Evaluation of selected antidiabetics in cardiovascular complications associated with cancer cachexia.

So far, the cardio-protective potential of antidiabetics is proved, but their effect on cardiovascular complications associated with cancer cachexia is not explored until now. Insulin resistance and glucose intolerance along with systemic inflammation are prominent in cachexia but the potential effect of antidiabetic agents especially those belonging to biguanide, DPP4 inhibitors and SGLT2 on the heart are not studied till now. In present study, the effect of metformin, vildagliptin, teneligliptin, dapagliflozin and empagliflozin on cardiovascular complications associated with cancer cachexia by using B16F1 induced metastatic cancer cachexia and urethane-induced cancer cachexia was studied. These antidiabetic agents proved to be beneficial against cachexia-induced atrophy of the heart, preserved ventricular weights, maintained cardiac hypertrophic index, preserved the wasting of cardiac muscles assessed by HE staining, Masson trichrome staining, periodic acid Schiff staining and picro-Sirius red staining. Altered cardiac gene expression was attenuated after treatment with selected antidiabetics, thus preventing cardiac atrophy. Also, antidiabetic agents treatment improved the serum creatinine kinase MB, Sodium potassium ATPase and collagen in the heart. Reduction in blood pressure and heart rate was observed after treatment with antidiabetic agents. Results of our study show that the selected antidiabetics prove to be beneficial in attenuating the cardiac atrophy and helps in regulation of hemodynamic stauts in cancer cachexia-induced cardiovascular complications. Our study provides some direction towards use of selected antidiabetic agents in the management of cardiovascular complications associated with cancer cachexia and the study outcomes can be useful in desiging clinical trials.

DNA damage induces STING mediated IL-6-STAT3 survival pathway in triple-negative breast cancer cells and decreased survival of breast cancer patients.

Triple-negative breast cancer is aggressive and metastatic breast cancer type and shows immune evasion, drug resistance, relapse and poor survival. Anti-cancer therapy like ionizing radiation and chemotherapeutic drug majorly induces DNA damage hence, alteration in DNA damage repair and downstream pathways may contribute to tumor cell survival. DNA damage during chemotherapy is sensed by cyclic GMP-AMP synthase(cGAS)-stimulator of interferon genes (STING), which determines the anti-tumor immune response by modulating the expression of programmed cell death ligand-1 (PD-L1), immune suppressor, in the tumor microenvironment. Triple-negative breast cancer cells are cGAS-STING positive and modulation of this pathway during DNA damage response for survival and immune escape mechanism is not well understood. Here we demonstrate that doxorubicin-mediated DNA damage induces STING mediated NF-κB activation in triple-negative as compared to ER/PR positive breast cancer cells. STING-mediated NF-κB induces the expression of IL-6 in triple-negative breast cancer cells and activates pSTAT3, which enhances cell survival and PD-L1 expression. Doxorubicin and STAT3 inhibitor act synergistically and inhibit cell survival and clonogenicity in triple-negative breast cancer cells. Knockdown of STING in triple-negative breast cancer cells enhances CD8 mediated immune cell death of breast cancer cells. The combinatorial treatment of triple-negative breast cells with doxorubicin and STAT3 inhibitor reduces PD-L1 expression and activates immune cell-mediated cancer cell death. Further STING and IL-6 levels show a positive correlation in breast cancer patients and poor survival outcomes. The study here strongly suggests that STING mediated activation of NF-κB enhances IL-6 mediated STAT3 in triple-negative breast cancer cells which induces cell survival and immune-suppressive mechanism.

The analog of cGAMP, c-di-AMP, activates STING mediated cell death pathway in estrogen-receptor negative breast cancer cells.

Immune adaptor protein like STING/MITA regulate innate immune response and plays a critical role in inflammation in the tumor microenvironment and regulation of metastasis including breast cancer. Chromosomal instability in highly metastatic cells releases fragmented chromosomal parts in the cytoplasm, hence the activation of STING via an increased level of cyclic dinucleotides (cDNs) synthesized by cGMP-AMP synthase (cGAS). Cyclic dinucleotides 2' 3'-cGAMP and it's analog can potentially activate STING mediated pathways leading to nuclear translocation of p65 and IRF-3 and transcription of inflammatory genes. The differential modulation of STING pathway via 2' 3'-cGAMP and its analog and its implication in breast tumorigenesis is still not well explored. In the current study, we demonstrated that c-di-AMP can activate type-1 IFN response in ER negative breast cancer cell lines which correlate with STING expression. c-di-AMP binds to STING and activates downstream IFN pathways in STING positive metastatic MDA-MB-231/MX-1 cells. Prolonged treatment of c-di-AMP induces cell death in STING positive metastatic MDA-MB-231/MX-1 cells mediated by IRF-3. c-di-AMP induces IRF-3 translocation to mitochondria and initiates Caspase-9 mediated cell death and inhibits clonogenicity of triple-negative breast cancer cells. This study suggests that c-di-AMP can activate and modulates STING pathway to induce mitochondrial mediated apoptosis in estrogen-receptor negative breast cancer cells.

