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1.
Proc Natl Acad Sci U S A ; 121(16): e2322924121, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38607933

RESUMO

Many Mendelian disorders, such as Huntington's disease (HD) and spinocerebellar ataxias, arise from expansions of CAG trinucleotide repeats. Despite the clear genetic causes, additional genetic factors may influence the rate of those monogenic disorders. Notably, genome-wide association studies discovered somewhat expected modifiers, particularly mismatch repair genes involved in the CAG repeat instability, impacting age at onset of HD. Strikingly, FAN1, previously unrelated to repeat instability, produced the strongest HD modification signals. Diverse FAN1 haplotypes independently modify HD, with rare genetic variants diminishing DNA binding or nuclease activity of the FAN1 protein, hastening HD onset. However, the mechanism behind the frequent and the most significant onset-delaying FAN1 haplotype lacking missense variations has remained elusive. Here, we illustrated that a microRNA acting on 3'-UTR (untranslated region) SNP rs3512, rather than transcriptional regulation, is responsible for the significant FAN1 expression quantitative trait loci signal and allelic imbalance in FAN1 messenger ribonucleic acid (mRNA), accounting for the most significant and frequent onset-delaying modifier haplotype in HD. Specifically, miR-124-3p selectively targets the reference allele at rs3512, diminishing the stability of FAN1 mRNA harboring that allele and consequently reducing its levels. Subsequent validation analyses, including the use of antagomir and 3'-UTR reporter vectors with swapped alleles, confirmed the specificity of miR-124-3p at rs3512. Together, these findings indicate that the alternative allele at rs3512 renders the FAN1 mRNA less susceptible to miR-124-3p-mediated posttranscriptional regulation, resulting in increased FAN1 levels and a subsequent delay in HD onset by mitigating CAG repeat instability.


Assuntos
Doença de Huntington , MicroRNAs , Humanos , Regiões 3' não Traduzidas/genética , Endodesoxirribonucleases , Exodesoxirribonucleases/genética , Estudo de Associação Genômica Ampla , Doença de Huntington/genética , MicroRNAs/genética , Enzimas Multifuncionais
2.
Hum Mol Genet ; 32(1): 30-45, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-35908190

RESUMO

Huntington's disease (HD) is a neurodegenerative disorder caused by an inherited unstable HTT CAG repeat that expands further, thereby eliciting a disease process that may be initiated by polyglutamine-expanded huntingtin or a short polyglutamine-product. Phosphorylation of selected candidate residues is reported to mediate polyglutamine-fragment degradation and toxicity. Here to support the discovery of phosphosites involved in the life-cycle of (full-length) huntingtin, we employed mass spectrometry-based phosphoproteomics to systematically identify sites in purified huntingtin and in the endogenous protein by proteomic and phosphoproteomic analyses of members of an HD neuronal progenitor cell panel. Our results bring total huntingtin phosphosites to 95, with more located in the N-HEAT domain relative to numbers in the Bridge and C-HEAT domains. Moreover, phosphorylation of C-HEAT Ser2550 by cAMP-dependent protein kinase (PKA), the top hit in kinase activity screens, was found to hasten huntingtin degradation, such that levels of the catalytic subunit (PRKACA) were inversely related to huntingtin levels. Taken together, these findings highlight categories of phosphosites that merit further study and provide a phosphosite kinase pair (pSer2550-PKA) with which to investigate the biological processes that regulate huntingtin degradation and thereby influence the steady state levels of huntingtin in HD cells.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico , Doença de Huntington , Humanos , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Temperatura Alta , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/metabolismo , Fosforilação , Domínios Proteicos , Proteômica
3.
Am J Hum Genet ; 109(5): 885-899, 2022 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-35325614

