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1.
Neurobiol Dis ; 134: 104637, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31614197

RESUMO

Huntington's disease (HD) is caused by CAG repeat expansion within the HTT gene, with the dysfunction and eventual loss of striatal medium spiny neurons a notable feature. Since medium spiny neurons receive high amounts of synaptic input, we hypothesised that this vulnerability originates from an inability to sustain presynaptic performance during intense neuronal activity. To test this hypothesis, primary cultures of either hippocampal or striatal neurons were prepared from either wild-type mice or a knock-in HD mouse model which contains 140 poly-glutamine repeats in the huntingtin protein (httQ140/Q140). We identified a striatum-specific defect in synaptic vesicle (SV) endocytosis in httQ140/Q140 neurons that was only revealed during high frequency stimulation. This dysfunction was also present in neurons that were heterozygous for the mutant HTT allele. Depletion of endogenous huntingtin using hydrophobically-modified siRNA recapitulated this activity-dependent defect in wild-type neurons, whereas depletion of mutant huntingtin did not rescue the effect in httQ140/Q140 neurons. Importantly, this SV endocytosis defect was corrected by overexpression of wild-type huntingtin in homozygous httQ140/Q140 neurons. Therefore, we have identified an activity-dependent and striatum-specific signature of presynaptic dysfunction in neurons derived from pre-symptomatic HD mice, which is due to loss of wild-type huntingtin function. This presynaptic defect may render this specific neuronal subtype unable to operate efficiently during high frequency activity patterns, potentially resulting in dysfunctional neurotransmission, synapse failure and ultimately degeneration.

2.
J Glob Health ; 9(3): 020703, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31673352

RESUMO

Background: Approximately 250 million children under the age of five in low and middle-income countries (LMICs) will not achieve their developmental potential due to poverty and stunting alone. Investments in programming to improve early childhood development (ECD) have the potential to disrupt the cycle of poverty and therefore should be prioritised. Support for ECD has increased in recent years. Nevertheless, donors and policies continue to neglect ECD, in part from lack of evidence to guide policy makers and donors about where they should focus policies and programmes. Identification and investment in research is needed to overcome these constraints and in order to achieve high quality implementation of programmes to improve ECD. Methods: The Child Health and Nutrition Research Initiative (CHNRI) priority setting methodology was applied in order to assess research priorities for improving ECD. A group of 348 global and local experts in ECD-related research were identified and invited to generate research questions. This resulted in 406 research questions which were categorised and refined by study investigators into 54 research questions across six thematic goals which were evaluated using five criteria: answerability, effectiveness, feasibility, impact, and effect on equity. Research options were ranked by their final research priority score multiplied by 100. Results: The top three research priority options from the LMIC experts came from the third thematic goal of improving the impact of interventions, whereas the top three research priority options from high-income country experts came from different goals: improving the integration of interventions, increasing the understanding of health economics and social protection strategies, and improving the impact of interventions. Conclusion: The results of this process highlight that priorities for future research should focus on the need for services and support to parents to provide nurturing care, and the training of health workers and non-specialists in implementation of interventions to improve ECD. Three of the six thematic goals of the present priority setting centred on interventions (ie, improving impact, implementation of interventions and improving the integration of interventions). In order to achieve higher coverage through sustainable interventions to improve ECD with equitable reach, interventions should be integrated and not be sector driven.


Assuntos
Desenvolvimento Infantil , Saúde Global , Pesquisa , Pré-Escolar , Humanos , Lactente , Desenvolvimento Sustentável
3.
Pediatrics ; 144(2)2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31262779

