Significant Mitochondrial Dysfunction Research Articles

Mitochondrial Dysfunction in Down Syndrome associated Alzheimer’s Disease

AbstractBackgroundMitochondrial dysfunction and Aβ accumulation are hallmarks of Alzheimer’s disease (AD). However, the role of these pathologies in Down Syndrome associated Alzheimer’s Disease (DSAD) is unknown. Decades of research describe a relationship between mitochondrial function and Aβ production. Amyloid precursor protein (APP), from which Aβ is generated, is found in mitochondria. APP and Aβ alter mitochondrial function, while mitochondrial function alters Aβ production from APP. How these interactions contribute to DSAD pathology and progression are unknown. Here we interrogated the association of full‐length APP with mitochondria, mitochondrial function, and AD pathological hallmarks.MethodND (n = 10, without DS) and DS associated Alzheimer’s Disease (DSAD, n = 10) postmortem brain tissue was obtained from the University of California Irvine. A human iPSC line was purchased from WiCell with Trisomy 21 and a isogenic control line which underwent Crispr/Cas9 genome editing to remove the extra chromosome 21 copy. iPSC models were differentiated into neurons, astrocytes, and cerebral organoids using StemCell Technologies reagents and protocols. We examined mitochondrial function using a Seahorse XF analyzer. We measured full‐length APP protein levels in whole cell extracts and mitochondrial fractions via Western Blotting. We measured Aβ levels with ELISA kits from ThermoFisher.ResultDSAD postmortem brain tissue had reduced mitochondrial function regardless of sex. Full‐length APP levels were significantly higher in mitochondrial fractions in DSAD brain tissue. Full‐length APP levels in mitochondrial fractions correlated with mitochondrial function. Higher mitochondrial APP (full‐length) levels associated with lower mitochondrial function. iPSC derived models showed similar phenotypes to postmortem brain tissues, including increased mitochondrial APP levels, and decreased mitochondrial function.ConclusionWe describe a relationship between mitochondrial APP accumulation, and mitochondrial function. These data support a centralized role for mitochondrial function in APP physiology and APP may play a role in modulating mitochondrial function. Further, DSAD postmortem tissue and iPSC models show significant mitochondrial dysfunction.

An Orai1 gain-of-function tubular aggregate myopathy mouse model phenocopies key features of the human disease

Tubular aggregate myopathy (TAM) is a heritable myopathy primarily characterized by progressive muscle weakness, elevated levels of creatine kinase (CK), hypocalcemia, exercise intolerance, and the presence of tubular aggregates (TAs). Here, we generated a knock-in mouse model based on a human gain-of-function mutation which results in a severe, early-onset form of TAM, by inducing a glycine-to-serine point mutation in the ORAI1 pore (Orai1G100S/+ or GS mice). By 8 months of age, GS mice exhibited significant muscle weakness, exercise intolerance, elevated CK levels, hypocalcemia, and robust TA presence. Unexpectedly, constitutive Ca2+ entry in mutant mice was observed in muscle only during early development and was abolished in adult skeletal muscle, partly due to reduced ORAI1 expression. Consistent with proteomic results, significant mitochondrial damage and dysfunction was observed in skeletal muscle of GS mice. Thus, GS mice represent a powerful model for investigation of the pathophysiological mechanisms that underlie key TAM symptoms, as well as those compensatory responses that limit the damaging effects of uncontrolled ORAI1-mediated Ca2+ influx.

Bisphenol A and bisphenol AF co-exposure induced apoptosis of human ovarian granulosa cells via mitochondrial dysfunction

Bisphenol A (BPA) is a synthetic chemical primarily utilized in the manufacturing of polycarbonate plastics and epoxy resins that are present in various consumer products. While the BPA impacts on female reproductive toxicity have been widely investigated, very little is currently identified about the mixed toxicity of BPA and bisphenol AF (BPAF), another common BPA derivative that is used in many industrial applications. In this study, we assessed the effect of co-exposure of BPA (30 and 50 μM) and BPAF (3 and 5 μM) on mitochondrial dysfunction in human granulosa cells (KGN cells) for 24 h. Our results exhibited that high-concentration bisphenol individual or their mixture exposure of KGN cells induced significant mitochondrial dysfunction by reducing mitochondrial mass, reducing ATP production, and damaging the mitochondrial respiratory chain. In addition, we found that the combination of BPA and BPAF significantly induced mitochondrial stress by increasing calcium levels and the production of ROS in mitochondria. Mitochondrial stress induced by BPA and BPAF was determined to be a mechanism that promoted cell apoptosis after pretreating the cells with the mitochondrial-targeted antioxidant and the calcium chelator. Our results provide novel evidence of the cytotoxicity of mixtures of different bisphenol compounds.

