The requirement of the mitochondrial protein NDUFS8 for angiogenesis.

Mitochondria are important for the activation of endothelial cells and the process of angiogenesis. NDUFS8 (NADH:ubiquinone oxidoreductase core subunit S8) is a protein that plays a critical role in the function of mitochondrial Complex I. We aimed to investigate the potential involvement of NDUFS8 in angiogenesis. In human umbilical vein endothelial cells (HUVECs) and other endothelial cell types, we employed viral shRNA to silence NDUFS8 or employed the CRISPR/Cas9 method to knockout (KO) it, resulting in impaired mitochondrial functions in the endothelial cells, causing reduction in mitochondrial oxygen consumption and Complex I activity, decreased ATP production, mitochondrial depolarization, increased oxidative stress and reactive oxygen species (ROS) production, and enhanced lipid oxidation. Significantly, NDUFS8 silencing or KO hindered cell proliferation, migration, and capillary tube formation in cultured endothelial cells. In addition, there was a moderate increase in apoptosis within NDUFS8-depleted endothelial cells. Conversely, ectopic overexpression of NDUFS8 demonstrated a pro-angiogenic impact, enhancing cell proliferation, migration, and capillary tube formation in HUVECs and other endothelial cells. NDUFS8 is pivotal for Akt-mTOR cascade activation in endothelial cells. Depleting NDUFS8 inhibited Akt-mTOR activation, reversible with exogenous ATP in HUVECs. Conversely, NDUFS8 overexpression boosted Akt-mTOR activation. Furthermore, the inhibitory effects of NDUFS8 knockdown on cell proliferation, migration, and capillary tube formation were rescued by Akt re-activation via a constitutively-active Akt1. In vivo experiments using an endothelial-specific NDUFS8 shRNA adeno-associated virus (AAV), administered via intravitreous injection, revealed that endothelial knockdown of NDUFS8 inhibited retinal angiogenesis. ATP reduction, oxidative stress, and enhanced lipid oxidation were detected in mouse retinal tissues with endothelial knockdown of NDUFS8. Lastly, we observed an increase in NDUFS8 expression in retinal proliferative membrane tissues obtained from human patients with proliferative diabetic retinopathy. Our findings underscore the essential role of the mitochondrial protein NDUFS8 in regulating endothelial cell activation and angiogenesis.

A Redox-Regulated, Heterodimeric NADH:cinnamate Reductase in Vibrio ruber.

Genes of putative reductases of α,ß-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the facultatively anaerobic marine bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists of the sjn56021-encoded subunit CrdB (NADH:flavin, FAD binding 2, and FMN bind domains) and an additional subunit CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 prediction). Each domain contains a flavin group (three FMNs and one FAD in total), one of the FMN groups being linked covalently by the flavin transferase. Crd readily reduces cinnamate, p-coumarate, caffeate, and ferulate under anaerobic conditions with NADH or methyl viologen as the electron donor, is moderately active against acrylate and practically inactive against urocanate and fumarate. Cinnamates induced Crd synthesis in V. ruber cells grown aerobically or anaerobically. The Crd-catalyzed reduction started by NADH demonstrated a time lag of several minutes, suggesting a redox regulation of the enzyme activity. The oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditions. Our findings identify Crd as a regulated NADH-dependent cinnamate reductase, apparently protecting V. ruber from (hydroxy)cinnamate poisoning.

NADH dehydrogenase-like complex L subunit improves salt tolerance by enhancing photosynthetic electron transport.