RESUMO

Genome-wide association studies (GWASs) of Huntington disease (HD) have identified six DNA maintenance gene loci (among others) as modifiers and implicated a two step-mechanism of pathogenesis: somatic instability of the causative HTT CAG repeat with subsequent triggering of neuronal damage. The largest studies have been limited to HD individuals with a rater-estimated age at motor onset. To capitalize on the wealth of phenotypic data in several large HD natural history studies, we have performed algorithmic prediction by using common motor and cognitive measures to predict age at other disease landmarks as additional phenotypes for GWASs. Combined with imputation with the Trans-Omics for Precision Medicine reference panel, predictions using integrated measures provided objective landmark phenotypes with greater power to detect most modifier loci. Importantly, substantial differences in the relative modifier signal across loci, highlighted by comparing common modifiers at MSH3 and FAN1, revealed that individual modifier effects can act preferentially in the motor or cognitive domains. Individual components of the DNA maintenance modifier mechanisms may therefore act differentially on the neuronal circuits underlying the corresponding clinical measures. In addition, we identified additional modifier effects at the PMS1 and PMS2 loci and implicated a potential second locus on chromosome 7. These findings indicate that broadened discovery and characterization of HD genetic modifiers based on additional quantitative or qualitative phenotypes offers not only the promise of in-human validated therapeutic targets but also a route to dissecting the mechanisms and cell types involved in both the somatic instability and toxicity components of HD pathogenesis.


Assuntos
Doença de Huntington , Cognição , DNA , Estudo de Associação Genômica Ampla , Humanos , Proteína Huntingtina/genética , Doença de Huntington/genética , Doença de Huntington/patologia , Expansão das Repetições de Trinucleotídeos
4.
Anal Chem ; 96(14): 5537-5545, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38545995

RESUMO

The chemical degradation of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-based aqueous energy storage and catalytic systems is pH sensitive. Herein, we voltammetrically monitor the local pH (pHlocal) at a Pt ultramicroelectrode (UME) upon electro-oxidation of imidazolium-linker functionalized TEMPO and show that its decrease is associated with the greater acidity of the cationic (oxidized) rather than radical (reduced) form of TEMPO. The protons that drive the decrease in pH arise from hydrolysis of the conjugated imidazolium-linker functional group of 4-[2-(N-methylimidazolium)acetoxy]-2,2,6,6-tetramethylpiperidine-1-oxyl chloride (MIMAcO-T), which was studied in comparison with 4-hydroxyl-TEMPO (4-OH-T). Voltammetric hysteresis is observed during the electrode oxidation of 4-OH-T and MIMAcO-T at a Pt UME in an unbuffered aqueous solution. The hysteresis arises from the pH-dependent formation and dissolution of Pt oxides, which interact with pHlocal in the vicinity of the UME. We find that electrogenerated MIMAcO-T+ significantly influences pHlocal, whereas 4-OH-T+ does not. Finite element analysis reveals that the thermodynamic and kinetic acid-base properties of MIMAcO-T+ are much more favorable than those of its reduced counterpart. Imidazolium-linker functionalized TEMPO molecules comprising different linking groups were also investigated. Reduced TEMPO molecules with carbonyl linkers behave as weak acids, whereas those with alkyl ether linkers do not. However, oxidized TEMPO+ molecules with alkyl ether linkers exhibit more facile acid-base kinetics than those with carbonyl ones. Density functional theory calculations confirm that OH- adduct formation on the imidazolium-linker functional group of TEMPO is responsible for the difference in the acid-base properties of the reduced and oxidized forms.

5.
Hum Mol Genet ; 30(3-4): 135-148, 2021 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-33432339

RESUMO

Huntington's disease pathogenesis involves a genetic gain-of-function toxicity mechanism triggered by the expanded HTT CAG repeat. Current therapeutic efforts aim to suppress expression of total or mutant huntingtin, though the relationship of huntingtin's normal activities to the gain-of-function mechanism and what the effects of huntingtin-lowering might be are unclear. Here, we have re-investigated a rare family segregating two presumed HTT loss-of-function (LoF) variants associated with the developmental disorder, Lopes-Maciel-Rodan syndrome (LOMARS), using whole-genome sequencing of DNA from cell lines, in conjunction with analysis of mRNA and protein expression. Our findings correct the muddled annotation of these HTT variants, reaffirm they are the genetic cause of the LOMARS phenotype and demonstrate that each variant is a huntingtin hypomorphic mutation. The NM_002111.8: c.4469+1G>A splice donor variant results in aberrant (exon 34) splicing and severely reduced mRNA, whereas, surprisingly, the NM_002111.8: c.8157T>A NP_002102.4: Phe2719Leu missense variant results in abnormally rapid turnover of the Leu2719 huntingtin protein. Thus, although rare and subject to an as yet unknown LoF intolerance at the population level, bona fide HTT LoF variants can be transmitted by normal individuals leading to severe consequences in compound heterozygotes due to huntingtin deficiency.