Tumor microenvironment-responsive intelligent nanoplatform with oxygen self-supply for synergistic chemotherapy/photodynamic therapy/photothermal therapy against hypoxic tumors

Nanomaterial-based multi-modal synergistic therapies are emerging as highly promising strategies for the treatment of malignant tumors. Herein, a novel nanoplatform modified with the peptide of cyclo(Arg-Gly-Asp-D-Phe-Lys) (cRGD) was designed for synergistic treatment of hypoxic tumors by the combination of chemotherapy (CT)/photodynamic therapy (PDT)/photothermal therapy (PTT). Firstly, Mn@APDA nanoparticles were synthesized for PTT and oxygen (O2) supply, consisting of an L-arginine-doped polydopamine (APDA) core and a manganese dioxide (MnO2) shell. Subsequently, the liposome nanoplatform (cRGD-Lipo-MART), loaded with Mn@APDA (MA), chemotherapy drug of deoxybouvardin dodecyl ester (RA, “R” for short), and mitochondrial targeted photosensitizer of triphenylphosphine-grafted protoporphyrin IX (TPPP, “T” for short), was developed using a film dispersion method. Upon targeted delivery to tumors, the nanoplatform disintegrated in response to the acidic and hydrogen peroxide (H2O2)-rich tumor microenvironment (TME) and near-infrared (NIR) laser irradiation, thereby facilitating controlled drug release and O2 supply to overcome hypoxia. The released RA could cause DNA damage and suppress the HIF-1α expression to sensitize PDT. Simultaneously, TPPP could generate singlet oxygen (1O2) and convert L-arginine (L-Arg) into nitric oxide (NO) for PDT, while APDA could induce hyperthermia for PTT. The combination of PDT and PTT resulted in significant mitochondrial dysfunction and lysosomal damage, ultimately leading to apoptosis. Furthermore, the chemotherapeutic effect of RA could be potentiated by the enhancement of cytoplasmic delivery caused by lysosomal disruption and the blocking of DNA damage repair pathway caused by mitochondrial dysfunction. Overall, cRGD-Lipo-MART, designed with a synergistic CT/PDT/PTT strategy, showed minimal systemic toxicity and could be used as an effective treatment for hypoxic tumors.

Abstract 440: BAP1 deubiquitinates ACLY to promote lipogenesis yet predisposing cholangiocarcinoma to iron overload and ferroptosis

