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1.
Stem Cells ; 39(9): 1253-1269, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33963799

RESUMO

Although previous studies suggest that neural stem cells (NSCs) exist in the adult olfactory bulb (OB), their location, identity, and capacity to generate mature neurons in vivo has been little explored. Here, we injected enhanced green fluorescent protein (EGFP)-expressing retroviral particles into the OB core of adult mice to label dividing cells and to track the differentiation/maturation of any neurons they might generate. EGFP-labeled cells initially expressed adult NSC markers on days 1 to 3 postinjection (dpi), including Nestin, GLAST, Sox2, Prominin-1, and GFAP. EGFP+ -doublecortin (DCX) cells with a migratory morphology were also detected and their abundance increased over a 7-day period. Furthermore, EGFP-labeled cells progressively became NeuN+ neurons, they acquired neuronal morphologies, and they became immunoreactive for OB neuron subtype markers, the most abundant representing calretinin expressing interneurons. OB-NSCs also generated glial cells, suggesting they could be multipotent in vivo. Significantly, the newly generated neurons established and received synaptic contacts, and they expressed presynaptic proteins and the transcription factor pCREB. By contrast, when the retroviral particles were injected into the subventricular zone (SVZ), nearly all (98%) EGFP+ -cells were postmitotic when they reached the OB core, implying that the vast majority of proliferating cells present in the OB are not derived from the SVZ. Furthermore, we detected slowly dividing label-retaining cells in this region that could correspond to the population of resident NSCs. This is the first time NSCs located in the adult OB core have been shown to generate neurons that incorporate into OB circuits in vivo.


Assuntos
Células-Tronco Neurais , Bulbo Olfatório , Animais , Diferenciação Celular/fisiologia , Interneurônios/metabolismo , Camundongos , Células-Tronco Neurais/metabolismo , Neurogênese , Neurônios/metabolismo
2.
Neurotox Res ; 30(1): 14-31, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26678495

RESUMO

NURR1 is an essential transcription factor for the differentiation, maturation, and maintenance of midbrain dopaminergic neurons (DA neurons) as it has been demonstrated using knock-out mice. DA neurons of the substantia nigra pars compacta degenerate in Parkinson's disease (PD) and mutations in the Nurr1 gene have been associated with this human disease. Thus, the study of NURR1 actions in vivo is fundamental to understand the mechanisms of neuron generation and degeneration in the dopaminergic system. Here, we present and discuss findings indicating that NURR1 is a valuable molecular tool for the in vitro generation of DA neurons which could be used for modeling and studying PD in cell culture and in transplantation approaches. Transduction of Nurr1 alone or in combination with other transcription factors such as Foxa2, Ngn2, Ascl1, and Pitx3, induces the generation of DA neurons, which upon transplantation have the capacity to survive and restore motor behavior in animal models of PD. We show that the survival of transplanted neurons is increased when the Nurr1-transduced olfactory bulb stem cells are treated with GDNF. The use of these and other factors with the induced pluripotent stem cell (iPSC)-based technology or the direct reprogramming of astrocytes or fibroblasts into human DA neurons has produced encouraging results for the study of the cellular and molecular mechanisms of neurodegeneration in PD and for the search of new treatments for this disease.


Assuntos
Neurônios Dopaminérgicos/fisiologia , Neurogênese/fisiologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/fisiologia , Animais , Células Cultivadas , Humanos , Camundongos Knockout , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/biossíntese , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Bulbo Olfatório/citologia , Transplante de Células-Tronco/métodos , Células-Tronco/fisiologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia
3.
Dev Neurobiol ; 75(8): 823-41, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25447275

