Impaired Oxidative Phosphorylation Research Articles

GTPBP8 plays a role in mitoribosome formation in human mitochondria

Mitochondrial gene expression relies on mitoribosomes to translate mitochondrial mRNAs. The biogenesis of mitoribosomes is an intricate process involving multiple assembly factors. Among these factors, GTP-binding proteins (GTPBPs) play important roles. In bacterial systems, numerous GTPBPs are required for ribosome subunit maturation, with EngB being a GTPBP involved in the ribosomal large subunit assembly. In this study, we focus on exploring the function of GTPBP8, the human homolog of EngB. We find that ablation of GTPBP8 leads to the inhibition of mitochondrial translation, resulting in significant impairment of oxidative phosphorylation. Structural analysis of mitoribosomes from GTPBP8 knock-out cells shows the accumulation of mitoribosomal large subunit assembly intermediates that are incapable of forming functional monosomes. Furthermore, fPAR-CLIP analysis reveals that GTPBP8 is an RNA-binding protein that interacts specifically with the mitochondrial ribosome large subunit 16 S rRNA. Our study highlights the role of GTPBP8 as a component of the mitochondrial gene expression machinery involved in mitochondrial large subunit maturation.

P-604 Underlying molecular mechanisms of Polycystic Ovary Syndrome and Morphology: focusing on the ovary

Abstract Study question What mechanisms are involved in the ovarian alterations of Polycystic Ovary Syndrome (PCOS) and Morphology? Summary answer PCOS patients associated a distinctive impairment of oxidative phosphorylation and dysregulated complement system in their follicular compartments not found in Polycystic Ovarian Morphology (PCOM) women. What is known already PCOS is a major cause of female infertility characterized by aberrant follicular development and hormonal imbalances leading to reproductive symptoms such as anovulation, poor oocyte quality, and hyperandrogenism. Likewise, PCOM is present in 16-25% of women with regular menses, but the clinical relevance of this ovarian phenotype and its correlation with PCOS remains unclear. Therefore, to establish the specific molecular mechanisms underlying the ovarian alterations in PCOS using High-throughput techniques will allow us to provide a better clinical management and a differential diagnosis with PCOM women. Study design, size, duration Cross-sectional molecular study including samples from 26 patients undergoing an IVF cycle between 2019-2022 at Hospital La Fe (Valencia, Spain). Eight women were considered PCOS, eleven PCOM and seven were control fertile patients. Granulosa cells (GCs) and follicular fluid (FF) samples were obtained during oocyte retrieval to perform RNA-seq and proteomic analysis, respectively. Participants/materials, setting, methods Women with two or more Rotterdam-criteria were considered as PCOS, while PCOM was defined by polycystic ovarian morphology on ultrasound with regular ovulatory cycles. RNA from GCs was obtained and sequenced using NextSeq500-Illumina to identify differentially expressed genes (DEGs) using DESeq2 (R-software). The proteome of FF samples was established by LC-MS/MS and differentially expressed proteins (DEPs) analyzed with limma package. Functional enrichment analysis of DEGs and DEPs (FDR< 0.05) was performed based on KEGG database. Main results and the role of chance RNA-seq data revealed clear distinctions between GCs from PCOS and control women, with 526 DEGs. By contrast, the transcriptomes of PCOS and PCOM were more similar, with only 34 DEGs. Notably, oxidative phosphorylation emerged as the most enriched pathway in both comparisons, showing a differential expression in PCOS. Indeed, 6 out of 12 shared DEGs (MT-TP, MT-ND4L, MT-ND2, MT-ATP8, MT-ND4, and MT-ATP6), were mitochondrial genes found upregulated in PCOS vs. PCOM and control samples. These findings suggest that granulosa cells from PCOS present a dysregulation in oxidative phosphorylation, potentially linked to alterations in those genes associated with the mitochondrial electron transport chain. The proteomic analysis showed 37 DEPs in the FF of PCOS compared with controls, being these DEPs mainly involved in the complement and coagulation cascades, PI3K-AKT pathway, oocyte meiosis and ferroptosis. Furthermore, the PCOS vs. PCOM analysis revealed 42 DEPs, significantly enriched also in complement and coagulation cascades, ferroptosis and glutathione metabolism. Interestingly, fifteen proteins were differentially expressed in both comparisons, with four of them related to the complement system (PROS1, C1S, C1QB and MASP2), suggesting that an alteration in this system at follicular fluid level might be one of the hallmarks of PCOS. Limitations, reasons for caution Further experimental studies are required to validate the specific changes identified in our study. Moreover, to establish how these alterations in the follicular compartments affect the oocytes of PCOS patients should also be explored. Wider implications of the findings Our study focused on specific mechanisms involved in PCOS ovarian alterations, providing new insights into potential biomarkers for an accurate diagnosis and management of this pathology. Dysregulations in oxidative phosphorylation and complement system might play a crucial role in PCOS multifactorial nature, contributing to impaired folliculogenesis and decreased oocyte quality. Trial registration number Not Applicable

Impaired polyamine metabolism causes behavioral and neuroanatomical defects in a mouse model of Snyder-Robinson syndrome.