Abstract Background: Cholangiocarcinoma (CCA) is a highly lethal and aggressive epithelial cell malignancy of the liver and biliary tract. CCA survival remains poor and response to chemotherapy or immunotherapy is limited. We AIM to identify metabolic vulnerabilities that render CCA susceptible to ferroptosis, an iron-dependent and caspase-independent cell death driven by lipid peroxidation. Materials and Methods: We treated human and mouse CCA cell lines with ferroptosis inducers (RSL3, Erastin), inhibitors (Fer-1, Lip-1), necroptosis inhibitor and a pan-caspase inhibitor. Cell viability, intracellular and mitochondrial iron, lipid peroxidation, mitochondrial structure, and function by cell-based and biochemical assays. Gain- and loss-of-function strategies, TURBO proximity-dependent biotin identification, and metabolic techniques were used for mechanistic studies. In vivo, orthotopically implanted C57B/6 mice were treated with RSL3 or vehicle intraperitoneally, followed by assessment of tumor burden and immunophenotyping analysis. Epcam-aptamer coated nanoparticles (tMNPs) packaged with siRNA against GPX4 were used for selective ferroptosis induction. Results: We identified ferroptosis-sensitive and -resistant human and mouse CCA cells. Ferroptosis-sensitive cells, rescued by ferroptosis inhibitors but not by necroptosis or pan-caspase inhibitors, exhibited elevated intracellular free iron and mitochondrial iron overload. This led to significant mitochondrial dysfunction and structural changes associated with ferroptosis. Mitochondrial free iron triggered reactive oxygen species generation and lipid peroxidation. We identified BRCA-associated protein 1 (BAP1) as a potential driver of ferroptosis sensitivity. BAP1, mutated in 22-25% of human CCAs, is a tumor suppressor and deubiquitinase (DUB). BAP1 knockdown by siRNA protected cells from ferroptosis, while re-expression of BAP1 sensitized cells to ferroptosis. Subcellular fractionation identified BAP1 in extranuclear compartments, including mitochondria and cytosol. We found that BAP1 interacts and deubiquitinates ATP citrate lyase (ACLY), which converts iron chelator citrate to oxaloacetate and acetyl-CoA. Inhibition of mitochondrial citrate export, protected cells from iron overload and ferroptosis. In vivo, RSL3-induced ferroptosis significantly reduced tumor burden, as compared to vehicle-treated mice. RSL3-induced ferroptosis promoted an immunogenic tumor immune microenvironment. Lastly, tMNPs with siGPX4 induced marked ferroptosis of mouse CCA cells and showed maximal enrichment in liver orthotopic tumors in vivo. Conclusions: Our findings indicate that BAP1 deubiquitination of ACLY predisposes CCA cells to iron overload by exhausting citrate to support lipogenesis. CCA vulnerability to ferroptosis opens up novel therapeutic opportunities for CCA. Citation Format: Peyton Classon, Sophia Jaramillo, Danielle Carlson, Irene Yan, Tushar Patel, Sumera I. Ilyas, Rory L. Smoot, Gregory J. Gores, Davide Povero. BAP1 deubiquitinates ACLY to promote lipogenesis yet predisposing cholangiocarcinoma to iron overload and ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 440.

3-Methyl-4-nitrophenol Exposure Deteriorates Oocyte Maturation by Inducing Spindle Instability and Mitochondrial Dysfunction.

3-methyl-4-nitrophenol (PNMC), a well-known constituent of diesel exhaust particles and degradation products of insecticide fenitrothion, is a widely distributed environmental contaminant. PNMC is toxic to the female reproductive system; however, how it affects meiosis progression in oocytes is unknown. In this study, in vitro maturation of mouse oocytes was applied to investigate the deleterious effects of PNMC. We found that exposure to PNMC significantly compromised oocyte maturation. PNMC disturbed the spindle stability; specifically, it decreased the spindle density and increased the spindle length. The weakened spindle pole location of microtubule-severing enzyme Fignl1 may result in a defective spindle apparatus in PNMC-exposed oocytes. PNMC exposure induced significant mitochondrial dysfunction, including mitochondria distribution, ATP production, mitochondrial membrane potential, and ROS accumulation. The mRNA levels of the mitochondria-related genes were also significantly impaired. Finally, the above-mentioned alterations triggered early apoptosis in the oocytes. In conclusion, PNMC exposure affected oocyte maturation and quality through the regulation of spindle stability and mitochondrial function.

Neuroendocrine and cellular mechanisms in stress resilience: From hormonal influence in the CNS to mitochondrial dysfunction and oxidative stress.

Recent advancements in neuroendocrinology challenge the long-held belief that hormonal effects are confined to perivascular tissues and do not extend to the central nervous system (CNS). This paradigm shift, propelled by groundbreaking research, reveals that synthetic hormones, notably in anti-inflammatory medications, significantly influence steroid psychosis, behavioural, and cognitive impairments, as well as neuropeptide functions. A seminal development in this field occurred in 1968 with McEven's proposal that rodent brains are responsive to glucocorticoids, fundamentally altering the understanding of how anxiety impacts CNS functionality and leading to the identification of glucocorticosteroids and mineralocorticoids as distinct corticotropic receptors. This paper focuses on the intricate roles of the neuroendocrine, immunological, and CNS in fostering stress resilience, underscored by recent animal model studies. These studies highlight active, compensatory, and passive strategies for resilience, supporting the concept that anxiety and depression are systemic disorders involving dysregulation across both peripheral and central systems. Resilience is conceptualized as a multifaceted process that enhances psychological adaptability to stress through adaptive mechanisms within the immunological system, brain, hypothalamo-pituitary-adrenal axis, and ANS Axis. Furthermore, the paper explores oxidative stress, particularly its origin from the production of reactive oxygen species (ROS) in mitochondria. The mitochondria's role extends beyond ATP production, encompassing lipid, heme, purine, and steroidogenesis synthesis. ROS-induced damage to biomolecules can lead to significant mitochondrial dysfunction and cell apoptosis, emphasizing the critical nature of mitochondrial health in overall cellular function and stress resilience. This comprehensive synthesis of neuroendocrinological and cellular biological research offers new insights into the systemic complexity of stress-related disorders and the imperative for multidisciplinary approaches in their study and treatment.

