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1.
Exp Neurol ; 323: 113095, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31712124

RESUMO

Currently, molecular, electrophysiological and structural studies delineate several neural subtypes in the hippocampus. However, the precise developmental mechanisms that lead to this diversity are still unknown. Here we show that alterations in a concrete hippocampal neuronal subpopulation during development specifically affect hippocampal-dependent spatial memory. We observed that the genetic deletion of the transcription factor Helios in mice, which is specifically expressed in developing hippocampal calbindin-positive CA1 pyramidal neurons (CB-CA1-PNs), induces adult alterations affecting spatial memory. In the same mice, CA3-CA1 synaptic plasticity and spine density and morphology in adult CB-CA1-PNs were severely compromised. RNAseq experiments in developing hippocampus identified an aberrant increase on the Visinin-like protein 1 (VSNL1) expression in the hippocampi devoid of Helios. This aberrant increase on VSNL1 levels was localized in the CB-CA1-PNs. Normalization of VSNL1 levels in CB-CA1-PNs devoid of Helios rescued their spine loss in vitro. Our study identifies a novel and specific developmental molecular pathway involved in the maturation and function of a CA1 pyramidal neuronal subtype.

2.
PLoS One ; 14(12): e0224901, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31790427

RESUMO

In Huntington's disease (HD), striatal medium spiny neurons (MSNs) are particularly sensitive to the presence of a CAG repeat in the huntingtin (HTT) gene. However, there are many evidences that cells from the peripheral immune system and central nervous system (CNS) immune cells, namely microglia, play an important role in the etiology and the progression of HD. However, it remains unclear whether MSNs neurodegeneration is mediated by a non-cell autonomous mechanism. The homeostasis in the healthy CNS is maintained by several mechanisms of interaction between all brain cells. Neurons can control microglia activation through several inhibitory mechanisms, such as the CD200-CD200R1 interaction. Due to the complete lack of knowledge about the CD200-CD200R1 system in HD, we determined the temporal patterns of CD200 and CD200R1 expression in the neocortex, hippocampus and striatum in the HD mouse models R6/1 and HdhQ111/7 from pre-symptomatic to manifest stages. In order to explore any alteration in the peripheral immune system, we also studied the levels of expression of CD200 and CD200R1 in whole blood. Although CD200R1 expression was not altered, we observed and increase in CD200 gene expression and protein levels in the brain parenchyma of all the regions we examined, along with HD pathogenesis in R6/1 mice. Interestingly, the expression of CD200 mRNA was also up-regulated in blood following a similar temporal pattern. These results suggest that canonical neuronal-microglial communication through CD200-CD200R1 interaction is not compromised, and CD200 up-regulation in R6/1 brain parenchyma could represent a neurotrophic signal to sustain or extend neuronal function in the latest stages of HD as pro-survival mechanism.

3.
Mol Ther Methods Clin Dev ; 12: 134-144, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30623002

RESUMO

Genetically modifying autologous T cells to express an anti-CD19 chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19+ B cell malignancies in several clinical trials (CTs). Making this treatment available to our patients prompted us to develop a novel CART19 based on our own anti-CD19 antibody (A3B1), followed by CD8 hinge and transmembrane region, 4-1BB- and CD3z-signaling domains. We show that A3B1 CAR T cells are highly cytotoxic and specific against CD19+ cells in vitro, inducing secretion of pro-inflammatory cytokines and CAR T cell proliferation. In vivo, A3B1 CAR T cells are able to fully control disease progression in an NOD.Cg-Prkdc scid Il2rd tm1Wjl /SzJ (NSG) xenograph B-ALL mouse model. Based on the pre-clinical data, we conclude that our CART19 is clearly functional against CD19+ cells, to a level similar to other CAR19s currently being used in the clinic. Concurrently, we describe the implementation of our CAR T cell production system, using lentiviral vector and CliniMACS Prodigy, within a medium-sized academic institution. The results of the validation phase show our system is robust and reproducible, while maintaining a low cost that is affordable for academic institutions. Our model can serve as a paradigm for similar institutions, and it may help to make CAR T cell treatment available to all patients.

4.
Pflugers Arch ; 470(9): 1359-1376, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29797067

RESUMO

Kv7 channels determine the resting membrane potential of neurons and regulate their excitability. Even though dysfunction of Kv7 channels has been linked to several debilitating childhood neuronal disorders, the ontogeny of the constituent genes, which encode Kv7 channels (KNCQ), and expression of their subunits have been largely unexplored. Here, we show that developmentally regulated expression of specific KCNQ mRNA and Kv7 channel subunits in mouse and human striatum is crucial to the functional maturation of mouse striatal neurons and human-induced pluripotent stem cell-derived neurons. This demonstrates their pivotal role in normal development and maturation, the knowledge of which can now be harnessed to synchronise and accelerate neuronal differentiation of stem cell-derived neurons, enhancing their utility for disease modelling and drug discovery.


Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Canal de Potássio KCNQ1/metabolismo , Neurônios/metabolismo , Regulação para Cima/fisiologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Humanos , Potenciais da Membrana/fisiologia , Camundongos , RNA Mensageiro/metabolismo
5.
J Neuroinflammation ; 14(1): 54, 2017 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-28302135

RESUMO

BACKGROUND: CCAAT/enhancer binding protein ß (C/EBPß) is a transcription factor that regulates the expression of important pro-inflammatory genes in microglia. Mice deficient for C/EBPß show protection against excitotoxic and ischemic CNS damage, but the involvement in this neuroprotective effect of the various C/EBPß-expressing cell types is not solved. Since C/EBPß-deficient microglia show attenuated neurotoxicity in culture, we hypothesized that specific C/EBPß deficiency in microglia could be neuroprotective in vivo. In this study, we have tested this hypothesis by generating mice with myeloid C/EBPß deficiency. METHODS: Mice with myeloid C/EBPß deficiency were generated by crossing LysMCre and C/EBPßfl/fl mice. Primary microglial cultures from C/EBPßfl/fl and LysMCre-C/EBPßfl/fl mice were treated with lipopolysaccharide ± interferon γ (IFNγ) for 6 h, and gene expression was analyzed by RNA sequencing. Gene expression and C/EBPß deletion were analyzed in vivo in microglia isolated from the brains of C/EBPßfl/fl and LysMCre-C/EBPßfl/fl mice treated systemically with lipolysaccharide or vehicle. Mice of LysMCre-C/EBPßfl/fl or control genotypes were subjected to experimental autoimmune encephalitis and analyzed for clinical signs for 52 days. One- or two-way ANOVA or Kruskal-Wallis with their appropriate post hoc tests were used. RESULTS: LysMCre-C/EBPßfl/fl mice showed an efficiency of C/EBPß deletion in microglia of 100 and 90% in vitro and in vivo, respectively. These mice were devoid of female infertility, perinatal mortality and reduced lifespan that are associated to full C/EBPß deficiency. Transcriptomic analysis of C/EBPß-deficient primary microglia revealed C/EBPß-dependent expression of 1068 genes, significantly enriched in inflammatory and innate immune responses GO terms. In vivo, microglial expression of the pro-inflammatory genes Cybb, Ptges, Il23a, Tnf and Csf3 induced by systemic lipopolysaccharide injection was also blunted by C/EBPß deletion. CNS expression of C/EBPß was upregulated in experimental autoimmune encephalitis and in multiple sclerosis samples. Finally, LysMCre-C/EBPßfl/fl mice showed robust attenuation of clinical signs in experimental autoimmune encephalitis. CONCLUSION: This study provides new data that support a central role for C/EBPß in the biology of activated microglia, and it offers proof of concept for the therapeutic potential of microglial C/EBPß inhibition in multiple sclerosis.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/deficiência , Encefalomielite Autoimune Experimental/patologia , Microglia/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Animais Recém-Nascidos , Ontologias Biológicas , Proteína beta Intensificadora de Ligação a CCAAT/genética , Antígeno CD11b/metabolismo , Células Cultivadas , Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/terapia , Feminino , Humanos , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos Transgênicos , Pessoa de Meia-Idade , Esclerose Múltipla/patologia , Glicoproteína Mielina-Oligodendrócito/toxicidade , Óxido Nítrico/metabolismo , Fragmentos de Peptídeos/toxicidade , Fagocitose/efeitos dos fármacos , Fagocitose/genética
6.
Development ; 144(8): 1566-1577, 2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28289129

RESUMO

Here, we unravel the mechanism of action of the Ikaros family zinc finger protein Helios (He) during the development of striatal medium spiny neurons (MSNs). He regulates the second wave of striatal neurogenesis involved in the generation of striatopallidal neurons, which express dopamine 2 receptor and enkephalin. To exert this effect, He is expressed in neural progenitor cells (NPCs) keeping them in the G1/G0 phase of the cell cycle. Thus, a lack of He results in an increase of S-phase entry and S-phase length of NPCs, which in turn impairs striatal neurogenesis and produces an accumulation of the number of cycling NPCs in the germinal zone (GZ), which end up dying at postnatal stages. Therefore, He-/- mice show a reduction in the number of dorso-medial striatal MSNs in the adult that produces deficits in motor skills acquisition. In addition, overexpression of He in NPCs induces misexpression of DARPP-32 when transplanted in mouse striatum. These findings demonstrate that He is involved in the correct development of a subset of striatopallidal MSNs and reveal new cellular mechanisms for neuronal development.