Assuntos
Regulação da Expressão Gênica , Proteína Huntingtina/genética , Mutação , Transtornos do Neurodesenvolvimento/genética , Sequência de Aminoácidos , Linhagem Celular , Criança , Pré-Escolar , Feminino , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/metabolismo , Mutação com Perda de Função , Masculino , Mutação de Sentido Incorreto , Transtornos do Neurodesenvolvimento/metabolismo , Linhagem , Fenótipo , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA
6.
Am J Hum Genet ; 107(1): 96-110, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32589923

RESUMO

A recent genome-wide association study of Huntington disease (HD) implicated genes involved in DNA maintenance processes as modifiers of onset, including multiple genome-wide significant signals in a chr15 region containing the DNA repair gene Fanconi-Associated Nuclease 1 (FAN1). Here, we have carried out detailed genetic, molecular, and cellular investigation of the modifiers at this locus. We find that missense changes within or near the DNA-binding domain (p.Arg507His and p.Arg377Trp) reduce FAN1's DNA-binding activity and its capacity to rescue mitomycin C-induced cytotoxicity, accounting for two infrequent onset-hastening modifier signals. We also idenified a third onset-hastening modifier signal whose mechanism of action remains uncertain but does not involve an amino acid change in FAN1. We present additional evidence that a frequent onset-delaying modifier signal does not alter FAN1 coding sequence but is associated with increased FAN1 mRNA expression in the cerebral cortex. Consistent with these findings and other cellular overexpression and/or suppression studies, knockout of FAN1 increased CAG repeat expansion in HD-induced pluripotent stem cells. Together, these studies support the process of somatic CAG repeat expansion as a therapeutic target in HD, and they clearly indicate that multiple genetic variations act by different means through FAN1 to influence HD onset in a manner that is largely additive, except in the rare circumstance that two onset-hastening alleles are present. Thus, an individual's particular combination of FAN1 haplotypes may influence their suitability for HD clinical trials, particularly if the therapeutic agent aims to reduce CAG repeat instability.


Assuntos
Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Doença de Huntington/genética , Enzimas Multifuncionais/genética , Linhagem Celular , Estudo de Associação Genômica Ampla/métodos , Células HEK293 , Haplótipos/genética , Humanos , Polimorfismo de Nucleotídeo Único/genética
7.
Hum Mol Genet ; 29(18): 3044-3053, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-32876667

RESUMO

Recent genome-wide association studies of age-at-onset in Huntington's disease (HD) point to distinct modes of potential disease modification: altering the rate of somatic expansion of the HTT CAG repeat or altering the resulting CAG threshold length-triggered toxicity process. Here, we evaluated the mouse orthologs of two HD age-at-onset modifier genes, FAN1 and RRM2B, for an influence on somatic instability of the expanded CAG repeat in Htt CAG knock-in mice. Fan1 knock-out increased somatic expansion of Htt CAG repeats, in the juvenile- and the adult-onset HD ranges, whereas knock-out of Rrm2b did not greatly alter somatic Htt CAG repeat instability. Simultaneous knock-out of Mlh1, the ortholog of a third HD age-at-onset modifier gene (MLH1), which suppresses somatic expansion of the Htt knock-in CAG repeat, blocked the Fan1 knock-out-induced acceleration of somatic CAG expansion. This genetic interaction indicates that functional MLH1 is required for the CAG repeat destabilizing effect of FAN1 loss. Thus, in HD, it is uncertain whether the RRM2B modifier effect on timing of onset may be due to a DNA instability mechanism. In contrast, the FAN1 modifier effects reveal that functional FAN1 acts to suppress somatic CAG repeat expansion, likely in genetic interaction with other DNA instability modifiers whose combined effects can hasten or delay onset and other CAG repeat length-driven phenotypes.


