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1.
Hum Mol Genet ; 28(18): 3126-3136, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31261385

RESUMO

Pyridox (am) ine 5'-phosphate oxidase (PNPO) is a rate-limiting enzyme in converting dietary vitamin B6 (VB6) to pyridoxal 5'-phosphate (PLP), the biologically active form of VB6 and involved in the synthesis of neurotransmitters including γ-aminobutyric acid (GABA), dopamine, and serotonin. In humans, PNPO mutations have been increasingly identified in neonatal epileptic encephalopathy and more recently also in early-onset epilepsy. Till now, little is known about the neurobiological mechanisms underlying PNPO-deficiency-induced seizures due to the lack of animal models. Previously, we identified a c.95 C>A missense mutation in sugarlethal (sgll)-the Drosophila homolog of human PNPO (hPNPO)-and found mutant (sgll95) flies exhibiting a lethal phenotype on a diet devoid of VB6. Here, we report the establishment of both sgll95 and ubiquitous sgll knockdown (KD) flies as valid animal models of PNPO-deficiency-induced epilepsy. Both sgll95 and sgll KD flies exhibit spontaneous seizures before they die. Electrophysiological recordings reveal that seizures caused by PNPO deficiency have characteristics similar to that in flies treated with the GABA antagonist picrotoxin. Both seizures and lethality are associated with low PLP levels and can be rescued by ubiquitous expression of wild-type sgll or hPNPO, suggesting the functional conservation of the PNPO enzyme between humans and flies. Results from cell type-specific sgll KD further demonstrate that PNPO in the brain is necessary for seizure prevention and survival. Our establishment of the first animal model of PNPO deficiency will lead to better understanding of VB6 biology, the PNPO gene and its mutations discovered in patients, and can be a cost-effective system to test therapeutic strategies.

2.
Nature ; 563(7730): 249-253, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30401835

RESUMO

N6-methyladenosine (m6A), the most prevalent internal RNA modification on mammalian messenger RNAs, regulates the fates and functions of modified transcripts through m6A-specific binding proteins1-5. In the nervous system, m6A is abundant and modulates various neural functions6-11. Whereas m6A marks groups of mRNAs for coordinated degradation in various physiological processes12-15, the relevance of m6A for mRNA translation in vivo remains largely unknown. Here we show that, through its binding protein YTHDF1, m6A promotes protein translation of target transcripts in response to neuronal stimuli in the adult mouse hippocampus, thereby facilitating learning and memory. Mice with genetic deletion of Ythdf1 show learning and memory defects as well as impaired hippocampal synaptic transmission and long-term potentiation. Re-expression of YTHDF1 in the hippocampus of adult Ythdf1-knockout mice rescues the behavioural and synaptic defects, whereas hippocampus-specific acute knockdown of Ythdf1 or Mettl3, which encodes the catalytic component of the m6A methyltransferase complex, recapitulates the hippocampal deficiency. Transcriptome-wide mapping of YTHDF1-binding sites and m6A sites on hippocampal mRNAs identified key neuronal genes. Nascent protein labelling and tether reporter assays in hippocampal neurons showed that YTHDF1 enhances protein synthesis in a neuronal-stimulus-dependent manner. In summary, YTHDF1 facilitates translation of m6A-methylated neuronal mRNAs in response to neuronal stimulation, and this process contributes to learning and memory.

3.
Neuron ; 99(2): 283-292.e5, 2018 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-30056831

RESUMO

N6-methyladenosine (m6A) regulates mRNA metabolism and translation, serving as an important source of post-transcriptional regulation. To date, the functional consequences of m6A deficiency within the adult brain have not been determined. To achieve m6A deficiency, we deleted Mettl14, an essential component of the m6A methyltransferase complex, in two related yet discrete mouse neuronal populations: striatonigral and striatopallidal. Mettl14 deletion reduced striatal m6A levels without altering cell numbers or morphology. Transcriptome-wide profiling of m6A-modified mRNAs in Mettl14-deleted striatum revealed downregulation of similar striatal mRNAs encoding neuron- and synapse-specific proteins in both neuronal types, but striatonigral and striatopallidal identity genes were uniquely downregulated in each respective manipulation. Upregulated mRNA species encoded non-neuron-specific proteins. These changes increased neuronal excitability, reduced spike frequency adaptation, and profoundly impaired striatal-mediated behaviors. Using viral-mediated, neuron-specific striatal Mettl14 deletion in adult mice, we further confirmed the significance of m6A in maintaining normal striatal function in the adult mouse.

