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1.
Nat Commun ; 10(1): 2748, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227709

RESUMO

The human amygdala grows during childhood, and its abnormal development is linked to mood disorders. The primate amygdala contains a large population of immature neurons in the paralaminar nuclei (PL), suggesting protracted development and possibly neurogenesis. Here we studied human PL development from embryonic stages to adulthood. The PL develops next to the caudal ganglionic eminence, which generates inhibitory interneurons, yet most PL neurons express excitatory markers. In children, most PL cells are immature (DCX+PSA-NCAM+), and during adolescence many transition into mature (TBR1+VGLUT2+) neurons. Immature PL neurons persist into old age, yet local progenitor proliferation sharply decreases in infants. Using single nuclei RNA sequencing, we identify the transcriptional profile of immature excitatory neurons in the human amygdala between 4-15 years. We conclude that the human PL contains excitatory neurons that remain immature for decades, a possible substrate for persistent plasticity at the interface of the hippocampus and amygdala.


Assuntos
Desenvolvimento do Adolescente/fisiologia , Complexo Nuclear Basolateral da Amígdala/crescimento & desenvolvimento , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Neurônios/fisiologia , Adolescente , Adulto , Idoso , Complexo Nuclear Basolateral da Amígdala/citologia , Núcleo Celular/genética , Criança , Pré-Escolar , Feto , Hipocampo/fisiologia , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Plasticidade Neuronal/fisiologia , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Adulto Jovem
2.
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.

3.
J Neurosci ; 38(19): 4598-4609, 2018 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-29661967

RESUMO

In the rodent olfactory system, neuroblasts produced in the ventricular-subventricular zone of the postnatal brain migrate tangentially in chain-like cell aggregates toward the olfactory bulb (OB) through the rostral migratory stream (RMS). After reaching the OB, the chains are dissociated and the neuroblasts migrate individually and radially toward their final destination. The cellular and molecular mechanisms controlling cell-cell adhesion during this detachment remain unclear. Here we report that Fyn, a nonreceptor tyrosine kinase, regulates the detachment of neuroblasts from chains in the male and female mouse OB. By performing chemical screening and in vivo loss-of-function and gain-of-function experiments, we found that Fyn promotes somal disengagement from the chains and is involved in neuronal migration from the RMS into the granule cell layer of the OB. Fyn knockdown or Dab1 (disabled-1) deficiency caused p120-catenin to accumulate and adherens junction-like structures to be sustained at the contact sites between neuroblasts. Moreover, a Fyn and N-cadherin double-knockdown experiment indicated that Fyn regulates the N-cadherin-mediated cell adhesion between neuroblasts. These results suggest that the Fyn-mediated control of cell-cell adhesion is critical for the detachment of chain-forming neuroblasts in the postnatal OB.SIGNIFICANCE STATEMENT In the postnatal brain, newly born neurons (neuroblasts) migrate in chain-like cell aggregates toward their destination, where they are dissociated into individual cells and mature. The cellular and molecular mechanisms controlling the detachment of neuroblasts from chains are not understood. Here we show that Fyn, a nonreceptor tyrosine kinase, promotes the somal detachment of neuroblasts from chains, and that this regulation is critical for the efficient migration of neuroblasts to their destination. We further show that Fyn and Dab1 (disabled-1) decrease the cell-cell adhesion between chain-forming neuroblasts, which involves adherens junction-like structures. Our results suggest that Fyn-mediated regulation of the cell-cell adhesion of neuroblasts is critical for their detachment from chains in the postnatal brain.

4.
Cell Stem Cell ; 22(1): 128-137.e9, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29276142

RESUMO

Radial glia (RG) are embryonic neural stem cells (NSCs) that produce neuroblasts and provide fibers that act as a scaffold for neuroblast migration during embryonic development. Although they normally disappear soon after birth, here we found that RG fibers can persist in injured neonatal mouse brains and act as a scaffold for postnatal ventricular-subventricular zone (V-SVZ)-derived neuroblasts that migrate to the lesion site. This injury-induced maintenance of RG fibers has a limited time window during post-natal development and promotes directional saltatory movement of neuroblasts via N-cadherin-mediated cell-cell contacts that promote RhoA activation. Transplanting an N-cadherin-containing scaffold into injured neonatal brains likewise promotes migration and maturation of V-SVZ-derived neuroblasts, leading to functional improvements in impaired gait behaviors. Together these results suggest that RG fibers enable postnatal V-SVZ-derived neuroblasts to migrate toward sites of injury, thereby enhancing neuronal regeneration and functional recovery from neonatal brain injuries.

