bims-midhyp 2023-07-09 papers

bims-midhyp

Biomed News

on Mitochondrial dysfunction and hypoxia

Issue of 2023‒07‒09
thirty papers selected by
Alia Abukiwan
University of Heidelberg

FOXA2 controls the anti-oxidant response in FH-deficient cells.
Metabolic reprogramming, oxidative stress, and pulmonary hypertension.
Prolonged hypoxia alleviates prolyl hydroxylation-mediated suppression of RIPK1 to promote necroptosis and inflammation.
MFN2 deficiency promotes cardiac response to hypobaric hypoxia by reprogramming cardiomyocyte metabolism.
Live Cell Imaging of ATP Levels Reveals Metabolic Compartmentalization within Motoneurons and Early Metabolic Changes in FUS ALS Motoneurons.
Mitochondrial regulation of local supply of energy in neurons.
Ferroptosis in mitochondrial cardiomyopathy.
Yeast NDI1 reconfigures neuronal metabolism and prevents the unfolded protein response in mitochondrial complex I deficiency.
Down-regulation of Hrd1 protects against myocardial ischemia-reperfusion injury by regulating PPARα to prevent oxidative stress, endoplasmic reticulum stress, and cellular apoptosis.
Viability of HepG2 and MCF-7 cells is not correlated with mitochondrial bioenergetics.
Mitochondria in endothelial cells angiogenesis and function: current understanding and future perspectives.
The Haves and Have-Nots: The Mitochondrial Permeability Transition Pore across Species.
Inhibition of pyruvate dehydrogenase kinase 4 ameliorates kidney ischemia-reperfusion injury by reducing succinate accumulation during ischemia and preserving mitochondrial function during reperfusion.
Novel insights into the role of mitochondria in diabetic cardiomyopathy: molecular mechanisms and potential treatments.
Chronic SIRT1 supplementation in diabetic mice improves endothelial function by suppressing oxidative stress.
Heart failure in patients is associated with downregulation of mitochondrial quality control genes.
Mitochondrial dysfunction in the offspring of obese mothers and it's transmission through damaged oocyte mitochondria: Integration of mechanisms.
miR181a/b-1 controls osteocyte metabolism and mechanical properties independently of bone morphology.
CDK4-E2F3 signals enhance oxidative skeletal muscle fiber numbers and function to affect myogenesis and metabolism.
Cardiac lipid metabolism, mitochondrial function and heart failure.
Respiratory complex I in mitochondrial membrane catalyzes oversized ubiquinones.
Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation.
Newly Developed Di-Block Copolymer-Based Cell Membrane Stabilizers Protect Mouse Coronary Artery Endothelial Cells against Hypoxia/Reoxygenation Injury.
Arf1 coordinates fatty acid metabolism and mitochondrial homeostasis.
Modeling conformational states of proteins with AlphaFold.
N-acetylcysteine modulates rotenone-induced mitochondrial Complex I dysfunction in THP-1 cells.
Alterations in synapses and mitochondria induced by acute or chronic intermittent hypoxia in the pre-Bötzinger complex of rats: an ultrastructural triple-labeling study with immunocytochemistry and histochemistry.
The Novel Role of Mitochondrial Citrate Synthase and Citrate in the Pathophysiology of Alzheimer's Disease.
Human Small Heat Shock Protein B8 Inhibits Protein Aggregation without Affecting the Native Folding Process.
ER-mitochondria contacts and cholesterol metabolism are disrupted by disease-associated tau protein.

Cell Rep. 2023 Jul 04. pii: S2211-1247(23)00762-3. [Epub ahead of print]42(7): 112751

FOXA2 controls the anti-oxidant response in FH-deficient cells.

Connor Rogerson, Marco Sciacovelli, Lucas A Maddalena, Andromachi Pouikli, Marc Segarra-Mondejar, Lorea Valcarcel-Jimenez, Christina Schmidt, Ming Yang, Elena Ivanova, Joshua Kent, Ariane Mora, Danya Cheeseman, Jason S Carroll, Gavin Kelsey, Christian Frezza.

  Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a cancer syndrome caused by inactivating germline mutations in fumarate hydratase (FH) and subsequent accumulation of fumarate. Fumarate accumulation leads to profound epigenetic changes and the activation of an anti-oxidant response via nuclear translocation of the transcription factor NRF2. The extent to which chromatin remodeling shapes this anti-oxidant response is currently unknown. Here, we explored the effects of FH loss on the chromatin landscape to identify transcription factor networks involved in the remodeled chromatin landscape of FH-deficient cells. We identify FOXA2 as a key transcription factor that regulates anti-oxidant response genes and subsequent metabolic rewiring cooperating without direct interaction with the anti-oxidant regulator NRF2. The identification of FOXA2 as an anti-oxidant regulator provides additional insights into the molecular mechanisms behind cell responses to fumarate accumulation and potentially provides further avenues for therapeutic intervention for HLRCC.

Keywords:  CP: Cancer; CP: Molecular biology; FOXA2; NRF2; anti-oxidant response; fumarate hydratase

DOI:  https://doi.org/10.1016/j.celrep.2023.112751
Redox Biol. 2023 Jun 24. pii: S2213-2317(23)00198-2. [Epub ahead of print]64 102797

Metabolic reprogramming, oxidative stress, and pulmonary hypertension.

Marissa D Pokharel, David P Marciano, Panfeng Fu, Maria Clara Franco, Hoshang Unwalla, Kim Tieu, Jeffrey R Fineman, Ting Wang, Stephen M Black.

  Mitochondria are highly dynamic organelles essential for cell metabolism, growth, and function. It is becoming increasingly clear that endothelial cell dysfunction significantly contributes to the pathogenesis and vascular remodeling of various lung diseases, including pulmonary arterial hypertension (PAH), and that mitochondria are at the center of this dysfunction. The more we uncover the role mitochondria play in pulmonary vascular disease, the more apparent it becomes that multiple pathways are involved. To achieve effective treatments, we must understand how these pathways are dysregulated to be able to intervene therapeutically. We know that nitric oxide signaling, glucose metabolism, fatty acid oxidation, and the TCA cycle are abnormal in PAH, along with alterations in the mitochondrial membrane potential, proliferation, and apoptosis. However, these pathways are incompletely characterized in PAH, especially in endothelial cells, highlighting the urgent need for further research. This review summarizes what is currently known about how mitochondrial metabolism facilitates a metabolic shift in endothelial cells that induces vascular remodeling during PAH.

Keywords:  Glycolysis; Metabolomics; Mitochondrial function; Mitochondrial remodeling; Pulmonary hypertension

DOI:  https://doi.org/10.1016/j.redox.2023.102797
Nat Cell Biol. 2023 Jul 03.

Prolonged hypoxia alleviates prolyl hydroxylation-mediated suppression of RIPK1 to promote necroptosis and inflammation.

Tao Zhang, Daichao Xu, Jianping Liu, Min Wang, Li-Juan Duan, Min Liu, Huyan Meng, Yuan Zhuang, Huibing Wang, Yingnan Wang, Mingming Lv, Zhengyi Zhang, Jia Hu, Linyu Shi, Rui Guo, Xingxing Xie, Hui Liu, Emily Erickson, Yaru Wang, Wenyu Yu, Fabin Dang, Dongxian Guan, Cong Jiang, Xiaoming Dai, Hiroyuki Inuzuka, Peiqiang Yan, Jingchao Wang, Mrigya Babuta, Gewei Lian, Zhenbo Tu, Ji Miao, Gyongyi Szabo, Guo-Hua Fong, Antoine E Karnoub, Yu-Ru Lee, Lifeng Pan, William G Kaelin, Junying Yuan, Wenyi Wei.

