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1.
Artigo em Inglês | MEDLINE | ID: mdl-31674811

RESUMO

Type I PKA regulatory subunit alpha (RIα; encoded by the Prkar1a gene) serves as the predominant inhibitor protein of the catalytic subunit of cAMP-dependent protein kinase (PKAc). However, recent evidence suggests that PKA signaling can be initiated by cAMP-independent events, especially within the context of cellular oxidative stress such as ischemia/reperfusion (I/R) injury. We determined whether RIα is actively involved in the regulation of PKA activity via Reactive Oxygen Species (ROS) dependent mechanisms during I/R stress in the heart. Induction of ex vivo global I/R injury in mouse hearts selectively down-regulated RIα protein expression, whereas RII subunit expression appears to remain unaltered. Cardiac myocyte cell culture models were used to determine that oxidant stimulus (i.e. H2O2) alone is sufficient to induce RIα protein down-regulation. Transient increase of RIα expression (via adenoviral overexpression) negatively affects cell survival and function upon oxidative stress as measured by increased induction of apoptosis and decreased mitochondrial respiration. Furthermore, analysis of mitochondrial subcellular fractions in heart tissue showed that PKA associated proteins are enriched in subsarcolemmal mitochondria (SSM) fractions, and that loss of RIα is most pronounced at SSM upon I/R injury. These data were supported via electron microscopy in AKAP1-KO mice, where loss of AKAP1 expression leads to aberrant mitochondrial morphology manifested in SSM but not interfibrillar mitochondria (IFM). Thus, we conclude that modification of RIα via ROS-dependent mechanisms induced by I/R injury has the potential to sensitize PKA signaling in the cell without the direct use of the canonical cAMP-dependent activation pathway.

2.
Sci Rep ; 9(1): 4734, 2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30894648

RESUMO

This study investigated the effects of elevated fatty acid (FA) supply from adipose tissue on the ultrastructure of cardiac lipid droplets (LDs) and the expression and organization of LD scaffold proteins perilipin-2 (PLIN2) and perilipin-5 (PLIN5). Stimulation of adipocyte lipolysis by fasting (24 h) or ß3-adrenergic receptor activation by CL316, 243 (CL) increased cardiac triacylglycerol (TAG) levels and LD size, whereas CL treatment also increased LD number. LDs were tightly associated with mitochondria, which was maintained during LD expansion. Electron tomography (ET) studies revealed continuity of LD and smooth endoplasmic reticulum (SER), suggesting interconnections among LDs. Under fed ad libitum conditions, the cristae of mitochondria that apposed LD were mostly organized perpendicularly to the tangent of the LD surface. Fasting significantly reduced, whereas CL treatment greatly increased, the perpendicular alignment of mitochondrial cristae. Fasting and CL treatment strongly upregulated PLIN5 protein and PLIN2 to a lesser extent. Immunofluorescence and immuno-electron microscopy demonstrated strong targeting of PLIN5 to the cardiac LD-mitochondrial interface, but not to the mitochondrial matrix. CL treatment augmented PLIN5 targeting to the LD-mitochondrial interface, whereas PLIN2 was not significantly affected. Together, our results support the concept that the interface between LD and cardiac mitochondria represents an organized and dynamic "metabolic synapse" that is highly responsive to FA trafficking.

3.
Biochem Biophys Res Commun ; 503(4): 2690-2697, 2018 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-30100066

RESUMO

Optineurin (OPTN) mutations are linked to glaucoma pathology and E50K mutation shows massive cell death in photoreceptor cells and retinal ganglion cells. However, little is known about E50K-mediated mitochondrial dysfunction in photoreceptor cell degeneration. We here show that overexpression of E50K expression triggered BDNF deficiency, leading to Bax activation in RGC-5 cells. BDNF deficiency induced mitochondrial dysfunction by decreasing mitochondrial maximal respiration and reducing intracellular ATP level in RGC-5 cells. However, BDNF deficiency did not alter mitochondrial dynamics. Also, BDNF deficiency resulted in LC3-mediated mitophagosome formation in RGC-5 cells. These results strongly suggest that E50K-mediated BDNF deficiency plays a critical role in compromised mitochondrial function in glaucomatous photoreceptor cell degeneration.