NLRX1 resides in mitochondrial RNA granules and regulates mitochondrial RNA processing and bioenergetic adaptation.

The role of mitochondria is emerging in regulation of innate immunity, inflammation and cell death beyond its primary role in energy metabolism. Mitochondria act as molecular platform for immune adaptor protein complexes, which participate in innate immune signaling. The mitochondrial localized immune adaptors are widely expressed in non-immune cells, however their role in regulation of mitochondrial function and metabolic adaption is not well understood. NLRX1, a member of NOD family receptor proteins, localizes to mitochondria and is a negative regulator of anti-viral signaling. However, the submitochondrial localization of NLRX1 and its implication in regulation of mitochondrial functions remains elusive. Here, we confirm that NLRX1 translocates to mitochondrial matrix and associates with mitochondrial FASTKD5 (Fas-activated serine-threonine kinase family protein-5), a bonafide component of mitochondrial RNA granules (MRGs). The association of NLRX1 with FASTKD5 negatively regulates the processing of mitochondrial genome encoded transcripts for key components of complex-I and complex-IV, to modulate its activity and supercomplexes formation. The evidences, here, suggest an important role of NLRX1 in regulating the post-transcriptional processing of mitochondrial RNA, which may have an important implication in bioenergetic adaptation during metabolic stress, oncogenic transformation and innate immunity.

Enforced lysosomal biogenesis rescues erythromycin- and clindamycin-induced mitochondria-mediated cell death in human cells.

Antibiotics are the front-line treatment against many bacterial infectious diseases in human. The excessive and long-term use of antibiotics in human cause several side effects. It is important to understand the underlying molecular mechanisms of action of antibiotics in the host cell to avoid the side effects due to the prevalent uses. In the current study, we investigated the crosstalk between mitochondria and lysosomes in the presence of widely used antibiotics: erythromycin (ERM) and clindamycin (CLDM), which target the 50S subunit of bacterial ribosomes. We report here that both ERM and CLDM induced caspase activation and cell death in several different human cell lines. The activity of the mitochondrial respiratory chain was compromised in the presence of ERM and CLDM leading to bioenergetic crisis and generation of reactive oxygen species. Antibiotics treatment impaired autophagy flux and lysosome numbers, resulting in decreased removal of damaged mitochondria through mitophagy, hence accumulation of defective mitochondria. We further show that over-expression of transcription factor EB (TFEB) increased the lysosome number, restored mitochondrial function and rescued ERM- and CLDM-induced cell death. These studies indicate that antibiotics alter mitochondria and lysosome interactions leading to apoptotsis and may develop a novel approach for targeting inter-organelle crosstalk to limit deleterious antibiotic-induced side effects.

Enhanced translocation of TRIM32 to mitochondria sensitizes dopaminergic neuronal cells to apoptosis during stress conditions in Parkinson's disease.

Parkinson's disease (PD) is a chronic neurodegenerative disease characterized by progressive loss of dopamine-producing neurons from the substantia nigra region of the brain. Mitochondrial dysfunction is one of the major causes of oxidative stress and neuronal cell death in PD. E3 ubiquitin ligases such as Parkin (PRKN) modulate mitochondrial quality control in PD; however, the role of other E3 ligases associated with mitochondria in the regulation of neuronal cell death in PD has not been explored. The current study investigated the role of TRIM32, RING E3 ligase, in sensitization to oxidative stress-induced neuronal apoptosis. The expression of TRIM32 sensitizes SH-SY5Y dopaminergic cells to rotenone and 6-OHDA-induced neuronal death, whereas the knockdown increased cell viability under PD stress conditions. The turnover of TRIM32 is enhanced under PD stress conditions and is mediated by autophagy. TRIM32 translocation to mitochondria is enhanced under PD stress conditions and localizes on the outer mitochondrial membrane. TRIM32 decreases complex-I assembly and activity as well as mitochondrial reactive oxygen species (ROS) and ATP levels under PD stress. Deletion of the RING domain of TRIM32 enhanced complex I activity and rescued ROS levels and neuronal viability under PD stress conditions. TRIM32 decreases the level of XIAP, and co-expression of XIAP with TRIM32 rescued the PD stress-induced cell death and mitochondrial ROS level. In conclusion, turnover of TRIM32 increases during stress conditions and translocation to mitochondria is enhanced, regulating mitochondrial functions and neuronal apoptosis by modulating the level of XIAP in PD.

