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1.
Neuroendocrinology ; 2019 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-31104058

RESUMO

In outbred mice, susceptibility or resistance to diet-induced obesity is associated with rapid changes in hypothalamic proopiomelanocortin (POMC) levels. Here, we evaluated three hypotheses that potentially explain the development of the different obesity phenotypes in outbred Swiss mice. First, rapid and differential changes in the gut microbiota in obese-prone (OP) and obese-resistant (OR) mice fed on a high-fat diet (HFD) might cause differential efficiencies in fatty acid harvesting leading to changes in systemic fatty acid concentrations that in turn affect POMC expression and processing. Second, independently of the gut microbiota, OP mice might have increased blood fatty acid levels after the introduction of a HFD, which could affect POMC expression and processing. Third, fatty acids might act directly in the hypothalamus to differentially regulate POMC expression and/or processing in OP and OR mice. We evaluated OP and OR, male Swiss mice using 16S rRNA sequencing for determination of gut microbiota; gas chromatography for blood lipid determination; immunoblot and real-time PCR for protein and transcript determination and indirect calorimetry. Some experiments were performed with human pluripotent stem cells differentiated into hypothalamic neurons. We did not find evidence supporting the first two hypotheses. However, we found that in OP but not in OR mice, palmitate induces a rapid increase in hypothalamic POMC, which is followed by increased expression of proprotein convertase subtilisin/kexin type 1 (PCSK1, PC1/3). Lentiviral inhibition of hypothalamic PC1/3 increased caloric intake and body mass in both OP and OR mice. In human stem cell-derived hypothalamic cells, we found that palmitate potently suppressed the production of POMC-derived peptides. Palmitate directly regulates PC1/3 in OP mice, and likely has a functional impact on POMC processing.

2.
Sex Transm Infect ; 95(6): 449-454, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30918121

RESUMO

OBJECTIVES: Pre-exposure prophylaxis (PrEP) is a highly effective method of HIV prevention for men who have sex with men (MSM). However, uncertainty remains around the optimal eligibility criteria for PrEP, specifically whether there are subgroups at low risk of HIV for whom PrEP might not be warranted. METHODS: PROUD was an open-label waitlist trial design that randomised MSM attending participating sexual health centres in England to receive PrEP immediately (IMM) or after a deferral period of 1 year (DEF). This analysis is based on participants who were randomised to the deferred arm, when they did not have access to PrEP. HIV incidence was compared between subgroups defined by baseline characteristics. RESULTS: Overall, 21 participants acquired HIV infection over 239.3 person-years (PY) follow-up, yielding an incidence rate of 8.8/100 PY (95% CI 5.4 to 13.4). Two highly significant predictors for HIV acquisition were identified. Men with a self-reported diagnosis of syphilis, rectal chlamydia (CT) or rectal gonorrhoea (GC) in the previous 12 months had an incidence of 17.2/100 PY (95% CI 9.7 to 28.5); those reporting receptive anal intercourse without a condom (ncRAI) with two or more partners in the previous 3 months had an incidence of 13.6/100 PY (95% CI 7.9 to 21.7). The incidence rate among participants lacking both of these risk factors was 1.1/100 PY (1/87.6, 95% CI 0.03 to 6.4). CONCLUSIONS: The high HIV incidence in PROUD suggests that most participants appropriately judged their need for PrEP. Eligibility criteria for a PrEP programme can therefore be broad, as in the current guidelines. However, a recent history of syphilis or rectal CT/GC, or multiple ncRAI partners indicates a high imminent risk of HIV infection. MSM with any of these characteristics should be offered PrEP as a matter of urgency.