Snyder-Robinson syndrome (SRS) is a rare X-linked recessive disorder caused by a mutation in the SMS gene, which encodes spermine synthase, and aberrant polyamine metabolism. SRS is characterized by intellectual disability, thin habitus, seizure, low muscle tone/hypotonia and osteoporosis. Progress towards understanding and treating SRS requires a model that recapitulates human gene variants and disease presentations. Here, we evaluated molecular and neurological presentations in the G56S mouse model, which carries a missense mutation in the Sms gene. The lack of SMS protein in the G56S mice resulted in increased spermidine/spermine ratio, failure to thrive, short stature and reduced bone density. They showed impaired learning capacity, increased anxiety, reduced mobility and heightened fear responses, accompanied by reduced total and regional brain volumes. Furthermore, impaired mitochondrial oxidative phosphorylation was evident in G56S cerebral cortex, G56S fibroblasts and Sms-null hippocampal cells, indicating that SMS may serve as a future therapeutic target. Collectively, our study establishes the suitability of the G56S mice as a preclinical model for SRS and provides a set of molecular and functional outcome measures that can be used to evaluate therapeutic interventions for SRS.

Baicalin Attenuates Diabetic Cardiomyopathy In Vivo and In Vitro by Inhibiting Autophagy and Cell Death through SENP1/SIRT3 Signaling Pathway Activation.

Diabetic heart damage can lead to cardiomyocyte death, which endangers human health. Baicalin (BAI) is a bioactive compound that plays an important role in cardiovascular diseases. Sentrin/SUMO-specific protease 1 (SENP1) regulates the de-small ubiquitin-like modifier (deSUMOylation) process of Sirtuin 3 (SIRT3) and plays a crucial role in regulating mitochondrial mass and preventing cell injury. Our hypothesis is that BAI regulates the deSUMOylation level of SIRT3 through SENP1 to enhance mitochondrial quality control and prevent cell death, ultimately improving diabetic cardiomyopathy (DCM). The protein expression of SENP1 decreased in cardiomyocytes induced by high glucose and in db/db mice. The cardioprotective effects of BAI were eliminated by silencing endogenous SENP1, while overexpression of SENP1 showed similar cardioprotective effects to those of BAI. Furthermore, Co-Immunoprecipitation (CO-IP) experiments showed that BAI's cardioprotective effect was due to the inhibition of the SUMOylation modification level of SIRT3 by SENP1. Inhibition of SENP1 expression resulted in an increase in SUMOylation of SIRT3. This led to increased acetylation of mitochondrial protein, accumulation of reactive oxygen species, impaired autophagy, impaired mitochondrial oxidative phosphorylation and increased cell death. None of these changes could be reversed by BAI. BAI improves DCM by promoting SIRT3 deSUMOylation through SENP1, restoring mitochondrial stability, and preventing the cell death of cardiomyocytes. This study proposes for the first time that SIRT3 SUMOylation modification is involved in the development of DCM, provides in vivo and in vitro data support that BAI inhibits cardiomyocyte ferroptosis and apoptosis in DCM through SENP1.

A positive feedback inhibition of isocitrate dehydrogenase 3β on paired-box gene 6 promotes Alzheimer-like pathology

Impaired brain glucose metabolism is an early indicator of Alzheimer’s disease (AD); however, the fundamental mechanism is unknown. In this study, we found a substantial decline in isocitrate dehydrogenase 3β (IDH3β) levels, a critical tricarboxylic acid cycle enzyme, in AD patients and AD-transgenic mice’s brains. Further investigations demonstrated that the knockdown of IDH3β induced oxidation-phosphorylation uncoupling, leading to reduced energy metabolism and lactate accumulation. The resulting increased lactate, a source of lactyl, was found to promote histone lactylation, thereby enhancing the expression of paired-box gene 6 (PAX6). As an inhibitory transcription factor of IDH3β, the elevated PAX6 in turn inhibited the expression of IDH3β, leading to tau hyperphosphorylation, synapse impairment, and learning and memory deficits resembling those seen in AD. In AD-transgenic mice, upregulating IDH3β and downregulating PAX6 were found to improve cognitive functioning and reverse AD-like pathologies. Collectively, our data suggest that impaired oxidative phosphorylation accelerates AD progression via a positive feedback inhibition loop of IDH3β-lactate-PAX6-IDH3β. Breaking this loop by upregulating IDH3β or downregulating PAX6 attenuates AD neurodegeneration and cognitive impairments.

Cytotoxicity and toxicoproteomics analysis of thiazolidinedione exposure in human-derived cardiomyocytes.