Assuntos
Proteínas de Ciclo Celular/genética , Endodesoxirribonucleases/genética , Exodesoxirribonucleases/genética , Proteína Huntingtina/genética , Doença de Huntington/genética , Enzimas Multifuncionais/genética , Proteína 1 Homóloga a MutL/genética , Ribonucleotídeo Redutases/genética , Idade de Início , Animais , Modelos Animais de Doenças , Genes Modificadores/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Doença de Huntington/patologia , Camundongos , Camundongos Knockout , Fenótipo , Expansão das Repetições de Trinucleotídeos/genética
8.
PLoS Genet ; 14(5): e1007274, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29750799

RESUMO

Modifiers of Mendelian disorders can provide insights into disease mechanisms and guide therapeutic strategies. A recent genome-wide association (GWA) study discovered genetic modifiers of Huntington's disease (HD) onset in Europeans. Here, we performed whole genome sequencing and GWA analysis of a Venezuelan HD cluster whose families were crucial for the original mapping of the HD gene defect. The Venezuelan HD subjects develop motor symptoms earlier than their European counterparts, implying the potential for population-specific modifiers. The main Venezuelan HD family inherits HTT haplotype hap.03, which differs subtly at the sequence level from European HD hap.03, suggesting a different ancestral origin but not explaining the earlier age at onset in these Venezuelans. GWA analysis of the Venezuelan HD cluster suggests both population-specific and population-shared genetic modifiers. Genome-wide significant signals at 7p21.2-21.1 and suggestive association signals at 4p14 and 17q21.2 are evident only in Venezuelan HD, but genome-wide significant association signals at the established European chromosome 15 modifier locus are improved when Venezuelan HD data are included in the meta-analysis. Venezuelan-specific association signals on chromosome 7 center on SOSTDC1, which encodes a bone morphogenetic protein antagonist. The corresponding SNPs are associated with reduced expression of SOSTDC1 in non-Venezuelan tissue samples, suggesting that interaction of reduced SOSTDC1 expression with a population-specific genetic or environmental factor may be responsible for modification of HD onset in Venezuela. Detection of population-specific modification in Venezuelan HD supports the value of distinct disease populations in revealing novel aspects of a disease and population-relevant therapeutic strategies.


Assuntos
Genes Modificadores/genética , Estudo de Associação Genômica Ampla/métodos , Doença de Huntington/genética , Sequenciamento Completo do Genoma/métodos , Proteínas Adaptadoras de Transdução de Sinal , Idade de Início , Saúde da Família , Feminino , Interação Gene-Ambiente , Genética Populacional , Haplótipos , Humanos , Proteína Huntingtina/genética , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Polimorfismo de Nucleotídeo Único , Proteínas/genética , Venezuela
9.
Bioorg Chem ; 102: 104055, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32663666

RESUMO

The UDP-2,3-diacylglucosamine pyrophosphate hydrolase LpxH is essential in lipid A biosynthesis and has emerged as a promising target for the development of novel antibiotics against multidrug-resistant Gram-negative pathogens. Recently, we reported the crystal structure of Klebsiella pneumoniae LpxH in complex with 1 (AZ1), a sulfonyl piperazine LpxH inhibitor. The analysis of the LpxH-AZ1 co-crystal structure and ligand dynamics led to the design of 2 (JH-LPH-28) and 3 (JH-LPH-33) with enhanced LpxH inhibition. In order to harness our recent findings, we prepared and evaluated a series of sulfonyl piperazine analogs with modifications in the phenyl and N-acetyl groups of 3. Herein, we describe the synthesis and structure-activity relationship of sulfonyl piperazine LpxH inhibitors. We also report the structural analysis of an extended N-acyl chain analog 27b (JH-LPH-41) in complex with K. pneumoniae LpxH, revealing that 27b reaches an untapped polar pocket near the di-manganese cluster in the active site of K. pneumoniae LpxH. We expect that our findings will provide designing principles for new LpxH inhibitors and establish important frameworks for the future development of antibiotics against multidrug-resistant Gram-negative pathogens.


