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1.
J Neurosci ; 2022 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-35970564

RESUMO

The mitochondrial anchor syntaphilin (SNPH) is a key mitochondrial protein normally expressed in axons to maintain neuronal health by positioning mitochondria along axons for metabolic needs. However, in 2019 we discovered a novel form of excitotoxicity that results when SNPH is misplaced into neuronal dendrites in disease models. A key unanswered question about this SNPH excitotoxicity is the pathologic molecules that trigger misplacement or intrusion of SNPH into dendrites. Here, we identified two different classes of pathologic molecules that interact to trigger dendritic SNPH intrusion. Using primary hippocampal neuronal cultures from mice of either sex, we demonstrated that the pro-inflammatory cytokine IL-1ß interacts with NMDA to trigger SNPH intrusion into dendrites. First, IL-1ß and NMDA each individually triggers dendritic SNPH intrusion. Second, IL-1ß and NMDA do not act independently but interact. Thus, blocking NMDAR by the antagonist MK-801 blocks IL-1ß from triggering dendritic SNPH intrusion. Further, de-coupling the known interaction between IL-1ß and NMDAR by tyrosine inhibitors prevents either IL-1ß or NMDA from triggering dendritic SNPH intrusion. Third, neuronal toxicity caused by IL-1ß or NMDA are strongly ameliorated in SNPH-/- neurons. Taken together, we hypothesize that the known bipartite IL-1ß/NMDAR crosstalk converges to trigger misplacement of SNPH in dendrites as a final common pathway to cause neurodegeneration. Targeting dendritic SNPH in this novel tripartite IL-1ß/NMDAR/SNPH interaction could be a strategic downstream locus for ameliorating neurotoxicity in inflammatory diseases.SIGNIFICANCE STATEMENTThe mitochondrial anchor Syntaphilin (SNPH) is a key mitochondrial protein normally expressed specifically in healthy axons to help position mitochondria along axons to match metabolic needs. In 2019, we discovered that misplacement of SNPH into neuronal dendrites causes a novel form of excitotoxicity in rodent models of multiple sclerosis. A key unanswered question about this new form of dendritic SNPH toxicity concerns pathologic molecules that trigger toxic misplacement of SNPH into dendrites. Here we identified two major categories of pathologic molecules, the pro-inflammatory cytokines and NMDA, that interact and converge to trigger toxic misplacement of SNPH into dendrites. We propose that dendritic mitochondrial anchor provides a novel, single common target for ameliorating diverse inflammatory and excitatory injuries in neurodegenerative diseases.

2.
J Neuroinflammation ; 19(1): 125, 2022 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-35624463

RESUMO

BACKGROUND: Ischemic stroke is a leading cause of mortality worldwide, largely due to the inflammatory response to brain ischemia during post-stroke reperfusion. Despite ongoing intensive research, there have not been any clinically approved drugs targeting the inflammatory component to stroke. Preclinical studies have identified T cells as pro-inflammatory mediators of ischemic brain damage, yet mechanisms that regulate the infiltration and phenotype of these cells are lacking. Further understanding of how T cells migrate to the ischemic brain and facilitate neuronal death during brain ischemia can reveal novel targets for post-stroke intervention. METHODS: To identify the population of T cells that produce IL-21 and contribute to stroke, we performed transient middle cerebral artery occlusion (tMCAO) in mice and performed flow cytometry on brain tissue. We also utilized immunohistochemistry in both mouse and human brain sections to identify cell types and inflammatory mediators related to stroke-induced IL-21 signaling. To mechanistically demonstrate our findings, we employed pharmacological inhibitor anti-CXCL13 and performed histological analyses to evaluate its effects on brain infarct damage. Finally, to evaluate cellular mechanisms of stroke, we exposed mouse primary neurons to oxygen glucose deprivation (OGD) conditions with or without IL-21 and measured cell viability, caspase activity and JAK/STAT signaling. RESULTS: Flow cytometry on brains from mice following tMCAO identified a novel population of cells IL-21 producing CXCR5+ CD4+ ICOS-1+ T follicular helper cells (TFH) in the ischemic brain early after injury. We observed augmented expression of CXCL13 on inflamed brain vascular cells and demonstrated that inhibition of CXCL13 protects mice from tMCAO by restricting the migration and influence of IL-21 producing TFH cells in the ischemic brain. We also illustrate that neurons express IL-21R in the peri-infarct regions of both mice and human stroke tissue in vivo. Lastly, we found that IL-21 acts on mouse primary ischemic neurons to activate the JAK/STAT pathway and induce caspase 3/7-mediated apoptosis in vitro. CONCLUSION: These findings identify a novel mechanism for how pro-inflammatory T cells are recruited to the ischemic brain to propagate stroke damage and provide a potential new therapeutic target for stroke.


