RESUMO
Tumor necrosis factor-α (TNF-α) is a major mediator of inflammation and its increased levels have been analyzed in vitiligo patients. Vitiligo is a depigmentary skin disarray caused due to disapperance of functional melanocytes. The aim of the study was to investigate the role of TNF-α in melanocyte biology, analyzing candidate molecules of melanocytes and immune homeostasis. Our results showed increased TNF-α transcripts in vitiligenous lesional and non-lesional skin. Melanocytes upon exogenous stimulation with TNF-α exhibited a significant reduction in cell viability with elevated cellular and mitochondrial ROS and compromised complex I activity. Moreover, we observed a reduction in melanin content via shedding of dendrites, down-regulation of MITF-M, TYR and up-regulation of TNFR1, IL6, ICAM1 expression, whereas TNFR2 levels remain unaltered. TNF-α exposure stimulated cell apoptosis at 48 h and autophagy at 12 h, elevating ATG12 and BECN1 transcripts. Our novel findings establish the functional link between autophagy and melanocyte destruction. Overall, our study suggests a key function of TNF-α in melanocyte homeostasis and autoimmune vitiligo pathogenesis.
Assuntos
Melaninas/metabolismo , Melanócitos/metabolismo , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Vitiligo/metabolismo , Apoptose/fisiologia , Proteína 12 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/metabolismo , Regulação para Baixo/fisiologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Pele/metabolismo , Regulação para Cima/fisiologiaRESUMO
Poly (ADP-ribose) polymerase-1 (PARP-1) is a multifunctional protein that is associated with various biological processes like chromatin remodeling, DNA damage, cell death etc. In Dictyostelium discoideum, PARP-1 has also been implicated in cellular differentiation and development. However, its interacting proteins during multicellular development are not yet explored. Hence, the present study aims to identify PARP-1 interacting proteins during multicellular development of D. discoideum. BRCA1 C-terminus (BRCT) domain of PARP-1, which is mainly involved in protein-protein interactions was cloned in pGEX4T1 vector and developmental interactome of PARP-1 were analyzed by affinity purification-mass spectrometry. These interactions were further confirmed by in-silico protein-protein docking analysis, which led to identification of the proteins that show high affinity for BRCT domain. Initially, the protein structures were modeled on SWISS MODEL and PHYRE2 servers, refined by 3Drefine and validated by PROCHECK. Further, interaction sites of BRCT and the conserved regions in all interacting proteins were predicted using cons-PPISP and ConSurf, respectively. Finally, protein-protein docking analysis was done by HADDOCK. Our results identified 19 possible BRCT interacting proteins during D. discoideum development. Furthermore, interacting residues involved in the interactions and functional regions were explored. This is the first report where PARP-1's developmental interactome in D. discoideum is well established. The current findings demonstrate PARP-1's developmental interactome in D. discoideum and provide the groundwork to understand its regulated functions in developmental biology which would undoubtedly extend our perception towards developmental diseases in higher complex organisms and their treatment.
Assuntos
Dictyostelium , Estágios do Ciclo de Vida/genética , Poli(ADP-Ribose) Polimerase-1 , Proteínas de Protozoários , Sítios de Ligação/genética , Bases de Dados de Proteínas , Dictyostelium/enzimologia , Dictyostelium/genética , Dictyostelium/crescimento & desenvolvimento , Espectrometria de Massas , Simulação de Acoplamento Molecular , Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Mapas de Interação de Proteínas/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
BACKGROUND INFORMATION: Poly(ADP-ribose) Polymerase-1 (PARP-1) is predominantly a nuclear protein and involved in various cellular processes like DNA repair, cell death, development, chromatin modulation etc. PARP-1 utilizes NAD+ and adds negatively charged PAR moieties on the target proteins. Over-activation of PARP-1 has been shown to cause energy crisis mediated cell death in which mitochondrial homeostasis is also affected. Moreover, the presence of mitochondrial NAD+ pools highlights the role of PARP-1 in mitochondria. The aim of present study is to understand the physiological role of PARP-1 in regulating mitochondrial functioning by varying the levels of PARP-1 in Dictyostelium discoideum. Intra-mitochondrial PARylation was analyzed by indirect immunofluorescence. Further, the effect of altered levels of PARP-1 i.e. overexpression, downregulation, knockout and its chemical inhibition was studied on mitochondrial respiration, reactive oxygen species (ROS) levels, ATP production, mitochondrial fission-fusion, mitochondrial morphology and mitochondrial DNA (mtDNA) content of D. discoideum. RESULTS: Our results show intra-mitochondrial PARylation under oxidative stress. Altered levels of PARP-1 caused impairment in the mitochondrial respiratory capacity, leading to elevated ROS levels and reduced ATP production. Moreover, PARP-1 affects the mitochondrial morphology and mtDNA content, alters the mitochondrial fission-fusion processes in lieu of preventing cell death under physiological conditions. CONCLUSION: The current study highlights the physiological role of PARP-1 in mitochondrial respiration, its morphology, fission-fusion processes and mtDNA maintenance in D. discoideum. SIGNIFICANCE: This study would provide new clues on the PARP-1's crucial role in mitochondrial homeostasis, exploring the therapeutic potential of PARP-1 in various mitochondrial diseases.
