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1.
EMBO J ; 43(4): 595-614, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38267654

RESUMO

Miro proteins are universally conserved mitochondrial calcium-binding GTPases that regulate a multitude of mitochondrial processes, including transport, clearance, and lipid trafficking. The exact role of Miro in these functions is unclear but involves binding to a variety of client proteins. How this binding is operated at the molecular level and whether and how it is important for mitochondrial health, however, remains unknown. Here, we show that known Miro interactors-namely, CENPF, Trak, and MYO19-all use a similar short motif to bind the same structural element: a highly conserved hydrophobic pocket in the first calcium-binding domain of Miro. Using these Miro-binding motifs, we identified direct interactors de novo, including MTFR1/2/1L, the lipid transporters Mdm34 and VPS13D, and the ubiquitin E3-ligase Parkin. Given the shared binding mechanism of these functionally diverse clients and its conservation across eukaryotes, we propose that Miro is a universal mitochondrial adaptor coordinating mitochondrial health.


Assuntos
Cálcio , Mitocôndrias , Humanos , Cálcio/metabolismo , Mitocôndrias/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Homeostase , Lipídeos , Proteínas Mitocondriais/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas/metabolismo
2.
EMBO J ; 40(14): e100715, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34152608

RESUMO

Clearance of mitochondria following damage is critical for neuronal homeostasis. Here, we investigate the role of Miro proteins in mitochondrial turnover by the PINK1/Parkin mitochondrial quality control system in vitro and in vivo. We find that upon mitochondrial damage, Miro is promiscuously ubiquitinated on multiple lysine residues. Genetic deletion of Miro or block of Miro1 ubiquitination and subsequent degradation lead to delayed translocation of the E3 ubiquitin ligase Parkin onto damaged mitochondria and reduced mitochondrial clearance in both fibroblasts and cultured neurons. Disrupted mitophagy in vivo, upon post-natal knockout of Miro1 in hippocampus and cortex, leads to a dramatic increase in mitofusin levels, the appearance of enlarged and hyperfused mitochondria and hyperactivation of the integrated stress response (ISR). Altogether, our results provide new insights into the central role of Miro1 in the regulation of mitochondrial homeostasis and further implicate Miro1 dysfunction in the pathogenesis of human neurodegenerative disease.


Assuntos
Mitocôndrias/metabolismo , Mitofagia/fisiologia , Neurônios/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/metabolismo , Doenças Neurodegenerativas/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia
3.
J Cell Sci ; 135(22)2022 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-36274588

RESUMO

Long-term changes in synaptic strength form the basis of learning and memory. These changes rely upon energy-demanding mechanisms, which are regulated by local Ca2+ signalling. Mitochondria are optimised for providing energy and buffering Ca2+. However, our understanding of the role of mitochondria in regulating synaptic plasticity is incomplete. Here, we have used optical and electrophysiological techniques in cultured hippocampal neurons and ex vivo hippocampal slices from mice with haploinsufficiency of the mitochondrial Ca2+ uniporter (MCU+/-) to address whether reducing mitochondrial Ca2+ uptake alters synaptic transmission and plasticity. We found that cultured MCU+/- hippocampal neurons have impaired Ca2+ clearance, and consequently enhanced synaptic vesicle fusion at presynapses occupied by mitochondria. Furthermore, long-term potentiation (LTP) at mossy fibre (MF) synapses, a process which is dependent on presynaptic Ca2+ accumulation, is enhanced in MCU+/- slices. Our results reveal a previously unrecognised role for mitochondria in regulating presynaptic plasticity of a major excitatory pathway involved in learning and memory.