Assuntos
Corpo Estriado/citologia , Proteínas de Ligação a DNA/metabolismo , Globo Pálido/citologia , Neurônios/citologia , Neurônios/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Recém-Nascidos , Contagem de Células , Pontos de Checagem do Ciclo Celular , Morte Celular , Proliferação de Células , Ciclina E/metabolismo , Fase G1 , Camundongos Knockout , Atividade Motora , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese , Fenótipo , Fase S
7.
Exp Neurol ; 291: 20-35, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28131724

RESUMO

Primary human fetal cells have been used in clinical trials of cell replacement therapy for the treatment of neurodegenerative disorders such as Huntington's disease (HD). However, human fetal primary cells are scarce and difficult to work with and so a renewable source of cells is sought. Human fetal neural stem cells (hfNSCs) can be generated from human fetal tissue, but little is known about the differences between hfNSCs obtained from different developmental stages and brain areas. In the present work we characterized hfNSCs, grown as neurospheres, obtained from three developmental stages: 4-5, 6-7 and 8-9weeks post conception (wpc) and four brain areas: forebrain, cortex, whole ganglionic eminence (WGE) and cerebellum. We observed that, as fetal brain development proceeds, the number of neural precursors is diminished and post-mitotic cells are increased. In turn, primary cells obtained from older embryos are more sensitive to the dissociation process, their viability is diminished and they present lower proliferation ratios compared to younger embryos. However, independently of the developmental stage of derivation proliferation ratios were very low in all cases. Improvements in the expansion rates were achieved by mechanical, instead of enzymatic, dissociation of neurospheres but not by changes in the seeding densities. Regardless of the developmental stage, neurosphere cultures presented large variability in the viability and proliferation rates during the initial 3-4 passages, but stabilized achieving significant expansion rates at passage 5 to 6. This was true also for all brain regions except cerebellar derived cultures that did not expand. Interestingly, the brain region of hfNSC derivation influences the expansion potential, being forebrain, cortex and WGE derived cells the most expandable compared to cerebellar. Short term expansion partially compromised the regional identity of cortical but not WGE cultures. Nevertheless, both expanded cultures were multipotent and kept the ability to differentiate to region specific mature neuronal phenotypes.


Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células-Tronco Fetais/citologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Análise de Variância , Encéfalo/citologia , Encéfalo/embriologia , Sobrevivência Celular , Células Cultivadas , Células-Tronco Fetais/fisiologia , Feto , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Idade Gestacional , Humanos , Antígeno Ki-67 , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/metabolismo
8.
Neuroscience ; 333: 320-30, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27475250

RESUMO

Human Dopamine- and cAMP-regulated phosphoprotein of molecular weight 32kDa (DARPP-32, also known as PPP1R1B) gene codes for different transcripts that are mainly translated into two DARPP-32 protein isoforms, full length (fl)-DARPP-32 and truncated (t)-DARPP. The t-DARPP lacks the first 36 residues at the N-terminal, which alters its function. In the central nervous system, fl-DARPP-32 is highly expressed in GABAergic striatal medium spiny neurons (MSNs), where it integrates dopaminergic and glutamatergic input signaling. However, no information about human DARPP-32 isoform expression during MSNs maturation is available. In this study, our aim is to determine the expression of the two DARPP-32 isoforms in human fetal and adult striatal samples. We show that DARPP-32 isoform expression is differentially regulated during human striatal development, with the t-DARPP isoform being virtually absent from whole ganglionic eminence (WGE) and highly induced in the adult striatum (in both caudate and putamen). We next compared the four most common anti-DARPP-32 antibodies used in human specimens, to study their recognition of the two isoforms in fetal and adult human striatal samples by western blot and immunohistochemistry. The four antibodies specifically identify the fl-DARPP-32 in both fetal and adult samples, while t-DARPP form was only detected in adult striatal samples. In addition, the lack of t-DARPP recognition in human adult striatum by the antibody generated against the full-length domain produces in turn different efficacy by immunohistochemical analysis. In conclusion, our results show that expression of human DARPP-32 protein isoforms depends on the striatal neurodevelopmental stage with t-DARPP being specific for the human adult striatum.


Assuntos
Corpo Estriado/crescimento & desenvolvimento , Corpo Estriado/metabolismo , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Corpo Estriado/citologia , Corpo Estriado/embriologia , Feminino , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos
9.
J Neurooncol ; 129(1): 67-76, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27324642