Trimetazidine Improves Mitochondrial Dysfunction in SOD1G93A Cellular Models of Amyotrophic Lateral Sclerosis through Autophagy Activation.

Amyotrophic Lateral Sclerosis (ALS) is considered the prototype of motor neuron disease, characterized by motor neuron loss and muscle waste. A well-established pathogenic hallmark of ALS is mitochondrial failure, leading to bioenergetic deficits. So far, pharmacological interventions for the disease have proven ineffective. Trimetazidine (TMZ) is described as a metabolic modulator acting on different cellular pathways. Its efficacy in enhancing muscular and cardiovascular performance has been widely described, although its molecular target remains elusive. We addressed the molecular mechanisms underlying TMZ action on neuronal experimental paradigms. To this aim, we treated murine SOD1G93A-model-derived primary cultures of cortical and spinal enriched motor neurons, as well as a murine motor-neuron-like cell line overexpressing SOD1G93A, with TMZ. We first characterized the bioenergetic profile of the cell cultures, demonstrating significant mitochondrial dysfunction that is reversed by acute TMZ treatments. We then investigated the effect of TMZ in promoting autophagy processes and its impact on mitochondrial morphology. Finally, we demonstrated the effectiveness of TMZ in terms of the mitochondrial functionality of ALS-rpatient-derived peripheral blood mononuclear cells (PBMCs). In summary, our results emphasize the concept that targeting mitochondrial dysfunction may represent an effective therapeutic strategy for ALS. The findings demonstrate that TMZ enhances mitochondrial performance in motor neuron cells by activating autophagy processes, particularly mitophagy. Although further investigations are needed to elucidate the precise molecular pathways involved, these results hold critical implications for the development of more effective and specific derivatives of TMZ for ALS treatment.

HOMER3 promotes non-small cell lung cancer growth and metastasis primarily through GABPB1-mediated mitochondrial metabolism

Cancer metabolism has emerged as a major target for cancer therapy, while the state of mitochondrial drugs has remained largely unexplored, partly due to an inadequate understanding of various mitochondrial functions in tumor contexts. Here, we report that HOMER3 is highly expressed in non-small cell lung cancer (NSCLC) and is closely correlated with poor prognosis. Lung cancer cells with low levels of HOMER3 are found to show significant mitochondrial dysfunction, thereby suppressing their proliferation and metastasis in vivo and in vitro. At the mechanistic level, we demonstrate that HOMER3 and platelet-activating factor acetylhydrolase 1b catalytic subunit 3 cooperate to upregulate the level of GA-binding protein subunit beta-1 (GABPB1), a key transcription factor involved in mitochondrial biogenesis, to control mitochondrial inner membrane genes and mitochondrial function. Concurrently, low levels of HOMER3 and its downstream target GABPB1 led to mitochondrial dysfunction and decreased proliferation and invasive activity of lung cancer cells, which raises the possibility that targeting mitochondrial synthesis is an important and promising therapeutic approach for NSCLC.

A genetic basis of mitochondrial DNAJA3 in nonalcoholic steatohepatitis-related hepatocellular carcinoma.