4.
J Neurosci ; 38(38): 8233-8242, 2018 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-30093535

RESUMO

Mitochondrial fission and fusion impact numerous cellular functions and neurons are particularly sensitive to perturbations in mitochondrial dynamics. Here we describe that male mice lacking the mitochondrial A-kinase anchoring protein 1 (AKAP1) exhibit increased sensitivity in the transient middle cerebral artery occlusion model of focal ischemia. At the ultrastructural level, AKAP1-/- mice have smaller mitochondria and increased contacts between mitochondria and the endoplasmic reticulum in the brain. Mechanistically, deletion of AKAP1 dysregulates complex II of the electron transport chain, increases superoxide production, and impairs Ca2+ homeostasis in neurons subjected to excitotoxic glutamate. Ca2+ deregulation in neurons lacking AKAP1 can be attributed to loss of inhibitory phosphorylation of the mitochondrial fission enzyme dynamin-related protein 1 (Drp1) at the protein kinase A (PKA) site Ser637. Our results indicate that inhibition of Drp1-dependent mitochondrial fission by the outer mitochondrial AKAP1/PKA complex protects neurons from ischemic stroke by maintaining respiratory chain activity, inhibiting superoxide production, and delaying Ca2+ deregulation. They also provide the first genetic evidence that Drp1 inhibition may be of therapeutic relevance for the treatment of stroke and neurodegeneration.SIGNIFICANCE STATEMENT Previous work suggests that activation of dynamin-related protein 1 (Drp1) and mitochondrial fission contribute to ischemic injury in the brain. However, the specificity and efficacy of the pharmacological Drp1 inhibitor mdivi-1 that was used has now been discredited by several high-profile studies. Our report is timely and highly impactful because it provides the first evidence that genetic disinhibition of Drp1 via knock-out of the mitochondrial protein kinase A (PKA) scaffold AKAP1 exacerbates stroke injury in mice. Mechanistically, we show that electron transport deficiency, increased superoxide production, and Ca2+ overload result from genetic disinhibition of Drp1. In summary, our work settles current controversies regarding the role of mitochondrial fission in neuronal injury, provides mechanisms, and suggests that fission inhibitors hold promise as future therapeutic agents.

5.
J Strength Cond Res ; 32(5): 1391-1403, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29309390

RESUMO

Liu, J, Lee, I, Feng, H-Z, Galen, SS, Hüttemann, PP, Perkins, GA, Jin, J-P, Hüttemann, M, and Malek, MH. Aerobic exercise preconception and during pregnancy enhances oxidative capacity in the hindlimb muscles of mice offspring. J Strength Cond Res 32(5): 1391-1403, 2018-Little is known about the effect of maternal exercise on offspring skeletal muscle health. The purpose of this study, therefore, was to determine whether maternal exercise (preconception and during pregnancy) alters offspring skeletal muscle capillarity and mitochondrial biogenesis. We hypothesized that offspring from exercised dams would have higher capillarity and mitochondrial density in the hindlimb muscles compared with offspring from sedentary dams. Female mice in the exercise condition had access to a running wheel in their individual cage 30 days before mating and throughout pregnancy, whereas the sedentary group did not have access to the running wheel before mating and during pregnancy. Male offspring from both groups were killed when they were 2 months old, and their tissues were analyzed. The results indicated no significant (p > 0.05) mean differences for capillarity density, capillarity-to-fiber ratio, or regulators of angiogenesis such as VEGF-A and TSP-1. Compared with offspring from sedentary dams, however, offspring from exercised dams had an increase in protein expression of myosin heavy chain type I (MHC I) (∼134%; p = 0.009), but no change in MHC II. For mitochondrial morphology, we found significant (all p-values ≤ 0.0124) increases in mitochondrial volume density (∼55%) and length (∼18%) as well as mitochondria per unit area (∼19%). For mitochondrial enzymes, there were also significant (all p-values ≤ 0.0058) increases in basal citrate synthase (∼79%) and cytochrome c oxidase activity (∼67%) in the nonoxidative muscle fibers as well as increases in basal (ATP) (∼52%). Last, there were also significant mean differences in protein expression for regulators (FIS1, Lon protease, and TFAM) of mitochondrial biogenesis. These findings suggest that maternal exercise before and during pregnancy enhances offspring skeletal muscle mitochondria functionality, but not capillarity.