Assuntos
Potenciação de Longa Duração , Fibras Musgosas Hipocampais , Camundongos , Animais , Fibras Musgosas Hipocampais/metabolismo , Potenciação de Longa Duração/fisiologia , Cálcio/metabolismo , Haploinsuficiência , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Mitocôndrias/metabolismo
4.
Brain ; 146(2): 727-738, 2023 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-35867861

RESUMO

The SARS-CoV-2 receptor, ACE2, is found on pericytes, contractile cells enwrapping capillaries that regulate brain, heart and kidney blood flow. ACE2 converts vasoconstricting angiotensin II into vasodilating angiotensin-(1-7). In brain slices from hamster, which has an ACE2 sequence similar to human ACE2, angiotensin II evoked a small pericyte-mediated capillary constriction via AT1 receptors, but evoked a large constriction when the SARS-CoV-2 receptor binding domain (RBD, original Wuhan variant) was present. A mutated non-binding RBD did not potentiate constriction. A similar RBD-potentiated capillary constriction occurred in human cortical slices, and was evoked in hamster brain slices by pseudotyped virions expressing SARS-CoV-2 spike protein. This constriction reflects an RBD-induced decrease in the conversion of angiotensin II to angiotensin-(1-7) mediated by removal of ACE2 from the cell surface membrane and was mimicked by blocking ACE2. The clinically used drug losartan inhibited the RBD-potentiated constriction. Thus, AT1 receptor blockers could be protective in COVID-19 by preventing pericyte-mediated blood flow reductions in the brain, and perhaps the heart and kidney.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , COVID-19/metabolismo , Pericitos/metabolismo , Angiotensina II/farmacologia , Angiotensina II/metabolismo , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Capilares , Constrição , Receptores Virais/química , Receptores Virais/metabolismo , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Ligação Proteica
5.
Nat Rev Neurosci ; 19(2): 63-80, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-29348666

RESUMO

Synapses enable neurons to communicate with each other and are therefore a prerequisite for normal brain function. Presynaptically, this communication requires energy and generates large fluctuations in calcium concentrations. Mitochondria are optimized for supplying energy and buffering calcium, and they are actively recruited to presynapses. However, not all presynapses contain mitochondria; thus, how might synapses with and without mitochondria differ? Mitochondria are also increasingly recognized to serve additional functions at the presynapse. Here, we discuss the importance of presynaptic mitochondria in maintaining neuronal homeostasis and how dysfunctional presynaptic mitochondria might contribute to the development of disease.


Assuntos
Mitocôndrias/fisiologia , Neurônios/fisiologia , Terminações Pré-Sinápticas/fisiologia , Transmissão Sináptica/fisiologia , Animais , Sinalização do Cálcio/fisiologia , Homeostase/fisiologia , Humanos
6.
J Biol Chem ; 296: 100364, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33539918

RESUMO

The K+/Cl- cotransporter KCC2 (SLC12A5) allows mature neurons in the CNS to maintain low intracellular Cl- levels that are critical in mediating fast hyperpolarizing synaptic inhibition via type A γ-aminobutyric acid receptors (GABAARs). In accordance with this, compromised KCC2 activity results in seizures, but whether such deficits directly contribute to the subsequent changes in neuronal structure and viability that lead to epileptogenesis remains to be assessed. Canonical hyperpolarizing GABAAR currents develop postnatally, which reflect a progressive increase in KCC2 expression levels and activity. To investigate the role that KCC2 plays in regulating neuronal viability and architecture, we have conditionally ablated KCC2 expression in developing and mature neurons. Decreasing KCC2 expression in mature neurons resulted in the rapid activation of the extrinsic apoptotic pathway. Intriguingly, direct pharmacological inhibition of KCC2 in mature neurons was sufficient to rapidly induce apoptosis, an effect that was not abrogated via blockade of neuronal depolarization using tetrodotoxin (TTX). In contrast, ablating KCC2 expression in immature neurons had no discernable effects on their subsequent development, arborization, or dendritic structure. However, removing KCC2 in immature neurons was sufficient to ablate the subsequent postnatal development of hyperpolarizing GABAAR currents. Collectively, our results demonstrate that KCC2 plays a critical role in neuronal survival by limiting apoptosis, and mature neurons are highly sensitive to the loss of KCC2 function. In contrast, KCC2 appears to play a minimal role in mediating neuronal development or architecture.