5.
Stem Cell Reports ; 9(1): 203-216, 2017 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-28648897

RESUMO

Neural stem cells (B1 astrocytes; NSCs) in the adult ventricular-subventricular-zone (V-SVZ) originate in the embryo. Surprisingly, recent work has shown that B1 cells remain largely quiescent. They are reactivated postnatally to function as primary progenitors for neurons destined for the olfactory bulb and some corpus callosum oligodendrocytes. The cellular and molecular properties of quiescent B1 cells remain unknown. Here we found that a subpopulation of B1 cells has a unique nuclear envelope invagination specialization similar to envelope-limited chromatin sheets (ELCS), reported in certain lymphocytes and some cancer cells. Using molecular markers, [3H]thymidine birth-dating, and Ara-C, we found that B1 cells with ELCS correspond to quiescent NSCs. ELCS begin forming in embryonic radial glia cells and represent a specific nuclear compartment containing particular epigenetic modifications and telomeres. These results reveal a unique nuclear compartment in quiescent NSCs, which is useful for identifying these primary progenitors and study their gene regulation.


Assuntos
Ventrículos Laterais/citologia , Células-Tronco Neurais/citologia , Membrana Nuclear/ultraestrutura , Células-Tronco Adultas/citologia , Animais , Astrócitos/citologia , Ciclo Celular , Células Cultivadas , Cromatina/química , Camundongos
6.
Neurology ; 88(13): 1235-1242, 2017 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-28251919

RESUMO

OBJECTIVE: To identify cell-surface antibodies in patients with neuromyotonia and to describe the main clinical implications. METHODS: Sera of 3 patients with thymoma-associated neuromyotonia and myasthenia gravis were used to immunoprecipitate and characterize neuronal cell-surface antigens using reported techniques. The clinical significance of antibodies against precipitated proteins was assessed with sera of 98 patients (neuromyotonia 46, myasthenia gravis 52, thymoma 42; 33 of them with overlapping syndromes) and 219 controls (other neurologic diseases, cancer, and healthy volunteers). RESULTS: Immunoprecipitation studies identified 3 targets, including the Netrin-1 receptors DCC (deleted in colorectal carcinoma) and UNC5A (uncoordinated-5A) as well as Caspr2 (contactin-associated protein-like 2). Cell-based assays with these antigens showed that among the indicated patients, 9 had antibodies against Netrin-1 receptors (7 with additional Caspr2 antibodies) and 5 had isolated Caspr2 antibodies. Only one of the 219 controls had isolated Caspr2 antibodies with relapsing myelitis episodes. Among patients with neuromyotonia and/or myasthenia gravis, the presence of Netrin-1 receptor or Caspr2 antibodies predicted thymoma (p < 0.05). Coexisting Caspr2 and Netrin-1 receptor antibodies were associated with concurrent thymoma, myasthenia gravis, and neuromyotonia, often with Morvan syndrome (p = 0.009). Expression of DCC, UNC5A, and Caspr2 proteins was demonstrated in paraffin-embedded thymoma samples (3) and normal thymus. CONCLUSIONS: Antibodies against Netrin-1 receptors (DCC and UNC5a) and Caspr2 often coexist and associate with thymoma in patients with neuromyotonia and myasthenia gravis. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that antibodies against Netrin-1 receptors can identify patients with thymoma (sensitivity 21.4%, specificity 100%).