Sildenafil as a Candidate Drug for Alzheimer's Disease: Real-World Patient Data Observation and Mechanistic Observations from Patient-Induced Pluripotent Stem Cell-Derived Neurons.

Background: Alzheimer's disease (AD) is a chronic neurodegenerative disease needing effective therapeutics urgently. Sildenafil, one of the approved phosphodiesterase-5 inhibitors, has been implicated as having potential effect in AD. Objective: To investigate the potential therapeutic benefit of sildenafil on AD. Methods: We performed real-world patient data analysis using the MarketScan® Medicare Supplemental and the Clinformatics® databases. We conducted propensity score-stratified analyses after adjusting confounding factors (i.e., sex, age, race, and comorbidities). We used both familial and sporadic AD patient induced pluripotent stem cells (iPSC) derived neurons to evaluate the sildenafil's mechanism-of-action. Results: We showed that sildenafil usage is associated with reduced likelihood of AD across four new drug compactor cohorts, including bumetanide, furosemide, spironolactone, and nifedipine. For instance, sildenafil usage is associated with a 54% reduced incidence of AD in MarketScan® (hazard ratio [HR] = 0.46, 95% CI 0.32- 0.66) and a 30% reduced prevalence of AD in Clinformatics® (HR = 0.70, 95% CI 0.49- 1.00) compared to spironolactone. We found that sildenafil treatment reduced tau hyperphosphorylation (pTau181 and pTau205) in a dose-dependent manner in both familial and sporadic AD patient iPSC-derived neurons. RNA-sequencing data analysis of sildenafil-treated AD patient iPSC-derived neurons reveals that sildenafil specifically target AD related genes and pathobiological pathways, mechanistically supporting the beneficial effect of sildenafil in AD. Conclusions: These real-world patient data validation and mechanistic observations from patient iPSC-derived neurons further suggested that sildenafil is a potential repurposable drug for AD. Yet, randomized clinical trials are warranted to validate the causal treatment effects of sildenafil in AD.

TNF-α induced NF-κB mediated LYRM7 expression modulates the tumor growth and metastatic ability in breast cancer.

Tumor microenvironment (TME) of solid tumors including breast cancer is complex and contains a distinct cytokine pattern including TNF-α, which determines the progression and metastasis of breast tumors. The metastatic potential of triple negative breast cancer subtypes is high as compared to other subtypes of breast cancer. NF-κB is key transcription factor regulating inflammation and mitochondrial bioenergetics including oxidative phosphorylation (OXPHOS) genes which determine its oxidative capacity and generating reducing equivalents for synthesis of key metabolites for proliferating breast cancer cells. The differential metabolic adaptation and OXPHOS function of breast cancer subtypes in inflammatory conditions and its contribution to metastasis is not well understood. Here we demonstrated that different subunits of NF-κB are differentially expressed in subtypes of breast cancer patients. RELA, one of the major subunits in regulation of the NF-κB pathway is positively correlated with high level of TNF-α in breast cancer patients. TNF-α induced NF-κB regulates the expression of LYRM7, an assembly factor for mitochondrial complex III. Downregulation of LYRM7 in MDA-MB-231 cells decreases mitochondrial super complex assembly and enhances ROS levels, which increases the invasion and migration potential of these cells. Further, in vivo studies using Infliximab, a monoclonal antibody against TNF-α showed decreased expression of LYRM7 in tumor tissue. Large scale breast cancer databases and human patient samples revealed that LYRM7 levels decreased in triple negative breast cancer patients compared to other subtypes and is determinant of survival outcome in patients. Our results indicate that TNF-α induced NF-κB is a critical regulator of LYRM7, a major factor for modulating mitochondrial functions under inflammatory conditions, which determines growth and survival of breast cancer cells.