The prolyl hydroxylation of hypoxia-inducible factor 1α (HIF-1α) mediated by the EGLN-pVHL pathway represents a classic signalling mechanism that mediates cellular adaptation under hypoxia. Here we identify RIPK1, a known regulator of cell death mediated by tumour necrosis factor receptor 1 (TNFR1), as a target of EGLN1-pVHL. Prolyl hydroxylation of RIPK1 mediated by EGLN1 promotes the binding of RIPK1 with pVHL to suppress its activation under normoxic conditions. Prolonged hypoxia promotes the activation of RIPK1 kinase by modulating its proline hydroxylation, independent of the TNFα-TNFR1 pathway. As such, inhibiting proline hydroxylation of RIPK1 promotes RIPK1 activation to trigger cell death and inflammation. Hepatocyte-specific Vhl deficiency promoted RIPK1-dependent apoptosis to mediate liver pathology. Our findings illustrate a key role of the EGLN-pVHL pathway in suppressing RIPK1 activation under normoxic conditions to promote cell survival and a model by which hypoxia promotes RIPK1 activation through modulating its proline hydroxylation to mediate cell death and inflammation in human diseases, independent of TNFR1.

DOI: https://doi.org/10.1038/s41556-023-01170-4
Acta Physiol (Oxf). 2023 Jul 04. e14018

MFN2 deficiency promotes cardiac response to hypobaric hypoxia by reprogramming cardiomyocyte metabolism.

Ru Zhang, Ailin Yang, Lin Zhang, Linjie He, Xiaoming Gu, Caiyong Yu, Zhenxing Lu, Chuang Wang, Feng Zhou, Fei Li, Lele Ji, Jinliang Xing, Haitao Guo.

  AIM: Under hypobaric hypoxia (HH), the heart triggers various defense mechanisms including metabolic remodeling against lack of oxygen. Mitofusin 2 (MFN2), located at the mitochondrial outer membrane, is closely involved in the regulation of mitochondrial fusion and cell metabolism. To date, however, the role of MFN2 in cardiac response to HH has not been explored.METHODS: Loss- and gain-of-function approaches were used to investigate the role of MFN2 in cardiac response to HH. In vitro, the function of MFN2 in the contraction of primary neonatal rat cardiomyocytes under hypoxia was examined. Non-targeted metabolomics and mitochondrial respiration analyses, as well as functional experiments were performed to explore underlying molecular mechanisms.
RESULTS: Our data demonstrated that, following 4 weeks of HH, cardiac-specific MFN2 knockout (MFN2 cKO) mice exhibited significantly better cardiac function than control mice. Moreover, restoring the expression of MFN2 clearly inhibited the cardiac response to HH in MFN2 cKO mice. Importantly, MFN2 knockout significantly improved cardiac metabolic reprogramming during HH, resulting in reduced capacity for fatty acid oxidation (FAO) and oxidative phosphorylation, and increased glycolysis and ATP production. In vitro data showed that down-regulation of MFN2 promoted cardiomyocyte contractility under hypoxia. Interestingly, increased FAO through palmitate treatment decreased contractility of cardiomyocyte with MFN2 knockdown under hypoxia. Furthermore, treatment with mdivi-1, an inhibitor of mitochondrial fission, disrupted HH-induced metabolic reprogramming and subsequently promoted cardiac dysfunction in MFN2-knockout hearts.
CONCLUSION: Our findings provide the first evidence that down-regulation of MFN2 preserves cardiac function in chronic HH by promoting cardiac metabolic reprogramming.

Keywords:  MFN2; cardiac response; hypobaric hypoxia; metabolic reprogramming; mitochondrial fusion

DOI:  https://doi.org/10.1111/apha.14018
Cells. 2023 May 09. pii: 1352. [Epub ahead of print]12(10):

Live Cell Imaging of ATP Levels Reveals Metabolic Compartmentalization within Motoneurons and Early Metabolic Changes in FUS ALS Motoneurons.

Vitaly L Zimyanin, Anna-Maria Pielka, Hannes Glaß, Julia Japtok, Dajana Großmann, Melanie Martin, Andreas Deussen, Barbara Szewczyk, Chris Deppmann, Eli Zunder, Peter M Andersen, Tobias M Boeckers, Jared Sterneckert, Stefanie Redemann, Alexander Storch, Andreas Hermann.

  Motoneurons are one of the most energy-demanding cell types and a primary target in Amyotrophic lateral sclerosis (ALS), a debilitating and lethal neurodegenerative disorder without currently available effective treatments. Disruption of mitochondrial ultrastructure, transport, and metabolism is a commonly reported phenotype in ALS models and can critically affect survival and the proper function of motor neurons. However, how changes in metabolic rates contribute to ALS progression is not fully understood yet. Here, we utilize hiPCS-derived motoneuron cultures and live imaging quantitative techniques to evaluate metabolic rates in fused in sarcoma (FUS)-ALS model cells. We show that differentiation and maturation of motoneurons are accompanied by an overall upregulation of mitochondrial components and a significant increase in metabolic rates that correspond to their high energy-demanding state. Detailed compartment-specific live measurements using a fluorescent ATP sensor and FLIM imaging show significantly lower levels of ATP in the somas of cells carrying FUS-ALS mutations. These changes lead to the increased vulnerability of diseased motoneurons to further metabolic challenges with mitochondrial inhibitors and could be due to the disruption of mitochondrial inner membrane integrity and an increase in its proton leakage. Furthermore, our measurements demonstrate heterogeneity between axonal and somatic compartments, with lower relative levels of ATP in axons. Our observations strongly support the hypothesis that mutated FUS impacts the metabolic states of motoneurons and makes them more susceptible to further neurodegenerative mechanisms.

Keywords:  amyotrophic lateral sclerosis; metabolism; mitochondria

DOI:  https://doi.org/10.3390/cells12101352
Curr Opin Neurobiol. 2023 Jun 29. pii: S0959-4388(23)00072-7. [Epub ahead of print]81 102747

Mitochondrial regulation of local supply of energy in neurons.

Guillermo López-Doménech, Josef T Kittler.

Brain computation is metabolically expensive and requires the supply of significant amounts of energy. Mitochondria are highly specialized organelles whose main function is to generate cellular energy. Due to their complex morphologies, neurons are especially dependent on a set of tools necessary to regulate mitochondrial function locally in order to match energy provision with local demands. By regulating mitochondrial transport, neurons control the local availability of mitochondrial mass in response to changes in synaptic activity. Neurons also modulate mitochondrial dynamics locally to adjust metabolic efficiency with energetic demand. Additionally, neurons remove inefficient mitochondria through mitophagy. Neurons coordinate these processes through signalling pathways that couple energetic expenditure with energy availability. When these mechanisms fail, neurons can no longer support brain function giving rise to neuropathological states like metabolic syndromes or neurodegeneration.

DOI: https://doi.org/10.1016/j.conb.2023.102747
Trends Cell Biol. 2023 Jul 05. pii: S0962-8924(23)00125-3. [Epub ahead of print]

Ferroptosis in mitochondrial cardiomyopathy.

Sofia Ahola, Thomas Langer.

  Ferroptosis is a form of necrotic cell death characterized by iron-dependent lipid peroxidation culminating in membrane rupture. Accumulating evidence links ferroptosis to multiple cardiac diseases and identifies mitochondria as important regulators of ferroptosis. Mitochondria are not only a major source of reactive oxygen species (ROS) but also counteract ferroptosis by preserving cellular redox balance and oxidative defense. Recent evidence has revealed that the mitochondrial integrated stress response limits oxidative stress and ferroptosis in oxidative phosphorylation (OXPHOS)-deficient cardiomyocytes and protects against mitochondrial cardiomyopathy. We summarize the multiple ways in which mitochondria modulate the susceptibility of cells to ferroptosis, and discuss the implications of ferroptosis for cardiomyopathies in mitochondrial disease.