Cyclic electron transport (CET) around photosystem I (PSI) mediated by the NADH dehydrogenase-like (NDH) complex is closely related to plant salt tolerance. However, whether overexpression of a core subunit of the NDH complex affects the photosynthetic electron transport under salt stress is currently unclear. Here, we expressed the NDH complex L subunit (Ndhl) genes ZmNdhl1 and ZmNdhl2 from C4 plant maize (Zea mays) or OsNdhl from C3 plant rice (Oryza sativa) using a constitutive promoter in rice. Transgenic rice lines expressing ZmNdhl1, ZmNdhl2, or OsNdhl displayed enhanced salt tolerance, as indicated by greater plant height, dry weight, and leaf relative water content, as well as lower malondialdehyde content compared to wild-type plants under salt stress. Fluorescence parameters such as post-illumination rise (PIR), the prompt chlorophyll a fluorescence transient (OJIP), modulated 820-nm reflection (MR), and delayed chlorophyll a fluorescence (DF) remained relatively normal in transgenic plants during salt stress. These results indicate that expression of ZmNdhl1, ZmNdhl2, or OsNdhl increases cyclic electron transport activity, slows down damage to linear electron transport, alleviates oxidative damage to the PSI reaction center and plastocyanin, and reduces damage to electron transport on the receptor side of PSI in rice leaves under salt stress. Thus, expression of Ndhl genes from maize or rice improves salt tolerance by enhancing photosynthetic electron transport in rice. Maize and rice Ndhl genes played a similar role in enhancing salinity tolerance and avoiding photosynthetic damage.

Neuroprotective mechanism of trans,trans-Farnesol in an ICV-STZ-induced rat model of Alzheimer's pathology.

BACKGROUND: Alzheimer's disease (AD) is a prominent cause of dementia, resulting in neurodegeneration and memory impairment. This condition imposes a considerable public health burden on both patients and their families due to the patients' functional impairments as well as the psychological and financial constraints. It has been well demonstrated that its aetiology involves proteinopathy, mitochondriopathies, and enhanced reactive oxygen species (ROS) generation, which are some of the key features of AD brains that further result in oxidative stress, excitotoxicity, autophagy, and mitochondrial dysfunction. OBJECTIVE: The current investigation was created with the aim of elucidating the neurological defence mechanism of trans,trans-Farnesol (TF) against intracerebroventricular-streptozotocin (ICV-STZ)-induced Alzheimer-like symptoms and related pathologies in rodents. MATERIALS AND METHODS: The current investigation involved male SD rats receiving TF (25-100 mg/kg, per oral) consecutively for 21 days in ICV-STZ-treated animals. An in silico study was carried out to explore the possible interaction between TF and NADH dehydrogenase and succinate dehydrogenase. Further, various behavioural (Morris water maze and novel object recognition test), biochemical (oxidants and anti-oxidant markers), activities of mitochondrial enzyme complexes and acetylcholinesterase (AChE), pro-inflammatory (tumor necrosis factor-alpha; TNF-α) levels, and histopathological studies were evaluated in specific brain regions. RESULTS: Rats administered ICV-STZ followed by treatment with TF (25, 50, and 100 mg/kg) for 21 days had significantly better mental performance (reduced escape latency to access platform, extended time spent in target quadrant, and improved differential index) in the Morris water maze test and new object recognition test models when compared to control (ICV-STZ)-treated groups. Further, TF treatment significantly restored redox proportion, anti-oxidant levels, regained mitochondrial capacities, attenuated altered AChE action, levels of TNF-α, and histopathological alterations in certain brain regions in comparison with control. In in silico analysis, TF caused greater interaction with NADH dehydrogenase and succinate dehydrogenase. CONCLUSION: The current work demonstrates the neuroprotective ability of TF in an experimental model with AD-like pathologies. The study further suggests that the neuroprotective impacts of TF may be related to its effects on TNF-α levels, oxidative stress pathways, and mitochondrial complex capabilities.

A Homozygous NDUFS6 Variant Associated with Neuropathy and Optic Atrophy.