RESUMO

The transcription factor Nurr1 is expressed in the mouse olfactory bulb (OB), although it remains unknown whether it influences the generation of dopaminergic neurons (DA) (DA neurons) in cells isolated from this brain region. We found that expressing Nurr1 in proliferating olfactory bulb stem cells (OBSCs) produces a marked inhibition of cell proliferation and the generation of immature neurons immunoreactive for tyrosine hydroxylase (TH) concomitant with marked upregulations of Th, Dat, Gad, and Fgfr2 transcripts. In long-term cultures, these cells develop neurochemical and synaptic markers of mature-like mesencephalic DA neurons, expressing GIRK2, VMAT2, DAT, calretinin, calbindin, synapsin-I, and SV2. Concurring with the increase in both Th and Gad expression, a subpopulation of induced cells was both TH- and GAD-immunoreactive indicating that they are dopaminergic-GABAergic neurons. Indeed, these cells could mature to express VGAT, suggesting they can uptake and store GABA in vesicles. Remarkably, the dopamine D1 receptor agonist SKF-38393 induced c-Fos in TH(+) cells and dopamine release was detected in these cultures under basal and KCl-evoked conditions. By contrast, cotransducing the Neurogenin2 and Nurr1 transcription factors produced a significant decrease in the number of TH-positive neurons. Our results indicate that Nurr1 overexpression in OBSCs induces the formation of two populations of mature dopaminergic neurons with features of the ventral mesencephalon or of the OB, capable of responding to functional dopaminergic stimuli and of releasing dopamine. They also suggest that the accumulation of Fgfr2 by Nurr1 in OBSCs may be involved in the generation of DA neurons.


Assuntos
Neurônios Dopaminérgicos/fisiologia , Neurônios GABAérgicos/fisiologia , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Bulbo Olfatório/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Neurônios Dopaminérgicos/efeitos dos fármacos , Fator de Crescimento Epidérmico/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Neurônios GABAérgicos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Mitose/efeitos dos fármacos , Mitose/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Bulbo Olfatório/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/metabolismo
4.
Neurobiol Dis ; 48(3): 271-81, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22820144

RESUMO

Tyrosine hydroxylase (TH)-immunoreactive (ir) neurons have been found in the striatum after dopamine depletion; however, little is known about the mechanism underlying their appearance or their functional significance. We previously showed an increase in striatal TH-ir neurons after L-DOPA treatment in mice with unilateral 6-OHDA lesions in the striatum. In the present study, we further examined the time-course and persistence of the effects of chronic L-DOPA treatment on the appearance and regulation of TH-ir neurons as well as their possible function. We found that the L-DOPA-induced increase in striatal TH-ir neurons is dose-dependent and persists for days after L-DOPA withdrawal, decreasing significantly 10 days after L-DOPA treatment ends. Using hemiparkinsonian D1 receptor knock-out (D1R-/-) and D2 receptor knock-out (D2R-/-) mice, we found that the D1R, but not the D2R, is required for the L-DOPA-induced appearance of TH-ir neurons in the dopamine-depleted striatum. Interestingly, our experiments in aphakia mice, which lack Pitx3 expression in the brain, indicate that the L-DOPA-dependent increase in the number of TH-ir neurons is independent of Pitx3, a transcription factor necessary for the development of mesencephalic dopaminergic neurons. To explore the possible function of L-DOPA-induced TH-ir neurons in the striatum, we examined dopamine overflow and forelimb use in L-DOPA-treated parkinsonian mice. These studies revealed a tight spatio-temporal correlation between the presence of striatal TH-ir neurons, the recovery of electrically stimulated dopamine overflow in the lesioned striatum, and the recovery of contralateral forelimb use with chronic L-DOPA treatment. Our results suggest that the presence of TH-ir neurons in the striatum may underlie the long-duration response to L-DOPA following withdrawal. Promotion of these neurons in the early stages of Parkinson's disease, when dopamine denervation is incomplete, may be beneficial for maintaining motor function.