Assuntos
Autoanticorpos/sangue , Miastenia Gravis/sangue , Fatores de Crescimento Neural/imunologia , Fatores de Crescimento Neural/metabolismo , Timoma/sangue , Neoplasias do Timo/sangue , Proteínas Supressoras de Tumor/imunologia , Proteínas Supressoras de Tumor/metabolismo , Adulto , Idoso , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Receptor DCC , Eletromiografia , Feminino , Células HEK293 , Humanos , Imunoprecipitação , Imagem por Ressonância Magnética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Miastenia Gravis/complicações , Miastenia Gravis/diagnóstico por imagem , Fatores de Crescimento Neural/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Netrina-1 , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Timoma/complicações , Timoma/diagnóstico por imagem , Neoplasias do Timo/complicações , Neoplasias do Timo/diagnóstico por imagem , Transfecção , Proteínas Supressoras de Tumor/genética
7.
J Med Genet ; 54(6): 371-380, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28289185

RESUMO

Oral-facial-digital syndromes (OFDS) gather rare genetic disorders characterised by facial, oral and digital abnormalities associated with a wide range of additional features (polycystic kidney disease, cerebral malformations and several others) to delineate a growing list of OFDS subtypes. The most frequent, OFD type I, is caused by a heterozygous mutation in the OFD1 gene encoding a centrosomal protein. The wide clinical heterogeneity of OFDS suggests the involvement of other ciliary genes. For 15 years, we have aimed to identify the molecular bases of OFDS. This effort has been greatly helped by the recent development of whole-exome sequencing (WES). Here, we present all our published and unpublished results for WES in 24 cases with OFDS. We identified causal variants in five new genes (C2CD3, TMEM107, INTU, KIAA0753 and IFT57) and related the clinical spectrum of four genes in other ciliopathies (C5orf42, TMEM138, TMEM231 and WDPCP) to OFDS. Mutations were also detected in two genes previously implicated in OFDS. Functional studies revealed the involvement of centriole elongation, transition zone and intraflagellar transport defects in OFDS, thus characterising three ciliary protein modules: the complex KIAA0753-FOPNL-OFD1, a regulator of centriole elongation; the Meckel-Gruber syndrome module, a major component of the transition zone; and the CPLANE complex necessary for IFT-A assembly. OFDS now appear to be a distinct subgroup of ciliopathies with wide heterogeneity, which makes the initial classification obsolete. A clinical classification restricted to the three frequent/well-delineated subtypes could be proposed, and for patients who do not fit one of these three main subtypes, a further classification could be based on the genotype.


Assuntos
Face/anormalidades , Síndromes Orofaciodigitais/genética , Anormalidades Múltiplas/genética , Transtornos da Motilidade Ciliar/genética , Encefalocele/genética , Feminino , Heterozigoto , Humanos , Masculino , Mutação/genética , Doenças Renais Policísticas/genética , Proteínas/genética , Retinite Pigmentosa
8.
Cell ; 168(1-2): 252-263.e14, 2017 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-28017328

RESUMO

Signaling receptors dynamically exit cilia upon activation of signaling pathways such as Hedgehog. Here, we find that when activated G protein-coupled receptors (GPCRs) fail to undergo BBSome-mediated retrieval from cilia back into the cell, these GPCRs concentrate into membranous buds at the tips of cilia before release into extracellular vesicles named ectosomes. Unexpectedly, actin and the actin regulators drebrin and myosin 6 mediate ectosome release from the tip of cilia. Mirroring signal-dependent retrieval, signal-dependent ectocytosis is a selective and effective process that removes activated signaling molecules from cilia. Congruently, ectocytosis compensates for BBSome defects as ectocytic removal of GPR161, a negative regulator of Hedgehog signaling, permits the appropriate transduction of Hedgehog signals in Bbs mutants. Finally, ciliary receptors that lack retrieval determinants such as the anorexigenic GPCR NPY2R undergo signal-dependent ectocytosis in wild-type cells. Our data show that signal-dependent ectocytosis regulates ciliary signaling in physiological and pathological contexts.