RESUMO

The initial aim of this study was to generate a transplantable glial tumour model of low-intermediate grade by disaggregation of a spontaneous tumour mass from genetically engineered models (GEM). This should result in an increased tumour incidence in comparison to GEM animals. An anaplastic oligoastrocytoma (OA) tumour of World Health Organization (WHO) grade III was obtained from a female GEM mouse with the S100ß-v-erbB/inK4a-Arf (+/-) genotype maintained in the C57BL/6 background. The tumour tissue was disaggregated; tumour cells from it were grown in aggregates and stereotactically injected into C57BL/6 mice. Tumour development was followed using Magnetic Resonance Imaging (MRI), while changes in the metabolomics pattern of the masses were evaluated by Magnetic Resonance Spectroscopy/Spectroscopic Imaging (MRS/MRSI). Final tumour grade was evaluated by histopathological analysis. The total number of tumours generated from GEM cells from disaggregated tumour (CDT) was 67 with up to 100 % penetrance, as compared to 16 % in the local GEM model, with an average survival time of 66 ± 55 days, up to 4.3-fold significantly higher than the standard GL261 glioblastoma (GBM) tumour model. Tumours produced by transplantation of cells freshly obtained from disaggregated GEM tumour were diagnosed as WHO grade III anaplastic oligodendroglioma (ODG) and OA, while tumours produced from a previously frozen sample were diagnosed as WHO grade IV GBM. We successfully grew CDT and generated tumours from a grade III GEM glial tumour. Freezing and cell culture protocols produced progression to grade IV GBM, which makes the developed transplantable model qualify as potential secondary GBM model in mice.


Assuntos
Animais Geneticamente Modificados , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/fisiopatologia , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL/genética , Oligodendroglioma/patologia , Oligodendroglioma/fisiopatologia , Animais , Neoplasias Encefálicas/diagnóstico por imagem , Linhagem Celular Tumoral , Feminino , Imagem por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Gradação de Tumores , Oligodendroglioma/diagnóstico por imagem , Análise de Sobrevida
10.
Am J Physiol Cell Physiol ; 310(7): C520-41, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26718628

RESUMO

Although numerous protocols have been developed for differentiation of neurons from a variety of pluripotent stem cells, most have concentrated on being able to specify effectively appropriate neuronal subtypes and few have been designed to enhance or accelerate functional maturity. Of those that have, most employ time courses of functional maturation that are rather protracted, and none have fully characterized all aspects of neuronal function, from spontaneous action potential generation through to postsynaptic receptor maturation. Here, we describe a simple protocol that employs the sequential addition of just two supplemented media that have been formulated to separate the two key phases of neural differentiation, the neurogenesis and synaptogenesis, each characterized by different signaling requirements. Employing these media, this new protocol synchronized neurogenesis and enhanced the rate of maturation of pluripotent stem cell-derived neural precursors. Neurons differentiated using this protocol exhibited large cell capacitance with relatively hyperpolarized resting membrane potentials; moreover, they exhibited augmented: 1) spontaneous electrical activity; 2) regenerative induced action potential train activity; 3) Na(+) current availability, and 4) synaptic currents. This was accomplished by rapid and uniform development of a mature, inhibitory GABAAreceptor phenotype that was demonstrated by Ca(2+) imaging and the ability of GABAAreceptor blockers to evoke seizurogenic network activity in multielectrode array recordings. Furthermore, since this protocol can exploit expanded and frozen prepatterned neural progenitors to deliver mature neurons within 21 days, it is both scalable and transferable to high-throughput platforms for the use in functional screens.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Meios de Cultura/química , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Western Blotting , Ciclo Celular/fisiologia , Linhagem Celular , Técnicas de Cocultura , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/metabolismo , Microscopia Eletrônica de Varredura , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Técnicas de Patch-Clamp , Receptores de GABA-A/metabolismo
11.
Mol Neurobiol ; 53(5): 2857-2868, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-25876513

RESUMO

RTP801 expression is induced by cellular stress and has a pro-apoptotic function in non-proliferating differentiated cells such as neurons. In several neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease, elevated levels of RTP801 have been observed, which suggests a role for RTP801 in neuronal death. Neuronal death is also a pathological hallmark in Huntington's disease (HD), an inherited neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. Currently, the exact mechanisms underlying mutant huntingtin (mhtt)-induced toxicity are still unclear. Here, we investigated whether RTP801 is involved in (mhtt)-induced cell death. Ectopic exon-1 mhtt elevated RTP801 mRNA and protein levels in nerve growth factor (NGF)-differentiated PC12 cells and in rat primary cortical neurons. In neuronal PC12 cells, mhtt also contributed to RTP801 protein elevation by reducing its proteasomal degradation rate, in addition to promoting RTP801 gene expression. Interestingly, silencing RTP801 expression with short hairpin RNAs (shRNAs) blocked mhtt-induced cell death in NGF-differentiated PC12 cells. However, RTP801 protein levels were not altered in the striatum of Hdh(Q7/Q111) and R6/1 mice, two HD models that display motor deficits but not neuronal death. Importantly, RTP801 protein levels were elevated in both neural telencephalic progenitors differentiated from HD patient-derived induced pluripotent stem cells and in the putamen and cerebellum of human HD postmortem brains. Taken together, our results suggest that RTP801 is a novel downstream effector of mhtt-induced toxicity and that it may be relevant to the human disease.


