RESUMO
Understanding the normal function of the Huntingtin (HTT) protein is of significance in the design and implementation of therapeutic strategies for Huntington's disease (HD). Expansion of the CAG repeat in the HTT gene, encoding an expanded polyglutamine (polyQ) repeat within the HTT protein, causes HD and may compromise HTT's normal activity contributing to HD pathology. Here, we investigated the previously defined role of HTT in autophagy specifically through studying HTT's association with ubiquitin. We find that HTT interacts directly with ubiquitin in vitro. Tandem affinity purification was used to identify ubiquitinated and ubiquitin-associated proteins that copurify with a HTT N-terminal fragment under basal conditions. Copurification is enhanced by HTT polyQ expansion and reduced by mimicking HTT serine 421 phosphorylation. The identified HTT-interacting proteins include RNA-binding proteins (RBPs) involved in mRNA translation, proteins enriched in stress granules, the nuclear proteome, the defective ribosomal products (DRiPs) proteome and the brain-derived autophagosomal proteome. To determine whether the proteins interacting with HTT are autophagic targets, HTT knockout (KO) cells and immunoprecipitation of lysosomes were used to investigate autophagy in the absence of HTT. HTT KO was associated with reduced abundance of mitochondrial proteins in the lysosome, indicating a potential compromise in basal mitophagy, and increased lysosomal abundance of RBPs which may result from compensatory up-regulation of starvation-induced macroautophagy. We suggest HTT is critical for appropriate basal clearance of mitochondrial proteins and RBPs, hence reduced HTT proteostatic function with mutation may contribute to the neuropathology of HD.
Assuntos
Proteína Huntingtina , Lisossomos , Mitocôndrias , Proteínas de Ligação a RNA , Ubiquitina , Proteína Huntingtina/metabolismo , Proteína Huntingtina/genética , Lisossomos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Humanos , Ubiquitina/metabolismo , Mitocôndrias/metabolismo , Autofagia , Animais , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Camundongos , Ligação Proteica , Doença de Huntington/metabolismo , Doença de Huntington/genética , Doença de Huntington/patologia , Peptídeos/metabolismoRESUMO
The human apolipoprotein E4 isoform (APOE4) is the strongest genetic risk factor for late-onset Alzheimer's disease (AD), and lysosomal dysfunction has been implicated in AD pathogenesis. We found, by examining cells stably expressing each APOE isoform, that APOE4 increases lysosomal trafficking, accumulates in enlarged lysosomes and late endosomes, alters autophagic flux and the abundance of autophagy proteins and lipid droplets, and alters the proteomic contents of lysosomes following internalization. We investigated APOE-related lysosomal trafficking further in cell culture, and found that APOE from the post-Golgi compartment is degraded through autophagy. We found that this autophagic process requires the lysosomal membrane protein LAMP2 in immortalized neuron-like and hepatic cells, and in mouse brain tissue. Several macroautophagy-associated proteins were also required for autophagic degradation and internalization of APOE in hepatic cells. The dysregulated autophagic flux and lysosomal trafficking of APOE4 that we observed suggest a possible novel mechanism that might contribute to AD pathogenesis. This article has an associated First Person interview with the first author of the paper.
Assuntos
Doença de Alzheimer , Proteômica , Doença de Alzheimer/genética , Animais , Apolipoproteína E4/genética , Apolipoproteínas E/genética , Autofagia , Lisossomos , Camundongos , Isoformas de Proteínas/genéticaRESUMO
Huntington's disease (HD), a genetic neurodegenerative disorder, primarily affects the striatum and cortex with progressive loss of medium-sized spiny neurons (MSNs) and pyramidal neurons, disrupting cortico-striatal circuitry. A promising regenerative therapeutic strategy of transplanting human neural stem cells (hNSCs) is challenged by the need for long-term functional integration. We previously described that, with short-term hNSC transplantation into the striatum of HD R6/2 mice, human cells differentiated into electrophysiologically active immature neurons, improving behavior and biochemical deficits. Here, we show that long-term (8 months) implantation of hNSCs into the striatum of HD zQ175 mice ameliorates behavioral deficits, increases brain-derived neurotrophic factor (BDNF) levels, and reduces mutant huntingtin (mHTT) accumulation. Patch clamp recordings, immunohistochemistry, single-nucleus RNA sequencing (RNA-seq), and electron microscopy demonstrate that hNSCs differentiate into diverse neuronal populations, including MSN- and interneuron-like cells, and form connections. Single-nucleus RNA-seq analysis also shows restoration of several mHTT-mediated transcriptional changes of endogenous striatal HD mouse cells. Remarkably, engrafted cells receive synaptic inputs, innervate host neurons, and improve membrane and synaptic properties. Overall, the findings support hNSC transplantation for further evaluation and clinical development for HD.