RESUMO

CONTEXT: Although adolescent mental health interventions are widely implemented, little consensus exists about elements comprising successful models. OBJECTIVE: We aimed to identify effective program components of interventions to promote mental health and prevent mental disorders and risk behaviors during adolescence and to match these components across these key health outcomes to inform future multicomponent intervention development. DATA SOURCES: A total of 14 600 records were identified, and 158 studies were included. STUDY SELECTION: Studies included universally delivered psychosocial interventions administered to adolescents ages 10 to 19. We included studies published between 2000 and 2018, using PubMed, Medline, PsycINFO, Scopus, Embase, and Applied Social Sciences Index Abstracts databases. We included randomized controlled, cluster randomized controlled, factorial, and crossover trials. Outcomes included positive mental health, depressive and anxious symptomatology, violence perpetration and bullying, and alcohol and other substance use. DATA EXTRACTION: Data were extracted by 3 researchers who identified core components and relevant outcomes. Interventions were separated by modality; data were analyzed by using a robust variance estimation meta-analysis model, and we estimated a series of single-predictor meta-regression models using random effects. RESULTS: Universally delivered interventions can improve adolescent mental health and reduce risk behavior. Of 7 components with consistent signals of effectiveness, 3 had significant effects over multiple outcomes (interpersonal skills, emotional regulation, and alcohol and drug education). LIMITATIONS: Most included studies were from high-income settings, limiting the applicability of these findings to low- and middle-income countries. Our sample included only trials. CONCLUSIONS: Three program components emerged as consistently effective across different outcomes, providing a basis for developing future multioutcome intervention programs.


Assuntos
Comportamento do Adolescente/psicologia , Saúde Mental , Comportamento de Redução do Risco , Assunção de Riscos , Adolescente , Humanos , Avaliação de Programas e Projetos de Saúde/métodos , Ensaios Clínicos Controlados Aleatórios como Assunto/métodos
4.
J Neurochem ; 151(1): 28-37, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31216055

RESUMO

The two most abundant molecules on synaptic vesicles (SVs) are synaptophysin and synaptobrevin-II (sybII). SybII is essential for SV fusion, whereas synaptophysin is proposed to control the trafficking of sybII after SV fusion and its retrieval during endocytosis. Despite controlling key aspects of sybII packaging into SVs, the absence of synaptophysin results in negligible effects on neurotransmission. We hypothesised that this apparent absence of effect may be because of the abundance of sybII on SVs, with the impact of inefficient sybII retrieval only revealed during periods of repeated SV turnover. To test this hypothesis, we subjected primary cultures of synaptophysin knockout neurons to repeated trains of neuronal activity, while monitoring SV fusion events and levels of vesicular sybII. We identified a significant decrease in both the number of SV fusion events (monitored using the genetically encoded reporter vesicular glutamate transporter-pHluorin) and vesicular sybII levels (via both immunofluorescence and Western blotting) using this protocol. This revealed that synaptophysin is essential to sustain both parameters during periods of repetitive SV turnover. This was confirmed by the rescue of presynaptic performance by the expression of exogenous synaptophysin. Importantly, the expression of exogenous sybII also fully restored SV fusion events in synaptophysin knockout neurons. The ability of additional copies of sybII to fully rescue presynaptic performance in these knockout neurons suggests that the principal role of synaptophysin is to mediate the efficient retrieval of sybII to sustain neurotransmitter release.

5.
J Neurochem ; 150(1): 88-106, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30716176

RESUMO

Treatment with the dopamine (DA) precursor l-3,4-dihydroxyphenylalanine (l-DOPA) provides symptomatic relief arising from DA denervation in Parkinson's disease. Mounting evidence that DA autooxidation to neurotoxic quinones is involved in Parkinson's disease pathogenesis has raised concern about potentiation of oxidative stress by l-DOPA. The rate of DA quinone formation increases in the presence of excess redox-active iron (Fe), which is a pathological hallmark of Parkinson's disease. Conversely, l-DOPA has pH-dependent Fe-chelating properties, and may act to 'redox silence' Fe and partially allay DA autoxidation. We examined the effects of l-DOPA in three murine models of parkinsonian neurodegeneration: early-life Fe overexposure in wild-type mice, transgenic human (h)A53T mutant α-synuclein (α-syn) over-expression, and a combined 'multi-hit' model of Fe-overload in hA53T mice. We found that l-DOPA was neuroprotective and prevented age-related Fe accumulation in the substantia nigra pars compacta (SNc), similar to the mild-affinity Fe chelator clioquinol. Chronic l-DOPA treatment showed no evidence of increased oxidative stress in wild-type midbrain and normalized motor performance, when excess Fe was present. Similarly, l-DOPA also did not exacerbate protein oxidation levels in hA53T mice, with or without excess nigral Fe, and showed evidence of neuroprotection. The effects of l-DOPA in Fe-fed hA53T mice were somewhat muted, suggesting that Fe-chelation alone is insufficient to attenuate neuron loss in an animal model also recapitulating altered DA metabolism. In summary, we found no evidence in any of our model systems that l-DOPA treatment accentuated neurodegeneration, suggesting DA replacement therapy does not contribute to oxidative stress in the Parkinson's disease brain.

