RESUMO
IMPORTANCE: The use of adeno-associated viruses (AAVs) as gene delivery vectors has vast potential for the treatment of many severe human diseases. Over one hundred naturally existing AAV capsid variants have been described and classified into phylogenetic clades based on their sequences. AAV8, AAV9, AAVrh.10, and other intensively studied capsids have been propelled into pre-clinical and clinical use, and more recently, marketed products; however, less-studied capsids may also have desirable properties (e.g., potency differences, tissue tropism, reduced immunogenicity, etc.) that have yet to be thoroughly described. These data will help build a broader structure-function knowledge base in the field, present capsid engineering opportunities, and enable the use of novel capsids with unique properties.
Assuntos
Dependovirus , Terapia Genética , Vetores Genéticos , Humanos , Capsídeo , Proteínas do Capsídeo/genética , Dependovirus/genética , Vetores Genéticos/genética , Filogenia , Distribuição TecidualRESUMO
Alzheimer's disease (AD) is the leading cause of age-related dementia. Neuropathological hallmarks of AD include brain deposition of ß-amyloid (Aß) plaques and accumulation of both hyperphosphorylated and acetylated tau. RGFP-966, a brain-penetrant and selective HDAC3 inhibitor, or HDAC3 silencing, increases BDNF expression, increases histone H3 and H4 acetylation, decreases tau phosphorylation and tau acetylation at disease-associated sites, reduces ß-secretase cleavage of the amyloid precursor protein (APP), and decreases Aß1-42 accumulation in HEK-293 cells overexpressing APP with the double Swedish mutation (HEK/APPsw). In the triple transgenic AD mouse model (3xTg-AD), repeated administration of 3 and 10 mg/kg of RGFP-966 reverses pathological tau phosphorylation at Thr181, Ser202, and Ser396, increases levels of the Aß degrading enzyme Neprilysin in plasma, decreases Aß1-42 protein levels in the brain and periphery, and improves spatial learning and memory. Finally, we show that RGFP-966 decreases Aß1-42 accumulation and both tau acetylation and phosphorylation at disease residues in neurons derived from induced pluripotent stem cells obtained from APOEε4-carrying AD patients. These data indicate that HDAC3 plays an important regulatory role in the expression and regulation of proteins associated with AD pathophysiology, supporting the notion that HDAC3 may be a disease-modifying therapeutic target.
Assuntos
Acrilamidas/farmacologia , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Memória/efeitos dos fármacos , Fenilenodiaminas/farmacologia , Aprendizagem Espacial/efeitos dos fármacos , Proteínas tau/metabolismo , Acetilação/efeitos dos fármacos , Doença de Alzheimer/tratamento farmacológico , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Inativação Gênica , Células HEK293 , Histona Desacetilases/genética , Histonas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Camundongos Transgênicos , Neprilisina/sangue , Neurônios/citologia , Fosforilação/efeitos dos fármacosRESUMO
Histone deacetylase (HDAC) inhibitors may have therapeutic utility in multiple neurological and psychiatric disorders, but the underlying mechanisms remain unclear. Here, we identify BRD4, a BET bromodomain reader of acetyl-lysine histones, as an essential component involved in potentiated expression of brain-derived neurotrophic factor (BDNF) and memory following HDAC inhibition. In in vitro studies, we reveal that pharmacological inhibition of BRD4 reversed the increase in BDNF mRNA induced by the class I/IIb HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). Knock-down of HDAC2 and HDAC3, but not other HDACs, increased BDNF mRNA expression, whereas knock-down of BRD4 blocked these effects. Using dCas9-BRD4, locus-specific targeting of BRD4 to the BDNF promoter increased BDNF mRNA. In additional studies, RGFP966, a pharmacological inhibitor of HDAC3, elevated BDNF expression and BRD4 binding to the BDNF promoter, effects that were abrogated by JQ1 (an inhibitor of BRD4). Examining known epigenetic targets of BRD4 and HDAC3, we show that H4K5ac and H4K8ac modifications and H4K5ac enrichment at the BDNF promoter were elevated following RGFP966 treatment. In electrophysiological studies, JQ1 reversed RGFP966-induced enhancement of LTP in hippocampal slice preparations. Last, in behavioral studies, RGFP966 increased subthreshold novel object recognition memory and cocaine place preference in male C57BL/6 mice, effects that were reversed by cotreatment with JQ1. Together, these data reveal that BRD4 plays a key role in HDAC3 inhibitor-induced potentiation of BDNF expression, neuroplasticity, and memory.SIGNIFICANCE STATEMENT Some histone deacetylase (HDAC) inhibitors are known to have neuroprotective and cognition-enhancing properties, but the underlying mechanisms have yet to be fully elucidated. In the current study, we reveal that BRD4, an epigenetic reader of histone acetylation marks, is necessary for enhancing brain-derived neurotrophic factor (BDNF) expression and improved memory following HDAC inhibition. Therefore, by identifying novel epigenetic regulators of BDNF expression, these data may lead to new therapeutic targets for the treatment of neuropsychiatric disorders.