RESUMO
CAG-repeat expansions in at least eight different genes cause neurodegeneration. The length of the extended polyglutamine stretches in the corresponding proteins is proportionally related to their aggregation propensity. Although these proteins are ubiquitously expressed, they predominantly cause toxicity to neurons. To understand this neuronal hypersensitivity, we generated induced pluripotent stem cell (iPSC) lines of spinocerebellar ataxia type 3 and Huntington's disease patients. iPSC generation and neuronal differentiation are unaffected by polyglutamine proteins and show no spontaneous aggregate formation. However, upon glutamate treatment, aggregates form in neurons but not in patient-derived neural progenitors. During differentiation, the chaperone network is drastically rewired, including loss of expression of the anti-amyloidogenic chaperone DNAJB6. Upregulation of DNAJB6 in neurons antagonizes glutamate-induced aggregation, while knockdown of DNAJB6 in progenitors results in spontaneous polyglutamine aggregation. Loss of DNAJB6 expression upon differentiation is confirmed in vivo, explaining why stem cells are intrinsically protected against amyloidogenesis and protein aggregates are dominantly present in neurons.
Assuntos
Proteínas Amiloidogênicas/genética , Diferenciação Celular/genética , Proteínas de Choque Térmico HSP40/genética , Chaperonas Moleculares/genética , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/metabolismo , Regulação da Expressão Gênica/genética , Técnicas de Inativação de Genes , Ácido Glutâmico/metabolismo , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/metabolismo , Doença de Machado-Joseph/patologia , Células-Tronco Neurais/patologia , Neurônios/metabolismo , Neurônios/patologia , Agregados Proteicos/genética , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Synaptic dysfunction and cognitive decline in Huntington's disease (HD) involve hyperactive A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10). To identify the molecular mechanisms through which ADAM10 is associated with synaptic dysfunction in HD, we performed an immunoaffinity purification-mass spectrometry (IP-MS) study of endogenous ADAM10 in the brains of wild-type and HD mice. We found that proteins implicated in synapse organization, synaptic plasticity, and vesicle and organelles trafficking interact with ADAM10, suggesting that it may act as hub protein at the excitatory synapse. Importantly, the ADAM10 interactome is enriched in presynaptic proteins and ADAM10 co-immunoprecipitates with piccolo (PCLO), a key player in the recycling and maintenance of synaptic vesicles. In contrast, reduced ADAM10/PCLO immunoprecipitation occurs in the HD brain, with decreased density of synaptic vesicles in the reserve and docked pools at the HD presynaptic terminal. Conditional heterozygous deletion of ADAM10 in the forebrain of HD mice reduces active ADAM10 to wild-type level and normalizes ADAM10/PCLO complex formation and synaptic vesicle density and distribution. The results indicate that presynaptic ADAM10 and PCLO are a relevant component of HD pathogenesis.
Assuntos
Proteína ADAM10/metabolismo , Proteínas do Citoesqueleto/metabolismo , Doença de Huntington/metabolismo , Neuropeptídeos/metabolismo , Vesículas Sinápticas/metabolismo , Proteína ADAM10/genética , Animais , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/ultraestrutura , Humanos , Doença de Huntington/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Terminações Pré-Sinápticas/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas/genética , Proteômica/métodos , Vesículas Sinápticas/ultraestrutura , Sinaptossomos/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
Evidence that Huntington's disease (HD) is characterized by impaired cholesterol biosynthesis in the brain has led to strategies to increase its level in the brain of the rapidly progressing R6/2 mouse model, with a positive therapeutic outcome. Here we tested the long-term efficacy of chronic administration of cholesterol to the brain of the slowly progressing zQ175DN knock-in HD mice in preventing ("early treatment") or reversing ("late treatment") HD symptoms. To do this we used the most advanced formulation of cholesterol loaded brain-permeable nanoparticles (NPs), termed hybrid-g7-NPs-chol, which were injected intraperitoneally. We show that one cycle of treatment with hybrid-g7-NPs-chol, administered in the presymptomatic ("early treatment") or symptomatic ("late treatment") stages is sufficient to normalize cognitive defects up to 5 months, as well as to improve other behavioral and neuropathological parameters. A multiple cycle treatment combining both early and late treatments ("2 cycle treatment") lasting 6 months generates therapeutic effects for more than 11 months, without severe adverse reactions. Sustained cholesterol delivery to the brain of zQ175DN mice also reduces mutant Huntingtin aggregates in both the striatum and cortex and completely normalizes synaptic communication in the striatal medium spiny neurons compared to saline-treated HD mice. Furthermore, through a meta-analysis of published and current data, we demonstrated the power of hybrid-g7-NPs-chol and other strategies able to increase brain cholesterol biosynthesis, to reverse cognitive decline and counteract the formation of mutant Huntingtin aggregates. These results demonstrate that cholesterol delivery via brain-permeable NPs is a therapeutic option to sustainably reverse HD-related behavioral decline and neuropathological signs over time, highlighting the therapeutic potential of cholesterol-based strategies in HD patients. DATA AVAILABILITY: This study does not include data deposited in public repositories. Data are available on request to the corresponding authors.