6.
Brain ; 141(9): 2576-2591, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30107533

RESUMO

Synaptotagmin 1 (SYT1) is a critical mediator of fast, synchronous, calcium-dependent neurotransmitter release and also modulates synaptic vesicle endocytosis. This paper describes 11 patients with de novo heterozygous missense mutations in SYT1. All mutations alter highly conserved residues, and cluster in two regions of the SYT1 C2B domain at positions Met303 (M303K), Asp304 (D304G), Asp366 (D366E), Ile368 (I368T) and Asn371 (N371K). Phenotypic features include infantile hypotonia, congenital ophthalmic abnormalities, childhood-onset hyperkinetic movement disorders, motor stereotypies, and developmental delay varying in severity from moderate to profound. Behavioural characteristics include sleep disturbance and episodic agitation. Absence of epileptic seizures and normal orbitofrontal head circumference are important negative features. Structural MRI is unremarkable but EEG disturbance is universal, characterized by intermittent low frequency high amplitude oscillations. The functional impact of these five de novo SYT1 mutations has been assessed by expressing rat SYT1 protein containing the equivalent human variants in wild-type mouse primary hippocampal cultures. All mutant forms of SYT1 were expressed at levels approximately equal to endogenous wild-type protein, and correctly localized to nerve terminals at rest, except for SYT1M303K, which was expressed at a lower level and failed to localize at nerve terminals. Following stimulation, SYT1I368T and SYT1N371K relocalized to nerve terminals at least as efficiently as wild-type SYT1. However, SYT1D304G and SYT1D366E failed to relocalize to nerve terminals following stimulation, indicative of impairments in endocytic retrieval and trafficking of SYT1. In addition, the presence of SYT1 variants at nerve terminals induced a slowing of exocytic rate following sustained action potential stimulation. The extent of disturbance to synaptic vesicle kinetics is mirrored by the severity of the affected individuals' phenotypes, suggesting that the efficiency of SYT1-mediated neurotransmitter release is critical to cognitive development. In summary, de novo dominant SYT1 missense mutations are associated with a recognizable neurodevelopmental syndrome, and further cases can now be diagnosed based on clinical features, electrophysiological signature and mutation characteristics. Variation in phenotype severity may reflect mutation-specific impact on the diverse physiological functions of SYT1.


Assuntos
Sinaptotagmina I/genética , Sinaptotagmina I/fisiologia , Potenciais de Ação , Adolescente , Animais , Cálcio/metabolismo , Criança , Pré-Escolar , Fenômenos Eletrofisiológicos , Endocitose , Feminino , Humanos , Deficiência Intelectual/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transtornos dos Movimentos/genética , Mutação de Sentido Incorreto/genética , Transtornos do Neurodesenvolvimento/metabolismo , Neurônios/metabolismo , Ratos , Transmissão Sináptica , Vesículas Sinápticas/genética , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/fisiologia , Adulto Jovem
7.
Methods Mol Biol ; 1847: 239-249, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30129022

RESUMO

This protocol utilizes lipophilic FM dyes to monitor membrane recycling in real time. FM dyes are virtually nonfluorescent in solution but when membrane bound are intensely fluorescent, combined with the flexibility of different emission wavelengths make these dyes an excellent choice for investigating clathrin-mediated endocytosis, among other membrane trafficking and recycling pathways.


Assuntos
Endocitose , Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Animais , Células Cultivadas , Clatrina/metabolismo , Neurônios/metabolismo
8.
Artigo em Inglês | MEDLINE | ID: mdl-26903854