Assuntos
Dictyostelium/enzimologia , Homeostase/efeitos dos fármacos , Mitocôndrias/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Trifosfato de Adenosina/biossíntese , Benzamidas/farmacologia , Morte Celular/efeitos dos fármacos , DNA Mitocondrial/metabolismo , Dictyostelium/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Dinâmica Mitocondrial/genética , Estresse Oxidativo/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Poly (ADP-ribose) polymerase-1 (PARP1) is a DNA damage sensor that gets activated in proportion to the damage, helping cells to determine whether to repair the damage or initiate cell death processes. We have previously shown PARP1's significance in the developmental processes of Dictyostelium discoideum in addition to its role in oxidative stress and UV-C stress induced cell death. In this study, we show the significance of ROS in PARP1 mediated responses of D. discoideum under different stress conditions. Interestingly, our results suggest differential kinetics of PARP1 activation and implications of ROS in starvation and cadmium induced cell death events. Increased accumulation of Poly (ADP-ribose), a product of PARP activation, could be detected within minutes post cadmium stress, whereas PARP1 activation was only a later event with starvation. Starvation induced PARP1 activation was supported by the depletion of ATP and NAD+, while PARP inhibitor confers protective effect during starvation. During starvation, cell death is induced in two phases, a primary ROS driven PARP1 independent early necrotic phase followed by a PARP1 driven ROS dependent paraptotic phase; both of which comprise mitochondrial changes. Cadmium (Cd) exerted a dose-dependent effect on cell death; a low dose of 0.2 mM Cd led to paraptosis and a higher dose of 0.5 mM Cd led to necrosis in D. discoideum cells within 24 h. Interestingly, glutathione (GSH) exposure could rescue cells from Cd stress mediated cell death. Besides unicellular cell death, the developmental arrest induced by cadmium and oxidative stress could be rescued by reinstating the redox equilibrium using GSH. In conclusion, we underscore the significant link between PARP1 and ROS in regulating the process of cell death and development in D. discoideum.
Assuntos
Morte Celular , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/metabolismo , Estresse Oxidativo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteínas de Protozoários/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Cádmio/toxicidade , Dictyostelium/efeitos dos fármacos , Mitocôndrias , Transdução de Sinais , Estresse FisiológicoRESUMO
BACKGROUND INFORMATION: Poly(ADP-ribose) polymerase-1 (PARP-1) has been attributed to varied roles in DNA repair, cell cycle, cell death, etc. Our previous reports demonstrate the role of PARP-1 during Dictyostelium discoideum development by its constitutive downregulation as well as by PARP-1 ortholog, ADP ribosyl transferase 1 A (ADPRT1A) overexpression. The current study analyses and strengthens the function of ADPRT1A in multicellular morphogenesis of D. discoideum. ADPRT1A was knocked out, and its effect was studied on cAMP signalling, chemotaxis and development of D. discoideum. RESULTS: We report that ADPRT1A is essential in multicellular development of D. discoideum, particularly at the aggregation stage. Genetic alterations of ADPRT1A and chemical inhibition of its activity affects the intracellular and extracellular cAMP levels during aggregation along with chemotaxis. Exogenous cAMP pulses could rescue this defect in the ADPRT1A knockout (ADPRT1A KO). Expression analysis of genes involved in cAMP signalling reveals altered transcript levels of four essential genes (PDSA, REGA, ACAA and CARA). Moreover, ADPRT1A KO affects prespore- and prestalk-specific gene expression and prestalk tendency is favoured in the ADPRT1A KO. CONCLUSION: ADPRT1A plays a definite role in regulating developmental morphogenesis via cAMP signalling. SIGNIFICANCE: This study helps in understanding the role of PARP-1 in multicellular development and differentiation in higher complex organisms.