Assuntos
Corpo Estriado/efeitos dos fármacos , Dopaminérgicos/farmacologia , Levodopa/farmacologia , Neurônios/efeitos dos fármacos , Transtornos Parkinsonianos/metabolismo , Animais , Corpo Estriado/metabolismo , Dopamina/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo
5.
Mol Cell Neurosci ; 46(1): 108-21, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20807572

RESUMO

The T-box brain 1 (Tbr1) gene encodes a transcription factor necessary for the maintenance and/or differentiation of glutamatergic cells in the olfactory bulb (OB) and cortex, although its precise function in the development of glutamatergic neurons is not known. Furthermore, Tbr1 has not been reported to regulate the formation of glial cells. We show that Tbr1 is expressed during the initial stages in the generation of glutamatergic mitral neurons from dividing progenitors in the E12.5 mouse OB. Retroviral-mediated overexpression of Tbr1 in cultured embryonic and adult OB stem cells (OBSC) produces a marked increase in the number of TuJ1(+) neurons (including VGLUT1(+) glutamatergic and GABA(+) neurons) and O4(+) oligodendrocytes. Moreover, transduction of Tbr1 inhibits the production of GFAP(+) astrocytes from both cultured OBSC and dividing progenitor cells in vivo. These results show that the expression of Tbr1 in neural stem and progenitor cells prevents them from following an astrocyte fate during OB development. Our findings suggest that the transduction of Tbr1 into neural stem cells could be useful to increase the production of neurons and oligodendrocytes in studies of neuroregeneration.


Assuntos
Astrócitos/fisiologia , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Neurais/fisiologia , Bulbo Olfatório/citologia , Bulbo Olfatório/embriologia , Animais , Astrócitos/citologia , Diferenciação Celular/fisiologia , Proliferação de Células , Proteínas de Ligação a DNA/genética , Ácido Glutâmico/metabolismo , Camundongos , Células-Tronco Neurais/citologia , Neurônios/citologia , Neurônios/fisiologia , Oligodendroglia/citologia , Oligodendroglia/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas com Domínio T , Ácido gama-Aminobutírico/metabolismo
6.
Cell Tissue Res ; 340(1): 1-11, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20182890

RESUMO

Adrenomedullin (AM) is a peptide hormone involved in the modulation of cellular growth, migration, apoptosis, and angiogenesis. These characteristics suggest that AM is involved in the control of neural stem/progenitor cell (NSPC) biology. To explore this hypothesis, we have obtained NSPC from the olfactory bulb of adult wild-type animals and brain conditional knockouts for adm, the gene that produces AM. Knockout NSPC contain higher levels of hyperpolymerized tubulin and more abundant filopodia than adm-containing cells, resulting in a different morphology in culture, whereas the size of the knockout neurospheres is smaller than that of the wild-types. Proliferation studies have demonstrated that adm-null NSPC incorporate less 5'-bromodeoxyuridine (BrdU) than their wild-type counterparts. In contrast, BrdU studies in the olfactory bulb of adult animals show more labeled cells in adm-null mice that in wild-types, suggesting that a compensatory mechanism exists that guarantees the sufficient production of neural cells in this organ. In NSPC differentiation tests, lack of adm results in significantly lower proportions of neurons and astrocytes and higher proportions of oligodendrocytes. The oligodendrocytes produced from adm-null neurospheres present an immature phenotype with fewer and shorter processes than adm-containing oligodendrocytes. Thus, AM is an important factor in regulating the proliferation and differentiation of adult NSPC and might be used to modulate stem cell renewal and fate in protocols destined to produce neural cells for regenerative therapies.


Assuntos
Adrenomedulina/genética , Encéfalo/anormalidades , Diferenciação Celular/genética , Malformações do Sistema Nervoso/genética , Neurogênese/genética , Células-Tronco/patologia , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiopatologia , Bromodesoxiuridina , Proliferação de Células , Forma Celular/genética , Células Cultivadas , Camundongos , Camundongos Knockout , Malformações do Sistema Nervoso/metabolismo , Malformações do Sistema Nervoso/fisiopatologia , Neuritos/metabolismo , Neuritos/patologia , Neurônios/metabolismo , Neurônios/patologia , Bulbo Olfatório/anormalidades , Bulbo Olfatório/crescimento & desenvolvimento , Bulbo Olfatório/fisiopatologia , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Fenótipo , Polissacarídeos , Células-Tronco/metabolismo
7.
Eur J Neurosci ; 30(5): 742-55, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19712103