Assuntos
Cílios/metabolismo , Vesículas Extracelulares/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular , Humanos , Rim/citologia , Rim/metabolismo , Camundongos , Microscopia Eletrônica de Varredura , Receptores de Somatostatina/metabolismo , Transdução de Sinais
9.
J Comp Neurol ; 524(15): 2982-92, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-26991819

RESUMO

In mammals, ventricular walls of the developing brain maintain a neurogenic niche, in which radial glial cells act as neural stem cells (NSCs) and generate new neurons in the embryo. In the adult brain, the neurogenic niche is maintained in the ventricular-subventricular zone (V-SVZ) of the lateral wall of lateral ventricles and the hippocampal dentate gyrus. In the neonatal V-SVZ, radial glial cells transform into astrocytic postnatal NSCs and multiciliated ependymal cells. On the other hand, in zebrafish, radial glial cells continue to cover the surface of the adult telencephalic ventricle and maintain a higher neurogenic potential in the adult brain. However, the cell composition of the neurogenic niche of the aged zebrafish brain has not been investigated. Here we show that multiciliated ependymal cells emerge in the neurogenic niche of the aged zebrafish telencephalon. These multiciliated cells appear predominantly in the dorsal part of the ventral telencephalic ventricular zone, which also contains clusters of migrating new neurons. Scanning electron microscopy and live imaging analyses indicated that these multiple cilia beat coordinately and generate constant fluid flow within the ventral telencephalic ventricle. Analysis of the cell composition by transmission electron microscopy revealed that the neurogenic niche in the aged zebrafish contains different types of cells, with ultrastructures similar to those of ependymal cells, transit-amplifying cells, and migrating new neurons in postnatal mice. These data suggest that the transformation capacity of radial glial cells is conserved but that its timing is different between fish and mice. J. Comp. Neurol. 524:2982-2992, 2016. © 2016 Wiley Periodicals, Inc.


Assuntos
Envelhecimento/fisiologia , Epêndima/citologia , Nicho de Células-Tronco/fisiologia , Telencéfalo/citologia , Peixe-Zebra/fisiologia , Envelhecimento/patologia , Animais , Animais Geneticamente Modificados , Movimento Celular/fisiologia , Cílios/ultraestrutura , Epêndima/crescimento & desenvolvimento , Epêndima/fisiologia , Epêndima/ultraestrutura , Imuno-Histoquímica , Microscopia Confocal , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Telencéfalo/crescimento & desenvolvimento , Telencéfalo/fisiologia , Telencéfalo/ultraestrutura , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/crescimento & desenvolvimento
10.
J Alzheimers Dis ; 51(3): 701-11, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26890773

RESUMO

Amyloid-ß (Aß) clearance from brain, which is decreased in Alzheimer's disease, is facilitated by apolipoprotein E (ApoE). ApoE is upregulated by activation of the retinoid X receptor moiety of the RXR/PPARγ dimeric receptor. Genistein, a non-toxic, well-tested, and inexpensive drug activates the other moiety of the receptor PPARγ. Treatment of an Alzheimer's disease mouse model with genistein results in a remarkable and rapid improvement in various parameters of cognition, such as hippocampal learning, recognition memory, implicit memory, and odor discrimination. This is associated with a lowering of Aß levels in brain, in the number and the area of amyloid plaques (confirmed in vivo by positron emission tomography) as well as in microglial reactivity. Finally, incubation of primary astrocytes with genistein results in a PPARγ-mediated increased release of ApoE. Our results strongly suggest that controlled clinical trials should be performed to test the effect of genistein as treatment of human Alzheimer's disease.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Apolipoproteínas E/metabolismo , Genisteína/farmacologia , Nootrópicos/farmacologia , PPAR gama/metabolismo , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Aprendizagem da Esquiva/efeitos dos fármacos , Bexaroteno , Encéfalo/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Habituação Psicofisiológica/efeitos dos fármacos , Aprendizagem em Labirinto , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fármacos Neuroprotetores/farmacologia , Percepção Olfatória/efeitos dos fármacos , Placa Amiloide/diagnóstico por imagem , Placa Amiloide/tratamento farmacológico , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Recognição (Psicologia)/efeitos dos fármacos , Tetra-Hidronaftalenos/farmacologia
11.
Front Cell Neurosci ; 9: 365, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26441536

RESUMO

Neural stem cells (NSCs) persist in the adult mammalian brain through life. The subventricular zone (SVZ) is the largest source of stem cells in the nervous system, and continuously generates new neuronal and glial cells involved in brain regeneration. During aging, the germinal potential of the SVZ suffers a widespread decline, but the causes of this turn down are not fully understood. This review provides a compilation of the current knowledge about the age-related changes in the NSC population, as well as the fate of the newly generated cells in the aged brain. It is known that the neurogenic capacity is clearly disrupted during aging, while the production of oligodendroglial cells is not compromised. Interestingly, the human brain seems to primarily preserve the ability to produce new oligodendrocytes instead of neurons, which could be related to the development of neurological disorders. Further studies in this matter are required to improve our understanding and the current strategies for fighting neurological diseases associated with senescence.