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/biossíntese , Inibidores de Histona Desacetilases/farmacologia , Memória/efeitos dos fármacos , Acrilamidas/farmacologia , Animais , Azepinas/farmacologia , Fator Neurotrófico Derivado do Encéfalo/efeitos dos fármacos , Epigênese Genética , Técnicas de Silenciamento de Genes , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Fenilenodiaminas/farmacologia , Ratos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Triazóis/farmacologia , Vorinostat/farmacologiaRESUMO
Alzheimer's disease (AD) comprises multifactorial ailments for which current therapeutic strategies remain insufficient to broadly address the underlying pathophysiology. Epigenetic gene regulation relies upon multifactorial processes that regulate multiple gene and protein pathways, including those involved in AD. We therefore took an epigenetic approach where a single drug would simultaneously affect the expression of a number of defined AD-related targets. We show that the small-molecule histone deacetylase inhibitor M344 reduces beta-amyloid (Aß), reduces tau Ser396 phosphorylation, and decreases both ß-secretase (BACE) and APOEε4 gene expression. M344 increases the expression of AD-relevant genes: BDNF, α-secretase (ADAM10), MINT2, FE65, REST, SIRT1, BIN1, and ABCA7, among others. M344 increases sAPPα and CTFα APP metabolite production, both cleavage products of ADAM10, concordant with increased ADAM10 gene expression. M344 also increases levels of immature APP, supporting an effect on APP trafficking, concurrent with the observed increase in MINT2 and FE65, both shown to increase immature APP in the early secretory pathway. Chronic i.p. treatment of the triple transgenic (APPsw/PS1M146V/TauP301L) mice with M344, at doses as low as 3 mg/kg, significantly prevented cognitive decline evaluated by Y-maze spontaneous alternation, novel object recognition, and Barnes maze spatial memory tests. M344 displays short brain exposure, indicating that brief pulses of daily drug treatment may be sufficient for long-term efficacy. Together, these data show that M344 normalizes several disparate pathogenic pathways related to AD. M344 therefore serves as an example of how a multitargeting compound could be used to address the polygenic nature of multifactorial diseases.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Memória/efeitos dos fármacos , Proteína ADAM10/genética , Proteína ADAM10/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Aprendizagem em Labirinto/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Memória/fisiologia , Camundongos Transgênicos , Fragmentos de Peptídeos/metabolismo , Proteínas Repressoras/genética , VorinostatRESUMO
Glutamate carboxypeptidase II (GCPII) inactivates the peptide neurotransmitter N-acetylaspartylglutamate (NAAG) following synaptic release. Inhibitors of GCPII increase extracellular NAAG levels and are efficacious in animal models of clinical disorders via NAAG activation of a group II metabotropic glutamate receptor. mGluR2 and mGluR3 knock-out (ko) mice were used to test the hypothesis that mGluR3 mediates the activity of GCPII inhibitors ZJ43 and 2-PMPA in animal models of memory and memory loss. Short- (1.5 h) and long- (24 h) term novel object recognition tests were used to assess memory. Treatment with ZJ43 or 2-PMPA prior to acquisition trials increased long-term memory in mGluR2, but not mGluR3, ko mice. Nine month-old triple transgenic Alzheimer's disease model mice exhibited impaired short-term novel object recognition memory that was rescued by treatment with a NAAG peptidase inhibitor. NAAG peptidase inhibitors and the group II mGluR agonist, LY354740, reversed the short-term memory deficit induced by acute ethanol administration in wild type mice. 2-PMPA also moderated the effect of ethanol on short-term memory in mGluR2 ko mice but failed to do so in mGluR3 ko mice. LY354740 and ZJ43 blocked ethanol-induced motor activation. Both GCPII inhibitors and LY354740 also significantly moderated the loss of motor coordination induced by 2.1 g/kg ethanol treatment. These data support the conclusion that inhibitors of glutamate carboxypeptidase II are efficacious in object recognition models of normal memory and memory deficits via an mGluR3 mediated process, actions that could have widespread clinical applications.