Background: The NADH dehydrogenase [ubiquinone] iron-sulfur protein 6 (NDUFS6) gene encodes for an accessory subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (complex I). Bi-allelic NDUFS6 variants have been linked with a severe disorder mostly reported as a lethal infantile mitochondrial disease (LMID) or Leigh syndrome (LS). Objective: Here, we identified a homozygous variant (c.309 + 5 G > A) in NDUFS6 in one male patient with axonal neuropathy accompanied by loss of small fibers in skin biopsy and further complicated by optic atrophy and borderline intellectual disability. Methods: To address the pathogenicity of the variant, biochemical studies (mtDNA copy number quantification, ELISA, Proteomic profiling) of patient-derived leukocytes were performed. Results: The analyses revealed loss of NDUFS6 protein associated with a decrease of three further mitochondrial NADH dehydrogenase subunit/assembly proteins (NDUFA12, NDUFS4 and NDUFV1). Mitochondrial copy number is not altered in leukocytes and the mitochondrial biomarker GDF15 is not significantly changed in serum. Conclusions: Hence, our combined clinical and biochemical data strengthen the concept of NDUFS6 being causative for a very rare form of axonal neuropathy associated with optic atrophy and borderline intellectual disability, and thus expand (i) the molecular genetic landscape of neuropathies and (ii) the clinical spectrum of NDUFS6-associated phenotypes.

AKAP1 in Renal Patients with AHF to Reduce Ferroptosis of Cardiomyocyte.

BACKGROUND: This study mainly investigated the mechanism and effects of AKAP1 in renal patients with acute heart failure (AHF). METHODS: Patients with renal patients with AHF and normal volunteers were collected. The left anterior descending arteries (LAD) of mice were ligated to induce myocardial infarction. RESULTS: AKAP1 messenger RNA (mRNA) expression was found to be down-regulated in renal patients with AHF. The serum levels of AKAP1 mRNA expression were negatively correlated with collagen I/III in patients. AKAP1 mRNA and protein expression in the heart tissue of mice with AHF were also found to be down-regulated in a time-dependent manner. Short hairpin (Sh)-AKAP1 promotes AHF in a mouse model. AKAP1 up-regulation reduces reactive oxygen species (ROS)-induced oxidative stress in an In Vitro model. AKAP1 up-regulation also reduces ROS-induced lipid peroxidation ferroptosis in an In Vitro model. AKAP1 induces NDUFS1 expression to increase GPX4 activity levels. AKAP1 protein interlinked with the NDUFS1 protein. Up-regulation of the AKAP1 gene reduced NDUFS1 ubiquitination, while down-regulation of the AKAP1 gene increased NDUFS1 ubiquitination in a model. In vivo imaging showed that the sh-AKAP1 virus reduced NDUFS1 expression in the heart of a mouse model. CONCLUSIONS: AKAP1 reduced ROS-induced lipid peroxidation ferroptosis through the inhibition of ubiquitination of NDUFS by mitochondrial damage in model of renal patients with AHF, suggest a novel target for AHF treatment.

Structural insights into respiratory complex I deficiency and assembly from the mitochondrial disease-related ndufs4-/- mouse.

Respiratory complex I (NADH:ubiquinone oxidoreductase) is essential for cellular energy production and NAD+ homeostasis. Complex I mutations cause neuromuscular, mitochondrial diseases, such as Leigh Syndrome, but their molecular-level consequences remain poorly understood. Here, we use a popular complex I-linked mitochondrial disease model, the ndufs4-/- mouse, to define the structural, biochemical, and functional consequences of the absence of subunit NDUFS4. Cryo-EM analyses of the complex I from ndufs4-/- mouse hearts revealed a loose association of the NADH-dehydrogenase module, and discrete classes containing either assembly factor NDUFAF2 or subunit NDUFS6. Subunit NDUFA12, which replaces its paralogue NDUFAF2 in mature complex I, is absent from all classes, compounding the deletion of NDUFS4 and preventing maturation of an NDUFS4-free enzyme. We propose that NDUFAF2 recruits the NADH-dehydrogenase module during assembly of the complex. Taken together, the findings provide new molecular-level understanding of the ndufs4-/- mouse model and complex I-linked mitochondrial disease.