Keywords:  Gpx4; ferroptosis; integrated stress response; mitochondrial cardiomyopathy

DOI:  https://doi.org/10.1016/j.tcb.2023.06.002
PLoS Genet. 2023 Jul 03. 19(7): e1010793

Yeast NDI1 reconfigures neuronal metabolism and prevents the unfolded protein response in mitochondrial complex I deficiency.

Lucy Granat, Debbra Y Knorr, Daniel C Ranson, Emma L Hamer, Ram Prosad Chakrabarty, Francesca Mattedi, Laura Fort-Aznar, Frank Hirth, Sean T Sweeney, Alessio Vagnoni, Navdeep S Chandel, Joseph M Bateman.

Mutations in subunits of the mitochondrial NADH dehydrogenase cause mitochondrial complex I deficiency, a group of severe neurological diseases that can result in death in infancy. The pathogenesis of complex I deficiency remain poorly understood, and as a result there are currently no available treatments. To better understand the underlying mechanisms, we modelled complex I deficiency in Drosophila using knockdown of the mitochondrial complex I subunit ND-75 (NDUFS1) specifically in neurons. Neuronal complex I deficiency causes locomotor defects, seizures and reduced lifespan. At the cellular level, complex I deficiency does not affect ATP levels but leads to mitochondrial morphology defects, reduced endoplasmic reticulum-mitochondria contacts and activation of the endoplasmic reticulum unfolded protein response (UPR) in neurons. Multi-omic analysis shows that complex I deficiency dramatically perturbs mitochondrial metabolism in the brain. We find that expression of the yeast non-proton translocating NADH dehydrogenase NDI1, which reinstates mitochondrial NADH oxidation but not ATP production, restores levels of several key metabolites in the brain in complex I deficiency. Remarkably, NDI1 expression also reinstates endoplasmic reticulum-mitochondria contacts, prevents UPR activation and rescues the behavioural and lifespan phenotypes caused by complex I deficiency. Together, these data show that metabolic disruption due to loss of neuronal NADH dehydrogenase activity cause UPR activation and drive pathogenesis in complex I deficiency.

DOI: https://doi.org/10.1371/journal.pgen.1010793
Eur J Pharmacol. 2023 Jun 29. pii: S0014-2999(23)00375-8. [Epub ahead of print]954 175864

Down-regulation of Hrd1 protects against myocardial ischemia-reperfusion injury by regulating PPARα to prevent oxidative stress, endoplasmic reticulum stress, and cellular apoptosis.

Boyu Xia, Qi Li, Koulong Zheng, Jingjing Wu, Chao Huang, Kun Liu, Qingsheng You, Xiaomei Yuan.

  The E3 ubiquitin ligase HMG-CoA reductase degradation protein 1 (Hrd1) is a key enzyme for ER-associated degradation of misfolded proteins. Its role in ischemic heart disease has not been fully elucidated. Here, we investigated its effect on oxidative status and cell survival in cardiac ischemia-reperfusion injury (MIRI). We found that virus-induced down-regulation of Hrd1 expression limited infarct size, decreased creatinine kinase (CK) and lactate dehydrogenase (LDH), and preserved cardiac function in mice subjected to left anterior descending coronary artery ligation and reperfusion. Silencing of the Hrd1 gene also prevented the ischemia/reperfusion (I/R)-induced (i) increase in dihydroethidium (DHE) intensity, mitochondrial production of reactive oxygen species (ROS), malondialdehyde (MDA), and nitric oxide (NO), (ii) decrease in total antioxidant capacity (T-AOC) and glutathione (GSH), (iii) disruption of mitochondrial membrane potential, and (iv) increase in the expression of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) in ischemic heart tissue. In addition, down-regulation of Hrd1 expression prevented the abnormally increased caspase-3/caspase-9/Bax expression and decreased Bcl-2 expression in ischemic heart tissue of I/R mice. Further analysis showed that the I/R stimulus reduced peroxisome proliferation activated receptor α (PPARα) expression in ischemic heart tissue, which was partially prevented by down-regulation of Hrd1. Pharmacological inhibition of PPARα was able to abolish the preventive effect of down-regulation of Hrd1 on oxidative stress, endoplasmic reticulum stress, and cellular apoptosis in ischemic heart tissue. These data suggest that down-regulation of Hrd1 protects the heart from I/R-induced damage by suppressing oxidative stress and cellular apoptosis likely through PPARα.

Keywords:  Apoptosis; Hrd1; MIRI; Oxidative stress; PPARα

DOI:  https://doi.org/10.1016/j.ejphar.2023.175864
Sci Rep. 2023 07 04. 13(1): 10822

Viability of HepG2 and MCF-7 cells is not correlated with mitochondrial bioenergetics.

Judit Doczi, Noemi Karnok, David Bui, Victoria Azarov, Gergely Pallag, Sara Nazarian, Bence Czumbel, Thomas N Seyfried, Christos Chinopoulos.

Alterations in metabolism are a hallmark of cancer. It is unclear if oxidative phosphorylation (OXPHOS) is necessary for tumour cell survival. In this study, we investigated the effects of severe hypoxia, site-specific inhibition of respiratory chain (RC) components, and uncouplers on necrotic and apoptotic markers in 2D-cultured HepG2 and MCF-7 tumour cells. Comparable respiratory complex activities were observed in both cell lines. However, HepG2 cells exhibited significantly higher oxygen consumption rates (OCR) and respiratory capacity than MCF-7 cells. Significant non-mitochondrial OCR was observed in MCF-7 cells, which was insensitive to acute combined inhibition of complexes I and III. Pre-treatment of either cell line with RC inhibitors for 24-72 h resulted in the complete abolition of respective complex activities and OCRs. This was accompanied by a time-dependent decrease in citrate synthase activity, suggesting mitophagy. High-content automated microscopy recordings revealed that the viability of HepG2 cells was mostly unaffected by any pharmacological treatment or severe hypoxia. In contrast, the viability of MCF-7 cells was strongly affected by inhibition of complex IV (CIV) or complex V (CV), severe hypoxia, and uncoupling. However, it was only moderately affected by inhibition of complexes I, II, and III. Cell death in MCF-7 cells induced by inhibition of complexes II, III, and IV was partially abrogated by aspartate. These findings indicate that OXPHOS activity and viability are not correlated in these cell lines, suggesting that the connection between OXPHOS and cancer cell survival is dependent on the specific cell type and conditions.

DOI: https://doi.org/10.1038/s41598-023-37677-x
J Transl Med. 2023 Jul 05. 21(1): 441

Mitochondria in endothelial cells angiogenesis and function: current understanding and future perspectives.

Zhen Luo, Jianbo Yao, Zhe Wang, Jianxiong Xu.

  Endothelial cells (ECs) angiogenesis is the process of sprouting new vessels from the existing ones, playing critical roles in physiological and pathological processes such as wound healing, placentation, ischemia/reperfusion, cardiovascular diseases and cancer metastasis. Although mitochondria are not the major sites of energy source in ECs, they function as important biosynthetic and signaling hubs to regulate ECs metabolism and adaptations to local environment, thus affecting ECs migration, proliferation and angiogenic process. The understanding of the importance and potential mechanisms of mitochondria in regulating ECs metabolism, function and the process of angiogenesis has developed in the past decades. Thus, in this review, we discuss the current understanding of mitochondrial proteins and signaling molecules in ECs metabolism, function and angiogeneic signaling, to provide new and therapeutic targets for treatment of diverse cardiovascular and angiogenesis-dependent diseases.