Thiazolidinediones (TZDs) (e.g. pioglitazone and rosiglitazone), known insulin sensitiser agents for type II diabetes mellitus, exhibit controversial effects on cardiac tissue. Despite consensus on their association with increased heart failure risk, limiting TZD use in diabetes management, the underlying mechanisms remain uncharacterised. Herein, we report a comprehensive in vitro investigation utilising a novel toxicoproteomics pipeline coupled with cytotoxicity assays in human adult cardiomyocytes to elucidate mechanistic insights into TZD cardiotoxicity. The cytotoxicity assay findings showed a significant loss of mitochondrial adenosine triphosphate production upon exposure to either TZD agents, which may underpin TZD cardiotoxicity. Our toxicoproteomics analysis revealed that mitochondrial dysfunction primarily stems from oxidative phosphorylation impairment, with distinct signalling mechanisms observed for both agents. The type of cell death differed strikingly between the two agents, with rosiglitazone exhibiting features of caspase-dependent apoptosis and pioglitazone implicating mitochondrial-mediated necroptosis, as evidenced by the protein upregulation in the phosphoglycerate mutase family 5-dynamin-related protein 1 axis. Furthermore, our analysis revealed additional mechanistic aspects of cardiotoxicity, showcasing drug specificity. The downregulation of various proteins involved in protein machinery and protein processing in the endoplasmic reticulum was observed in rosiglitazone-treated cells, implicating proteostasis in the rosiglitazone cardiotoxicity. Regarding pioglitazone, the findings suggested the potential activation of the interplay between the complement and coagulation systems and the disruption of the cytoskeletal architecture, which was primarily mediated through the integrin-signalling pathways responsible for pioglitazone-induced myocardial contractile failure. Collectively, this study unlocks substantial mechanistic insight into TZD cardiotoxicity, providing the rationale for future optimisation of antidiabetic therapies.

Molecular mechanisms of metabolic dysregulation in diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM), one of the most serious complications of diabetes mellitus, has become recognized as a cardiometabolic disease. In normoxic conditions, the majority of the ATP production (>95%) required for heart beating comes from mitochondrial oxidative phosphorylation of fatty acids (FAs) and glucose, with the remaining portion coming from a variety of sources, including fructose, lactate, ketone bodies (KB) and branched chain amino acids (BCAA). Increased FA intake and decreased utilization of glucose and lactic acid were observed in the diabetic hearts of animal models and diabetic patients. Moreover, the polyol pathway is activated, and fructose metabolism is enhanced. The use of ketones as energy sources in human diabetic hearts also increases significantly. Furthermore, elevated BCAA levels and impaired BCAA metabolism were observed in the hearts of diabetic mice and patients. The shift in energy substrate preference in diabetic hearts results in increased oxygen consumption and impaired oxidative phosphorylation, leading to diabetic cardiomyopathy. However, the precise mechanisms by which impaired myocardial metabolic alterations result in diabetes mellitus cardiac disease are not fully understood. Therefore, this review focuses on the molecular mechanisms involved in alterations of myocardial energy metabolism. It not only adds more molecular targets for the diagnosis and treatment, but also provides an experimental foundation for screening novel therapeutic agents for diabetic cardiomyopathy.

A comparative modeling study of the mitochondrial function of the proximal tubule and thick ascending limb cells in the rat kidney.

To reabsorb >99% of the glomerular filtrate, the metabolic demand of the kidney is high. Interestingly, renal blood flow distribution exhibits marked inhomogeneity, with typical tissue oxygen tension (Po2) of 50-60 mmHg in the well-perfused cortex and 10-20 mmHg in the inner medulla. Cellular fluid composition and acidity also varies substantially. To understand how different renal epithelial cells adapt to their local environment, we have developed and applied computational models of mitochondrial function of proximal convoluted tubule cell (baseline Po2 = 50 mmHg, cytoplasmic pH = 7.20) and medullary thick ascending limb (mTAL) cell (baseline Po2 = 10 mmHg, cytoplasmic pH = 6.85). The models predict key cellular quantities, including ATP generation, P/O (phosphate/oxygen) ratio, proton motive force, electrical potential gradient, oxygen consumption, the redox state of key electron carriers, and ATP consumption. Model simulations predict that close to their respective baseline conditions, the proximal tubule and mTAL mitochondria exhibit qualitatively similar behaviors. Nonetheless, because the mTAL mitochondrion has adapted to a much lower Po2, it can sustain a sufficiently high ATP production at Po2 as low as 4-5 mmHg, whereas the proximal tubule mitochondria would not. Also, because the mTAL cytosol is already acidic under baseline conditions, the proton motive force (pmf) exhibits higher sensitivity to further acidification. Among the different pathways that lead to oxidative phosphorylation impairment, the models predict that both the proximal tubule and mTAL mitochondria are most sensitive to reductions in Complex III activity.NEW & NOTEWORTHY Tissue fluid composition varies substantially within the mammalian kidney. The renal cortex is well perfused and pH neutral, whereas some medullary regions are hypoxic and acidic. How do these environments affect the mitochondrial function of proximal convoluted tubule and medullary thick ascending limb cells, which reside in the cortex and medulla, respectively? This computational modeling study demonstrates that these mitochondria can adapt to their contrasting environments and exhibit different sensitivities to perturbations to local environments.