Systematic characterization of multi-omics landscape between gut microbial metabolites and GPCRome in Alzheimer's disease.

Shifts in the magnitude and nature of gut microbial metabolites have been implicated in Alzheimer's disease (AD), but the host receptors that sense and respond to these metabolites are largely unknown. Here, we develop a systems biology framework that integrates machine learning and multi-omics to identify molecular relationships of gut microbial metabolites with non-olfactory G-protein-coupled receptors (termed the "GPCRome"). We evaluate 1.09 million metabolite-protein pairs connecting 408 human GPCRs and 335 gut microbial metabolites. Using genetics-derived Mendelian randomization and integrative analyses of human brain transcriptomic and proteomic profiles, we identify orphan GPCRs (i.e., GPR84) as potential drug targets in AD and that triacanthine experimentally activates GPR84. We demonstrate that phenethylamine and agmatine significantly reduce tau hyperphosphorylation (p-tau181 and p-tau205) in AD patient induced pluripotent stem cell-derived neurons. This study demonstrates a systems biology framework to uncover the GPCR targets of human gut microbiota in AD and other complex diseases if broadly applied.

Neuronal exosomal miRNAs modulate mitochondrial functions and cell death in bystander neuronal cells under Parkinson's disease stress conditions.

Parkinson's Disease (PD) is a chronic neurodegenerative disorder characterized by progressive loss of midbrain dopaminergic neurons in the substantia nigra part of the brain. Pathology spread to numerous brain regions and cell types suggests that intercellular communication is essential to PD progression. Exosomes mediate intercellular communication between neurons, glia, and other cell types throughout PD-relevant brain regions. However, the mechanism remains unclear, and its implication in PD pathology, is not well understood. In the current study, we explored the role of exosomes in modulating the response to PD-relevant toxicants. In cellular models of PD, neuronal cell-derived exosomes are readily internalized by recipient neuronal cells as intact vesicles. Internalized exosomes in bystander neuronal cells localize to mitochondria and dysregulate mitochondrial functions, leading to cell death under PD stress conditions. NGS analysis of exosomes released by neuronal cells subjected to PD stress conditions showed that levels of specific miRNAs were altered in exosomes under PD stress conditions. Bioinformatic analysis of the miRNA targets revealed enriched pathways related to neuronal processes and morphogenesis, apoptosis and ageing. Levels of two miRNAs, hsa-miR-30a-5p and hsa-miR-181c-5p, were downregulated in exosomes under PD stress conditions. Expression of the identified miRNAs in neuronal cells led to their enrichment in exosomes, and exosome uptake in neuronal cells ameliorated mitochondrial dysfunction induced by PD stress conditions and rescued cell death. In conclusion, loss of enrichment of specific miRNAs, including miR-30a-5p and miR-181c-5p, under PD stress conditions causes mitochondrial dysfunction and neuronal death, and hence may lead to progression of PD.

Altered trafficking of miRNAs at mitochondria modulates mitochondrial functions and cell death in brain ischemia.

Stroke is one of the major causes of death and disabilities worldwide. The rapid induction of cell death by necrosis and apoptosis is observed at the ischemic core, while long lasting apoptosis and brain inflammation continue in the penumbra. The emerging evidence suggests a critical role of mitochondria in acute and chronic inflammation and cell death. Mitochondrial dysfunction may result in the release of mitokines and/or mitochondrial DNA into the cytoplasm and activate multiple cytosolic pathways which in turn triggers inflammation. The role of miRNA, specifically mitochondria-associated miRNAs (mitomiRs) in the regulation of mitochondrial functions is emerging. In the current study, we hypothesized that ischemia-induced mitomiRs may modulate the mitochondrial functions and such alterations under stress conditions may lead to mitochondrial dysfunction and cell death. We have demonstrated the specific pattern of miRNAs associated with mitochondria that is altered under ischemic condition induced by transient middle artery occlusion (tMCAo) in rats. The putative targets of altered miRNAs include several mitochondrial proteins which signifies their involvement in maintaining mitochondrial homeostasis. The alteration of selected miRNAs in mitochondria was further detected in a cellular models when hypoxia was induced using a chemical agent CoCl2, in three cell lines. Two candidate mitomiRs, hsa-miR-149-3p and hsa-miR-204-5p were further analyzed for their functional role during in vitro hypoxia by transfecting mitomiR mimics into cells and determining critical mitochondrial functions and cell viability. The results here emphasize the role of certain mitomiRs as an important modulator of mitochondrial function under the ischemic condition.