Keywords:  Angiogenesis; Cardiovascular diseases; Endothelial cells; Mitochondrial protein; Signaling pathways

DOI:  https://doi.org/10.1186/s12967-023-04286-1
Cells. 2023 May 17. pii: 1409. [Epub ahead of print]12(10):

The Haves and Have-Nots: The Mitochondrial Permeability Transition Pore across Species.

Elena Frigo, Ludovica Tommasin, Giovanna Lippe, Michela Carraro, Paolo Bernardi.

  The demonstration that F1FO (F)-ATP synthase and adenine nucleotide translocase (ANT) can form Ca2+-activated, high-conductance channels in the inner membrane of mitochondria from a variety of eukaryotes led to renewed interest in the permeability transition (PT), a permeability increase mediated by the PT pore (PTP). The PT is a Ca2+-dependent permeability increase in the inner mitochondrial membrane whose function and underlying molecular mechanisms have challenged scientists for the last 70 years. Although most of our knowledge about the PTP comes from studies in mammals, recent data obtained in other species highlighted substantial differences that could be perhaps attributed to specific features of F-ATP synthase and/or ANT. Strikingly, the anoxia and salt-tolerant brine shrimp Artemia franciscana does not undergo a PT in spite of its ability to take up and store Ca2+ in mitochondria, and the anoxia-resistant Drosophila melanogaster displays a low-conductance, selective Ca2+-induced Ca2+ release channel rather than a PTP. In mammals, the PT provides a mechanism for the release of cytochrome c and other proapoptotic proteins and mediates various forms of cell death. In this review, we cover the features of the PT (or lack thereof) in mammals, yeast, Drosophila melanogaster, Artemia franciscana and Caenorhabditis elegans, and we discuss the presence of the intrinsic pathway of apoptosis and of other forms of cell death. We hope that this exercise may help elucidate the function(s) of the PT and its possible role in evolution and inspire further tests to define its molecular nature.

Keywords:  ATP synthase; adenine nucleotide translocase; calcium signaling; cell death; channels; mitochondria; permeability transition

DOI:  https://doi.org/10.3390/cells12101409
Kidney Int. 2023 Jul 01. pii: S0085-2538(23)00477-5. [Epub ahead of print]

Inhibition of pyruvate dehydrogenase kinase 4 ameliorates kidney ischemia-reperfusion injury by reducing succinate accumulation during ischemia and preserving mitochondrial function during reperfusion.

Chang Joo Oh, Min-Ji Kim, Ji-Min Lee, Dong Hun Kim, Il-Young Kim, Sanghee Park, Yeongmin Kim, Kyung-Bok Lee, Sang-Hee Lee, Chae Won Lim, Myeongjin Kim, Jung-Yi Lee, Haushabhau S Pagire, Suvarna H Pagire, Myung Ae Bae, Dipanjan Chanda, Themis Thoudam, Ah Reum Khang, Robert A Harris, Jin Hee Ahn, Jae-Han Jeon, In-Kyu Lee.

  Ischemia-reperfusion (IR) injury, a leading cause of acute kidney injury (AKI), is still without effective therapies. Succinate accumulation during ischemia followed by its oxidation during reperfusion leads to excessive reactive oxygen species (ROS) and severe kidney damage. Consequently, the targeting of succinate accumulation may represent a rational approach to the prevention of IR-induced kidney injury. Since ROS are generated primarily in mitochondria, which are abundant in the proximal tubule of the kidney, we explored the role of pyruvate dehydrogenase kinase 4 (PDK4), a mitochondrial enzyme, in IR-induced kidney injury using proximal tubule cell-specific Pdk4 knockout (Pdk4ptKO) mice. Knockout or pharmacological inhibition of PDK4 ameliorated IR-induced kidney damage. Succinate accumulation during ischemia, which is responsible for mitochondrial ROS production during reperfusion, was reduced by PDK4 inhibition. PDK4 deficiency established conditions prior to ischemia resulting in less succinate accumulation, possibly because of a reduction in electron flow reversal in complex II, which provides electrons for the reduction of fumarate to succinate by succinate dehydrogenase during ischemia. The administration of dimethyl succinate a cell-permeable form of the Complex II inhibitor malonate, attenuated the beneficial effects of PDK4 deficiency, suggesting that the kidney-protective effect is succinate-dependent. Finally, genetic or pharmacological inhibition of PDK4 prevented IR-induced mitochondrial damage in mice and normalized mitochondrial function in an in vitro model of IR injury. Thus, inhibition of PDK4 represents a novel means of preventing IR-induced kidney injury, and involves the inhibition of ROS-induced kidney-toxicity through reduction in succinate accumulation and mitochondrial dysfunction.

Keywords:  Acute renal failure; ischemia-reperfusion injury; mitochondrial dysfunction; pyruvate dehydrogenase kinase 4; reactive oxygen species; succinate accumulation

DOI:  https://doi.org/10.1016/j.kint.2023.06.022
Cell Stress Chaperones. 2023 Jul 05.

Novel insights into the role of mitochondria in diabetic cardiomyopathy: molecular mechanisms and potential treatments.

Fumin Zhi, Qian Zhang, Li Liu, Xing Chang, Hongtao Xu.

  Diabetic cardiomyopathy describes decreased myocardial function in diabetic patients in the absence of other heart diseases such as myocardial ischemia and hypertension. Recent studies have defined numerous molecular interactions and signaling events that may account for deleterious changes in mitochondrial dynamics and functions influenced by hyperglycemic stress. A metabolic switch from glucose to fatty acid oxidation to fuel ATP synthesis, mitochondrial oxidative injury resulting from increased mitochondrial ROS production and decreased antioxidant capacity, enhanced mitochondrial fission and defective mitochondrial fusion, impaired mitophagy, and blunted mitochondrial biogenesis are major signatures of mitochondrial pathologies during diabetic cardiomyopathy. This review describes the molecular alterations underlying mitochondrial abnormalities associated with hyperglycemia and discusses their influence on cardiomyocyte viability and function. Based on basic research findings and clinical evidence, diabetic treatment standards and their impact on mitochondrial function, as well as mitochondria-targeted therapies of potential benefit for diabetic cardiomyopathy patients, are also summarized.

Keywords:  Diabetic cardiomyopathy; Mitochondria; Mitochondrial biogenesis; Mitochondrial fission/fusion; Mitochondrial metabolism; Mitochondrial oxidative stress; Mitophagy

DOI:  https://doi.org/10.1007/s12192-023-01361-w
Cardiovasc Res. 2023 Jul 04. pii: cvad102. [Epub ahead of print]

Chronic SIRT1 supplementation in diabetic mice improves endothelial function by suppressing oxidative stress.

Kangmin Yang, Srividya Velagapudi, Alexander Akhmedov, Simon Kraler, Tetiana Lapikova-Bryhinska, Martin O Schmiady, Xiaoping Wu, Leiluo Geng, Giovanni G Camici, Aimin Xu, Thomas F Lüscher.