Assuntos
Doença de Huntington , Camundongos , Animais , Doença de Huntington/tratamento farmacológico , Doença de Huntington/patologia , Encéfalo/patologia , Colesterol , Corpo Estriado/patologia , Cognição , Modelos Animais de Doenças , Camundongos TransgênicosRESUMO
Brain cholesterol is produced mainly by astrocytes and is important for neuronal function. Its biosynthesis is severely reduced in mouse models of Huntington's disease. One possible mechanism is a diminished nuclear translocation of the transcription factor sterol regulatory element-binding protein 2 (SREBP2) and, consequently, reduced activation of SREBP2-controlled genes in the cholesterol biosynthesis pathway. Here we evaluated the efficacy of a gene therapy based on the unilateral intra-striatal injection of a recombinant adeno-associated virus 2/5 (AAV2/5) targeting astrocytes specifically and carrying the transcriptionally active N-terminal fragment of human SREBP2 (hSREBP2). Robust hSREBP2 expression in striatal glial cells in R6/2 Huntington's disease mice activated the transcription of cholesterol biosynthesis pathway genes, restored synaptic transmission, reversed dopamine receptor D2 (Drd2) transcript levels decline, cleared mutant huntingtin aggregates and attenuated behavioural deficits. We conclude that glial SREBP2 participates in Huntington's disease brain pathogenesis in vivo and that AAV-based delivery of SREBP2 to astrocytes counteracts key features of the disease.
Assuntos
Astrócitos/metabolismo , Corpo Estriado/metabolismo , Técnicas de Transferência de Genes , Terapia Genética/métodos , Doença de Huntington/terapia , Proteína de Ligação a Elemento Regulador de Esterol 2/administração & dosagem , Animais , Astrócitos/patologia , Corpo Estriado/patologia , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Masculino , Camundongos , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Fenótipo , Proteína de Ligação a Elemento Regulador de Esterol 2/biossíntese , Proteína de Ligação a Elemento Regulador de Esterol 2/genéticaRESUMO
In vitro models of corticogenesis from pluripotent stem cells (PSCs) have greatly improved our understanding of human brain development and disease. Among these, 3D cortical organoid systems are able to recapitulate some aspects of in vivo cytoarchitecture of the developing cortex. Here, we tested three cortical organoid protocols for brain regional identity, cell type specificity and neuronal maturation. Overall, all protocols gave rise to organoids that displayed a time-dependent expression of neuronal maturation genes such as those involved in the establishment of synapses and neuronal function. Comparatively, guided differentiation methods without WNT activation generated the highest degree of cortical regional identity, whereas default conditions produced the broadest range of cell types such as neurons, astrocytes and hematopoietic-lineage-derived microglia cells. These results suggest that cortical organoid models produce diverse outcomes of brain regional identity and cell type specificity and emphasize the importance of selecting the correct model for the right application.