Assuntos
Mitocôndrias Musculares/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Condicionamento Físico Animal/fisiologia , Cuidado Pré-Concepcional/métodos , Animais , Feminino , Membro Posterior , Extremidade Inferior/fisiologia , Masculino , Camundongos , Mitocôndrias/metabolismo , Cadeias Pesadas de Miosina/fisiologia , Oxirredução , Estresse Oxidativo , Gravidez , Trombospondina 1/metabolismo
6.
J Cell Sci ; 130(19): 3248-3260, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28808085

RESUMO

Each mitochondrial compartment contains varying protein compositions that underlie a diversity of localized functions. Insights into the localization of mitochondrial intermembrane space-bridging (MIB) components will have an impact on our understanding of mitochondrial architecture, dynamics and function. By using the novel visualizable genetic tags miniSOG and APEX2 in cultured mouse cardiac and human astrocyte cell lines and performing electron tomography, we have mapped at nanoscale resolution three key MIB components, Mic19, Mic60 and Sam50 (also known as CHCHD3, IMMT and SAMM50, respectively), in the environment of structural landmarks such as cristae and crista junctions (CJs). Tagged Mic19 and Mic60 were located at CJs, distributed in a network pattern along the mitochondrial periphery and also enriched inside cristae. We discovered an association of Mic19 with cytochrome c oxidase subunit IV. It was also found that tagged Sam50 is not uniformly distributed in the outer mitochondrial membrane and appears to incompletely overlap with Mic19- or Mic60-positive domains, most notably at the CJs.


Assuntos
Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Linhagem Celular Transformada , Humanos , Proteínas de Membrana/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética
7.
J Cell Biol ; 216(8): 2315-2327, 2017 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-28663346

RESUMO

Intestinal stem cells (ISCs) maintain the midgut epithelium in Drosophila melanogaster Proper cellular turnover and tissue function rely on tightly regulated rates of ISC division and appropriate differentiation of daughter cells. However, aging and epithelial injury cause elevated ISC proliferation and decreased capacity for terminal differentiation of daughter enteroblasts (EBs). The mechanisms causing functional decline of stem cells with age remain elusive; however, recent findings suggest that stem cell metabolism plays an important role in the regulation of stem cell activity. Here, we investigate how alterations in mitochondrial homeostasis modulate stem cell behavior in vivo via RNA interference-mediated knockdown of factors involved in mitochondrial dynamics. ISC/EB-specific knockdown of the mitophagy-related genes Pink1 or Parkin suppresses the age-related loss of tissue homeostasis, despite dramatic changes in mitochondrial ultrastructure and mitochondrial damage in ISCs/EBs. Maintenance of tissue homeostasis upon reduction of Pink1 or Parkin appears to result from reduction of age- and stress-induced ISC proliferation, in part, through induction of ISC senescence. Our results indicate an uncoupling of cellular, tissue, and organismal aging through inhibition of ISC proliferation and provide insight into strategies used by stem cells to maintain tissue homeostasis despite severe damage to organelles.


Assuntos
Envelhecimento/metabolismo , Proliferação de Células , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Intestinos/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Células-Tronco/enzimologia , Estresse Fisiológico , Ubiquitina-Proteína Ligases/metabolismo , Envelhecimento/genética , Animais , Animais Geneticamente Modificados , Senescência Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Homeostase , Intestinos/ultraestrutura , Mitocôndrias/enzimologia , Mitocôndrias/ultraestrutura , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Transdução de Sinais , Células-Tronco/ultraestrutura , Fatores de Tempo , Ubiquitina-Proteína Ligases/genética
8.
Am J Physiol Renal Physiol ; 313(2): F282-F290, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28331062