Assuntos
Doença de Huntington , Células-Tronco Neurais , Humanos , Camundongos , Animais , Doença de Huntington/genética , Doença de Huntington/terapia , Corpo Estriado , Neurônios , Fenótipo , Modelos Animais de Doenças , Camundongos Transgênicos , Proteína Huntingtina/genéticaRESUMO
DNA damage repair genes are modifiers of disease onset in Huntington's disease (HD), but how this process intersects with associated disease pathways remains unclear. Here we evaluated the mechanistic contributions of protein inhibitor of activated STAT-1 (PIAS1) in HD mice and HD patient-derived induced pluripotent stem cells (iPSCs) and find a link between PIAS1 and DNA damage repair pathways. We show that PIAS1 is a component of the transcription-coupled repair complex, that includes the DNA damage end processing enzyme polynucleotide kinase-phosphatase (PNKP), and that PIAS1 is a SUMO E3 ligase for PNKP. Pias1 knockdown (KD) in HD mice had a normalizing effect on HD transcriptional dysregulation associated with synaptic function and disease-associated transcriptional coexpression modules enriched for DNA damage repair mechanisms as did reduction of PIAS1 in HD iPSC-derived neurons. KD also restored mutant HTT-perturbed enzymatic activity of PNKP and modulated genomic integrity of several transcriptionally normalized genes. The findings here now link SUMO modifying machinery to DNA damage repair responses and transcriptional modulation in neurodegenerative disease.
Assuntos
Enzimas Reparadoras do DNA/genética , Reparo do DNA , DNA/genética , Proteína Huntingtina/genética , Doença de Huntington/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Inibidoras de STAT Ativados/genética , Processamento de Proteína Pós-Traducional , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Animais , Diferenciação Celular , DNA/metabolismo , Dano ao DNA , Enzimas Reparadoras do DNA/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/metabolismo , Neurônios/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/patologia , Cultura Primária de Células , Proteínas Inibidoras de STAT Ativados/antagonistas & inibidores , Proteínas Inibidoras de STAT Ativados/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/antagonistas & inibidores , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Transcrição GênicaRESUMO
Transcriptional and epigenetic alterations occur early in Huntington's disease (HD), and treatment with epigenetic modulators is beneficial in several HD animal models. The drug JQ1, which inhibits histone acetyl-lysine reader bromodomains, has shown promise for multiple cancers and neurodegenerative disease. We tested whether JQ1 could improve behavioral phenotypes in the R6/2 mouse model of HD and modulate HD-associated changes in transcription and epigenomics. R6/2 and non-transgenic (NT) mice were treated with JQ1 daily from 5 to 11 weeks of age and behavioral phenotypes evaluated over this period. Following the trial, cortex and striatum were isolated and subjected to mRNA-seq and ChIP-seq for the histone marks H3K4me3 and H3K27ac. Initially, JQ1 enhanced motor performance in NT mice. In R6/2 mice, however, JQ1 had no effect on rotarod or grip strength but exacerbated weight loss and worsened performance on the pole test. JQ1-induced gene expression changes in NT mice were distinct from those in R6/2 and primarily involved protein translation and bioenergetics pathways. Dysregulation of HD-related pathways in striatum was exacerbated by JQ1 in R6/2 mice, but not in NTs, and JQ1 caused a corresponding increase in the formation of a mutant huntingtin protein-dependent high molecular weight species associated with pathogenesis. This study suggests that drugs predicted to be beneficial based on their mode of action and effects in wild-type or in other neurodegenerative disease models may have an altered impact in the HD context. These observations have important implications in the development of epigenetic modulators as therapies for HD.