  AIMS: Enhancing SIRT1 activity exerts beneficial cardiovascular effects. In diabetes, plasma SIRT1 levels are reduced. We aimed to investigate the therapeutic potential of chronic recombinant murine SIRT1 (rmSIRT1) supplementation in diabetic mice (db/db) to alleviate endothelial and vascular dysfunction.METHODS AND RESULTS: Left-internal mammary arteries from patients undergoing coronary artery bypass grafting (CABG) with or without a diagnosis of diabetes were assayed for SIRT1 protein levels. Twelve-week-old male db/db mice and db/+ controls were treated with vehicle or rmSIRT1 i.p. for 4 weeks, after which carotid artery pulse wave velocity (PWV) and energy expenditure/activity were assessed by ultrasound and metabolic cages, respectively. Aorta, carotid, and mesenteric arteries were isolated to determine endothelial and vascular function using myograph system.Arteries obtained from diabetic patients had significantly lower levels of SIRT1 than non-diabetic controls. Likewise, aortic SIRT1 levels were reduced in db/db mice compared to db/+ mice, while rmSIRT1 supplementation restored it to levels of controls. Mice treated with rmSIRT1 displayed increased physical activity and improved vascular compliance as reflected by reduced PWV and attenuated collagen deposition. Aorta of rmSIRT1-treated mice exhibited increased endothelial nitric oxide (eNOS) activity, and endothelium-dependent contractions of their carotid arteries were significantly decreased, while mesenteric resistance arteries showed preserved hyperpolarization. Ex-vivo incubation with reactive oxygen species scavenger (ROS) Tiron and NADPH oxidase inhibitor apocynin revealed that rmSIRT1 preserved vascular function by supressing NADPH oxidase (NOX)-related ROS sythesis. Chronic rmSIRT1 treatment suppressed the expression of NOX-1 and NOX-4, in line with a reduction in aortic protein carbonylation and plasma nitrotyrosine levels.
CONCLUSIONS: In diabetic conditions, arterial SIRT1 is reduced. Chronic rmSIRT1 supplementation improves endothelial function and vascular compliance by enhancing eNOS activity and suppresses NOX-related oxidative stress. Thus, SIRT1 supplementation may be a novel therapeutic strategy preventing diabetic vascular disease.
TRANSLATIONAL PERSPECTIVE: The growing burden of obesity and diabetes drives an increasing portion of atherosclerotic cardiovascular disease, and represents a major challenge to public health. Herein, we probe the efficacy of recombinant SIRT1 supplementation to preserve endothelial function and vascular compliance during diabetic conditions. Notably, SIRT1 levels were diminished in diabetic arteries of mice and humans alike, while recombinant SIRT1 delivery improved energy metabolism and vascular function by suppressing oxidative stress. Our study deepens mechanistic insights into the vasculo-protective effects conferred by recombinant SIRT1 supplementation and opens therapeutic avenues to mitigate vascular disease in diabetic patients.

Keywords:  Endothelial Function; NOX1-NOX4; Nitric Oxide; Reactive Oxidative Species; Sirtuin

DOI:  https://doi.org/10.1093/cvr/cvad102
Eur J Clin Invest. 2023 Jul 04. e14054

Heart failure in patients is associated with downregulation of mitochondrial quality control genes.

T Svagusa, S Sikiric, M Milavic, A Sepac, S Seiwerth, D Milicic, H Gasparovic, B Biocina, I Rudez, Z Sutlic, S Manola, J Varvodic, M Udovicic, M Urlic, S Ivankovic, S Plestina, F Paic, A Kulic, P Bakovic, F Sedlic.

  BACKGROUND: Mitochondrial dysfunction is one of key factors causing heart failure. We performed a comprehensive analysis of expression of mitochondrial quality control (MQC) genes in heart failure.METHODS: Myocardial samples were obtained from patients with ischemic and dilated cardiomyopathy in a terminal stage of heart failure and donors without heart disease. Using quantitative real-time PCR, we analysed a total of 45 MQC genes belonging to mitochondrial biogenesis, fusion-fission balance, mitochondrial unfolded protein response (UPRmt), translocase of the inner membrane (TIM) and mitophagy. Protein expression was analysed by ELISA and immunohistochemistry.
RESULTS: The following genes were downregulated in ischemic and dilated cardiomyopathy: COX1, NRF1, TFAM, SIRT1, MTOR, MFF, DNM1L, DDIT3, UBL5, HSPA9, HSPE1, YME1L, LONP1, SPG7, HTRA2, OMA1, TIMM23, TIMM17A, TIMM17B, TIMM44, PAM16, TIMM22, TIMM9, TIMM10, PINK1, PARK2, ROTH1, PARL, FUNDC1, BNIP3, BNIP3L, TPCN2, LAMP2, MAP1LC3A and BECN1. Moreover, MT-ATP8, MFN2, EIF2AK4 and ULK1 were downregulated in heart failure from dilated, but not ischemic cardiomyopathy. VDAC1 and JUN were only genes that exhibited significantly different expression between ischemic and dilated cardiomyopathy. Expression of PPARGC1, OPA1, JUN, CEBPB, EIF2A, HSPD1, TIMM50 and TPCN1 was not significantly different between control and any form of heart failure. TOMM20 and COX proteins were downregulated in ICM and DCM.
CONCLUSIONS: Heart failure in patients with ischemic and dilated cardiomyopathy is associated with downregulation of large number of UPRmt, mitophagy, TIM and fusion-fission balance genes. This indicates multiple defects in MQC and represents one of potential mechanisms underlying mitochondrial dysfunction in patients with heart failure.

Keywords:  MQC; TIM; UPRmt; heart; mitochondria; mitophagy

DOI:  https://doi.org/10.1111/eci.14054
Biochim Biophys Acta Mol Basis Dis. 2023 Jul 04. pii: S0925-4439(23)00168-0. [Epub ahead of print] 166802

Mitochondrial dysfunction in the offspring of obese mothers and it's transmission through damaged oocyte mitochondria: Integration of mechanisms.

A L Elías-López, O Vázquez-Mena, A N Sferruzzi-Perri.

  In vivo and in vitro studies demonstrate that mitochondria in the oocyte, are susceptible to damage by suboptimal pre/pregnancy conditions, such as obesity. These suboptimal conditions have been shown to induce mitochondrial dysfunction (MD) in multiple tissues of the offspring, suggesting that mitochondria of oocytes that pass from mother to offspring, can carry information that can programme mitochondrial and metabolic dysfunction of the next generation. They also suggest that transmission of MD could increase the risk of obesity and other metabolic diseases in the population inter- and trans-generationally. In this review, we examined whether MD observed in offspring tissues of high energetic demand, is the result of the transmission of damaged mitochondria from obese mothers' oocytes to the offspring. The contribution of genome-independent mechanisms (namely mitophagy) in this transmission were also explored. Finally, potential interventions aimed at improving oocyte/embryo health were investigated, to see if they may provide an opportunity to halter the generational effects of MD.

Keywords:  Downregulated mitophagy; Maternal obesity; Metabolic dysfunction; Milpa diet; Mitochondrial metabolic dysfunction; Mitophagy; Offspring mitochondrial dysfunction; Oocyte mitochondria; Preconceptional interventions; Traditional diet

DOI:  https://doi.org/10.1016/j.bbadis.2023.166802
Bone. 2023 Jul 04. pii: S8756-3282(23)00169-2. [Epub ahead of print] 116836

miR181a/b-1 controls osteocyte metabolism and mechanical properties independently of bone morphology.

Jihee Yoon, Serra Kaya, Gen Matsumae, Neha Dole, Tamara Alliston.