4.
Neuron ; 97(2): 313-325.e6, 2018 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-29346752

RESUMO

N6-methyladenosine (m6A) affects multiple aspects of mRNA metabolism and regulates developmental transitions by promoting mRNA decay. Little is known about the role of m6A in the adult mammalian nervous system. Here we report that sciatic nerve lesion elevates levels of m6A-tagged transcripts encoding many regeneration-associated genes and protein translation machinery components in the adult mouse dorsal root ganglion (DRG). Single-base resolution m6A-CLIP mapping further reveals a dynamic m6A landscape in the adult DRG upon injury. Loss of either m6A methyltransferase complex component Mettl14 or m6A-binding protein Ythdf1 globally attenuates injury-induced protein translation in adult DRGs and reduces functional axon regeneration in the peripheral nervous system in vivo. Furthermore, Pten deletion-induced axon regeneration of retinal ganglion neurons in the adult central nervous system is attenuated upon Mettl14 knockdown. Our study reveals a critical epitranscriptomic mechanism in promoting injury-induced protein synthesis and axon regeneration in the adult mammalian nervous system.

5.
Cell ; 171(4): 877-889.e17, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-28965759

RESUMO

N6-methyladenosine (m6A), installed by the Mettl3/Mettl14 methyltransferase complex, is the most prevalent internal mRNA modification. Whether m6A regulates mammalian brain development is unknown. Here, we show that m6A depletion by Mettl14 knockout in embryonic mouse brains prolongs the cell cycle of radial glia cells and extends cortical neurogenesis into postnatal stages. m6A depletion by Mettl3 knockdown also leads to a prolonged cell cycle and maintenance of radial glia cells. m6A sequencing of embryonic mouse cortex reveals enrichment of mRNAs related to transcription factors, neurogenesis, the cell cycle, and neuronal differentiation, and m6A tagging promotes their decay. Further analysis uncovers previously unappreciated transcriptional prepatterning in cortical neural stem cells. m6A signaling also regulates human cortical neurogenesis in forebrain organoids. Comparison of m6A-mRNA landscapes between mouse and human cortical neurogenesis reveals enrichment of human-specific m6A tagging of transcripts related to brain-disorder risk genes. Our study identifies an epitranscriptomic mechanism in heightened transcriptional coordination during mammalian cortical neurogenesis.


Assuntos
Neurogênese , Prosencéfalo/embriologia , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Animais , Ciclo Celular , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Humanos , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Camundongos Knockout , Células-Tronco Neurais/metabolismo , Organoides/metabolismo , Prosencéfalo/citologia , Prosencéfalo/metabolismo , Estabilidade de RNA
6.
Science ; 357(6357): 1255-1261, 2017 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-28882997

RESUMO

Mitochondrial and lysosomal dysfunction have been implicated in substantia nigra dopaminergic neurodegeneration in Parkinson's disease (PD), but how these pathways are linked in human neurons remains unclear. Here we studied dopaminergic neurons derived from patients with idiopathic and familial PD. We identified a time-dependent pathological cascade beginning with mitochondrial oxidant stress leading to oxidized dopamine accumulation and ultimately resulting in reduced glucocerebrosidase enzymatic activity, lysosomal dysfunction, and α-synuclein accumulation. This toxic cascade was observed in human, but not in mouse, PD neurons at least in part because of species-specific differences in dopamine metabolism. Increasing dopamine synthesis or α-synuclein amounts in mouse midbrain neurons recapitulated pathological phenotypes observed in human neurons. Thus, dopamine oxidation represents an important link between mitochondrial and lysosomal dysfunction in PD pathogenesis.