NAFLD is the most common form of liver disease worldwide, but only a subset of individuals with NAFLD may progress to NASH. While NASH is an important etiology of HCC, the underlying mechanisms responsible for the conversion of NAFLD to NASH and then to HCC are poorly understood. We aimed to identify genetic risk genes that drive NASH and NASH-related HCC. We searched genetic alleles among the 24 most significant alleles associated with body fat distribution from a genome-wide association study of 344,369 individuals and validated the top allele in 3 independent cohorts of American and European patients (N=1380) with NAFLD/NASH/HCC. We identified an rs3747579-TT variant significantly associated with NASH-related HCC and demonstrated that rs3747579 is expression quantitative trait loci of a mitochondrial DnaJ Heat Shock Protein Family (Hsp40) Member A3 ( DNAJA3 ). We also found that rs3747579-TT and a previously identified PNPLA3 as a functional variant of NAFLD to have significant additional interactions with NASH/HCC risk. Patients with HCC with rs3747579-TT had a reduced expression of DNAJA3 and had an unfavorable prognosis. Furthermore, mice with hepatocyte-specific Dnaja3 depletion developed NASH-dependent HCC either spontaneously under a normal diet or enhanced by diethylnitrosamine. Dnaja3 -deficient mice developed NASH/HCC characterized by significant mitochondrial dysfunction, which was accompanied by excessive lipid accumulation and inflammatory responses. The molecular features of NASH/HCC in the Dnaja3 -deficient mice were closely associated with human NASH/HCC. We uncovered a genetic basis of DNAJA3 as a key player of NASH-related HCC.

Mitochondrial dysfunction and oxidative stress in diabetic wound.

Diabetic wounds nowadays have become a major health challenge with the changes of the disease spectrum. Mitochondria are closely associated with stubborn nonhealing diabetic wounds for their vital role in energy metabolism, redox homeostasis, and signal transduction. There is significant mitochondrial dysfunction and oxidative stress in diabetic wounds. However, the contribution of mitochondrial dysfunction in oxidative stress induced nonhealing diabetic wound is still not fully understood. In this review, we will briefly summarize the current knowledge of the reported signaling pathways and therapeutic strategies involved in mitochondrial dysfunction in diabetic wounds. The findings provide further understanding of strategies that focus on mitochondria in diabetic wound treatment.

SKQ1 Improves Trimethylamine-N-Oxide-Induced Endothelial Dysfunction by Attenuating Oxidative Stress and Restoring Junctional Proteins

In recent years, the role of microbiota and microbial metabolites in the progress of aging and age-related diseases has been attracting increased attention. In this context, trimethylamine N-oxide (TMAO) is a pro-atherogenic and pro-inflammatory metabolite derived from the gut microbial metabolism of trimethylamine and is found to be elevated in patients with age-related cardiovascular disorders. We identified that TMAO treatment in mice has enhanced mitochondrial ROS production, endoplasmic reticulum stress and caused significant endothelial dysfunction. The present study investigated whether mitochondria-targeted antioxidant SKQ1 protected against TMAO-induced endothelial dysfunction. Endothelial cells were treated with or without TMAO (30μM) in the cell culture media. TMAO treated cells showed significant increase in both total ROS as well as mito-ROS production compared to untreated control group (n=8/group; p<0.05). In addition, western blot analysis demonstrated that TMAO treatment significantly decreased tight junction proteins such as occludin, ZO-1 and VE-Cadherin which were restored upon SKQ1 treatment (150nM, 12h). TMAO (30μM) treatment for 24 h induced significant mitochondrial dysfunction as evidenced by decreased mitochondrial membrane potential, increased mitochondrial ROS levels, and enhanced endothelial cell permeability compared to the control group (n=8/group; p<0.05). Treatment with a SKQ1 (150nM, 12h) significantly inhibited TMAO-induced ROS production and protected against increased endothelial cell permeability and mitochondrial ROS production (n=8/group; p<0.05). In conclusion, mitochondria-targeted antioxidant SKQ1 protects against TMAO induced endothelial dysfunction by attenuating ROS production and disruption of tight junction proteins. This work is supported by NIH R01 HL148711 award to Saisudha Koka This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

High-fat diet-induced mitochondrial dysfunction is associated with loss of protection from ischemic preconditioning in renal ischemia reperfusion.