Assuntos
Lesões Encefálicas , Isquemia Encefálica , Acidente Vascular Cerebral , Animais , Lesões Encefálicas/metabolismo , Isquemia Encefálica/metabolismo , Quimiocina CXCL13/metabolismo , Humanos , Infarto da Artéria Cerebral Média/patologia , Mediadores da Inflamação/metabolismo , Interleucinas , Isquemia/patologia , Janus Quinases/metabolismo , Camundongos , Neurônios/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Acidente Vascular Cerebral/patologia
3.
J Neurosci ; 35(13): 5293-306, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25834054

RESUMO

The demyelinating disease multiple sclerosis (MS) has an early inflammatory phase followed by an incurable progressive phase with subdued inflammation and poorly understood neurodegenerative mechanism. In this study, we identified various parallelisms between progressive MS and the dysmyelinating mouse model Shiverer and then genetically deleted a major neuron-specific mitochondrial anchoring protein Syntaphilin (SNPH) from the mouse. Prevailing evidence suggests that deletion of SNPH is harmful in demyelination. Surprisingly, SNPH deletion produces striking benefits in the Shiverer by prolonging survival, reducing cerebellar damage, suppressing oxidative stress, and improving mitochondrial health. In contrast, SNPH deletion does not benefit clinical symptoms in experimental autoimmune encephalomyelitis (EAE), a model for early-phase MS. We propose that deleting mitochondrial anchoring is a novel, specific treatment for progressive MS.


Assuntos
Modelos Animais de Doenças , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Mitocôndrias/metabolismo , Esclerose Múltipla Crônica Progressiva/genética , Animais , Cerebelo/patologia , Cerebelo/ultraestrutura , Encefalomielite Autoimune Experimental/genética , Substância Cinzenta/patologia , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/genética , Esclerose Múltipla Crônica Progressiva/terapia , Proteínas do Tecido Nervoso , Estresse Oxidativo/genética , Análise de Sobrevida , Substância Branca/patologia
4.
J Neurosci ; 33(8): 3514-25, 2013 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-23426679

RESUMO

We report a focal disturbance in myelination of the optic nerve in the osteopetrotic (op/op) mouse, which results from a spontaneous compression of the nerve resulting from stenosis of the optic canal. The growth of the op/op optic nerve was significantly affected, being maximally suppressed at postnatal day 30 (P30; 33% of age matched control). Myelination of the nerve in the optic canal was significantly delayed at P15, and myelin was almost completely absent at P30. The size of nerves and myelination were conserved both in the intracranial and intraorbital segments at P30, suggesting that the axons in the compressed site are spared in all animals at P30. Interestingly, we observed recovery both in the nerve size and the density of myelinated axons at 7 months in almost half of the optic nerves examined, although some nerves lost axons and became atrophic. In vivo and ex vivo electrophysiological examinations of P30 op/op mice showed that nerve conduction was significantly delayed but not blocked with partial recovery in some mice by 7 months. Transcardial perfusion of FITC-labeled albumin suggested that local ischemia was at least in part the cause of this myelination failure. These results suggest that the primary abnormality is dysmyelination of the optic nerve in early development. This noninvasive model system will be a valuable tool to study the effects of nerve compression on the function and survival of oligodendrocyte progenitor cells/oligodendrocytes and axons and to explore the mechanism of redistribution of oligodendrocyte progenitor cells with compensatory myelination.


Assuntos
Doenças Desmielinizantes/genética , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Síndromes de Compressão Nervosa/patologia , Doenças do Nervo Óptico/genética , Doenças do Nervo Óptico/patologia , Nervo Óptico/patologia , Animais , Camundongos , Camundongos Mutantes Neurológicos , Síndromes de Compressão Nervosa/genética , Fibras Nervosas Mielinizadas/patologia , Fibras Nervosas Mielinizadas/fisiologia , Condução Nervosa/genética , Oligodendroglia/patologia , Oligodendroglia/fisiologia , Nervo Óptico/fisiologia , Osteopetrose/genética , Células-Tronco/patologia , Células-Tronco/fisiologia
5.
J Neurosci ; 30(10): 3555-66, 2010 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-20219989