Different platforms for mitomiRs in mitochondria: Emerging facets in regulation of mitochondrial functions.

Mitochondria are one of the central organelles involved in cellular energy metabolism and play a regulatory role in various human pathologies ranging from inborn errors of metabolism, cancer, inflammation, and infections. Mitochondrial DNA encodes limited number of genes that is not sufficient for its optimal functioning. Hence, mitochondria import ∼1500 of proteins and ncRNAs from the nucleus depending on energy requirement of cell, tissue size, complexity and diversity of functions. Mitochondrial outer membrane can serve as a platform for regulation of local translation of nuclear-encoded mRNAs of mitochondrial proteins (nmRNAmp); however, underlying molecular mechanism for translational regulation of nmRNAmp at mitochondria is unexplored. Emerging evidence now suggest that mitochondria are enriched with specific miRNAs known as mitomiRs, which may be nuclear or mitochondrial DNA encoded. MitomiRs may modulate mitochondrial function and metabolism by fine-tuning protein levels related to mitochondria. The discovery of mitomiRs raised the questions of elucidating molecular pathways for their biogenesis, translocation, action sites and mechanism of action. Here, we have reviewed the existing reports describing the role of mitomiRs in sub mitochondrial compartments and discussed possible molecular mechanisms of mitomiRs in the regulation of nmRNAmp and mitogenome encoded transcripts. Further understanding of mitomiRs will uncover their implication in various pathophysiological conditions associated with mitochondria.

TNF-α-induced E3 ligase, TRIM15 inhibits TNF-α-regulated NF-κB pathway by promoting turnover of K63 linked ubiquitination of TAK1.

Ubiquitin E3-ligases are recruited at different steps of TNF-α-induced NF-κB activation; however, their role in temporal regulation of the pathway remains elusive. The study systematically identified TRIMs as potential feedback regulators of the TNF-α-induced NF-κB pathway. We further observed that TRIM15 is "late" response TNF-α-induced gene and inhibits the TNF-α-induced NF-κB pathway in several human cell lines. TRIM15 promotes turnover of K63-linked ubiquitin chains in a PRY/SPRY domain-dependent manner. TRIM15 interacts with TAK1 and inhibits its K63-linked ubiquitination, thus NF-κB activity. Further, TRIM15 interacts with TRIM8 and inhibits cytosolic translocation to antagonize TRIM8 modualted NF-κB. TRIM8 and TRIM15 also show functionally inverse correlation in psoriasis condition. In conclusion, TRIM15 is TNF-α-induced late response gene and inhibits TNF-α induced NF-κB pathway hence a feedback modulator to keep the proinflammatory NF-κB pathway under control.

Mitohormesis; Potential implications in neurodegenerative diseases.

Mitochondrial dysfunction is known to be associated with neurodegenerative diseases (NDDs), which is a major burden on the society. Therefore, understanding the regulation of mitochondrial dysfunctions and its implication in neurodegeneration has been major goal for exploiting these mechanisms to rescue neuronal death. The crosstalk between mitochondria and nucleus is important for different neuronal functions including axonal branching, energy homeostasis, neuroinflammation and neuronal survival. The decreased mitochondria capacity during progressive neurodegeneration leads to the altered OXPHOS activity and generation of ROS. The ROS levels in narrow physiological range can reprogram nuclear gene expression to enhance the cellular survival by phenomenon called mitohormesis. Here, we have systematically reviewed the existing reports of mitochondrial dysfunctions causing altered ROS levels in NDDs. We further discussed the role of ROS in regulating mitohormesis and emphasized the importance of mitohormesis in neuronal homeostasis. The emerging role of mitohormesis highlights its importance in future studies on intracellular ROS mediated rescue of mitochondrial dysfunction along with other prevailing mechanisms to alleviate neurodegeneration.