Preneoplastic cells switch to Warburg metabolism from their inception exposing multiple vulnerabilities for targeted elimination

Otto Warburg described tumour cells as displaying enhanced aerobic glycolysis whilst maintaining defective oxidative phosphorylation (OXPHOS) for energy production almost 100 years ago [1, 2]. Since then, the ‘Warburg effect’ has been widely accepted as a key feature of rapidly proliferating cancer cells [3–5]. What is not clear is how early “Warburg metabolism” initiates in cancer and whether changes in energy metabolism might influence tumour progression ab initio. We set out to investigate energy metabolism in the HRASG12V driven preneoplastic cell (PNC) at inception, in a zebrafish skin PNC model. We find that, within 24 h of HRASG12V induction, PNCs upregulate glycolysis and blocking glycolysis reduces PNC proliferation, whilst increasing available glucose enhances PNC proliferation and reduces apoptosis. Impaired OXPHOS accompanies enhanced glycolysis in PNCs, and a mild complex I inhibitor, metformin, selectively suppresses expansion of PNCs. Enhanced mitochondrial fragmentation might be underlining impaired OXPHOS and blocking mitochondrial fragmentation triggers PNC apoptosis. Our data indicate that altered energy metabolism is one of the earliest events upon oncogene activation in somatic cells, which allows a targeted and effective PNC elimination.

COX6C expression driven by copy amplification of 8q22.2 regulates cell proliferation via mediation of mitosis by ROS-AMPK signaling in lung adenocarcinoma

Copy number variations (CNVs) play a vital role in regulating genes expression and tumorigenesis. We explored the copy number alterations in early-stage lung adenocarcinoma using high-throughput sequencing and nucleic acid flight mass spectrometry technology, and found that 8q22.1-22.2 is frequently amplified in lung adenocarcinoma tissues. COX6C localizes on the region and its expression is notably enhanced that driven by amplification in lung adenocarcinoma. Knockdown of COX6C significantly inhibits the cell proliferation, and induces S-G2/M cell cycle arrest, mitosis deficiency and apoptosis. Moreover, COX6C depletion causes a deficiency in mitochondrial fusion, and impairment of oxidative phosphorylation. Mechanistically, COX6C-induced mitochondrial deficiency stimulates ROS accumulation and activates AMPK pathway, then leading to abnormality in spindle formation and chromosome segregation, activating spindle assemble checkpoint, causing mitotic arrest, and ultimately inducing cell apoptosis. Collectively, we suggested that copy amplification-mediated COX6C upregulation might serves as a prospective biomarker for prognosis and targeting therapy in patients with lung adenocarcinoma.

Cellular and Molecular Responses to Mitochondrial DNA Deletions in Kearns-Sayre Syndrome: Some Underlying Mechanisms.

Kearns-Sayre syndrome (KSS) is a rare multisystem mitochondrial disorder. It is caused by mitochondrial DNA (mtDNA) rearrangements, mostly large-scale deletions of 1.1-10kb. These deletions primarily affect energy supply through impaired oxidative phosphorylation and reduced ATP production. This impairment gives rise to dysfunction of several tissues, in particular those with high energy demand like brain and muscles. Over the past decades, changes in respiratory chain complexes and energy metabolism have been emphasized, whereas little attention has been paid to other reports on ROS overproduction, protein synthesis inhibition, myelin vacuolation, demyelination, autophagy, apoptosis, and involvement of lipid raft and oligodendrocytes in KSS. Therefore, this paper draws attention towards these relatively underemphasized findings that might further clarify the pathologic cascades following deletions in the mtDNA.

NADPH oxidase overexpression and mitochondrial OxPhos impairment are more profound in human hearts donated after circulatory death than brain death.

This study investigated cardiac stress and mitochondrial oxidative phosphorylation (OxPhos) in human donation after circulatory death (DCD) hearts regarding warm ischemic time (WIT) and subsequent cold storage and compared them with that of human brain death donor (DBD) hearts. A total of 24 human hearts were procured for the research study-6 in the DBD group and 18 in the DCD group. DCD group was divided into three groups (n = 6) based on different WITs (20, 40, and 60 min). All hearts received del Nido cardioplegia before being placed in normal saline cold storage for 6 h. Left ventricular biopsies were performed at hours 0, 2, 4, and 6. Cardiac stress [nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits: 47-kDa protein of phagocyte oxidase (p47phox), 91-kDa glycoprotein of phagocyte oxidase (gp91phox)] and mitochondrial oxidative phosphorylation [OxPhos, complex I (NADH dehydrogenase) subunit of ETC (CI)-complex V (ATP synthase) subunit of ETC (CV)] proteins were measured in cardiac tissue and mitochondria respectively. Modulation of cardiac stress and mitochondrial dysfunction were observed in both DCD and DBD hearts. However, DCD hearts suffered more cardiac stress (overexpressed NADPH oxidase subunits) and diminished mitochondrial OxPhos than DBD hearts. The severity of cardiac stress and impaired oxidative phosphorylation in DCD hearts correlated with the longer WIT and subsequent cold storage time. More drastic changes were evident in DCD hearts with a WIT of 60 min or more. Activation of NADPH oxidase via overproduction of p47phox and gp91phox proteins in cardiac tissue may be responsible for cardiac stress leading to diminished mitochondrial oxidative phosphorylation. These protein changes can be used as biomarkers for myocardium damage and might help assess DCD and DBD heart transplant suitability.NEW & NOTEWORTHY First human DCD heart research studied cardiac stress and mitochondrial dysfunction concerning WIT and the efficacy of del Nido cardioplegia as an organ procurement solution and subsequent cold storage. Mild to moderate cardiac stress and mitochondrial dysfunction were noticed in DCD hearts with WIT 20 and 40 min and cold storage for 4 and 2 h, respectively. These changes can serve as biomarkers, allowing interventions to preserve mitochondria and extend WIT in DCD hearts.