RESUMO

The pathophysiology of chronic kidney disease (CKD) is driven by alterations in surviving nephrons to sustain renal function with ongoing nephron loss. Oxygen supply-demand mismatch, due to hemodynamic adaptations, with resultant hypoxia, plays an important role in the pathophysiology in early CKD. We sought to investigate the underlying mechanisms of this mismatch. We utilized the subtotal nephrectomy (STN) model of CKD to investigate the alterations in renal oxygenation linked to sodium (Na) transport and mitochondrial function in the surviving nephrons. Oxygen delivery was significantly reduced in STN kidneys because of lower renal blood flow. Fractional oxygen extraction was significantly higher in STN. Tubular Na reabsorption was significantly lower per mole of oxygen consumed in STN. We hypothesized that decreased mitochondrial bioenergetic capacity may account for this and uncovered significant mitochondrial dysfunction in the early STN kidney: higher oxidative metabolism without an attendant increase in ATP levels, elevated superoxide levels, and alterations in mitochondrial morphology. We further investigated the effect of activation of hypoxia-inducible factor-1α (HIF-1α), a master regulator of cellular hypoxia response. We observed significant improvement in renal blood flow, glomerular filtration rate, and tubular Na reabsorption per mole of oxygen consumed with HIF-1α activation. Importantly, HIF-1α activation significantly lowered mitochondrial oxygen consumption and superoxide production and increased mitochondrial volume density. In conclusion, we report significant impairment of renal oxygenation and mitochondrial function at the early stages of CKD and demonstrate the beneficial role of HIF-1α activation on renal function and metabolism.


Assuntos
Aminoácidos Dicarboxílicos/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Rim/irrigação sanguínea , Rim/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Oxigênio/sangue , Insuficiência Renal Crônica/tratamento farmacológico , Trifosfato de Adenosina/metabolismo , Animais , Hipóxia Celular , Modelos Animais de Doenças , Metabolismo Energético/efeitos dos fármacos , Taxa de Filtração Glomerular/efeitos dos fármacos , Rim/metabolismo , Rim/ultraestrutura , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Ratos Wistar , Circulação Renal/efeitos dos fármacos , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/fisiopatologia , Reabsorção Renal/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sódio/metabolismo , Superóxidos/metabolismo , Fatores de Tempo
10.
J Cell Biol ; 215(4): 531-542, 2016 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-27872255

RESUMO

Hereditary spastic paraplegia (HSP) is a neurological syndrome characterized by degeneration of central nervous system (CNS) axons. Mutated HSP proteins include myelin proteolipid protein (PLP) and axon-enriched proteins involved in mitochondrial function, smooth endoplasmic reticulum (SER) structure, and microtubule (MT) stability/function. We characterized axonal mitochondria, SER, and MTs in rodent optic nerves where PLP is replaced by the peripheral nerve myelin protein, P0 (P0-CNS mice). Mitochondrial pathology and degeneration were prominent in juxtaparanodal axoplasm at 1 mo of age. In wild-type (WT) optic nerve axons, 25% of mitochondria-SER associations occurred on extensions of the mitochondrial outer membrane. Mitochondria-SER associations were reduced by 86% in 1-mo-old P0-CNS juxtaparanodal axoplasm. 1-mo-old P0-CNS optic nerves were more sensitive to oxygen-glucose deprivation and contained less adenosine triphosphate (ATP) than WT nerves. MT pathology and paranodal axonal ovoids were prominent at 6 mo. These data support juxtaparanodal mitochondrial degeneration, reduced mitochondria-SER associations, and reduced ATP production as causes of axonal ovoid formation and axonal degeneration.


Assuntos
Axônios/metabolismo , Mitocôndrias/metabolismo , Proteína Proteolipídica de Mielina/deficiência , Bainha de Mielina/metabolismo , Trifosfato de Adenosina/biossíntese , Animais , Axônios/ultraestrutura , Transporte Biológico , Retículo Endoplasmático/metabolismo , Metabolismo Energético , Camundongos Transgênicos , Microtúbulos/metabolismo , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Proteína Proteolipídica de Mielina/metabolismo , Bainha de Mielina/ultraestrutura , Nervo Óptico , Fosforilação , Proteínas tau/metabolismo
11.
Sci Rep ; 6: 33830, 2016 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-27654856

RESUMO

Mutations in optineurin (OPTN) are linked to the pathology of primary open angle glaucoma (POAG) and amyotrophic lateral sclerosis. Emerging evidence indicates that OPTN mutation is involved in accumulation of damaged mitochondria and defective mitophagy. Nevertheless, the role played by an OPTN E50K mutation in the pathogenic mitochondrial mechanism that underlies retinal ganglion cell (RGC) degeneration in POAG remains unknown. We show here that E50K expression induces mitochondrial fission-mediated mitochondrial degradation and mitophagy in the axons of the glial lamina of aged E50K-tg mice in vivo. While E50K activates the Bax pathway and oxidative stress, and triggers dynamics alteration-mediated mitochondrial degradation and mitophagy in RGC somas in vitro, it does not affect transport dynamics and fission of mitochondria in RGC axons in vitro. These results strongly suggest that E50K is associated with mitochondrial dysfunction in RGC degeneration in synergy with environmental factors such as aging and/or oxidative stress.

