RESUMO
Cancer is influenced by its microenvironment, yet broader, environmental effects also play a role but remain poorly defined. We report here that mice living in an enriched housing environment show reduced tumor growth and increased remission. We found this effect in melanoma and colon cancer models, and that it was not caused by physical activity alone. Serum from animals held in an enriched environment (EE) inhibited cancer proliferation in vitro and was markedly lower in leptin. Hypothalamic brain-derived neurotrophic factor (BDNF) was selectively upregulated by EE, and its genetic overexpression reduced tumor burden, whereas BDNF knockdown blocked the effect of EE. Mechanistically, we show that hypothalamic BDNF downregulated leptin production in adipocytes via sympathoneural beta-adrenergic signaling. These results suggest that genetic or environmental activation of this BDNF/leptin axis may have therapeutic significance for cancer.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Neoplasias do Colo/metabolismo , Hipotálamo/metabolismo , Leptina/metabolismo , Melanoma/metabolismo , Transdução de Sinais , Meio Social , Adipócitos/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Neoplasias do Colo/genética , Neoplasias do Colo/fisiopatologia , Genes APC , Abrigo para Animais , Hipotálamo/citologia , Imunocompetência , Melanoma/genética , Melanoma/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Processos Neoplásicos , Distribuição Aleatória , Receptores Adrenérgicos beta/metabolismoRESUMO
Recombinant adeno-associated virus (rAAV) vectors are attractive vehicles for gene therapy. Gene delivery to the adipose tissue using naturally occurring AAV serotypes is less successful compared to liver and muscle. Here, we demonstrate that oral administration of an engineered serotype Rec2 led to preferential transduction of brown fat with absence of transduction in the gastrointestinal track. Among the six natural and engineered serotypes being compared, Rec2 was the most efficient serotype achieving high level transduction at a dose 1~2 orders lower than reported doses for systemic administration. Overexpressing vascular endothelial growth factor (VEGF) in brown fat via oral administration of Rec2-VEGF vector increased the brown fat mass and enhanced thermogenesis. In contrast, knockdown VEGF in brown fat of VEGF (loxP) mice via Rec2-Cre vector hampered cold response and decreased brown fat mass. Oral administration of Rec2 vector provides a novel tool to genetically manipulate brown fat for research and therapeutic applications.
Assuntos
Tecido Adiposo Marrom/efeitos dos fármacos , Vetores Genéticos/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/genética , Tecido Adiposo Marrom/metabolismo , Administração Oral , Animais , Dependovirus/genética , Técnicas de Transferência de Genes , Camundongos , Especificidade de Órgãos , Termogênese/efeitos dos fármacos , Transdução GenéticaRESUMO
In Parkinson's disease, long-term dopamine replacement therapy is complicated by the appearance of L-DOPA-induced dyskinesia (LID). One major hypothesis is that LID results from an aberrant transcriptional program in striatal neurons induced by L-DOPA and triggered by the activation of ERK. To identify these genes, we performed transcriptome analyses in the striatum in 6-hydroxydopamine-lesioned mice. A time course analysis (0-6 h after treatment with L-DOPA) identified an acute signature of 709 genes, among which genes involved in protein phosphatase activity were overrepresented, suggesting a negative feedback on ERK activation by l-DOPA. l-DOPA-dependent deregulation of 28 genes was blocked by pretreatment with SL327, an inhibitor of ERK activation, and 26 genes were found differentially expressed between highly and weakly dyskinetic animals after treatment with L-DOPA. The intersection list identified five genes: FosB, Th, Nptx2, Nedd4l, and Ccrn4l. Nptx2 encodes neuronal pentraxin II (or neuronal activity-regulated pentraxin, Narp), which is involved in the clustering of glutamate receptors. We confirmed increased Nptx2 expression after L-DOPA and its blockade by SL327 using quantitative RT-PCR in independent experiments. Using an escalating L-DOPA dose protocol, LID severity was decreased in Narp knock-out mice compared with their wild-type littermates or after overexpression of a dominant-negative form of Narp in the striatum. In conclusion, we have identified a molecular signature induced by L-DOPA in the dopamine-denervated striatum that is dependent on ERK and associated with LID. Here, we demonstrate the implication of one of these genes, Nptx2, in the development of LID.