Assuntos
Antinematódeos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Piperazina/síntese química , Piperazina/uso terapêutico , Antinematódeos/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Piperazina/farmacologia , Relação Estrutura-Atividade
10.
J Environ Manage ; 260: 110001, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-31941640

RESUMO

An acid-recovering nanofiltration (NF) membrane with both acid resistance and selective acid permeability was fabricated via a water-based coating process for the recovery of hydrochloric acid. To achieve this, a thermally cross-linked branched-polyethyleneimine (b-PEI) layer was introduced to a loose polyethersulfone NF membrane by dip-coating of b-PEI and an epoxy linker and heat treatment in a sealed oven with a high-humidity atmosphere. The resulting membrane displayed a positive surface charge with a zeta potential, and exhibited a rejection performance order of MgCl2> MgSO4> NaCl > Na2SO4 characteristic of positive-charge-separation membranes. Mg rejection and Cl permeation experiments showed that the selective permeation of hydrochloric acid was achieved with Mg rejection above 95% and Cl permeation above 70%, and this allowed the acid to be recovered by obtaining permeate at the same pH as the feed. Moreover, the NF membrane maintained selective separation performance and flow rate for a month.


Assuntos
Filtração , Poluentes Químicos da Água , Ácido Clorídrico , Membranas Artificiais , Permeabilidade
11.
Hum Mol Genet ; 26(7): 1258-1267, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28165127

RESUMO

Huntington's disease (HD) reflects dominant consequences of a CAG repeat expansion mutation in HTT. Expanded CAG repeat size is the primary determinant of age at onset and age at death in HD. Although HD pathogenesis is driven by the expanded CAG repeat, whether the mutation influences the expression levels of mRNA and protein from the disease allele is not clear due to the lack of sensitive allele-specific quantification methods and the presence of confounding factors. To determine the impact of CAG expansion at the molecular level, we have developed novel allele-specific HTT mRNA and protein quantification methods based on principles of multiplex ligation-dependent probe amplification and targeted MS/MS parallel reaction monitoring, respectively. These assays, exhibiting high levels of specificity and sensitivity, were designed to distinguish allelic products based upon expressed polymorphic variants in HTT, including rs149 109 767. To control for other cis-haplotype variations, we applied allele-specific quantification assays to a panel of HD lymphoblastoid cell lines, each carrying the major European disease haplotype (i.e. hap.01) on the mutant chromosome. We found that steady state levels of HTT mRNA and protein were not associated with expanded CAG repeat length. Rather, the products of mutant and normal alleles, both mRNA and protein, were balanced, thereby arguing that a cis-regulatory effect of the expanded CAG repeat is not a critical component of the underlying mechanism of HD. These robust allele-specific assays could prove valuable for monitoring the impact of allele-specific gene silencing strategies currently being explored as therapeutic interventions in HD.


Assuntos
Encéfalo/metabolismo , Proteína Huntingtina/biossíntese , Doença de Huntington/genética , Expansão das Repetições de Trinucleotídeos/genética , Adulto , Idade de Início , Alelos , Autopsia , Encéfalo/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Proteína Huntingtina/genética , Doença de Huntington/patologia , Masculino , RNA Mensageiro/biossíntese
12.
Hum Mol Genet ; 26(19): 3859-3867, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28934397

RESUMO

Huntington's disease (HD) is a dominantly inherited neurodegenerative disease caused by an expanded CAG repeat in HTT. Many clinical characteristics of HD such as age at motor onset are determined largely by the size of HTT CAG repeat. However, emerging evidence strongly supports a role for other genetic factors in modifying the disease pathogenesis driven by mutant huntingtin. A recent genome-wide association analysis to discover genetic modifiers of HD onset age provided initial evidence for modifier loci on chromosomes 8 and 15 and suggestive evidence for a locus on chromosome 3. Here, genotyping of candidate single nucleotide polymorphisms in a cohort of 3,314 additional HD subjects yields independent confirmation of the former two loci and moves the third to genome-wide significance at MLH1, a locus whose mouse orthologue modifies CAG length-dependent phenotypes in a Htt-knock-in mouse model of HD. Both quantitative and dichotomous association analyses implicate a functional variant on ∼32% of chromosomes with the beneficial modifier effect that delays HD motor onset by 0.7 years/allele. Genomic DNA capture and sequencing of a modifier haplotype localize the functional variation to a 78 kb region spanning the 3'end of MLH1 and the 5'end of the neighboring LRRFIP2, and marked by an isoleucine-valine missense variant in MLH1. Analysis of expression Quantitative Trait Loci (eQTLs) provides modest support for altered regulation of MLH1 and LRRFIP2, raising the possibility that the modifier affects regulation of both genes. Finally, polygenic modification score and heritability analyses suggest the existence of additional genetic modifiers, supporting expanded, comprehensive genetic analysis of larger HD datasets.