13.
Cell Tissue Res ; 363(3): 693-712, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26572539

RESUMO

Chromogranin A (CgA) is a prohormone and granulogenic factor in neuroendocrine tissues with a regulated secretory pathway. The impact of CgA depletion on secretory granule formation has been previously demonstrated in cell culture. However, studies linking the structural effects of CgA deficiency with secretory performance and cell metabolism in the adrenomedullary chromaffin cells in vivo have not previously been reported. Adrenomedullary content of the secreted adrenal catecholamines norepinephrine (NE) and epinephrine (EPI) was decreased 30-40 % in Chga-KO mice. Quantification of NE and EPI-storing dense core (DC) vesicles (DCV) revealed decreased DCV numbers in chromaffin cells in Chga-KO mice. For both cell types, the DCV diameter in Chga-KO mice was less (100-200 nm) than in WT mice (200-350 nm). The volume density of the vesicle and vesicle number was also lower in Chga-KO mice. Chga-KO mice showed an ~47 % increase in DCV/DC ratio, implying vesicle swelling due to increased osmotically active free catecholamines. Upon challenge with 2 U/kg insulin, there was a diminution in adrenomedullary EPI, no change in NE and a very large increase in the EPI and NE precursor dopamine (DA), consistent with increased catecholamine biosynthesis during prolonged secretion. We found dilated mitochondrial cristae, endoplasmic reticulum and Golgi complex, as well as increased synaptic mitochondria, synaptic vesicles and glycogen granules in Chga-KO mice compared to WT mice, suggesting that decreased granulogenesis and catecholamine storage in CgA-deficient mouse adrenal medulla is compensated by increased VMAT-dependent catecholamine update into storage vesicles, at the expense of enhanced energy expenditure by the chromaffin cell.


Assuntos
Catecolaminas/metabolismo , Grânulos Cromafim/metabolismo , Cromogranina A/deficiência , Metabolismo Energético , Glândulas Suprarrenais/efeitos dos fármacos , Glândulas Suprarrenais/metabolismo , Animais , Western Blotting , Grânulos Cromafim/efeitos dos fármacos , Grânulos Cromafim/ultraestrutura , Cromogranina A/metabolismo , Dopamina/metabolismo , Endocitose/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Metabolismo Energético/efeitos dos fármacos , Epinefrina/metabolismo , Exocitose/efeitos dos fármacos , Glucose/metabolismo , Glicogênio/metabolismo , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Humanos , Insulina/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Norepinefrina/metabolismo , Nervos Esplâncnicos/efeitos dos fármacos , Nervos Esplâncnicos/metabolismo , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/metabolismo
14.
Synapse ; 69(5): 268-82, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25683026

RESUMO

A key goal in neurobiology is to generate a theoretical framework that merges structural, physiological, and molecular explanations of brain function. These categories of explanation do not advance in synchrony; advances in one category define new experiments in other categories. For example, the synapse was defined physiologically and biochemically before it was visualized using electron microscopy. Indeed, the original descriptions of synapses in the 1950s were lent credence by the presence of spherical vesicles in presynaptic terminals that were considered to be the substrate for quantal neurotransmission. In the last few decades, our understanding of synaptic function has again been driven by physiological and molecular techniques. The key molecular players for synaptic vesicle structure, mobility and fusion were identified and applications of the patch clamp technique permitted physiological estimation of neurotransmitter release and receptor properties. These advances demand higher resolution structural images of synapses. During the 1990s a second renaissance in cell biology driven by EM was fueled by improved techniques for electron tomography (ET) with the ability to compute virtual images with nm resolution between image planes. Over the last 15 years, ET has been applied to the presynaptic terminal with special attention to the active zone and organelles of the nerve terminal. In this review, we first summarize the technical improvements that have led to a resurgence in utilization of ET and then we summarize new insights gained by the application of ET to reveal the high-resolution structure of the nerve terminal.