RESUMO

The reformation of synaptic vesicles (SVs) during endocytosis is essential for the maintenance of neurotransmission in central nerve terminals. Newly formed SVs must be generated with the correct protein cargo in the correct stoichiometry to be functional for exocytosis. Classical clathrin adaptor protein complexes play a key role in sorting and clustering synaptic vesicle cargo in this regard. However it is becoming increasingly apparent that additional "fail-safe" mechanisms exist to ensure the accurate retrieval of essential cargo molecules. For example, the monomeric adaptor proteins AP180/CALM and stonin-2 are required for the efficient retrieval of synaptobrevin II (sybII) and synaptotagmin-1 respectively. Furthermore, recent studies have revealed that sybII and synaptotagmin-1 interact with other SV cargoes to ensure a high fidelity of retrieval. These cargoes are synaptophysin (for sybII) and SV2A (for synaptotagmin-1). In this review, we summarize current knowledge regarding the retrieval mechanisms for both sybII and synaptotagmin-1 during endocytosis. We also define and set criteria for a new functional group of SV molecules that facilitate the retrieval of their interaction partners. We have termed these molecules intrinsic trafficking partners (iTRAPs) and we discuss how the function of this group impacts on presynaptic performance in both health and disease.

9.
PLoS One ; 11(2): e0149457, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26871701

RESUMO

Synaptobrevin II (sybII) is a vesicular soluble NSF attachment protein receptor (SNARE) protein that is essential for neurotransmitter release, and thus its correct trafficking to synaptic vesicles (SVs) is critical to render them fusion competent. The SV protein synaptophysin binds to sybII and facilitates its retrieval to SVs during endocytosis. Synaptophysin and sybII are the two most abundant proteins on SVs, being present in a 1:2 ratio. Synaptophysin and sybII are proposed to form a large multimeric complex, and the copy number of the proteins in this complex is also in a 1:2 ratio. We investigated the importance of this ratio between these proteins for the localisation and trafficking of sybII in central neurons. SybII was overexpressed in mouse hippocampal neurons at either 1.6 or 2.15-2.35-fold over endogenous protein levels, in the absence or presence of varying levels of synaptophysin. In the absence of exogenous synaptophysin, exogenous sybII was dispersed along the axon, trapped on the plasma membrane and retrieved slowly during endocytosis. Co-expression of exogenous synaptophysin rescued all of these defects. Importantly, the expression of synaptophysin at nerve terminals in a 1:2 ratio with sybII was sufficient to fully rescue normal sybII trafficking. These results demonstrate that the balance between synaptophysin and sybII levels is critical for the correct targeting of sybII to SVs and suggests that small alterations in synaptophysin levels might affect the localisation of sybII and subsequent presynaptic performance.


Assuntos
Hipocampo/citologia , Neurônios/citologia , Vesículas Sinápticas/metabolismo , Sinaptofisina/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Animais , Células Cultivadas , Endocitose , Feminino , Hipocampo/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Sinaptofisina/análise , Proteína 2 Associada à Membrana da Vesícula/análise
10.
J Neurosci ; 35(6): 2492-507, 2015 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-25673844

RESUMO

Synaptic vesicle protein 2A (SV2A) is a ubiquitous component of synaptic vesicles (SVs). It has roles in both SV trafficking and neurotransmitter release. We demonstrate that Casein kinase 1 family members, including isoforms of Tau-tubulin protein kinases (TTBK1 and TTBK2), phosphorylate human SV2A at two constellations of residues, namely Cluster-1 (Ser42, Ser45, and Ser47) and Cluster-2 (Ser80, Ser81, and Thr84). These residues are also phosphorylated in vivo, and the phosphorylation of Thr84 within Cluster-2 is essential for triggering binding to the C2B domain of human synaptotagmin-1. We show by crystallographic and other analyses that the phosphorylated Thr84 residue binds to a pocket formed by three conserved Lys residues (Lys314, Lys326, and Lys328) on the surface of the synaptotagmin-1 C2B domain. Finally, we observed dysfunctional synaptotagmin-1 retrieval during SV endocytosis by ablating its phospho-dependent interaction with SV2A, knockdown of SV2A, or rescue with a phosphorylation-null Thr84 SV2A mutant in primary cultures of mouse neurons. This study reveals fundamental details of how phosphorylation of Thr84 on SV2A controls its interaction with synaptotagmin-1 and implicates SV2A as a phospho-dependent chaperone required for the specific retrieval of synaptotagmin-1 during SV endocytosis.