Assuntos
Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo , Doença de Parkinson/metabolismo , Animais , Antioxidantes/farmacologia , Inibidores de Calcineurina/farmacologia , Linhagem Celular , Modelos Animais de Doenças , Glucosilceramidase/deficiência , Humanos , Melaninas/metabolismo , Mesencéfalo/enzimologia , Mesencéfalo/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Proteína Desglicase DJ-1/genética , Substância Negra/enzimologia , Substância Negra/metabolismo , Tacrolimo/farmacologia , alfa-Sinucleína/metabolismo
7.
Genome Biol Evol ; 9(5): 1357-1369, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28472322

RESUMO

Foraging behavior is critical for the fitness of individuals. However, the genetic basis of variation in foraging behavior and the evolutionary forces underlying such natural variation have rarely been investigated. We developed a systematic approach to assay the variation in survival rate in a foraging environment for adult flies derived from a wild Drosophila melanogaster population. Despite being such an essential trait, there is substantial variation of foraging behavior among D. melanogaster strains. Importantly, we provided the first evaluation of the potential caveats of using inbred Drosophila strains to perform genome-wide association studies on life-history traits, and concluded that inbreeding depression is unlikely a major contributor for the observed large variation in adult foraging behavior. We found that adult foraging behavior has a strong genetic component and, unlike larval foraging behavior, depends on multiple loci. Identified candidate genes are enriched in those with high expression in adult heads and, demonstrated by expression knock down assay, are involved in maintaining normal functions of the nervous system. Our study not only identified candidate genes for foraging behavior that is relevant to individual fitness, but also shed light on the initial stage underlying the evolution of the behavior.


Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Comportamento Alimentar , Genes de Insetos , Variação Genética , Animais , Comportamento Animal , Encéfalo/metabolismo , Drosophila melanogaster/fisiologia , Estudos de Associação Genética , Genoma de Inseto , Estudo de Associação Genômica Ampla , Depressão por Endogamia , Locos de Características Quantitativas , Taxa de Sobrevida
8.
J Neurosci ; 36(19): 5228-40, 2016 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-27170121

RESUMO

UNLABELLED: Although dopamine receptor antagonism has long been associated with impairments in motor performance, more recent studies have shown that dopamine D2 receptor (D2R) antagonism, paired with a motor task, not only impairs motor performance concomitant with the pharmacodynamics of the drug, but also impairs future motor performance once antagonism has been relieved. We have termed this phenomenon "aberrant motor learning" and have suggested that it may contribute to motor symptoms in movement disorders such as Parkinson's disease (PD). Here, we show that chronic nicotine (cNIC), but not acute nicotine, treatment mitigates the acquisition of D2R-antagonist-induced aberrant motor learning in mice. Although cNIC mitigates D2R-mediated aberrant motor learning, cNIC has no effect on D1R-mediated motor learning. ß2-containing nicotinic receptors in dopamine neurons likely mediate the protective effect of cNIC against aberrant motor learning, because selective deletion of ß2 nicotinic subunits in dopamine neurons reduced D2R-mediated aberrant motor learning. Finally, both cNIC treatment and ß2 subunit deletion blunted postsynaptic responses to D2R antagonism. These results suggest that a chronic decrease in function or a downregulation of ß2-containing nicotinic receptors protects the striatal network against aberrant plasticity and aberrant motor learning induced by motor experience under dopamine deficiency. SIGNIFICANCE STATEMENT: Increasingly, aberrant plasticity and aberrant learning are recognized as contributing to the development and progression of movement disorders. Here, we show that chronic nicotine (cNIC) treatment or specific deletion of ß2 nicotinic receptor subunits in dopamine neurons mitigates aberrant motor learning induced by dopamine D2 receptor (D2R) blockade in mice. Moreover, both manipulations also reduced striatal dopamine release and blunt postsynaptic responses to D2R antagonists. These results suggest that chronic downregulation of function and/or receptor expression of ß2-containing nicotinic receptors alters presynaptic and postsynaptic striatal signaling to protect against aberrant motor learning. Moreover, these results suggest that cNIC treatment may alleviate motor symptoms and/or delay the deterioration of motor function in movement disorders by blocking aberrant motor learning.


