Mitochondrial Oxygen Consumption Rate Research Articles

Reversal of High-Fat Diet-Induced Non-Alcoholic Fatty Liver Disease by Metformin Combined with PGG, an Inducer of Glycine N-Methyltransferase.

Nonalcoholic fatty liver disease (NAFLD) is a major cause of liver-related morbidities and mortality, and no effective drug treatment currently exists. We aimed to develop a novel treatment strategy to induce the expression of glycine N-methyltransferase (GNMT), which is an important enzyme regulating S-adenosylmethionine metabolism whose expression is downregulated in patients with NAFLD. Because 1,2,3,4,6-pentagalloyl glucose (PGG) is a GNMT inducer, and metformin was shown to upregulate liver mitochondrial GNMT protein expression, the effect of PGG and metformin was evaluated. Biochemical analysis, histopathological examination, immunohistochemical staining, reverse transcription-quantitative PCR (RT-qPCR), Western blotting (WB), proteomic analysis and Seahorse XF Cell Mito Stress Test were performed. The high-fat diet (HFD)-induced NAFLD mice were treated with PGG and metformin. Combination of PGG and metformin nearly completely reversed weight gain, elevation of serum aminotransferases, and hepatic steatosis and steatohepatitis. In addition, the downregulated GNMT expression in liver tissues of HFD-induced NAFLD mice was restored. The GNMT expression was further confirmed by RT-qPCR and WB analysis using both in vitro and in vivo systems. In addition, PGG treatment was shown to increase oxygen consumption rate (OCR) maximum capacity in a dose-dependent manner, and was capable of rescuing the suppression of mitochondrial OCR induced by metformin. Proteomic analysis identified increased expression of glutathione S-transferase mu 4 (GSTM4), heat shock protein 72 (HSP72), pyruvate carboxylase (PYC) and 40S ribosomal protein S28 (RS28) in the metformin plus PGG treatment group. Our findings show that GNMT expression plays an important role in the pathogenesis of NAFLD, and combination of an inducer of GNMT and metformin can be of therapeutic potential for patients with NAFLD.

Piezo1 stimulates mitochondrial function via cAMP signaling.

Mechanical signals stimulate mitochondrial function but the molecular mechanisms are not clear. Here, we show that the mechanically sensitive ion channel Piezo1 plays a critical role in mitochondrial adaptation to mechanical stimulation. The activation of Piezo1 induced mitochondrial calcium uptake and oxidative phosphorylation (OXPHOS). In contrast, loss of Piezo1 reduced the mitochondrial oxygen consumption rate (OCR) and adenosine triphosphate (ATP) production in calvarial cells and these changes were associated with increased expression of the phosphodiesterases Pde4a and lower cyclic AMP (cAMP) levels. In addition, Piezo1 increased cAMP production and the activation of a cAMP-responsive transcriptional reporter. Consistent with this, cAMP was sufficient to increase mitochondrial OCR and the inhibition of phosphodiesterases augmented the increase in OCR induced by Piezo1. Moreover, the inhibition of cAMP production or activity of protein kinase A, a kinase activated by cAMP, prevented the increase in OCR induced by Piezo1. These results demonstrate that cAMP signaling contributes to the increase in mitochondrial OXPHOS induced by activation of Piezo1.

P12–20 Presentation of clear guidance for measuring mitochondrial oxygen consumption rate as a cardiotoxic indicator

P12–20 Presentation of clear guidance for measuring mitochondrial oxygen consumption rate as a cardiotoxic indicator

SZC-6, a small-molecule activator of SIRT3, attenuates cardiac hypertrophy in mice.