Assuntos
Azepinas/farmacologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Triazóis/farmacologia , Acetilação , Animais , Escala de Avaliação Comportamental , Sintomas Comportamentais/tratamento farmacológico , Córtex Cerebral/patologia , Sequenciamento de Cromatina por Imunoprecipitação , Corpo Estriado/patologia , Modelos Animais de Doenças , Metabolismo Energético/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Ontologia Genética , Histonas/metabolismo , Proteína Huntingtina/genética , Doença de Huntington/tratamento farmacológico , Doença de Huntington/genética , Doença de Huntington/patologia , Masculino , Camundongos , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , RNA-Seq , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Huntington's disease is associated with a reactive microglial response and consequent inflammation. To address the role of these cells in disease pathogenesis, we depleted microglia from R6/2 mice, a rapidly progressing model of Huntington's disease marked by behavioural impairment, mutant huntingtin (mHTT) accumulation, and early death, through colony-stimulating factor 1 receptor inhibition (CSF1Ri) with pexidartinib (PLX3397) for the duration of disease. Although we observed an interferon gene signature in addition to downregulated neuritogenic and synaptic gene pathways with disease, overt inflammation was not evident by microglial morphology or cytokine transcript levels in R6/2 mice. Nonetheless, CSF1Ri-induced microglial elimination reduced or prevented disease-related grip strength and object recognition deficits, mHTT accumulation, astrogliosis, and striatal volume loss, the latter of which was not associated with reductions in cell number but with the extracellular accumulation of chondroitin sulphate proteoglycans (CSPGs)-a primary component of glial scars. A concurrent loss of proteoglycan-containing perineuronal nets was also evident in R6/2 mice, and microglial elimination not only prevented this but also strikingly increased perineuronal nets in the brains of naïve littermates, suggesting a new role for microglia as homeostatic regulators of perineuronal net formation and integrity.
Assuntos
Aminopiridinas/farmacologia , Matriz Extracelular/efeitos dos fármacos , Proteína Huntingtina/efeitos dos fármacos , Doença de Huntington/metabolismo , Microglia/efeitos dos fármacos , Neostriado/efeitos dos fármacos , Pirróis/farmacologia , Reconhecimento Psicológico/efeitos dos fármacos , Animais , Astrócitos/efeitos dos fármacos , Proteoglicanas de Sulfatos de Condroitina/efeitos dos fármacos , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Citocinas/efeitos dos fármacos , Citocinas/genética , Modelos Animais de Doenças , Regulação para Baixo , Matriz Extracelular/metabolismo , Força da Mão , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/patologia , Doença de Huntington/fisiopatologia , Inflamação , Camundongos , Camundongos Transgênicos , Neostriado/patologia , Neuritos/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Sinapses/efeitos dos fármacos , TranscriptomaRESUMO
Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the first exon of the HTT gene encoding huntingtin. Prior reports have established a correlation between CAG expanded HTT and altered gene expression. However, the mechanisms leading to disruption of RNA processing in HD remain unclear. Here, our analysis of the reported HTT protein interactome identifies interactions with known RNA-binding proteins (RBPs). Total, long-read sequencing and targeted RASL-seq of RNAs from cortex and striatum of the HD mouse model R6/2 reveals increased exon skipping which is confirmed in Q150 and Q175 knock-in mice and in HD human brain. We identify the RBP TDP-43 and the N6-methyladenosine (m6A) writer protein methyltransferase 3 (METTL3) to be upstream regulators of exon skipping in HD. Along with this novel mechanistic insight, we observe decreased nuclear localization of TDP-43 and cytoplasmic accumulation of phosphorylated TDP-43 in HD mice and human brain. In addition, TDP-43 co-localizes with HTT in human HD brain forming novel nuclear aggregate-like bodies distinct from mutant HTT inclusions or previously observed TDP-43 pathologies. Binding of TDP-43 onto RNAs encoding HD-associated differentially expressed and aberrantly spliced genes is decreased. Finally, m6A RNA modification is reduced on RNAs abnormally expressed in striatum from HD R6/2 mouse brain, including at clustered sites adjacent to TDP-43 binding sites. Our evidence supports TDP-43 loss of function coupled with altered m6A modification as a novel mechanism underlying alternative splicing/unannotated exon usage in HD and highlights the critical nature of TDP-43 function across multiple neurodegenerative diseases.