Assuntos
Proteína Huntingtina/genética , Proteína 1 Homóloga a MutL/genética , Alelos , Animais , Cromossomos Humanos Par 15 , Cromossomos Humanos Par 8 , Modelos Animais de Doenças , Genes Modificadores/genética , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Camundongos , Proteína 1 Homóloga a MutL/metabolismo , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Repetições de Trinucleotídeos
13.
Hum Mol Genet ; 26(5): 913-922, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28334820

RESUMO

Huntington's disease is a dominantly inherited neurodegenerative disease caused by the expansion of a CAG repeat in the HTT gene. In addition to the length of the CAG expansion, factors such as genetic background have been shown to contribute to the age at onset of neurological symptoms. A central challenge in understanding the disease progression that leads from the HD mutation to massive cell death in the striatum is the ability to characterize the subtle and early functional consequences of the CAG expansion longitudinally. We used dense time course sampling between 4 and 20 postnatal weeks to characterize early transcriptomic, molecular and cellular phenotypes in the striatum of six distinct knock-in mouse models of the HD mutation. We studied the effects of the HttQ111 allele on the C57BL/6J, CD-1, FVB/NCr1, and 129S2/SvPasCrl genetic backgrounds, and of two additional alleles, HttQ92 and HttQ50, on the C57BL/6J background. We describe the emergence of a transcriptomic signature in HttQ111/+ mice involving hundreds of differentially expressed genes and changes in diverse molecular pathways. We also show that this time course spanned the onset of mutant huntingtin nuclear localization phenotypes and somatic CAG-length instability in the striatum. Genetic background strongly influenced the magnitude and age at onset of these effects. This work provides a foundation for understanding the earliest transcriptional and molecular changes contributing to HD pathogenesis.


Assuntos
Corpo Estriado/metabolismo , Proteína Huntingtina/genética , Doença de Huntington/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Corpo Estriado/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Introdução de Genes , Patrimônio Genético , Instabilidade Genômica/genética , Humanos , Proteína Huntingtina/biossíntese , Doença de Huntington/patologia , Camundongos , Mutação/genética , Neurônios/metabolismo , Neurônios/patologia , Fenótipo , Transcriptoma/genética
14.
Am J Hum Genet ; 98(2): 287-98, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26849111

RESUMO

Huntington disease (HD) is caused by an expanded HTT CAG repeat that leads in a length-dependent, completely dominant manner to onset of a characteristic movement disorder. HD also displays early mortality, so we tested whether the expanded CAG repeat exerts a dominant influence on age at death and on the duration of clinical disease. We found that, as with clinical onset, HD age at death is determined by expanded CAG-repeat length and has no contribution from the normal CAG allele. Surprisingly, disease duration is independent of the mutation's length. It is also unaffected by a strong genetic modifier of HD motor onset. These findings suggest two parsimonious alternatives. (1) HD pathogenesis is driven by mutant huntingtin, but before or near motor onset, sufficient CAG-driven damage occurs to permit CAG-independent processes and then lead to eventual death. In this scenario, some pathological changes and their clinical correlates could still worsen in a CAG-driven manner after disease onset, but these CAG-related progressive changes do not themselves determine duration. Alternatively, (2) HD pathogenesis is driven by mutant huntingtin acting in a CAG-dependent manner with different time courses in multiple cell types, and the cellular targets that lead to motor onset and death are different and independent. In this scenario, processes driven by HTT CAG length lead directly to death but not via the striatal pathology associated with motor manifestations. Each scenario has important ramifications for the design and testing of potential therapeutics, especially those aimed at preventing or delaying characteristic motor manifestations.