Consumption of high-fat diet (HFD) promotes mitochondrial dysfunction and the latter act as a critical factor in determining the severity of ischemia-reperfusion (IR) injury in different cell types. Ischemic preconditioning (IPC), a well-known protocol that render IR protection in kidney works via mitochondria. In the present study, we evaluated how HFD kidney with underlying mitochondrial changes respond to precondition protocol after IR induction. Wistar male rats were used in this study and were divided into two groups: SD (standard diet; n = 18) and HFD (high-fat diet; n = 18), which were further subdivided into sham, ischemia-reperfusion, and precondition groups at the end of the dietary regimen. Blood biochemistry, renal injury marker, creatinine clearance (CrCl), mitochondrial quality (fission, fusion, and phagy), mitochondrial function via ETC enzyme activities and respiration, and signalling pathway were analysed. Sixteen weeks of HFD administration to the rat deteriorated the renal mitochondrial health measured via 10% decline in mitochondrial respiration index ADP/O (in GM), reduced mitochondrial copy number (55%), biogenesis (56%), low bioenergetics potential (19% complex I + III and 15% complex II + III), increased oxidative stress, and reduced expression of mitochondrial fusion genes compared with SD rats. IR procedure in HFD rat kidney inflicted significant mitochondrial dysfunction and further deteriorated copy number along with impaired mitophagy and mitochondrial dynamics. IPC could effectively ameliorate the renal ischemia injury in normal rat but failed to provide similar kind of protection in HFD rat kidney. Even though the IR-associated mitochondrial dysfunction in both normal and HFD rats were similar, the magnitude of overall dysfunction and corresponding renal injury and compromised physiology was high in HFD rats. This observation was further confirmed via in vitro protein translation assay in isolated mitochondria from normal and HFD rat kidney that showed significantly reduction in the response ability of mitochondria in HFD. In conclusion, the deteriorated mitochondrial function and its quality along with low mitochondrial copy number and downregulation of mitochondrial dynamic gene exhibited by HFD rat kidney augments the sensitivity of renal tissue towards the IR injury which leads to the compromised protective ability by ischemic preconditioning.

Mitochondrial Inhibitor Rotenone Triggers and Enhances Neuronal Ferroptosis Following Intracerebral Hemorrhage.

Ferroptosis, a form of regulatory non-apoptotic cell death driven by iron-dependent lipid peroxidation, accounts for more than 80% of the total types of neuronal death in the acute phase of intracerebral hemorrhage (ICH). Mitochondria have essential roles in energy production, macromolecule synthesis, cellular metabolism, and cell death regulation. However, its role in ferroptosis remains unclear and somewhat controversial, especially in ICH. This study aimed to investigate whether damaged mitochondria could trigger and enhance neuronal ferroptosis in ICH. The isobaric tag for relative and absolute quantitation proteomics on human ICH samples suggested that ICH caused significant damage to the mitochondria, which presented ferroptosis-like morphology under electron microscopy. Subsequently, use of the mitochondrial special inhibitor Rotenone (Rot) to induce mitochondrial damage showed that it has significant dose-dependent toxicity on primary neurons. Single Rot administration markedly inhibited neuronal viability, promoted iron accumulation, increased malondialdehyde (MDA) contents, decreased total superoxide dismutase (SOD) activity, and downregulated ferroptosis-related proteins RPL8, COX-2, xCT, ASCL4, and GPX4 in primary neurons. Moreover, Rot enhanced these changes via hemin and autologous blood administration in primary neurons and mice, mimicking the in vitro and in vivo ICH models, respectively. Furthermore, Rot exacerbated the ICH-induced hemorrhagic volumes, brain edema, and neurological deficits in mice. Together, our data revealed that ICH induced significant mitochondrial dysfunction and that mitochondrial inhibitor Rot can trigger and enhance neuronal ferroptosis.

High Glucose-Induced Cardiomyocyte Damage Involves Interplay between Endothelin ET-1/ETA/ETB Receptor and mTOR Pathway.

Patients with type two diabetes mellitus (T2DM) are at increased risk for cardiovascular diseases. Impairments of endothelin-1 (ET-1) signaling and mTOR pathway have been implicated in diabetic cardiomyopathies. However, the molecular interplay between the ET-1 and mTOR pathway under high glucose (HG) conditions in H9c2 cardiomyoblasts has not been investigated. We employed MTT assay, qPCR, western blotting, fluorescence assays, and confocal microscopy to assess the oxidative stress and mitochondrial damage under hyperglycemic conditions in H9c2 cells. Our results showed that HG-induced cellular stress leads to a significant decline in cell survival and an impairment in the activation of ETA-R/ETB-R and the mTOR main components, Raptor and Rictor. These changes induced by HG were accompanied by a reactive oxygen species (ROS) level increase and mitochondrial membrane potential (MMP) loss. In addition, the fragmentation of mitochondria and a decrease in mitochondrial size were observed. However, the inhibition of either ETA-R alone by ambrisentan or ETA-R/ETB-R by bosentan or the partial blockage of the mTOR function by silencing Raptor or Rictor counteracted those adverse effects on the cellular function. Altogether, our findings prove that ET-1 signaling under HG conditions leads to a significant mitochondrial dysfunction involving contributions from the mTOR pathway.