12.
J Neurosci ; 35(31): 11153-68, 2015 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-26245976

RESUMO

UNLABELLED: Directional beating of ependymal (E) cells' cilia in the walls of the ventricles in the brain is essential for proper CSF flow. E cells display two forms of planar cell polarity (PCP): rotational polarity of individual cilium and translational polarity (asymmetric positioning of cilia in the apical area). The orientation of individual E cells varies according to their location in the ventricular wall (location-specific PCP). It has been hypothesized that hydrodynamic forces on the apical surface of radial glia cells (RGCs), the embryonic precursors of E cells, could guide location-specific PCP in the ventricular epithelium. However, the detection mechanisms for these hydrodynamic forces have not been identified. Here, we show that the mechanosensory proteins polycystic kidney disease 1 (Pkd1) and Pkd2 are present in primary cilia of RGCs. Ablation of Pkd1 or Pkd2 in Nestin-Cre;Pkd1(flox/flox) or Nestin-Cre;Pkd2(flox/flox) mice, affected PCP development in RGCs and E cells. Early shear forces on the ventricular epithelium may activate Pkd1 and Pkd2 in primary cilia of RGCs to properly polarize RGCs and E cells. Consistently, Pkd1, Pkd2, or primary cilia on RGCs were required for the proper asymmetric localization of the PCP protein Vangl2 in E cells' apical area. Analyses of single- and double-heterozygous mutants for Pkd1 and/or Vangl2 suggest that these genes function in the same pathway to establish E cells' PCP. We conclude that Pkd1 and Pkd2 mechanosensory proteins contribute to the development of brain PCP and prevention of hydrocephalus. SIGNIFICANCE STATEMENT: This study identifies key molecules in the development of planar cell polarity (PCP) in the brain and prevention of hydrocephalus. Multiciliated ependymal (E) cells within the brain ventricular epithelium generate CSF flow through ciliary beating. E cells display location-specific PCP in the orientation and asymmetric positioning of their cilia. Defects in this PCP can result in hydrocephalus. Hydrodynamic forces on radial glial cells (RGCs), the embryonic progenitors of E cells, have been suggested to guide PCP. We show that the mechanosensory proteins Pkd1 and Pkd2 localize to primary cilia in RGCs, and their ablation disrupts the development of PCP in E cells. Early shear forces on RGCs may activate Pkd1 and Pkd2 in RGCs' primary cilia to properly orient E cells. This study identifies key molecules in the development of brain PCP and prevention of hydrocephalus.


Assuntos
Polaridade Celular/genética , Ventrículos Cerebrais/metabolismo , Cílios/metabolismo , Células Ependimogliais/metabolismo , Canais de Cátion TRPP/genética , Animais , Epêndima/citologia , Epêndima/metabolismo , Células Ependimogliais/citologia , Camundongos , Camundongos Knockout , Canais de Cátion TRPP/metabolismo
13.
J Appl Toxicol ; 35(7): 737-51, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25256750

RESUMO

Ochratoxin A (OTA), a mycotoxin that was discovered as a secondary metabolite of the fungal species Aspergillus and Penicillium, is a common contaminant in food and animal feed. This mycotoxin has been described as teratogenic, carcinogenic, genotoxic, immunotoxic and has been proven a potent neurotoxin. Other authors have previously reported the effects of OTA in different structures of the central nervous system as well as in some neurogenic regions. However, the impact of OTA exposure in the subventricular zone (SVZ) has not been assessed yet. To elucidate whether OTA affects neural precursors of the mouse SVZ we investigated, in vitro and in vivo, the effects of OTA exposure on the SVZ and on the neural precursors obtained from this neurogenic niche. In this work, we prove the cumulative effect of OTA exposure on proliferation, differentiation and depletion of neural stem cells cultured from the SVZ. In addition, we corroborated these results in vivo by immunohistochemistry and electron microscopy. As a result, we found a significant alteration in the proliferation process, which was evidenced by a decrease in the number of 5-bromo-2-deoxyuridine-positive cells and glial cells, as well as, a significant decrease in the number of neuroblasts in the SVZ. To summarize, in this study we demonstrate how OTA could be a threat to the developing and the adult SVZ through its impact in cell viability, proliferation and differentiation in a dose-dependent manner.