Assuntos
Caseína Quinase I/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sinaptotagmina I/metabolismo , Sequência de Aminoácidos , Animais , Química Encefálica/genética , Células Cultivadas , Endocitose/fisiologia , Células HEK293 , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Neurônios/metabolismo , Fosforilação , Sinaptotagmina I/química , Sinaptotagmina I/isolamento & purificação
11.
J Clin Invest ; 125(4): 1670-8, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25705886

RESUMO

Synaptotagmin-1 (SYT1) is a calcium-binding synaptic vesicle protein that is required for both exocytosis and endocytosis. Here, we describe a human condition associated with a rare variant in SYT1. The individual harboring this variant presented with an early onset dyskinetic movement disorder, severe motor delay, and profound cognitive impairment. Structural MRI was normal, but EEG showed extensive neurophysiological disturbances that included the unusual features of low-frequency oscillatory bursts and enhanced paired-pulse depression of visual evoked potentials. Trio analysis of whole-exome sequence identified a de novo SYT1 missense variant (I368T). Expression of rat SYT1 containing the equivalent human variant in WT mouse primary hippocampal cultures revealed that the mutant form of SYT1 correctly localizes to nerve terminals and is expressed at levels that are approximately equal to levels of endogenous WT protein. The presence of the mutant SYT1 slowed synaptic vesicle fusion kinetics, a finding that agrees with the previously demonstrated role for I368 in calcium-dependent membrane penetration. Expression of the I368T variant also altered the kinetics of synaptic vesicle endocytosis. Together, the clinical features, electrophysiological phenotype, and in vitro neuronal phenotype associated with this dominant negative SYT1 mutation highlight presynaptic mechanisms that mediate human motor control and cognitive development.


Assuntos
Deficiência Intelectual/genética , Transtornos das Habilidades Motoras/genética , Transtornos dos Movimentos/genética , Mutação de Sentido Incorreto , Mutação Puntual , Terminações Pré-Sinápticas/fisiologia , Vesículas Sinápticas/fisiologia , Sinaptotagmina I/fisiologia , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Células Cultivadas , Criança , Endocitose/genética , Endocitose/fisiologia , Potenciais Evocados Visuais , Exocitose/genética , Exocitose/fisiologia , Genes Dominantes , Hipocampo/citologia , Humanos , Cinética , Masculino , Fusão de Membrana , Camundongos , Dados de Sequência Molecular , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sinaptotagmina I/genética
12.
Dis Model Mech ; 7(6): 711-22, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24764192

RESUMO

Mutations in RAB18 have been shown to cause the heterogeneous autosomal recessive disorder Warburg Micro syndrome (WARBM). Individuals with WARBM present with a range of clinical symptoms, including ocular and neurological abnormalities. However, the underlying cellular and molecular pathogenesis of the disorder remains unclear, largely owing to the lack of any robust animal models that phenocopy both the ocular and neurological features of the disease. We report here the generation and characterisation of a novel Rab18-mutant mouse model of WARBM. Rab18-mutant mice are viable and fertile. They present with congenital nuclear cataracts and atonic pupils, recapitulating the characteristic ocular features that are associated with WARBM. Additionally, Rab18-mutant cells exhibit an increase in lipid droplet size following treatment with oleic acid. Lipid droplet abnormalities are a characteristic feature of cells taken from WARBM individuals, as well as cells taken from individuals with other neurodegenerative conditions. Neurological dysfunction is also apparent in Rab18-mutant mice, including progressive weakness of the hind limbs. We show that the neurological defects are, most likely, not caused by gross perturbations in synaptic vesicle recycling in the central or peripheral nervous system. Rather, loss of Rab18 is associated with widespread disruption of the neuronal cytoskeleton, including abnormal accumulations of neurofilament and microtubule proteins in synaptic terminals, and gross disorganisation of the cytoskeleton in peripheral nerves. Global proteomic profiling of peripheral nerves in Rab18-mutant mice reveals significant alterations in several core molecular pathways that regulate cytoskeletal dynamics in neurons. The apparent similarities between the WARBM phenotype and the phenotype that we describe here indicate that the Rab18-mutant mouse provides an important platform for investigation of the disease pathogenesis and therapeutic interventions.