RESUMO
The complexity of affected brain regions and cell types is a challenge for Huntington's disease (HD) treatment. Here we use single nucleus RNA sequencing to investigate molecular pathology in the cortex and striatum from R6/2 mice and human HD post-mortem tissue. We identify cell type-specific and -agnostic signatures suggesting oligodendrocytes (OLs) and oligodendrocyte precursors (OPCs) are arrested in intermediate maturation states. OL-lineage regulators OLIG1 and OLIG2 are negatively correlated with CAG length in human OPCs, and ATACseq analysis of HD mouse NeuN-negative cells shows decreased accessibility regulated by OL maturation genes. The data implicates glucose and lipid metabolism in abnormal cell maturation and identify PRKCE and Thiamine Pyrophosphokinase 1 (TPK1) as central genes. Thiamine/biotin treatment of R6/1 HD mice to compensate for TPK1 dysregulation restores OL maturation and rescues neuronal pathology. Our insights into HD OL pathology spans multiple brain regions and link OL maturation deficits to abnormal thiamine metabolism.
Assuntos
Biotina , Doença de Huntington , Oligodendroglia , Tiamina , Animais , Humanos , Camundongos , Biotina/metabolismo , Biotina/farmacologia , Suplementos Nutricionais , Modelos Animais de Doenças , Doença de Huntington/metabolismo , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Oligodendroglia/metabolismo , Núcleo Solitário/metabolismo , Tiamina/metabolismo , Tiamina/farmacologiaRESUMO
BACKGROUND & AIMS: Intestinal thiamin uptake process is vital for maintaining normal body homeostasis of the vitamin; in vitro studies suggest that both thiamin transporter-1 (THTR-1) and -2 (THTR-2) are involved. Mutations in THTR-1 cause thiamin-responsive megaloblastic anemia, a tissue-specific disease associated with diabetes mellitus, megaloblastic anemia, and sensorineural deafness. However, in patients with thiamin-responsive megaloblastic anemia, plasma thiamin levels are within normal range, indicating that THTR-2 (or another carrier) could provide sufficient intestinal thiamin absorption. We tested this possibility and examined the role of THTR-2 in uptake of thiamin in the intestine of mice. METHODS: THTR-2-deficient mice were generated by SLC19A3 gene knockout and used to examine intestinal uptake of thiamin in vitro (isolated cells) and in vivo (intact intestinal loops). We also examined intestinal thiamin uptake in THTR-1-deficient mice. RESULTS: Intestine of THTR-2-deficient mice had reduced uptake of thiamin compared with those of wild-type littermate mice (P < .01); this reduction was associated with a decrease (P < .01) in blood thiamin levels in THTR-2-deficient mice. However, intestinal uptake of thiamin in THTR-1-deficient mice was not significantly different from that of wild-type littermate animals. Level of expression of THTR-1 was not altered in the intestine of THTR-2-deficient mice, but level of expression of THTR-2 was up-regulated in the intestine of THTR-1-deficient mice. CONCLUSIONS: THTR-2 is required for normal uptake of thiamin in the intestine and can fulfill normal levels of uptake in conditions associated with THTR-1 dysfunction.
