Altered Mitochondrial Dynamics Research Articles

N-oleoylethanolamide treatment of lymphoblasts deficient in Tafazzin improves cell growth and mitochondrial morphology and dynamics.

Barth syndrome (BTHS) is caused by mutations in the TAZ gene encoding the cardiolipin remodeling enzyme, Tafazzin. The study objective was to quantitatively examine growth characteristics and mitochondrial morphology of transformed lymphoblast cell lines derived from five patients with BTHS relative to five healthy controls, as well as the therapeutic potential of oleoylethanolamide (OEA) and linoleoylethanolamide (LEA). These bioactive lipids both activate PPARα, which may be therapeutic. BTHS lymphoblasts grew more slowly than controls, suggesting lymphopenia merits clinical investigation. Treatment of BTHS lymphoblasts with OEA, but not LEA, significantly restored mitochondrial membrane potential, as well as colony growth in all BTHS lymphoblast lines, although a full growth rescue was not achieved. Quantification analysis of electron micrographs from three BTHS and healthy lymphoblast donors indicated similar numbers of mitochondria per cell, but lower average cristae length per mitochondrion, and higher mitochondrial density. Additionally, BTHS lymphoblasts had larger mitochondria, and a higher percentage of abnormally large mitochondria (> 1 μm2) than healthy controls. Notably, OEA treatment significantly restored mitochondrial size, without affecting density or cristae lengths. Cardiolipin total content, relative linoleic acid content and monolysocardiolipin:cardiolipin ratios were not improved by OEA, indicating that effects on growth, and mitochondrial morphology and function, occurred without resolving this deficit. However, immunoblotting showed higher levels of OPA1, a biomarker for mitochondrial fusion, in BTHS lymphoblasts, which was attenuated by OEA treatment, implicating altered mitochondrial dynamics in the pathology and treatment of BTHS.

Chronic inhibition of the mitochondrial ATP synthase in skeletal muscle triggers sarcoplasmic reticulum distress and tubular aggregates

Tubular aggregates (TA) are honeycomb-like arrays of sarcoplasmic-reticulum (SR) tubules affecting aged glycolytic fibers of male individuals and inducing severe sarcomere disorganization and muscular pain. TA develop in skeletal muscle from Tubular Aggregate Myopathy (TAM) patients as well as in other disorders including endocrine syndromes, diabetes, and ageing, being their primary cause unknown. Nowadays, there is no cure for TA. Intriguingly, both hypoxia and calcium dyshomeostasis prompt TA formation, pointing to a possible role for mitochondria in their setting. However, a functional link between mitochondrial dysfunctions and TA remains unknown. Herein, we investigate the alteration in muscle-proteome of TAM patients, the molecular mechanism of TA onset and a potential therapy in a preclinical mouse model of the disease. We show that in vivo chronic inhibition of the mitochondrial ATP synthase in muscle causes TA. Upon long-term restrained oxidative phosphorylation (OXPHOS), oxidative soleus experiments a metabolic and structural switch towards glycolytic fibers, increases mitochondrial fission, and activates mitophagy to recycle damaged mitochondria. TA result from the overresponse of the fission controller DRP1, that upregulates the Store-Operate-Calcium-Entry and increases the mitochondria-SR interaction in a futile attempt to buffer calcium overloads upon prolonged OXPHOS inhibition. Accordingly, hypoxic muscles cultured ex vivo show an increase in mitochondria/SR contact sites and autophagic/mitophagic zones, where TA clusters grow around defective mitochondria. Moreover, hypoxia triggered a stronger TA formation upon ATP synthase inhibition, and this effect was reduced by the DRP1 inhibitor mDIVI. Remarkably, the muscle proteome of TAM patients displays similar alterations in mitochondrial dynamics and in ATP synthase contents. In vivo edaravone treatment in mice with restrained OXPHOS restored a healthy phenotype by prompting mitogenesis and mitochondrial fusion. Altogether, our data provide a functional link between the ATP synthase/DRP1 axis and the setting of TA, and repurpose edaravone as a possible treatment for TA-associated disorders.

Altered Mitochondrial Dynamics in Hepatocytes Induces Nonalcoholic Fatty Liver Disease Progression