Assuntos
Anormalidades Múltiplas/fisiopatologia , Catarata/congênito , Córnea/anormalidades , Citoesqueleto/fisiologia , Modelos Animais de Doenças , Olho/crescimento & desenvolvimento , Hipogonadismo/fisiopatologia , Deficiência Intelectual/fisiopatologia , Microcefalia/fisiopatologia , Neurônios/fisiologia , Atrofia Óptica/fisiopatologia , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Catarata/fisiopatologia , Córnea/fisiopatologia , Camundongos , Camundongos Knockout , Proteínas rab de Ligação ao GTP/genética
13.
Traffic ; 15(3): 245-54, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24279465

RESUMO

Synaptobrevin II (sybII) is a key fusogenic molecule on synaptic vesicles (SVs) therefore the active maintenance of both its conformation and location in sufficient numbers on this organelle is critical in both mediating and sustaining neurotransmitter release. Recently three proteins have been identified having key roles in the presentation, trafficking and retrieval of sybII during the fusion and endocytosis of SVs. The nerve terminal protein α-synuclein catalyses sybII entry into SNARE complexes, whereas the monomeric adaptor protein AP-180 is required for sybII retrieval during SV endocytosis. Overarching these events is the tetraspan SV protein synaptophysin, which is a major sybII interaction partner on the SV. This review will evaluate recent studies to propose working models for the control of sybII traffic by synaptophysin and other Sybtraps (sybII trafficking partners) and suggest how dysfunction in sybII traffic may contribute to human disease.


Assuntos
Proteínas Monoméricas de Montagem de Clatrina/metabolismo , Proteínas R-SNARE/metabolismo , Sinaptofisina/metabolismo , alfa-Sinucleína/metabolismo , Animais , Humanos , Ligação Proteica , Transporte Proteico
14.
J Neurosci ; 33(34): 13695-700, 2013 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-23966691

RESUMO

Synaptophysin is an integral synaptic vesicle (SV) protein that accounts for ∼10% of total SV protein cargo. Deletion of synaptophysin results in the defective retrieval of synaptobrevin II (sybII) from the plasma membrane during endocytosis, coupled with a slowing in the speed of endocytosis. Synaptophysin has been implicated in X-linked intellectual disability, with a recent study identifying four separate synaptophysin gene mutations in families affected by the disorder. To determine how these mutations may affect synaptophysin function, we expressed them in cultured neurons derived from synaptophysin knock-out mice. Two distinct truncating mutants were mislocalized throughout the axon and phenocopied the arrest of sybII retrieval in synaptophysin knock-out cultures. The remaining two mutants displayed a nerve terminal localization but did not support efficient sybII retrieval. Interestingly, one mutant fully rescued SV endocytosis kinetics, suggesting that sybII retrieval and endocytosis speed are independent from each other. These studies suggest that the efficient retrieval of sybII by synaptophysin may be key to maintaining synaptic health and perturbation of this event may contribute to the pathogenesis underlying neurodevelopmental disorders such as X-linked intellectual disability.


Assuntos
Neurônios/metabolismo , Mutação Puntual/genética , Sinaptofisina/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Potenciais de Ação/genética , Animais , Axônios/fisiologia , Células Cultivadas , Córtex Cerebral/citologia , Embrião de Mamíferos , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Técnicas de Patch-Clamp , Sinaptofisina/deficiência , Transfecção , Vesículas Transportadoras/genética , Vesículas Transportadoras/metabolismo , Proteínas Vesiculares de Transporte de Glutamato/metabolismo
15.
J Neurosci ; 31(39): 14032-6, 2011 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-21957264

RESUMO

The integral synaptic vesicle (SV) protein synaptophysin forms ∼10% of total SV protein content, but has no known function in SV physiology. Synaptobrevin (sybII) is another abundant integral SV protein with an essential role in SV exocytosis. Synaptophysin and sybII form a complex in nerve terminals, suggesting this interaction may have a key role in presynaptic function. To determine how synaptophysin controls sybII traffic in nerve terminals, we used a combination of optical imaging techniques in cultures derived from synaptophysin knock-out mice. We show that synaptophysin is specifically required for the retrieval of the pH-sensitive fluorescent reporter sybII-pHluorin from the plasma membrane during endocytosis. The retrieval of other SV protein cargo reporters still occurred; however, their recapture proceeded with slower kinetics. This slowing of SV retrieval kinetics in the absence of synaptophysin did not impact on global SV turnover. These results identify a specific and selective requirement for synaptophysin in the retrieval of sybII during SV endocytosis and suggest that their interaction may act as an adjustable regulator of SV retrieval efficiency.