Assuntos
Absorção Intestinal , Jejuno/metabolismo , Proteínas de Membrana Transportadoras/deficiência , Tiamina/metabolismo , Animais , Comportamento Animal , Transporte Biológico , Regulação da Expressão Gênica , Genótipo , Mucosa Intestinal/metabolismo , Rim/metabolismo , Rim/patologia , Fígado/metabolismo , Fígado/patologia , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , RNA Mensageiro/metabolismo , Tiamina/sangueRESUMO
Chronic cellular stress associated with neurodegenerative disease can result in the persistence of stress granule (SG) structures, membraneless organelles that form in response to cellular stress. In Huntington's disease (HD), chronic expression of mutant huntingtin generates various forms of cellular stress, including activation of the unfolded protein response and oxidative stress. However, it has yet to be determined whether SGs are a feature of HD neuropathology. We examined the miRNA composition of extracellular vesicles (EVs) present in the cerebrospinal fluid (CSF) of patients with HD and show that a subset of their target mRNAs were differentially expressed in the prefrontal cortex. Of these targets, SG components were enriched, including the SG-nucleating Ras GTPase-activating protein-binding protein 1 (G3BP1). We investigated localization and levels of G3BP1 and found a significant increase in the density of G3BP1-positive granules in the cortex and hippocampus of R6/2 transgenic mice and in the superior frontal cortex of the brains of patients with HD. Intriguingly, we also observed that the SG-associated TAR DNA-binding protein 43 (TDP43), a nuclear RNA/DNA binding protein, was mislocalized to the cytoplasm of G3BP1 granule-positive HD cortical neurons. These findings suggest that G3BP1 SG dynamics may play a role in the pathophysiology of HD.
Assuntos
Grânulos Citoplasmáticos/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Hipocampo/metabolismo , Doença de Huntington/metabolismo , Neurônios/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Córtex Pré-Frontal/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Animais , Grânulos Citoplasmáticos/patologia , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Feminino , Hipocampo/patologia , Humanos , Doença de Huntington/genética , Doença de Huntington/patologia , Masculino , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Neurônios/patologia , Proteínas de Ligação a Poli-ADP-Ribose/genética , Córtex Pré-Frontal/patologia , Transporte Proteico/genética , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/genéticaRESUMO
The human sodium-dependent vitamin C transporters (hSVCT1 and hSVCT2) mediate cellular uptake of ascorbic acid. Both these transporters contain potential sites for N-glycosylation in their extracellular domains (Asn-138, Asn-144 [hSVCT1]; Asn-188, Asn-196 [hSVCT2]), however the role of N-glycosylation in transporter function is unexplored. On the basis of the result that tunicamycin decreased (14)C-ascorbic acid uptake in HepG2 cells, we systematically ablated all consensus N-glycosylation sites in hSVCT1 and hSVCT2 to resolve any effects on ascorbic acid uptake, transporter expression and targeting. We show that removal of individual N-glycosylation sites significantly impairs protein expression and consequently ascorbic acid uptake for hSVCT1 mutants (N138Q is retained intracellularly) and for hSVCT2 mutants (all of which reach the cell surface). N-Glycosylation is therefore essential for vitamin C transporter functionality.
Assuntos
Ácido Ascórbico/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Linhagem Celular , Membrana Celular/metabolismo , Glicosilação , Humanos , Mutação , Transportadores de Ânions Orgânicos Dependentes de Sódio/antagonistas & inibidores , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Estrutura Secundária de Proteína , Transportadores de Sódio Acoplados à Vitamina C , Simportadores/antagonistas & inibidores , Simportadores/genética , Tunicamicina/farmacologiaRESUMO
The principal symptoms of Huntington's disease (HD), chorea, cognitive deficits, and psychiatric symptoms are associated with the massive loss of striatal and cortical projection neurons. As current drug therapies only partially alleviate symptoms, finding alternative treatments has become peremptory. Cell replacement using stem cells is a rapidly expanding field that offers such an alternative. In this review, we examine recent studies that use mesenchymal cells, as well as pluripotent, cell-derived products in animal models of HD. Additionally, we provide further electrophysiological characterization of a human neural stem cell line, ESI-017, which has already demonstrated disease-modifying properties in two mouse models of HD. Overall, the field of regenerative medicine represents a viable and promising avenue for the treatment of neurodegenerative disorders including HD.
Assuntos
Doença de Huntington/fisiopatologia , Doença de Huntington/terapia , Transplante de Células-Tronco , Animais , Modelos Animais de Doenças , Humanos , RoedoresRESUMO
Huntington's disease (HD) is an inherited neurodegenerative disorder with no disease-modifying treatment. Expansion of the glutamine-encoding repeat in the Huntingtin (HTT) gene causes broad effects that are a challenge for single treatment strategies. Strategies based on human stem cells offer a promising option. We evaluated efficacy of transplanting a good manufacturing practice (GMP)-grade human embryonic stem cell-derived neural stem cell (hNSC) line into striatum of HD modeled mice. In HD fragment model R6/2 mice, transplants improve motor deficits, rescue synaptic alterations, and are contacted by nerve terminals from mouse cells. Furthermore, implanted hNSCs are electrophysiologically active. hNSCs also improved motor and late-stage cognitive impairment in a second HD model, Q140 knockin mice. Disease-modifying activity is suggested by the reduction of aberrant accumulation of mutant HTT protein and expression of brain-derived neurotrophic factor (BDNF) in both models. These findings hold promise for future development of stem cell-based therapies.