Nonalcoholic fatty liver disease (NAFLD) is a hepatic manifestation of metabolic syndrome that affects approximately 25% of adults globally. Although several pharmacological therapies have been reported to improve NAFLD, the therapeutic ranges of the drugs are limited. Mitochondrial dysfunction could be a key factor involved in NAFLD progression. However, the association between mitochondrial dynamics, which involves mitochondrial fission and fusion, and NAFLD development has not been fully characterized to date. Herein, we examined the effects of alterations of mitochondrial dynamics in hepatocytes and liver sinusoidal endothelial cells (LSECs), the most abundant non‐parenchymal cells in the liver, on NAFLD development and hypothesized that rebalancing of mitochondrial dynamics in these cells could attenuate NAFLD progression. Mice were fed a choline‐deficient, L‐amino acid‐defined diet (CDAA) for 1 or 4 weeks to recapitulate human NAFLD development. Malformed mitochondria, such as megamitochondria and mitochondria with deformed cristae, were substantially increased in both, hepatocytes and LSECs, of mice that were fed CDAA diet, and were associated with NAFLD development. In addition, decreased mitochondrial content and increased mitochondrial number, suggesting excessive mitochondrial fission, were induced in hepatocytes, but not in LSECs, after four weeks of CDAA feeding. These unfavorable changes in the mitochondria of hepatocytes, but not of LSECs, were attenuated by mdivi1, a chemical mitochondrial fission inhibitor. Liver inflammation and fibrosis were also attenuated by mdivi1, as assessed by serum biochemical and histochemical analyses. The expression of several inflammatory and fibrogenic genes (Tnfα, Il6, and Col3α1) tended to decrease with mdivi1 treatment. The expression of genes related to mitochondrial biogenesis, fission, fusion, and mitophagy was also significantly reduced by mdivi1 (p<0.05), suggesting rebalanced mitochondrial dynamics. Ultrastructural analyses revealed that endoplasmic reticulum stress and autophagy in hepatocytes were mitigated by mdivi1 administration. Taken together, these findings suggest that excessive mitochondrial fission in hepatocytes induces alterations in mitochondrial dynamics and other organellar injuries, and subsequently, liver inflammation and fibrogenesis. Therefore, rebalancing mitochondrial dynamics in hepatocytes by inhibiting excessive fission is a promising therapeutic strategy for NAFLD.

The pathological progression of repetitive and mild traumatic brain injury in mice

Traumatic brain injury (TBI) is defined as an impact to the head by an external force that causes brain alterations and subsequent long‐term functional deficits. TBI contributes to an economic burden of $17 billion USD annually and is a leading cause of death and disability for individuals under 45. The severity of TBI varies from mild to severe with repetitive and mild (rm) TBI, and accounts for the highest percentage of TBI‐cases, leading to long‐term cognitive impairment. There are no current treatment(s) for repetitive and mild TBI, therefore, we sought to identify novel signaling molecules/pathways that could contribute to TBI.We employed a clinically relevant (non‐surgical) closed‐head impact model of engineered rotational acceleration (CHIMERA) that allows free rotation of the head upon impact generated from an air‐compressed piston. Our data suggest a novel role for PRMT7 (protein arginine methyltransferases) in the disease progression as indicated by the temporal decrease in protein expression post‐rmTBI. Our central hypothesis is that the loss of PRMT7, due to repetitive and mild TBI, mediates excitotoxicity, increased cellular death, disturbed mitochondrial dynamics and contributes to behavioral deficits. PRMTs are novel targets that catalyze the methylation of arginine residues (a constitutive post‐translational modification) involved in transcription, translation, receptor trafficking, and protein stability. There are currently 11 known PRMT isoforms (PRMT1‐11), with PRMT7 gene deletion in human patients causing neurological deficits such as intellectual disability, microcephaly, and brachydactyly, along with hyperexcitability and impaired social behaviors in murine in vivo models.We assessed diffuse axonal injury in our model of mild and repetitive TBI (via CHIMERA) to suggest enhanced silver deposition (dark stained regions) throughout the brain, similar to human pathology. Next, we measured PRMT7 protein levels that were decreased in the cortex and hippocampus 7‐days post‐rmTBI. Relative PRMT7 mRNA (via real‐time qPCR) was enhanced in the cortex 1‐day post‐rmTBI. Using LC‐MS, we measured excitatory neurotransmitters to suggest that glutamate was enhanced in the hippocampus 3‐day post‐rmTBI. In addition, mitochondrial fission and fusion was assessed by measuring DRP1 and OPA1 and our results indicated increased polarization towards fission as indicated by significant increase of DRP1 protein expression 1,3,7 days post rmTBI. Furthermore, mitochondrial oxygen consumption rates were analyzed via Seahorse XF analyzer and indicated dynamic changes in ATP‐linked respiration and maximal respiration 1 and 3 days post‐rmTBI. Finally, learning, working memory, and locomotor skills were significantly impaired as indicated by decreased alternation ratios via T‐maze, novel object recognition, and rotarod assessment. Overall, our results suggest that PRMT7 can mediate neuronal hyperexcitability, altered mitochondrial dynamics and can affect functional outcomes post‐rmTBI.

Differential regulation of L84F SOD1 in motor neurons and skeletal muscles: an insight into ALS pathology

Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disorder characterized by progressive deterioration of motor neurons leading to skeletal muscle denervation. Recent evidences have highlighted early involvement of neuromuscular junction (NMJ) in ALS pathogenesis. NMJ is a specialized tripartite chemical synapse which involves a well‐coordinated communication among the presynaptic motor neuron, postsynaptic skeletal muscle and terminal Schwann cells. Despite several evidences, series of events triggering NMJ disassembly in ALS are still obscure.The aim of our study is to investigate the cellular and molecular mechanisms of NMJ dysfunction in an in vitro Superoxide Dismutase 1 (SOD1) induced model of familial ALS. We have generated an ALS cell model based on a novel SOD1 mutation (L84F) identified previously in a family with a history of ALS. This mutation caused decrease in neurite outgrowth which led us to hypothesize that it may be involved in neuromuscular junction disassembly in ALS pathology.We have characterized cellular components of NMJ by differentiating hybrid spinal cord cell line (NSC‐34) into motor neurons and myoblast cell line (C2C12) into myotubes. Expression of mutant SOD1 led to decrease in mitochondrial footprints in axons and synaptic terminals and altered mitochondrial dynamics in motor neurons. However mitochondrial fission/fusion is not altered in muscles suggesting differential consequences of mutant protein. L84F SOD1 mutant readily formed detergent‐resistant aggregates in motor neurons but failed to accumulate in skeletal muscles. Skeletal muscles displayed increased levels of LC3‐II compared to motor neurons suggesting a robust proteasome machinery of muscles is responsible for clearance of mutant protein. Furthermore, motor neurons exhibited increased cell death compared to skeletal muscles when subjected to exogenous stress. Interestingly, we observed that condition media from skeletal muscles was able to rescue motor neurons from exogenous stress. We are currently identifying paracrine neuroprotective factors secreted by the muscles. It is noteworthy that mutant SOD1 led to altered expression of post synaptic acetylcholine receptor complex which might hamper neuromuscular transmission in ALS.To conclude, molecular cues from both neurons and muscles appear to play a role in compromising NMJ integrity in ALS.

Mitofusins Mfn1 and Mfn2 Are Required to Preserve Glucose- but Not Incretin-Stimulated β-Cell Connectivity and Insulin Secretion.

Mitochondrial glucose metabolism is essential for stimulated insulin release from pancreatic β-cells. Whether mitofusin gene expression, and hence, mitochondrial network integrity, is important for glucose or incretin signaling has not previously been explored. Here, we generated mice with β-cell–selective, adult-restricted deletion knock-out (dKO) of the mitofusin genes Mfn1 and Mfn2 (βMfn1/2 dKO). βMfn1/2-dKO mice displayed elevated fed and fasted glycemia and a more than fivefold decrease in plasma insulin. Mitochondrial length, glucose-induced polarization, ATP synthesis, and cytosolic and mitochondrial Ca2+ increases were all reduced in dKO islets. In contrast, oral glucose tolerance was more modestly affected in βMfn1/2-dKO mice, and glucagon-like peptide 1 or glucose-dependent insulinotropic peptide receptor agonists largely corrected defective glucose-stimulated insulin secretion through enhanced EPAC-dependent signaling. Correspondingly, cAMP increases in the cytosol, as measured with an Epac-camps–based sensor, were exaggerated in dKO mice. Mitochondrial fusion and fission cycles are thus essential in the β-cell to maintain normal glucose, but not incretin, sensing. These findings broaden our understanding of the roles of mitofusins in β-cells, the potential contributions of altered mitochondrial dynamics to diabetes development, and the impact of incretins on this process.

Chemotherapy impairs skeletal muscle mitochondrial homeostasis in early breast cancer patients.

BackgroundChemotherapy is extensively used to treat breast cancer and is associated with skeletal muscle deconditioning, which is known to reduce patients' quality of life, treatment efficiency, and overall survival. To date, skeletal muscle mitochondrial alterations represent a major aspect explored in breast cancer patients; nevertheless, the cellular mechanisms remain relatively unknown. This study was dedicated to investigating overall skeletal muscle mitochondrial homeostasis in early breast cancer patients undergoing chemotherapy, including mitochondrial quantity, function, and dynamics.MethodsWomen undergoing (neo)adjuvant anthracycline‐cyclophosphamide and taxane‐based chemotherapy participated in this study (56 ± 12 years). Two muscle biopsies were collected from the vastus lateralis muscle before the first and after the last chemotherapy administration. Mitochondrial respiratory capacity, reactive oxygen species production, and western blotting analyses were performed.ResultsAmong the 11 patients, we found a decrease in key markers of mitochondrial quantity, reaching −52.0% for citrate synthase protein levels (P = 0.02) and −38.2% for VDAC protein levels (P = 0.04). This mitochondrial content loss is likely explained by reduced mitochondrial biogenesis, as evidenced by a decrease in PGC‐1α1 protein levels (−29.5%; P = 0.04). Mitochondrial dynamics were altered, as documented by a decrease in MFN2 protein expression (−33.4%; P = 0.01), a key marker of mitochondrial outer membrane fusion. Mitochondrial fission is a prerequisite for mitophagy activation, and no variation was found in either key markers of mitochondrial fission (Fis1 and DRP1) or mitophagy (Parkin, PINK1, and Mul1). Two contradictory hypotheses arise from these results: defective mitophagy, which probably increases the number of damaged and fragmented mitochondria, or a relative increase in mitophagy through elevated mitophagic potential (Parkin/VDAC ratio; +176.4%; P < 0.02). Despite no change in mitochondrial respiratory capacity and COX IV protein levels, we found an elevation in H2O2 production (P < 0.05 for all substrate additions) without change in antioxidant enzymes. We investigated the apoptosis pathway and found an increase in the protein expression of the apoptosis initiation marker Bax (+72.0%; P = 0.04), without variation in the anti‐apoptotic protein Bcl‐2.ConclusionsThis study demonstrated major mitochondrial alterations subsequent to chemotherapy in early breast cancer patients: (i) a striking reduction in mitochondrial biogenesis, (ii) altered mitochondrial dynamics and potential mitophagy defects, (iii) exacerbated H2O2 production, and (iv) increased initiation of apoptosis. All of these alterations likely explain, at least in part, the high prevalence of skeletal muscle and cardiorespiratory deconditioning classically observed in breast cancer patients.