Assuntos
Antiparkinsonianos/toxicidade , Proteína C-Reativa/biossíntese , Proteína C-Reativa/genética , Discinesia Induzida por Medicamentos/genética , Discinesia Induzida por Medicamentos/metabolismo , Levodopa/toxicidade , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Animais , Discinesia Induzida por Medicamentos/patologia , Feminino , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Anorexia and weight loss are part of the wasting syndrome of late-stage cancer, are a major cause of morbidity and mortality in cancer, and are thought to be cytokine mediated. Macrophage inhibitory cytokine-1 (MIC-1) is produced by many cancers. Examination of sera from individuals with advanced prostate cancer showed a direct relationship between MIC-1 abundance and cancer-associated weight loss. In mice with xenografted prostate tumors, elevated MIC-1 levels were also associated with marked weight, fat and lean tissue loss that was mediated by decreased food intake and was reversed by administration of antibody to MIC-1. Additionally, normal mice given systemic MIC-1 and transgenic mice overexpressing MIC-1 showed hypophagia and reduced body weight. MIC-1 mediates its effects by central mechanisms that implicate the hypothalamic transforming growth factor-beta receptor II, extracellular signal-regulated kinases 1 and 2, signal transducer and activator of transcription-3, neuropeptide Y and pro-opiomelanocortin. Thus, MIC-1 is a newly defined central regulator of appetite and a potential target for the treatment of both cancer anorexia and weight loss, as well as of obesity.
Assuntos
Anorexia/metabolismo , Citocinas/fisiologia , Família Multigênica/imunologia , Neoplasias da Próstata/metabolismo , Redução de Peso , Animais , Anorexia/genética , Anorexia/imunologia , Anorexia/fisiopatologia , Anticorpos/administração & dosagem , Anticorpos/fisiologia , Linhagem Celular Tumoral , Citocinas/sangue , Citocinas/genética , Citocinas/imunologia , Fator 15 de Diferenciação de Crescimento , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Neoplasias da Próstata/sangue , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/fisiopatologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/fisiologia , Redução de Peso/genética , Redução de Peso/imunologiaRESUMO
Narp knockout (KO) mice demonstrate an impaired extinction of morphine conditioned place preference (CPP). Because the medial prefrontal cortex (mPFC) has been implicated in extinction learning, we tested whether Narp cells in this region play a role in the extinction of morphine CPP. We found that intracranial injections of adenoassociated virus (AAV) expressing wild-type (WT) Narp into the mPFC of Narp KO mice rescued the extinction and the injection of AAV expressing a dominant negative form of Narp (NarpN) into the mPFC of WT mice impaired the extinction of morphine CPP. These findings suggest that Narp in the mPFC mediates the extinction of morphine CPP.
Assuntos
Proteína C-Reativa/metabolismo , Condicionamento Operante/fisiologia , Extinção Psicológica/fisiologia , Morfina/administração & dosagem , Entorpecentes/administração & dosagem , Proteínas do Tecido Nervoso/metabolismo , Córtex Pré-Frontal/metabolismo , Análise de Variância , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteína C-Reativa/deficiência , Dependovirus/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microinjeções , Proteínas do Tecido Nervoso/deficiênciaRESUMO
The medial prefrontal cortex (mPFC) plays a key role in extinction learning. Previously, we found that expression of a neuronal activity-regulated pentraxin (Narp) dominant-negative construct in the mPFC of mice blocked extinction of morphine-conditioned place preference. To further investigate the role of mPFC Narp in the extinction of drug seeking, we tested whether mPFC Narp is necessary for the extinction of heroin self-administration in rats. Specifically, we injected an adeno-associated viral vector expressing a dominant-negative form of Narp (NarpN) into the infralimbic region of the mPFC of rats and compared lever presses during extinction to those of rats injected with a control virus. In contrast to our previous study, we found that injection of NarpN did not affect extinction of heroin self-administration. Our findings suggest that mPFC Narp is necessary for extinction of opiate seeking in the Pavlovian-conditioned place preference paradigm but not in the operant paradigm of drug self-administration.