Assuntos
Cognição , Doença de Huntington/terapia , Atividade Motora , Células-Tronco Neurais/transplante , Recuperação de Função Fisiológica , Animais , Linhagem Celular , Modelos Animais de Doenças , Xenoenxertos , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/patologia , Humanos , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Doença de Huntington/fisiopatologia , Camundongos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologiaRESUMO
Uptake of vitamin C occurs through the Na+ -dependent vitamin C transporters (SVCT1 and 2), the products of two separate genes. In cultured human vascular smooth muscle cells (hVSMC), we found expression of only the hSVCT2 transcript and identified an additional 5'-UTR transcript variant that we termed exon 1b, in addition to the previously described exon 1a. We cloned and tested the promoter functionality of the two genomic regions of the hSVCT2 upstream of these alternative first exons in hVSMC. Both demonstrated activity, and deletion constructs demonstrated that the minimal promoter regions were within approximately 100 bp relative to their adjacent exons.
Assuntos
Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Sequências Reguladoras de Ácido Nucleico/genética , Simportadores/genética , Regiões 5' não Traduzidas/genética , Sequência de Bases , Células CACO-2 , Células Cultivadas , Éxons/genética , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transportadores de Sódio Acoplados à Vitamina CRESUMO
The disruption of protein quality control networks is central to pathology in Huntington's disease (HD) and other neurodegenerative disorders. The aberrant accumulation of insoluble high-molecular-weight protein complexes containing the Huntingtin (HTT) protein and SUMOylated protein corresponds to disease manifestation. We previously identified an HTT-selective E3 SUMO ligase, PIAS1, that regulates HTT accumulation and SUMO modification in cells. Here we investigated whether PIAS1 modulation in neurons alters HD-associated phenotypes in vivo. Instrastriatal injection of a PIAS1-directed miRNA significantly improved behavioral phenotypes in rapidly progressing mutant HTT (mHTT) fragment R6/2 mice. PIAS1 reduction prevented the accumulation of mHTT and SUMO- and ubiquitin-modified proteins, increased synaptophysin levels, and normalized key inflammatory markers. In contrast, PIAS1 overexpression exacerbated mHTT-associated phenotypes and aberrant protein accumulation. These results confirm the association between aberrant accumulation of expanded polyglutamine-dependent insoluble protein species and pathogenesis, and they link phenotypic benefit to reduction of these species through PIAS1 modulation.
Assuntos
Encéfalo/metabolismo , Doença de Huntington/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Animais , Modelos Animais de Doenças , Humanos , Proteína Huntingtina/genética , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , FenótipoRESUMO
The liver is an important site for thiamin metabolism, utilization, and storage. Little is known about the mechanism of thiamin uptake by the human liver. In this study, we examined cellular and molecular aspects of the human liver thiamin uptake process using the human-derived liver HepG2 cells as a model system. Our studies showed that the initial rate of thiamin uptake to be: (1) Na(+)-independent and occurs with no detectable metabolic alterations in the transported substrate, (2) highly pH-dependent with diminished uptake upon decreasing incubation buffer pH from 8.0 to 5.0, (3) higher following cell acidification compared to unacidified control cells, (4) saturable as a function of concentration with an apparent K(m) of 7.7+/-1.6 microM, (5) inhibited by the thiamin structural analogues oxythiamin and amprolium but not by the unrelated organic cations tetraethylammonium (TEA) and N-methylnicotinamide (NMN), and (6) inhibited in a concentration-dependent manner by the membrane transport inhibitor amiloride. Both of the recently cloned human thiamin transporters, i.e., SLC19A2 and SLC19A3, were found to be expressed in liver HepG2 cells with the former being the predominant form. High promoter activity of the predominant form, i.e., SLC19A2, was detected in HepG2 cells, and the minimal region of the SLC19A2 promoter required for its basal activity in these cells was found to be encoded in a sequence between -356 and -36 and has multiple putative cis-regulatory elements. Mutation of a number of these putative cis-elements diminished promoter activity of the SLC19A2 minimal region. These results show the involvement of a specialized carrier-mediated mechanism for thiamin uptake by human liver HepG2 cells. In addition, SLC19A2 was found to be the predominant thiamin uptake carrier expressed in these cells and its promoter displays a high level of activity in them.