Sirtuin3 (SIRT3), a class III histone deacetylase, is implicated in various cardiovascular diseases as a novel therapeutic target. SIRT3 has been proven to be cardioprotective in a model of Ang II-induced cardiac hypertrophy. However, a few small-molecule compounds targeting deacetylases could activate SIRT3. In this study, we generated a novel SIRT3 activator, 3-(2-bromo-4-hydroxyphenyl)-7-hydroxy-2H-chromen-2-one (SZC-6), through structural optimization of the first SIRT3 agonist C12. We demonstrated that SZC-6 directly bound to SIRT3 with Kd value of 15 μM, and increased SIRT3 deacetylation activity with EC50 value of 23.2 ± 3.3 µM. In neonatal rat cardiomyocytes (NRCMs), pretreatment with SZC-6 (10, 20, 40 µM) dose-dependently attenuated isoproterenol (ISO)-induced hypertrophic responses. Administration of SZC-6 (20, 40 and 60 mg·kg-1·d-1, s.c.) for 2 weeks starting from one week prior ISO treatment dose-dependently reversed ISO-induced impairment of diastolic and systolic cardiac function in wild-type mice, but not in SIRT3 knockdown mice. We showed that SZC-6 (10, 20, 40 µM) dose-dependently inhibited cardiac fibroblast proliferation and differentiation into myofibroblasts, which was abolished in SIRT3-knockdown mice. We further revealed that activation of SIRT3 by SZC-6 increased ATP production and rate of mitochondrial oxygen consumption, and reduced ROS, improving mitochondrial function in ISO-treated NRCMs. We also found that SZC-6 dose-dependently enhanced LKB1 phosphorylation, thereby promoting AMPK activation to inhibit Drp1-dependent mitochondrial fragmentation. Taken together, these results demonstrate that SZC-6 is a novel SIRT3 agonist with potential value in the treatment of cardiac hypertrophy partly through activation of the LKB1-AMPK pathway.

Loss of mitochondrial fatty acid β-oxidation protein short-chain Enoyl-CoA hydratase disrupts oxidative phosphorylation protein complex stability and function.

Short-chain enoyl-CoA hydratase 1 (ECHS1) is involved in the second step of mitochondrial fatty acid β-oxidation (FAO), catalysing the hydration of short-chain enoyl-CoA esters to short-chain 3-hyroxyl-CoA esters. Genetic deficiency in ECHS1 (ECHS1D) is associated with a specific subset of Leigh Syndrome, a disease typically caused by defects in oxidative phosphorylation (OXPHOS). Here, we examined the molecular pathogenesis of ECHS1D using a CRISPR/Cas9 edited human cell 'knockout' model and fibroblasts from ECHS1D patients. Transcriptome analysis of ECHS1 'knockout' cells showed reductions in key mitochondrial pathways, including the tricarboxylic acid cycle, receptor-mediated mitophagy and nucleotide biosynthesis. Subsequent proteomic analyses confirmed these reductions and revealed additional defects in mitochondrial oxidoreductase activity and fatty acid β-oxidation. Functional analysis of ECHS1 'knockout' cells showed reduced mitochondrial oxygen consumption rates when metabolising glucose or OXPHOS complex I-linked substrates, as well as decreased complex I and complex IV enzyme activities. ECHS1 'knockout' cells also exhibited decreased OXPHOS protein complex steady-state levels (complex I, complex III2 , complex IV, complex V and supercomplexes CIII2 /CIV and CI/CIII2 /CIV), which were associated with a defect in complex I assembly. Patient fibroblasts exhibit varied reduction of mature OXPHOS complex steady-state levels, with defects detected in CIII2 , CIV, CV and the CI/CIII2 /CIV supercomplex. Overall, these findings highlight the contribution of defective OXPHOS function, in particular complex I deficiency, to the molecular pathogenesis of ECHS1D.

GOLM1 depletion modifies cellular sphingolipid metabolism and adversely affects cell growth

Golgi membrane protein 1 (GOLM1) is a Golgi-resident type 2 transmembrane protein known to be overexpressed in several cancers, including hepatocellular carcinoma (HCC), as well as in viral infections. However, the role of GOLM1 in lipid metabolism remains enigmatic. In this study, we employed siRNA-mediated GOLM1 depletion in Huh-7 HCC cells to study the role of GOLM1 in lipid metabolism. Mass spectrometric lipidomic analysis in GOLM1 knockdown cells showed an aberrant accumulation of sphingolipids, such as ceramides, hexosylceramides, dihexosylceramides, sphinganine, sphingosine, and ceramide phosphate, along with cholesteryl esters. Furthermore, we observed a reduction in phosphatidylethanolamines and lysophosphatidylethanolamines. In addition, Seahorse extracellular flux analysis indicated a reduction in mitochondrial oxygen consumption rate upon GOLM1 depletion. Finally, alterations in Golgi structure and distribution were observed both by electron microscopy imaging and immunofluorescence microscopy analysis. Importantly, we found that GOLM1 depletion also affected cell proliferation and cell cycle progression in Huh-7 HCC cells. The Golgi structural defects induced by GOLM1 reduction might potentially affect the trafficking of proteins and lipids leading to distorted intracellular lipid homeostasis, which may result in organelle dysfunction and altered cell growth. In conclusion, we demonstrate that GOLM1 depletion affects sphingolipid metabolism, mitochondrial function, Golgi structure, and proliferation of HCC cells.