Assuntos
Dopamina/deficiência , Aprendizagem/efeitos dos fármacos , Atividade Motora , Nicotina/farmacologia , Receptores de Dopamina D2/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nicotina/administração & dosagem , Agonistas Nicotínicos/farmacologia , Potenciais Sinápticos
9.
ACS Nano ; 10(3): 3486-95, 2016 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-26950644

RESUMO

Photodynamic therapy (PDT) offers an alternative for cancer treatment by using ultraviolet or visible light in the presence of a photosensitizer and molecular oxygen, which can produce highly reactive oxygen species that ultimately leading to the ablation of tumor cells by multifactorial mechanisms. However, this technique is limited by the penetration depth of incident light, the hypoxic environment of solid tumors, and the vulnerability of photobleaching reduces the efficiency of many imaging agents. In this work, we reported a cellular level dual-functional imaging and PDT nanosystem BMEPC-loaded DNA origami for photodynamic therapy with high efficiency and stable photoreactive property. The carbazole derivative BMEPC is a one- and two-photon imaging agent and photosensitizer with large two-photon absorption cross section, which can be fully excited by near-infrared light, and is also capable of destroying targets under anaerobic condition by generating reactive intermediates of Type I photodynamic reactions. However, the application of BMEPC was restricted by its poor solubility in aqueous environment and its aggregation caused quenching. We observed BMEPC-loaded DNA origami effectively reduced the photobleaching of BMEPC within cells. Upon binding to DNA origami, the intramolecular rotation of BMEPC became proper restricted, which intensify fluorescence emission and radicals production when being excited. After the BMEPC-loaded DNA origami are taken up by tumor cells, upon irradiation, BMEPC could generate free radicals and be released due to DNA photocleavage as well as the following partially degradation. Apoptosis was then induced by the generation of free radicals. This functional nanosystem provides an insight into the design of photosensitizer-loaded DNA origami for effective intracellular imaging and photodynamic therapy.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Mama/efeitos dos fármacos , Carbazóis/administração & dosagem , DNA/química , Portadores de Fármacos/química , Nanoestruturas/química , Fármacos Fotossensibilizantes/administração & dosagem , Mama/patologia , Neoplasias da Mama/patologia , Carbazóis/uso terapêutico , Feminino , Humanos , Células MCF-7 , Modelos Moleculares , Fotodegradação , Fotoquimioterapia , Fármacos Fotossensibilizantes/uso terapêutico
10.
Biol Psychiatry ; 79(11): 887-97, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-26281715

RESUMO

BACKGROUND: The dopamine D2 receptor (D2R) has received much attention in obesity studies. Data indicate that D2R is reduced in obesity and that the TaqA1 D2R variant may be more prevalent among obese persons. It is often suggested that reduced D2R generates a reward deficiency and altered appetitive motivation that induces compulsive eating and contributes to obesity. Although dopamine is known to regulate physical activity, it is often neglected in these studies, leaving open the question of whether reduced D2R contributes to obesity through alterations in energy expenditure and activity. METHODS: We generated a D2R knockdown (KD) mouse line and assessed both energy expenditure and appetitive motivation under conditions of diet-induced obesity. RESULTS: The KD mice did not gain more weight or show increased appetitive motivation compared with wild-type mice in a standard environment; however, in an enriched environment with voluntary exercise opportunities, KD mice exhibited dramatically lower activity and became more obese than wild-type mice, obtaining no protective benefit from exercise opportunities. CONCLUSIONS: These data suggest the primary contribution of altered D2R signaling to obesity lies in altered energy expenditure rather than the induction of compulsive overeating.