Assuntos
Neurônios/metabolismo , Simportadores/metabolismo , Animais , Apoptose , Cloretos/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurogênese/efeitos dos fármacos , Neurônios/fisiologia , Potássio/metabolismo , Cultura Primária de Células , Receptores de GABA/metabolismo , Convulsões , Simportadores/fisiologia , Ácido gama-Aminobutírico/metabolismo , Cotransportadores de K e Cl-
7.
EMBO J ; 37(3): 321-336, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29311115

RESUMO

In the current model of mitochondrial trafficking, Miro1 and Miro2 Rho-GTPases regulate mitochondrial transport along microtubules by linking mitochondria to kinesin and dynein motors. By generating Miro1/2 double-knockout mouse embryos and single- and double-knockout embryonic fibroblasts, we demonstrate the essential and non-redundant roles of Miro proteins for embryonic development and subcellular mitochondrial distribution. Unexpectedly, the TRAK1 and TRAK2 motor protein adaptors can still localise to the outer mitochondrial membrane to drive anterograde mitochondrial motility in Miro1/2 double-knockout cells. In contrast, we show that TRAK2-mediated retrograde mitochondrial transport is Miro1-dependent. Interestingly, we find that Miro is critical for recruiting and stabilising the mitochondrial myosin Myo19 on the mitochondria for coupling mitochondria to the actin cytoskeleton. Moreover, Miro depletion during PINK1/Parkin-dependent mitophagy can also drive a loss of mitochondrial Myo19 upon mitochondrial damage. Finally, aberrant positioning of mitochondria in Miro1/2 double-knockout cells leads to disruption of correct mitochondrial segregation during mitosis. Thus, Miro proteins can fine-tune actin- and tubulin-dependent mitochondrial motility and positioning, to regulate key cellular functions such as cell proliferation.


Assuntos
Dineínas/metabolismo , Cinesinas/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Miosinas/metabolismo , Proteínas rho de Ligação ao GTP/genética , Actinas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Animais , Transporte Biológico , Proteínas de Transporte/metabolismo , Linhagem Celular Transformada , Proliferação de Células/genética , Desenvolvimento Embrionário/genética , Camundongos , Camundongos Knockout , Microtúbulos/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
8.
EMBO Rep ; 21(2): e49865, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-31894645

RESUMO

Peroxisomes are essential for a number of cellular functions, including reactive oxygen species metabolism, fatty acid ß-oxidation and lipid synthesis. To ensure optimal functionality, peroxisomal size, shape and number must be dynamically maintained; however, many aspects of how this is regulated remain poorly characterised. Here, we show that the localisation of Miro1 and Miro2-outer mitochondrial membrane proteins essential for mitochondrial trafficking-to peroxisomes is not required for basal peroxisomal distribution and long-range trafficking, but rather for the maintenance of peroxisomal size and morphology through peroxisomal fission. Mechanistically, this is achieved by Miro negatively regulating Drp1-dependent fission, a function that is shared with the mitochondria. We further find that the peroxisomal localisation of Miro is regulated by its first GTPase domain and is mediated by an interaction through its transmembrane domain with the peroxisomal-membrane protein chaperone, Pex19. Our work highlights a shared regulatory role of Miro in maintaining the morphology of both peroxisomes and mitochondria, supporting a crosstalk between peroxisomal and mitochondrial biology.