Impact of metabolic stress induced by diets, aging and fasting on tissue oxygen consumption

ObjectiveAlterations in mitochondrial function play an important role in the development of various diseases, such as obesity, insulin resistance, steatohepatitis, atherosclerosis and cancer. However, accurate assessment of mitochondrial respiration ex vivo is limited and remains highly challenging. Using our novel method, we measured mitochondrial oxygen consumption (OCR) and extracellular acidification rate (ECAR) of metabolically relevant tissues ex vivo to investigate the impact of different metabolic stressors on mitochondrial function. MethodsComparative analyses of OCR and ECAR were performed in tissue biopsies of young mice fed 12 weeks standard-control (STD), high-fat (HFD), high-sucrose (HSD), or western diet (WD), matured mice with HFD, and 2year-old mice aged on STD with and without fasting. ResultsWhile diets had only marginal effects on mitochondrial respiration, respiratory chain complexes II and IV were reduced in adipose tissue (AT). Moreover, matured HFD-fed mice showed a decreased hepatic metabolic flexibility and prolonged aging increased OCR in brown AT. Interestingly, fasting boosted pancreatic and hepatic OCR while decreasing weight of those organs. Furthermore, ECAR measurements in AT could indicate its lipolytic capacity. ConclusionUsing ex vivo tissue measurements, we could extensively analyze mitochondrial function of liver, AT, pancreas and heart revealing effects of metabolic stress, especially aging.

Abstract P3029: Role Of Histone Deacetylase 9 In The Development Of Adipose Tissue Senescence And Mitochondrial Dysfunction

Background: The prevalence of obesity increases with age and is a major risk factor for the development of cardiovascular disease. Our previous studies showed that histone deacetylase 9 (HDAC9) gene deletion protected mice against obesity-associated metabolic disorder. Here, we hypothesized that HDAC9 expression during aging plays an important role in the development of adipose tissue senescence and mitochondrial dysfunction. Methods and Results: Visceral adipose tissue from 20-month-old wild type (WT) mice showed increased expression of HDAC9 (2-fold), p16 (5-fold) and p21 (2-fold) compared to 3-month-old mice. In addition, HDAC9 expression in human subcutaneous adipose tissue positively correlated with age (r 2 =0.088, p=0.034). Furthermore, 1-year-old HDAC9 knockout (KO) mice were protected against the development of age-associated weight gain compared to age-matched WT, in conjunction with reduced levels of senescence-associated beta-galactosidase (SABG) staining and lower expression of p16 (0.7-fold) and p21 (0.5-fold). Interestingly, preadipocytes, which reside in adipose tissue and replenish aged adipocytes, were protected against H2O2 and UV-light induced senescence as evaluated by SABG staining, p16 and p21 expression. Next, we measured adipose tissue mitochondrial function, which is mechanistically linked to senescence, using the Seahorse analyzer. HDAC9 KO significantly increased adipose tissue mitochondrial oxygen consumption rate (e.g., increased basal respiration, proton leak and ATP production). To gain further mechanistic insight, we performed RNA-sequencing of WT and KO adipose tissues and identified thiosulfate sulfurtransferase (TST), an enzyme that is reduced in human obesity and modulates mitochondrial function, as one of the potential mediators for adipose tissue senescence and metabolism. Interestingly, TST expression was reduced in 1-year-old compared to 3-month-old mice (0.2-fold) and enriched in KO compared to WT (4-fold) adipose tissue by qPCR analysis. Conclusion: HDAC9 plays a crucial role in the development of adipose tissue senescence, potentially through regulation of TST and mitochondrial function and may be a promising target to maintain healthy adipose tissue during aging.