Assuntos
Doença de Huntington/genética , Mutação , Proteínas do Tecido Nervoso/genética , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Alelos , Criança , Pré-Escolar , Estudos de Coortes , Corpo Estriado/metabolismo , Haplótipos , Humanos , Proteína Huntingtina , Doença de Huntington/mortalidade , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismo , Adulto Jovem
15.
J Hum Genet ; 64(10): 995-1004, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31296921

RESUMO

Huntington's disease (HD) is caused by an expanded CAG trinucleotide repeat in the first exon of the huntingtin gene (HTT). Since the entire course of the disease starts from this dominant gain-of-function mutation, lowering total or mutant huntingtin mRNA/protein has emerged as an appealing therapeutic strategy. We reasoned that endogenous mechanisms underlying HTT gene regulation may inform strategies to target the source of the disease. As part of our investigation to understand how the expression of HTT is controlled, we performed (1) complete sequencing analysis for mutant HTT 3'-UTR and (2) unbiased screening assays to identify naturally-occurring miRNAs that could lower the HTT mRNA levels. By sequencing HD families inheriting the major European mutant haplotype, we determined the full sequence of HTT 3'-UTRs of the most frequent mutant (i.e., hap.01) and normal (i.e., hap.08) haplotypes, revealing 5 sites with alternative alleles. In subsequent miRNA activity assays using the full-length hap.01 and hap.08 3'-UTR reporter vectors and follow-up validation experiments, hsa-miR-4324 and hsa-miR-4756-5p significantly reduced HTT 3'-UTR reporter activity and endogenous HTT protein levels. However, those miRNAs did not show strong haplotype-specific effects. Nevertheless, our data highlighting full sequences of HTT 3'-UTR haplotypes, effects of miRNAs on HTT levels, and potential interaction sites provide rationale and promising targets for total and mutant-specific HTT lowering intervention strategies using endogenous and artificial miRNAs, respectively.


Assuntos
Proteína Huntingtina/genética , Doença de Huntington/genética , MicroRNAs/genética , Regiões 3' não Traduzidas/genética , Alelos , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Haplótipos , Humanos , Proteína Huntingtina/metabolismo , Mutação
16.
Chemphyschem ; 19(14): 1711-1715, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-29719110

RESUMO

We examined how to enhance the lifetime of organic light-emitting diodes (OLEDs) based on bipolar host molecules ET-HT, where ET and HT refer to electron- and hole-transporting units, respectively, by analyzing their thermodynamic and kinetic stabilities. Our DFT calculations reveal that the thermodynamic stability of ET-HT is determined by that of its anion, which is difficult to improve by chemical modifications of ET and HT. The kinetic stability of ET-HT can be enhanced by the spiroconjugation between ET and HT, which occurs when their π-frameworks are extended and have an orthogonal arrangement. Green OLED devices were fabricated by using ET-HTs with and without spiroconjugation, to find that the device with spiroconjugation has a lifetime that is approximately 6 times longer than the one without spiroconjugation.

17.
J Environ Manage ; 223: 652-657, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-29975892

RESUMO

We used a nanofiltration (NF) membrane process to produce purified aqueous sulfuric acid from copper-refining sulfuric acid wastewater. Wastewater generated from a copper-refining process was used to explore the membrane performances and acid stabilities of six commercial NF membranes. A combination of permeate flux, sulfate permeation, and metal ion rejection clearly showed that two polyamide membranes and a polyacrylonitrile-based membrane achieved recovery of a purified sulfuric acid solution. Acid-stability and long-term performance tests showed that the polyamide membranes were unsuitable for copper-refining wastewater treatment because of their low acid stabilities. In contrast, the polyacrylonitrile-based composite membrane showed excellent acid stability, and gave greater than 90% metal ion rejection, with the exception of calcium ions, for 430 d. We also evaluated the recovery performance in 1 ton/d pilot-scale process using wastewater from copper-refining process; 90% metal ion rejection was achieved, with the exception of calcium ions, even at 95% recovery rate.