Assuntos
Tomografia com Microscopia Eletrônica/métodos , Terminações Pré-Sinápticas/ultraestrutura , Animais , Humanos , Vesículas Sinápticas/ultraestrutura
15.
Glia ; 63(5): 736-53, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25557093

RESUMO

Abnormal structure and function of astrocytes have been observed within the lamina cribrosa region of the optic nerve head (ONH) in glaucomatous neurodegeneration. Glutamate excitotoxicity-mediated mitochondrial alteration has been implicated in experimental glaucoma. However, the relationships among glutamate excitotoxicity, mitochondrial alteration and ONH astrocytes in the pathogenesis of glaucoma remain unknown. We found that functional N-methyl-d-aspartate (NMDA) receptors (NRs) are present in human ONH astrocytes and that glaucomatous human ONH astrocytes have increased expression levels of NRs and the glutamate aspartate transporter. Glaucomatous human ONH astrocytes exhibit mitochondrial fission that is linked to increased expression of dynamin-related protein 1 and its phosphorylation at Serine 616. In BAC ALDH1L1 eGFP or Thy1-CFP transgenic mice, NMDA treatment induced axon loss as well as hypertrophic morphology and mitochondrial fission in astrocytes of the glial lamina. In human ONH astrocytes, NMDA treatment in vitro triggered mitochondrial fission by decreasing mitochondrial length and number, thereby reducing mitochondrial volume density. However, blocking excitotoxicity by memantine (MEM) prevented these alterations by increasing mitochondrial length, number and volume density. In glaucomatous DBA/2J (D2) mice, blocking excitotoxicity by MEM inhibited the morphological alteration as well as increased mitochondrial number and volume density in astrocytes of the glial lamina. However, blocking excitotoxicity decreased autophagosome/autolysosome volume density in both astrocytes and axons in the glial lamina of glaucomatous D2 mice. These findings provide evidence that blocking excitotoxicity prevents ONH astrocyte dysfunction in glaucomatous neurodegeneration by increasing mitochondrial fission, increasing mitochondrial volume density and length, and decreasing autophagosome/autolysosome formation. GLIA 2015;63:736-753.


Assuntos
Astrócitos , Glaucoma/patologia , Ácido Glutâmico/farmacologia , Mitocôndrias/patologia , Dinâmica Mitocondrial/efeitos dos fármacos , Disco Óptico/patologia , Idoso , Idoso de 80 Anos ou mais , Aldeído Desidrogenase/genética , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Astrócitos/fisiologia , Contagem de Células , Células Cultivadas , Antagonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Pressão Intraocular/efeitos dos fármacos , Memantina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , N-Metilaspartato/farmacologia , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/efeitos dos fármacos
16.
Methods Enzymol ; 547: 165-79, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25416358

RESUMO

In this chapter, we provide details along with considerations and future directions for the use of miniSOG (for mini Singlet Oxygen Generator), a versatile label for correlated light and electron microscopy of genetically tagged proteins in cells, tissues, and organisms. This new visualizable genetic tag improves the ability of biologists to locate specific proteins at nanoscale resolution and to see these tagged proteins in the environment of structural landmarks that we are used to navigating by, such as mitochondrial membranes and compartments. miniSOG provides high-quality ultrastructural preservation and permits three-dimensional protein localization via electron tomography or serial section block-face scanning electron microscopy. miniSOG is now doing for electron microscopy what the family of green fluorescent protein did for fluorescence microscopy.


Assuntos
Tomografia com Microscopia Eletrônica/métodos , Microscopia Eletrônica/métodos , Proteínas Mitocondriais/análise , Proteínas Mitocondriais/metabolismo , Proteínas Recombinantes/metabolismo , Oxigênio Singlete/metabolismo , Animais , Canais de Cálcio/análise , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Células Cultivadas , Luz , Microscopia Eletrônica de Varredura/métodos , Microtomia/métodos , Proteínas Mitocondriais/genética , Oxirredução , Fototropinas/genética , Fototropinas/metabolismo , Proteínas Recombinantes/genética , Roedores , Transfecção/métodos
17.
J Clin Invest ; 123(10): 4294-308, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24091324