Assuntos
Intoxicação Alcoólica/metabolismo , Doença de Alzheimer/metabolismo , Glutamato Carboxipeptidase II/metabolismo , Memória/fisiologia , Receptores de Glutamato Metabotrópico/deficiência , Ureia/análogos & derivados , Intoxicação Alcoólica/genética , Doença de Alzheimer/genética , Animais , Modelos Animais de Doenças , Etanol/administração & dosagem , Antagonistas de Aminoácidos Excitatórios/farmacologia , Glutamato Carboxipeptidase II/antagonistas & inibidores , Glutamato Carboxipeptidase II/genética , Masculino , Memória/efeitos dos fármacos , Transtornos da Memória/genética , Transtornos da Memória/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Ureia/farmacologiaRESUMO
A substantial body of data was reported between 1984 and 2000 demonstrating that the neuropeptide N-acetylaspartylglutamate (NAAG) not only functions as a neurotransmitter but also is the third most prevalent transmitter in the mammalian nervous system behind glutamate and GABA. By 2005, this conclusion was validated further through a series of studies in vivo and in vitro. The primary enzyme responsible for the inactivation of NAAG following its synaptic release had been cloned, characterized and knocked out. Potent inhibitors of this enzyme were developed and their efficacy has been extensively studied in a series of animal models of clinical conditions, including stroke, peripheral neuropathy, traumatic brain injury, inflammatory and neuropathic pain, cocaine addiction, and schizophrenia. Considerable progress also has been made in defining further the mechanism of action of these peptidase inhibitors in elevating synaptic levels of NAAG with the consequent inhibition of transmitter release via the activation of pre-synaptic metabotropic glutamate receptor 3 by this peptide. Very recent discoveries include identification of two different nervous system enzymes that mediate the synthesis of NAAG from N-acetylaspartate and glutamate and the finding that one of these enzymes also mediates the synthesis of a second member of the NAAG family of neuropeptides, N-acetylaspartylglutamylglutamate.
