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1.
Redox Biol ; 48: 102186, 2021 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-34801863

RESUMO

When ROS production exceeds the cellular antioxidant capacity, the cell needs to eliminate the defective mitochondria responsible for excessive ROS production. It has been proposed that the removal of these defective mitochondria involves mitophagy, but the mechanism of this regulation remains unclear. Here, we demonstrate that moderate mitochondrial superoxide and hydrogen peroxide production oxidates KEAP1, thus breaking the interaction between this protein and PGAM5, leading to the inhibition of its proteasomal degradation. Accumulated PGAM5 interferes with the processing of the PINK1 in the mitochondria leading to the accumulation of PINK1 on the outer mitochondrial membrane. In turn, PINK1 promotes Parkin recruitment to mitochondria and sensitizes mitochondria for autophagic removal. We also demonstrate that inhibitors of the KEAP1-PGAM5 protein-protein interaction (including CPUY192018) mimic the effect of mitochondrial ROS and sensitize mitophagy machinery, suggesting that these inhibitors could be used as pharmacological regulators of mitophagy. Together, our results show that KEAP1/PGAM5 complex senses mitochondrially generated superoxide/hydrogen peroxide to induce mitophagy.

2.
Autophagy ; 15(5): 930-931, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30806158

RESUMO

The Parkinson disease-associated proteins PINK1 and PRKN coordinate the ubiquitination of mitochondrial outer membrane proteins to tag them either for degradation or for autophagic clearance of the mitochondrion. The proteins include the mitochondrial trafficking proteins RHOT1 and RHOT2, the removal of which may be required for immobilization of mitochondria prior to mitophagy. Here, we demonstrate that RHOT1 and RHOT2 are not only substrates for PINK1-PRKN-dependent degradation but that they also play an active role in the process of mitophagy. RHOT1, and likely also RHOT2, may act as a docking site for inactive PRKN prior to mitochondrial damage, thus keeping PRKN in close proximity to its potential substrates and thereby facilitating mitophagy. We also show that RHOT1 functions as a calcium-sensing docking site for PRKN, and we suggest that calcium binding to RHOT is a key step in the calcium-dependent activation of mitophagy machinery.


Assuntos
Autofagia , Mitofagia , Proteínas de Transporte , Mitocôndrias , Proteínas Mitocondriais , Proteínas Quinases , Ubiquitina-Proteína Ligases
3.
EMBO J ; 38(2)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30504269

RESUMO

The Parkinson's disease-associated protein kinase PINK1 and ubiquitin ligase Parkin coordinate the ubiquitination of mitochondrial proteins, which marks mitochondria for degradation. Miro1, an atypical GTPase involved in mitochondrial trafficking, is one of the substrates tagged by Parkin after mitochondrial damage. Here, we demonstrate that a small pool of Parkin interacts with Miro1 before mitochondrial damage occurs. This interaction does not require PINK1, does not involve ubiquitination of Miro1 and also does not disturb Miro1 function. However, following mitochondrial damage and PINK1 accumulation, this initial pool of Parkin becomes activated, leading to the ubiquitination and degradation of Miro1. Knockdown of Miro proteins reduces Parkin translocation to mitochondria and suppresses mitophagic removal of mitochondria. Moreover, we demonstrate that Miro1 EF-hand domains control Miro1's ubiquitination and Parkin recruitment to damaged mitochondria, and they protect neurons from glutamate-induced mitophagy. Together, our results suggest that Miro1 functions as a calcium-sensitive docking site for Parkin on mitochondria.


Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Mitofagia , Domínios Proteicos , Transporte Proteico , Proteólise , Ratos , Ubiquitinação , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/genética
4.
PLoS Biol ; 14(7): e1002511, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27434582