NPC1 is required for postnatal islet β cell differentiation by maintaining mitochondria turnover.

Rationale: NPC1 is a protein localized on the lysosome membrane regulating intracellular cholesterol transportation and maintaining normal lysosome function. GWAS studies have found that NPC1 variants in T2D was a pancreatic islet expression quantitative trait locus, suggesting a potential role of NPC1 in T2D islet pathophysiology. Methods: Two-week-old Npc1-/- mice and wild type littermates were employed to examine pancreatic β cell morphology and functional changes induced by loss of Npc1. Single cell RNA sequencing was conducted on primary islets. Npc1-/- Min6 cell line was generated using CRISPR/Cas9 gene editing. Seahorse XF24 was used to analyze primary islet and Min6 cell mitochondria respiration. Ultra-high-resolution cell imaging with Lattice SIM2 and electron microscope imaging were used to observe mitochondria and lysosome in primary islet β and Min6 cells. Mitophagy Dye and mt-Keima were used to measure β cell mitophagy. Results: In Npc1-/- mice, we found that β cell survival and pancreatic β cell mass expansion as well as islet glucose induced insulin secretion in 2-week-old mice were reduced. Npc1 loss retarded postnatal β cell differentiation and growth as well as impaired mitochondria oxidative phosphorylation (OXPHOS) function to increase mitochondrial superoxide production, which might be attributed to impaired autophagy flux particularly mitochondria autophagy (mitophagy) induced by dysfunctional lysosome in Npc1 null β cells. Conclusion: Our study revealed that NPC1 played an important role in maintaining normal lysosome function and mitochondria turnover, which ensured establishment of sufficient mitochondria OXPHOS for islet β cells differentiation and maturation.

Mitochondrial dysfunction and metabolic reprogramming induce macrophage pro-inflammatory phenotype switch and atherosclerosis progression in aging.

Aging increases the risk of atherosclerotic vascular disease and its complications. Macrophages are pivotal in the pathogenesis of vascular aging, driving inflammation and atherosclerosis progression. NOX4 (NADPH oxidase 4) expression increases with age, correlating with mitochondrial dysfunction, inflammation, and atherosclerosis. We hypothesized that the NOX4-dependent mitochondrial oxidative stress promotes aging-associated atherosclerosis progression by causing metabolic dysfunction and inflammatory phenotype switch in macrophages. We studied atherosclerotic lesion morphology and macrophage phenotype in young (5-month-old) and aged (16-month-old) Nox4 -/-/Apoe -/- and Apoe -/- mice fed Western diet. Young Nox4-/-/Apoe-/- and Apoe-/- mice had comparable aortic and brachiocephalic artery atherosclerotic lesion cross-sectional areas. Aged mice showed significantly increased lesion area compared with young mice. Aged Nox4-/-/Apoe-/- had significantly lower lesion areas than Apoe-/- mice. Compared with Apoe-/- mice, atherosclerotic lesions in aged Nox4-/-/Apoe-/- showed reduced cellular and mitochondrial ROS and oxidative DNA damage, lower necrotic core area, higher collagen content, and decreased inflammatory cytokine expression. Immunofluorescence and flow cytometry analysis revealed that aged Apoe-/- mice had a higher percentage of classically activated pro-inflammatory macrophages (CD38+CD80+) in the lesions. Aged Nox4-/-/Apoe-/- mice had a significantly higher proportion of alternatively activated pro-resolving macrophages (EGR2+/CD163+CD206+) in the lesions, with an increased CD38+/EGR2+ cell ratio compared with Apoe-/- mice. Mitochondrial respiration assessment revealed impaired oxidative phosphorylation and increased glycolytic ATP production in macrophages from aged Apoe-/- mice. In contrast, macrophages from Nox4-/-/Apoe-/- mice were less glycolytic and more aerobic, with preserved basal and maximal respiration and mitochondrial ATP production. Macrophages from Nox4-/-/Apoe-/- mice also had lower mitochondrial ROS levels and reduced IL1β secretion; flow cytometry analysis showed fewer CD38+ cells after IFNγ+LPS treatment and more EGR2+ cells after IL4 treatment than in Apoe-/- macrophages. In aged Apoe-/- mice, inhibition of NOX4 activity using GKT137831 significantly reduced macrophage mitochondrial ROS and improved mitochondrial function, resulting in decreased CD68+CD80+ and increased CD163+CD206+ lesion macrophage proportion and attenuated atherosclerosis. Our findings suggest that increased NOX4 in aging drives macrophage mitochondrial dysfunction, glycolytic metabolic switch, and pro-inflammatory phenotype, advancing atherosclerosis. Inhibiting NOX4 or mitochondrial dysfunction could alleviate vascular inflammation and atherosclerosis, preserving plaque integrity.