Assuntos
Dipeptídeos/fisiologia , Neuropeptídeos/fisiologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/fisiologia , Lesões Encefálicas/tratamento farmacológico , Dipeptídeos/genética , Dipeptídeos/metabolismo , Glutamato Carboxipeptidase II/antagonistas & inibidores , Humanos , Hiperalgesia/tratamento farmacológico , Neuralgia/tratamento farmacológico , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Neurotransmissores/fisiologia , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Doenças do Sistema Nervoso Periférico/metabolismo , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Esquizofrenia/tratamento farmacológico , Transtornos Relacionados ao Uso de Substâncias/terapiaRESUMO
Emerging evidence indicates that molecular aging may follow nonlinear or discontinuous trajectories. Whether this occurs in human neuromuscular tissue, particularly for the noncoding transcriptome, and independent of metabolic and aerobic capacities, is unknown. Applying our novel RNA method to quantify tissue coding and long noncoding RNA (lncRNA), we identified ~800 transcripts tracking with age up to ~60 years in human muscle and brain. In silico analysis demonstrated that this temporary linear "signature" was regulated by drugs, which reduce mortality or extend life span in model organisms, including 24 inhibitors of the IGF-1/PI3K/mTOR pathway that mimicked, and 5 activators that opposed, the signature. We profiled Rapamycin in nondividing primary human myotubes (n = 32 HTA 2.0 arrays) and determined the transcript signature for reactive oxygen species in neurons, confirming that our age signature was largely regulated in the "pro-longevity" direction. Quantitative network modeling demonstrated that age-regulated ncRNA equaled the contribution of protein-coding RNA within structures, but tended to have a lower heritability, implying lncRNA may better reflect environmental influences. Genes ECSIT, UNC13, and SKAP2 contributed to a network that did not respond to Rapamycin, and was associated with "neuron apoptotic processes" in protein-protein interaction analysis (FDR = 2.4%). ECSIT links inflammation with the continued age-related downwards trajectory of mitochondrial complex I gene expression (FDR < 0.01%), implying that sustained inhibition of ECSIT may be maladaptive. The present observations link, for the first time, model organism longevity programs with the endogenous but temporary genome-wide responses to aging in humans, revealing a pattern that may ultimately underpin personalized rates of health span.
Assuntos
Envelhecimento/genética , Envelhecimento/metabolismo , Longevidade/genética , RNA Longo não Codificante/genética , Transcriptoma , Adulto , Córtex Cerebral/metabolismo , Redes Reguladoras de Genes , Humanos , Fibras Musculares Esqueléticas/metabolismo , Neurônios/metabolismo , RNA-Seq , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Ativação Transcricional/efeitos dos fármacos , Gêmeos Monozigóticos/genéticaRESUMO
In all eukaryotic organisms, chromatin, the physiological template of all genetic information, is essential for heredity. Chromatin is subject to an array of diverse posttranslational modifications (PTMs) that mostly occur in the amino termini of histone proteins (i.e., histone tail) and regulate the accessibility and functional state of the underlying DNA. Histone tails extend from the core of the nucleosome and are subject to the addition of acetyl groups by histone acetyltransferases (HATs) and the removal of acetyl groups by histone deacetylases (HDACs) during cellular growth and differentiation. Specific acetylation patterns on lysine (K) residues on histone tails determine a dynamic homeostasis between transcriptionally active or transcriptionally repressed chromatin by (1) influencing the core histone assembly and (2) recruiting synergistic or antagonistic chromatin-associated proteins to the transcription site. The fundamental regulatory mechanism of the complex nature of histone tail PTMs influences the majority of chromatin-templated processes and results in changes in cell maturation and differentiation in both normal and pathological development. The goal of the current report is to provide novices with an efficient method to purify core histone proteins from cells and brain tissue and to reliably quantify acetylation marks on histones H3 and H4.
Assuntos
Encéfalo/metabolismo , Epigenômica/métodos , Histonas/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Acetilação , Encéfalo/citologia , HumanosRESUMO
MicroRNA 184 (miR-184) is known to play a key role in neurological development and apoptosis and is highly expressed in mouse brain, mouse corneal epithelium, zebrafish lens and human retinal pigment epithelium (RPE). However, the role of miR-184 in RPE is largely unknown. We investigated the role of miR-184 in RPE and its possible implication in age-related macular degeneration (AMD). Proteomic analysis identified the ezrin (EZR) gene as a target of miR-184 in human RPE. EZR is a membrane cytoskeleton crosslinker that is also known to bind to lysosomal-associated membrane protein 1 (LAMP-1) during the formation of phagocytic vacuoles. In adult retinal pigment epithelium 19 (ARPE19) cells, inhibition of miR-184 resulted in upregulation of EZR mRNA and EZR protein, and induced downregulation of LAMP-1. The inhibition of miR-184 decreased EZR-bound LAMP-1 protein levels and affected phagocytic activity in ARPE19 cells. In primary culture of human RPE isolated from eyes of AMD donors (AMD RPE), miR-184 was significantly downregulated compared with control (normal) RPE. Downregulation of miR-184 was consistent with significantly lower levels of LAMP-1 protein in AMD RPE, and overexpression of MIR-184 in AMD RPE was able to rescue LAMP-1 protein expression to normal levels. Altogether, these observations suggest a novel role for miR-184 in RPE health and support a model proposing that downregulation of miR-184 expression during aging may result in dysregulation of RPE function, contributing to retinal degeneration.