Assuntos
Quimiotaxia , Dictyostelium/crescimento & desenvolvimento , Poli(ADP-Ribose) Polimerase-1/fisiologia , Proteínas de Protozoários/fisiologia , AMP Cíclico/metabolismo , Dictyostelium/genética , Dictyostelium/fisiologia , Técnicas de Inativação de Genes , Isoenzimas/genética , Isoenzimas/fisiologia , Morfogênese , Poli(ADP-Ribose) Polimerase-1/genética , Proteínas de Protozoários/genética , Transdução de Sinais , TranscriptomaRESUMO
Gene regulation mediates the processes of cellular development and differentiation leading to the origin of different cell types each having their own signature gene expression profile. However, the compact chromatin structure and the timely recruitment of molecules involved in various signaling pathways are of prime importance for temporal and spatial gene regulation that eventually contribute towards cell type and specificity. Poly (ADP-ribose) polymerase-1 (PARP-1), a 116-kDa nuclear multitasking protein is involved in modulation of chromatin condensation leading to altered gene expression. In response to activation signals, it adds ADP-ribose units to various target proteins including itself, thus regulating various key cellular processes like DNA repair, cell death, transcription, mRNA splicing etc. This review provides insights into the role of PARP-1 in gene regulation, cell differentiation and multicellular morphogenesis. In addition, the review also explores involvement of PARP-1 in immune cells development and therapeutic possibilities to treat various human diseases.
Assuntos
Diferenciação Celular/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Transcrição Gênica , Animais , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica , Humanos , Modelos BiológicosRESUMO
The unicellular, as well as multicellular stages of Dictyostelium discoideum's life cycle, make it an excellent model system for cell type determination, differentiation, development, and cell death studies. Our preliminary results show the involvement of poly (ADP-ribose) polymerase-1 (PARP-1) during D. discoideum growth by its constitutive downregulation as well as by its ortholog overexpression. The current study now analyzes and strengthens the role of the PARP-1 ortholog in cellular proliferation of D. discoideum. ADPRT1A was knocked out (KO) from D. discoideum and studied for its effect on cell growth, cell cycle, morphology, and oxidative stress. The present findings show that ADPRT1A KO ( A KO) cells exhibited reduced cellular proliferation, stressed phenotype, and cell cycle arrest in G2-M phase. Under oxidative stress, A KO cells exhibited slower growth and DNA damage. This is the first report where the involvement of ADPRT1A in growth in D. discoideum is established.
Assuntos
Proliferação de Células/fisiologia , Dictyostelium/metabolismo , Dictyostelium/fisiologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/fisiologia , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/fisiologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologiaRESUMO
Apoptosis Inducing Factor (AIF), a phylogenetically conserved mitochondrial inter-membrane space flavoprotein has an important role in caspase independent cell death. Nevertheless, AIF is also essential for cell survival. It is required for mitochondrial organization and energy metabolism. Upon apoptotic stimulation, AIF induces DNA fragmentation after its mitochondrio-nuclear translocation. Although it executes critical cellular functions in a coordinated manner, the exact mechanism still remains obscure. The present study aims to understand AIF's role in cell survival, growth and development by its down-regulation in an interesting unicellular eukaryote, D. discoideum which exhibits multicellularity upon starvation. Constitutive AIF down-regulated (dR) cells exhibited slower growth and delayed developmental morphogenesis. Also, constitutive AIF dR cells manifested high intracellular ROS, oxidative DNA damage and calcium levels with lower ATP content. Interestingly, constitutive AIF dR cells showed amelioration in cell growth upon antioxidant treatment, strengthening its role as ROS regulator. Under oxidative stress, AIF dR cells showed early mitochondrial membrane depolarization followed by AIF translocation from mitochondria to nucleus and exhibited necrotic cell death as compared to paraptoptic cell death of control cells. Thus, the results of this study provide an exemplar where AIF is involved in growth and development by regulating ROS levels and maintaining mitochondrial function in D. discoideum, an evolutionarily significant model organism exhibiting caspase independent apoptosis.