RESUMO

The node of Ranvier is a tiny segment of a myelinated fiber with various types of specializations adapted for generation of high-speed nerve impulses. It is ionically specialized with respect to ion channel segregation and ionic fluxes, and metabolically specialized in ionic pump expression and mitochondrial density augmentation. This report examines the interplay of three important parameters (calcium fluxes, Na pumps, mitochondrial motility) at nodes of Ranvier in frog during normal nerve activity. First, we used calcium dyes to resolve a highly localized elevation in axonal calcium at a node of Ranvier during action potentials, and showed that this calcium elevation retards mitochondrial motility during nerve impulses. Second, we found, surprisingly, that physiologic activation of the Na pumps retards mitochondrial motility. Blocking Na pumps alone greatly prevents action potentials from retarding mitochondrial motility, which reveals that mitochondrial motility is coupled to Na/K-ATPase. In conclusion, we suggest that during normal nerve activity, Ca elevation and activation of Na/K-ATPase act, possibly in a synergistic manner, to recruit mitochondria to a node of Ranvier to match metabolic needs.


Assuntos
Potenciais de Ação/fisiologia , Cálcio/fisiologia , Mitocôndrias/enzimologia , Fibras Nervosas Mielinizadas/enzimologia , Nós Neurofibrosos/enzimologia , ATPase Trocadora de Sódio-Potássio/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fibras Nervosas Mielinizadas/efeitos dos fármacos , Fibras Nervosas Mielinizadas/metabolismo , Nós Neurofibrosos/efeitos dos fármacos , Nós Neurofibrosos/metabolismo , Xenopus laevis
6.
J Biol Chem ; 285(45): 35155-68, 2010 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-20817726

RESUMO

Neuronal dendrites are vulnerable to injury under diverse pathological conditions. However, the underlying mechanisms for dendritic Na(+) overload and the selective dendritic injury remain poorly understood. Our current study demonstrates that activation of NHE-1 (Na(+)/H(+) exchanger isoform 1) in dendrites presents a major pathway for Na(+) overload. Neuronal dendrites exhibited higher pH(i) regulation rates than soma as a result of a larger surface area/volume ratio. Following a 2-h oxygen glucose deprivation and a 1-h reoxygenation, NHE-1 activity was increased by ∼70-200% in dendrites. This elevation depended on activation of p90 ribosomal S6 kinase. Moreover, stimulation of NHE-1 caused dendritic Na(+)(i) accumulation, swelling, and a concurrent loss of Ca(2+)(i) homeostasis. The Ca(2+)(i) overload in dendrites preceded the changes in soma. Inhibition of NHE-1 or the reverse mode of Na(+)/Ca(2+) exchange prevented these changes. Mitochondrial membrane potential in dendrites depolarized 40 min earlier than soma following oxygen glucose deprivation/reoxygenation. Blocking NHE-1 activity not only attenuated loss of dendritic mitochondrial membrane potential and mitochondrial Ca(2+) homeostasis but also preserved dendritic membrane integrity. Taken together, our study demonstrates that NHE-1-mediated Na(+) entry and subsequent Na(+)/Ca(2+) exchange activation contribute to the selective dendritic vulnerability to in vitro ischemia.


Assuntos
Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Dendritos/metabolismo , Homeostase , Hidrogênio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Sódio/metabolismo , Animais , Hipóxia Celular , Células Cultivadas , Dendritos/patologia , Concentração de Íons de Hidrogênio , Transporte de Íons , Camundongos , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Trocador 1 de Sódio-Hidrogênio
7.
Dev Biol ; 324(1): 10-7, 2008 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-18804104

RESUMO

The establishment of prostatic budding patterns occurs early in prostate development but mechanisms responsible for this event are poorly understood. We investigated the role of WNT5A in patterning prostatic buds as they emerge from the fetal mouse urogenital sinus (UGS). Wnt5a mRNA was expressed in UGS mesenchyme during budding and was focally up-regulated as buds emerged from the anterior, dorsolateral, and ventral UGS regions. We observed abnormal UGS morphology and prostatic bud patterns in Wnt5a null male fetuses, demonstrated that prostatic bud number was decreased by recombinant mouse WNT5A protein during wild type UGS morphogenesis in vitro, and showed that ventral prostate development was selectively impaired when these WNT5A-treated UGSs were grafted under under kidney capsules of immunodeficient mice and grown for 28 d. Moreover, a WNT5A inhibitory antibody, added to UGS organ culture media, rescued prostatic budding from inhibition by a ventral prostatic bud inhibitor, 2,3,8,7-tetrachlorodibenzo-p-dioxin, and restored ventral prostate morphogenesis when these tissues were grafted under immunodeficient mouse kidney capsules and grown for 28 d. These results suggest that WNT5A participates in prostatic bud patterning by restricting mouse ventral prostate development.