Assuntos
Proteína C-Reativa/metabolismo , Extinção Psicológica/efeitos dos fármacos , Heroína/administração & dosagem , Entorpecentes/administração & dosagem , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Córtex Pré-Frontal/citologia , Análise de Variância , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteína C-Reativa/genética , Condicionamento Clássico/efeitos dos fármacos , Dependovirus/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Autoadministração , Fatores de Tempo , Transdução GenéticaRESUMO
BACKGROUND: The combination of gene therapy and plastic surgery may have the potential to improve the specificity that is needed to achieve clinically applicable treatment regimens. Our goal was to develop a method for gene modification that would yield sustainable production of gene products but would be less time consuming than existing protocols. METHODS: An adenoassociated virus was used to deliver gene products to pectoralis muscle flaps. Gene modification was accomplished via either direct injection or novel fat grafting techniques. RESULTS: The production of gene product was observable by both in vivo imaging and immunohistochemical staining. Gene products were not detected in tissues that were not in contact with the fat grafts that were incubated with the viral vector, indicating that the transduction stayed local to the flap. CONCLUSIONS: Using novel recombinant adenoassociated virus vectors, we have developed a method for gene delivery that is highly efficient and applicable to muscle flaps.
Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Procedimentos de Cirurgia Plástica/métodos , Gordura Subcutânea/transplante , Retalhos Cirúrgicos , Animais , Vetores Genéticos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gordura Subcutânea/virologia , Retalhos Cirúrgicos/virologia , Transfecção/métodosRESUMO
Axon degeneration is a hallmark of neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Such degeneration is not a passive event but rather an active process mediated by mechanisms that are distinct from the canonical pathways of programmed cell death that mediate destruction of the cell soma. Little is known of the diverse mechanisms involved, particularly those of retrograde axon degeneration. We have previously observed in living animal models of degeneration in the nigrostriatal projection that a constitutively active form of the kinase, myristoylated Akt (Myr-Akt), demonstrates an ability to suppress programmed cell death and preserve the soma of dopamine neurons. Here, we show in both neurotoxin and physical injury (axotomy) models that Myr-Akt is also able to preserve dopaminergic axons due to suppression of acute retrograde axon degeneration. This cellular phenotype is associated with increased mammalian target of rapamycin (mTor) activity and can be recapitulated by a constitutively active form of the small GTPase Rheb, an upstream activator of mTor. Axon degeneration in these models is accompanied by the occurrence of macroautophagy, which is suppressed by Myr-Akt. Conditional deletion of the essential autophagy mediator Atg7 in adult mice also achieves striking axon protection in these acute models of retrograde degeneration. The protection afforded by both Myr-Akt and Atg7 deletion is robust and lasting, because it is still observed as protection of both axons and dopaminergic striatal innervation weeks after injury. We conclude that acute retrograde axon degeneration is regulated by Akt/Rheb/mTor signaling pathways.
Assuntos
Autofagia/fisiologia , Axônios/metabolismo , Dopamina/metabolismo , Neurônios/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Degeneração Retrógrada/metabolismo , Degeneração Retrógrada/patologia , Animais , Autofagia/efeitos dos fármacos , Proteína 7 Relacionada à Autofagia , Axônios/efeitos dos fármacos , Axônios/ultraestrutura , Dependovirus/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/genética , Feixe Prosencefálico Mediano/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia Eletrônica de Transmissão/métodos , Proteínas Associadas aos Microtúbulos/metabolismo , Oxidopamina/efeitos adversos , Proteínas Proto-Oncogênicas c-akt/genética , Degeneração Retrógrada/etiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Substância Negra/patologia , Serina-Treonina Quinases TOR/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
OBJECTIVE: A prevailing concept in neuroscience has been that the adult mammalian central nervous system is incapable of restorative axon regeneration. Recent evidence, however, has suggested that reactivation of intrinsic cellular programs regulated by protein kinase B (Akt)/mammalian target of rapamycin (mTor) signaling may restore this ability. METHODS: To assess this possibility in the brain, we have examined the ability of adenoassociated virus (AAV)-mediated transduction of dopaminergic neurons of the substantia nigra (SN) with constitutively active forms of the kinase Akt and the GTPase Ras homolog enriched in brain (Rheb) to induce regrowth of axons after they have been destroyed by neurotoxin lesion. RESULTS: Both constitutively active myristoylated Akt and hRheb(S16H) induce regrowth of axons from dopaminergic neurons to their target, the striatum. Histological analysis demonstrates that these new axons achieve morphologically accurate reinnervation. In addition, functional reintegration into target circuitry is achieved, as indicated by partial behavioral recovery. INTERPRETATION: We conclude that regrowth of axons within the adult nigrostriatal projection, a system that is prominently affected in Parkinson's disease, can be achieved by activation of Akt/mTor signaling in surviving endogenous mesencephalic dopaminergic neurons by viral vector transduction.