Assuntos
Fator de Indução de Apoptose/metabolismo , Evolução Biológica , Dictyostelium/citologia , Dictyostelium/metabolismo , Trifosfato de Adenosina/metabolismo , Anexina A5/metabolismo , Cálcio/metabolismo , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/ultraestrutura , Regulação para Baixo/efeitos dos fármacos , Citometria de Fluxo , Fluoresceína-5-Isotiocianato/metabolismo , Fluorometria , Glucose/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , NAD/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Propídio/metabolismo , Transporte Proteico/efeitos dos fármacos , RNA Antissenso/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Coloração e RotulagemRESUMO
Poly (ADP-ribose) polymerase (PARP)-1 regulates various biological processes like DNA repair, cell death etc. However, the role of PARP-1 in growth and differentiation still remains elusive. The present study has been undertaken to understand the role of PARP-1 in growth and development of a unicellular eukaryote, Dictyostelium discoideum. In silico analysis demonstrates ADPRT1A as the ortholog of human PARP-1 in D. discoideum. The present study shows that ADPRT1A overexpression (A OE) led to slow growth of D. discoideum and significant population of AOE cells were in S and G2/M phase. Also, AOE cells exhibited high endogenous PARP activity, significant NAD(+) depletion and also significantly lower ADPRT1B and ADPRT2 transcript levels. Moreover, AOE cells are intrinsically stressed and also exhibited susceptibility to oxidative stress. AOE also affected development of D. discoideum predominantly streaming, aggregation and formation of early culminant which are concomitant with reports on PARP's role in D. discoideum development. In addition, under developmental stimuli, increased PARP activity was seen along with developmentally regulated transcript levels of ADPRT1A during D. discoideum multicellularity. Thus the present study suggests that PARP-1 regulates growth as well as the developmental morphogenesis of D. discoideum, thereby opening new avenues to understand the same in higher eukaryotes.
Assuntos
ADP Ribose Transferases/metabolismo , Diferenciação Celular , Dictyostelium/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Estresse Oxidativo , Poli(ADP-Ribose) Polimerase-1/metabolismo , ADP Ribose Transferases/genética , Sequência de Aminoácidos , Animais , Apoptose , Western Blotting , Ciclo Celular , Células Cultivadas , Dictyostelium/enzimologia , Dictyostelium/metabolismo , Potencial da Membrana Mitocondrial , Filogenia , Poli(ADP-Ribose) Polimerase-1/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
Apoptosis involves a cascade of caspase activation leading to the ordered dismantling of critical cell components. However, little is known about the dismantling process in non-apoptotic cell death where caspases are not involved. Dictyostelium discoideum is a good model system to study caspase-independent cell death where experimental accessibility of non-apoptotic cell death is easier and molecular redundancy is reduced compared with other animal models. Poly(ADP-ribose) polymerase (PARP) is one of the key players in cell death. We have previously reported the role of PARP in development and the oxidative stress-induced cell death of D. discoideum. D. discoideum possesses nine PARP genes and does not have a caspase gene, and thus it provides a better model system to dissect the role of PARP in caspase-independent cell death. The current study shows that non-apoptotic cell death in D. discoideum occurs in a programmed fashion where proteases cause mitochondrial membrane potential changes followed by plasma membrane rupture and early loss of plasma membrane integrity. Furthermore, the results suggest that calpains and cathepsin D, which are instrumental in dismantling the cell, act downstream of PARP. Thus, PARP, apoptosis inducing factor, calpains and cathepsin D are the key players in D. discoideum caspase-independent cell death, acting in a sequential manner.