The mitochondrial NAD+ transporter SLC25A51 is a fasting-induced gene affecting SIRT3 functions

IntroductionNicotinamide adenine dinucleotide (NAD) is a coenzyme central to metabolism and energy production. NAD+-dependent deacetylase sirtuin 3 (SIRT3) regulates the acetylation levels of mitochondrial proteins that are involved in mitochondrial homeostasis. Fasting up-regulates hepatic SIRT3 activity, which requires mitochondrial NAD+. What is the mechanism, then, to transport more NAD+ into mitochondria to sustain enhanced SIRT3 activity during fasting? ObjectiveSLC25A51 is a recently discovered mitochondrial NAD+ transporter. We tested the hypothesis that, during fasting, increased expression of SLC25A51 is needed for enhanced mitochondrial NAD+ uptake to sustain SIRT3 activity. Because the fasting-fed cycle and circadian rhythm are closely linked, we further tested the hypothesis that SLC25A51 is a circadian regulated gene. MethodsWe examined Slc25a51 expression in the liver of fasted mice, and examined its circadian rhythm in wild-type mice and those with liver-specific deletion of the clock gene BMAL1 (LKO). We suppressed Slc25a51 expression in hepatocytes and the mouse liver using shRNA-mediated knockdown, and then examined mitochondrial NAD+ levels, SIRT3 activities, and acetylation levels of SIRT3 target proteins (IDH2 and ACADL). We measured mitochondrial oxygen consumption rate using Seahorse analysis in hepatocytes with reduced Slc25a51 expression. ResultsWe found that fasting induced the hepatic expression of Slc25a51, and its expression showed a circadian rhythm-like pattern that was disrupted in LKO mice. Reduced expression of Slc25a51 in hepatocytes decreased mitochondrial NAD+ levels and SIRT3 activity, reflected by increased acetylation of SIRT3 targets. Slc25a51 knockdown reduced the oxygen consumption rate in intact hepatocytes. Mice with reduced Slc25a51 expression in the liver manifested reduced hepatic mitochondrial NAD+ levels, hepatic steatosis and hypertriglyceridemia. ConclusionsSlc25a51 is a fasting-induced gene that is needed for hepatic SIRT3 functions.

ARID5B influences B-cell development and function in mouse.

There is growing evidence for an inherited basis of susceptibility to childhood acute lymphoblastic leukemia. Genomewide association studies by us and others have identified non-coding acute lymphoblastic leukemia risk variants at the ARID5B gene locus, but the molecular mechanisms linking ARID5B to normal and malignant hematopoiesis remain largely unknown. Using a Vav1-driven transgenic mouse model, we characterized the role of Arid5b in hematopoiesis in vivo. Arid5b overexpression resulted in a dramatic reduction in the proportion of circulating B cells, immature, and mature Bcell fractions in the peripheral blood and the bone marrow, and also a decrease of follicular B cells in the spleen. There were significant defects in B-cell activation upon Arid5b overexpression in vitro with hyperactivation of B-cell receptor signaling at baseline. In addition, increased mitochondrial oxygen consumption rate of naïve or stimulated B cells of Arid5b-overexpressing mice was observed, compared to the rate of wild-type counterparts. Taken together, our results indicate that ARID5B may play an important role in B-cell development and function.

Pharmaceutical Agents for Contractile-Metabolic Dysfunction After Volumetric Muscle Loss.

Volumetric muscle loss (VML) injuries represent a majority of military service member casualties and are common in civilian populations following blunt and/or penetrating traumas. Characterized as a skeletal muscle injury with permanent functional impairments, there is currently no standard for rehabilitation, leading to lifelong disability. Toward developing rehabilitative strategies, previous research demonstrates that the remaining muscle after a VML injury lacks similar levels of plasticity or adaptability as healthy, uninjured skeletal muscle. This may be due, in part, to impaired innervation and vascularization of the remaining muscle, as well as disrupted molecular signaling cascades commonly associated with muscle adaptation. The primary objective of this study was to assess the ability of four pharmacological agents with a strong record of modulating muscle contractile and metabolic function to improve functional deficits in a murine model of VML injury. Male C57BL/6 mice underwent a 15% multimuscle VML injury of the posterior hindlimb and were randomized into drug treatment groups (formoterol [FOR], 5-aminoimidazole-4-carboxamide riboside [AICAR], pioglitazone [PIO], or sildenafil [SIL]) or untreated VML group. At the end of 60 days, the injury model was first validated by comparison to age-matched injury-naive mice. Untreated VML mice had 22% less gastrocnemius muscle mass, 36% less peak-isometric torque, and 27% less maximal mitochondrial oxygen consumption rate compared to uninjured mice (p < 0.01). Experimental drug groups were, then, compared to VML untreated, and there was minimal evidence of efficacy for AICAR, PIO, or SIL in improving contractile and metabolic functional outcomes. However, FOR-treated VML mice had 18% greater peak isometric torque (p < 0.01) and permeabilized muscle fibers had 36% greater State III mitochondrial oxygen consumption rate (p < 0.01) compared to VML untreated mice, suggesting an overall improvement in muscle condition. There was minimal evidence that these benefits came from greater mitochondrial biogenesis and/or mitochondrial complex protein content, but could be due to greater enzyme activity levels for complex I and complex II. These findings suggest that FOR treatment is candidate to pair with a rehabilitative approach to maximize functional improvements in VML-injured muscle. Impact statement Volumetric muscle loss (VML) injuries result in deficiencies in strength and mobility, which have a severe impact on patient quality of life. Despite breakthroughs in tissue engineering, there are currently no treatments available that can restore function to the affected limb. Our data show that treatment of VML injuries with clinically available and FDA-approved formoterol (FOR), a beta-agonist, significantly improves strength and metabolism of VML-injured muscle. FOR is therefore a promising candidate for combined therapeutic approaches (i.e., regenerative rehabilitation) such as pairing FOR with structured rehabilitation or cell-seeded biomaterials as it may provide greater functional improvements than either strategy alone.