Assuntos
Próstata/embriologia , Proteínas Wnt/fisiologia , Animais , Padronização Corporal , Masculino , Camundongos , Camundongos Knockout , Organogênese , Próstata/metabolismo , Técnicas de Cultura de Tecidos , Proteína Wnt-5a
8.
J Cell Biol ; 162(6): 1149-60, 2003 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-12963709

RESUMO

In myelinated axons, K+ channels are concealed under the myelin sheath in the juxtaparanodal region, where they are associated with Caspr2, a member of the neurexin superfamily. Deletion of Caspr2 in mice by gene targeting revealed that it is required to maintain K+ channels at this location. Furthermore, we show that the localization of Caspr2 and clustering of K+ channels at the juxtaparanodal region depends on the presence of TAG-1, an immunoglobulin-like cell adhesion molecule that binds Caspr2. These results demonstrate that Caspr2 and TAG-1 form a scaffold that is necessary to maintain K+ channels at the juxtaparanodal region, suggesting that axon-glia interactions mediated by these proteins allow myelinating glial cells to organize ion channels in the underlying axonal membrane.


Assuntos
Axônios/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas de Membrana , Fibras Nervosas Mielinizadas/metabolismo , Proteínas do Tecido Nervoso/deficiência , Canais de Potássio/metabolismo , Nós Neurofibrosos/metabolismo , Animais , Axônios/ultraestrutura , Comunicação Celular/genética , Contactina 2 , Marcação de Genes , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Mutação/genética , Fibras Nervosas Mielinizadas/ultraestrutura , Proteínas do Tecido Nervoso/genética , Condução Nervosa/genética , Neuroglia/metabolismo , Neuroglia/ultraestrutura , Canais de Potássio/genética , Nós Neurofibrosos/ultraestrutura , Superfamília Shaker de Canais de Potássio
9.
Cell Rep ; 29(3): 685-696.e5, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31618636

RESUMO

Syntaphilin (SNPH) is a major mitochondrial anchoring protein targeted to axons and excluded from dendrites. In this study, we provide in vivo evidence that this spatial specificity is lost in Shiverer (Shi) mice, a model for progressive multiple sclerosis (MS), resulting in inappropriate intrusion of SNPH into dendrites of cerebellar Purkinje cells with neurodegenerative consequences. Thus, reconstituting dendritic SNPH intrusion in SNPH-KO mice by viral transduction greatly sensitizes Purkinje cells to excitotoxicity when the glutamatergic climbing fibers are stimulated. Finally, we demonstrate in vitro that overexpression of SNPH in dendrites compromises neuronal viability by inducing N-methyl-D-aspartate (NMDA) excitotoxicity, reducing mitochondrial calcium uptake, and interfering with quality control of mitochondria by blocking somal mitophagy. Collectively, we propose that inappropriate immobilization of dendritic mitochondria by SNPH intrusion produces excitotoxicity and suggest that interception of dendritic SNPH intrusion is a therapeutic strategy to combat neurodegeneration.


Assuntos
Dendritos/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Axônios/metabolismo , Cálcio/metabolismo , Células Cultivadas , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitofagia/efeitos dos fármacos , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , N-Metilaspartato/farmacologia , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurônios/metabolismo , Células de Purkinje/citologia , Células de Purkinje/metabolismo
10.
BMC Biol ; 5: 42, 2007 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-17925011

RESUMO

BACKGROUND: Shaker codes for a Drosophila voltage-dependent potassium channel. Flies carrying Shaker null or hypomorphic mutations sleep 3-4 h/day instead of 8-14 h/day as their wild-type siblings do. Shaker-like channels are conserved across species but it is unknown whether they affect sleep in mammals. To address this issue, we studied sleep in Kcna2 knockout (KO) mice. Kcna2 codes for Kv1.2, the alpha subunit of a Shaker-like voltage-dependent potassium channel with high expression in the mammalian thalamocortical system. RESULTS: Continuous (24 h) electroencephalograph (EEG), electromyogram (EMG), and video recordings were used to measure sleep and waking in Kcna2 KO, heterozygous (HZ) and wild-type (WT) pups (P17) and HZ and WT adult mice (P67). Sleep stages were scored visually based on 4-s epochs. EEG power spectra (0-20 Hz) were calculated on consecutive 4-s epochs. KO pups die by P28 due to generalized seizures. At P17 seizures are either absent or very rare in KO pups (< 1% of the 24-h recording time), and abnormal EEG activity is only present during the seizure. KO pups have significantly less non-rapid eye movement (NREM) sleep (-23%) and significantly more waking (+21%) than HZ and WT siblings with no change in rapid eye movement (REM) sleep time. The decrease in NREM sleep is due to an increase in the number of waking episodes, with no change in number or duration of sleep episodes. Sleep patterns, daily amounts of sleep and waking, and the response to 6 h sleep deprivation are similar in HZ and WT adult mice. CONCLUSION: Kv1.2, a mammalian homologue of Shaker, regulates neuronal excitability and affects NREM sleep.