Assuntos
Morte Celular , Dictyostelium/efeitos dos fármacos , Dictyostelium/fisiologia , Estresse Oxidativo , Peptídeo Hidrolases/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Membrana Celular/fisiologia , Dictyostelium/enzimologia , Potencial da Membrana Mitocondrial , Membranas Mitocondriais/fisiologiaRESUMO
BACKGROUND: Myocardial infarction (MI) is a significant cause of death in diabetic patients. Growing evidence suggests that mitochondrial dysfunction contributes to heart failure in diabetes. However, the molecular mechanisms of mitochondrial dysfunction mediating heart failure in diabetes are still poorly understood. METHODS: We examined MRPL12 levels in right atrial appendage tissues from diabetic patients undergoing coronary artery bypass graft (CABG) surgery. Using AC-16 cells overexpressing MRPL12 under normal and hyperglycemic conditions we performed mitochondrial functional assays OXPHOS, bioenergetics, mitochondrial membrane potential, ATP production and cell death. RESULTS: We observed elevated MRPL12 levels in heart tissue samples from diabetic patients with ischemic heart disease compared to non-diabetic patients. Overexpression of MRPL12 under hyperglycemic conditions did not affect oxidative phosphorylation (OXPHOS) levels, cellular ATP levels, or cardiomyocyte cell death. However, notable impairment in mitochondrial membrane potential (MMP) was observed under hyperglycemic conditions, along with alterations in both basal respiration oxygen consumption rate (OCR) and maximal respiratory capacity OCR. CONCLUSIONS: Overall, our results suggest that MRPL12 may have a compensatory role in the diabetic myocardium with ischemic heart disease, suggesting that MRPL12 may implicate in the pathophysiology of MI in diabetes.
Assuntos
Proteínas de Ciclo Celular , Potencial da Membrana Mitocondrial , Isquemia Miocárdica , Proteínas Nucleares , Fosforilação Oxidativa , Proteínas Ribossômicas , Idoso , Animais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Trifosfato de Adenosina/metabolismo , Apêndice Atrial/metabolismo , Apêndice Atrial/patologia , Ponte de Artéria Coronária , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/complicações , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Mitocôndrias Cardíacas/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Isquemia Miocárdica/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMO
Age-related atrophy of skeletal muscle, is characterized by loss of mass, strength, endurance, and oxidative capacity during aging. Notably, bioenergetics and protein turnover studies have shown that mitochondria mediate this decline in function. Although exercise has been the only therapy to mitigate sarcopenia, the mechanisms that govern how exercise serves to promote healthy muscle aging are unclear. Mitochondrial aging is associated with decreased mitochondrial capacity, so we sought to investigate how aging affects mitochondrial structure and potential age-related regulators. Specifically, the three-dimensional (3D) mitochondrial structure associated with morphological changes in skeletal muscle during aging requires further elucidation. We hypothesized that aging causes structural remodeling of mitochondrial 3D architecture representative of dysfunction, and this effect is mitigated by exercise. We used serial block-face scanning electron microscopy to image human skeletal tissue samples, followed by manual contour tracing using Amira software for 3D reconstruction and subsequent analysis of mitochondria. We then applied a rigorous in vitro and in vivo exercise regimen during aging. Across 5 human cohorts, we correlate differences in magnetic resonance imaging, mitochondria 3D structure, exercise parameters, and plasma immune markers between young (under 50 years) and old (over 50 years) individuals. We found that mitochondria we less spherical and more complex, indicating age-related declines in contact site capacity. Additionally, aged samples showed a larger volume phenotype in both female and male humans, indicating potential mitochondrial swelling. Concomitantly, muscle area, exercise capacity, and mitochondrial dynamic proteins showed age-related losses. Exercise stimulation restored mitofusin 2 (MFN2), one such of these mitochondrial dynamic proteins, which we show is required for the integrity of mitochondrial structure. Furthermore, we show that this pathway is evolutionarily conserved as Marf, the MFN2 ortholog in Drosophila, knockdown alters mitochondrial morphology and leads to the downregulation of genes regulating mitochondrial processes. Our results define age-related structural changes in mitochondria and further suggest that exercise may mitigate age-related structural decline through modulation of mitofusin 2.