Mitochondrial Dynamics in the Metabolic Memory of Diabetic Retinopathy.

Mitochondria play a central role in the development of diabetic retinopathy and in the metabolic memory associated with its continued progression. Mitochondria have a regulated fusion fission process, which is essential for their homeostasis. One of the major fission proteins, dynamin-related protein 1 (Drp1), is recruited to the mitochondria by fission protein 1 (Fis1) to initiate fragmentation. Our aim is to investigate the role of Drp1 in the altered mitochondrial dynamics in the continued progression of diabetic retinopathy. Methods. Drp1 activation, mitochondrial transport, and Drp1-Fis1 interactions were analyzed in retinal endothelial cells incubated in 20 mM glucose (HG), followed by 5 mM glucose (NG), for four days each (HG-NG group). The results were confirmed in retinal microvessels from streptozotocin-induced diabetic rats with poor glycemia (>350 mg/dl blood glucose, PC group), followed by normal glycemia (~100 mg/dl), for four months each (PC-GC group). Results. GTPase activity of Drp1, Fis1-Drp1 interactions, mitochondrial levels of Drp1, and fragmentation of the mitochondria were elevated in HG group. Mitochondrial Division Inhibitor 1 (Mdiv) or Drp1-siRNA attenuated Drp1 activation, mitochondrial fragmentation, and DNA damage. In HG-NG group, NG failed to ameliorate Drp1 activation and Drp1-Fis1 interactions, and the mitochondria remained fragmented. However, Mdiv supplementation in normal glucose, which had followed four days of high glucose (HG-NG/Mdiv group), inhibited Drp1 activation, mitochondrial fragmentation, and increase in ROS and prevented mitochondrial damage. Retinal microvessels from the rats in PC and PC-GC groups had similar Drp1 activation. Conclusion. Thus, Drp1 plays a major role in mitochondrial homeostasis in diabetic retinopathy and in the metabolic memory phenomenon associated with its continued progression. Supplementation of normal glycemia with a Drp1 inhibitor could retard development and further progression of diabetic retinopathy.

Chronic exposure to ozone induces cardiac antioxidant response and overexpression of either mitochondrial fision protein DRP1 and hipertrophyc-related proteins.

Pollution is considered a risk factor for cardiovascular disease; however, the mechanisms to explain this relationship are not well understood; ozone is one of the most abundant and studied air contaminants. Our study aimed to evaluate the effect of chronic exposition of rats to controlled low doses of ozone on oxidative stress, apoptosis, mitochondrial dynamics, and cardiac hypertrophy. Male Wistar rats were daily exposed to low ozone doses during 7, 15, 30, and 60days, 4h/day. Hearts were dissected, and homogenates were prepared. Oxidative stress was evaluated by TBARS and protein nitrosylation in addition to Superoxide dismutase 1 (SOD1) and Catalase levels; the apoptosis related-proteins caspase 3, caspase 9, Bax, Bcl-2, and the mitochondrial dynamic-associated proteins Fis1, Drp1, OPA1, and Mfn1 were quantified by western blot among the cardiac hypertrophy indicator alpha-actin (cardiac actin). There were no changes in the oxidative stress markers, however SOD1 expression increases. Caspase 3 expression decreased, whereas caspase 9 increased without changes in Bax or Bcl-2. Mitochondrial fission may be favored according to the increased expression of Drp1 but not changes in fusion-related proteins OPA1 and Mfn1. Finally, the molecular marker for cardiac hypertrophy was overexpressed after 30 and 60days of ozone exposition. The chronic exposition to ozone induces a deleterious effect on cardiac mitochondria. Antioxidant defenses also show changes in relation to exposure time, as well as an apparent pro-hypertrophic effect associated with altered mitochondrial dynamics.