Assuntos
Endocitose/fisiologia , Vesículas Sinápticas/metabolismo , Sinaptofisina/fisiologia , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Potenciais de Ação/genética , Potenciais de Ação/fisiologia , Animais , Membrana Celular/genética , Membrana Celular/fisiologia , Células Cultivadas , Endocitose/genética , Feminino , Genes Reporter , Masculino , Camundongos , Camundongos Knockout , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/fisiologia , Transporte Proteico/genética
16.
Biochemistry ; 50(9): 1545-55, 2011 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-21235236

RESUMO

Tyrosine hydroxylase (TH) performs the first and rate-limiting step in the synthesis of catecholamines, which feed back to regulate the enzyme by irreversibly binding to a high-affinity site and inhibiting TH activity. Phosphorylation of Ser40 relieves this inhibition by allowing dissociation of catecholamine. We have recently documented the existence of a low-affinity catecholamine binding which is dissociable, is not abolished by phosphorylation, and inhibits TH by competing with the essential cofactor, tetrahydrobiopterin. Here, we have substituted a number of active site residues to determine the structural nature of the low- and high-affinity sites. E332D and Y371F increased the IC(50) of dopamine for the low-affinity site 10-fold and 7 0-fold, respectively, in phosphorylated TH, indicating dramatic reductions in affinity. Only 2-4-fold increases in IC(50) were measured in the nonphosphorylated forms of E332D and Y371F and also in L294A and F300Y. This suggests that while the magnitude of low-affinity site inhibition in wild-type TH remains the same upon TH phosphorylation as previously shown, the active site structure changes to place greater importance on E332 and Y371. Changes to high affinity binding were also measured, including a loss of competition with tetrahydrobiopterin for E332D, A297L, and Y371F and a decreased ability to inhibit catalysis (V(max)) for A297L and Y371F. The common roles of E332 and Y371 indicate that the low- and high-affinity catecholamine binding sites are colocalized in the active site, but due to simultaneous binding, may exist in separate monomers of the TH tetramer.


Assuntos
Catecolaminas/metabolismo , Análise Mutacional de DNA , Tirosina 3-Mono-Oxigenase/metabolismo , Domínio Catalítico , Catecolaminas/química , Catecolaminas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Enzimológica da Expressão Gênica , Ligação Proteica , Tirosina 3-Mono-Oxigenase/química , Tirosina 3-Mono-Oxigenase/genética
17.
Toxicol Sci ; 113(1): 150-7, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19815691

RESUMO

In this study, we demonstrate that human neuroblastoma SH-SY5Y cells transfected with human tyrosine hydroxylase isoform 1 (SH + TH cells) were substantially more resistant to cell death induced by hydrogen peroxide and 6-hydroxydopamine when compared to wild-type SH-SY5Y cells (SH cells). SH + TH cells exhibit increased levels of dopamine (DA) compared to SH cells. Incubation with hydrogen peroxide or 6-hydroxydopamine (10-100microM) for 24 h caused a significant reduction in cell viability and increased apoptosis in both cell types. However, these effects were significantly reduced in the SH + TH cells when compared to the SH cells. The SH + TH cells showed an improved ability to detoxify peroxide, which correlated with an increase in glutathione peroxidase and glutathione reductase activities, while catalase activity was unchanged. Our data suggest that a preconditioning-like mechanism linked to higher DA levels increased the resistance of SH + TH cells against oxidative insults, which is at least in part related to an augmentation in the activity of glutathione-related antioxidant enzymes.


Assuntos
Apoptose , Dopamina/metabolismo , Neuroblastoma/enzimologia , Estresse Oxidativo , Tirosina 3-Mono-Oxigenase/metabolismo , Apoptose/efeitos dos fármacos , Carmustina/farmacologia , Catalase/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Glutationa Peroxidase/antagonistas & inibidores , Glutationa Peroxidase/metabolismo , Glutationa Redutase/antagonistas & inibidores , Glutationa Redutase/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Neuroblastoma/genética , Neuroblastoma/patologia , Estresse Oxidativo/efeitos dos fármacos , Oxidopamina/toxicidade , Tiomalatos/farmacologia , Transfecção , Tirosina 3-Mono-Oxigenase/genética
18.
Biochim Biophys Acta ; 1793(12): 1860-7, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19833152