Assuntos
Canal de Potássio Kv1.2/genética , Sono/genética , Animais , Eletroencefalografia , Eletromiografia , Análise de Fourier , Heterozigoto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sono REM/genética , Vigília/genética
11.
Sci Rep ; 8(1): 15726, 2018 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-30356079

RESUMO

The anti-proliferative activity of dietary flavonoid fisetin has been validated in various cancer models. Establishing its precise mechanism of action has proved somewhat challenging given the multiplicity of its targets. We demonstrated that YB-1 promotes epithelial-to-mesenchymal transition and its inhibition suppressed tumor cell proliferation and invasion. The p90 ribosomal S6 kinase (RSK), an important ERK effector, activates YB-1 to drive melanoma growth. We found that fisetin treatment of monolayer/3-D melanoma cultures resulted in YB-1 dephosphorylation and reduced transcript levels. In parallel, fisetin suppressed mesenchymal markers and matrix-metalloproteinases in melanoma cells. Data from cell-free/cell-based systems indicated that fisetin inhibited RSK activity through binding to the kinase. Affinity studies for RSK isoforms evaluated stronger interaction for RSK2 than RSK1. Competition assays performed to monitor binding responses revealed that YB-1 and RSK2 do not compete, rather binding of fisetin to RSK2 promotes its binding to YB-1. Fisetin suppressed YB-1/RSK signaling independent of its effect on ERK, and reduced MDR1 levels. Comparable efficacy of fisetin and vemurafenib for inhibiting melanoma growth was noted albeit through divergent modulation of ERK. Our studies provide insight into additional modes of regulation through which fisetin interferes with melanoma growth underscoring its potential therapeutic efficacy in disease progression.


Assuntos
Flavonoides/farmacologia , Melanoma/tratamento farmacológico , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteína 1 de Ligação a Y-Box/antagonistas & inibidores , Antineoplásicos/farmacologia , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Flavonóis , Humanos , Sistema de Sinalização das MAP Quinases , Melanoma/patologia , Fosforilação , Transdução de Sinais , Transcrição Gênica/efeitos dos fármacos , Proteína 1 de Ligação a Y-Box/metabolismo
12.
Sci Rep ; 8(1): 8251, 2018 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-29844467

RESUMO

Brown adipose tissue (BAT) is a specialized thermogenic organ in mammals. The ability of BAT mitochondria to generate heat in response to cold-challenge to maintain core body temperature is essential for organismal survival. While cold activated BAT mitochondrial biogenesis is recognized as critical for thermogenic adaptation, the contribution of mitochondrial quality control to this process remains unclear. Here, we show mitophagy is required for brown adipocyte mitochondrial homeostasis during thermogenic adaptation. Mitophagy is significantly increased in BAT from cold-challenged mice (4 °C) and in ß-agonist treated brown adipocytes. Blockade of mitophagy compromises brown adipocytes mitochondrial oxidative phosphorylation (OX-PHOS) capacity, as well as BAT mitochondrial integrity. Mechanistically, cold-challenge induction of BAT mitophagy is UCP1-dependent. Furthermore, our results indicate that mitophagy coordinates with mitochondrial biogenesis, maintaining activated BAT mitochondrial homeostasis. Collectively, our in vivo and in vitro findings identify mitophagy as critical for brown adipocyte mitochondrial homeostasis during cold adaptation.


Assuntos
Adipócitos/fisiologia , Tecido Adiposo Marrom/fisiologia , Hipotermia/metabolismo , Mitocôndrias/metabolismo , Mitofagia , Termogênese , Proteína Desacopladora 1/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Animais , Células Cultivadas , Temperatura Baixa , Homeostase , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Biogênese de Organelas , Fosforilação Oxidativa , Proteína Desacopladora 1/genética
13.
Cell Rep ; 20(4): 923-934, 2017 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-28746876