Assuntos
Proteína Huntingtina/toxicidade , Proteínas Mutantes/toxicidade , Proteínas Repressoras/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Modelos Animais de Doenças , Feminino , Humanos , Doença de Huntington/patologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células PC12 , Proteólise/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Repressoras/genética , Regulação para Cima/efeitos dos fármacos
12.
EMBO Mol Med ; 7(12): 1529-46, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26516212

RESUMO

The epigenomic landscape of Parkinson's disease (PD) remains unknown. We performed a genomewide DNA methylation and a transcriptome studies in induced pluripotent stem cell (iPSC)-derived dopaminergic neurons (DAn) generated by cell reprogramming of somatic skin cells from patients with monogenic LRRK2-associated PD (L2PD) or sporadic PD (sPD), and healthy subjects. We observed extensive DNA methylation changes in PD DAn, and of RNA expression, which were common in L2PD and sPD. No significant methylation differences were present in parental skin cells, undifferentiated iPSCs nor iPSC-derived neural cultures not-enriched-in-DAn. These findings suggest the presence of molecular defects in PD somatic cells which manifest only upon differentiation into the DAn cells targeted in PD. The methylation profile from PD DAn, but not from controls, resembled that of neural cultures not-enriched-in-DAn indicating a failure to fully acquire the epigenetic identity own to healthy DAn in PD. The PD-associated hypermethylation was prominent in gene regulatory regions such as enhancers and was related to the RNA and/or protein downregulation of a network of transcription factors relevant to PD (FOXA1, NR3C1, HNF4A, and FOSL2). Using a patient-specific iPSC-based DAn model, our study provides the first evidence that epigenetic deregulation is associated with monogenic and sporadic PD.


Assuntos
Neurônios Dopaminérgicos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Doença de Parkinson/genética , Reprogramação Celular , Metilação de DNA , Epigenômica , Perfilação da Expressão Gênica , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Doença de Parkinson/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo
13.
Mol Ther Methods Clin Dev ; 2: 15030, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26417608

RESUMO

A systematic characterization of the spatio-temporal gene expression during human neurodevelopment is essential to understand brain function in both physiological and pathological conditions. In recent years, stem cell technology has provided an in vitro tool to recapitulate human development, permitting also the generation of human models for many diseases. The correct differentiation of human pluripotent stem cell (hPSC) into specific cell types should be evaluated by comparison with specific cells/tissue profiles from the equivalent adult in vivo organ. Here, we define by a quantitative high-throughput gene expression analysis the subset of specific genes of the whole ganglionic eminence (WGE) and adult human striatum. Our results demonstrate that not only the number of specific genes is crucial but also their relative expression levels between brain areas. We next used these gene profiles to characterize the differentiation of hPSCs. Our findings demonstrate a temporal progression of gene expression during striatal differentiation of hPSCs from a WGE toward an adult striatum identity. Present results establish a gene expression profile to qualitatively and quantitatively evaluate the telencephalic hPSC-derived progenitors eventually used for transplantation and mature striatal neurons for disease modeling and drug-screening.

14.
Hum Mol Genet ; 24(17): 4958-70, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26063761

RESUMO

Huntington's disease (HD) is a hereditary neurodegenerative disorder characterized by motor and cognitive impairments, involving striatum, cortex and hippocampus. Synaptic and memory dysfunction in HD mouse models have been related to low levels of brain-derived neurotrophic factor (BDNF) and imbalance between TrkB and p75(NTR) receptors. In addition, astrocyte over-activation has also been suggested to contribute to HD cognitive deficits. Fingolimod (FTY720), a modulator of sphingosine-1 phosphate (S1P) receptors, has been shown to increase BDNF levels and to reduce astrogliosis, proving its potential to regulate trophic support and inflammatory response. In this view, we have investigated whether FTY720 improves synaptic plasticity and memory in the R6/1 mouse model of HD, through regulation of BDNF signaling and astroglial reactivity. Chronic administration of FTY720 from pre-symptomatic stages ameliorated long-term memory deficits and dendritic spine loss in CA1 hippocampal neurons from R6/1 mice. Furthermore, FTY720 delivery prevented astrogliosis and over-activation of nuclear factor kappa beta (NF-κB) signaling in the R6/1 hippocampus, reducing tumor necrosis factor alpha (TNFα) and induced nitric oxide synthase (iNOS) levels. TNFα decrease correlated with the normalization of p75(NTR) expression in the hippocampus of FTY720-treated R6/1 mice, thus preventing p75(NTR)/TrkB imbalance. In addition, FTY720 increased cAMP levels and promoted phosphorylation of CREB and RhoA in the hippocampus of R6/1 mice, further supporting its role in the enhancement of synaptic plasticity. Our findings provide new insights into the mechanism of action of FTY720 and reveal a novel therapeutic strategy to treat memory deficits in HD.