Assuntos
Proteínas do Citoesqueleto/fisiologia , Proteínas de Membrana Lisossomal/fisiologia , Degeneração Macular/etiologia , MicroRNAs/fisiologia , Fagocitose , Epitélio Pigmentado da Retina/metabolismo , Adolescente , Idoso , Idoso de 80 Anos ou mais , Células Cultivadas , Criança , Proteínas do Citoesqueleto/genética , Regulação para Baixo , Feminino , Humanos , Proteínas de Membrana Lisossomal/genética , Degeneração Macular/genética , Degeneração Macular/metabolismo , Masculino , Pessoa de Meia-Idade , Segmento Externo das Células Fotorreceptoras da Retina/metabolismoRESUMO
The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) is inactivated by the extracellular enzyme glutamate carboxypeptidase II. Inhibitors of this enzyme reverse dizocilpine (MK-801)-induced impairment of short-term memory in the novel object recognition test. The objective of this study was to test the hypothesis that NAAG peptidase inhibition enhances long-term (24h delay) memory of C57BL mice. These mice and mice in which glutamate carboxypeptidase II had been knocked out were presented with two identical objects to explore for 10min on day 1 and tested with one of these familiar objects and one novel object on day 2. Memory was assessed as the degree to which the mice recalled the familiar object and explored the novel object to a greater extent on day 2. Uninjected mice or mice injected with saline prior to the acquisition session on day 1 demonstrated a lack of memory of the acquisition experience by exploring the familiar and novel objects to the same extent on day 2. Mice treated with glutamate carboxypeptidase II inhibitors ZJ43 or 2-PMPA prior to the acquisition trial explored the novel object significantly more time than the familiar object on day 2. Consistent with these results, mice in which glutamate carboxypeptidase II had been knocked out distinguished the novel from the familiar object on day 2 while their heterozygous colony mates did not. Inhibition of glutamate carboxypeptidase II enhances recognition memory, a therapeutic action that might be useful in treatment of memory deficits related to age and neurological disorders.
Assuntos
Deleção de Genes , Glutamato Carboxipeptidase II/antagonistas & inibidores , Glutamato Carboxipeptidase II/genética , Memória/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Reconhecimento Psicológico/efeitos dos fármacos , Reconhecimento Psicológico/fisiologia , Animais , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Técnicas de Inativação de Genes , Glutamato Carboxipeptidase II/deficiência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Compostos Organofosforados/farmacologia , Ureia/análogos & derivados , Ureia/farmacologiaRESUMO
Proteolytic fragments of the pigment cell-specific glycoprotein, PMEL, form the amyloid fibrillar matrix underlying melanins in melanosomes. The fibrils form within multivesicular endosomes to which PMEL is selectively sorted and that serve as melanosome precursors. GPNMB is a tissue-restricted glycoprotein with substantial sequence homology to PMEL, but no known function, and was proposed to localize to non-fibrillar domains of distinct melanosome subcompartments in melanocytes. Here we confirm that GPNMB localizes to compartments distinct from the PMEL-containing multivesicular premelanosomes or late endosomes in melanocytes and HeLa cells, respectively, and is largely absent from fibrils. Using domain swapping, the unique PMEL localization is ascribed to its polycystic kidney disease (PKD) domain, whereas the homologous PKD domain of GPNMB lacks apparent sorting function. The difference likely reflects extensive modification of the GPNMB PKD domain by N-glycosylation, nullifying its sorting function. These results reveal the molecular basis for the distinct trafficking and morphogenetic properties of PMEL and GPNMB and support a deterministic function of the PMEL PKD domain in both protein sorting and amyloidogenesis.