Assuntos
Proteínas Mitocondriais , Proteínas rho de Ligação ao GTP , Animais , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Peroxissomos/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
9.
Cell Mol Life Sci ; 78(5): 1929-1941, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33141311

RESUMO

Peroxisomes are organelles that perform a wide range of essential metabolic processes. To ensure that peroxisomes are optimally positioned in the cell, they must be transported by both long- and short-range trafficking events in response to cellular needs. Here, we review our current understanding of the mechanisms by which the cytoskeleton and organelle contact sites alter peroxisomal distribution. Though the focus of the review is peroxisomal transport in mammalian cells, findings from flies and fungi are used for comparison and to inform the gaps in our understanding. Attention is given to the apparent overlap in regulatory mechanisms for mitochondrial and peroxisomal trafficking, along with the recently discovered role of the mitochondrial Rho-GTPases, Miro, in peroxisomal dynamics. Moreover, we outline and discuss the known pathological and pharmacological conditions that perturb peroxisomal positioning. We conclude by highlighting several gaps in our current knowledge and suggest future directions that require attention.


Assuntos
Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Peroxissomos/metabolismo , Animais , Transporte Biológico , Humanos , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Proteínas rho de Ligação ao GTP/metabolismo
10.
J Cell Sci ; 132(24)2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31757889

RESUMO

Binding of motor proteins to cellular cargoes is regulated by adaptor proteins. HAP1 and GRIP1 are kinesin-1 adaptors that have been implicated individually in the transport of vesicular cargoes in the dendrites of neurons. We find that HAP1a and GRIP1 form a protein complex in the brain, and co-operate to activate the kinesin-1 subunit KIF5C in vitro Based upon this co-operative activation of kinesin-1, we propose a modification to the kinesin activation model that incorporates stabilisation of the central hinge region known to be critical to autoinhibition of kinesin-1.


Assuntos
Proteínas de Transporte/metabolismo , Cinesinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Células COS , Proteínas de Transporte/genética , Chlorocebus aethiops , Células HeLa , Humanos , Cinesinas/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
11.
Development ; 144(13): 2445-2455, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28533206

RESUMO

Growth factors of the TGFß superfamily play key roles in regulating neuronal and muscle function. Myostatin (or GDF8) and GDF11 are potent negative regulators of skeletal muscle mass. However, expression of myostatin and its cognate receptors in other tissues, including brain and peripheral nerves, suggests a potential wider biological role. Here, we show that Myoglianin (MYO), the Drosophila homolog of myostatin and GDF11, regulates not only body weight and muscle size, but also inhibits neuromuscular synapse strength and composition in a Smad2-dependent manner. Both myostatin and GDF11 affected synapse formation in isolated rat cortical neuron cultures, suggesting an effect on synaptogenesis beyond neuromuscular junctions. We also show that MYO acts in vivo to inhibit synaptic transmission between neurons in the escape response neural circuit of adult flies. Thus, these anti-myogenic proteins act as important inhibitors of synapse function and neuronal growth.


Assuntos
Forma Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Miostatina/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Sinapses/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Peso Corporal , Regulação para Baixo/genética , Drosophila melanogaster/citologia , Inativação Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Fatores de Diferenciação de Crescimento/metabolismo , Humanos , Larva/metabolismo , Células Musculares/metabolismo , Neuroglia/metabolismo , Junção Neuromuscular/metabolismo , Ratos , Transdução de Sinais , Transmissão Sináptica
12.
EMBO Rep ; 18(2): 231-240, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28039205

RESUMO

Mitochondrial trafficking is influenced by neuronal activity, but it remains unclear how mitochondrial positioning influences neuronal transmission and plasticity. Here, we use live cell imaging with the genetically encoded presynaptically targeted Ca2+ indicator, SyGCaMP5, to address whether presynaptic Ca2+ responses are altered by mitochondria in synaptic terminals. We find that presynaptic Ca2+ signals, as well as neurotransmitter release, are significantly decreased in terminals containing mitochondria. Moreover, the localisation of mitochondria at presynaptic sites can be altered during long-term activity changes, dependent on the Ca2+-sensing function of the mitochondrial trafficking protein, Miro1. In addition, we find that Miro1-mediated activity-dependent synaptic repositioning of mitochondria allows neurons to homeostatically alter the strength of presynaptic Ca2+ signals in response to prolonged changes in neuronal activity. Our results support a model in which mitochondria are recruited to presynaptic terminals during periods of raised neuronal activity and are involved in rescaling synaptic signals during homeostatic plasticity.