Assuntos
Astrócitos/metabolismo , Cloridrato de Fingolimode/farmacologia , Hipocampo/metabolismo , Doença de Huntington/metabolismo , Doença de Huntington/fisiopatologia , Memória/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , AMP Cíclico/metabolismo , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/patologia , Cloridrato de Fingolimode/administração & dosagem , Expressão Gênica , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Doença de Huntington/genética , Doença de Huntington/patologia , Inflamação/metabolismo , Inflamação/patologia , Camundongos , RNA Mensageiro/genética , Receptor trkB/genética , Receptor trkB/metabolismo , Receptores de Fator de Crescimento Neural , Regulação para Cima
15.
Hum Mol Genet ; 22(19): 3869-82, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-23720495

RESUMO

Huntington's disease (HD) is a neurodegenerative disorder caused by an abnormal expansion of a CAG repeat encoding a polyglutamine tract in the huntingtin (Htt) protein. The mutation leads to neuronal death through mechanisms which are still unknown. One hypothesis is that mitochondrial defects may play a key role. In support of this, the activity of mitochondrial complex II (C-II) is preferentially reduced in the striatum of HD patients. Here, we studied C-II expression in different genetic models of HD expressing N-terminal fragments of mutant Htt (mHtt). Western blot analysis showed that the expression of the 30 kDa Iron-Sulfur (Ip) subunit of C-II was significantly reduced in the striatum of the R6/1 transgenic mice, while the levels of the FAD containing catalytic 70 kDa subunit (Fp) were not significantly changed. Blue native gel analysis showed that the assembly of C-II in mitochondria was altered early in N171-82Q transgenic mice. Early loco-regional reduction in C-II activity and Ip protein expression was also demonstrated in a rat model of HD using intrastriatal injection of lentiviral vectors encoding mHtt. Infection of the rat striatum with a lentiviral vector coding the C-II Ip or Fp subunits induced a significant overexpression of these proteins that led to significant neuroprotection of striatal neurons against mHtt neurotoxicity. These results obtained in vivo support the hypothesis that structural and functional alterations of C-II induced by mHtt may play a critical role in the degeneration of striatal neurons in HD and that mitochondrial-targeted therapies may be useful in its treatment.


Assuntos
Corpo Estriado/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Doença de Huntington/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Células Cultivadas , Corpo Estriado/fisiopatologia , Modelos Animais de Doenças , Complexo II de Transporte de Elétrons/genética , Feminino , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Doença de Huntington/fisiopatologia , Masculino , Camundongos , Camundongos Transgênicos , Mitocôndrias/genética , Proteínas Mutantes/metabolismo , Mutação , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley
16.
Brain ; 136(Pt 4): 1161-76, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23518711

RESUMO

Activating transcription factor 5 (ATF5) is a basic-leucine-zipper transcription factor of the ATF/CREB family. The Atf5 gene generates two transcripts, Atf5α and Atf5ß, of which Atf5α is known to be selectively translated upon endoplasmic reticulum stress response in non-neuronal cells. ATF5 is highly expressed in the developing brain where it modulates proliferation of neural progenitor cells. These cells show a high level of ATF5 that has to decrease to allow them to differentiate into mature neurons or glial cells. This has led to the extended notion that differentiated neural cells do not express ATF5 unless they undergo tumourigenic transformation. However, no systematic analysis of the distribution of ATF5 in adult brain or of its potential role in neuronal endoplasmic reticulum stress response has been reported. By immunostaining here we confirm highest ATF5 levels in neuroprogenitor cells of the embryonic and adult subventricular zone but also found ATF5 in a large variety of neurons in adult mouse brain. By combining Atf5 in situ hybridization and immunohistochemistry for the neuronal marker NeuN we further confirmed Atf5 messenger RNA in adult mouse neurons. Quantitative reverse transcriptase polymerase chain reaction demonstrated that Atf5α is the most abundant transcript in adult mouse encephalon and injection of the endoplasmic reticulum stress inducer tunicamycin into adult mouse brain increased neuronal ATF5 levels. Accordingly, ATF5 levels increased in hippocampal neurons of a mouse model of status epilepticus triggered by intra-amygdala injection of kainic acid, which leads to abnormal hippocampal neuronal activity and endoplasmic reticulum stress. Interestingly, ATF5 upregulation occurred mainly in hippocampal neuronal fields that do not undergo apoptosis in this status epilepticus model such as CA1 and dentate gyrus, thus suggesting a neuroprotective role. This was confirmed in a primary neuronal culture model in which ATF5 overexpression resulted in decreased endoplasmic reticulum stress-induced apoptosis and the opposite result was achieved by Atf5 RNA interference. Furthermore, in vivo administration of the eIF2α phosphatase inhibitor salubrinal resulted in increased ATF5 hippocampal levels and attenuated status epilepticus-induced neuronal death in the vulnerable CA3 subfield. In good agreement with the neuroprotective effect of increased ATF5, we found that apoptosis-resistant epileptogenic foci from patients with temporal lobe epilepsy also showed increased levels of ATF5. Thus, our results demonstrate that adult neurons express ATF5 and that they increase its levels upon endoplasmic reticulum stress as a pro-survival mechanism, thus opening a new field for neuroprotective strategies focused on ATF5 modulation.