RESUMO

Ischemic damage is recognized to cause cardiomyocyte (CM) death and myocardial dysfunction, but the role of cell-matrix interactions and integrins in this process has not been extensively studied. Expression of α7ß1D integrin, the dominant integrin in normal adult CMs, increases during ischemia/reperfusion (I/R), while deficiency of ß1 integrins increases ischemic damage. We hypothesized that the forced overexpression of integrins on the CM would offer protection from I/R injury. Tg mice with CM-specific overexpression of integrin α7ß1D exposed to I/R had a substantial reduction in infarct size compared with that of α5ß1D-overexpressing mice and WT littermate controls. Using isolated CMs, we found that α7ß1D preserved mitochondrial membrane potential during hypoxia/reoxygenation (H/R) injury via inhibition of mitochondrial Ca2+ overload but did not alter H/R effects on oxidative stress. Therefore, we assessed Ca2+ handling proteins in the CM and found that ß1D integrin colocalized with ryanodine receptor 2 (RyR2) in CM T-tubules, complexed with RyR2 in human and rat heart, and specifically bound to RyR2 amino acids 165-175. Integrins stabilized the RyR2 interdomain interaction, and this stabilization required integrin receptor binding to its ECM ligand. These data suggest that α7ß1D integrin modifies Ca2+ regulatory pathways and offers a means to protect the myocardium from ischemic injury.


Assuntos
Integrinas/metabolismo , Isquemia Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Hipóxia Celular , Células Cultivadas , Humanos , Integrinas/química , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Isquemia Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/patologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Subunidades Proteicas/metabolismo , Ratos , Ratos Sprague-Dawley , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
18.
Mol Cell Proteomics ; 12(12): 3744-58, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24030101

RESUMO

Insulin resistance plays a major role in the development of type 2 diabetes and obesity and affects a number of biological processes such as mitochondrial biogenesis. Though mitochondrial dysfunction has been linked to the development of insulin resistance and pathogenesis of type 2 diabetes, the precise mechanism linking the two is not well understood. We used high fat diet (HFD)-induced obesity dependent diabetes mouse models to gain insight into the potential pathways altered with metabolic disease, and carried out quantitative proteomic analysis of liver mitochondria. As previously reported, proteins involved in fatty acid oxidation, branched chain amino acid degradation, tricarboxylic acid cycle, and oxidative phosphorylation were uniformly up-regulated in the liver of HFD fed mice compared with that of normal diet. Further, our studies revealed that retinol metabolism is distinctly down-regulated and the mitochondrial structural proteins-components of mitochondrial inter-membrane space bridging (MIB) complex (Mitofilin, Sam50, and ChChd3), and Tim proteins-essential for protein import, are significantly up-regulated in HFD fed mice. Structural and functional studies on HFD and normal diet liver mitochondria revealed remodeling of HFD mitochondria to a more condensed form with increased respiratory capacity and higher ATP levels compared with normal diet mitochondria. Thus, it is likely that the structural remodeling is essential to accommodate the increased protein content in presence of HFD: the mechanism could be through the MIB complex promoting contact site and crista junction formation and in turn facilitating the lipid and protein uptake.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Mitocôndrias Hepáticas/metabolismo , Proteínas Mitocondriais/metabolismo , Obesidade/metabolismo , Proteoma/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Ciclo do Ácido Cítrico/genética , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/genética , Dieta Hiperlipídica , Gorduras na Dieta/administração & dosagem , Regulação da Expressão Gênica , Resistência à Insulina , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/genética , Mitocôndrias Hepáticas/ultraestrutura , Proteínas Mitocondriais/genética , Anotação de Sequência Molecular , Obesidade/induzido quimicamente , Obesidade/genética , Fosforilação Oxidativa , Mapeamento de Interação de Proteínas , Proteoma/genética , Transdução de Sinais , Espectrometria de Massas em Tandem , Vitamina A/metabolismo
19.
PLoS One ; 8(4): e60804, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23577164