Assuntos
Comportamento Alimentar/fisiologia , Motivação/fisiologia , Atividade Motora/fisiologia , Obesidade/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Glicemia , Peso Corporal , Calorimetria Indireta , Comportamento de Escolha/fisiologia , Condicionamento Operante/fisiologia , Modelos Animais de Doenças , Metabolismo Energético/fisiologia , Feminino , Predisposição Genética para Doença , Abrigo para Animais , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Obesidade/psicologia , Receptores de Dopamina D2/genética
11.
Bioorg Med Chem Lett ; 25(17): 3458-63, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26195136

RESUMO

A series of pyrimidine alkynyl derivatives were designed and synthesized as new Bcr-Abl inhibitors by hybriding the structural moieties from GNF-7, ponatinib and nilotinib. One of the most potent compounds 4e strongly suppresses Bcr-Abl(WT) and Bcr-Abl(T315I) kinase with IC50 values of 5.0 and 9.0 nM, and inhibits the proliferation of K562 and murine Ba/F3 cells ectopically expressing Bcr-Abl(T315I) cells with IC50 values of 2 and 50 nM, respectively. It also displays good pharmacokinetics properties with an oral bioavailability of 35.3% and T(1/2) value of 48.7 h, and demonstrates significantly suppression on tumor growth in xenografted mice of K562 and Ba/F3 cells expressing Bcr-Abl(T315I). These inhibitors may serve as lead compounds for further developing new anticancer drugs overcoming the clinically acquired resistance against current Bcr-Abl inhibitors.


Assuntos
Proteínas de Fusão bcr-abl/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Pirimidinas/química , Proliferação de Células , Modelos Moleculares , Mutação
12.
ACS Med Chem Lett ; 6(5): 543-7, 2015 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-26005530

RESUMO

A series of N-(3-ethynyl-2,4-difluorophenyl)sulfonamides were identified as new selective Raf inhibitors. The compounds potently inhibit B-Raf(V600E) with low nanomolar IC50 values and exhibit excellent target specificity in a selectivity profiling investigation against 468 kinases. They strongly suppress proliferation of a panel of human cancer cell lines and patient-derived melanoma cells with B-Raf(V600E) mutation while being significantly less potent to the cells with B-Raf(WT). The compounds also display favorable pharmacokinetic properties with a preferred example (3s) demonstrating significant in vivo antitumor efficacy in a xenograft mouse model of B-Raf(V600E) mutated Colo205 human colorectal cancer cells, supporting it as a promising lead compound for further anticancer drug discovery.

13.
Bioorg Med Chem ; 23(13): 3751-60, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25910584

RESUMO

The estrogen-related receptor γ (ERRγ) is a potential molecular target for the development of small molecules to stimulate the adipose browning process, which may represent a novel attractive strategy to treat obesity related disorders. The receptor possesses a very small ligand binding cavity and therefore identification of small molecule ERRγ modulators is a considerable challenge. We have successfully designed and synthesized a series of 1-benzyl-4-phenyl-1H-1,2,3-triazoles and demonstrated that they improve the transcriptional functions of ERRγ, potently elevating both the mRNA levels and the protein levels of ERRγ downstream targets. One of the most promising compounds, 4-(1-(4-iso-propylbenzyl)-1H-1,2,3-triazol-4-yl)benzene-1,2-diol (2e) was further shown to directly bind with the ERRγ ligand binding domain (ERRγ-LBD) in an isothermal calorimetric (ITC) assay and to thermally stabilize ERRγ-LBD protein by increasing its melting temperature (Tm) as demonstrated by circular dichroism (CD) spectroscopy. Furthermore, 2e potently stimulates the adipocyte browning process and induces mitochondrial biogenesis both in vitro and in vivo, suggesting the considerable therapeutic potential of this compound for the treatment of obesity and related disorders.