Assuntos
Ventrículos Laterais/efeitos dos fármacos , Micotoxinas/toxicidade , Ocratoxinas/toxicidade , Animais , Astrócitos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Ventrículos Laterais/patologia , Ventrículos Laterais/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Células-Tronco Neurais/efeitos dos fármacos , Neuroglia/efeitos dos fármacos
14.
Neuron ; 83(3): 558-71, 2014 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-25043421

RESUMO

Defects in ependymal (E) cells, which line the ventricle and generate cerebrospinal fluid flow through ciliary beating, can cause hydrocephalus. Dishevelled genes (Dvls) are essential for Wnt signaling, and Dvl2 has been shown to localize to the rootlet of motile cilia. Using the hGFAP-Cre;Dvl1(-/-);2(flox/flox);3(+/-) mouse, we show that compound genetic ablation of Dvls causes hydrocephalus. In hGFAP-Cre;Dvl1(-/-);2(flox/flox);3(+/-) mutants, E cells differentiated normally, but the intracellular and intercellular rotational alignments of ependymal motile cilia were disrupted. As a consequence, the fluid flow generated by the hGFAP-Cre;Dvl1(-/-);2(flox/flox);3(+/-) E cells was significantly slower than that observed in control mice. Dvls were also required for the proper positioning of motile cilia on the apical surface. Tamoxifen-induced conditional removal of Dvls in adult mice also resulted in defects in intracellular rotational alignment and positioning of ependymal motile cilia. These results suggest that Dvls are continuously required for E cell planar polarity and may prevent hydrocephalus.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Polaridade Celular/fisiologia , Cílios/patologia , Epêndima/patologia , Hidrocefalia/etiologia , Fosfoproteínas/genética , Transdução de Sinais/fisiologia , Animais , Polaridade Celular/genética , Cílios/genética , Proteínas Desgrenhadas , Hidrocefalia/genética , Hidrocefalia/patologia , Camundongos , Camundongos Transgênicos , Transdução de Sinais/genética
15.
Nat Genet ; 46(8): 905-11, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24997988

RESUMO

Centrioles are microtubule-based, barrel-shaped structures that initiate the assembly of centrosomes and cilia. How centriole length is precisely set remains elusive. The microcephaly protein CPAP (also known as MCPH6) promotes procentriole growth, whereas the oral-facial-digital (OFD) syndrome protein OFD1 represses centriole elongation. Here we uncover a new subtype of OFD with severe microcephaly and cerebral malformations and identify distinct mutations in two affected families in the evolutionarily conserved C2CD3 gene. Concordant with the clinical overlap, C2CD3 colocalizes with OFD1 at the distal end of centrioles, and C2CD3 physically associates with OFD1. However, whereas OFD1 deletion leads to centriole hyperelongation, loss of C2CD3 results in short centrioles without subdistal and distal appendages. Because C2CD3 overexpression triggers centriole hyperelongation and OFD1 antagonizes this activity, we propose that C2CD3 directly promotes centriole elongation and that OFD1 acts as a negative regulator of C2CD3. Our results identify regulation of centriole length as an emerging pathogenic mechanism in ciliopathies.