Assuntos
Axônios/fisiologia , Dopamina/fisiologia , Proteínas Monoméricas de Ligação ao GTP/administração & dosagem , Regeneração Nervosa/fisiologia , Neuropeptídeos/administração & dosagem , Proteínas Proto-Oncogênicas c-akt/administração & dosagem , Transdução de Sinais , Animais , Axônios/metabolismo , Dependovirus/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Monoméricas de Ligação ao GTP/genética , Regeneração Nervosa/genética , Neurônios/citologia , Neurônios/patologia , Neurônios/virologia , Neuropeptídeos/genética , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Transdução de Sinais/genética , Substância Negra/metabolismo , Substância Negra/patologia , Substância Negra/virologiaRESUMO
Activation-induced cytidine deaminase (AID) is an enzyme essential for the generation of Ab diversity in B cells and is considered to be a general gene mutator. In addition, AID expression was also implicated in the pathogenesis of human B cell malignancies and associated with poor prognosis. In this study, we report that small interfering RNA silencing of AID in plasmacytoma dramatically increased its susceptibility to immunotherapy by CTLs. AID silencing did not decrease the mutation frequencies of tumor Ag gene P1A. Gene-array analysis showed dramatically altered expression of a number of genes in AID-silenced plasmacytoma cells, and upregulation of CD200 was shown to be in favor of tumor eradication by CTLs. Taken together, we demonstrate a novel function of AID in tumor evasion of CTL therapy and that targeting AID should be beneficial in the immunotherapy of AID-positive tumors.
Assuntos
Citidina Desaminase/metabolismo , Marcação de Genes , Imunoterapia Adotiva , Linfócitos T Citotóxicos/imunologia , Evasão Tumoral/imunologia , Animais , Linhagem Celular Tumoral , Citidina Desaminase/antagonistas & inibidores , Citidina Desaminase/genética , Citidina Desaminase/fisiologia , Citotoxicidade Imunológica/genética , Marcação de Genes/métodos , Imunoterapia Adotiva/métodos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Plasmocitoma/enzimologia , Plasmocitoma/genética , Plasmocitoma/imunologia , RNA Interferente Pequeno/fisiologia , Linfócitos T Citotóxicos/metabolismo , Evasão Tumoral/genéticaRESUMO
Extensive clinical data from liver-mediated gene therapy trials have shown that dose-dependent immune responses against the vector capsid may impair or even preclude transgene expression if not managed successfully with prompt immune suppression. The goal of this preclinical study was to generate an adeno-associated viral (AAV) vector capable of expressing therapeutic levels of B-domain deleted factor VIII (FVIII) at the lowest possible vector dose to minimize the potential Risk of a capsid-mediated immune response in the clinical setting. Here, we describe the studies that identified the investigational agent SPK-8011, currently being evaluated in a phase 1/2 study (NCT03003533) in individuals with hemophilia A. In particular, the potency of our second-generation expression cassettes was evaluated in mice and in non-human primates using two different bioengineered capsids (AAV-Spark100 and AAV-Spark200). At 2 weeks after gene transfer, primates transduced with 2 × 1012 vg/kg AAV-Spark100-FVIII or AAV-Spark200-FVIII expressed FVIII antigen levels of 13% ± 2% and 22% ± 6% of normal, respectively. Collectively, these preclinical results validate the feasibility of lowering the AAV capsid dose for a gene-based therapeutic approach for hemophilia A to a dose level orders of magnitude lower than the first-generation vectors in the clinic.
RESUMO
Mucopolysaccharidosis (MPS) IIIB is a lysosomal storage disease with severe neurological manifestations due to alpha-N-acetylglucosaminidase (NaGlu) deficiency. The mechanism of neuropathology in MPS IIIB is unclear. This study investigates the role of immune responses in neurological disease of MPS IIIB in mice. By means of gene expression microarrays and real-time quantitative reverse transcriptase-polymerase chain reaction, we demonstrated significant up-regulation of numerous immune-related genes in MPS IIIB mouse brain involving a broad range of immune cells and molecules, including T cells, B cells, microglia/macrophages, complement, major histocompatibility complex class I, immunoglobulin, Toll-like receptors, and molecules essential for antigen presentation. The significantly enlarged spleen and lymph nodes in MPS IIIB mice were due to an increase in splenocytes/lymphocytes, and functional assays indicated that the T cells were activated. An autoimmune component to the disease was further suggested by the presence of putative autoantigen or autoantigens in brain extracts that reacted specifically with serum IgG from MPS IIIB mice. We also demonstrated for the first time that immunosuppression with prednisolone alone can significantly slow the central nervous system disease progression. Our data indicate that immune responses contribute greatly to the neuropathology of MPS IIIB and should be considered as an adjunct treatment in future therapeutic developments for optimal therapeutic effect.