RESUMO

The major human tyrosine hydroxylase isoforms (hTH1 and 2) differ in their ability to be phosphorylated in vitro. hTH1 is phosphorylated at Ser31 by extracellular signal-regulated kinase (ERK). This kinase is not capable of phosphorylating hTH2 at Ser35 (the residue that corresponds to Ser31 in hTH1). We have stably transfected SH-SY5Y cells with hTH1 or hTH2 to determine if hTH2 can be phosphorylated at Ser35 in situ. Forskolin increased the phosphorylation of Ser40 in hTH1 and Ser44 in hTH2. Muscarine increased the phosphorylation of both Ser19 and Ser40/44 in both hTH1 and hTH2. EGF increased the phosphorylation of Ser31 in hTH1. Phosphorylation of Ser35 in hTH2 was not detected under any of the conditions tested. Inhibition of ERK by UO126 decreased the phosphorylation of Ser31 and this lead to a 50% decrease in the basal level of phosphorylation of Ser40 in hTH1. The basal level of Ser44 phosphorylation in hTH2 was not altered by treatment with UO126. Therefore, phosphorylation of Ser31 contributes to the phosphorylation of Ser40 in hTH1 in situ; however, this effect is absent in hTH2. This represents a major difference between the two human TH isoforms, and has implications for the regulation of catecholamine synthesis in vivo.


Assuntos
Serina/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Butadienos/farmacologia , Catecolaminas/biossíntese , Catecolaminas/genética , Linhagem Celular , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Nitrilos/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Serina/genética , Tirosina 3-Mono-Oxigenase/genética
19.
Neurochem Res ; 34(10): 1830-7, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19448984

RESUMO

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is inhibited in vitro by catecholamines binding to two distinct sites on the enzyme. The N-terminal regulatory domain of TH contributes to dopamine binding to the high affinity site of the enzyme. We prepared an N-terminal deletion mutant of TH to examine the role of the N-terminal domain in dopamine binding to the low affinity site. Deletion of the N-terminus of TH removes the high affinity dopamine binding site, but does not affect dopamine binding to the low affinity site. The role of the low affinity site in situ was examined by incubating PC12 cells with L-DOPA to increase the cytosolic catecholamine concentration. This resulted in an inhibition of TH activity in situ under both basal conditions and conditions that promoted the phosphorylation of Ser40. Therefore the low affinity site is active in situ regardless of the phosphorylation status of Ser40.


Assuntos
Dopamina/química , Dopamina/metabolismo , Tirosina 3-Mono-Oxigenase/química , Tirosina 3-Mono-Oxigenase/metabolismo , Animais , Sítios de Ligação/genética , Ativação Enzimática/genética , Células PC12 , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação/genética , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Ratos , Deleção de Sequência , Serina/genética , Serina/metabolismo , Tirosina 3-Mono-Oxigenase/genética
20.
J Neurochem ; 106(4): 1614-23, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18513370

RESUMO

Tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of the catecholamines dopamine, noradrenaline and adrenaline, is regulated acutely by feedback inhibition by the catecholamines and relief of this inhibition by phosphorylation of serine 40 (Ser40). Phosphorylation of serine 40 abolishes the binding of dopamine to a high affinity (K(D) < 4 nM) site on TH, thereby increasing the activity of the enzyme. We have found that TH also contains a second low affinity (K(D) = 90 nM) dopamine-binding site, which is present in both the non-phosphorylated and the Ser40-phosphorylated forms of the enzyme. Binding of dopamine to the high-affinity site decreases V(max) and increases the K(m) for the cofactor tetrahydrobiopterin, while binding of dopamine to the low-affinity site regulates TH activity by increasing the K(m) for tetrahydrobiopterin. Kinetic analysis indicates that both sites are present in each of the four human TH isoforms. Dissociation of dopamine from the low-affinity site increases TH activity 12-fold for the non-phosphorylated enzyme and 9-fold for the Ser40-phosphorylated enzyme. The low-affinity dopamine-binding site has the potential to be the primary mechanism responsible for the regulation of catecholamine synthesis under most conditions.


Assuntos
Dopamina/fisiologia , Tirosina 3-Mono-Oxigenase/metabolismo , Animais , Sítios de Ligação/fisiologia , Catecolaminas/biossíntese , Dopamina/metabolismo , Ativação Enzimática/fisiologia , Humanos , Fosforilação , Ratos , Serina/metabolismo
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