Assuntos
Organoides , Células-Tronco Pluripotentes , Humanos , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular , Neurônios/metabolismo , EncéfaloRESUMO
Objective: The present work aimed to evaluate: (a) the psychometric properties of the Centrality of Event Scale in Italian primiparous and multiparous women; (b) individual differences in those demographic variables that influence change in women's identity and the maternal role acquisition during pregnancy; (c) the association between the extent to which pregnancy has an impact on woman's life story and identity and prenatal attachment; (c) how the centrality of the pregnancy event is related to the experience of PTSD during pregnancy.Background: Pregnancy is a crucial phase in women's life that involves many changes for a woman's role and identity.Methods 319 pregnant women were assessd during the third trimester of pregnancy.Results: Exploratory Factor Analyses confirmed a one-factor solution of the CES. Moreover, the perception of pregnancy as central in women's lives is significantly related to prenatal attachment. Finally, the perception of pregnancy as central in women's lives is positively correlated to PTSD symptoms.Conclusion: Our findings provide evidence on the validity of the scale with pregnant women samples, which may contribute for a better understanding of the impact of pregnancy on women's identity and life story, as well as the underlying psychological challenges related to pregnancy.
Assuntos
Família , Gestantes , Análise Fatorial , Feminino , Humanos , Gravidez , Terceiro Trimestre da Gravidez , PsicometriaRESUMO
RUES2 cell lines represent the first collection of isogenic human embryonic stem cells (hESCs) carrying different pathological CAG lengths in the HTT gene. However, their neuronal differentiation potential has yet to be thoroughly evaluated. Here, we report that RUES2 during ventral telencephalic differentiation is biased towards medial ganglionic eminence (MGE). We also show that HD-RUES2 cells exhibit an altered MGE transcriptional signature in addition to recapitulating known HD phenotypes, with reduced expression of the neurodevelopmental regulators NEUROD1 and BDNF and increased cleavage of synaptically enriched N-cadherin. Finally, we identified the transcription factor SP1 as a common potential detrimental co-partner of muHTT by de novo motif discovery analysis on the LGE, MGE, and cortical genes differentially expressed in HD human pluripotent stem cells in our and additional datasets. Taken together, these observations suggest a broad deleterious effect of muHTT in the early phases of neuronal development that may unfold through its altered interaction with SP1.
Assuntos
Biomarcadores Tumorais/metabolismo , Diferenciação Celular/fisiologia , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Pluripotentes/citologia , Receptores Imunológicos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/patologia , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismoRESUMO
Allele-specific silencing by RNA interference (ASP-siRNA) holds promise as a therapeutic strategy for downregulating a single mutant allele with minimal suppression of the corresponding wild-type allele. This approach has been effectively used to target autosomal dominant mutations and single nucleotide polymorphisms linked with aberrantly expanded trinucleotide repeats. Here, we propose ASP-siRNA as a preferable choice to target duplicated disease genes, avoiding potentially harmful excessive downregulation. As a proof-of-concept, we studied autosomal dominant adult-onset demyelinating leukodystrophy (ADLD) due to lamin B1 (LMNB1) duplication, a hereditary, progressive and fatal disorder affecting myelin in the CNS. Using a reporter system, we screened the most efficient ASP-siRNAs preferentially targeting one of the alleles at rs1051644 (average minor allele frequency: 0.45) located in the 3' untranslated region of the gene. We identified four siRNAs with a high efficacy and allele-specificity, which were tested in ADLD patient-derived fibroblasts. Three of the small interfering RNAs were highly selective for the target allele and restored both LMNB1 mRNA and protein levels close to control levels. Furthermore, small interfering RNA treatment abrogates the ADLD-specific phenotypes in fibroblasts and in two disease-relevant cellular models: murine oligodendrocytes overexpressing human LMNB1, and neurons directly reprogrammed from patients' fibroblasts. In conclusion, we demonstrated that ASP-silencing by RNA interference is a suitable and promising therapeutic option for ADLD. Moreover, our results have a broad translational value extending to several pathological conditions linked to gene-gain in copy number variations.