Assuntos
Fatores Ativadores da Transcrição/biossíntese , Estresse do Retículo Endoplasmático/fisiologia , Neurônios/metabolismo , Fármacos Neuroprotetores/metabolismo , Estado Epiléptico/metabolismo , Estado Epiléptico/patologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Cinamatos/administração & dosagem , Cinamatos/farmacologia , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/patologia , Estado Epiléptico/tratamento farmacológico , Tioureia/administração & dosagem , Tioureia/análogos & derivados , Tioureia/farmacologia
17.
Exp Neurol ; 237(2): 335-45, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22776425

RESUMO

Brain-derived neurotrophic factor (BDNF) is a key player in learning and memory processes. However, little is known about brain area-specific functions of this neurotrophin. Here we investigated whether BDNF could differently affect motor neocortical and hippocampal-related cognitive and plastic morphologic changes in young (12-week-old) and middle-aged (30-week-old) BDNF heterozygous (BDNF⁺/⁻) and wild type (wt) mice. We found that at 30 weeks of age, BDNF⁺/⁻ mice showed impaired performance in accelerating rotarod and grasping tests while preserved spatial learning in a T-maze and recognition memory in an object recognition task compared with wt mice suggesting a specific neocortical dysfunction. Accordingly, a significant reduction of synaptic markers (PSD-95 and GluR1) and corresponding puncta was observed in motor neocortex but not in hippocampus of BDNF⁺/⁻ mice. Interestingly, 30-week-old BDNF⁺/⁻ mice displayed increased TrkB levels in the hippocampus but not in the motor neocortex, which suggests specific hippocampal compensatory mechanisms as a consequence of BDNF decrease. In conclusion, our data indicates that BDNF could differentially regulate the neuronal micro-structures and cognition in a region-specific and in an age-dependent manner.


Assuntos
Envelhecimento/fisiologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Encéfalo/metabolismo , Guanilato Quinases/biossíntese , Proteínas de Membrana/biossíntese , Receptor trkB/biossíntese , Animais , Western Blotting , Fator Neurotrófico Derivado do Encéfalo/genética , Espinhas Dendríticas/metabolismo , Proteína 4 Homóloga a Disks-Large , Ensaio de Imunoadsorção Enzimática , Heterozigoto , Aprendizagem/fisiologia , Memória/fisiologia , Camundongos , Microscopia Confocal , Plasticidade Neuronal/fisiologia
18.
EMBO Mol Med ; 4(5): 380-95, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22407749

RESUMO

Induced pluripotent stem cells (iPSC) offer an unprecedented opportunity to model human disease in relevant cell types, but it is unclear whether they could successfully model age-related diseases such as Parkinson's disease (PD). Here, we generated iPSC lines from seven patients with idiopathic PD (ID-PD), four patients with familial PD associated to the G2019S mutation in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene (LRRK2-PD) and four age- and sex-matched healthy individuals (Ctrl). Over long-time culture, dopaminergic neurons (DAn) differentiated from either ID-PD- or LRRK2-PD-iPSC showed morphological alterations, including reduced numbers of neurites and neurite arborization, as well as accumulation of autophagic vacuoles, which were not evident in DAn differentiated from Ctrl-iPSC. Further induction of autophagy and/or inhibition of lysosomal proteolysis greatly exacerbated the DAn morphological alterations, indicating autophagic compromise in DAn from ID-PD- and LRRK2-PD-iPSC, which we demonstrate occurs at the level of autophagosome clearance. Our study provides an iPSC-based in vitro model that captures the patients' genetic complexity and allows investigation of the pathogenesis of both sporadic and familial PD cases in a disease-relevant cell type.


Assuntos
Dopamina/metabolismo , Neurônios/patologia , Neurônios/fisiologia , Doença de Parkinson/patologia , Doença de Parkinson/fisiopatologia , Células-Tronco Pluripotentes/patologia , Células-Tronco Pluripotentes/fisiologia , Autofagia , Células Cultivadas , Humanos , Receptores Dopaminérgicos/metabolismo , Vacúolos/metabolismo , Vacúolos/ultraestrutura
19.
Stem Cells Dev ; 21(12): 2239-51, 2012 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-22142223