3.
Lifetime Data Anal ; 2019 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-30811019

RESUMO

CD4-based multi-state back-calculation methods are key for monitoring the HIV epidemic, providing estimates of HIV incidence and diagnosis rates by disentangling their inter-related contribution to the observed surveillance data. This paper, extends existing approaches to age-specific settings, permitting the joint estimation of age- and time-specific incidence and diagnosis rates and the derivation of other epidemiological quantities of interest. This allows the identification of specific age-groups at higher risk of infection, which is crucial in directing public health interventions. We investigate, through simulation studies, the suitability of various bivariate splines for the non-parametric modelling of the latent age- and time-specific incidence and illustrate our method on routinely collected data from the HIV epidemic among gay and bisexual men in England and Wales.

4.
Clin Infect Dis ; 69(7): 1136-1143, 2019 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-30534981

RESUMO

BACKGROUND: Drug-resistant minority variants (DRMinVs) detected in patients who recently acquired human immunodeficiency virus type 1 (HIV-1) can be transmitted, generated de novo through virus replication, or technical errors. The first form is likely to persist and result in treatment failure, while the latter two could be stochastic and transient. METHODS: Ultradeep sequencing of plasma samples from 835 individuals with recent HIV-1 infection in the United Kingdom was performed to detect DRMinVs at a mutation frequency between 2% and 20%. Sequence alignments including >110 000 HIV-1 partial pol consensus sequences from the UK HIV Drug Resistance Database (UK-HDRD), linked to epidemiological and clinical data from the HIV and AIDS Reporting System, were used for transmission cluster analysis. Transmission clusters were identified using Cluster Picker with a clade support of >90% and maximum genetic distances of 4.5% or 1.5%, the latter to limit detection to likely direct transmission events. RESULTS: Drug-resistant majority variants (DRMajVs) were detected in 66 (7.9%) and DRMinVs in 84 (10.1%) of the recently infected individuals. High levels of clustering to sequences in UK-HDRD were observed for both DRMajV (n = 48; 72.7%) and DRMinV (n = 63; 75.0%) sequences. Of these, 43 (65.2%) with DRMajVs were in a transmission cluster with sequences that harbored the same DR mutation compared to only 3 (3.6%) sequences with DRMinVs (P < .00001, Fisher exact test). Evidence of likely direct transmission of DRMajVs was observed for 25/66 (37.9%), whereas none were observed for the DRMinVs (P < .00001). CONCLUSIONS: Using a densely sampled HIV-infected population, we show no evidence of DRMinV transmission among recently infected individuals.

5.
Mol Metab ; 17: 82-97, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30201275

RESUMO

OBJECTIVE: The lack of pro-opiomelanocortin (POMC)-derived melanocortin peptides results in hypoadrenalism and severe obesity in both humans and rodents that is treatable with synthetic melanocortins. However, there are significant differences in POMC processing between humans and rodents, and little is known about the relative physiological importance of POMC products in the human brain. The aim of this study was to determine which POMC-derived peptides are present in the human brain, to establish their relative concentrations, and to test if their production is dynamically regulated. METHODS: We analysed both fresh post-mortem human hypothalamic tissue and hypothalamic neurons derived from human pluripotent stem cells (hPSCs) using liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine the sequence and quantify the production of hypothalamic neuropeptides, including those derived from POMC. RESULTS: In both in vitro and in vivo hypothalamic cells, LC-MS/MS revealed the sequence of hundreds of neuropeptides as a resource for the field. Although the existence of ß-melanocyte stimulating hormone (MSH) is controversial, we found that both this peptide and desacetyl α-MSH (d-α-MSH) were produced in considerable excess of acetylated α-MSH. In hPSC-derived hypothalamic neurons, these POMC derivatives were appropriately trafficked, secreted, and their production was significantly (P < 0.0001) increased in response to the hormone leptin. CONCLUSIONS: Our findings challenge the assumed pre-eminence of α-MSH and suggest that in humans, d-α-MSH and ß-MSH are likely to be the predominant physiological products acting on melanocortin receptors.