RESUMO

Deficiency of the protein Wolfram syndrome 1 (WFS1) is associated with multiple neurological and psychiatric abnormalities similar to those observed in pathologies showing alterations in mitochondrial dynamics. The aim of this study was to examine the hypothesis that WFS1 deficiency affects neuronal function via mitochondrial abnormalities. We show that down-regulation of WFS1 in neurons leads to dramatic changes in mitochondrial dynamics (inhibited mitochondrial fusion, altered mitochondrial trafficking, and augmented mitophagy), delaying neuronal development. WFS1 deficiency induces endoplasmic reticulum (ER) stress, leading to inositol 1,4,5-trisphosphate receptor (IP3R) dysfunction and disturbed cytosolic Ca2+ homeostasis, which, in turn, alters mitochondrial dynamics. Importantly, ER stress, impaired Ca2+ homeostasis, altered mitochondrial dynamics, and delayed neuronal development are causatively related events because interventions at all these levels improved the downstream processes. Our data shed light on the mechanisms of neuronal abnormalities in Wolfram syndrome and point out potential therapeutic targets. This work may have broader implications for understanding the role of mitochondrial dynamics in neuropsychiatric diseases.


Assuntos
Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Neurogênese , Neurônios/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Encéfalo/metabolismo , Cálcio/metabolismo , Células Cultivadas , Estresse do Retículo Endoplasmático/genética , Transferência Ressonante de Energia de Fluorescência , Homeostase , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Potencial da Membrana Mitocondrial/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Knockout , Microscopia Confocal , Mitocôndrias/genética , Mitofagia/genética , Neurônios/citologia , Células PC12 , Interferência de RNA , Ratos , Ratos Wistar , Imagem com Lapso de Tempo/métodos , Síndrome de Wolfram/genética , Síndrome de Wolfram/metabolismo
6.
Pharmacol Res ; 113(Pt B): 723-730, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27168226

RESUMO

This Info article offers an overview on the main historical facts and the current perspectives of the scientific and educational competence in field of pharmacology in three European countries on Baltic sea East coast: Estonia, Latvia and Lithuania. The research areas have changed constantly due to economical and political reasons during the last 200 years and today do cover quite different pharmacological areas in each of Baltic countries and are recognized internationally. Today the main topics of studies in Estonia are the pharmacology of neurodegenerative diseases, mood disorders and brain plasticity; the role of mitochondria in neurodegenerative diseases, and the epigenetics of drug dependence. In Latvia, the primary research areas include molecular, neuropharmacology, particularly search for novel medicines capable to halt neurodegenerative diseases as well as cardiovascular pharmacology. In Lithuania the main focus is on clinical pharmacology, rational use of drugs, pharmacoepidemiology and pharmacoeconomy, in experimental pharmacology on regenerative medicine and nephropharmacology. All three countries have their own active Societies of Pharmacology.


Assuntos
Farmacologia/educação , Estônia , Humanos , Letônia , Lituânia
7.
Development ; 143(11): 1981-92, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27122166

RESUMO

During early development, neurons undergo complex morphological rearrangements to assemble into neuronal circuits and propagate signals. Rapid growth requires a large quantity of building materials, efficient intracellular transport and also a considerable amount of energy. To produce this energy, the neuron should first generate new mitochondria because the pre-existing mitochondria are unlikely to provide a sufficient acceleration in ATP production. Here, we demonstrate that mitochondrial biogenesis and ATP production are required for axonal growth and neuronal development in cultured rat cortical neurons. We also demonstrate that growth signals activating the CaMKKß, LKB1-STRAD or TAK1 pathways also co-activate the AMPK-PGC-1α-NRF1 axis leading to the generation of new mitochondria to ensure energy for upcoming growth. In conclusion, our results suggest that neurons are capable of signalling for upcoming energy requirements. Earlier activation of mitochondrial biogenesis through these pathways will accelerate the generation of new mitochondria, thereby ensuring energy-producing capability for when other factors for axonal growth are synthesized.


Assuntos
Axônios/metabolismo , Biogênese de Organelas , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Adenilato Quinase/metabolismo , Animais , Animais Recém-Nascidos , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Proliferação de Células , Células Cultivadas , Córtex Cerebral/citologia , Metabolismo Energético , MAP Quinase Quinase Quinases/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Neurogênese , Fator 1 Nuclear Respiratório/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Ratos Wistar , Fator de Crescimento Transformador beta/metabolismo
8.
Cell Calcium ; 58(1): 79-85, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25467968