RESUMO

Regulated inter-mitochondrial fusion/fission is essential for maintaining optimal mitochondrial respiration and control of apoptosis and autophagy. In mammals, mitochondrial fusion is controlled by outer membrane GTPases MFN1 and MFN2 and by inner membrane (IM) GTPase OPA1. Disordered mitochondrial fusion/fission contributes to various pathologies, and MFN2 or OPA1 mutations underlie neurodegenerative diseases. Here, we show that the WBSCR16 protein is primarily associated with the outer face of the inner mitochondrial membrane and is important for mitochondrial fusion. We provide evidence of a WBSCR16/OPA1 physical interaction in the intact cell and of a WBSCR16 function as an OPA1-specific guanine nucleotide exchange factor (GEF). Homozygosity for a Wbscr16 mutation causes early embryonic lethality, whereas neurons of mice heterozygous for the mutation have mitochondria with reduced membrane potential and increased susceptibility to fragmentation upon exposure to stress, suggesting roles for WBSCR16 deficits in neuronal pathologies.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Feminino , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Células HeLa , Humanos , Imunoprecipitação , Masculino , Camundongos , Camundongos Mutantes , Dinâmica Mitocondrial/genética , Dinâmica Mitocondrial/fisiologia , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Ligação Proteica
14.
Lab Chip ; 5(1): 97-101, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15616746

RESUMO

A device for cell culture is presented that combines MEMS technology and liquid-phase photolithography to create a microfluidic chip that influences and records electrical cellular activity. A photopolymer channel network is formed on top of a multichannel microelectrode array. Preliminary results indicated successful local thermal control within microfluidic channels and control of lamina position over the electrode array. To demonstrate the biological application of such a device, adult dissociated dorsal root ganglion neurons with a subpopulation of thermally-sensitive cells are attached onto the electrode array. Using laminar flow, dynamic control of local temperature of the neural cells was achieved while maintaining a constant chemical culture medium. Recording the expected altered cellular activity confirms the success of the integrated device.


Assuntos
Gânglios Espinais/citologia , Microeletrodos , Técnicas Analíticas Microfluídicas , Neurônios Aferentes/citologia , Temperatura , Potenciais de Ação , Animais , Desenho de Equipamento , Gânglios Espinais/fisiologia , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Neurônios Aferentes/fisiologia , Técnicas de Patch-Clamp
15.
Oncotarget ; 5(9): 2462-74, 2014 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-24770864

RESUMO

Epithelial-to-mesenchymal transition (EMT) plays an important role in prostate cancer (PCa) metastasis. The transcription/translation regulatory Y-box binding protein-1 (YB-1) is known to be associated with cancer metastasis. We observed that YB-1 expression increased with tumor grade and showed an inverse relationship with E-cadherin in a human PCa tissue array. Forced YB-1 expression induced a mesenchymal morphology that was associated with down regulation of epithelial markers. Silencing of YB-1 reversed mesenchymal features and decreased cell proliferation, migration and invasion in PCa cells. YB-1 is activated directly via Akt mediated phosphorylation at Ser102 within the cold shock domain (CSD). We next identified fisetin as an inhibitor of YB-1 activation. Computational docking and molecular dynamics suggested that fisetin binds on the residues from ß1 - ß4 strands of CSD, hindering Akt's interaction with YB-1. Calculated free binding energy ranged from -11.9845 to -9.6273 kcal/mol. Plasmon Surface Resonance studies showed that fisetin binds to YB-1 with an affinity of approximately 35 µM, with both slow association and dissociation. Fisetin also inhibited EGF induced YB-1 phosphorylation and markers of EMT both in vitro and in vivo. Collectively our data suggest that YB-1 induces EMT in PCa and identify fisetin as an inhibitor of its activation.


Assuntos
Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Flavonoides/farmacologia , Próstata/citologia , Neoplasias da Próstata/patologia , Proteína 1 de Ligação a Y-Box/antagonistas & inibidores , Proteína 1 de Ligação a Y-Box/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Flavonóis , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Nus , Gradação de Tumores , Fosforilação/efeitos dos fármacos , Próstata/efeitos dos fármacos , Próstata/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise Serial de Tecidos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína 1 de Ligação a Y-Box/genética
16.
J Neurosci Methods ; 207(1): 51-8, 2012 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-22484559