6.
Mol Metab ; 10: 14-27, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29439854

RESUMO

OBJECTIVE: Dietary proteins are sensed by hypothalamic neurons and strongly influence multiple aspects of metabolic health, including appetite, weight gain, and adiposity. However, little is known about the mechanisms by which hypothalamic neural circuits controlling behavior and metabolism sense protein availability. The aim of this study is to characterize how neurons from the mediobasal hypothalamus respond to a signal of protein availability: the amino acid l-leucine. METHODS: We used primary cultures of post-weaning murine mediobasal hypothalamic neurons, hypothalamic neurons derived from human induced pluripotent stem cells, and calcium imaging to characterize rapid neuronal responses to physiological changes in extracellular l-Leucine concentration. RESULTS: A neurochemically diverse subset of both mouse and human hypothalamic neurons responded rapidly to l-leucine. Consistent with l-leucine's anorexigenic role, we found that 25% of mouse MBH POMC neurons were activated by l-leucine. 10% of MBH NPY neurons were inhibited by l-leucine, and leucine rapidly reduced AGRP secretion, providing a mechanism for the rapid leucine-induced inhibition of foraging behavior in rodents. Surprisingly, none of the candidate mechanisms previously implicated in hypothalamic leucine sensing (KATP channels, mTORC1 signaling, amino-acid decarboxylation) were involved in the acute activity changes produced by l-leucine. Instead, our data indicate that leucine-induced neuronal activation involves a plasma membrane Ca2+ channel, whereas leucine-induced neuronal inhibition is mediated by inhibition of a store-operated Ca2+ current. CONCLUSIONS: A subset of neurons in the mediobasal hypothalamus rapidly respond to physiological changes in extracellular leucine concentration. Leucine can produce both increases and decreases in neuronal Ca2+ concentrations in a neurochemically-diverse group of neurons, including some POMC and NPY/AGRP neurons. Our data reveal that leucine can signal through novel mechanisms to rapidly affect neuronal activity.

7.
Sex Transm Infect ; 94(1): 67-71, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28490580

RESUMO

BACKGROUND: Evidence suggests that sexual transmission between men has replaced foreign travel as the predominant mode of Shigella transmission in England. However, sexuality and HIV status are not routinely recorded for laboratory-reported Shigella, and the role of HIV in the Shigella epidemic is not well understood. METHODS: The Modular Open Laboratory Information System containing all Shigella cases reported to Public Health England (PHE) and the PHE HIV and AIDS Reporting System holding all adults living with diagnosed HIV in England were matched using a combination of Soundex code, date of birth and gender. RESULTS: From 2004 to 2015, 88 664 patients were living with HIV, and 10 269 Shigella cases were reported in England; 9% (873/10 269) of Shigella cases were diagnosed with HIV, of which 93% (815/873) were in men. Shigella cases without reported travel history were more likely to be living with HIV than those who had travelled (14% (751/5427) vs 3% (134/4854); p<0.01). From 2004 to 2015, the incidence of Shigella in men with HIV rose from 47/100 000 to 226/100 000 (p<0.01) peaking in 2014 at 265/100 000, but remained low in women throughout the study period (0-24/100 000). Among Shigella cases without travel and with HIV, 91% (657/720) were men who have sex with men (MSM). HIV preceded Shigella diagnosis in 86% (610/720), and 65% (237/362) had an undetectable viral load (<50 copies/mL). DISCUSSION: We observed a sustained increase in the national rate of shigellosis in MSM with HIV, who may experience more serious clinical disease. Sexual history, HIV status and STI risk might require sensitive investigation in men presenting with gastroenteritis.