RESUMO
The liver, the largest internal organ and a metabolic hub, undergoes significant declines due to aging, affecting mitochondrial function and increasing the risk of systemic liver diseases. How the mitochondrial three-dimensional (3D) structure changes in the liver across aging, and the biological mechanisms regulating such changes confers remain unclear. In this study, we employed Serial Block Face-Scanning Electron Microscopy (SBF-SEM) to achieve high-resolution 3D reconstructions of murine liver mitochondria to observe diverse phenotypes and structural alterations that occur with age, marked by a reduction in size and complexity. We also show concomitant metabolomic and lipidomic changes in aged samples. Aged human samples reflected altered disease risk. To find potential regulators of this change, we examined the Mitochondrial Contact Site and Cristae Organizing System (MICOS) complex, which plays a crucial role in maintaining mitochondrial architecture. We observe that the MICOS complex is lost during aging, but not Sam50. Sam50 is a component of the sorting and assembly machinery (SAM) complex that acts in tandem with the MICOS complex to modulate cristae morphology. In murine models subjected to a high-fat diet, there is a marked depletion of the mitochondrial protein SAM50. This reduction in Sam50 expression may heighten the susceptibility to liver disease, as our human biobank studies corroborate that Sam50 plays a genetically regulated role in the predisposition to multiple liver diseases. We further show that changes in mitochondrial calcium dysregulation and oxidative stress accompany the disruption of the MICOS complex. Together, we establish that a decrease in mitochondrial complexity and dysregulated metabolism occur with murine liver aging. While these changes are partially be regulated by age-related loss of the MICOS complex, the confluence of a murine high-fat diet can also cause loss of Sam50, which contributes to liver diseases. In summary, our study reveals potential regulators that affect age-related changes in mitochondrial structure and metabolism, which can be targeted in future therapeutic techniques.
RESUMO
The kidney filters nutrient waste and bodily fluids from the bloodstream, in addition to secondary functions of metabolism and hormone secretion, requiring an astonishing amount of energy to maintain its functions. In kidney cells, mitochondria produce adenosine triphosphate (ATP) and help maintain kidney function. Due to aging, the efficiency of kidney functions begins to decrease. Dysfunction in mitochondria and cristae, the inner folds of mitochondria, is a hallmark of aging. Therefore, age-related kidney function decline could be due to changes in mitochondrial ultrastructure, increased reactive oxygen species (ROS), and subsequent alterations in metabolism and lipid composition. We sought to understand if there is altered mitochondrial ultrastructure, as marked by 3D morphological changes, across time in tubular kidney cells. Serial block facing-scanning electron microscope (SBF-SEM) and manual segmentation using the Amira software were used to visualize murine kidney samples during the aging process at 3 months (young) and 2 years (old). We found that 2-year mitochondria are more fragmented, compared to the 3-month, with many uniquely shaped mitochondria observed across aging, concomitant with shifts in ROS, metabolomics, and lipid homeostasis. Furthermore, we show that the mitochondrial contact site and cristae organizing system (MICOS) complex is impaired in the kidney due to aging. Disruption of the MICOS complex shows altered mitochondrial calcium uptake and calcium retention capacity, as well as generation of oxidative stress. We found significant, detrimental structural changes to aged kidney tubule mitochondria suggesting a potential mechanism underlying why kidney diseases occur more readily with age. We hypothesize that disruption in the MICOS complex further exacerbates mitochondrial dysfunction, creating a vicious cycle of mitochondrial degradation and oxidative stress, thus impacting kidney health.
RESUMO
Mitochondria play a critical role in energy metabolism and signal transduction, which is tightly regulated by proteins, metabolites, and ion fluxes. Metabolites and ion homeostasis are mainly mediated by channels and transporters present on mitochondrial membranes. Mitochondria comprise two distinct compartments, the outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM), which have differing permeabilities to ions and metabolites. The OMM is semipermeable due to the presence of non-selective molecular pores, while the IMM is highly selective and impermeable due to the presence of specialized channels and transporters which regulate ion and metabolite fluxes. These channels and transporters are modulated by various post-translational modifications (PTMs), including phosphorylation, oxidative modifications, ions, and metabolites binding, glycosylation, acetylation, and others. Additionally, the mitochondrial protein quality control (MPQC) system plays a crucial role in ensuring efficient molecular flux through the mitochondrial membranes by selectively removing mistargeted or defective proteins. Inefficient functioning of the transporters and channels in mitochondria can disrupt cellular homeostasis, leading to the onset of various pathological conditions. In this review, we provide a comprehensive overview of the current understanding of mitochondrial channels and transporters in terms of their functions, PTMs, and quality control mechanisms.