RESUMO

While insulin-like growth factor-I (IGF-I) supports neuronal and glial differentiation in the CNS, it is largely unknown whether IGF-I also influences neuronal migration and positioning. We show here that the pattern of olfactory bulb (OB) layering is altered in Igf-I (-/-) mice. In these animals, Tbr1(+)-glutamatergic neurons are misplaced in the mitral cell layer (ML) and the external plexiform layer (EPL). In addition, there are fewer interneurons in the glomerular layer and the EPL of the Igf-I (-/-) mice, and fewer newborn neurons are incorporated into the OB from the forebrain subventricular zone (SVZ). Indeed, neuroblasts accumulate in the postnatal/adult SVZ of Igf-I (-/-) mice. Significantly, the positioning of Tbr1(+)-cells in a primitive ML is stimulated by IGF-I in cultured embryonic OB slices, an effect that is partially repressed by the phosphoinositide 3-kinase (PI3K) inhibitor. In OB cell cultures, IGF-I increases the phosphorylation of disabled1 (P-Dab1), an adaptor protein that is a target of Src family kinases (SFK) in the reelin signalling pathway, whereas reduced P-Dab1 levels were found in Igf-I (-/-) mice. Neuroblast migration from the rostral migratory stream (RMS) explants of postnatal Igf-I (-/-) was similar to that from Igf-I (+/+) explants. However, cell migration was significantly enhanced by IGF-I added to the explants, an effect that was repressed by PI3K and SFK inhibitors. These findings suggest that IGF-I promotes neuronal positioning in the OB and support a role for IGF-I in stimulating neuroblast exit from the SVZ into the RMS, thereby promoting the incorporation of newly formed neurons into the OB.


Assuntos
Movimento Celular/fisiologia , Fator de Crescimento Insulin-Like I/metabolismo , Bulbo Olfatório/fisiologia , Prosencéfalo/fisiologia , Animais , Apoptose/fisiologia , Western Blotting , Contagem de Células , Células Cultivadas , Imunofluorescência , Ácido Glutâmico/metabolismo , Hibridização In Situ , Fator de Crescimento Insulin-Like I/genética , Interneurônios/metabolismo , Interneurônios/fisiologia , Camundongos , Camundongos Knockout , Células Neuroepiteliais/metabolismo , Neurogênese , Neurônios/metabolismo , Neurônios/fisiologia , Bulbo Olfatório/citologia , Bulbo Olfatório/metabolismo , Técnicas de Cultura de Órgãos , Fosforilação/fisiologia , Prosencéfalo/citologia , Prosencéfalo/metabolismo , Proteína Reelina , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Tirosina 3-Mono-Oxigenase/metabolismo
8.
Development ; 133(21): 4367-79, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17038521

RESUMO

During the embryonic period, many olfactory bulb (OB) interneurons arise in the lateral ganglionic eminence (LGE) from precursor cells expressing Dlx2, Gsh2 and Er81 transcription factors. Whether GABAergic and dopaminergic interneurons are also generated within the embryonic OB has not been studied thoroughly. In contrast to abundant Dlx2 and Gsh2 expression in ganglionic eminences (GE), Dlx2 and Gsh2 proteins are not expressed in the E12.5-13.5 mouse OB, whereas the telencephalic pallial domain marker Pax6 is abundant. We found GABAergic and dopaminergic neurons originating from dividing precursor cells in E13.5 OB and in short-term dissociated cultures prepared from the rostral half of E13.5 OB. In OB cultures, 22% of neurons were GAD+, of which 53% were Dlx2+, whereas none expressed Gsh2. By contrast, 70% of GAD+ cells in GE cultures were Dlx2+ and 16% expressed Gsh2. In E13.5 OB slices transplanted with EGFP-labeled E13.5 OB precursor cells, 31.7% of EGFP+ cells differentiated to GABAergic neurons. OB and LGE precursors transplanted into early postnatal OB migrated and differentiated in distinct patterns. Transplanted OB precursors gave rise to interneurons with dendritic spines in close proximity to synaptophysin-positive boutons. Interneurons were also abundant in differentiating OB neural stem cell cultures; the neurons responded to the neurotrophin Bdnf and expressed presynaptic proteins. In vivo, the Bdnf receptor TrkB colocalized with synaptic proteins at the glomeruli. These findings suggest that, in addition to receiving interneurons from the LGE, the embryonic OB contains molecularly distinct local precursor cells that generate mature GABAergic and dopaminergic neurons.