Pharmacologicallyactive chemical compositeof Musa balbisiana ameliorates oxidative stress, mitochondrial cellular respiration, and thereby metabolic dysfunction.

The ripe fruit pulp of different Musa species is known for its excellent source of nutrient contents. Musa balbisiana (MB) is one such variety of Musa species, mainly found in the southern part of Asia, especially in the North-eastern part of India, remains unexplored despite its continuous use by the local traditional healers. The present study focuses on identifying and quantifying the active chemicals present in the ripe fruit pulp of Musa balbisiana (RFPMB) to understand its combined efficacy and nutritional benefit to control human metabolic complications specially related to diabetes and cardiovascular disorder. Characterization and confirmation through targeted LC-MS and HPLC-PDA based assays followed by quantitative analysis led us to identify the major bioactive compounds in RFPMB as shikimic acid, p-hydroxybenzoic acid, vanillic acid, ferulic acid, sinapic acid, caffeic acid, syringic acid, chlorogenic acid, trans-cinnamic acid, and two essential fatty acids; linolenic acid and linoleic acid. The ripe fruit pulp is further analyzed to understand the nutritional and mineral content and found a substantial presence of calcium and potassium (15.74 ± 0.43 and 395.20 ± 9.5mg/100 g of raw pulp, respectively) compared to other reported varieties. The active portion of RFPMB reduces the production of ROS, the expression of inflammatory marker genes TNF-α and TGF-β, and accelerates the mitochondrial oxygen consumption rate (OCR) by enhancing the basal respiration, maximal respiration, and ATP production capacity of the targeted cells. The present study concluded that, a particular phytopharmaceutical composition of RFPMB with 11-biomarker compounds might be an efficacious formulation for developing a value-added nutraceutical product in managing metabolic complications and its related oxidative stress. PRACTICAL APPLICATIONS: This study has provided the prior information regarding the potential nutraceutical and phytochemical advantages of Musa balbisiana (MB) fruit pulp over other reported banana varieties. The HPLC-based quantification will give a clear understanding of the food values in comparison of bioactive compounds present in the active fraction of RFPMB, which can be an effective phytopharmaceutical in combating metabolic disorders and oxidative stress. Overall this study will help to commercialize a value-added product from this variety of banana with proper scientific validation.

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

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

Associations between fatigue and cellular metabolism in breast cancer patients: A longitudinal study

PurposeFatigue is frequently experienced during treatment for cancer and persists for months to years after treatment completion in a subset of patients. The underlying mechanisms remain poorly understood. We postulated that reduced cellular energy metabolism may underlie fatigue in cancer patients and survivors and tested this hypothesis in a sample of patients newly diagnosed with early-stage breast cancer (n = 49) followed for approximately 1 year from before the start of neoadjuvant chemotherapy (NACT) till after treatment completion. MethodsPatient-reported fatigue was assessed with the Checklist Individual Strength, and blood samples were obtained before, during, and shortly after NACT. A final assessment was completed after surgery and radiation therapy, 4–6 months after NACT. At each study time point, mitochondrial oxygen consumption and glycolytic activity were measured in peripheral blood mononuclear cells (PBMC). Associations of these measures of PBMC energy metabolism with fatigue were assessed in multilevel models. ResultsBefore NACT, higher mitochondrial oxygen consumption and glycolytic activity were associated with higher fatigue, whereas after completion of all primary treatment, these assessments were associated with lower fatigue. ConclusionThese findings suggest that lower cellular energy metabolism after treatment may be a novel target for interventions aimed at preventing or reducing persistent fatigue. Earlier studies investigated the use of supplements for maintaining mitochondrial health during treatment, with mixed results; when proven to be safe, such interventions may be more effective after treatment and in individuals with reduced mitochondrial oxygen consumption rates.