Dystonia, spastic tetraplegia, and ataxia due to a novel mutation in the dynamin domain of OPA1.

Movement disorders manifest in various hereditary neurodegenerative diseases. We reported a young man who presented with progressive upper limb dystonia, spastic tetraplegia, and ataxia. Whole-exome sequencing (WES) revealed a novel variant, c.2357A > G, in the dynamin domain of OPA1. No mtDNA deletion was detected in muscle by long-range PCR. Atrophy and decreased glucose metabolism of the basal ganglia were discovered. Decreased mtDNA copy number, fragmented mitochondria, slightly impaired oxidative phosphorylation, and increased autophagy were detected in mutant fibroblasts. Evident oxidative phosphorylation impairment and mtDNA deletions were not involved in the pathogenicity of this mutation unlike mutations in the GTPase domain of OPA1.

Glucose-mediated mitochondrial reprogramming by cholesterol export at TM4SF5-enriched mitochondria-lysosome contact sites.

Transmembrane 4 L six family member 5 (TM4SF5) translocates subcellularly and functions metabolically, although it is unclear how intracellular TM4SF5 translocation is linked to metabolic contexts. It is thus of interests to understand how the traffic dynamics of TM4SF5 to subcellular endosomal membranes are correlated to regulatory roles of metabolisms. Here, we explored the metabolic significance of TM4SF5 localization at mitochondria-lysosome contact sites (MLCSs), using in vitro cells and in vivo animal systems, via approaches by immunofluorescence, proximity labelling based proteomics analysis, organelle reconstitution etc. RESULTS: Upon extracellular glucose repletion following depletion, TM4SF5 became enriched at MLCSs via an interaction between mitochondrial FK506-binding protein 8 (FKBP8) and lysosomal TM4SF5. Proximity labeling showed molecular clustering of phospho-dynamic-related protein I (DRP1) and certain mitophagy receptors at TM4SF5-enriched MLCSs, leading to mitochondrial fission and autophagy. TM4SF5 bound NPC intracellular cholesterol transporter 1 (NPC1) and free cholesterol, and mediated export of lysosomal cholesterol to mitochondria, leading to impaired oxidative phosphorylation but intact tricarboxylic acid (TCA) cycle and β-oxidation. In mouse models, hepatocyte Tm4sf5 promoted mitophagy and cholesterol transport to mitochondria, both with positive relations to liver malignancy. Our findings suggested that TM4SF5-enriched MLCSs regulate glucose catabolism by facilitating cholesterol export for mitochondrial reprogramming, presumably while hepatocellular carcinogenesis, recapitulating aspects for hepatocellular carcinoma metabolism with mitochondrial reprogramming to support biomolecule synthesis in addition to glycolytic energetics.

Transcriptome analysis reveals therapeutic potential of NAMPT in protecting against abdominal aortic aneurysm in human and mouse

Abdominal Aortic Aneurysm (AAA) is a life-threatening vascular disease characterized by the weakening and ballooning of the abdominal aorta, which has no effective therapeutic approaches due to unclear molecular mechanisms. Using single-cell RNA sequencing, we analyzed the molecular profile of individual cells within control and AAA abdominal aortas. We found cellular heterogeneity, with increased plasmacytoid dendritic cells and reduced endothelial cells and vascular smooth muscle cells (VSMCs) in AAA. Up-regulated genes in AAA were associated with muscle tissue development and apoptosis. Genes controlling VSMCs aberrant switch from contractile to synthetic phenotype were significantly enriched in AAA. Additionally, VSMCs in AAA exhibited cell senescence and impaired oxidative phosphorylation. Similar observations were made in a mouse model of AAA induced by Angiotensin II, further affirming the relevance of our findings to human AAA. The concurrence of gene expression changes between human and mouse highlighted the impairment of oxidative phosphorylation as a potential target for intervention. Nicotinamide phosphoribosyltransferase (NAMPT, also named VISFATIN) signaling emerged as a signature event in AAA. NAMPT was significantly downregulated in AAA. NAMPT-extracellular vesicles (EVs) derived from mesenchymal stem cells restored NAMPT levels, and offered protection against AAA. Furthermore, NAMPT-EVs not only repressed injuries, such as cell senescence and DNA damage, but also rescued impairments of oxidative phosphorylation in both mouse and human AAA models, suggesting NAMPT supplementation as a potential therapeutic approach for AAA treatment. These findings shed light on the cellular heterogeneity and injuries in AAA, and offered promising therapeutic intervention for AAA treatment.

Multimodal assessment of mitochondrial function in Parkinson's disease.