MnTE-2-PyP protects fibroblast mitochondria from hyperglycemia and radiation exposure

Radiation is a common anticancer therapy for prostate cancer, which transforms tumor-associated normal fibroblasts to myofibroblasts, resulting in fibrosis. Oxidative stress caused by radiation-mediated mitochondrial damage is one of the major contributors to fibrosis. As diabetics are oxidatively stressed, radiation-mediated reactive oxygen species cause severe treatment failure, treatment-related side effects, and significantly reduced survival for diabetic prostate cancer patients as compared to non-diabetic prostate cancer patients. Hyperglycemia and enhanced mitochondrial damage significantly contribute to oxidative damage and disease progression after radiation therapy among diabetic prostate cancer patients. Therefore, reduction of mitochondrial damage in normal prostate fibroblasts after radiation should improve the overall clinical state of diabetic prostate cancer patients. We previously reported that MnTE-2-PyP, a manganese porphyrin, reduces oxidative damage in irradiated hyperglycemic prostate fibroblasts by scavenging superoxide and activating NRF2. In the current study, we have investigated the potential role of MnTE-2-PyP to protect mitochondrial health in irradiated hyperglycemic prostate fibroblasts. This study revealed that hyperglycemia and radiation increased mitochondrial ROS via blocking the mitochondrial electron transport chain, altered mitochondrial dynamics, and reduced mitochondrial biogenesis. Increased mitochondrial damage preceeded an increase in myofibroblast differentiation. MnTE-2-PyP reduced myofibroblast differentiation, improved mitochondrial health by releasing the block on the mitochondrial electron transport chain, enhanced ATP production efficiency, and restored mitochondrial dynamics and metabolism in the irradiated-hyperglycemic prostate fibroblasts. Therefore, we are proposing that one of the mechanisms that MnTE-2-PyP protects prostate fibroblasts from irradiation and hyperglycemia-mediated damage is by protecting the mitochondrial health in diabetic prostate cancer patients.

Sirt3 ameliorates mitochondrial dysfunction and oxidative stress through regulating mitochondrial biogenesis and dynamics in cardiomyoblast.

Sirtuins are the endogenously present anti-aging protein deacetylases that regulate the mitochondrial biogenesis and function. Especially Sirt3, a mitochondrial sirtuin, is well known for maintaining mitochondrial function and health. In the present study, we have explored the novel role of Sirt3 in mitochondrial biogenesis and shown the role of Sirt3 in mito-nuclear communication through AMPK-α in Sirt3 knockdown and Sirt3 overexpressed H9c2 cells. The study found that impaired mitochondrial function in Sirt3-knockdown H9c2 cells was associated with decreased expression of mitochondrial DNA encoded genes, reduced SOD2 expression and activity. The study also revealed that Sirt3 knockdown affects mitochondrial biogenesis and dynamics. To further confirm the role of Sirt3 on mitochondrial biogenesis and health, we did Sirt3 overexpression in H9c2 cells. Sirt3 overexpression enhanced the expression of mitochondrial DNA encoded genes, increased SOD2 activity and altered mitochondrial dynamics. Sirt3 overexpression also caused an increase in mitochondrial biogenesis gene and protein (PGC-1α and TFAM) expression. All these changes were confirmed with mitochondrial functional parameters like basal respiration, maximal respiratory capacity, spare respiratory capacity and ATP production. We found decreased mitochondrial function in Sirt3-knockdown H9c2 cells when compared to control H9c2 cells. Together our data conclude that Sirt3 regulates cardiac mitochondrial health and function through the Sirt3-AMPKα-PGC-1α axis.

The Interplay between Cell-Extracellular Matrix Interaction and Mitochondria Dynamics in Cancer.

Simple SummaryMitochondria are essential cellular organelles, involved in controlling energy production, cell metabolism, cell growth, and cell death. Since cellular functions are de-regulated in cancer, it is not surprising that mitochondria dysfunctions have been observed in this disease. One key aspect in controlling tumor formation and progression is the interaction between the cancer cells and their surrounding environment, known as the tumor microenvironment. The extracellular matrix is an abundant component of the tumor microenvironment and it has been shown to affect tumor initiation and progression. Here, we will explore how the interaction between cancer cells and the extracellular matrix impinges on mitochondria function during cancer progression.The tumor microenvironment, in particular the extracellular matrix (ECM), plays a pivotal role in controlling tumor initiation and progression. In particular, the interaction between cancer cells and the ECM promotes cancer cell growth and invasion, leading to the formation of distant metastasis. Alterations in cancer cell metabolism is a key hallmark of cancer, which is often associated with alterations in mitochondrial dynamics. Recent research highlighted that, changes in mitochondrial dynamics are associated with cancer migration and metastasis—these has been extensively reviewed elsewhere. However, less is known about the interplay between the extracellular matrix and mitochondria functions. In this review, we will highlight how ECM remodeling associated with tumorigenesis contribute to the regulation of mitochondrial function, ultimately promoting cancer cell metabolic plasticity, able to fuel cancer invasion and metastasis.

Alterations in hippocampal mitochondrial dynamics areassociated with neurodegeneration and recognition memory decline in old male mice.