Assuntos
Adipócitos/efeitos dos fármacos , Obesidade/tratamento farmacológico , Receptores Estrogênicos/genética , Transcrição Genética/efeitos dos fármacos , Triazóis/farmacologia , Adipócitos/citologia , Adipócitos/metabolismo , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/patologia , Animais , Embrião de Mamíferos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Humanos , Ligantes , Luciferases/genética , Luciferases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , Plasmídeos/química , Plasmídeos/metabolismo , Cultura Primária de Células , Receptores Estrogênicos/química , Receptores Estrogênicos/metabolismo , Transdução de Sinais , Transfecção , Triazóis/síntese química
14.
J Neurosci ; 35(3): 890-905, 2015 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-25609609

RESUMO

In vitro evidence suggests that the inefficient removal of damaged mitochondria by macroautophagy contributes to Parkinson's disease (PD). Using a tissue-specific gene amplification strategy, we generated a transgenic mouse line with human α-synuclein A53T overexpression specifically in dopamine (DA) neurons. Transgenic mice showed profound early-onset mitochondria abnormalities, characterized by macroautophagy marker-positive cytoplasmic inclusions containing mainly mitochondrial remnants, which preceded the degeneration of DA neurons. Genetic deletion of either parkin or PINK1 in these transgenic mice significantly worsened mitochondrial pathologies, including drastically enlarged inclusions and loss of total mitochondria contents. These data suggest that mitochondria are the main targets of α-synuclein and their defective autophagic clearance plays a significant role during pathogenesis. Moreover, endogenous PINK1 or parkin is indispensable for the proper autophagic removal of damaged mitochondria. Our data for the first time establish an essential link between mitochondria macroautophagy impairments and DA neuron degeneration in an in vivo model based on known PD genetics. The model, its well-defined pathologies, and the demonstration of a main pathogenesis pathway in the present study have set the stage and direction of emphasis for future studies.


Assuntos
Autofagia/genética , Neurônios Dopaminérgicos/metabolismo , Mitocôndrias/genética , Degeneração Neural/genética , alfa-Sinucleína/genética , Animais , Contagem de Células , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Modelos Animais de Doenças , Neurônios Dopaminérgicos/patologia , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , alfa-Sinucleína/metabolismo
15.
Nat Commun ; 5: 5244, 2014 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-25370169

RESUMO

Mitochondrial dysfunction has been reported in both familial and sporadic Parkinson's disease (PD). However, effective therapy targeting this pathway is currently inadequate. Recent studies suggest that manipulating the processes of mitochondrial fission and fusion has considerable potential for treating human diseases. To determine the therapeutic impact of targeting these pathways on PD, we used two complementary mouse models of mitochondrial impairments as seen in PD. We show here that blocking mitochondrial fission is neuroprotective in the PTEN-induced putative kinase-1 deletion (PINK1(-/-)) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse models. Specifically, we show that inhibition of the mitochondrial fission GTPase dynamin-related protein-1 (Drp1) using gene-based and small-molecule approaches attenuates neurotoxicity and restores pre-existing striatal dopamine release deficits in these animal models. These results suggest Drp1 inhibition as a potential treatment for PD.


Assuntos
Dopamina/metabolismo , Dinaminas/antagonistas & inibidores , Dinâmica Mitocondrial , Doença de Parkinson/terapia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Animais , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/metabolismo , Doença de Parkinson/metabolismo , Proteínas Quinases/genética
16.
ACS Nano ; 8(11): 11715-23, 2014 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-25375351

RESUMO

The structural arrangement of amino acid residues in a native enzyme provides a blueprint for the design of artificial enzymes. One challenge of mimicking the catalytic center of a native enzyme is how to arrange the essential amino acid residues in an appropriate position. In this study, we designed an artificial hydrolase via self-assembly of short peptides to catalyze ester hydrolysis. When the assembled hydrolase catalytic sites were embedded in a matrix of peptide nanofibers, they exhibited much higher catalytic efficiency than the peptide nanofibers without the catalytic sites, suggesting that this well-ordered nanostructure is an attractive scaffold for developing new artificial enzymes. Furthermore, the cytotoxicity of the assembled hydrolase was evaluated with human cells, and the novel artificial biological enzyme showed excellent biocompatibility.