NDUFA8 is transcriptionally regulated by EP300/H3K27ac and promotes mitochondrial respiration to support proliferation and inhibit apoptosis in cervical cancer.

Cervical cancer, a common malignancy in women, poses a significant health burden worldwide. In this study, we aimed to investigate the expression, function, and potential mechanisms of NADH: ubiquinone oxidoreductase subunit A8 (NDUFA8) in cervical cancer. The Gene Expression Profiling Interactive Analysis (GEPIA) database and immunohistochemical scoring were used to analyze NDUFA8 expression in cervical cancer tissues and normal tissues. Quantitative real-time PCR and Western blot analyses were performed to assess the expression level of NDUFA8 in cervical cancer cell lines. NDUFA8 knockdown or overexpression experiments were conducted to evaluate its impact on cell proliferation and apoptosis. The mitochondrial respiratory status was analyzed by measuring cellular oxygen consumption, adenosine triphosphate (ATP) levels, and the expression levels of Mitochondrial Complex I activity, and Mitochondrial Complex IV-associated proteins Cytochrome C Oxidase Subunit 5B (COX5B) and COX6C. NDUFA8 exhibited high expression levels in cervical cancer tissues, and these levels were correlated with reduced survival rates. A significant upregulation of NDUFA8 expression was observed in cervical cancer cell lines compared to normal cells. Silencing NDUFA8 hindered cell proliferation, promoted apoptosis, and concurrently suppressed cellular mitochondrial respiration, resulting in decreased levels of available ATP. Conversely, NDUFA8 overexpression induced the opposite effects. Herein, we also found that E1A Binding Protein P300 (EP300) overexpression facilitated Histone H3 Lysine 27 (H3K27) acetylation enrichment, enhancing the activity of the NDUFA8 promoter region. NDUFA8, which is highly expressed in cervical cancer, is regulated by transcriptional control via EP300/H3K27 acetylation. By promoting mitochondrial respiration, NDUFA8 contributes to cervical cancer cell proliferation and apoptosis. These findings provide novel insights into NDUFA8 as a therapeutic target in cervical cancer.

Regulatory NADH dehydrogenase-like complex optimizes C4 photosynthetic carbon flow and cellular redox in maize.

C4 plants typically operate a CO2 concentration mechanism from mesophyll (M) cells into bundle sheath (BS) cells. NADH dehydrogenase-like (NDH) complex is enriched in the BS cells of many NADP-malic enzyme (ME) type C4 plants and is more abundant in C4 than in C3 plants, but to what extent it is involved in the CO2 concentration mechanism remains to be experimentally investigated. We created maize and rice mutants deficient in NDH function and then used a combination of transcriptomic, proteomic, and metabolomic approaches for comparative analysis. Considerable decreases in growth, photosynthetic activities, and levels of key photosynthetic proteins were observed in maize but not rice mutants. However, transcript abundance for many cyclic electron transport (CET) and Calvin-Benson cycle components, as well as BS-specific C4 enzymes, was increased in maize mutants. Metabolite analysis of the maize ndh mutants revealed an increased NADPH : NADP ratio, as well as malate, ribulose 1,5-bisphosphate (RuBP), fructose 1,6-bisphosphate (FBP), and photorespiration intermediates. We suggest that by optimizing NADPH and malate levels and adjusting NADP-ME activity, NDH functions to balance metabolic and redox states in the BS cells of maize (in addition to ATP supply), coordinating photosynthetic transcript abundance and protein content, thus directly regulating the carbon flow in the two-celled C4 system of maize.

TCA cycle tailoring facilitates optimal growth of proton-pumping NADH dehydrogenase-dependent Escherichia coli.