Exosome Release Is Modulated by the Mitochondrial-Lysosomal Crosstalk in Parkinson's Disease Stress Conditions.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN) pars compacta region of the brain. The main pathological hallmark involves cytoplasmic inclusions of α-synuclein and mitochondrial dysfunction, which is observed in other part of the central nervous system other than SN suggesting the spread of pathogenesis to bystander neurons. The inter-neuronal communication through exosomes may play an important role in the spread of the disease; however, the mechanisms are not well elucidated. Mitochondria and its role in inter-organellar crosstalk with multivesicular body (MVB) and lysosome and its role in modulation of exosome release in PD is not well understood. In the current study, we investigated the mitochondria-lysosome crosstalk modulating the exosome release in neuronal and glial cells. We observed that PD stress showed enhanced release of exosomes in dopaminergic neurons and glial cells. The PD stress condition in these cells showed fragmented network and mitochondrial dysfunction which further leads to functional deficit of lysosomes and hence inhibition of autophagy flux. Neuronal and glial cells treated with rapamycin showed enhanced autophagy and inhibited the exosomal release. The results here suggest that maintenance of mitochondrial function is important for the lysosomal function and hence exosomal release which is important for the pathogenesis of PD.

Expression of expanded FMR1-CGG repeats alters mitochondrial miRNAs and modulates mitochondrial functions and cell death in cellular model of FXTAS.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a progressive neurodegenerative disorder caused by an expansion of 55 to 200 CGG repeats located within 5'UTR of FMR1.These CGG repeats are transcribed into RNAs, which sequester several RNA binding proteins and alter the processing of miRNAs. CGG repeats are also translated into a toxic polyglycine-containing protein, FMRpolyG, that affects mitochondrial and nuclear functions reported in cell and animal models and patient studies. Nuclear-encoded small non-coding RNAs, including miRNAs, are transported to mitochondria; however, the role of mitochondrial miRNAs in FXTAS pathogenesis is not understood. Here, we analyzed mitochondrial miRNAs from HEK293 cells expressing expanded CGG repeats and their implication in the regulation of mitochondrial functions. The analysis of next generation sequencing (NGS) data of small RNAs from HEK293 cells expressing CGG premutation showed decreased level of cellular miRNAs and an altered pattern of association of miRNAs with mitochondria (mito-miRs). Among such mito-miRs, miR-320a was highly enriched in mitoplast and RNA immunoprecipitation of Ago2 (Argonaute-2) followed by Droplet digital PCR (ddPCR)suggested that miR-320a may form a complex with Ago2 and mitotranscripts. Finally, transfection of miR-320a mimic in cells expressing CGG permutation recovers mitochondrial functions and rescues cell death. Overall, this work reveals an altered translocation of miRNAs to mitochondria and the role of miR-320a in FXTAS pathology.

TNF-α differentially modulates subunit levels of respiratory electron transport complexes of ER/PR +ve/-ve breast cancer cells to regulate mitochondrial complex activity and tumorigenic potential.

BACKGROUND: Tumor necrosis factor-α (TNF-α) is an immunostimulatory cytokine that is consistently high in the breast tumor microenvironment (TME); however, its differential role in mitochondrial functions and cell survival in ER/PR +ve and ER/PR -ve breast cancer cells is not well understood. METHODS: In the current study, we investigated TNF-α modulated mitochondrial proteome using high-resolution mass spectrometry and identified the differentially expressed proteins in two different breast cancer cell lines, ER/PR positive cell line; luminal, MCF-7 and ER/PR negative cell line; basal-like, MDA-MB-231 and explored its implication in regulating the tumorigenic potential of breast cancer cells. We also compared the activity of mitochondrial complexes, ATP, and ROS levels between MCF-7 and MDA-MB-231 in the presence of TNF-α. We used Tumor Immune Estimation Resource (TIMER) webserver to analyze the correlation between TNF-α and mitochondrial proteins in basal and luminal breast cancer patients. Kaplan-Meier method was used to analyze the correlation between mitochondrial protein expression and survival of breast cancer patients. RESULTS: The proteome analysis revealed that TNF-α differentially altered the level of critical proteins of mitochondrial respiratory chain complexes both in MCF-7 and MDA-MB-231, which correlated with differential assembly and activity of mitochondrial ETC complexes. The inhibition of the glycolytic pathway in the presence of TNF-α showed that glycolysis is indispensable for the proliferation and clonogenic ability of MDA-MB-231 cells (ER/PR -ve) as compared to MCF-7 cells (ER/PR +ve). The TIMER database showed a negative correlation between the expressions of TNF-α and key regulators of mitochondrial OXPHOS complexes in basal breast vs lobular carcinoma. Conversely, patient survival analysis showed an improved relapse-free survival with increased expression of identified proteins of ETC complexes and survival of the breast cancer patients. CONCLUSION: The evidence presented in our study convincingly demonstrates that TNF-α regulates the survival and proliferation of aggressive tumor cells by modulating the levels of critical assembly factors and subunits involved in mitochondrial respiratory chain supercomplexes organization and function. This favors the rewiring of mitochondrial metabolism towards anaplerosis to support the survival and proliferation of breast cancer cells. Collectively, the results strongly suggest that TNF-α differentially regulates metabolic adaptation in ER/PR +ve (MCF-7) and ER/PR -ve (MDA-MB-231) cells by modulating the mitochondrial supercomplex assembly and activity.