Assuntos
Disenteria Bacilar/epidemiologia , Disenteria Bacilar/transmissão , Epidemias , Infecções por HIV/epidemiologia , Infecções por HIV/transmissão , Minorias Sexuais e de Gênero , Adolescente , Adulto , Coinfecção/epidemiologia , Coinfecção/microbiologia , Coinfecção/virologia , Disenteria Bacilar/microbiologia , Disenteria Bacilar/virologia , Inglaterra/epidemiologia , Infecções por HIV/microbiologia , Infecções por HIV/virologia , Homossexualidade Masculina , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Comportamento Sexual , Parceiros Sexuais , Shigella/isolamento & purificação , Shigella flexneri/isolamento & purificação , Shigella sonnei/isolamento & purificação , Viagem , Adulto Jovem
8.
Curr Protoc Neurosci ; 81: 3.33.1-3.33.24, 2017 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-29064566

RESUMO

Neurons in the hypothalamus orchestrate homeostatic physiological processes and behaviors essential for life. Defects in the function of hypothalamic neurons cause a spectrum of human diseases, including obesity, infertility, growth defects, sleep disorders, social disorders, and stress disorders. These diseases have been studied in animal models such as mice, but the rarity and relative inaccessibility of mouse hypothalamic neurons and species-specific differences between mice and humans highlight the need for human cellular models of hypothalamic diseases. We and others have developed methods to differentiate human pluripotent stem cells (hPSCs) into hypothalamic neurons and related cell types, such as astrocytes. This protocol builds on published studies by providing detailed step-by-step instructions for neuronal differentiation, quality control, long-term neuronal maintenance, and the functional interrogation of hypothalamic cells by calcium imaging. Together, these protocols should enable any group with appropriate facilities to generate and study human hypothalamic cells. © 2017 by John Wiley & Sons, Inc.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Hipotálamo/citologia , Neurogênese/fisiologia , Neurônios/fisiologia , Proteína Relacionada com Agouti/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Humanos , Hormônios Hipotalâmicos/metabolismo , Hipotálamo/embriologia , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/fisiologia , Pró-Opiomelanocortina/metabolismo , Transdução de Sinais/fisiologia , Fatores de Tempo
9.
AIDS ; 31(17): 2403-2413, 2017 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-28857827

RESUMO

OBJECTIVES: Tuberculosis (TB) is common in people living with HIV, leading to worse clinical outcomes including increased mortality. We investigated risk factors for developing TB following HIV diagnosis. DESIGN: Adults aged at least 15 years first presenting to health services for HIV care in England, Wales or Northern Ireland from 2000 to 2014 were identified from national HIV surveillance data and linked to TB surveillance data. METHODS: We calculated incidence rates for TB occurring more than 91 days after HIV diagnosis and investigated risk factors using multivariable Poisson regression. RESULTS: A total of 95 003 adults diagnosed with HIV were followed for 635 591 person-years; overall incidence of TB was 344 per 100 000 person-years (95% confidence interval 330-359). TB incidence was high for people who acquired HIV through injecting drugs [PWID; men 876 (696-1104), women 605 (365-945)] and black Africans born in high TB incidence countries [644 (612-677)]. The adjusted incidence rate ratio for TB amongst PWID was 4.79 (3.35-6.85) for men and 6.18 (3.49-10.93) for women, compared with MSM. The adjusted incidence rate ratio for TB in black Africans from high-TB countries was 4.27 (3.42-5.33), compared with white UK-born individuals. Lower time-updated CD4 cell count was associated with increased rates of TB. CONCLUSION: PWID had the greatest risk of TB; incidence rates were comparable with those in black Africans from high TB incidence countries. Most TB cases in PWID were UK-born, and likely acquired TB through transmission within the United Kingdom. Earlier HIV diagnosis and quicker initiation of antiretroviral therapy should reduce TB incidence in these populations.


Assuntos
Infecções por HIV/complicações , Abuso de Substâncias por Via Intravenosa/complicações , Tuberculose/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Inglaterra/epidemiologia , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Irlanda do Norte/epidemiologia , Fatores de Risco , País de Gales/epidemiologia , Adulto Jovem
10.
Euro Surveill ; 22(25)2017 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-28662762

RESUMO

Since October 2015 up to September 2016, HIV diagnoses fell by 32% compared with October 2014-September 2015 among men who have sex with men (MSM) attending selected London sexual health clinics. This coincided with high HIV testing volumes and rapid initiation of treatment on diagnosis. The fall was most apparent in new HIV testers. Intensified testing of high-risk populations, combined with immediately received anti-retroviral therapy and a pre-exposure prophylaxis (PrEP) programme, may make elimination of HIV achievable.