Assuntos
Fígado/metabolismo , Tiamina/metabolismo , Sequência de Bases , Linhagem Celular , Primers do DNA , Humanos , Fígado/citologia , Proteínas de Membrana Transportadoras/genética , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The molecular mechanism and regulation of the intestinal uptake process of dietary thiamine is not well understood. Previous studies have established the involvement of a carrier-mediated system for thiamine uptake in the human intestine. Recently a human thiamine transporter, SLC19A2, was cloned from a number of human tissues. Little, however, is known about expression of the SLC19A2 message along the native human gastrointestinal tract, and no analysis of its promoter in intestinal tissue is available. Therefore, the current studies were aimed at investigating the expression of SLC19A2 in the human gastrointestinal tract and at analyzing the promoter of this potential intestinal thiamine transporter. First we cloned SLC19A2 cDNA from a human intestinal cell line (Caco-2) by reverse transcriptase-polymerase chain reaction, then used this cDNA as a probe in Northern blot analysis. SLC19A2 message was found to be expressed in all gastrointestinal tissues in the following order: liver>stomach>duodenum>jejunum>colon>cecum>rectum>ileum. SLC19A2 was also expressed at the protein level in Caco-2 cells and in native human small intestine by Western blot analysis. We also cloned the 5'-regulatory region of the SLC19A2 gene and confirmed activity of its promoter following transfection into intestinal epithelial Caco-2 cells. Furthermore, we identified the minimal promoter region required for basal activity of SLC19A2 in these cells which was found to be mainly encoded in a sequence between -356 and -36, and included multiple cis-regulatory elements. Transcription initiation sites of the SLC19A2 gene in intestinal epithelial Caco-2 cells were also identified by 5'-rapid amplification of cDNA ends. These results demonstrate that SLC19A2 is expressed in various regions of the human gastrointestinal tract. In addition, the results provide the first characterization of the SLC19A2 promoter. These findings raise the possibility that SLC19A2 may play a role in the normal intestinal thiamine absorption process.
Assuntos
Mucosa Intestinal/metabolismo , Proteínas de Membrana Transportadoras/genética , Regiões Promotoras Genéticas , Sequência de Bases , Células CACO-2 , DNA Complementar/biossíntese , Humanos , Jejuno/metabolismo , Fígado/metabolismo , Proteínas de Membrana Transportadoras/biossíntese , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Tiamina/metabolismoRESUMO
Ascorbic acid, the active form of vitamin C, is a vital antioxidant in the human liver, yet the molecular mechanisms involved in the regulation of ascorbic acid transporters [human sodium-dependent vitamin C transporters (hSVCT) 1 and 2] in liver cells are poorly understood. Therefore, we characterized the minimal promoter regions of hSVCT1 and 2 in cultured human liver epithelial cells (HepG2) and examined the effects of ascorbic acid deprivation and supplementation on activity and regulation of the transport systems. Identified minimal promoters required for basal activity were found to include multiple cis regulatory elements, whereas mutational analysis demonstrated that HNF-1 sites in the hSVCT1 promoter and KLF/Sp1 sites in the hSVCT2 promoter were essential for activities. When cultured in ascorbic acid deficient or supplemented media, HepG2 cells demonstrated significant (P<.01) and specific reciprocal changes in [(14)C]-Ascorbic acid uptake, and in hSVCT1 mRNA and protein levels as well as hSVCT1 promoter activity. However, no significant changes in hSVCT2 expression or promoter activity were observed during ascorbic acid deficient or supplemented conditions. We mapped the ascorbic acid responsive region in the hSVCT1 promoter and determined that HNF-1 sites are important for the adaptive regulation response. The results of these studies further characterize the hSVCT1 and 2 promoters establish that ascorbic acid uptake by human liver epithelial cells is adaptively regulated and show that transcriptional mechanisms via HNF-1 in the hSVCT1 promoter may, in part, be involved in this regulation.