The heterogenous aetiology of Parkinson's disease is increasingly recognized; both mitochondrial and lysosomal dysfunction have been implicated. Powerful, clinically applicable tools are required to enable mechanistic stratification for future precision medicine approaches. The aim of this study was to characterize bioenergetic dysfunction in Parkinson's disease by applying a multimodal approach, combining standardized clinical assessment with midbrain and putaminal 31-phosphorus magnetic resonance spectroscopy (31P-MRS) and deep phenotyping of mitochondrial and lysosomal function in peripheral tissue in patients with recent-onset Parkinson's disease and control subjects. Sixty participants (35 patients with Parkinson's disease and 25 healthy controls) underwent 31P-MRS for quantification of energy-rich metabolites [ATP, inorganic phosphate (Pi) and phosphocreatine] in putamen and midbrain. In parallel, skin biopsies were obtained from all research participants to establish fibroblast cell lines for subsequent quantification of total intracellular ATP and mitochondrial membrane potential (MMP) as well as mitochondrial and lysosomal morphology, using high content live cell imaging. Lower MMP correlated with higher intracellular ATP (r = -0.55, P = 0.0016), higher mitochondrial counts (r = -0.72, P < 0.0001) and higher lysosomal counts (r = -0.62, P = 0.0002) in Parkinson's disease patient-derived fibroblasts only, consistent with impaired mitophagy and mitochondrial uncoupling. 31P-MRS-derived posterior putaminal Pi/ATP ratio variance was considerably greater in Parkinson's disease than in healthy controls (F-tests, P = 0.0036). Furthermore, elevated 31P-MRS-derived putaminal, but not midbrain Pi/ATP ratios (indicative of impaired oxidative phosphorylation) correlated with both greater mitochondrial (r = 0.37, P = 0.0319) and lysosomal counts (r = 0.48, P = 0.0044) as well as lower MMP in both short (r = -0.52, P = 0.0016) and long (r = -0.47, P = 0.0052) mitochondria in Parkinson's disease. Higher 31P-MRS midbrain phosphocreatine correlated with greater risk of rapid disease progression (r = 0.47, P = 0.0384). Our data suggest that impaired oxidative phosphorylation in the striatal dopaminergic nerve terminals exceeds mitochondrial dysfunction in the midbrain of patients with early Parkinson's disease. Our data further support the hypothesis of a prominent link between impaired mitophagy and impaired striatal energy homeostasis as a key event in early Parkinson's disease.

Serine Phosphorylation Marks Proteins for Degradation By the Mitochondrial Serine Protease Complex, Clpxp

ClpXP, a serine protease located in the mitochondrial matrix, regulates mitochondrial proteostasis by degrading damaged or aggregated proteins, including respiratory chain subunits. ClpXP is a bipartite protein complex comprised of the ClpX regulatory particle that caps each end of the ClpP degradation chamber. ClpX recognizes substrates, unfolds them, and feeds them into ClpP for proteolysis. Genetic or chemical inhibition of ClpXP leads to impaired oxidative phosphorylation function and leukemic cell death in vitro and in vivo. Importantly, however, the degradation marker of human ClpXP remains elusive. Since the bacterial homologue of ClpXP recognizes phosphorylated arginine (pArg)-tagged protein for degradation, we hypothesized that human ClpXP might also recognize phosphorylated amino acids as degrons. We first investigated whether phosphorylation influences substrate degradation by ClpXP using a model substrate, α-casein. Recombinant human ClpXP preferentially degraded phosphorylated α-casein compared to dephosphorylated α-casein. To further elucidate the effects of phosphorylation, we screened a panel of phosphorylated amino acids for their ability to impede ClpXP-mediated degradation of FITC-tagged α-casein. Phosphorylated serine (pSer) and phosphorylated threonine (pThr) free amino acids, or short peptides containing pSer or pThr, inhibited α-casein degradation by ClpXP in a dose-dependent manner. In contrast, phosphorylated pArg, and phosphorylated tyrosine (pTyr), as well as free unmodified Ser or Thr, did not impact ClpXP protease activity. Next, we applied a thermal shift assay to measure the binding capabilities of pSer and pThr to ClpX. We discovered that these phosphorylated amino acids, whether free or incorporated into short peptides, successfully associated with ClpX. In contrast, their dephosphorylated counterparts did not exhibit the same binding activity. Notably, pSer did not affect enzyme activity of the related LonP1 mitochondrial matrix serine protease, thereby demonstrating the specificity of pSer for ClpXP. Extending our studies to intact cells, we analyzed a proteomic dataset of post-translational modifications in Jurkat cells with and without treatment with bortezomib (1 µM), a proteasome inhibitor that inhibits ClpXP at this concentration. Notably, a global enrichment of phosphorylation was observed for mitochondrial proteins upon bortezomib treatment. We overlaid observed phosphorylated proteins with the ClpXP interactome as determined by BioID, and identified 9 phosphorylated mitochondrial proteins that also interact with ClpXP. Of these proteins, respiratory chain II complex subunit, SDHA, was the top hit, where serine phosphorylation was increased over 4 folds. Next we determined how depletion of ClpXP affected levels of phosphorylated substrate in intact cells. Knockdown of ClpX or ClpP in OCI-AML2 cells increased levels of serine phosphorylated SDHA (pSer-SDHA). We then added recombinant ClpXP protein to mitochondrial lysates and observed selective degradation of pSer-SDHA. To determine if pSer-SDHA are damaged proteins, we partitioned mitochondria into soluble and insoluble fractions using digitonin. pSer-SDHA was enriched in the detergent-insoluble fraction. In addition, we induced mitochondrial proteolytic stress by treating OCI-AML2 cells with antimycin to increase mitochondrial ROS or through heat shock by culturing cells at 42 oC. Both antimycin and heat shock increased pSer-SDHA in the detergent-insoluble fraction, further supporting pSer marking damaged proteins. Finally, we developed small molecule pSer mimics. We showed that these compounds bound ClpX in thermal shift assays. In addition, these compounds inhibited ClpXP protease activity in cell-free enzymatic assays. In summary, ClpXP recognizes serine phosphorylation as a degradation marker for damaged mitochondrial proteins. Small molecules that inhibit ClpX could be leads for novel anti-cancer agents.