The emerging molecular mechanisms for mitochondrial dysfunctions in FXTAS.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an inherited neurodegenerative disorder caused by an expansion of 55-200 CGG repeats at 5UTR of FMR1 gene, known as premutation. The main clinical and neuropathological features of FXTAS include progressive intention tremor, gait ataxia, neuronal cell loss and presence of ubiquitin-positive intranuclear inclusions in neurons and astrocytes. Various mitochondrial dysfunctions are reported in in vitro/vivo models of FXTAS; however, the molecular mechanisms underlying such mitochondrial dysfunctions are unclear. CGG expansions are pathogenic through distinct mechanisms involving RNA gain of function, impaired DNA damage repair and FMRpolyG toxicity. Here, we have systematically reviewed the reports of mitochondrial dysfunctions under premutation condition. We have also focused on potential emerging mechanisms to understand mitochondrial associated pathology in FXTAS. This review highlights the important role of mitochondria in FXTAS and other related disorders; and suggests focus of future studies on mitochondrial dysfunction along with other prevailing mechanisms to alleviate neurodegeneration.

TRIM32 regulates mitochondrial mediated ROS levels and sensitizes the oxidative stress induced cell death.

Emerging evidence suggests that ubiquitin mediated post translational modification is a critical regulatory process involved in diverse cellular pathways including cell death. During ubiquitination, E3 ligases recognize target proteins and determine the topology of ubiquitin chains. Recruitment of E3 ligases to targets proteins under stress conditions including oxidative stress and their implication in cell death have not been systemically explored. In the present study, we characterized the role of TRIM32 as an E3 ligase in regulation of oxidative stress induced cell death. TRIM32 is ubiquitously expressed in cell lines of different origin and form cytoplasmic speckle like structures that transiently interact with mitochondria under oxidative stress conditions. The ectopic expression of TRIM32 sensitizes cell death induced by oxidative stress whereas TRIM32 knockdown shows a protective effect. The turnover of TRIM32 is enhanced during oxidative stress and its expression induces ROS generation, loss of mitochondrial transmembrane potential and decrease in complex-I activity. The pro-apoptotic effect was rescued by pan-caspase inhibitor or antioxidant treatment. E3 ligase activity of TRIM32 is essential for oxidative stress induced apoptotic cell death. Furthermore, TRIM32 decreases X-linked inhibitor of apoptosis (XIAP) level and overexpression of XIAP rescued cells from TRIM32 mediated oxidative stress and cell death. Overall, the results of this study provide the first evidence supporting the role of TRIM32 in regulating oxidative stress induced cell death, which has implications in numerous pathological conditions including cancer and neurodegeneration.

FMRpolyG alters mitochondrial transcripts level and respiratory chain complex assembly in Fragile X associated tremor/ataxia syndrome [FXTAS].

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an inherited neurodegenerative disorder caused by an expansion of 55 to 200 CGG repeats (premutation) in FMR1. These CGG repeats are Repeat Associated non-ATG (RAN) translated into a small and pathogenic protein, FMRpolyG. The cellular and molecular mechanisms of FMRpolyG toxicity are unclear. Various mitochondrial dysfunctions have been observed in FXTAS patients and animal models. However, the causes of these mitochondrial alterations are not well understood. In the current study, we investigated interaction of FMRpolyG with mitochondria and its role in modulating mitochondrial functions. Beside nuclear inclusions, FMRpolyG also formed small cytosolic aggregates that interact with mitochondria both in cell and mouse model of FXTAS. Importantly, expression of FMRpolyG reduces ATP levels, mitochondrial transmembrane potential, mitochondrial supercomplexes assemblies and activities and expression of mitochondrial DNA encoded transcripts in cell and animal model of FXTAS, as well as in FXTAS patient brain tissues. Overall, these results suggest that FMRpolyG alters mitochondrial functions, bioenergetics and initiates cell death. The further study in this direction will help to establish the role of mitochondria in FXTAS conditions.