Assuntos
Alelos , Duplicação Gênica/efeitos dos fármacos , Inativação Gênica , Doenças Genéticas Inatas/tratamento farmacológico , Lamina Tipo B/metabolismo , Doença de Pelizaeus-Merzbacher/tratamento farmacológico , RNA Interferente Pequeno/uso terapêutico , Animais , Estudos de Casos e Controles , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Vetores Genéticos , Humanos , Lentivirus , Neurônios/metabolismo , RatosRESUMO
DACH1 is the human homolog of the Drosophila dachshund gene, which is involved in the development of the eye, nervous system, and limbs in the fly. Here, we systematically investigate DACH1 expression patterns during human neurodevelopment, from 5 to 21 postconceptional weeks. By immunodetection analysis, we found that DACH1 is highly expressed in the proliferating neuroprogenitors of the developing cortical ventricular and subventricular regions, while it is absent in the more differentiated cortical plate. Single-cell global transcriptional analysis revealed that DACH1 is specifically enriched in neuroepithelial and ventricular radial glia cells of the developing human neocortex. Moreover, we describe a previously unreported DACH1 expression in the human striatum, in particular in the striatal medium spiny neurons. This finding qualifies DACH1 as a new striatal projection neuron marker, together with PPP1R1B, BCL11B, and EBF1. We finally compared DACH1 expression profile in human and mouse forebrain, where we observed spatio-temporal similarities in its expression pattern thus providing a precise developmental description of DACH1 in the 2 mammalian species.
Assuntos
Corpo Estriado/embriologia , Corpo Estriado/metabolismo , Proteínas do Olho/metabolismo , Neocórtex/embriologia , Neocórtex/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Fatores de Transcrição/metabolismo , Feto Abortado/embriologia , Feto Abortado/metabolismo , Células Ependimogliais/metabolismo , Idade Gestacional , Humanos , Ventrículos Laterais/embriologia , Ventrículos Laterais/metabolismo , Células-Tronco Neurais/metabolismo , Células Neuroepiteliais/metabolismo , Prosencéfalo/embriologia , Prosencéfalo/metabolismo , Especificidade da EspécieRESUMO
Young cancer survivors often wish to bear a child after oncological treatments, as they might not have started or completed their families. As young cancer survivors have a higher risk of developing psychological difficulties, this study investigated whether there were significant differences in psychological aspects between pregnant women who received a cancer diagnosis in the past and pregnant women without a history of cancer. A total of 123 pregnant women, of which 36 were cancer survivors and 87 women without a history of cancer, were recruited during their last trimester at different hospitals in Northern Italy. Patients were asked to complete a socio-demographic profile and questionnaires measuring mood states, post-traumatic symptoms, centrality of the pregnancy and cancer event, quality of life, and prenatal attachment. Cancer survivors had significantly higher levels of PTSD symptoms, perceived pregnancy as more central to their identity and life story, perceived lower quality of life and had lower intensity of prenatal attachment compared with the control group. Centrality of the cancer event did not correlate with any psychological variables. Preliminary results suggest that a past cancer diagnosis can influence the mother's psychological functioning and the development of the relationship with their child.
Assuntos
Sobreviventes de Câncer/psicologia , Neoplasias/psicologia , Complicações Neoplásicas na Gravidez/psicologia , Adaptação Psicológica , Adulto , Estudos de Casos e Controles , Saúde da Família , Feminino , Humanos , Neoplasias/genética , Gravidez , Qualidade de Vida , Inquéritos e QuestionáriosRESUMO
Posttranslational modifications can have profound effects on the biological and biophysical properties of proteins associated with misfolding and aggregation. However, their detection and quantification in clinical samples and an understanding of the mechanisms underlying the pathological properties of misfolding- and aggregation-prone proteins remain a challenge for diagnostics and therapeutics development. We have applied an ultrasensitive immunoassay platform to develop and validate a quantitative assay for detecting a posttranslational modification (phosphorylation at residue T3) of a protein associated with polyglutamine repeat expansion, namely Huntingtin, and characterized its presence in a variety of preclinical and clinical samples. We find that T3 phosphorylation is greatly reduced in samples from Huntington's disease models and in Huntington's disease patients, and we provide evidence that bona-fide T3 phosphorylation alters Huntingtin exon 1 protein conformation and aggregation properties. These findings have significant implications for both mechanisms of disease pathogenesis and the development of therapeutics and diagnostics for Huntington's disease.