RESUMO

There are a number of known and suspected channels and exchangers in the endoplasmic reticulum that may participate in potassium flux across its membrane. They include trimeric intracellular cation channels permeable for potassium, ATP-sensitive potassium channels, calcium-activated potassium channels and the potassium-hydrogen exchanger. Apart from trimeric intracellular cation channels, which are specific to the endoplasmic reticulum, other potassium channels are also expressed in the plasma membrane and/or mitochondria, and their specific role in the endoplasmic reticulum has not yet been fully established. In addition to these potassium-selective channels, the ryanodine receptor and, potentially, the inositol 1,4,5-trisphosphate receptor are permeable to potassium ions. Also, the role of potassium fluxes across the endoplasmic reticulum membrane has remained elusive. It has been proposed that their main role is to balance the charge movement that occurs during calcium release and uptake from or to the endoplasmic reticulum. This review aims to summarize current knowledge on endoplasmic reticulum potassium channels and fluxes and their potential role in endoplasmic reticulum calcium uptake and release.


Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Potássio/metabolismo , Animais , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Canais de Potássio/química , Canais de Potássio/metabolismo , Antiportadores de Potássio-Hidrogênio/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
9.
Autophagy ; 10(6): 1105-19, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24879156

RESUMO

The autophagy protein BECN1/Beclin 1 is known to play a central role in autophagosome formation and maturation. The results presented here demonstrate that BECN1 interacts with the Parkinson disease-related protein PARK2. This interaction does not require PARK2 translocation to mitochondria and occurs mostly in cytosol. However, our results suggest that BECN1 is involved in PARK2 translocation to mitochondria because loss of BECN1 inhibits CCCP- or PINK1 overexpression-induced PARK2 translocation. Our results also demonstrate that the observed PARK2-BECN1 interaction is functionally important. Measurements of the level of MFN2 (mitofusin 2), a PARK2 substrate, demonstrate that depletion of BECN1 prevents PARK2 translocation-induced MFN2 ubiquitination and loss. BECN1 depletion also rescues the MFN2 loss-induced suppression of mitochondrial fusion. In sum, our results demonstrate that BECN1 interacts with PARK2 and regulates PARK2 translocation to mitochondria as well as PARK2-induced mitophagy prior to autophagosome formation.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Mitofagia/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Autofagia , Proteína Beclina-1 , Transporte Biológico Ativo , Células Cultivadas , GTP Fosfo-Hidrolases , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Células PC12 , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , RNA Interferente Pequeno/genética , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
10.
J Cell Sci ; 125(Pt 3): 625-33, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22331352

RESUMO

Calcium pumping into the endoplasmic reticulum (ER) lumen is thought to be coupled to a countertransport of protons through sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) and the members of the ClC family of chloride channels. However, pH in the ER lumen remains neutral, which suggests a mechanism responsible for proton re-entry. We studied whether cation-proton exchangers could act as routes for such a re-entry. ER Ca(2+) uptake was measured in permeabilized immortalized hypothalamic neurons, primary rat cortical neurons and mouse cardiac fibers. Replacement of K(+) in the uptake solution with Na(+) or tetraethylammonium led to a strong inhibition of Ca(2+) uptake in neurons and cardiomyocytes. Furthermore, inhibitors of the potassium-proton exchanger (quinine or propranolol) but not of the sodium-proton exchanger reduced ER Ca(2+) uptake by 56-82%. Externally added nigericin, a potassium-proton exchanger, attenuated the inhibitory effect of propranolol. Inhibitors of small conductance calcium-sensitive K(+) (SK(Ca)) channels (UCL 1684, dequalinium) blocked the uptake of Ca(2+) by the ER in all preparations by 48-94%, whereas inhibitors of other K(+) channels (IK(Ca), BK(Ca) and K(ATP)) had no effect. Fluorescence microscopy and western blot analysis revealed the presence of both SK(Ca) channels and the potassium-proton exchanger leucine zipper-EF-hand-containing transmembrane protein 1 (LETM1) in ER in situ and in the purified ER fraction. The data obtained demonstrate that SK(Ca) channels and LETM1 reside in the ER membrane and that their activity is essential for ER Ca(2+) uptake.