Assuntos
Sinalização do Cálcio , Homeostase , Mitocôndrias/metabolismo , Plasticidade Neuronal , Terminações Pré-Sinápticas/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Expressão Gênica , Camundongos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Neurônios/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
13.
J Biol Chem ; 291(2): 613-29, 2016 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-26553875

RESUMO

The DISC1 protein is implicated in major mental illnesses including schizophrenia, depression, bipolar disorder, and autism. Aberrant mitochondrial dynamics are also associated with major mental illness. DISC1 plays a role in mitochondrial transport in neuronal axons, but its effects in dendrites have yet to be studied. Further, the mechanisms of this regulation and its role in neuronal development and brain function are poorly understood. Here we have demonstrated that DISC1 couples to the mitochondrial transport and fusion machinery via interaction with the outer mitochondrial membrane GTPase proteins Miro1 and Miro2, the TRAK1 and TRAK2 mitochondrial trafficking adaptors, and the mitochondrial fusion proteins (mitofusins). Using live cell imaging, we show that disruption of the DISC1-Miro-TRAK complex inhibits mitochondrial transport in neurons. We also show that the fusion protein generated from the originally described DISC1 translocation (DISC1-Boymaw) localizes to the mitochondria, where it similarly disrupts mitochondrial dynamics. We also show by super resolution microscopy that DISC1 is localized to endoplasmic reticulum contact sites and that the DISC1-Boymaw fusion protein decreases the endoplasmic reticulum-mitochondria contact area. Moreover, disruption of mitochondrial dynamics by targeting the DISC1-Miro-TRAK complex or upon expression of the DISC1-Boymaw fusion protein impairs the correct development of neuronal dendrites. Thus, DISC1 acts as an important regulator of mitochondrial dynamics in both axons and dendrites to mediate the transport, fusion, and cross-talk of these organelles, and pathological DISC1 isoforms disrupt this critical function leading to abnormal neuronal development.


Assuntos
Dendritos/metabolismo , Dinâmica Mitocondrial , Morfogênese , Proteínas do Tecido Nervoso/metabolismo , Animais , Axônios/metabolismo , Transporte Biológico , Células COS , Chlorocebus aethiops , Retículo Endoplasmático/metabolismo , Humanos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas do Tecido Nervoso/química , Ligação Proteica , RNA Longo não Codificante , Proteínas Recombinantes de Fusão/metabolismo , Esquizofrenia/metabolismo , Relação Estrutura-Atividade
14.
J Neurosci ; 35(48): 15996-6011, 2015 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-26631479

RESUMO

It is fast emerging that maintaining mitochondrial function is important for regulating astrocyte function, although the specific mechanisms that govern astrocyte mitochondrial trafficking and positioning remain poorly understood. The mitochondrial Rho-GTPase 1 protein (Miro1) regulates mitochondrial trafficking and detachment from the microtubule transport network to control activity-dependent mitochondrial positioning in neurons. However, whether Miro proteins are important for regulating signaling-dependent mitochondrial dynamics in astrocytic processes remains unclear. Using live-cell confocal microscopy of rat organotypic hippocampal slices, we find that enhancing neuronal activity induces transient mitochondrial remodeling in astrocytes, with a concomitant, transient reduction in mitochondrial trafficking, mediated by elevations in intracellular Ca(2+). Stimulating neuronal activity also induced mitochondrial confinement within astrocytic processes in close proximity to synapses. Furthermore, we show that the Ca(2+)-sensing EF-hand domains of Miro1 are important for regulating mitochondrial trafficking in astrocytes and required for activity-driven mitochondrial confinement near synapses. Additionally, activity-dependent mitochondrial positioning by Miro1 reciprocally regulates the levels of intracellular Ca(2+) in astrocytic processes. Thus, the regulation of intracellular Ca(2+) signaling, dependent on Miro1-mediated mitochondrial positioning, could have important consequences for astrocyte Ca(2+) wave propagation, gliotransmission, and ultimately neuronal function.