En masse organoid phenotyping informs metabolic-associated genetic susceptibility to NASH

Genotype-phenotype associations for common diseases are often compounded by pleiotropy and metabolic state. Here, we devised a pooled human organoid-panel of steatohepatitis to investigate the impact of metabolic status on genotype-phenotype association. En masse population-based phenotypic analysis under insulin insensitive conditions predicted key non-alcoholic steatohepatitis (NASH)-genetic factors including the glucokinase regulatory protein (GCKR)-rs1260326:C>T. Analysis of NASH clinical cohorts revealed that GCKR-rs1260326-T allele elevates disease severity only under diabetic state but protects from fibrosis under non-diabetic states. Transcriptomic, metabolomic, and pharmacological analyses indicate significant mitochondrial dysfunction incurred by GCKR-rs1260326, which was not reversed with metformin. Uncoupling oxidative mechanisms mitigated mitochondrial dysfunction and permitted adaptation to increased fatty acid supply while protecting against oxidant stress, forming a basis for future therapeutic approaches for diabetic NASH. Thus, "in-a-dish" genotype-phenotype association strategies disentangle the opposing roles of metabolic-associated gene variant functions and offer a rich mechanistic, diagnostic, and therapeutic inference toolbox toward precision hepatology. VIDEO ABSTRACT.

Mitochondria-targeted nanoplatforms building for in situ ROS generating photodynamic tumor therapy through reinforcing mitochondria apoptotic pathway

Mitochondria-targeted photodynamic therapy (PDT) represents an attractive therapeutic strategy for antitumor therapy. There is growing evidence indicating that in situ generation of reactive oxygen species (ROS) in mitochondria plays a critical role in significant mitochondrial dysfunction and cell apoptosis. Nevertheless, survivin, a member of the inhibitor of apoptosis protein (IAP) family, is overexpressed by PDT stimulation, which inhibit cysteine aspartic acid specific protease-9 (caspase-9) activation and inhibits apoptosis. The corresponding overexpression of Livin protein in tumor cells can inhibit cysteine aspartic acid specific protease-3 (caspase-3) activity, leading to apoptosis inhibition and promoting tumorigenesis and tumor progression. Thus, a precise combination of mitochondria-targeted photodynamic photosensitizers and IAP inhibitor is expected to significantly augment the PDT efficacy. For proof-of-concept, a therapeutic nanoplatform MSNs/GM/I3C-BSA/MnO2-IR775 (MGIBR) was ingeniously constructed for synergistic cancer therapy. MGIBR can effectively target mitochondria due to the presence of IR775. Under NIR irradiation, MGIBR can generate in situ 1O2 in mitochondria. Importantly, geldanamycin (GM) and indole-3-carbinol (I3C) can inhibit IAP, synergistic enhance PDT and activate mitochondrial apoptosis pathways, leading to effectively cell death. Such a strategy of MGIBR enabled mitochondrial dysfunction based on in situ generation of ROS in mitochondria and IAP inhibitor synergistically, provides a promising paradigm for highly effective cancer therapeutics.

Age-Dependent Behavioral and Metabolic Assessment of App NL-G-F/NL-G-F Knock-in (KI) Mice.