  Bone derives its ability to resist fracture from bone mass and quality concurrently; however, many questions about the molecular mechanisms controlling bone quality remain unanswered, limiting the development of diagnostics and therapeutics. Despite the increasing evidence on the importance of miR181a/b-1 in bone homeostasis and disease, whether and how osteocyte-intrinsic miR181a/b-1 controls bone quality remains elusive. Osteocyte-intrinsic deletion of miR181a/b-1 in osteocytes in vivo resulted in compromised overall bone mechanical behavior in both sexes, although the parameters affected by miR181a/b-1 varied distinctly based on sex. Furthermore, impaired fracture resistance in both sexes was unexplained by cortical bone morphology, which was altered in female mice and intact in male mice with miR181a/b-1-deficient osteocytes. The role of miR181a/b-1 in the regulation of osteocyte metabolism was apparent in bioenergetic testing of miR181a/b-1-deficient OCY454 osteocyte-like cells and transcriptomic analysis of cortical bone from mice with osteocyte-intrinsic ablation of miR181a/b-1. Altogether, this study demonstrates the sexually dimorphic regulation of cortical bone morphology and mechanical properties and the control of osteocyte bioenergetics by miR181a/b-1, hinting at the role of osteocyte metabolism in the regulation of mechanical behavior.

Keywords:  Bone quality; Cell metabolism; Mechanical behavior; Microrna; Osteocyte

DOI:  https://doi.org/10.1016/j.bone.2023.116836
J Clin Invest. 2023 07 03. pii: e162479. [Epub ahead of print]133(13):

CDK4-E2F3 signals enhance oxidative skeletal muscle fiber numbers and function to affect myogenesis and metabolism.

Young Jae Bahn, Hariom Yadav, Paolo Piaggi, Brent S Abel, Oksana Gavrilova, Danielle A Springer, Ioannis Papazoglou, Patricia M Zerfas, Monica C Skarulis, Alexandra C McPherron, Sushil G Rane.

  Understanding how skeletal muscle fiber proportions are regulated is vital to understanding muscle function. Oxidative and glycolytic skeletal muscle fibers differ in their contractile ability, mitochondrial activity, and metabolic properties. Fiber-type proportions vary in normal physiology and disease states, although the underlying mechanisms are unclear. In human skeletal muscle, we observed that markers of oxidative fibers and mitochondria correlated positively with expression levels of PPARGC1A and CDK4 and negatively with expression levels of CDKN2A, a locus significantly associated with type 2 diabetes. Mice expressing a constitutively active Cdk4 that cannot bind its inhibitor p16INK4a, a product of the CDKN2A locus, were protected from obesity and diabetes. Their muscles exhibited increased oxidative fibers, improved mitochondrial properties, and enhanced glucose uptake. In contrast, loss of Cdk4 or skeletal muscle-specific deletion of Cdk4's target, E2F3, depleted oxidative myofibers, deteriorated mitochondrial function, and reduced exercise capacity, while increasing diabetes susceptibility. E2F3 activated the mitochondrial sensor PPARGC1A in a Cdk4-dependent manner. CDK4, E2F3, and PPARGC1A levels correlated positively with exercise and fitness and negatively with adiposity, insulin resistance, and lipid accumulation in human and rodent muscle. All together, these findings provide mechanistic insight into regulation of skeletal muscle fiber-specification that is of relevance to metabolic and muscular diseases.

Keywords:  Metabolism; Mitochondria; Muscle Biology; Skeletal muscle

DOI:  https://doi.org/10.1172/JCI162479
Cardiovasc Res. 2023 Jul 01. pii: cvad100. [Epub ahead of print]

Cardiac lipid metabolism, mitochondrial function and heart failure.

Lorenzo Da Dalt, Ainara G Cabodevilla, Ira J Goldberg, Giuseppe Danilo Norata.

  A fine balance between uptake, storage and the use of high energy fuels, like lipids, is crucial in the homeostasis of different metabolic tissues. Nowhere is this balance more important and more precarious than in the heart. This highly energy demanding muscle normally oxidizes almost all the available substrates to generate energy, with fatty acids being the preferred source under physiological conditions. In patients with cardiomyopathies and heart failure, changes in the main energetic substrate are observed; these hearts often prefer to utilize glucose rather than oxidizing fatty acids. An imbalance between uptake and oxidation of fatty acid can result in cellular lipid accumulation and cytotoxicity. In this review we will focus on the sources and uptake pathways used to direct fatty acids to cardiomyocytes. We will then discuss the intracellular machinery used to either store or oxidize these lipids and explain how disruptions in homeostasis can lead to mitochondrial dysfunction and heart failure. Moreover, we will also discuss the role of cholesterol accumulation in cardiomyocytes. Our discussion will attempt to weave in vitro experiments and in vivo data from mice and humans and use several human diseases to illustrate metabolism gone haywire as a cause of or accomplice to cardiac dysfunction.

Keywords:  Cholesterol and Heart failure; Lipids; Lipoprotein

DOI:  https://doi.org/10.1093/cvr/cvad100
J Biol Chem. 2023 Jun 30. pii: S0021-9258(23)02029-X. [Epub ahead of print] 105001

Respiratory complex I in mitochondrial membrane catalyzes oversized ubiquinones.

Ryo Ikunishi, Ryohei Otani, Takahiro Masuya, Kyoko Shinzawa-Itoh, Tomoo Shiba, Masatoshi Murai, Hideto Miyoshi.

  NADH-ubiquinone (UQ) oxidoreductase (complex I) couples electron transfer from NADH to UQ with proton translocation in its membrane part. The UQ reduction step is key to triggering proton translocation. Structural studies have identified a long, narrow, tunnel-like cavity within complex I, through which UQ may access a deep reaction site. To elucidate the physiological relevance of this UQ-accessing tunnel, we previously investigated whether a series of oversized UQs (OS-UQs), whose tail moiety is too large to enter and transit the narrow tunnel, can be catalytically reduced by complex I using the native enzyme in bovine heart submitochondrial particles (SMPs) and the isolated enzyme reconstituted into liposomes. Nevertheless, the physiological relevance remained unclear because some amphiphilic OS-UQs were reduced in SMPs but not in proteoliposomes, and investigation of extremely hydrophobic OS-UQs was not possible in SMPs. To uniformly assess the electron transfer activities of all OS-UQs with the native complex I, here we present a new assay system using SMPs, which were fused with liposomes incorporating OS-UQ and supplemented with a parasitic quinol oxidase to recycle reduced OS-UQ. In this system, all OS-UQs tested were reduced by the native enzyme, and the reduction was coupled with proton translocation. This finding does not support the canonical tunnel model. We propose that the UQ reaction cavity is flexibly open in the native enzyme to allow OS-UQs to access the reaction site, but their access is obstructed in the isolated enzyme as the cavity is altered by detergent-solubilizing from the mitochondrial membrane.

Keywords:  bioenergetics; chemical biology; complex I; respiratory enzymes; ubiquinone

DOI:  https://doi.org/10.1016/j.jbc.2023.105001
Nucleic Acids Res. 2023 Jul 05. pii: gkad562. [Epub ahead of print]

Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation.

Carlos Guzman, Sascha Duttke, Yixin Zhu, Camila De Arruda Saldanha, Nicholas L Downes, Christopher Benner, Sven Heinz.

Cis-regulatory elements (CREs) can be classified by the shapes of their transcription start site (TSS) profiles, which are indicative of distinct regulatory mechanisms. Massively parallel reporter assays (MPRAs) are increasingly being used to study CRE regulatory mechanisms, yet the degree to which MPRAs replicate individual endogenous TSS profiles has not been determined. Here, we present a new low-input MPRA protocol (TSS-MPRA) that enables measuring TSS profiles of episomal reporters as well as after lentiviral reporter chromatinization. To sensitively compare MPRA and endogenous TSS profiles, we developed a novel dissimilarity scoring algorithm (WIP score) that outperforms the frequently used earth mover's distance on experimental data. Using TSS-MPRA and WIP scoring on 500 unique reporter inserts, we found that short (153 bp) MPRA promoter inserts replicate the endogenous TSS patterns of ∼60% of promoters. Lentiviral reporter chromatinization did not improve fidelity of TSS-MPRA initiation patterns, and increasing insert size frequently led to activation of extraneous TSS in the MPRA that are not active in vivo. We discuss the implications of our findings, which highlight important caveats when using MPRAs to study transcription mechanisms. Finally, we illustrate how TSS-MPRA and WIP scoring can provide novel insights into the impact of transcription factor motif mutations and genetic variants on TSS patterns and transcription levels.