IMPORTANCE: Energy generation pathways are a potential avenue for the development of novel antibiotics. However, bacteria possess remarkable resilience due to the compensatory pathways, which presents a challenge in this direction. NADH, the primary reducing equivalent, can transfer electrons to two distinct types of NADH dehydrogenases. Type I NADH dehydrogenase is an enzyme complex comprising multiple subunits and can generate proton motive force (PMF). Type II NADH dehydrogenase does not pump protons but plays a crucial role in maintaining the turnover of NAD+. To study the adaptive rewiring of energy metabolism, we evolved an Escherichia coli mutant lacking type II NADH dehydrogenase. We discovered that by modifying the flux through the tricarboxylic acid (TCA) cycle, E. coli could mitigate the growth impairment observed in the absence of type II NADH dehydrogenase. This research provides valuable insights into the intricate mechanisms employed by bacteria to compensate for disruptions in energy metabolism.

A narrative review of the effects of dexamethasone on traumatic brain injury in clinical and animal studies: focusing on inflammation.

Traumatic brain injury (TBI) is a type of brain injury resulting from a sudden physical force to the head. TBI can range from mild, such as a concussion, to severe, which might result in long-term complications or even death. The initial impact or primary injury to the brain is followed by neuroinflammation, excitotoxicity, and oxidative stress, which are the hallmarks of the secondary injury phase, that can further damage the brain tissue. Dexamethasone (DXM) has neuroprotective effects. It reduces neuroinflammation, a critical factor in secondary injury-associated neuronal damage. DXM can also suppress the microglia activation and infiltrated macrophages, which are responsible for producing pro-inflammatory cytokines that contribute to neuroinflammation. Considering the outcomes of this research, some of the effects of DXM on TBI include: (1) DXM-loaded hydrogels reduce apoptosis, neuroinflammation, and lesion volume and improves neuronal cell survival and motor performance, (2) DXM treatment elevates the levels of Ndufs2, Gria3, MAOB, and Ndufv2 in the hippocampus following TBI, (3) DXM decreases the quantity of circulating endothelial progenitor cells, (4) DXM reduces the expression of IL1, (5) DXM suppresses the infiltration of RhoA + cells into primary lesions of TBI and (6) DXM treatment led to an increase in fractional anisotropy values and a decrease in apparent diffusion coefficient values, indicating improved white matter integrity. According to the study, the findings show that DXM treatment has neuroprotective effects in TBI. This indicates that DXM is a promising therapeutic approach to treating TBI.

Quantitative proteomics reveals the neurotoxicity of trimethyltin chloride on mitochondria in the hippocampus of mice.

Trimethyltin chloride (TMT) is a potent neurotoxin widely used as a constituent of polyvinyl chloride plastic in the industrial and agricultural fields. However, the underlying mechanisms by which TMT leads to neurotoxicity remain elusive. In the present study, we constructed a dose and time dependent neurotoxic mouse model of TMT exposure to explore the molecular mechanisms involved in TMT-induced neurological damage. Based on this model, the cognitive ability of TMT exposed mice was assessed by the Morris water maze test and a passive avoidance task. The ultrastructure of hippocampus was analyzed by the transmission electron microscope. Subsequently, proteomics integrated with bioinformatics and experimental verification were employed to reveal potential mechanisms of TMT-induced neurotoxicity. Gene ontology (GO) and pathway enrichment analysis were done by using Metascape and GeneCards database respectively. Our results demonstrated that TMT-exposed mice exhibited cognitive disorder, and mitochondrial respiratory chain abnormality of the hippocampus. Proteomics data showed that a total of 7303 proteins were identified in hippocampus of mice of which 224 ones displayed a 1.5-fold increase or decrease in TMT exposed mice compared with controls. Further analysis indicated that these proteins were mainly involved in tricarboxylic acid (TCA) cycle and respiratory electron transport, proteasome degradation, and multiple metabolic pathways as well as inflammatory signaling pathways. Some proteins, including succinate-CoA ligase subunit (Suclg1), NADH dehydrogenase subunit 5 (Nd5), NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 4-like 2 (Ndufa4l2) and cytochrome c oxidase assembly factor 7 (Coa7), which were closely related to mitochondrial respiratory electron transport, showed TMT dose and time dependent changes in the hippocampus of mice. Moreover, apoptotic molecules Bax and cleaved caspase-3 were up-regulated, while anti-apoptotic Bcl-2 was down-regulated compared with controls. In conclusion, our findings suggest that impairment of mitochondrial respiratory chain transport and promotion of apoptosis are the potential mechanisms of TMT induced hippocampus toxicity in mice.