Assuntos
Centríolos/genética , Proteínas Associadas aos Microtúbulos/genética , Síndromes Orofaciodigitais/genética , Linhagem Celular , Pré-Escolar , Predisposição Genética para Doença , Células HEK293 , Humanos , Masculino , Microcefalia/genética , Proteínas/genética
16.
Exp Neurol ; 261: 236-44, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24858805

RESUMO

Hydrocephalus can develop secondarily to a disturbance in production, flow and/or absorption of cerebrospinal fluid. Experimental models of hydrocephalus, especially subacute and chronic hydrocephalus, are few and limited, and the effects of hydrocephalus on the subventricular zone are unclear. The aim of this study was to analyze the effects of long-term obstructive hydrocephalus on the subventricular zone, which is the neurogenic niche lining the lateral ventricles. We developed a new method to induce hydrocephalus by obstructing the aqueduct of Sylvius in the mouse brain, thus simulating aqueductal stenosis in humans. In 120-day-old rodents (n=18 per group), the degree of ventricular dilatation and cellular composition of the subventricular zone were studied by immunofluorescence and transmission electron microscopy. In adult patients (age>18years), the sizes of the subventricular zone, corpus callosum, and internal capsule were analyzed by magnetic resonance images obtained from patients with and without aqueductal stenosis (n=25 per group). Mice with 60-day hydrocephalus had a reduced number of Ki67+ and doublecortin+cells on immunofluorescence, as well as decreased number of neural progenitors and neuroblasts in the subventricular zone on electron microscopy analysis as compared to non-hydrocephalic mice. Remarkably, a number of extracellular matrix structures (fractones) contacting the ventricular lumen and blood vessels were also observed around the subventricular zone in mice with hydrocephalus. In humans, the widths of the subventricular zone, corpus callosum, and internal capsule in patients with aqueductal stenosis were significantly smaller than age and gender-matched patients without aqueductal stenosis. In summary, supratentorial hydrocephalus reduces the proliferation rate of neural progenitors and modifies the cytoarchitecture and extracellular matrix compounds of the subventricular zone. In humans, this similar process reduces the subventricular niche as well as the width of corpus callosum and internal capsule.


Assuntos
Hidrocefalia/patologia , Ventrículos Laterais/metabolismo , Ventrículos Laterais/patologia , Adulto , Animais , Estudos de Coortes , Corpo Caloso/metabolismo , Corpo Caloso/patologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Hidrocefalia/fisiopatologia , Cápsula Interna/metabolismo , Cápsula Interna/patologia , Antígeno Ki-67/metabolismo , Ventrículos Laterais/ultraestrutura , Imagem por Ressonância Magnética , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Associadas aos Microtúbulos/metabolismo , Neuropeptídeos/metabolismo , Fatores de Tempo , Adulto Jovem
17.
Chemistry ; 19(40): 13538-46, 2013 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-23943097

RESUMO

A simple and straightforward method for the orthogonal functionalisation of upconverting NaYF4 nanocrystals (UCNCs)-doped withYb(3+) and Er(3+)-based on N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide/N-hydroxysuccinimide (EDC/NHS) selective reactions between two dyes and two different reactive groups present at the periphery of the upconverting nanocrystals is reported. Organic-soluble UCNCs of 10 and 50 nm in size are encapsulated efficiently in a 1:1 mixture of two commercial 3000 Da poly(ethylene glycol) derivatives with two different reactive groups (amino and carboxylic groups). The water-dispersible UCNCs are non-cytotoxic, stable in the physiological environment, and present free amine and carboxylic reactive groups on their periphery, allowing rapid, selective, and modular covalent conjugation to payloads through EDC/NHS reactions. PEG-encapsulated UCNCs with and without covalent conjugation to payloads are characterised in vitro through spectroscopic, dynamic light scattering, and electron microscopy measurements. Living cell analyses coupled with TEM measurements confirm the uptake and low cytotoxicity of the coated UCNCs. They are linked covalently to two different dyes, internalised by living cells, and analysed by confocal microscopy. The related colocalisation measurements prove the reactivity of both amines and carboxylic acids on the periphery of the nanocrystals. This approach demonstrates that it is possible to produce water-dispersible and cyto-compatible dual-functional UCNCs.