Biophysical and modeling-based approach for the identification of inhibitors against DOHH from Leishmania donovani.

The amino acid hypusine (Nε-4-amino-2-hydroxybutyl(lysine)) occurs only in isoforms of eukaryotic translation factor 5A (eIF5A) and has a role in initiating protein translation. Hypusinated eIF5A promotes translation and modulates mitochondrial function and oxygen consumption rates. The hypusination of eIF5A involves two enzymes, deoxyhypusine synthase and deoxyhypusine hydroxylase (DOHH). DOHH is the second enzyme that completes the synthesis of hypusine and the maturation of eIF5A. Our current study aims to identify inhibitors against DOHH from Leishmania donovani (LdDOHH), an intracellular protozoan parasite causing Leishmaniasis in humans. The LdDOHH protein was produced heterologously in Escherichia coli BL21(DE3) cells and characterized biochemically. The three-dimensional structure was predicted, and the compounds folic acid, scutellarin and homoarbutin were selected as top hits in virtual screening. These compounds were observed to bind in the active site of LdDOHH stabilizing the structure by making hydrogen bonds in the active site, as observed by the docking and molecular dynamics simulation studies. These results pave the path for further investigation of these molecules for their anti-leishmanial activities.

A Non-Canonical Role for IRE1α Links ER and Mitochondria as Key Regulators of Astrocyte Dysfunction: Implications in Methamphetamine use and HIV-Associated Neurocognitive Disorders

Astrocytes are one of the most numerous glial cells in the central nervous system (CNS) and provide essential support to neurons to ensure CNS health and function. During a neuropathological challenge, such as during human immunodeficiency virus (HIV)-1 infection or (METH)amphetamine exposure, astrocytes shift their neuroprotective functions and can become neurotoxic. Identifying cellular and molecular mechanisms underlying astrocyte dysfunction are of heightened importance to optimize the coupling between astrocytes and neurons and ensure neuronal fitness against CNS pathology, including HIV-1-associated neurocognitive disorders (HAND) and METH use disorder. Mitochondria are essential organelles for regulating metabolic, antioxidant, and inflammatory profiles. Moreover, endoplasmic reticulum (ER)-associated signaling pathways, such as calcium and the unfolded protein response (UPR), are important messengers for cellular fate and function, including inflammation and mitochondrial homeostasis. Increasing evidence supports that the three arms of the UPR are involved in the direct contact and communication between ER and mitochondria through mitochondria-associated ER membranes (MAMs). The current study investigated the effects of HIV-1 infection and chronic METH exposure on astrocyte ER and mitochondrial homeostasis and then examined the three UPR messengers as potential regulators of astrocyte mitochondrial dysfunction. Using primary human astrocytes infected with pseudotyped HIV-1 or exposed to low doses of METH for 7 days, astrocytes had increased mitochondrial oxygen consumption rate (OCR), cytosolic calcium flux and protein expression of UPR mediators. Notably, inositol-requiring protein 1α (IRE1α) was most prominently upregulated following both HIV-1 infection and chronic METH exposure. Moreover, pharmacological inhibition of the three UPR arms highlighted IRE1α as a key regulator of astrocyte metabolic function. To further explore the regulatory role of astrocyte IRE1α, astrocytes were transfected with an IRE1α overexpression vector followed by activation with the proinflammatory cytokine interleukin 1β. Overall, our findings confirm IRE1α modulates astrocyte mitochondrial respiration, glycolytic function, morphological activation, inflammation, and glutamate uptake, highlighting a novel potential target for regulating astrocyte dysfunction. Finally, these findings suggest both canonical and non-canonical UPR mechanisms of astrocyte IRE1α. Thus, additional studies are needed to determine how to best balance astrocyte IRE1α functions to both promote astrocyte neuroprotective properties while preventing neurotoxic properties during CNS pathologies.

Selective Probiotic Treatment Positively Modulates the Microbiota-Gut-Brain Axis in the BTBR Mouse Model of Autism.