PRMT1 methylation of WTAP promotes multiple myeloma tumorigenesis by activating oxidative phosphorylation via m6A modification of NDUFS6.

Epigenetic modifications play important roles during the pathogenesis of multiple myeloma (MM). Herein, we found that protein arginine methyltransferase 1 (PRMT1) was highly expressed in MM patients, which was positively correlated with MM stages. High PRMT1 expression was correlated with adverse prognosis in MM patients. We further showed that silencing PRMT1 inhibited MM proliferation and tumorigenesis in vitro and in vivo. Mechanistically, we revealed that the knockdown of PRMT1 reduced the oxidative phosphorylation (OXPHOS) of MM cells through NDUFS6 downregulation. Meanwhile, we identified that WTAP, a key component of the m6A methyltransferase complex, was methylated by PRMT1, and NDUFS6 was identified as a bona fide m6A target of WTAP. Finally, we found that the combination of PRMT1 inhibitor and bortezomib synergistically inhibited MM progression. Collectively, our results demonstrate that PRMT1 plays a crucial role during MM tumorigenesis and suggeste that PRMT1 could be a potential therapeutic target in MM.

Mitochondrial integrated stress response controls lung epithelial cell fate.

Alveolar epithelial type 1 (AT1) cells are necessary to transfer oxygen and carbon dioxide between the blood and air. Alveolar epithelial type 2 (AT2) cells serve as a partially committed stem cell population, producing AT1 cells during postnatal alveolar development and repair after influenza A and SARS-CoV-2 pneumonia1-6. Little is known about the metabolic regulation of the fate of lung epithelial cells. Here we report that deleting the mitochondrial electron transport chain complex I subunit Ndufs2 in lung epithelial cells during mouse gestation led to death during postnatal alveolar development. Affected mice displayed hypertrophic cells with AT2 and AT1 cell features, known as transitional cells. Mammalian mitochondrial complex I, comprising 45 subunits, regenerates NAD+ and pumps protons. Conditional expression of yeast NADH dehydrogenase (NDI1) protein that regenerates NAD+ without proton pumping7,8 was sufficient to correct abnormal alveolar development and avert lethality. Single-cell RNA sequencing revealed enrichment of integrated stress response (ISR) genes in transitional cells. Administering an ISR inhibitor9,10 or NAD+ precursor reduced ISR gene signatures in epithelial cells and partially rescued lethality in the absence of mitochondrial complex I function. Notably, lung epithelial-specific loss of mitochondrial electron transport chain complex II subunit Sdhd, which maintains NAD+ regeneration, did not trigger high ISR activation or lethality. These findings highlight an unanticipated requirement for mitochondrial complex I-dependent NAD+ regeneration in directing cell fate during postnatal alveolar development by preventing pathological ISR induction.

Membrane Proteomics to Understand Enhancement Effects of Millimeter-Wave Irradiation on Wheat Root under Flooding Stress.