RESUMO
Mitochondrial calcium overload contributes to neurodegenerative disease development and progression. We recently reported that loss of the mitochondrial sodium/calcium exchanger (NCLX), the primary mechanism of mCa2+ efflux, promotes mCa2+ overload, metabolic derangement, redox stress, and cognitive decline in models of Alzheimer's disease (AD). However, whether disrupted mCa2+ signaling contributes to neuronal pathology and cognitive decline independent of pre-existing amyloid or tau pathology remains unknown. Here, we generated mice with neuronal deletion of the mitochondrial sodium/calcium exchanger (NCLX, Slc8b1 gene), and evaluated age-associated changes in cognitive function and neuropathology. Neuronal loss of NCLX resulted in an age-dependent decline in spatial and cued recall memory, moderate amyloid deposition, mild tau pathology, synaptic remodeling, and indications of cell death. These results demonstrate that loss of NCLX-dependent mCa2+ efflux alone is sufficient to induce an Alzheimer's disease-like pathology and highlights the promise of therapies targeting mCa2+ exchange.
RESUMO
BACKGROUND: Nuclear poly(ADP-ribose) polymerase-1 (PARP-1) is a well characterised protein that accounts for the majority of PARylation reactions using NAD+ as a substrate, regulating diverse cellular functions. In addition to its nuclear functions, several recent studies have identified localization of PARP-1 in mitochondria and emphasized its possible role in maintaining mitochondrial homeostasis. Various reports suggest that nuclear PARP-1 has been implicated in diverse mitochondria-specific communication processes. SCOPE OF REVIEW: The present review emphasizes on the potential role of PARP-1 in mitochondrial processes such as bioenergetics, mtDNA maintenance, cell death and mitophagy. MAJOR CONCLUSIONS: The origin of mitochondrial PARP-1 is still an enigma; however researchers are trying to establish the cross-talk between nuclear and mitochondrial PARP-1 and how these PARP-1 pools modulate mitochondrial activity. GENERAL SIGNIFICANCE: A better understanding of the possible role of PARP-1 in mitochondrial homeostasis helps us to explore the potential therapeutic targets to protect mitochondrial dysfunctions.
Assuntos
Mitocôndrias/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Animais , DNA Mitocondrial/metabolismo , Metabolismo Energético , Homeostase , Humanos , Mitocôndrias/patologia , Mitofagia , Poli(ADP-Ribose) Polimerase-1/análiseRESUMO
Apoptosis Inducing Factor (AIF), a nuclear encoded mitochondrial inter-membrane space flavoprotein with intrinsic NADH oxidase activity, plays an important role in inducing cell death mechanisms. In response to cell death signals, it undergoes mitochondrio-nuclear translocation leading to DNA fragmentation. In addition to its role in cell death, AIF has a pro-survival role, wherein it contributes to the maintenance of mitochondrial structure and function in a coordinated manner. However, its exact mechanism of controlling mitochondrial homeostasis is unclear. The current study aims to explore the protective functions of AIF by its downregulation and overexpression in Dictyostelium discoideum. Constitutive AIF downregulated (dR) cells exhibited compromised oxidative phosphorylation along with elevated levels of cellular ROS. Interestingly, constitutive AIF dR cells showed amelioration in the activity of the ETC complexes upon antioxidant treatment, strengthening AIF's role as an ROS regulator, by virtue of its oxidoreductase property. Also, constitutive AIF dR cells showed lower transcript levels of the various subunits of ETC. Moreover, loss of AIF affected mtDNA content and mitochondrial fusion-fission mechanism, which subsequently caused morphometric mitochondrial alterations. Constitutive AIF overexpressed (OE) cells also showed higher cellular ROS and mitochondrial fission genes transcript levels along with reduced mitochondrial fusion genes transcript levels and mtDNA content. Thus, the results of the current study provide a paradigm where AIF is implicated in cell survival by maintaining mitochondrial bioenergetics, morphology and fusion-fission mechanism in D. discoideum, an evolutionarily significant model organism for mitochondrial diseases.