Assuntos
Astrócitos/ultraestrutura , Sinalização do Cálcio/fisiologia , Espaço Intracelular/metabolismo , Mitocôndrias/fisiologia , Proteínas Mitocondriais/metabolismo , Sinapses/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Dependovirus/genética , Embrião de Mamíferos , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Ácido Glutâmico/farmacologia , Hipocampo/citologia , Técnicas In Vitro , Espaço Intracelular/genética , Masculino , Proteínas Mitocondriais/genética , Neurônios/fisiologia , Técnicas de Cultura de Órgãos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Ratos , Ratos Sprague-Dawley , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteínas rho de Ligação ao GTP/genética
15.
Glia ; 64(7): 1252-64, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27189737

RESUMO

The astrocytic GLT-1 (or EAAT2) is the major glutamate transporter for clearing synaptic glutamate. While the diffusion dynamics of neurotransmitter receptors at the neuronal surface are well understood, far less is known regarding the surface trafficking of transporters in subcellular domains of the astrocyte membrane. Here, we have used live-cell imaging to study the mechanisms regulating GLT-1 surface diffusion in astrocytes in dissociated and brain slice cultures. Using GFP-time lapse imaging, we show that GLT-1 forms stable clusters that are dispersed rapidly and reversibly upon glutamate treatment in a transporter activity-dependent manner. Fluorescence recovery after photobleaching and single particle tracking using quantum dots revealed that clustered GLT-1 is more stable than diffuse GLT-1 and that glutamate increases GLT-1 surface diffusion in the astrocyte membrane. Interestingly, the two main GLT-1 isoforms expressed in the brain, GLT-1a and GLT-1b, are both found to be stabilized opposed to synapses under basal conditions, with GLT-1b more so. GLT-1 surface mobility is increased in proximity to activated synapses and alterations of neuronal activity can bidirectionally modulate the dynamics of both GLT-1 isoforms. Altogether, these data reveal that astrocytic GLT-1 surface mobility, via its transport activity, is modulated during neuronal firing, which may be a key process for shaping glutamate clearance and glutamatergic synaptic transmission. GLIA 2016;64:1252-1264.


Assuntos
Astrócitos/fisiologia , Transporte Biológico/fisiologia , Córtex Cerebral/citologia , Transportador 2 de Aminoácido Excitatório/metabolismo , Neurônios/fisiologia , 4-Aminopiridina/farmacologia , Anestésicos Locais/farmacologia , Animais , Animais Recém-Nascidos , Ácido Aspártico/análogos & derivados , Ácido Aspártico/farmacologia , Astrócitos/efeitos dos fármacos , Transporte Biológico/genética , Células Cultivadas , Técnicas de Cocultura , Embrião de Mamíferos , Transportador 2 de Aminoácido Excitatório/genética , Ácido Glutâmico/farmacologia , Hipocampo/citologia , Neurônios/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Transgênicos , Tetrodotoxina/farmacologia
16.
Neurobiol Dis ; 90: 27-34, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26707701