DOI: https://doi.org/10.1093/nar/gkad562
Cells. 2023 May 15. pii: 1394. [Epub ahead of print]12(10):

Newly Developed Di-Block Copolymer-Based Cell Membrane Stabilizers Protect Mouse Coronary Artery Endothelial Cells against Hypoxia/Reoxygenation Injury.

Zhu Li, Mukesh K Gupta, Matthew B Barajas, Takuro Oyama, Craig L Duvall, Matthias L Riess.

  Reperfusion after ischemia causes additional cellular damage, known as reperfusion injury, for which there is still no effective remedy. Poloxamer (P)188, a tri-block copolymer-based cell membrane stabilizer (CCMS), has been shown to provide protection against hypoxia/reoxygenation (HR) injury in various models by reducing membrane leakage and apoptosis and improving mitochondrial function. Interestingly, substituting one of its hydrophilic poly-ethylene oxide (PEO) blocks with a (t)ert-butyl terminus added to the hydrophobic poly-propylene oxide (PPO) block yields a di-block compound (PEO-PPOt) that interacts better with the cell membrane lipid bi-layer and exhibits greater cellular protection than the gold standard tri-block P188 (PEO75-PPO30-PEO75). For this study, we custom-made three different new di-blocks (PEO113-PPO10t, PEO226-PPO18t and PEO113-PPO20t) to systemically examine the effects of the length of each polymer block on cellular protection in comparison to P188. Cellular protection was assessed by cell viability, lactate dehydrogenase release, and uptake of FM1-43 in mouse artery endothelial cells (ECs) following HR injury. We found that di-block CCMS were able to provide the same or better EC protection than P188. Our study provides the first direct evidence that custom-made di-block CCMS can be superior to P188 in improving EC membrane protection, raising their potential in treating cardiac reperfusion injury.

Keywords:  LDH; P188; apoptosis; heart; ischemia reperfusion injury; murine; myocardial; poloxamer; tert-butyl; tri-block

DOI:  https://doi.org/10.3390/cells12101394
Nat Cell Biol. 2023 Jul 03.

Arf1 coordinates fatty acid metabolism and mitochondrial homeostasis.

Ludovic Enkler, Viktoria Szentgyörgyi, Mirjam Pennauer, Cristina Prescianotto-Baschong, Isabelle Riezman, Aneta Wiesyk, Reut Ester Avraham, Martin Spiess, Einat Zalckvar, Roza Kucharczyk, Howard Riezman, Anne Spang.

Lipid mobilization through fatty acid β-oxidation is a central process essential for energy production during nutrient shortage. In yeast, this catabolic process starts in the peroxisome from where β-oxidation products enter mitochondria and fuel the tricarboxylic acid cycle. Little is known about the physical and metabolic cooperation between these organelles. Here we found that expression of fatty acid transporters and of the rate-limiting enzyme involved in β-oxidation is decreased in cells expressing a hyperactive mutant of the small GTPase Arf1, leading to an accumulation of fatty acids in lipid droplets. Consequently, mitochondria became fragmented and ATP synthesis decreased. Genetic and pharmacological depletion of fatty acids phenocopied the arf1 mutant mitochondrial phenotype. Although β-oxidation occurs in both mitochondria and peroxisomes in mammals, Arf1's role in fatty acid metabolism is conserved. Together, our results indicate that Arf1 integrates metabolism into energy production by regulating fatty acid storage and utilization, and presumably organelle contact sites.

DOI: https://doi.org/10.1038/s41556-023-01180-2
Curr Opin Struct Biol. 2023 Jun 29. pii: S0959-440X(23)00119-7. [Epub ahead of print]81 102645

Modeling conformational states of proteins with AlphaFold.

D Sala, F Engelberger, H S Mchaourab, J Meiler.

  Many proteins exert their function by switching among different structures. Knowing the conformational ensembles affiliated with these states is critical to elucidate key mechanistic aspects that govern protein function. While experimental determination efforts are still bottlenecked by cost, time, and technical challenges, the machine-learning technology AlphaFold showed near experimental accuracy in predicting the three-dimensional structure of monomeric proteins. However, an AlphaFold ensemble of models usually represents a single conformational state with minimal structural heterogeneity. Consequently, several pipelines have been proposed to either expand the structural breadth of an ensemble or bias the prediction toward a desired conformational state. Here, we analyze how those pipelines work, what they can and cannot predict, and future directions.

Keywords:  AlphaFold; Conformational changes; Conformational ensemble; Protein structures

DOI:  https://doi.org/10.1016/j.sbi.2023.102645
Mitochondrion. 2023 Jul 05. pii: S1567-7249(23)00056-9. [Epub ahead of print]

N-acetylcysteine modulates rotenone-induced mitochondrial Complex I dysfunction in THP-1 cells.

Winston Tse-Hou Kwok, Haejin Angela Kwak, Ana Cristina Andreazza.

  Mitochondrial Complex I dysfunction and oxidative stress have been part of the pathophysiology of several diseases ranging from mitochondrial disease to chronic diseases such as diabetes, mood disorders and Parkinson's Disease. Nonetheless, to investigate the potential of mitochondria-targeted therapeutic strategies for these conditions, there is a need further our understanding on how cells respond and adapt in the presence of Complex I dysfunction. In this study, we used low doses of rotenone, a classical inhibitor of mitochondrial complex I, to mimic peripheral mitochondrial dysfunction in THP-1 cells, a human monocytic cell line, and explored the effects of N-acetylcysteine on preventing this rotenone-induced mitochondrial dysfunction. Our results show that in THP-1 cells, rotenone exposure led to increases in mitochondrial superoxide, levels of cell-free mitochondrial DNA, and protein levels of the NDUFS7 subunit. N-acetylcysteine (NAC) pre-treatment ameliorated the rotenone-induced increase of cell-free mitochondrial DNA and NDUFS7 protein levels, but not mitochondrial superoxide. Furthermore, rotenone exposure did not affect protein levels of the NDUFV1 subunit but induced NDUFV1 glutathionylation. In summary, NAC may help to mitigate the effects of rotenone on Complex I and preserve the normal function of mitochondria in THP-1 cells.

Keywords:  Cell model; Complex I; Mitochondrial; N-acetylcysteine; Reactive oxygen species; Rotenone

DOI:  https://doi.org/10.1016/j.mito.2023.07.001
Front Cell Neurosci. 2023 ;17 1132241

Alterations in synapses and mitochondria induced by acute or chronic intermittent hypoxia in the pre-Bötzinger complex of rats: an ultrastructural triple-labeling study with immunocytochemistry and histochemistry.

Junjun Kang, Naining Lu, Shoujing Yang, Baolin Guo, Yuanyuan Zhu, Shengxi Wu, Xiaofeng Huang, Margaret T T Wong-Riley, Ying-Ying Liu.