RESUMO

Development of the nervous system is finely regulated by consecutive expression of cell-specific transcription factors. Here we show that Helios, a member of the Ikaros transcription factor family, is expressed in ectodermal and neuroectodermal-derived tissues. During embryonic development, Helios is expressed by several brain structures including the lateral ganglionic eminence (LGE, the striatal anlage); the cingulated, insular and retrosplenial cortex; the hippocampus; and the accessory olfactory bulb. Moreover, Helios is also expressed by Purkinje neurons during postnatal cerebellar development. Within the LGE, Helios expression follows a dynamic spatio-temporal pattern starting at embryonic stages (E14.5), peaking at E18.5, and completely disappearing during postnatal development. Helios is expressed by a small population of nestin-positive neural progenitor cells located in the subventricular zone as well as by a larger population of immature neurons distributed throughout the mantle zone. In the later, Helios is preferentially expressed in the matrix compartment, where it colocalizes with Bcl11b and Foxp1, well-known markers of striatal projection neurons. In addition, we observed that Helios expression is not detected in Dlx1/2 and Gsx2 null mutants, while its expression is maintained in Ascl1 mutants. These findings allow us to introduce a new transcription factor in the cascade of events that take part of striatal development postulating the existence of at least 4 different neural progenitors in the LGE. An Ascl1-independent but Gsx2- & Dlx1/2-dependent precursor will express Helios defining a new lineage for a subset of matrix striatal neurons.


Assuntos
Corpo Estriado/citologia , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/fisiologia , Neurônios/metabolismo , Fatores de Transcrição/fisiologia , Animais , Cerebelo/metabolismo , Corpo Estriado/embriologia , Corpo Estriado/crescimento & desenvolvimento , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/metabolismo , Nestina , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
20.
Neural Dev ; 5: 21, 2010 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-20735826

RESUMO

BACKGROUND: Nolz1 is a zinc finger transcription factor whose expression is enriched in the lateral ganglionic eminence (LGE), although its function is still unknown. RESULTS: Here we analyze the role of Nolz1 during LGE development. We show that Nolz1 expression is high in proliferating neural progenitor cells (NPCs) of the LGE subventricular zone. In addition, low levels of Nolz1 are detected in the mantle zone, as well as in the adult striatum. Similarly, Nolz1 is highly expressed in proliferating LGE-derived NPC cultures, but its levels rapidly decrease upon cell differentiation, pointing to a role of Nolz1 in the control of NPC proliferation and/or differentiation. In agreement with this hypothesis, we find that Nolz1 over-expression promotes cell cycle exit of NPCs in neurosphere cultures and negatively regulates proliferation in telencephalic organotypic cultures. Within LGE primary cultures, Nolz1 over-expression promotes the acquisition of a neuronal phenotype, since it increases the number of ß-III tubulin (Tuj1)- and microtubule-associated protein (MAP)2-positive neurons, and inhibits astrocyte generation and/or differentiation. Retinoic acid (RA) is one of the most important morphogens involved in striatal neurogenesis, and regulates Nolz1 expression in different systems. Here we show that Nolz1 also responds to this morphogen in E12.5 LGE-derived cell cultures. However, Nolz1 expression is not regulated by RA in E14.5 LGE-derived cell cultures, nor is it affected during LGE development in mouse models that present decreased RA levels. Interestingly, we find that Gsx2, which is necessary for normal RA signaling during LGE development, is also required for Nolz1 expression, which is lost in Gsx2 knockout mice. These findings suggest that Nolz1 might act downstream of Gsx2 to regulate RA-induced neurogenesis. Keeping with this hypothesis, we show that Nolz1 induces the selective expression of the RA receptor (RAR)ß without altering RARα or RARγ. In addition, Nozl1 over-expression increases RA signaling since it stimulates the RA response element. This RA signaling is essential for Nolz1-induced neurogenesis, which is impaired in a RA-free environment or in the presence of a RAR inverse agonist. It has been proposed that Drosophila Gsx2 and Nolz1 homologues could cooperate with the transcriptional co-repressors Groucho-TLE to regulate cell proliferation. In agreement with this view, we show that Nolz1 could act in collaboration with TLE-4, as they are expressed at the same time in NPC cultures and during mouse development. CONCLUSIONS: Nolz1 promotes RA signaling in the LGE, contributing to the striatal neurogenesis during development.


Assuntos
Antineoplásicos/farmacologia , Proteínas de Transporte/metabolismo , Corpo Estriado/citologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Transdução de Sinais/fisiologia , Tretinoína/farmacologia , Animais , Proteínas de Transporte/genética , Contagem de Células , Proliferação de Células , Células Cultivadas , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Corpos Geniculados/embriologia , Proteínas de Homeodomínio/genética , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos CBA , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Neurogênese/fisiologia , Neuroglia/efeitos dos fármacos , Neuroglia/fisiologia , Neurônios/fisiologia , Proteínas Nucleares/genética , Gravidez , Retinal Desidrogenase/deficiência , Transdução de Sinais/genética , Tubulina (Proteína)/metabolismo
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