Assuntos
Nanofibras/química , Peptídeos/química , Catálise , Dicroísmo Circular , Hidrólise , Microscopia Eletrônica de Transmissão , Estrutura Secundária de Proteína
17.
Bioorg Med Chem Lett ; 24(17): 4250-3, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25091926

RESUMO

Emerging drug resistance and other drawbacks limit tubulin inhibitors' therapeutic applications and developing novel tubulin inhibitors still attracts intensive efforts. We describe the discovery and structure-activity relationship study of a series of benzimidazole-2-urea derivatives as novel ß tubulin inhibitors. The representative compound 6o potently suppressed the proliferation of a panel of human cancer cells (NCI-H460, Colo205, K562, A431, HepG2, Hela, MDA-MB-435S) with IC50 values of 0.040, 0.050, 0.006, 0.026, 1.774, 0.452 and 0.052 µM, respectively. Compound 6o obviously inhibited NCI-H460 spindles formation and induced cell cycle arrest at G2/M phase at 0.10 µM. Computational study suggested that 6o interacts with ß tubulin in a novel binding mode. Our results suggested that benzimidazole-2-urea derivatives might be promising tubulin inhibitors with novel binding mode for further development.


Assuntos
Antineoplásicos/farmacologia , Benzimidazóis/farmacologia , Compostos de Metilureia/farmacologia , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Benzimidazóis/síntese química , Benzimidazóis/química , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Fase G2/efeitos dos fármacos , Células HeLa , Células Hep G2 , Humanos , Células K562 , Compostos de Metilureia/síntese química , Compostos de Metilureia/química , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Moduladores de Tubulina/síntese química
18.
J Neurosci ; 34(19): 6692-9, 2014 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-24806695

RESUMO

Recent experimental evidence suggests that the low dopamine conditions in Parkinson's disease (PD) cause motor impairment through aberrant motor learning. Those data, along with computational models, suggest that this aberrant learning results from maladaptive corticostriatal plasticity and learned motor inhibition. Dopaminergic modulation of both corticostriatal long-term depression (LTD) and long-term potentiation (LTP) is proposed to be critical for these processes; however, the regulatory mechanisms underlying bidirectional corticostriatal plasticity are not fully understood. Previously, we demonstrated a key role for cAMP signaling in corticostriatal LTD. In this study, mouse brain slices were used to perform a parametric experiment that tested the impact of varying both intracellular cAMP levels and the strength of excitatory inputs on corticostriatal plasticity. Using slice electrophysiology in the dorsolateral striatum, we demonstrate that both LTP and LTD can be sequentially induced in the same D2-expressing neuron and that LTP was strongest with high intracellular cAMP and LFS, whereas LTD required low intracellular cAMP and high-frequency stimulation. Our results provide a molecular and cellular basis for regulating bidirectional corticostriatal synaptic plasticity and may help to identify novel therapeutic targets for blocking or reversing the aberrant synaptic plasticity that likely contributes to motor deficits in PD.


Assuntos
AMP Cíclico/fisiologia , Globo Pálido/fisiologia , Neostriado/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios Aferentes/fisiologia , Sinapses/fisiologia , Animais , Dopamina/fisiologia , Estimulação Elétrica , Fenômenos Eletrofisiológicos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Globo Pálido/citologia , Técnicas In Vitro , Potenciação de Longa Duração/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neostriado/citologia , Técnicas de Patch-Clamp
19.
G3 (Bethesda) ; 4(6): 1147-54, 2014 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-24739647