Assuntos
Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Imunoensaio/métodos , Animais , Células Cultivadas , Éxons , Células HEK293 , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/genética , Camundongos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fosforilação , Conformação Proteica , Processamento de Proteína Pós-Traducional , Sensibilidade e EspecificidadeRESUMO
Medium spiny neurons (MSNs) are a key population in the basal ganglia network, and their degeneration causes a severe neurodegenerative disorder, Huntington's disease. Understanding how ventral neuroepithelial progenitors differentiate into MSNs is critical for regenerative medicine to develop specific differentiation protocols using human pluripotent stem cells. Studies performed in murine models have identified some transcriptional determinants, including GS Homeobox 2 (Gsx2) and Early B-cell factor 1 (Ebf1). Here, we have generated human embryonic stem (hES) cell lines inducible for these transcription factors, with the aims of (i) studying their biological role in human neural progenitors and (ii) incorporating TF conditional expression in a developmental-based protocol for generating MSNs from hES cells. Using this approach, we found that Gsx2 delays cell-cycle exit and reduces Pax6 expression, whereas Ebf1 promotes neuronal differentiation. Moreover, we found that Gsx2 and Ebf1 combined overexpression in hES cells achieves high yields of MSNs, expressing Darpp32 and Ctip2, in vitro as well in vivo after transplantation. We show that hES-derived striatal progenitors can be transplanted in animal models and can differentiate and integrate into the host, extending fibers over a long distance.
Assuntos
Diferenciação Celular/genética , Proteínas de Homeodomínio/genética , Células-Tronco Embrionárias Humanas/metabolismo , Neurônios/metabolismo , Transativadores/genética , Animais , Ciclo Celular/genética , Linhagem Celular , Fosfoproteína 32 Regulada por cAMP e Dopamina/genética , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Células-Tronco Embrionárias Humanas/transplante , Humanos , Camundongos Nus , Neurônios/citologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transplante de Células-Tronco/métodos , Telencéfalo/citologia , Transativadores/metabolismo , Transplante Heterólogo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The Huntingtin (HTT) gene contains a CAG repeat in exon 1, whose expansion beyond 39 repeats consistently leads to Huntington's disease (HD), whereas normal-to-intermediate alleles seemingly modulate brain structure, function and behavior. The role of the CAG repeat in Autism Spectrum Disorder (ASD) was investigated applying both family-based and case-control association designs, with the SCA3 repeat as a negative control. Significant overtransmission of "long" CAG alleles (≥17 repeats) to autistic children and of "short" alleles (≤16 repeats) to their unaffected siblings (all p < 10-5 ) was observed in 612 ASD families (548 simplex and 64 multiplex). Surprisingly, both 193 population controls and 1,188 neurological non-HD controls have significantly lower frequencies of "short" CAG alleles compared to 185 unaffected siblings and higher rates of "long" alleles compared to 548 ASD patients from the same families (p < .05-.001). The SCA3 CAG repeat displays no association. "Short" HTT alleles seemingly exert a protective effect from clinically overt autism in families carrying a genetic predisposition for ASD, while "long" alleles may enhance autism risk. Differential penetrance of autism-inducing genetic/epigenetic variants may imply atypical developmental trajectories linked to HTT functions, including excitation/inhibition imbalance, cortical neurogenesis and apoptosis, neuronal migration, synapse formation, connectivity and homeostasis.
Assuntos
Transtorno Autístico/genética , Proteína Huntingtina/genética , Adulto , Alelos , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Transtorno Autístico/metabolismo , Encéfalo , Estudos de Casos e Controles , Criança , Pré-Escolar , Família , Feminino , Frequência do Gene/genética , Humanos , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Neurogênese , Penetrância , Fatores de Risco , Expansão das Repetições de Trinucleotídeos/genética , Repetições de Trinucleotídeos/genéticaRESUMO
Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the gene encoding for huntingtin protein. A lot has been learned about this disease since its first description in 1872 and the identification of its causative gene and mutation in 1993. We now know that the disease is characterized by several molecular and cellular abnormalities whose precise timing and relative roles in pathogenesis have yet to be understood. HD is triggered by the mutant protein, and both gain-of-function (of the mutant protein) and loss-of-function (of the normal protein) mechanisms are involved. Here we review the data that describe the emergence of the ancient huntingtin gene and of the polyglutamine trait during the last 800 million years of evolution. We focus on the known functions of wild-type huntingtin that are fundamental for the survival and functioning of the brain neurons that predominantly degenerate in HD. We summarize data indicating how the loss of these beneficial activities reduces the ability of these neurons to survive. We also review the different mechanisms by which the mutation in huntingtin causes toxicity. This may arise both from cell-autonomous processes and dysfunction of neuronal circuitries. We then focus on novel therapeutical targets and pathways and on the attractive option to counteract HD at its primary source, i.e., by blocking the production of the mutant protein. Strategies and technologies used to screen for candidate HD biomarkers and their potential application are presented. Furthermore, we discuss the opportunities offered by intracerebral cell transplantation and the likely need for these multiple routes into therapies to converge at some point as, ideally, one would wish to stop the disease process and, at the same time, possibly replace the damaged neurons.