RESUMO

Neurons are highly polarised cells with an elaborate and diverse cytoarchitecture. But this complex architecture presents a major problem: how to appropriately distribute metabolic resources where they are most needed within the cell. The solution comes in the form of mitochondria: highly dynamic organelles subject to a repertoire of trafficking, fission/fusion and quality control systems which work in concert to orchestrate a precisely distributed and healthy mitochondrial network. Mitochondria are critical for maintaining local energy supply and buffering Ca(2+) flux within neurons, and are increasingly recognised as being essential for healthy neuronal function. Mitochondrial movements are facilitated by their coupling to microtubule-based transport via kinesin and dynein motors. Adaptor proteins are required for this coupling and the mitochondrial Rho GTPases Miro1 and Miro2 are core components of this machinery. Both Miros have Ca(2+)-sensing and GTPase domains, and are therefore ideally suited to coordinating mitochondrial dynamics with intracellular signalling pathways and local energy turnover. In this review, we focus on Miro's role in mediating mitochondrial transport in neurons, and the relevance of these mechanisms to neuronal health and disease.


Assuntos
Dinâmica Mitocondrial/fisiologia , Proteínas Mitocondriais/metabolismo , Neurônios/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Humanos
17.
J Biol Chem ; 289(21): 14569-82, 2014 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-24671417

RESUMO

Mitochondrial transport plays an important role in matching mitochondrial distribution to localized energy production and calcium buffering requirements. Here, we demonstrate that Miro1, an outer mitochondrial membrane (OMM) protein crucial for the regulation of mitochondrial trafficking and distribution, is a substrate of the PINK1/Parkin mitochondrial quality control system in human dopaminergic neuroblastoma cells. Moreover, Miro1 turnover on damaged mitochondria is altered in Parkinson disease (PD) patient-derived fibroblasts containing a pathogenic mutation in the PARK2 gene (encoding Parkin). By analyzing the kinetics of Miro1 ubiquitination, we further demonstrate that mitochondrial damage triggers rapid (within minutes) and persistent Lys-27-type ubiquitination of Miro1 on the OMM, dependent on PINK1 and Parkin. Proteasomal degradation of Miro1 is then seen on a slower time scale, within 2-3 h of the onset of ubiquitination. We find Miro ubiquitination in dopaminergic neuroblastoma cells is independent of Miro1 phosphorylation at Ser-156 but is dependent on the recently identified Ser-65 residue within Parkin that is phosphorylated by PINK1. Interestingly, we find that Miro1 can stabilize phospho-mutant versions of Parkin on the OMM, suggesting that Miro is also part of a Parkin receptor complex. Moreover, we demonstrate that Ser-65 in Parkin is critical for regulating Miro levels upon mitochondrial damage in rodent cortical neurons. Our results provide new insights into the ubiquitination-dependent regulation of the Miro-mediated mitochondrial transport machinery by PINK1/Parkin and also suggest that disruption of this regulation may be implicated in Parkinson disease pathogenesis.


Assuntos
Proteínas Mitocondriais/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Células Cultivadas , Chlorocebus aethiops , Feminino , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisina/genética , Lisina/metabolismo , Masculino , Microscopia Confocal , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Mutação , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Interferência de RNA , Ratos Sprague-Dawley , Serina/genética , Serina/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas rho de Ligação ao GTP/genética
18.
J Neurosci ; 33(40): 15767-78, 2013 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-24089484

RESUMO

Shank3, which encodes a scaffolding protein at glutamatergic synapses, is a genetic risk factor for autism. In this study, we examined the impact of Shank3 deficiency on the NMDA-type glutamate receptor, a key player in cognition and mental illnesses. We found that knockdown of Shank3 with a small interfering RNA (siRNA) caused a significant reduction of NMDAR-mediated ionic or synaptic current, as well as the surface expression of NR1 subunits, in rat cortical cultures. The effect of Shank3 siRNA on NMDAR currents was blocked by an actin stabilizer, and was occluded by an actin destabilizer, suggesting the involvement of actin cytoskeleton. Since actin dynamics is regulated by the GTPase Rac1 and downstream effector p21-activated kinase (PAK), we further examined Shank3 regulation of NMDARs when Rac1 or PAK was manipulated. We found that the reducing effect of Shank3 siRNA on NMDAR currents was mimicked and occluded by specific inhibitors for Rac1 or PAK, and was blocked by constitutively active Rac1 or PAK. Immunocytochemical data showed a strong reduction of F-actin clusters after Shank3 knockdown, which was occluded by a PAK inhibitor. Inhibiting cofilin, the primary downstream target of PAK and a major actin depolymerizing factor, prevented Shank3 siRNA from reducing NMDAR currents and F-actin clusters. Together, these results suggest that Shank3 deficiency induces NMDAR hypofunction by interfering with the Rac1/PAK/cofilin/actin signaling, leading to the loss of NMDAR membrane delivery or stability. It provides a potential mechanism for the role of Shank3 in cognitive deficit in autism.