Assuntos
Retículo Endoplasmático/metabolismo , Miócitos Cardíacos/metabolismo , Neurônios/metabolismo , Antiportadores de Potássio-Hidrogênio/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Transporte de Íons/efeitos dos fármacos , Camundongos , Modelos Biológicos , Miócitos Cardíacos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Nigericina/farmacologia , Antiportadores de Potássio-Hidrogênio/antagonistas & inibidores , Propranolol/farmacologia , Ratos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/antagonistas & inibidores
11.
J Biol Chem ; 286(12): 10814-24, 2011 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-21252228

RESUMO

Parkinson disease is characterized by the accumulation of aggregated α-synuclein as the major component of the Lewy bodies. α-Synuclein accumulation in turn leads to compensatory effects that may include the up-regulation of autophagy. Another common feature of Parkinson disease (PD) is mitochondrial dysfunction. Here, we provide evidence that the overactivation of autophagy may be a link that connects the intracellular accumulation of α-synuclein with mitochondrial dysfunction. We found that the activation of macroautophagy in primary cortical neurons that overexpress mutant A53T α-synuclein leads to massive mitochondrial destruction and loss, which is associated with a bioenergetic deficit and neuronal degeneration. No mitochondrial removal or net loss was observed when we suppressed the targeting of mitochondria to autophagosomes by silencing Parkin, overexpressing wild-type Mitofusin 2 and dominant negative Dynamin-related protein 1 or blocking autophagy by silencing autophagy-related genes. The inhibition of targeting mitochondria to autophagosomes or autophagy was also partially protective against mutant A53T α-synuclein-induced neuronal cell death. These data suggest that overactivated mitochondrial removal could be one of the contributing factors that leads to the mitochondrial loss observed in PD models.


Assuntos
Autofagia , Mitocôndrias/metabolismo , Mutação de Sentido Incorreto , Neurônios/metabolismo , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Substituição de Aminoácidos , Animais , Modelos Animais de Doenças , GTP Fosfo-Hidrolases , Inativação Gênica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Células PC12 , Doença de Parkinson/genética , Ratos , Ratos Wistar , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , alfa-Sinucleína/genética
12.
Cardiovasc Res ; 87(1): 83-91, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20124402

RESUMO

AIMS: The myofibrillar and nuclear compartments in cardiomyocytes are known to be sensitive to extracellular mechanical stimuli. Recently, we have shown that alterations in the mitochondrial ionic balance in cells in situ are associated with considerably increased mitochondrial volume. Theoretically, this swelling of mitochondria could impose mechanical constraints on the myofibrils and nuclei in their vicinity. Thus, we studied whether modulation of mitochondrial volume in cardiomyocytes in situ has a mechanical effect on the myofibrillar and nuclear compartments. METHODS AND RESULTS: We used the measurement of passive force developed by saponin-permeabilized mouse ventricular fibres as a sensor for compression of the myofibrils. Osmotic compression induced by dextran caused an increase in passive force. Similarly, mitochondrial swelling induced by drugs that alter ionic homeostasis (alamethicin and propranolol) markedly augmented passive force (confirmed by confocal microscopy). Diazoxide, a mitochondrial ATP-sensitive potassium channel opener known to cause moderate mitochondrial swelling, also increased passive force (by 28 +/- 5% at 10% stretch, P < 0.01). This effect was completely blocked by 5-hydroxydecanoate (5-HD), a putative specific inhibitor of these channels. Mitochondrial swelling induced by alamethicin and propranolol led to significant nuclear deformation, which was visualized by confocal microscopy. Furthermore, diazoxide decreased nuclear volume, calculated using three-dimensional reconstructed images, in a 5-HD-dependent manner by 12 +/- 2% (P < 0.05). This corresponds to an increase in intracellular pressure of 2.1 +/- 0.3 kPa. CONCLUSION: This study is the first to demonstrate that mitochondria are able to generate internal pressure, which can mechanically affect the morphological and functional properties of intracellular organelles.