Assuntos
Dopamina/metabolismo , Interneurônios/fisiologia , Bulbo Olfatório , Células-Tronco , Ácido gama-Aminobutírico/metabolismo , Animais , Biomarcadores/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Diferenciação Celular , Transplante de Células , Células Cultivadas , Proteínas do Olho/metabolismo , Feminino , Idade Gestacional , Proteínas de Homeodomínio/metabolismo , Interneurônios/citologia , Camundongos , Neurônios/citologia , Neurônios/fisiologia , Bulbo Olfatório/química , Bulbo Olfatório/citologia , Bulbo Olfatório/embriologia , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/metabolismo , Gravidez , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , Células-Tronco/química , Células-Tronco/citologia , Células-Tronco/fisiologia , Fatores de Transcrição/metabolismo
9.
J Cell Sci ; 119(Pt 13): 2739-48, 2006 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-16787946

RESUMO

Neural stem cells depend on insulin-like growth factor I (IGF-I) for differentiation. We analysed how activation and inhibition of the PI 3-kinase-Akt signalling affects the number and differentiation of mouse olfactory bulb stem cells (OBSCs). Stimulation of the pathway with insulin and/or IGF-I, led to an increase in Akt phosphorylated on residues Ser473 and Thr308 (P-Akt(Ser473) and P-Akt(Thr308), respectively) in proliferating OBSCs, and in differentiating cells. Conversely, P-Akt(Ser473) levels decreased by 50% in the OB of embryonic day 16.5-18.5 IGF-I knockout mouse embryos. Overexpression of PTEN, a negative regulator of the PI 3-kinase pathway, caused a reduction in the basal levels of P-Akt(Ser473) and P-Akt(Thr308) and a minor reduction in IGF-I-stimulated P-Akt(Ser473). Although PTEN overexpression decreased the proportion of neurons and astrocytes in the absence of insulin/IGF-I, it did not alter the proliferation or survival of OBSCs. Accordingly, overexpression of a catalytically inactive PTEN mutant promoted OBSCs differentiation. Inhibition of PI 3-kinase by LY294002 produced strong and moderate reductions in IGF-I-stimulated P-Akt(Ser473) and P-Akt(Thr308), respectively. Consequently, LY294002 reduced the proliferation of OBSCs and the number of neurons and astrocytes, and also augmented cell death. These findings indicate that OBSC differentiation is more sensitive to lower basal levels of P-Akt than proliferation or death. By regulating P-Akt levels in opposite ways, IGF-I and PTEN contribute to the fine control of neurogenesis in the olfactory bulb.


Assuntos
Indução Embrionária , Fator de Crescimento Insulin-Like I/fisiologia , Neurônios/fisiologia , Proteína Oncogênica v-akt/metabolismo , PTEN Fosfo-Hidrolase/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Células-Tronco/fisiologia , Animais , Astrócitos/fisiologia , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cromonas/farmacologia , Embrião de Mamíferos , Técnicas In Vitro , Camundongos , Camundongos Knockout , Morfolinas/farmacologia , Proteínas Mutantes/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Transdução de Sinais
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