Assuntos
Doença de Huntington/genética , Doença de Huntington/terapia , Animais , Biomarcadores/metabolismo , Encéfalo/fisiopatologia , Sobrevivência Celular , Modelos Animais de Doenças , Marcação de Genes , Humanos , Proteína Huntingtina , Doença de Huntington/fisiopatologia , Mutação , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMO
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 SexuaisRESUMO
At the time of writing, the Italian Parliament is debating a new law that would make it legal to practice an unproven stem cell treatment in public hospitals. The treatment, offered by a private non-medical organization, may not be safe, lacks a rationale, and violates current national laws and European regulations. This case raises multiple concerns, most prominently the urgent need to protect patients who are severely ill, exposed to significant risks, and vulnerable to exploitation. The scientific community must consider the context-social, financial, medical, legal-in which stem cell science is currently situated and the need for stringent regulation. Additional concerns are emerging. These emanate from the novel climate, created within science itself, and stem cell science in particular, by the currently prevailing model of 'translational medicine'. Only rigorous science and rigorous regulation can ensure translation of science into effective therapies rather than into ineffective market products, and mark, at the same time, the sharp distinction between the striving for new therapies and the deceit of patients.
Assuntos
Transplante de Células-Tronco/legislação & jurisprudência , Europa (Continente) , Humanos , Itália , Segurança do Paciente , Transplante de Células-Tronco/efeitos adversos , Transplante de Células-Tronco/normas , Células-Tronco , Pesquisa Translacional BiomédicaRESUMO
Huntington disease is a neurodegenerative disorder caused by a gene (HTT) with a unique feature of trinucleotide repeats ranging from 10 to 35 in healthy people; when expanded beyond 39 repeats, Huntington disease develops. Animal models demonstrate that HTT is vital to brain development; however, this has not been studied in humans. Moreover, evidence suggests that triplet repeat genes may have been vital in evolution of the human brain. Here we evaluate brain structure using magnetic resonance imaging and brain function using cognitive tests in a sample of school-aged children ages 6 to 18 years old. DNA samples were processed to quantify the number of CAG repeats within HTT. We find that the number of repeats in HTT, below disease threshold, confers advantageous changes in brain structure and general intelligence (IQ): the higher the number of repeats, the greater the change in brain structure, and the higher the IQ. The pattern of structural brain changes associated with HTT is strikingly different between males and females. HTT may confer an advantage or a disadvantage depending on the repeat length, playing a key role in either the evolution of a superior human brain or development of a uniquely human brain disease. © 2016 Wiley Periodicals, Inc.
Assuntos
Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Proteína Huntingtina/genética , Inteligência/genética , Caracteres Sexuais , Repetições de Trinucleotídeos/genética , Adolescente , Encéfalo/diagnóstico por imagem , Criança , Feminino , Humanos , Doença de Huntington/diagnóstico por imagem , Doença de Huntington/genética , Doença de Huntington/patologia , Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Masculino , Testes Neuropsicológicos , Adulto JovemRESUMO
Medium-sized spiny neurons (MSNs) are the only neostriatum projection neurons, and their degeneration underlies some of the clinical features of Huntington's disease. Using knowledge of human developmental biology and exposure to key neurodevelopmental molecules, human pluripotent stem (hPS) cells were induced to differentiate into MSNs. In a feeder-free adherent culture, ventral telencephalic specification is induced by BMP/TGFß inhibition and subsequent SHH/DKK1 treatment. The emerging FOXG1(+)/GSX2(+) telencephalic progenitors are then terminally differentiated, resulting in the systematic line-independent generation of FOXP1(+)/FOXP2(+)/CTIP2(+)/calbindin(+)/DARPP-32(+) MSNs. Similar to mature MSNs, these neurons carry dopamine and A2a receptors, elicit a typical firing pattern and show inhibitory postsynaptic currents, as well as dopamine neuromodulation and synaptic integration ability in vivo. When transplanted into the striatum of quinolinic acid-lesioned rats, hPS-derived neurons survive and differentiate into DARPP-32(+) neurons, leading to a restoration of apomorphine-induced rotation behavior. In summary, hPS cells can be efficiently driven to acquire a functional striatal fate using an ontogeny-recapitulating stepwise method that represents a platform for in vitro human developmental neurobiology studies and drug screening approaches.