RESUMO

Mitochondrial remodeling (replication, fission/fusion) is a dynamically regulated process with diverse functions in neurons. A myelinated axon is an extension from the cell soma of a fully differentiated neuron. Mitochondria, once synthesized in the cell body, enter the axon displaying robust trafficking and accumulation at nodes of Ranvier to match metabolic needs. This long-distance deployment of mitochondria to axons raises the issue of whether myelinated axons can function independently of the cell body to execute mitochondrial remodeling to match local demands. Mitochondrial fusion has been suggested to occur in axons in simple neuronal cultures in vitro. However, whether such events occur in vivo in an intact nervous system remains unanswered. Here we describe a novel technique which allows monitoring of mitochondrial fusion in intact sciatic nerve of frog (Xenopus laevis). Mitochondrial population was labeled by injecting two different MitoTracker dyes (Red and Green), spatially apart along sciatic nerves surgically and then allow to "meet"in vivo. At 24h post-surgery, the sciatic nerves were taken out for mitochondrial imaging at the half-way point. During the post-injection periods, the anterograde-directed Green mitochondria meet with the retrograde-directed Red mitochondria. If fusion occurs, the merged of Green and Red fluorophores in the same mitochondrion will produce a Yellow color in merged images. The labeled mitochondria were observed with a Nikon A1 confocal microscope. Our new mitochondrial imaging method opens an avenue to separately assess the role of local axonal mitochondrial fusion, independent of the cell body of nerve fibers.


Assuntos
Axônios/fisiologia , Mitocôndrias/fisiologia , Fibras Nervosas Mielinizadas/fisiologia , Coloração e Rotulagem/métodos , Animais , Corantes , Feminino , Microscopia Confocal , Nervo Isquiático/citologia , Nervo Isquiático/fisiologia , Xenopus laevis
17.
Integr Biol (Camb) ; 2(1): 58-64, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20473413

RESUMO

Regulation of intracellular pH (pH(i)) in neurons is crucial to maintain their physiological function. In the current study, newly-developed polydimethylsiloxane (PDMS) microfluidic devices were used to independently investigate pH(i) regulation in neuronal soma and neurites. Embryonic cortical neurons were cultured in PDMS microfluidic devices with soma growing in one chamber (seeded) and neurites extending through a set of perpendicular microchannels into the opposite parallel chamber (non-seeded). Neurons in the microchambers were characterized by the vital dye calcein-red, polarized mitochondria, and expression of neuronal specific beta-tubulin (type-III), axonal Tau-1 protein, dendritic microtubule associated protein (MAP-2), and Na(+)/H(+) exchanger isoform 1 (NHE-1). Neurites exhibited higher resting pH(i) than soma (7.16 +/- 0.09 vs. 6.90 +/- 0.15). The neurites had a proton extrusion rate 3.7-fold faster than in soma following NH(4)Cl prepulse-mediated acidification (p < 0.05). The difference in the pH(i) regulation rates between neurites and soma can be accounted for by the larger surface area to volume ratio in the neurites. Interestingly, pharmacological inhibition of NHE-1 activity blocked the pH(i) regulation in soma and in neurites by approximately 70% (p < 0.05). Taken together, our study demonstrated that the microfluidic devices provide a useful tool to study neuronal pH(i) regulation in soma and their neurites. We conclude that NHE-1 plays an important role in regulation of pH(i) in both compartments.


Assuntos
DNA Glicosilases/metabolismo , Técnicas Analíticas Microfluídicas/métodos , Neuritos/metabolismo , Neurônios/metabolismo , ATPase Trocadora de Sódio-Potássio/fisiologia , Animais , Técnicas de Cultura de Células/métodos , Células Cultivadas , Concentração de Íons de Hidrogênio , Ratos
18.
J Neurophysiol ; 98(3): 1501-25, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17634333

RESUMO

Genes Kcna1 and Kcna2 code for the voltage-dependent potassium channel subunits Kv1.1 and Kv1.2, which are coexpressed in large axons and commonly present within the same tetramers. Both contribute to the low-voltage-activated potassium current I Kv1, which powerfully limits excitability and facilitates temporally precise transmission of information, e.g., in auditory neurons of the medial nucleus of the trapezoid body (MNTB). Kcna1-null mice lacking Kv1.1 exhibited seizure susceptibility and hyperexcitability in axons and MNTB neurons, which also had reduced I Kv1. To explore whether a lack of Kv1.2 would cause a similar phenotype, we created and characterized Kcna2-null mice (-/-). The -/- mice exhibited increased seizure susceptibility compared with their +/+ and +/- littermates, as early as P14. The mRNA for Kv1.1 and Kv1.2 increased strongly in +/+ brain stems between P7 and P14, suggesting the increasing importance of these subunits for limiting excitability. Surprisingly, MNTB neurons in brain stem slices from -/- and +/- mice were hypoexcitable despite their Kcna2 deficit, and voltage-clamped -/- MNTB neurons had enlarged I Kv1. This contrasts strikingly with the Kcna1-null MNTB phenotype. Toxin block experiments on MNTB neurons suggested Kv1.2 was present in every +/+ Kv1 channel, about 60% of +/- Kv1 channels, and no -/- Kv1 channels. Kv1 channels lacking Kv1.2 activated at abnormally negative potentials, which may explain why MNTB neurons with larger proportions of such channels had larger I Kv1. If channel voltage dependence is determined by how many Kv1.2 subunits each contains, neurons might be able to fine-tune their excitability by adjusting the Kv1.1:Kv1.2 balance rather than altering Kv1 channel density.