Recent studies have shown promise for the use of probiotics in modulating behaviour through the microbiota–gut–brain axis. In the present study, we assessed the impact of two probiotic strains in mitigating autism-related symptomology in the BTBR T+ Itpr3tf/J mouse model of autism spectrum disorder (ASD). Male juvenile BTBR mice were randomized into: (1) control, (2) Lr probiotic (1 × 109 CFU/mL Lacticaseibacillus rhamnosus HA-114), and (3) Ls probiotic groups (1 × 109 CFU/mL Ligilactobacillus salivarius HA-118) (n = 18–21/group), receiving treatments in drinking water for 4 weeks. Gut microbiota profiling by 16S rRNA showed Lr, but not Ls supplementation, to increase microbial richness and phylogenetic diversity, with a rise in potential anti-inflammatory and butyrate-producing taxa. Assessing serum and brain metabolites, Lr and Ls supplementation produced distinct metabolic profiles, with Lr treatment elevating concentrations of potentially beneficial neuroactive compounds, such as 5-aminovaleric acid and choline. As mitochondrial dysfunction is often observed in ASD, we assessed mitochondrial oxygen consumption rates in the prefrontal cortex and hippocampus. No differences were observed for either treatment. Both Lr and Ls treatment reduced behavioural deficits in social novelty preference. However, no changes in hyperactivity, repetitive behaviour, and sociability were observed. Results show Lr to impart positive changes along the microbiota–gut–brain axis, exhibiting beneficial effects on selected behaviour, gut microbial diversity, and metabolism in BTBR mice.

Ginsenoside Rb1 inhibits astrocyte activation and promotes transfer of astrocytic mitochondria to neurons against ischemic stroke

Astrocytes activation in response to stroke results in altered mitochondrial exchange with neurons. Ginsenoside Rb1is a major ginsenoside of Panax ginseng particularly known for its neuroprotective potential. This work aimed to investigate if Rb1 could rescue neurons from ischemic insult via astrocyte inactivation and mitochondrial transfer. We prepared conditioned astrocytes-derived medium for co-culture with neurons and examined the role of Rb1 in mitochondrial transfer from astrocytes to neurons. The neuroprotective potential of Rb1 was further confirmed in vivo using a mouse model of brain ischemia. In response to oxygen-glucose deprivation and reperfusion (OGD/R), astrocytes were reactivated and produced reactive oxygen species (ROS), an action that was blocked by Rb1. Mechanistically, Rb1 inhibited NADH dehydrogenase in mitochondrial complex I to block reverse electron transport-derived ROS production from complex I, and thus inactivated astrocytes to protect the mitochondria. Mitochondrial signal, mitochondrial membrane potential and ATP production detected in conditioned astrocyte-derived medium indicated that Rb1 protected functional mitochondria and facilitated their transfer. When neurons were injured by OGD/R insult, co-culturing with conditioned medium increased mitochondrial membrane potential and oxygen consumption rate within the neurons, indicating the protection conferred on them by Rb1 via mitochondrial transfer from astrocytes. Using the ischemic mouse brain model, CD38 knockdown in the cerebral ventricles diminished the neuroprotective effects of Rb1, providing evidence in support of the role of astrocyte mitochondrial transfer. Transient inhibition of mitochondrial complex I by Rb1 reduced mitochondrial ROS production and consequently avoided astrocyte activation. Astrocyte mitochondrial transfer therefore seemed a means by which Rb1 could promote neuronal survival and function. Different from the neurocentric view, these findings suggest the astrocytes may be a promising target for pharmacological interventions in ischemic brain injury.

463-P: Effects of Imeglimin, a New Oral Hyperglycemic Agent, on High Glucose and Low Glucose–Induced Cell Death and Mitochondrial Dysfunction in Schwann Cells