Assuntos
Infecções por HIV/diagnóstico , Infecções por HIV/prevenção & controle , Homossexualidade Masculina/estatística & dados numéricos , Programas de Rastreamento/tendências , Profilaxia Pré-Exposição , Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Humanos , Londres , Masculino , Vigilância da População , Comportamento Sexual , Saúde Sexual , Parceiros Sexuais
11.
Clin Infect Dis ; 64(3): 335-342, 2017 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-27927870

RESUMO

BACKGROUND: An urgent UK investigation was launched to assess risk of invasive Mycobacterium chimaera infection in cardiothoracic surgery and a possible association with cardiopulmonary bypass heater-cooler units following alerts in Switzerland and The Netherlands. METHODS: Parallel investigations were pursued: (1) identification of cardiopulmonary bypass-associated M. chimaera infection through national laboratory and hospital admissions data linkage; (2) cohort study to assess patient risk; (3) microbiological and aerobiological investigations of heater-coolers in situ and under controlled laboratory conditions; and (4) whole-genome sequencing of clinical and environmental isolates. RESULTS: Eighteen probable cases of cardiopulmonary bypass-associated M. chimaera infection were identified; all except one occurred in adults. Patients had undergone valve replacement in 11 hospitals between 2007 and 2015, a median of 19 months prior to onset (range, 3 months to 5 years). Risk to patients increased after 2010 from <0.2 to 1.65 per 10000 person-years in 2013, a 9-fold rise for infections within 2 years of surgery (rate ratio, 9.08 [95% CI, 1.81-87.76]). Endocarditis was the most common presentation (n = 11). To date, 9 patients have died. Investigations identified aerosol release through breaches in heater-cooler tanks. Mycobacterium chimaera and other pathogens were recovered from water and air samples. Phylogenetic analysis found close clustering of strains from probable cases. CONCLUSIONS: We identified low but escalating risk of severe M. chimaera infection associated with heater-coolers with cases in a quarter of cardiothoracic centers. Our investigations strengthen etiological evidence for the role of heater-coolers in transmission and raise the possibility of an ongoing, international point-source outbreak. Active management of heater-coolers and heightened clinical awareness are imperative given the consequences of infection.


Assuntos
Ponte Cardiopulmonar/efeitos adversos , Contaminação de Equipamentos , Infecções por Micobactéria não Tuberculosa/epidemiologia , Micobactérias não Tuberculosas/isolamento & purificação , Equipamentos Cirúrgicos/microbiologia , Infecção da Ferida Cirúrgica/epidemiologia , Infecção da Ferida Cirúrgica/microbiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Microbiologia do Ar , Criança , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Micobactéria não Tuberculosa/microbiologia , Infecções por Micobactéria não Tuberculosa/mortalidade , Infecções por Micobactéria não Tuberculosa/transmissão , Micobactérias não Tuberculosas/classificação , Micobactérias não Tuberculosas/genética , Estudos Retrospectivos , Fatores de Risco , Infecção da Ferida Cirúrgica/mortalidade , Reino Unido/epidemiologia , Microbiologia da Água
12.
BMC Med ; 13: 297, 2015 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-26654248