Assuntos
Fluoretos/química , Nanopartículas/química , Polietilenoglicóis/química , Succinimidas/química , Ítrio/química , Estrutura Molecular
18.
Brain Res ; 1307: 177-94, 2010 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-19833108

RESUMO

The leucine-rich glioma inactivated (LGI) gene subfamily contains four highly conserved members (LGI1, 2, 3 and 4), which have been described in human, mouse and other mammalians. Although their main roles remain unknown, LGI1 gene mutations have been found in human partial temporal lobe epilepsy. Moreover, previous studies showed that the products of these genes exert their function in the nervous system. The anatomical distribution of these gene transcripts in the brain might give some insight to elucidate their possible function. In this study, the pattern of expression of the four LGI genes was assessed in the brain of C57BL/6J adult mice by in situ hybridization. We found that the LGI1 transcript is mainly expressed in the dentate gyrus and CA3 field of the hippocampus. LGI2 and LGI4 genes, which showed a similar pattern of distribution with minor differences, were mostly expressed in the medial septal area, thalamic reticular nucleus and substantia nigra pars compacta. LGI3-expressing cells were distributed widespread, but were more consistently observed in the hippocampal formation, thalamic and hypothalamic nuclei, substantia nigra and reticular formation. In summary, LGI1 gene expression is very restricted to intrahippocampal circuitry, which might be related to its involvement in temporal lobe epilepsy. The patterns of expression of LGI2 and LGI4 genes are very similar and their distribution in the vertical limb of the diagonal band and in putative hippocampal interneurons suggests that the function of these genes might be related to the generation of hippocampal theta rhythm. Finally, LGI3 gene widespread expression in the brain suggests that its transcripts might be involved in a common cellular process present in different neuronal types.


Assuntos
Encéfalo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas/metabolismo , Animais , Encéfalo/anatomia & histologia , Mapeamento Encefálico , Hibridização In Situ/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas/genética
19.
Hum Mol Genet ; 15(23): 3436-45, 2006 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-17067999

RESUMO

Autosomal dominant lateral temporal epilepsy (ADTLE) is a partial epilepsy caused by mutations in LGI1, a multidomain protein of unknown function. To begin to understand the biological function of LGI1, we have determined its pattern of glycosylation, subcellular expression and capacity for secretion. LGI1 is expressed as two different isoforms in the brain, and we show that the long isoform is a secreted protein, whereas the short isoform is retained in an intracellular pool. ADLTE-related mutants of the long form are defective for secretion and are retained in the endoplasmic reticulum and Golgi complex. Finally, we show that normal secreted LGI1 specifically binds to the cell surface of differentiated PC12 cells. We propose that LGI1 is a secreted factor important for neuronal development and that ADTLE is a disease that results from the loss of regulation in the protein available either extracellular or intracellularly.


Assuntos
Epilepsia do Lobo Temporal/genética , Glicoproteínas/metabolismo , Proteínas/genética , Proteínas/metabolismo , Animais , Membrana Celular/química , Células Cultivadas , Ativação Enzimática , Glicoproteínas/análise , Glicoproteínas/genética , Glicosilação , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Células PC12 , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Proteínas/análise , Ratos
20.
Eur J Neurosci ; 23(3): 659-66, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16487147

RESUMO

The leucine-rich repeat kinase 2 (LRRK2) gene was recently found to have multiple mutations that are causative for autosomal dominant inherited Parkinson's disease (PD). Previously, we used Northern blot analysis to show that this gene was expressed in the cerebellum, cerebral cortex, medulla, spinal cord, occipital pole, frontal lobe, temporal lobe and caudate putamen. However, a more comprehensive map of LRRK2 mRNA localization in the central nervous system is still lacking. In this study we have mapped the distribution of the mRNA encoding for LRRK2 using nonradioactive in situ hybridization. We detected a moderate expression of this PD-related gene throughout the adult B2B6 mouse brain. A stronger hybridization signal was observed in deep cerebral cortex layers, superficial cingulate cortex layers, the piriform cortex, hippocampal formation, caudate putamen, substantia nigra, the basolateral and basomedial anterior amygdala nuclei, reticular thalamic nucleus and also in the cerebellar granular cell layer. Given that LRRK2 mRNA is highly enriched in motor systems and also is expressed in other systems, we may conclude that mutations in LRRK2 may affect several motor and nonmotor structures that may play an important role in the development of PD.


Assuntos
Encéfalo/metabolismo , Expressão Gênica/fisiologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Encéfalo/patologia , Mapeamento Encefálico , Modelos Animais de Doenças , Hibridização In Situ/métodos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Masculino , Camundongos , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/metabolismo
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