RESUMO

DIDS is a commonly used anion channel antagonist that is putatively cytoprotective against ischemic insult. However, recent reports indicate potentially deleterious secondary effects of DIDS. To assess the impact of DIDS on cellular viability comprehensively we examined neuronal morphology and function through 24 hours treatment with ACSF ± DIDS (40 or 400 µM). Control cells were unchanged, whereas DIDS induced an apoptotic phenotype (chromatin condensation, nuclear fragmentation and cleavage of the nuclear membrane protein lamin A, expression of pro-apoptotic proteins c-Jun N-terminal kinase 3, caspase 3, and cytochrome C, Annexin V staining, RNA degradation, and oligonucleosomal DNA cleavage). These deleterious effects were mediated by DIDS in a dose- and time-dependant manner, such that higher [DIDS] induced apoptosis more rapidly while apoptosis was observed at lower [DIDS] with prolonged exposure. In an apparent paradox, despite a clear overall apoptotic phenotype, certain hallmarks of apoptosis were not present in DIDS treated cells, including mitochondrial fission and loss of plasma membrane integrity. We conclude that DIDS induces apoptosis in cultured hippocampal neurons, in spite of the fact that some common hallmarks of cell death pathways are prevented. These contradictory effects may cause false-positive results in certain assays and future evaluations of DIDS as a neuroprotective agent should incorporate multiple viability assays.


Assuntos
Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Apoptose/efeitos dos fármacos , Isquemia Encefálica/metabolismo , Hipocampo/citologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Biomarcadores/metabolismo , Isquemia Encefálica/patologia , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Hipocampo/patologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Propídio/metabolismo , Fatores de Tempo
20.
Clin Sci (Lond) ; 124(11): 663-74, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23252598

RESUMO

Alternative approaches to reduce congenital muscle dysfunction are needed in cases where the ability to exercise is limited. (-)-Epicatechin is found in cocoa and may stimulate capillarity and mitochondrial proliferation in skeletal muscle. A total of 21 male rats bred for LCR (low running capacity) from generation 28 were randomized into three groups: vehicle for 30 days (control); (-)-epicatechin for 30 days; and (-)-epicatechin for 30 days followed by 15 days without (-)-epicatechin. Groups 2 and 3 received 1.0 mg of (-)-epicatechin/kg of body mass twice daily, whereas water was given to the control group. The plantaris muscle was harvested for protein and morphometric analyses. In addition, in vitro experiments were conducted to examine the role of (-)-epicatechin on mitochondrial respiratory kinetics at different incubation periods. Treatment for 30 days with (-)-epicatechin increased capillarity (P<0.001) and was associated with increases in protein expression of VEGF (vascular endothelial growth factor)-A with a concomitant decrease in TSP-1 (thrombospondin-1) and its receptor, which remained after 15 days of (-)-epicatechin cessation. Analyses of the p38 MAPK (mitogen-activated protein kinase) signalling pathway indicated an associated increase in phosphorylation of MKK3/6 (MAPK kinase 3/6) and p38 and increased protein expression of MEF2A (myocyte enhancer factor 2A). In addition, we observed significant increases in protein expression of PGC-1α (peroxisome-proliferator-activated receptor γ co-activator 1α), PGC-1ß, Tfam and cristae abundance. Interestingly, these increases associated with (-)-epicatechin treatment remained after 15 days of cessation. Lastly, in vitro experiments indicated that acute exposure of LCR muscle to (-)-epicatechin incubation was not sufficient to increase mitochondrial respiration. The results suggest that increases in skeletal muscle capillarity and mitochondrial biogenesis are associated with 30 days of (-)-epicatechin treatment and sustained for 15 days following cessation of treatment. Clinically, the use of this natural compound may have potential application in populations that experience muscle fatigue and are unable to perform endurance exercise.


Assuntos
Indutores da Angiogênese/farmacologia , Catequina/farmacologia , Mitocôndrias/efeitos dos fármacos , Corrida/fisiologia , Transdução de Sinais/efeitos dos fármacos , Animais , Antígenos CD36/metabolismo , Antígeno CD47/metabolismo , Capilares/efeitos dos fármacos , Catequina/fisiologia , Metabolismo Energético/efeitos dos fármacos , Membro Posterior/metabolismo , Cinética , Proteínas de Domínio MADS/metabolismo , MAP Quinase Quinase 3/metabolismo , MAP Quinase Quinase 6/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fatores de Transcrição MEF2 , Masculino , Mitocôndrias/metabolismo , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/metabolismo , Fatores de Regulação Miogênica/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fosforilação , Resistência Física , Proteínas de Ligação a RNA/metabolismo , Ratos , Trombospondina 1/metabolismo , Fatores de Transcrição/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
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