RESUMO

BACKGROUND: Recent studies have identified HIV infection as a potential risk factor for invasive meningococcal disease (IMD), suggesting that HIV-infected individuals could benefit from meningococcal vaccination to reduce their risk of this rare, but severe and potentially fatal infection. In the United Kingdom, as in most industrialised countries, HIV is not considered a risk factor for IMD. METHODS: IMD incidence and relative risk by age group and meningococcal capsular group in HIV-positive compared with HIV-uninfected individuals was estimated through data linkage of national datasets in England between 2011 and 2013. RESULTS: IMD incidence among persons diagnosed with HIV was 6.6 per 100,000 compared to 1.5 per 100,000 among HIV-negative individuals, with a relative risk of 4.5 (95 % CI, 2.7-7.5). All but one case occurred in adults aged 16-64 years, who had a 22.7-fold (95 % CI, 12.4-41.6; P <0.001) increased risk compared with the HIV-negative adults. IMD risk by capsular group varied with age. HIV-positive children and adolescents had a higher risk of meningococcal group B disease, while adults were at increased risk of groups C, W and Y disease. Most HIV-positive individuals had been born in Africa, had acquired HIV through heterosexual contact, and were known to be HIV-positive and receiving antiretroviral treatment at IMD diagnosis. The most common clinical presentation was septicemia and, although intensive care admission was common, none died of IMD. CONCLUSIONS: HIV-positive children and adults are at significantly increased risk of IMD, providing an evidence base for policy makers to consider HIV as a risk factor for meningococcal vaccination.


Assuntos
Infecções por HIV/complicações , Infecções Meningocócicas/epidemiologia , Adolescente , Adulto , África , Criança , Estudos de Coortes , Emigrantes e Imigrantes , Inglaterra/epidemiologia , Feminino , Humanos , Incidência , Masculino , Vacinas Meningocócicas , Pessoa de Meia-Idade , Fatores de Risco , Adulto Jovem
13.
Development ; 142(18): 3178-87, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26395144

RESUMO

A key aspect of nervous system development, including that of the cerebral cortex, is the formation of higher-order neural networks. Developing neural networks undergo several phases with distinct activity patterns in vivo, which are thought to prune and fine-tune network connectivity. We report here that human pluripotent stem cell (hPSC)-derived cerebral cortex neurons form large-scale networks that reflect those found in the developing cerebral cortex in vivo. Synchronised oscillatory networks develop in a highly stereotyped pattern over several weeks in culture. An initial phase of increasing frequency of oscillations is followed by a phase of decreasing frequency, before giving rise to non-synchronous, ordered activity patterns. hPSC-derived cortical neural networks are excitatory, driven by activation of AMPA- and NMDA-type glutamate receptors, and can undergo NMDA-receptor-mediated plasticity. Investigating single neuron connectivity within PSC-derived cultures, using rabies-based trans-synaptic tracing, we found two broad classes of neuronal connectivity: most neurons have small numbers (<10) of presynaptic inputs, whereas a small set of hub-like neurons have large numbers of synaptic connections (>40). These data demonstrate that the formation of hPSC-derived cortical networks mimics in vivo cortical network development and function, demonstrating the utility of in vitro systems for mechanistic studies of human forebrain neural network biology.


Assuntos
Córtex Cerebral/crescimento & desenvolvimento , Técnicas In Vitro/métodos , Rede Nervosa/crescimento & desenvolvimento , Células-Tronco Pluripotentes/fisiologia , Córtex Cerebral/citologia , Espinhas Dendríticas/ultraestrutura , Humanos , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Rede Nervosa/citologia , Plasticidade Neuronal/fisiologia , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Célula Única , Gravação em Vídeo
14.
Cell Rep ; 11(5): 689-96, 2015 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-25921538