Assuntos
Encéfalo/imunologia , Imunidade Inata , Mucopolissacaridose III/imunologia , Animais , Astrócitos/fisiologia , Autoanticorpos , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Doenças Desmielinizantes/fisiopatologia , Modelos Animais de Doenças , Progressão da Doença , Regulação da Expressão Gênica , Imunossupressores/uso terapêutico , Linfonodos/patologia , Linfonodos/fisiopatologia , Ativação Linfocitária , Linfócitos/fisiologia , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Knockout , Microglia/fisiologia , Mucopolissacaridose III/tratamento farmacológico , Mucopolissacaridose III/genética , Mucopolissacaridose III/fisiopatologia , Doenças Neurodegenerativas/fisiopatologia , Prednisolona/uso terapêutico , Baço/patologia , Baço/fisiopatologiaRESUMO
This corrects the article DOI: 10.1038/ncb3514.
RESUMO
Adipocytes undergo considerable volumetric expansion in the setting of obesity. It has been proposed that such marked increases in adipocyte size may be sensed via adipocyte-autonomous mechanisms to mediate size-dependent intracellular signalling. Here, we show that SWELL1 (LRRC8a), a member of the Leucine-Rich Repeat Containing protein family, is an essential component of a volume-sensitive ion channel (VRAC) in adipocytes. We find that SWELL1-mediated VRAC is augmented in hypertrophic murine and human adipocytes in the setting of obesity. SWELL1 regulates adipocyte insulin-PI3K-AKT2-GLUT4 signalling, glucose uptake and lipid content via SWELL1 C-terminal leucine-rich repeat domain interactions with GRB2/Cav1. Silencing GRB2 in SWELL1 KO adipocytes rescues insulin-pAKT2 signalling. In vivo, shRNA-mediated SWELL1 knockdown and adipose-targeted SWELL1 knockout reduce adiposity and adipocyte size in obese mice while impairing systemic glycaemia and insulin sensitivity. These studies identify SWELL1 as a cell-autonomous sensor of adipocyte size that regulates adipocyte growth, insulin sensitivity and glucose tolerance.
Assuntos
Adipócitos/metabolismo , Tamanho Celular , Metabolismo Energético , Glucose/metabolismo , Insulina/metabolismo , Proteínas de Membrana/metabolismo , Obesidade/metabolismo , Transdução de Sinais , Adipócitos/patologia , Adiposidade , Animais , Células Cultivadas , Canais de Cloreto/metabolismo , Modelos Animais de Doenças , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/metabolismo , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Homeostase , Humanos , Resistência à Insulina , Ativação do Canal Iônico , Masculino , Potenciais da Membrana , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Obesidade/genética , Obesidade/patologia , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Fatores de Tempo , TransfecçãoRESUMO
Living in an enriched environment (EE) decreases adiposity, increases energy expenditure, causes resistance to diet induced obesity, and induces brown-like (beige) cells in white fat via activating a hypothalamic-adipocyte axis. Here we report that EE stimulated vascular endothelial growth factor (VEGF) expression in a fat depot-specific manner prior to the emergence of beige cells. The VEGF up-regulation was independent of hypoxia but required intact sympathetic tone to the adipose tissue. Targeted adipose overexpression of VEGF reproduced the browning effect of EE. Adipose-specific VEGF knockout or pharmacological VEGF blockade with antibodies abolished the induction of beige cell by EE. Hypothalamic brain-derived neurotrophic factor stimulated by EE regulated the adipose VEGF expression, and VEGF signaling was essential to the hypothalamic brain-derived neurotrophic factor-induced white adipose tissue browning. Furthermore, VEGF signaling was essential to the beige cells induction by exercise, a ß3-adrenergic agonist, and a peroxisome proliferator-activated receptor-γ ligand, suggesting a common downstream pathway integrating diverse upstream mechanisms. Exploiting this pathway may offer potential therapeutic interventions to obesity and metabolic diseases.