Assuntos
Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Neurônios/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Adesão Celular , Diferenciação Celular , Linhagem da Célula , Sobrevivência Celular , Transplante de Células , Células-Tronco Embrionárias/citologia , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Neurônios GABAérgicos/metabolismo , Humanos , Doença de Huntington/metabolismo , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Técnicas de Patch-Clamp , Ácido Quinolínico/farmacologia , RNA/metabolismo , Ratos , Células-Tronco/citologia , Fatores de TempoRESUMO
Human cytomegalovirus (HCMV) is known to exert suppressive effects on the host immune system through expression of various viral genes, thus directly and indirectly affecting antiviral immunity of the infected individuals. We report here that HCMV UL10 encodes a protein (pUL10) with immunosuppressive properties. UL10 has been classified as a member of the HCMV RL11 gene family. Although pUL10 is known to be dispensable for viral replication in cultured cells, its amino-acid sequence is well conserved among different HCMV isolates, suggesting that the protein has a crucial role in viral survival in the host environment. We show that pUL10 is cleaved from the cell surface of fibroblasts as well as epithelial cells and interacts with a cellular receptor ubiquitously expressed on the surface of human leukocytes, demonstrated by ex vivo cell-based assays and flow cytometric analyses on both lymphoid cell lines and primary blood cells. Furthermore, preincubation of peripheral blood mononuclear cells with purified pUL10 ectodomain results in significantly impaired proliferation and substantially reduced pro-inflammatory cytokine production, in particular in CD4+ T cells upon in vitro T-cell stimulation. The inhibitory effect of pUL10 is also observed on antigen receptor-mediated intracellular tyrosine phosphorylation in a T-cell line. Based on these observations, we suggest that pUL10 is a newly identified immunomodulatory protein encoded by HCMV. Further elucidation of interactions between pUL10 and the host immune system during HCMV may contribute to finding ways towards new therapies for HCMV infection.
Assuntos
Proteínas do Capsídeo/metabolismo , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Sequência de Aminoácidos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Proteínas do Capsídeo/química , Linhagem Celular , Membrana Celular/metabolismo , Proliferação de Células , Citocinas/biossíntese , Glicosilação , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
Huntington disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by an increased number of CAG repeats in the HTT gene coding for huntingtin. Decreased neurotrophic support and increased mitochondrial and excitotoxic stress have been reported in HD striatal and cortical neurons. The members of the class O forkhead (FOXO) transcription factor family, including FOXO3a, act as sensor proteins that are activated upon decreased survival signals and/or increased cellular stress. Using immunocytochemical screening in mouse striatal Hdh(7/7) (wild type), Hdh(7/109) (heterozygous for HD mutation), and Hdh(109/109) (homozygous for HD mutation) cells, we identified FOXO3a as a differentially regulated transcription factor in HD. We report increased nuclear FOXO3a levels in mutant Hdh cells. Additionally, we show that treatment with mitochondrial toxin 3-nitropropionic acid results in enhanced nuclear localization of FOXO3a in wild type Hdh(7/7) cells and in rat primary cortical neurons. Furthermore, mRNA levels of Foxo3a are increased in mutant Hdh cells compared with wild type cells and in 3-nitropropionic acid-treated primary neurons compared with untreated neurons. A similar increase was observed in the cortex of R6/2 mice and HD patient post-mortem caudate tissue compared with controls. Using chromatin immunoprecipitation and reporter assays, we demonstrate that FOXO3a regulates its own transcription by binding to the conserved response element in Foxo3a promoter. Altogether, the findings of this study suggest that FOXO3a levels are increased in HD cells as a result of overactive positive feedback loop.