RESUMO

Accumulation of Aß peptide fragments of the APP protein and neurofibrillary tangles of the microtubule-associated protein tau are the cellular hallmarks of Alzheimer's disease (AD). To investigate the relationship between APP metabolism and tau protein levels and phosphorylation, we studied human-stem-cell-derived forebrain neurons with genetic forms of AD, all of which increase the release of pathogenic Aß peptides. We identified marked increases in intracellular tau in genetic forms of AD that either mutated APP or increased its dosage, suggesting that APP metabolism is coupled to changes in tau proteostasis. Manipulating APP metabolism by ß-secretase and γ-secretase inhibition, as well as γ-secretase modulation, results in specific increases and decreases in tau protein levels. These data demonstrate that APP metabolism regulates tau proteostasis and suggest that the relationship between APP processing and tau is not mediated solely through extracellular Aß signaling to neurons.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Córtex Cerebral/metabolismo , Neurônios/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Substituição de Aminoácidos , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Dosagem de Genes , Humanos , Fosforilação , Transdução de Sinais
15.
Development ; 141(11): 2216-24, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24821983

RESUMO

Generation of neurons from patient fibroblasts using a combination of developmentally defined transcription factors has great potential in disease modelling, as well as ultimately for use in regeneration and repair. However, generation of physiologically mature neurons in vitro remains problematic. Here we demonstrate the cell-cycle-dependent phosphorylation of a key reprogramming transcription factor, Ascl1, on multiple serine-proline sites. This multisite phosphorylation is a crucial regulator of the ability of Ascl1 to drive neuronal differentiation and maturation in vivo in the developing embryo; a phosphomutant form of Ascl1 shows substantially enhanced neuronal induction activity in Xenopus embryos. Mechanistically, we see that this un(der)phosphorylated Ascl1 is resistant to inhibition by both cyclin-dependent kinase activity and Notch signalling, both of which normally limit its neurogenic potential. Ascl1 is a central component of reprogramming transcription factor cocktails to generate neurons from human fibroblasts; the use of phosphomutant Ascl1 in place of the wild-type protein significantly promotes neuronal maturity after human fibroblast reprogramming in vitro. These results demonstrate that cell-cycle-dependent post-translational modification of proneural proteins directly regulates neuronal differentiation in vivo during development, and that this regulatory mechanism can be harnessed to promote maturation of neurons obtained by transdifferentiation of human cells in vitro.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Proteínas de Xenopus/metabolismo , Animais , Técnicas de Cultura de Células , Ciclo Celular , Linhagem Celular , Transdiferenciação Celular/fisiologia , Fibroblastos/metabolismo , Células HEK293 , Humanos , Neurogênese , Neurônios/metabolismo , Fosforilação , Prolina/metabolismo , Processamento de Proteína Pós-Traducional , Receptores Notch/metabolismo , Serina/metabolismo , Transdução de Sinais , Xenopus laevis
16.
Nat Protoc ; 7(10): 1836-46, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22976355

RESUMO

Efficient derivation of human cerebral neocortical neural stem cells (NSCs) and functional neurons from pluripotent stem cells (PSCs) facilitates functional studies of human cerebral cortex development, disease modeling and drug discovery. Here we provide a detailed protocol for directing the differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) to all classes of cortical projection neurons. We demonstrate an 80-d, three-stage process that recapitulates cortical development, in which human PSCs (hPSCs) first differentiate to cortical stem and progenitor cells that then generate cortical projection neurons in a stereotypical temporal order before maturing to actively fire action potentials, undergo synaptogenesis and form neural circuits in vitro. Methods to characterize cortical neuron identity and synapse formation are described.


Assuntos
Técnicas de Cultura de Células , Diferenciação Celular , Córtex Cerebral/citologia , Rede Nervosa , Neurônios/citologia , Células-Tronco Pluripotentes/citologia , Linhagem Celular , Humanos , Sinapses
17.
Sci Transl Med ; 4(124): 124ra29, 2012 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-22344463

