin mediated pre-constriction is only involved in midzone fission.

cell biology↗