Mitochondria play a crucial role in Alzheimer's disease (AD) onset and progression. Traditional transgenic AD mouse models which were widely used in the past decades share a common limitation: The overexpression of APP and overproduction of amyloid-beta (Aβ) are accompanied by other APP peptide fragments, which could introduce artificial and non-clinically relevant phenotypes. Here, we performed an in-depth and time-resolved behavioral and metabolic characterization of a clinically relevant AD mouse model engineered to express normal physiological levels of APP harboring humanized Swedish (K670N/M671L), Beyreuther/Iberian (I716F), and Arctic (E693G) mutations (AppNL−G−F/NL−G−F), termed APP knock-in (APPKI) mice. Our result showed that APPKI mice exhibited fear learning deficits at 6-m age and contextual memory deficit at 12-m age. Histopathological analysis revealed mild amyloidosis (6E10) accompanied by microgliosis (Iba1) as early as 3 months, which progressed significantly together with significant astrocytosis at 6 and 12 m. We further analyzed hippocampal mitochondrial dysfunction by multiple assays, while 3-m APPKI mice brain mitochondrial function remains a similar level as WT mice. Significant mitochondrial dysfunction characterized by decreased ATP production and higher membrane potential with subsequent overproduction of reactive oxygen species (ROS) was observed in mitochondria isolated from 7-m APPKI mice hippocampal tissue. Morphologically, these mitochondria were larger in volume with a decreased level of mitochondrial fusion protein mitofusin-2 (MFN2). At 12 months, APPKI mice exhibit a significantly decreased total mitochondrial oxygen consumption rate (OCR) in isolated hippocampal mitochondria detected by high-resolution respirometry. These data indicate early mitochondrial dysfunction in the brain at pre-symptomatic age in the AppNL−G−F/NL−G−mice, which may play a key role in the progression of the disease. Moreover, the identified behavioral and bioenergetic alterations in this clinically relevant AD mouse model provide a valuable tool to optimize the temporal component for therapeutic interventions to treat AD.

Preserving mitochondrial function by inhibiting GRP75 ameliorates neuron injury under ischemic stroke.

Ischemic stroke is a life-threatening disease, which is closely related to neuron damage during ischemia. Mitochondrial dysfunction is essentially involved in the pathophysiological process of ischemic stroke. Mitochondrial calcium overload contributes to the development of mitochondrial dysfunction. However, the underlying mechanisms of mitochondrial calcium overload are far from being fully revealed. In the present study, middle cerebral artery obstruction (MCAO) was performed in vivo and oxygen and glucose deprivation (OGD) in vitro. The results indicated that both MCAO and OGD induced significant mitochondrial dysfunction in vivo and in vitro. The mitochondria became fragmented under hypoxia conditions, accompanied with upregulation of the heat shock protein 75 kDa glucose-regulated protein (GRP75). Inhibition of GRP75 was able to effectively ameliorate mitochondrial calcium overload and preserve mitochondrial function, which may provide evidence for further translational studies of ischemic diseases.

L-arginine Ameliorated Mitochondrial Oxidative Damage Induced by Sub-chronic Exposure to Cadmium in Mice Kidney

Background:Cadmium is a heavy metal that can cause various injuries in the body, including nephrotoxicity. L-Arginine is a metal chelator that can prevent oxidative damage caused by oxygen free radicals.
 Objectives:This study aimed to investigate the effect of L-arginine in inhibiting mitochondrial toxicity induced by subchronic cadmium exposure in the kidney of male mice.
 Methods: A total of 42 male mice were randomly divided into six groups (n=6): control (normal saline), cadmium (2 mg/kg), cadmium (2 mg/kg) plus three doses of L-arginine (50, 100, and 200 mg/kg) and finally cadmium (2 mg/kg) plus vitamin C (500 mg/kg). After 42 days, the animals were anesthetized with ketamine/xylazine. Their kidney tissues were removed, and mitochondrial fractions were isolated. Oxidative stress factors and mitochondrial damage parameters (MTT, swelling, and mitochondrial membrane potential) were measured in renal isolated mitochondria. Also, evaluation of Blood Urea Nitrogen (BUN) and Creatinine (Cr) tests were done.
 Results: Significant rise in BUN and Cr were observed in cadmium-treated mice (P<0.05). Cadmium enhanced oxidative stress in the kidney via increasing lipid peroxidation and oxidation of protein and glutathione. It caused significant mitochondrial dysfunction, mitochondrial membrane potential collapse, and swelling in isolated mitochondria (P<0.05). L-Arginine significantly ameliorated cadmium-induced oxidative stress and mitochondrial damage (P<0.05). Furthermore, a significant reduction in serum BUN and Cr were observed in L-arginine received group (P<0.05).
 Conclusion: The results showed that L-arginine has significant protective effects against cadmium-induced renal toxicity in male mice.