RESUMO

Human cellular models of Alzheimer's disease (AD) pathogenesis would enable the investigation of candidate pathogenic mechanisms in AD and the testing and developing of new therapeutic strategies. We report the development of AD pathologies in cortical neurons generated from human induced pluripotent stem (iPS) cells derived from patients with Down syndrome. Adults with Down syndrome (caused by trisomy of chromosome 21) develop early-onset AD, probably due to increased expression of a gene on chromosome 21 that encodes the amyloid precursor protein (APP). We found that cortical neurons generated from iPS cells and embryonic stem cells from Down syndrome patients developed AD pathologies over months in culture, rather than years in vivo. These cortical neurons processed the transmembrane APP protein, resulting in secretion of the pathogenic peptide fragment amyloid-ß42 (Aß42), which formed insoluble intracellular and extracellular amyloid aggregates. Production of Aß peptides was blocked by a γ-secretase inhibitor. Finally, hyperphosphorylated tau protein, a pathological hallmark of AD, was found to be localized to cell bodies and dendrites in iPS cell-derived cortical neurons from Down syndrome patients, recapitulating later stages of the AD pathogenic process.


Assuntos
Doença de Alzheimer/patologia , Síndrome de Down/patologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Diferenciação Celular , Linhagem Celular , Síndrome de Down/metabolismo , Eletrofisiologia , Células-Tronco Embrionárias/citologia , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas tau/metabolismo
18.
Nat Neurosci ; 15(3): 477-86, S1, 2012 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-22306606

RESUMO

Efforts to study the development and function of the human cerebral cortex in health and disease have been limited by the availability of model systems. Extrapolating from our understanding of rodent cortical development, we have developed a robust, multistep process for human cortical development from pluripotent stem cells: directed differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells to cortical stem and progenitor cells, followed by an extended period of cortical neurogenesis, neuronal terminal differentiation to acquire mature electrophysiological properties, and functional excitatory synaptic network formation. We found that induction of cortical neuroepithelial stem cells from human ES cells and human iPS cells was dependent on retinoid signaling. Furthermore, human ES cell and iPS cell differentiation to cerebral cortex recapitulated in vivo development to generate all classes of cortical projection neurons in a fixed temporal order. This system enables functional studies of human cerebral cortex development and the generation of individual-specific cortical networks ex vivo for disease modeling and therapeutic purposes.


Assuntos
Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Células-Tronco Embrionárias/citologia , Neurogênese/fisiologia , Células-Tronco Pluripotentes/citologia , Sinapses/fisiologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Proteína 4 Homóloga a Disks-Large , Células-Tronco Embrionárias/fisiologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feto , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Antígeno Ki-67/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Técnicas de Patch-Clamp , Piperidinas/farmacologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/fisiologia , Bloqueadores dos Canais de Potássio/farmacologia , RNA Mensageiro , Retinoides/farmacologia , Transdução de Sinais/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Sinapses/efeitos dos fármacos , Potenciais Sinápticos/efeitos dos fármacos , Potenciais Sinápticos/fisiologia , Tetrodotoxina/farmacologia , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
19.
J Interprof Care ; 26(3): 226-31, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22233262

RESUMO

This study explored the different values of staff from two care homes for older people in which the managers had different qualifications (social worker vs. nursing). Their views were examined to explore whether the values of the staff might reflect any value difference originating in the professional backgrounds of the managers. There was little evidence of awareness in either home of the ethical principles underlying day-to-day decisions. However, a distinction based on care qualification did appear with "care-qualified" staff (defined in terms of qualification requirements for this care work) demonstrating a more reflective response and fewer ageist assumptions than their non-care-qualified colleagues. The study found no difference in values between the nursing and social worker-led homes. All respondents, regardless of the profession of their manager, were keenly aware that they have a "duty of care" and overwhelmingly they defined that as their duty to keep the resident safe, as opposed to allowing her to exercise autonomy. The study results suggest that value base constitutes a commonality between professions involved in the care of older people rather than a barrier to collaboration, as is sometimes posited.


Assuntos
Ética em Enfermagem , Geriatria/ética , Preconceito , Serviço Social/ética , Adulto , Idoso , Tomada de Decisões , Feminino , Instituição de Longa Permanência para Idosos/ética , Humanos , Masculino , Pessoa de Meia-Idade , Casas de Saúde/ética
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