Assuntos
Actinas/metabolismo , Córtex Cerebral/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Células Cultivadas , Córtex Cerebral/citologia , Células HEK293 , Humanos , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Interferência de RNA , RNA Interferente Pequeno , Ratos , Receptores de N-Metil-D-Aspartato/genética , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
19.
Hum Mol Genet ; 21(9): 2017-28, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22291444

RESUMO

Disrupted in Schizophrenia 1 (DISC1) is a key susceptibility gene implicated in major mental illnesses, such as schizophrenia, depression, bipolar disorder and autism, but the link between this protein and the pathology of these diseases remains unclear. Recently, DISC1 has been demonstrated to form insoluble protein aggregates in vitro and in human post-mortem brain tissue but the cellular dynamics of these DISC1 aggregates and their effects on neuronal function are unknown. Using a combination of biochemistry and live cell confocal and video microscopy, we characterize the properties of DISC1 aggregates and their effects on cellular function. We demonstrate that DISC1 protein aggregates are recruited to the aggresome and degraded there by the autophagic pathway. We show that there is a compromised exchange between DISC1 in aggresomes and the cytosolic DISC1 pool, and that the large DISC1 aggregates, which can also co-recruit endogenous soluble DISC1, exhibit altered trafficking. Moreover, we demonstrate that large DISC1 aggregates have a pathological effect in neurons by causing the disruption of intracellular transport of key organellar cargo, such as mitochondria. These data, therefore, show that DISC1 is recruited to aggresomes with negative effects on neuronal function, and suggests a novel DISC1-based mechanism for neuronal pathology.


Assuntos
Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Animais , Autofagia , Transporte Biológico Ativo , Encéfalo/metabolismo , Encéfalo/patologia , Células COS , Chlorocebus aethiops , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Espaço Intracelular/metabolismo , Microscopia de Vídeo , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Neurônios/patologia , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Esquizofrenia/genética , Esquizofrenia/metabolismo , Esquizofrenia/patologia , Solubilidade
20.
Biochem Soc Trans ; 42(5): 1302-10, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25233407

RESUMO

Astrocytes exhibit cellular excitability through variations in their intracellular calcium (Ca²âº) levels in response to synaptic activity. Astrocyte Ca²âº elevations can trigger the release of neuroactive substances that can modulate synaptic transmission and plasticity, hence promoting bidirectional communication with neurons. Intracellular Ca²âº dynamics can be regulated by several proteins located in the plasma membrane, within the cytosol and by intracellular organelles such as mitochondria. Spatial dynamics and strategic positioning of mitochondria are important for matching local energy provision and Ca²âº buffering requirements to the demands of neuronal signalling. Although relatively unresolved in astrocytes, further understanding the role of mitochondria in astrocytes may reveal more about the complex bidirectional relationship between astrocytes and neurons in health and disease. In the present review, we discuss some recent insights regarding mitochondrial function, transport and turnover in astrocytes and highlight some important questions that remain to be answered.


Assuntos
Astrócitos/metabolismo , Dinâmica Mitocondrial , Modelos Biológicos , Animais , Astrócitos/citologia , Astrócitos/patologia , Sinalização do Cálcio , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia
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