Mitochondrial dynamics is a key process that modulates the ultrastructure, quality and function of mitochondria. It is disrupted in numerous major neurodegenerative disorders including Parkinson's, Alzheimer's and Huntington's disease. Mitochondrial dysfunction has been correlated with the loss of memory. Previous studies suggest the involvement of Vdac1 and Drp1 in outer mitochondrial membrane permeabilization and promotion of mitochondrial fragmentation through Drp1 phosphorylation at S616. However, alterations in mitochondrial dynamics with respect to aging, memory loss and neurodegeneration remain unexplored. Therefore, the present study focuses on the involvement of mitochondrial dynamics in neurodegeneration and recognition memory decline during aging. The recognition memory decline was validated by the novel object recognition test and measurement of hippocampal Arc protein level during aging. The ultrastructure analysis revealed a decline in mitochondrial length and area, while an increase in the number of fragmented, round and disrupted mitochondria in the hippocampus during aging. Disruption was also evident in mitochondrial cristae and membrane with advancing age. The change in mitochondrial morphology was corroborated by an increase in the expression of phospho-Drp1 (S616) and Cyt-c proteins but decline in Mfn2, LC3B, Vdac1, Bcl-XL and Bcl-2 proteins in the hippocampus during aging. Taken together, our findings reveal that an increase in the expression of phospho-Drp1 (S616) and decrease in Mfn2 and LC3B proteins in the hippocampus bring about a reduction in mitochondrial length and area, and rise in mitochondrial fragmentation leading to reduced neuronal cell density, increased neurodegeneration and recognition memory decline in old male mice. Diagram depicts the increase in hippocampal mitochondrial fragmentation during aging of mice. Increased mitochondrial fragmentation causes distorted mitochondrial function such as decrease in ATP/ADP transportation due to decrease in Vdac1 protein level and increase in oxidative damage. These alterations result in hippocampal neurodegeneration and consequently impairment in recognition memory during aging.

Characterization of Mitochondrial Proteome and Function in Luminal A and Basal-like Breast Cancer Subtypes Reveals Alteration in Mitochondrial Dynamics and Bioenergetics Relevant to Their Diagnosis.

Breast cancer (BC) is the most prevalent cancer and the one with the highest mortality among women worldwide. Although the molecular classification of BC has been a helpful tool for diagnosing and predicting the treatment of BC, developments are still being made to improve the diagnosis and find new therapeutic targets. Mitochondrial dysfunction is a crucial feature of cancer, which can be associated with cancer aggressiveness. Although the importance of mitochondrial dynamics in cancer is well recognized, its involvement in the mitochondrial function and bioenergetics context in BC molecular subtypes has been scantly explored. In this study, we combined mitochondrial function and bioenergetics experiments in MCF7 and MDA-MB-231 cell lines with statistical and bioinformatics analyses of the mitochondrial proteome of luminal A and basal-like tumors. We demonstrate that basal-like tumors exhibit a vicious cycle between mitochondrial fusion and fission; impaired but not completely inactive mitochondrial function; and the Warburg effect, associated with decreased oxidative phosphorylation (OXPHOS) complexes I and III. Together with the results obtained in the cell lines and the mitochondrial proteome analysis, two mitochondrial signatures were proposed: one signature reflecting alterations in mitochondrial functions and a second signature exclusively of OXPHOS, which allow us to distinguish between luminal A and basal-like tumors.

Nrf2 Deficiency Attenuates Testosterone Efficiency in Ameliorating Mitochondrial Function of the Substantia Nigra in Aged Male Mice.

Reduced testosterone level is a common feature of aging in men. Aging, as a risk factor for several neurodegenerative disorders, shows declined mitochondrial function and downregulated mitochondrial biogenesis and mitochondrial dynamics. Mitochondrial biogenesis and mitochondrial dynamics are crucial in maintaining proper mitochondrial function. Supplementation with testosterone is conducive to improving mitochondrial function of males during aging. Nuclear factor erythroid 2-related factor 2 (Nrf2), a regulator of redox homeostasis, is involved in the ameliorative effects of testosterone supplementation upon aging. To explore Nrf2 role in the effects of testosterone supplementation on mitochondrial function during aging, we studied the efficiency of testosterone supplementation in improving mitochondrial function of Nrf2 knockout- (KO-) aged male mice by analyzing the changes of mitochondrial biogenesis and mitochondrial dynamics. It was found that wild-type- (WT-) aged male mice showed low mitochondrial function and expression levels of PGC-1α, NRF-1\\NRF-2, and TFAM regulating mitochondrial biogenesis, as well as Drp1, Mfn1, and OPA1 controlling mitochondrial dynamics in the substantia nigra (SN). Nrf2 KO aggravated the defects above in SN of aged male mice. Testosterone supplementation to WT-aged male mice significantly ameliorated mitochondrial function and upregulated mitochondrial biogenesis and mitochondrial dynamics, which were not shown in Nrf2 KO-aged male mice due to Nrf2 deficiency. Testosterone deficiency by gonadectomy (GDX) decreased mitochondrial function, downregulated mitochondrial biogenesis, and altered mitochondrial dynamics balance in young male mice. Supplementation with testosterone to Nrf2 KO-GDX mice only ameliorated the alterations above but did not reverse them to sham level. Nrf2 deficiency attenuated testosterone efficiency in ameliorating mitochondrial function in the SN of aged male mice through mitochondrial biogenesis and mitochondrial dynamics to some extent. Activation of Nrf2 might contribute to testosterone-upregulating mitochondrial biogenesis and mitochondrial dynamics in the SN during aging to produce efficient mitochondria for ATP production.