Assuntos
Mecanotransdução Celular , Mitocôndrias/metabolismo , Dilatação Mitocondrial , Miócitos Cardíacos/metabolismo , Alameticina/farmacologia , Animais , Núcleo Celular/metabolismo , Forma do Núcleo Celular , Tamanho do Núcleo Celular , Ácidos Decanoicos/farmacologia , Diazóxido/farmacologia , Hidroxiácidos/farmacologia , Masculino , Mecanotransdução Celular/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Mitocôndrias/efeitos dos fármacos , Dilatação Mitocondrial/efeitos dos fármacos , Contração Miocárdica , Miócitos Cardíacos/efeitos dos fármacos , Miofibrilas/metabolismo , Pressão Osmótica , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/metabolismo , Propranolol/farmacologia , Ratos
13.
J Biol Chem ; 284(32): 21379-85, 2009 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-19542216

RESUMO

Recent studies indicate that regulation of cellular oxidative capacity through enhancing mitochondrial biogenesis may be beneficial for neuronal recovery and survival in human neurodegenerative disorders. The peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) has been shown to be a master regulator of mitochondrial biogenesis and cellular energy metabolism in muscle and liver. The aim of our study was to establish whether PGC-1alpha and PGC-1beta control mitochondrial density also in neurons and if these coactivators could be up-regulated by deacetylation. The results demonstrate that PGC-1alpha and PGC-1beta control mitochondrial capacity in an additive and independent manner. This effect was observed in all studied subtypes of neurons, in cortical, midbrain, and cerebellar granule neurons. We also observed that endogenous neuronal PGC-1alpha but not PGC-1beta could be activated through its repressor domain by suppressing it. Results demonstrate also that overexpression of SIRT1 deacetylase or suppression of GCN5 acetyltransferase activates transcriptional activity of PGC-1alpha in neurons and increases mitochondrial density. These effects were mediated exclusively via PGC-1alpha, since overexpression of SIRT1 or suppression of GCN5 was ineffective where PGC-1alpha was suppressed by short hairpin RNA. Moreover, the results demonstrate that overexpression of PGC-1beta or PGC-1alpha or activation of the latter by SIRT1 protected neurons from mutant alpha-synuclein- or mutant huntingtin-induced mitochondrial loss. These evidences demonstrate that activation or overexpression of the PGC-1 family of coactivators could be used to compensate for neuronal mitochondrial loss and suggest that therapeutic agents activating PGC-1 would be valuable for treating neurodegenerative diseases in which mitochondrial dysfunction and oxidative damage play an important pathogenic role.


Assuntos
Regulação da Expressão Gênica , Mitocôndrias/metabolismo , Neurônios/metabolismo , Proteínas de Ligação a RNA/fisiologia , Fatores de Transcrição/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Animais Recém-Nascidos , Autofagia , Humanos , Oxigênio/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Proteínas de Ligação a RNA/metabolismo , Ratos , Ratos Wistar , Sirtuína 1 , Sirtuínas/biossíntese , Fatores de Transcrição/metabolismo , Fatores de Transcrição de p300-CBP/biossíntese
14.
Cardiovasc Res ; 83(1): 89-96, 2009 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-19389722

RESUMO

AIMS: Increased diastolic sarcoplasmic reticulum (SR) Ca(2+) loss could depress contractility in heart failure. Since the failing myocardium has impaired energetics, we investigated whether Ca(2+) loss is linked to changes in energetic pathways. METHODS AND RESULTS: Leakage from SR in mouse permeabilized preparations was assessed using exogenous ATP, ATP + phosphocreatine (activation of bound creatine kinase, CK), ATP + mitochondrial substrates (mitochondrial activation), or with all of these together (optimal energetic conditions) in Ca(2+)-free solution. In ventricular fibres caffeine-induced tension transients under optimal energetic conditions were used to estimate SR [Ca(2+)]. In cardiomyocytes, intra-SR Ca(2+) was monitored by use of the fluorescent marker Mag-fluo 4. In fibres, SR Ca(2+) content after 5 min incubation strongly depended on energy supply (100%-optimal energetic conditions; 27 +/- 5%-exogenous ATP only, 52 +/- 5%-endogenous CK activation; 88 +/- 8%-mitochondrial activation, P < 0.01 vs. CK system). The significant loss with only exogenous ATP was not inhibited by the ryanodine receptor blockers tetracaine or ruthenium red. However, the SR Ca(2+)-ATPase (SERCA) inhibitors cyclopiazonic acid or 2,5-di(tert-butyl)-1,4-benzohydroquinone significantly decreased Ca(2+) loss. At 100 nM external [Ca(2+)], the SR Ca(2+) loss was also energy dependent and was not significantly inhibited by tetracaine. In cardiomyocytes, the decline in SR [Ca(2+)] at zero external [Ca(2+)] was almost two times slower under optimal energetic conditions than in the presence of exogenous ATP only. CONCLUSION: At low extra-reticular [Ca(2+)], the main leak pathway is an energy-sensitive backward Ca(2+) pump, and direct mitochondrial-SERCA ATP channelling is more effective in leak prevention than local ATP generation by bound CK.