  Introduction: The pre-Bötzinger complex (pre-BötC), a kernel of inspiratory rhythmogenesis, is a heterogeneous network with excitatory glutamatergic and inhibitory GABAergic and glycinergic neurons. Inspiratory rhythm generation relies on synchronous activation of glutamatergic neuron, whilst inhibitory neurons play a critical role in shaping the breathing pattern, endowing the rhythm with flexibility in adapting to environmental, metabolic, and behavioral needs. Here we report ultrastructural alterations in excitatory, asymmetric synapses (AS) and inhibitory, symmetric synapses (SS), especially perforated synapses with discontinuous postsynaptic densities (PSDs) in the pre-BötC in rats exposed to daily acute intermittent hypoxia (dAIH) or chronic (C) IH.Methods: We utilized for the first time a combination of somatostatin (SST) and neurokinin 1 receptor (NK1R) double immunocytochemistry with cytochrome oxidase histochemistry, to reveal synaptic characteristics and mitochondrial dynamic in the pre-BötC.
Results: We found perforated synapses with synaptic vesicles accumulated in distinct pools in apposition to each discrete PSD segments. dAIH induced significant increases in the PSD size of macular AS, and the proportion of perforated synapses. AS were predominant in the dAIH group, whereas SS were in a high proportion in the CIH group. dAIH significantly increased SST and NK1R expressions, whereas CIH led to a decrease. Desmosome-like contacts (DLC) were characterized for the first time in the pre-BötC. They were distributed alongside of synapses, especially SS. Mitochondria appeared in more proximity to DLC than synapses, suggestive of a higher energy demand of the DLC. Findings of single spines with dual AS and SS innervation provide morphological evidence of excitation-inhibition interplay within a single spine in the pre-BötC. In particular, we characterized spine-shaft microdomains of concentrated synapses coupled with mitochondrial positioning that could serve as a structural basis for synchrony of spine-shaft communication. Mitochondria were found within spines and ultrastructural features of mitochondrial fusion and fission were depicted for the first time in the pre-BötC.
Conclusion: We provide ultrastructural evidence of excitation-inhibition synapses in shafts and spines, and DLC in association with synapses that coincide with mitochondrial dynamic in their contribution to respiratory plasticity in the pre-BötC.

Keywords:  desmosome-like contact; intermittent hypoxia; mitochondria; neuroplasticity; pre-Bötzinger complex; synapse; ultrastructure

DOI:  https://doi.org/10.3389/fncel.2023.1132241
J Alzheimers Dis. 2023 Jun 27.

The Novel Role of Mitochondrial Citrate Synthase and Citrate in the Pathophysiology of Alzheimer's Disease.

Neeraj Chhimpa, Neha Singh, Nikkita Puri, Hanuman Prasad Kayath.

  Citrate synthase is a key mitochondrial enzyme that utilizes acetyl-CoA and oxaloacetate to form citrate in the mitochondrial membrane, which participates in energy production in the TCA cycle and linked to the electron transport chain. Citrate transports through a citrate malate pump and synthesizes acetyl-CoA and acetylcholine (ACh) in neuronal cytoplasm. In a mature brain, acetyl-CoA is mainly utilized for ACh synthesis and is responsible for memory and cognition. Studies have shown low citrate synthase in different regions of brain in Alzheimer's disease (AD) patients, which reduces mitochondrial citrate, cellular bioenergetics, neurocytoplasmic citrate, acetyl-CoA, and ACh synthesis. Reduced citrate mediated low energy and favors amyloid-β (Aβ) aggregation. Citrate inhibits Aβ25-35 and Aβ1-40 aggregation in vitro. Hence, citrate can be a better therapeutic option for AD by improving cellular energy and ACh synthesis, and inhibiting Aβ aggregation, which prevents tau hyperphosphorylation and glycogen synthase kinase-3 beta. Therefore, we need to study if citrate reverses Aβ deposition by balancing mitochondrial energy pathway and neurocytoplasmic ACh production. Furthermore, in AD's silent phase pathophysiology, when neuronal cells are highly active, they shift ATP utilization from oxidative phosphorylation to glycolysis and prevent excessive generation of hydrogen peroxide and reactive oxygen species (oxidative stress) as neuroprotective action, which upregulates glucose transporter-3 (GLUT3) and pyruvate dehydrogenase kinase-3 (PDK3). PDK3 inhibits pyruvate dehydrogenase, which decreases mitochondrial-acetyl-CoA, citrate, and cellular bioenergetics, and decreases neurocytoplasmic citrate, acetyl-CoA, and ACh formation, thus initiating AD pathophysiology. Therefore, GLUT3 and PDK3 can be biomarkers for silent phase of AD.

Keywords:  APOEɛ4; Acetyl-CoA; Alzheimer’s disease; GLUT3; acetylcholine; amyloid-beta; cellular bioenergetics; citrate; citrate synthase

DOI:  https://doi.org/10.3233/JAD-220514
J Am Chem Soc. 2023 Jul 06.

Human Small Heat Shock Protein B8 Inhibits Protein Aggregation without Affecting the Native Folding Process.

Dhawal Choudhary, Laura Mediani, Mario J Avellaneda, Sveinn Bjarnason, Simon Alberti, Edgar E Boczek, Pétur O Heidarsson, Alessandro Mossa, Serena Carra, Sander J Tans, Ciro Cecconi.

Small Heat Shock Proteins (sHSPs) are key components of our Protein Quality Control system and are thought to act as reservoirs that neutralize irreversible protein aggregation. Yet, sHSPs can also act as sequestrases, promoting protein sequestration into aggregates, thus challenging our understanding of their exact mechanisms of action. Here, we employ optical tweezers to explore the mechanisms of action of the human small heat shock protein HSPB8 and its pathogenic mutant K141E, which is associated with neuromuscular disease. Through single-molecule manipulation experiments, we studied how HSPB8 and its K141E mutant affect the refolding and aggregation processes of the maltose binding protein. Our data show that HSPB8 selectively suppresses protein aggregation without affecting the native folding process. This anti-aggregation mechanism is distinct from previous models that rely on the stabilization of unfolded polypeptide chains or partially folded structures, as has been reported for other chaperones. Rather, it appears that HSPB8 selectively recognizes and binds to aggregated species formed at the early stages of aggregation, preventing them from growing into larger aggregated structures. Consistently, the K141E mutation specifically targets the affinity for aggregated structures without impacting native folding, and hence impairs its anti-aggregation activity.

DOI: https://doi.org/10.1021/jacs.3c02022
EMBO Rep. 2023 Jul 04. e57499

ER-mitochondria contacts and cholesterol metabolism are disrupted by disease-associated tau protein.

Leonora Szabo, Nadia Cummins, Paolo Paganetti, Alex Odermatt, Andreas Papassotiropoulos, Celeste Karch, Jürgen Götz, Anne Eckert, Amandine Grimm.

  Abnormal tau protein impairs mitochondrial function, including transport, dynamics, and bioenergetics. Mitochondria interact with the endoplasmic reticulum (ER) via mitochondria-associated ER membranes (MAMs), which coordinate and modulate many cellular functions, including mitochondrial cholesterol metabolism. Here, we show that abnormal tau loosens the association between the ER and mitochondria in vivo and in vitro. Especially, ER-mitochondria interactions via vesicle-associated membrane protein-associated protein (VAPB)-protein tyrosine phosphatase-interacting protein 51 (PTPIP51) are decreased in the presence of abnormal tau. Disruption of MAMs in cells with abnormal tau alters the levels of mitochondrial cholesterol and pregnenolone, indicating that conversion of cholesterol into pregnenolone is impaired. Opposite effects are observed in the absence of tau. Besides, targeted metabolomics reveals overall alterations in cholesterol-related metabolites by tau. The inhibition of GSK3β decreases abnormal tau hyperphosphorylation and increases VAPB-PTPIP51 interactions, restoring mitochondrial cholesterol and pregnenolone levels. This study is the first to highlight a link between tau-induced impairments in the ER-mitochondria interaction and cholesterol metabolism.

Keywords:  GSK3β; cholesterol; endoplasmic reticulum; mitochondria; tau protein

DOI:  https://doi.org/10.15252/embr.202357499