Drp1/Fis1-Dependent Pathologic Fission and Associated Damaged Extracellular Mitochondria Contribute to Macrophage Dysfunction in Endotoxin Tolerance.

Recent publications have shown that mitochondrial dynamics can govern the quality and quantity of extracellular mitochondria subsequently impacting immune phenotypes. This study aims to determine if pathologic mitochondrial fission mediated by Drp1/Fis1 interaction impacts extracellular mitochondrial content and macrophage function in sepsis-induced immunoparalysis. Laboratory investigation. University laboratory. C57BL/6 and BALB/C mice. Using in vitro and murine models of endotoxin tolerance (ET), we evaluated changes in Drp1/Fis1-dependent pathologic fission and simultaneously measured the quantity and quality of extracellular mitochondria. Next, by priming mouse macrophages with isolated healthy mitochondria (MC) and damaged mitochondria, we determined if damaged extracellular mitochondria are capable of inducing tolerance to subsequent endotoxin challenge. Finally, we determined if inhibition of Drp1/Fis1-mediated pathologic fission abrogates release of damaged extracellular mitochondria and improves macrophage response to subsequent endotoxin challenge. When compared with naïve macrophages (NMs), endotoxin-tolerant macrophages (ETM) demonstrated Drp1/Fis1-dependent mitochondrial dysfunction and higher levels of damaged extracellular mitochondria (Mitotracker-Green + events/50 μL: ETM = 2.42 × 106 ± 4,391 vs NM = 5.69 × 105 ± 2,478; p < 0.001). Exposure of NMs to damaged extracellular mitochondria (MH) induced cross-tolerance to subsequent endotoxin challenge, whereas MC had minimal effect (tumor necrosis factor [TNF]-α [pg/mL]: NM = 668 ± 3, NM + MH = 221 ± 15, and NM + Mc = 881 ± 15; p < 0.0001). Inhibiting Drp1/Fis1-dependent mitochondrial fission using heptapeptide (P110), a selective inhibitor of Drp1/Fis1 interaction, improved extracellular mitochondrial function (extracellular mitochondrial membrane potential, JC-1 [R/G] ETM = 7 ± 0.5 vs ETM + P110 = 19 ± 2.0; p < 0.001) and subsequently improved immune response in ETMs (TNF-α [pg/mL]; ETM = 149 ± 1 vs ETM + P110 = 1,150 ± 4; p < 0.0001). Similarly, P110-treated endotoxin tolerant mice had lower amounts of damaged extracellular mitochondria in plasma (represented by higher extracellular mitochondrial membrane potential, TMRM/MT-G: endotoxin tolerant [ET] = 0.04 ± 0.02 vs ET + P110 = 0.21 ± 0.02; p = 0.03) and improved immune response to subsequent endotoxin treatment as well as cecal ligation and puncture. Inhibition of Drp1/Fis1-dependent mitochondrial fragmentation improved macrophage function and immune response in both in vitro and in vivo models of ET. This benefit is mediated, at least in part, by decreasing the release of damaged extracellular mitochondria, which contributes to endotoxin cross-tolerance. Altogether, these data suggest that alterations in mitochondrial dynamics may play an important role in sepsis-induced immunoparalysis.

Observation of Inflammation, Oxidative Stress, Mitochondrial Dynamics, and Apoptosis in Dental Pulp following a Diagnosis of Irreversible Pulpitis.

Mitochondrial dynamics play a pivotal role in maintaining the homeostasis of the dental pulp. Inflammation and oxidative stress can trigger changes in mitochondrial dynamics, leading to cell death in the dental pulp. This study aimed to investigate inflammation, oxidative stress, mitochondrial dynamic alterations, and cell death in inflamed pulpal tissues compared to healthy pulp tissues. Pulpal tissues were collected (n=15 per group) from: 1) healthy people as the control and 2) people with clinically diagnosed irreversible pulpitis. Proteins indicating inflammation, oxidative stress, mitochondrial dynamics, and cell death markers were investigated by western blot analysis. A Student's t-test was used to analyse differences between the healthy and irreversible pulpitis groups. A probability of 0.05 was used to indicate statistical significance (p<0.05). The expression of the proteins, tumour necrosis factor-alpha (TNF-α) and nuclear factor kappa-lightchain-enhancer, by activated B cells (NF-κB) from inflamed pulp tissues were significantly higher than those of control. Compared to controls, 4 hydroxynonenal (4HNE) and dynamin-related protein 1 (Drp1) were significantly higher, while mitofusin 2 (MFN2) and optic atrophy type 1 (OPA1) were significantly lower in inflamed pulp tissues. Bcl-2-associated X protein (Bax), cleaved caspase-3, and cytochrome c were significantly higher in inflamed pulpal tissues compared to controls. In inflamed pulpal tissues, we found a significant increase in the expression of receptor-interacting serine or threonine-protein kinase 1 (RIPK1) but not receptor-interacting serine or threonine-protein kinase 3 (RIPK3). Irreversible pulpitis is associated with inflammation, oxidative stress, alterations in mitochondrial dynamics, and apoptosis in pulpal tissues. (EEJ-2022-01-014).