It is known that hyperglycemia-induced oxidative stress is a major cause of the pathogenesis of diabetic neuropathy, and hyperglycemia-induced mitochondrial ROS production is considered as one pivotal mechanism of increased oxidative stress. On the other hand, imeglimin is a new class of oral hypoglycemic agents called “glymines” containing tetrahydrotriazines, which targets mitochondrial dysfunction. We investigated the effects of imeglimin on cell death and mitochondrial dysfunction induced by high glucose and low glucose in Schwann cells. Immortalized adult mouse Schwann (IMS32) cells were cultured for 1 hour in the medium with normal glucose concentration of 5.5 mM, low glucose of 2.5 mM, and high glucose of 25 mM. Compared to normal glucose, not only high glucose but also low glucose decreased cell viability of IMS32 cells and increased mitochondrial oxidative stress. Mitochondrial membrane potential was also decreased either by high glucose or low glucose. In addition, mitochondrial oxygen consumption rate (OCR) was increased by low glucose as well as high glucose. Mitochondria isolated from IMS32 cells exposed to high glucose and low glucose for 1 hour exhibited increased activity of complex I. The expression of complex I protein was unchanged by high glucose for 1 hour. Imeglimin ameliorated the decrease in cell viability and improved the mitochondrial dysfunctions via inhibiting the increase of mitochondrial oxidative stress, OCR and the activity of complex I caused by high glucose and low glucose. These results indicate that hyperglycemia and hypoglycemia induce mitochondrial dysfunction in nerves, and that imeglimin may prevent against diabetic neuropathy by attenuation mitochondrial dysfunction and cell death in Schwann cells. Disclosure T. Himeno: None. J. Nakamura: None. H. Kamiya: Research Support; Abbott Japan Co., Ltd., Eli Lilly and Company, Japan Tobacco Inc., Kissei Pharmaceutical Co., Ltd., Kowa Company, Ltd., Merck &amp; Co., Inc., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk, Ono Pharmaceutical Co., Ltd., Sanwa Kagaku Kenkyusho, Sumitomo Dainippon Pharma Co., Ltd., Taiho Pharmaceutical Co. Ltd., Takeda Pharmaceutical Company Limited, Terumo Corporation. Speaker's Bureau; Astellas Pharma Inc., AstraZeneca, Boehringer Ingelheim International GmbH, Daiichi Sankyo, Eli Lilly and Company, Kissei Pharmaceutical Co., Ltd., Kowa Company, Ltd., Merck &amp; Co., Inc., Mitsubishi Tanabe Pharma Corporation, Novartis Pharma K.K., Novo Nordisk, Ono Pharmaceutical Co., Ltd., Sanofi K.K., Sanwa Kagaku Kenkyusho, Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Holdings Co., Ltd. K. Sango: None. K. Kato: None.

Implementation of a combined CDK inhibition and arginine-deprivation approach to target arginine-auxotrophic glioblastoma multiforme cells

Constitutive activation of cyclin-dependent kinases (CDKs) or arginine auxotrophy are hallmarks of Glioblastoma multiforme (GBM). The latter metabolic defect renders tumor cells vulnerable to arginine-depleting substances, such as arginine deiminase from Streptococcus pyogenes (SpyADI). Previously, we confirmed the susceptibility of patient-derived GBM cells towards SpyADI as well as CDK inhibitors (CDKis). To improve therapeutic effects, we here applied a combined approach based on SpyADI and CDKis (dinaciclib, abemaciclib). Three arginine-auxotrophic patient-derived GBM lines with different molecular characteristics were cultured in 2D and 3D and effects of this combined SpyADI/CDKi approach were analyzed in-depth. All CDKi/SpyADI combinations yielded synergistic antitumoral effects, especially when given sequentially (SEQ), i.e., CDKi in first-line and most pronounced in the 3D models. SEQ application demonstrated impaired cell proliferation, invasiveness, and viability. Mitochondrial impairment was demonstrated by increasing mitochondrial membrane potential and decreasing oxygen consumption rate and extracellular acidification rate after SpyADI/abemaciclib monotherapy or its combination regimens. The combined treatment even induced autophagy in target cells (abemaciclib/SpyADI > dinaciclib/SpyADI). By contrast, the unfolded protein response and p53/p21 induced senescence played a minor role. Transmission electron microscopy confirmed damaged mitochondria and endoplasmic reticulum together with increased vacuolization under CDKi mono- and combination therapy. SEQ-abemaciclib/SpyADI treatment suppressed the DSB repair system via NHEJ and HR, whereas SEQ-dinaciclib/SpyADI treatment increased γ-H2AX accumulation and induced Rad51/Ku80. The latter combination also activated the stress sensor GADD45 and β-catenin antagonist AXIN2 and induced expression changes of genes involved in cellular/cytoskeletal integrity. This study highlights the strong antitumoral potential of a combined arginine deprivation and CDK inhibition approach via complex effects on mitochondrial dysfunction, invasiveness as well as DNA-damage response. This provides a good starting point for further in vitro and in vivo proof-of-concept studies to move forward with this strategy.