Mitochondrial tRNA Pseudouridylation Regulates Erythropoiesis Via the mTOR Signaling Pathway: Implications for Mlasa and Treatment Strategies

Pseudouridine, the most abundant RNA modification found in various RNA molecules, is crucial for cellular functions. Pseudouridylation is the process of converting uridine to pseudouridine, catalyzed by pseudouridine synthases (PUSs), and abnormal pseudouridylation has been linked to human diseases, including mitochondrial myopathy, lactic acidosis, and sideroblastic anemia syndrome (MLASA). MLASA is associated with genetic mutations in the PUS1 gene, leading to progressive exercise intolerance, sideroblastic anemia, hyperlactatemia, and muscle weakness. The underlying mechanism of anemia in MLASA remains unclear. In this study, we identified a novel homozygous frameshift deletion (c.523delC, p.P175fs*8) in the PUS1 gene in an MLASA patient with progressive anemia in our hospital. We investigated the role of pseudouridylation in erythropoiesis and its impact on MLASA using in vitro and in vivo approaches. We established an MLASA patient-derived inducible pluripotent stem cell (iPSC) line (referred to as MLASA-iPSCs) with the mutation, as well as a repaired line (MLASA-Res iPSC), and a corresponding mutant mouse model for the investigation. During erythroid differentiation, MLASA-iPSCs showed a significant decrease in the production of erythroblasts compared to the repaired line. Also, MLASA-iPSCs exhibited a lower ratio of mitochondrial membrane potential to mitochondrial mass, decreased oxygen consumption rates, reduced ATP production, and elevated levels of ROS. Attenuated activities of NADH dehydrogenase (Complex I) and cytochrome c reductase (Complex III) were also observed in MLASA-iPSCs, indicating compromised mitochondrial functions. Further analysis revealed a reduction in mitochondrial tRNA levels and abnormal mitochondrial translation in MLASA-iPSCs due to the specific loss of pseudouridylation. Screening of mitochondrial supplements, which could boost mitochondrial function or modulating protein synthesis, revealed that nicotinamide ribose (NR) and mitoquinone (MitoQ) improved mitochondrial function but failed to promote erythroid differentiation in MLASA-iPSCs. Interestingly, rapamycin, an mTOR inhibitor, improved erythroid differentiation in MLASA-iPSCs. Furthermore, MLASA-iPSCs showed activated mTOR signaling, increased protein synthesis and higher translation efficiency of mTOR1 targets, all of which could be rescued by rapamycin treatment, suggesting mTOR-associated translation stress. Consistently, the corresponding PUS1 mutant mouse model exhibited anemia with blocked erythropoiesis at four weeks of age. Serial competitive transplantation experiments revealed an intrinsic impairment in the reconstitution of red blood cells. Mutant erythroblasts showed impaired oxidative phosphorylation and aberrant activation of the mTOR signaling pathway. Rapamycin treatment partially alleviated the anemia phenotype in mice. Significantly, in the MLASA patient, sirolimus (rapamycin) administration under strict supervision and medical guidance effectively alleviated anemia symptoms and improved blood routine profile. In summary, our findings provide insights into the critical role of mitochondrial tRNA pseudouridylation in governing erythropoiesis via mTOR signaling pathway. We propose rapamycin as potential therapeutic strategies for anemia patients experiencing protein translation pressure, such as those with MLASA. #These authors contributed equally: Chu Y, Wang B, Shi D and Lian Y. Correspondence to: wpyuan@ihcams.ac.cn, shijun@ihcams.ac.cn Fig. 1 Schematic illustration of erythropoiesis regulated by PUS1-mediated pseudouridylation. Under physiological conditions (left), PUS1 regulates mt-tRNA abundance by catalyzing the pseudouridine synthesis of specific mt-tRNAs, thereby regulating mitochondrial translation, and ultimately affecting mitochondrial function and erythroid differentiation. In MLASA disease states (right), PUS1 deficiency causes the loss of pseudouridine modifications of specific tRNAs and thus affects the amount of PUS1 targeted tRNAs and its corresponding mitochondrial protein products, leading to mitochondrial dysfunction. The defects resulted in aberrant activation of the mTOR signaling pathway and accelerated global translation rate, ultimately leading to a blockage in erythroid differentiation, which could be relieved with rapamycin treatment.