Assuntos
Cobre , Ácidos Sulfúricos/química , Águas Residuárias , Purificação da Água , Filtração , Membranas Artificiais , Nanotecnologia , Poluentes Químicos da Água
18.
J Environ Manage ; 206: 740-748, 2018 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-29161676

RESUMO

We develop mesoporous magnetic clusters (MMCs) functionalized with hydrophilic branched polyethylenimine (b-PEI), later called b-MG, and MMCs functionalized with positively charged b-PEI (p-MG). These materials efficiently remove Pb(II) and Cr(VI) from wastewater. Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, thermogravimetric analysis, and nitrogen adsorption-desorption analysis results clearly indicate that hydrophilic b-PEI and positively charged b-PEI are successfully attached to the MMC surfaces. Wide-angle X-ray diffraction, high-resolution transmission electron microscopy, and field-emission scanning electron microscopy analyses confirm that the crystal structures and morphologies of the MMCs are maintained well even when wet chemical modification processes are used to introduce hydrophilic b-PEI and positively charged b-PEI to the MMC surfaces. Langmuir and Sips isotherm models are applied to describe Pb(II) adsorption behavior of the b-MG and Cr(VI) adsorption behavior of the p-MG. The isotherm models indicate that the maximum adsorption capacities of b-MG and p-MG, respectively, are 216.3 and 334.1 mg g-1, respectively. These are higher than have previously been found for other adsorbents. In reusability tests, using magnetic separation and controlling the pH, the Pb(II) recovery efficiency of the b-MG is 95.6% and the Cr(VI) recovery efficiency of the p-MG is 68.0% even after the third cycle.


Assuntos
Cromo/isolamento & purificação , Chumbo/isolamento & purificação , Poluentes Químicos da Água/isolamento & purificação , Adsorção , Cromo/química , Cinética , Chumbo/química , Polietilenoimina , Águas Residuárias , Poluentes Químicos da Água/química
19.
Molecules ; 23(1)2018 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-29361735

RESUMO

Multi-target-directed ligands (MTDLs) offer new hope for the treatment of multifactorial complex diseases such as Alzheimer's Disease (AD). Herein, we present compounds aimed at targeting the NMDA and the P2X7 receptors, which embody a different approach to AD therapy. On one hand, we are seeking to delay neurodegeneration targeting the glutamatergic NMDA receptors; on the other hand, we also aim to reduce neuroinflammation, targeting P2X7 receptors. Although the NMDA receptor is a widely recognized therapeutic target in treating AD, the P2X7 receptor remains largely unexplored for this purpose; therefore, the dual inhibitor presented herein-which is open to further optimization-represents the first member of a new class of MTDLs.


Assuntos
Antagonistas do Receptor Purinérgico P2X/química , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X/química , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Linhagem Celular Tumoral , Técnicas de Química Sintética , Relação Dose-Resposta a Droga , Desenho de Fármacos , Descoberta de Drogas , Humanos , Concentração Inibidora 50 , Ligantes , Estrutura Molecular , Antagonistas do Receptor Purinérgico P2X/síntese química , Receptores Purinérgicos P2X/metabolismo
20.
Neurobiol Dis ; 98: 66-76, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27913290

RESUMO

Cholesterol precursors and cholesterol levels are reduced in brain regions of Huntington's disease (HD) mice. Here we quantified the rate of in vivo de novo cholesterol biosynthesis in the HD brain. Samples from different brain regions and blood of the heterozygous knock-in mouse model carrying 175 CAG repeats (Q175) at different phenotypic stages were processed independently by two research units to quantify cholesterol synthesis rate by 2H2O labeling and measure the concentrations of lathosterol, cholesterol and its brain-specific cholesterol catabolite 24-hydroxy-cholesterol (24OHC) by isotope dilution mass spectrometry. The daily synthesis rate of cholesterol and the corresponding concentration of lathosterol were significantly reduced in the striatum of heterozygous Q175 mice early in the disease course. We also report that the decrease in lathosterol was inversely correlated with CAG-size at symptomatic stage, as observed in striatal samples from an allelic series of HD mice. There was also a significant correlation between the fractional synthesis rates of total cholesterol and 24OHC in brain of wild-type (WT) and Q175 mice, supporting the evidence that plasma 24OHC may reflect cholesterol synthesis in the adult brain. This comprehensive analysis demonstrates consistent cholesterol biosynthesis defects in HD mouse models and suggests that plasma 24OHC may serve as a biomarker of brain cholesterol metabolism.


Assuntos
Encéfalo/metabolismo , Colesterol/biossíntese , Doença de Huntington/metabolismo , Animais , Biomarcadores/sangue , Modelos Animais de Doenças , Progressão da Doença , Feminino , Técnicas de Introdução de Genes , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Caracteres Sexuais
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