Assuntos
Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Simportadores/genética , Ácido Ascórbico/metabolismo , Ácido Ascórbico/farmacologia , Deficiência de Ácido Ascórbico/fisiopatologia , Regulação da Expressão Gênica , Células Hep G2 , Fator 1 Nuclear de Hepatócito/fisiologia , Humanos , Regiões Promotoras Genéticas/fisiologia , Transportadores de Sódio Acoplados à Vitamina CRESUMO
Differentiation of intestinal epithelial cells is accompanied by alterations in levels of expression of many genes, including those involved in nutrient uptake. Effects of differentiation of intestinal epithelial cells on the physiological and molecular parameters of the intestinal folate uptake process are not well characterized. To address this issue, we used two models, Caco-2 cells and native mouse intestine. Studies with Caco-2 cells showed a significant increase in the initial rate of carrier-mediated folic acid uptake during differentiation (i.e., as the cells transitioned from preconfluent to confluent and then to postconfluent stages). This increase was associated with an increase in the level of expression of the human reduced folate carrier (hRFC) and the human proton-coupled folate transporter (hPCFT) both at the protein and mRNA levels with differentiation; it was also associated with a significant increase in activity of the hRFC and hPCFT promoters. Studies with native mouse intestine showed a significantly higher folate uptake in villus compared with crypt cells, which was again associated with a significantly higher level of expression of the mouse RFC and PCFT at the protein and mRNA levels. Together, these studies demonstrate that the intestinal folate uptake process undergoes differentiation-dependent regulation and that this regulation is mediated via changes in the level of expression of both the RFC and PCFT. In addition, the studies suggest the possible involvement (at least in part) of a transcriptional mechanism(s) in this type of regulation of the intestinal folate uptake process.
Assuntos
Diferenciação Celular , Células Epiteliais/metabolismo , Ácido Fólico/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Animais , Células CACO-2 , Diferenciação Celular/genética , Proliferação de Células , Humanos , Cinética , Proteínas de Membrana Transportadoras/genética , Camundongos , Regiões Promotoras Genéticas , Transportador de Folato Acoplado a Próton , RNA Mensageiro/metabolismo , Proteína Carregadora de Folato Reduzido , Transcrição Gênica , Regulação para CimaRESUMO
Humans use two sodium-ascorbate cotransporters (hSVCT1 and hSVCT2) for transporting the dietary essential micronutrient ascorbic acid, the reduced and active form of vitamin C. Although the human liver plays a pivotal role in regulating and maintaining vitamin C homeostasis, vitamin C transport physiology and regulation of the hSVCT systems in this organ have not been well defined. Thus, this research used a human hepatic cell line (HepG2), confirming certain results with primary human hepatocytes and determined the initial rate of ascorbic acid uptake to be Na(+) gradient, pH dependent, and saturable as a function of concentration over low and high micromolar ranges. Additionally, hSVCT2 protein and mRNA are expressed at higher levels in HepG2 cells and native human liver, and the cloned hSVCT2 promoter has more activity in HepG2 cells. Results using short interfering RNA suggest that in HepG2 cells, decreasing hSVCT2 message levels reduces the overall ascorbic acid uptake process more than decreasing hSVCT1 message levels. Activation of PKC intracellular regulatory pathways caused a downregulation in ascorbic acid uptake not mediated by a single predicted PKC-specific amino acid phosphorylation site in hSVCT1 or hSVCT2. However, PKC activation causes internalization of hSVCT1 but not hSVCT2. Examination of other intracellular regulatory pathways on ascorbic acid uptake determined that regulation also potentially occurs by PKA, PTK, and Ca(2+)/calmodulin, but not by nitric oxide-dependent pathways. These studies are the first to determine the overall ascorbic acid uptake process and relative expression, regulation, and contribution of the hSVCT systems in human liver epithelial cells.