Assuntos
Canal de Potássio Kv1.2/deficiência , Canal de Potássio Kv1.2/fisiologia , Convulsões/genética , Superfamília Shaker de Canais de Potássio/fisiologia , Envelhecimento , Animais , Tronco Encefálico/fisiologia , Tronco Encefálico/fisiopatologia , Vetores Genéticos , Genoma , Genótipo , Expectativa de Vida , Camundongos , Camundongos Knockout , Neurônios/fisiologia , Fases de Leitura Aberta , Mapeamento por Restrição
19.
J Neurophysiol ; 96(2): 695-709, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16835363

RESUMO

The myelin sheath enables saltatory conduction by demarcating the axon into a narrow nodal region for excitation and an extended, insulated internodal region for efficient spread of passive current. This anatomical demarcation produces a dramatic heterogeneity in ionic fluxes during excitation, a classical example being the restriction of Na influx at the node. Recent studies have revealed that action potentials also induce calcium influx into myelinated axons of mammalian optic nerves. Does calcium influx in myelinated axons show spatial heterogeneity during nerve excitation? To address this, we analyzed spatial profiles of axonal calcium transients during action potentials by selectively staining axons with calcium indicators and subjected the data to theoretical analysis with parameters for axial calcium diffusion empirically determined using photolysis of caged compounds. The results show surprisingly that during action potentials, calcium influx occurs uniformly along an axon of a fully myelinated mouse optic nerve.


Assuntos
Potenciais de Ação/fisiologia , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Bainha de Mielina/fisiologia , Nervo Óptico/metabolismo , Algoritmos , Animais , Axônios/metabolismo , Cádmio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/fisiologia , Compostos de Cálcio , Simulação por Computador , Difusão , Eletrofisiologia , Técnicas In Vitro , Camundongos , Microscopia Confocal , Modelos Neurológicos , Nervo Óptico/citologia , Fotólise , Sódio/fisiologia
20.
J Neurophysiol ; 88(2): 802-16, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12163532

RESUMO

Axonal populations in neonatal and mature optic nerves were selectively stained with calcium dyes for analysis of calcium homeostasis and its possible coupling to axonal Na. Repetitive nerve stimulation causes a rise in axonal [Ca(2+)](i) the posttetanus recovery of which is impeded by increasing the number of action potentials in the tetanus. This effect is augmented in 4-aminopyridine (4-AP; 1 mM), which dramatically increases the calcium and presumably sodium load during the tetanus. Increasing axonal [Na](i) with the Na-ionophore monensin (4-50 microM) and ouabain (30 microM) retards posttetanus calcium decline, suggesting that efficient calcium clearance depends on a low level of axonal [Na](i). Posttetanus calcium clearance is not affected by K-mediated depolarization. To further examine coupling between axonal [Na](i) and [Ca(2+)](i), the resting axonal [Ca(2+)](i) was monitored as axonal [Na(+)](i) was elevated with ouabain, veratridine, and monensin. In all cases, elevation of axonal [Na(+)](i) evokes a calcium influx into axons. This influx is unrelated to activation of calcium channels but is consistent with calcium influx via reversal of the Na/Ca exchanger expected as a consequence of axonal [Na(+)](i) elevation. In conclusion, this study demonstrates that calcium homeostasis in the axons of the optic nerve is strongly coupled to axonal [Na(+)](i) in a manner consistent with the Na/Ca exchanger playing a major role in extruding calcium following nerve activity.


Assuntos
Axônios/metabolismo , Cálcio/metabolismo , Nervo Óptico/metabolismo , Sódio/metabolismo , 4-Aminopiridina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Adenosina/farmacologia , Envelhecimento , Animais , Animais Recém-Nascidos , Axônios/efeitos dos fármacos , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Estimulação Elétrica , Eletrofisiologia , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes , Homeostase/efeitos dos fármacos , Ionóforos/farmacologia , Camundongos , Monensin/farmacologia , Nervo Óptico/efeitos dos fármacos , Ouabaína/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Trocador de Sódio e Cálcio/farmacologia , Veratridina/farmacologia
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