Millimeter-wave irradiation of wheat seeds enhances the growth of roots under flooding stress, but its mechanism is not clearly understood. To understand the role of millimeter-wave irradiation on root-growth enhancement, membrane proteomics was performed. Membrane fractions purified from wheat roots were evaluated for purity. H+-ATPase and calnexin, which are protein markers for membrane-purification efficiency, were enriched in a membrane fraction. A principal-component analysis of the proteomic results indicated that the millimeter-wave irradiation of seeds affects membrane proteins in grown roots. Proteins identified using proteomic analysis were confirmed using immunoblot or polymerase chain reaction analyses. The abundance of cellulose synthetase, which is a plasma-membrane protein, decreased under flooding stress; however, it increased with millimeter-wave irradiation. On the other hand, the abundance of calnexin and V-ATPase, which are proteins in the endoplasmic reticulum and vacuolar, increased under flooding stress; however, it decreased with millimeter-wave irradiation. Furthermore, NADH dehydrogenase, which is found in mitochondria membranes, was upregulated due to flooding stress but downregulated following millimeter-wave irradiation even under flooding stress. The ATP content showed a similar trend toward change in NADH dehydrogenase expression. These results suggest that millimeter-wave irradiation improves the root growth of wheat via the transitions of proteins in the plasma membrane, endoplasmic reticulum, vacuolar, and mitochondria.

Ocular stress enhances contralateral transfer of lenadogene nolparvovec gene therapy through astrocyte networks.

Lenadogene nolparvovec (GS010) was developed to treat a point mutation in mitochondrial ND4 that causes Leber hereditary optic neuropathy. GS010 delivers human cDNA encoding wild-type ND4 packaged into an rAAV2/2 vector that transduces retinal ganglion cells, to induce allotopic expression of hybrid mitochondrial ND4. GS010 clinical trials improved best-corrected visual acuity (BCVA) up to 5 years after treatment. Interestingly, unilateral treatment improved BCVA bilaterally. Subsequent studies revealed GS010 DNA in visual tissues contralateral to the injected eye, suggesting migration. Here we tested whether unilateral intraocular pressure (IOP) elevation could influence the transfer of viral ND4 RNA in contralateral tissues after GS010 delivery to the IOP-elevated eye and probed a potential mechanism mediating translocation in mice. We found IOP elevation enhanced viral ND4 RNA transcripts in contralateral visual tissues, including retinas. Using conditional transgenic mice, we depleted astrocytic gap junction connexin 43 (Cx43), required for distant redistribution of metabolic resources between astrocytes during stress. After unilateral IOP elevation and GS010 injection, Cx43 knockdown eradicated ND4 RNA transcript detection in contralateral retinal tissues, while transcript was still detectable in optic nerves. Overall, our study indicates long-range migration of GS010 product to contralateral visual tissues is enhanced by Cx43-linked astrocyte networks.

Proteomic Analysis of Skeletal Muscle and White Adipose Tissue after Aerobic Exercise Training in High Fat Diet Induced Obese Mice.

Obesity is associated with excessive fat accumulation in adipose tissue and other organs, such as skeletal muscle, whereas aerobic exercise (AE) plays an important role in managing obesity through profound protein regulation. Our study aimed to investigate the impact of AE on proteomic changes in both the skeletal muscle and the epididymal fat pad (EFP) of high-fat-diet-induced obese mice. Bioinformatic analyses were performed on differentially regulated proteins using gene ontology enrichment analysis and ingenuity pathway analysis. Eight weeks of AE significantly reduced body weight, increased the serum FNDC5 level, and improved the homeostatic model assessment of insulin resistance. A high-fat diet caused alterations in a subset of proteins involved in the sirtuin signaling pathway and the production of reactive oxygen species in both skeletal muscle and EFP, leading to insulin resistance, mitochondrial dysfunction, and inflammation. On the other hand, AE upregulated skeletal muscle proteins (NDUFB5, NDUFS2, NDUFS7, ETFD, FRDA, and MKNK1) that enhance mitochondrial function and insulin sensitivity. Additionally, the upregulation of LDHC and PRKACA and the downregulation of CTBP1 in EFP can promote the browning of white adipose tissue with the involvement of FNDC5/irisin in the canonical pathway. Our study provides insights into AE-induced molecular responses and may help further develop exercise-mimicking therapeutic targets.

A novel NADH-dependent leucine dehydrogenase for multi-step cascade synthesis of L-phosphinothricin.