Monitoring Mitochondrial Morphology and Respiration in Doxorubicin-Induced Cardiomyopathy.

Doxorubicin (DOX)-induced cardiomyopathy constitutes dose-dependent cardiac toxicity, culminating in fatal heart failure progression. Cardiac toxicity limits effective and subsequent use of DOX in chemotherapy regimens in pediatric, adult, and recurrent cancer patients. DOX-induced profound alterations in mitochondrial morphology, dynamics, and defects in mitochondrial energy metabolism in the heart comprise key stressors in DOX-induced cardiotoxicity. Hence, the discovery of novel molecular targets and therapeutics to mitigate DOX-induced mitochondrial dysfunctions are imperative. Herein, we provided two laboratory protocols to monitor DOX-induced alterations in mitochondrial morphology and respiration in isolated primary neonatal rat cardiomyocytes. Neonatal rat cardiomyocytes are extensively used to monitor signaling mechanisms regulating cardiomyopathy in vitro. Therefore, these protocols will help researchers study the effects of novel pharmacological and genetic manipulations against DOX-induced alterations in mitochondrial morphology and energy metabolism in cardiomyocytes.

Exercise Preconditioning Blunts Early Atrogenes Expression and Atrophy in Gastrocnemius Muscle of Hindlimb Unloaded Mice.

A large set of FoxOs-dependent genes play a primary role in controlling muscle mass during hindlimb unloading. Mitochondrial dysfunction can modulate such a process. We hypothesized that endurance exercise before disuse can protect against disuse-induced muscle atrophy by enhancing peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) expression and preventing mitochondrial dysfunction and energy-sensing AMP-activated protein kinase (AMPK) activation. We studied cross sectional area (CSA) of muscle fibers of gastrocnemius muscle by histochemistry following 1, 3, 7, and 14 days of hindlimb unloading (HU). We used Western blotting and qRT-PCR to study mitochondrial dynamics and FoxOs-dependent atrogenes’ expression at 1 and 3 days after HU. Preconditioned animals were submitted to moderate treadmill exercise for 7 days before disuse. Exercise preconditioning protected the gastrocnemius from disuse atrophy until 7 days of HU. It blunted alterations in mitochondrial dynamics up to 3 days after HU and the expression of most atrogenes at 1 day after disuse. In preconditioned mice, the activation of atrogenes resumed 3 days after HU when mitochondrial dynamics, assessed by profusion and pro-fission markers (mitofusin 1, MFN1, mitofusin 2, MFN2, optic atrophy 1, OPA1, dynamin related protein 1, DRP1 and fission 1, FIS1), PGC1α levels, and AMPK activation were at a basal level. Therefore, the normalization of mitochondrial dynamics and function was not sufficient to prevent atrogenes activation just a few days after HU. The time course of sirtuin 1 (SIRT1) expression and content paralleled the time course of atrogenes’ expression. In conclusion, seven days of endurance exercise counteracted alterations of mitochondrial dynamics and the activation of atrogenes early into disuse. Despite the normalization of mitochondrial dynamics, the effect on atrogenes’ suppression died away within 3 days of HU. Interestingly, muscle protection lasted until 7 days of HU. A longer or more intense exercise preconditioning may prolong atrogenes suppression and muscle protection.

A New Insight into an Alternative Therapeutic Approach to Restore Redox Homeostasis and Functional Mitochondria in Neurodegenerative Diseases.

Neurodegenerative diseases are accompanied by oxidative stress and mitochondrial dysfunction, leading to a progressive loss of neuronal cells, formation of protein aggregates, and a decrease in cognitive or motor functions. Mitochondrial dysfunction occurs at the early stage of neurodegenerative diseases. Protein aggregates containing oxidatively damaged biomolecules and other misfolded proteins and neuroinflammation have been identified in animal models and patients with neurodegenerative diseases. A variety of neurodegenerative diseases commonly exhibits decreased activity of antioxidant enzymes, lower amounts of antioxidants, and altered cellular signalling. Although several molecules have been approved clinically, there is no known cure for neurodegenerative diseases, though some drugs are focused on improving mitochondrial function. Mitochondrial dysfunction is caused by oxidative damage and impaired cellular signalling, including that of peroxisome proliferator-activated receptor gamma coactivator 1α. Mitochondrial function can also be modulated by mitochondrial biogenesis and the mitochondrial fusion/fission cycle. Mitochondrial biogenesis is regulated mainly by sirtuin 1, NAD+, AMP-activated protein kinase, mammalian target of rapamycin, and peroxisome proliferator-activated receptor γ. Altered mitochondrial dynamics, such as increased fission proteins and decreased fusion products, are shown in neurodegenerative diseases. Due to the restrictions of a target-based approach, a phenotype-based approach has been performed to find novel proteins or pathways. Alternatively, plasma membrane redox enzymes improve mitochondrial function without the further production of reactive oxygen species. In addition, inducers of antioxidant response elements can be useful to induce a series of detoxifying enzymes. Thus, redox homeostasis and metabolic regulation can be important therapeutic targets for delaying the progression of neurodegenerative diseases.