Assuntos
Cálcio/metabolismo , Metabolismo Energético/fisiologia , Miócitos Cardíacos/metabolismo , Retículo Sarcoplasmático/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Indóis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Miócitos Cardíacos/citologia , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , Transdução de Sinais/fisiologia , Tetracaína/farmacologia
15.
J Biol Chem ; 282(45): 32821-6, 2007 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-17785462

RESUMO

Organelle transport in neuronal processes is central to the organization, developmental fate, and functions of neurons. Organelles must be transported through the slender, highly branched neuronal processes, making the axonal transport vulnerable to any perturbation. However, some intracellular structures like mitochondria are able to considerably modify their volume. We therefore hypothesized that swollen mitochondria could impair the traffic of other organelles in neurite shafts. To test this hypothesis, we have investigated the effects of mitochondrial swellers on the organelle traffic. Our data demonstrate that treatment of neurons with potassium ionophore valinomycin led to the fast time-dependent inhibition of organelle movement in cerebellar granule neurons. Similar inhibition was observed in neurons treated with the inhibitors of the mitochondrial respiratory chain, sodium azide and antimycin, which also induced swelling. No decrease in the motility of organelles was observed in cultures treated with inhibitors of ATP production or transport, oligomycin or bongkrekic acid, suggesting that inhibition of the ATP-generating activity itself without swelling does not affect the motility of organelles. The effect of swellers on the traffic was more important in thin processes, thus indicating the role of steric hindrance of swollen mitochondria. We propose that the size and morphology of the transported cargo is also relevant for seamless axonal transport and speculate that mitochondrial swelling could be one of the reasons for impaired organelle transport in neuronal processes.


Assuntos
Cerebelo/citologia , Dilatação Mitocondrial/fisiologia , Neurônios/citologia , Organelas/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Dilatação Mitocondrial/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Organelas/efeitos dos fármacos , Ratos , Ratos Wistar , Técnicas de Cultura de Tecidos
16.
Epilepsia ; 48(4): 752-7, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17319918

RESUMO

Unverricht-Lundborg disease (EPM1) has been considered to be an autosomal-recessive disease related with loss of function mutations in the gene encoding cystatin B. Although heterozygous carriers are generally asymptomatic, earlier studies in Finnish EPM1 families have reported minor symptoms together with slight changes in the EEG recordings also in near relatives of patients. Here we tested the hypothesis that EPM1 phenotype is expressed also in heterozygous subjects using 17-month-old cystatin B deficient mice as a model of disease. Western blot analysis demonstrated a 50% decrease in cystatin B expression in the cerebellum of these animals. Heterozygous mice showed significantly impaired rotarod performance and were weaker in the grid test. Also the total seizure-rating score of heterozygous animals was higher than in wild-type mice. The stereological analysis revealed a significant decrease in the number of neurons in cerebral cortex and the granule cell layer of cerebellum. These results suggest that partial decrease in cystatin B expression in heterozygous mice could lead to the development of mild EPM1 phenotype.


Assuntos
Cistatinas/deficiência , Cistatinas/genética , Heterozigoto , Síndrome de Unverricht-Lundborg/genética , Animais , Ataxia/diagnóstico , Ataxia/genética , Comportamento Animal/fisiologia , Encéfalo/patologia , Contagem de Células , Morte Celular/genética , Cerebelo/patologia , Córtex Cerebral/patologia , Cistatina B , Modelos Animais de Doenças , Feminino , Finlândia/epidemiologia , Manobra Psicológica , Masculino , Camundongos , Camundongos Mutantes , Mutação , Neurônios/patologia , Fenótipo , Teste de Desempenho do Rota-Rod , Convulsões/diagnóstico , Convulsões/genética , Síndrome de Unverricht-Lundborg/epidemiologia , Síndrome de Unverricht-Lundborg/patologia
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