L-Phosphinothricin (L-PPT) is the effective constituent in racemic PPT (a high-efficiency and broad-spectrum herbicide), and the exploitation of green and sustainable synthesis route for L-PPT has always been the focus in pesticide industry. In recent years, "one-pot, two-step" enzyme-mediated cascade strategy is a mainstream pathway to obtain L-PPT. Herein, RgDAAO and BsLeuDH were applied to expand "one-pot, two-step" process. Notably, a NADH-dependent leucine dehydrogenase from Bacillus subtilis (BsLeuDH) was firstly characterized and attempted to generate L-PPT, achieving an excellent enantioselectivity (99.9% ee). Meanwhile, a formate dehydrogenase from Pichia pastoris (PpFDH) was utilized to implement NADH cofactor regeneration and only CO2 was by-product. Sufficient amount of the corresponding keto acid precursor PPO was obtained by oxidation of D-PPT relying on a D-amino acid oxidase from Rhodotorula gracilis (RgDAAO) with content conversion (46.1%). L-PPT was ultimately prepared from racemized PPT via oxidative deamination catalyzed by RgDAAO and reductive amination catalyzed by BsLeuDH, achieving 80.3% overall yield and > 99.9% ee value.

Loss of respiratory complex I subunit NDUFB10 affects complex I assembly and supercomplex formation.

The orchestrated activity of the mitochondrial respiratory or electron transport chain (ETC) and ATP synthase convert reduction power (NADH, FADH2) into ATP, the cell's energy currency in a process named oxidative phosphorylation (OXPHOS). Three out of the four ETC complexes are found in supramolecular assemblies: complex I, III, and IV form the respiratory supercomplexes (SC). The plasticity model suggests that SC formation is a form of adaptation to changing conditions such as energy supply, redox state, and stress. Complex I, the NADH-dehydrogenase, is part of the largest supercomplex (CI + CIII2 + CIVn). Here, we demonstrate the role of NDUFB10, a subunit of the membrane arm of complex I, in complex I and supercomplex assembly on the one hand and bioenergetics function on the other. NDUFB10 knockout was correlated with a decrease of SCAF1, a supercomplex assembly factor, and a reduction of respiration and mitochondrial membrane potential. This likely is due to loss of proton pumping since the CI P P -module is downregulated and the P D -module is completely abolished in NDUFB10 knock outs.

The Interactions of the 70 kDa Fragment of Cell Adhesion Molecule L1 with Topoisomerase 1, Peroxisome Proliferator-Activated Receptor γ and NADH Dehydrogenase (Ubiquinone) Flavoprotein 2 Are Involved in Gene Expression and Neuronal L1-Dependent Functions.

The cell adhesion molecule L1 is essential not only for neural development, but also for synaptic functions and regeneration after trauma in adulthood. Abnormalities in L1 functions cause developmental and degenerative disorders. L1's functions critically depend on proteolysis which underlies dynamic cell interactions and signal transduction. We showed that a 70 kDa fragment (L1-70) supports mitochondrial functions and gene transcription. To gain further insights into L1-70's functions, we investigated several binding partners. Here we show that L1-70 interacts with topoisomerase 1 (TOP1), peroxisome proliferator-activated receptor γ (PPARγ) and NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2). TOP1, PPARγ and NDUFV2 siRNAs reduced L1-dependent neurite outgrowth, and the topoisomerase inhibitors topotecan and irinotecan inhibited L1-dependent neurite outgrowth, neuronal survival and migration. In cultured neurons, L1 siRNA reduces the expression levels of the long autism genes neurexin-1 (Nrxn1) and neuroligin-1 (Nlgn1) and of the mitochondrially encoded gene NADH:ubiquinone oxidoreductase core subunit 2 (ND2). In mutant mice lacking L1-70, Nrxn1 and Nlgn1, but not ND2, mRNA levels are reduced. Since L1-70's interactions with TOP1, PPARγ and NDUFV2 contribute to the expression of two essential long autism genes and regulate important neuronal functions, we propose that L1 may not only ameliorate neurological problems, but also psychiatric dysfunctions.