RESUMO

The concept of auxotrophic complementation has been proposed as an approach to identify genes in essential metabolic pathways in Drosophila melanogaster. However, it has achieved limited success to date, possibly due to the low probability of finding mutations fit with the chemically defined profile. Instead of using the chemically defined culture media lacking specific nutrients, we used bare minimum culture medium, i.e., 4% sucrose, for adult Drosophila. We identified a nutritional conditional lethal mutant and localized a c.95C > A mutation in the Drosophila pyridoxine 5'-phosphate oxidase gene [dPNPO or sugarlethal (sgll)] using meiotic recombination mapping, deficiency mapping, and whole genome sequencing. PNPO converts dietary vitamin B6 such as pyridoxine to its active form pyridoxal 5'-phosphate (PLP). The missense mutation (sgll(95)) results in the substitution of alanine to aspartate (p.Ala32Asp). The sgll(95) flies survive well on complete medium but all die within 6 d on 4% sucrose only diet, which can be rescued by pyridoxine or PLP supplement, suggesting that the mutation does not cause the complete loss of PNPO activity. The sgll knockdown further confirms its function as the Drosophila PNPO. Because better tools for positional cloning and cheaper whole genome sequencing have made the identification of point mutations much easier than before, alleviating the necessity to pinpoint specific metabolic pathways before gene identification, we propose that nutritional conditional screens based on bare minimum growth media like ours represent promising approaches for discovering important genes and mutations in metabolic pathways, thereby accelerating the establishment of in vivo models that recapitulate human metabolic diseases.


Assuntos
Drosophila melanogaster/genética , Genes Letais , Mutação , Piridoxaminafosfato Oxidase/deficiência , Alelos , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas da Membrana Bacteriana Externa , Mapeamento Cromossômico , Cromossomos , Cruzamentos Genéticos , Análise Mutacional de DNA , Elementos de DNA Transponíveis , Drosophila melanogaster/metabolismo , Técnicas de Silenciamento de Genes , Genótipo , Masculino , Meiose/genética , Dados de Sequência Molecular , Fenótipo , Fosfato de Piridoxal/metabolismo , Piridoxina/metabolismo , Recombinação Genética , Alinhamento de Sequência , Sacarose/metabolismo , Vitamina B 6
20.
J Neurogenet ; 28(1-2): 112-21, 2014 Mar-Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24673634

RESUMO

Although both cocaine and amphetamine mainly target the dopamine transporter (DAT) and cause psychomotor effects, they have very different mechanisms of actions. The authors examined whether responses to cocaine and amphetamine were affected differentially by changes in DAT expression levels using transgenic mice with different DAT expression levels. In the constitutive DAT knockdown mice, reduced DAT expression enhanced cocaine's locomotor stimulatory effects and at the same time diminished amphetamine's locomotor stimulatory effects. Similar effects were observed in the inducible DAT knockdown mice, ruling out the contribution of developmental compensations in DAT knockdown mice. Extracellular dopamine levels in response to psychostimulants were assessed by in vivo microdialysis. Whereas amphetamine-induced increase in extracellular dopamine was drastically diminished in constitutive DAT knockdown mice, cocaine-induced increase in extracellular dopamine had a faster onset in knockdown mice compared with wild-type controls. Postsynaptically, D1 agonist-stimulated c-fos expression was significantly attenuated in constitutive DAT knockdown mice compared with wild-type controls. The authors propose that responses to cocaine and amphetamine depend on psychostimulant drug type, drug dose, as well as DAT expression level. DAT expression level affects presynaptic responses to psychostimulants directly and postsynaptic responses to psychostimulants indirectly via changes in receptor signaling. These data imply that individual differences in DAT expression (either genetically or pharmacologically induced) may affect susceptibility to addiction of different types of psychostimulants.


Assuntos
Anfetamina/farmacologia , Cocaína/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Administração Oral , Inibidores da Captação Adrenérgica/farmacologia , Animais , Benzazepinas/farmacologia , Dopamina/metabolismo , Agonistas de Dopamina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Relação Dose-Resposta a Droga , Doxiciclina/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , N-Metil-3,4-Metilenodioxianfetamina/farmacologia
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