Assuntos
Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Tecido Adiposo/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Adipose tissue plays an essential role in metabolic homeostasis, and holds promise as an alternative depot organ in gene therapy. However, efficient methods of gene transfer into adipose tissue in vivo have yet to be established. Here we assessed the transduction efficiency to fat depots by a family of novel engineered hybrid capsid serotypes (Rec1~4) recombinant AAV vectors in comparison with natural serotypes AAV1, AAV8, and AAV9. Rec2 serotype led to widespread transduction in both brown fat and white fat with the highest efficiency among the seven serotypes tested. As a proof-of-efficacy, Rec2 serotype was used to deliver Cre recombinase to adipose tissues of insulin receptor floxed animals. Insulin receptor knockdown led to decreased fat pad mass, morphological and molecular changes in the targeted depot. These novel hybrid AAV vectors can serve as powerful tools to genetically manipulate adipose tissue and provide valuable vehicles to gene therapy targeting adipose tissue.
RESUMO
Dopamine signaling in the nucleus accumbens is critical in mediating the effects of cocaine. There are two splice variants of dopamine D2 receptors, D2L and D2S, which are believed to have different functional roles. Here, we show, that knocking down D2L selectively using viral-mediated short-hairpin RNA led to a slight but significant decrease in basal locomotor activity with no significant change in cocaine-induced stimulation of locomotion. The knockdown appears to produce a trend of reduced conditioned place preference to cocaine but the difference was not statistically significant. Our results demonstrated that the splice variants of D2 receptors can be selectively manipulated in vivo in specific brain regions allowing more specific studies of each D2 receptor isoform.
Assuntos
Comportamento Animal/efeitos dos fármacos , Cocaína/farmacologia , Atividade Motora , Receptores de Dopamina D2/genética , Animais , Condicionamento Psicológico/efeitos dos fármacos , Condicionamento Psicológico/fisiologia , Corpo Estriado/metabolismo , Corpo Estriado/fisiologia , Dopamina/farmacologia , Antagonistas dos Receptores de Dopamina D2 , Técnicas de Silenciamento de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Núcleo Accumbens/efeitos dos fármacos , Isoformas de Proteínas/genética , RNA Interferente Pequeno , Receptores de Dopamina D2/fisiologiaRESUMO
Mice lacking the vesicular glutamate transporter-3 (VGLUT3) are congenitally deaf due to loss of glutamate release at the inner hair cell afferent synapse. Cochlear delivery of VGLUT3 using adeno-associated virus type 1 (AAV1) leads to transgene expression in only inner hair cells (IHCs), despite broader viral uptake. Within 2 weeks of AAV1-VGLUT3 delivery, auditory brainstem response (ABR) thresholds normalize, along with partial rescue of the startle response. Lastly, we demonstrate partial reversal of the morphologic changes seen within the afferent IHC ribbon synapse. These findings represent a successful restoration of hearing by gene replacement in mice, which is a significant advance toward gene therapy of human deafness.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/genética , Surdez/genética , Surdez/terapia , Potenciais Evocados Auditivos do Tronco Encefálico/genética , Terapia Genética/métodos , Reflexo de Sobressalto/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Surdez/metabolismo , Dependovirus/genética , Ácido Glutâmico/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Testes Auditivos , Camundongos , Camundongos Knockout , Sinapses/metabolismo , Transmissão Sináptica/genéticaRESUMO
Living in an enriched environment with complex physical and social stimulation leads to improved cognitive and metabolic health. In white fat, enrichment induced the upregulation of the brown fat cell fate determining gene Prdm16, brown fat-specific markers, and genes involved in thermogenesis and ß-adrenergic signaling. Moreover, pockets of cells with prototypical brown fat morphology and high UCP1 levels were observed in the white fat of enriched mice associated with resistance to diet-induced obesity. Hypothalamic overexpression of BDNF reproduced the enrichment-associated activation of the brown fat gene program and lean phenotype. Inhibition of BDNF signaling by genetic knockout or dominant-negative trkB reversed this phenotype. Our genetic and pharmacologic data suggest a mechanism whereby induction of hypothalamic BDNF expression in response to environmental stimuli leads to selective sympathoneural modulation of white fat to induce "browning" and increased energy dissipation.