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17 páginas, 7 figuras., 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., This work was funded by grants from the Spanish “Ministerio de Economía y Competitividad and Ministerio de Ciencia, Innovacion y Universidades ” (MINECO and MICIU: SAF2013-4759R and SAF2016-80419-R, PID2019-109059RB-I00 to C.V., SAF2015-70433-R to H.M., BFU2016-80870-P to A.C., and CIBERNED CB06/05/0065 to C.V.), from the “Comunidad de Madrid” (S2011/BMD-2336) to C.V. and H.M., from the “Generalitat Valenciana” (PROMETEO/2018/055) to H.M., and from the CSIC Intramural program (Refs. 201220E098 and 201320E054) to C.V. Additional support was provided by the European Research Council (ERC) 2012-StG (311736-PD-HUMMODEL), the Instituto de Salud Carlos III - ISCIII/FEDER (Red de Terapia Celular - TerCel RD16/0011/0024 and PIE14/00061) to A.C. Ç.D. received a short-term fellowship from the Red Olfativa Española (ROE). M.M.-E, M.D.-M., A.H.-C., and V.N.-E. were supported by FPI Fellowships from the MICINN and MINECO.

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.

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.

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.

10 páginas, 8 figuras -- PAGS nros. 2739-2748, 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-AktSer473 and P-AktThr308, respectively) in proliferating OBSCs, and in differentiating cells. Conversely, P-AktSer473 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-AktSer473 and P-AktThr308 and a minor reduction in IGF-I-stimulated P-AktSer473. 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-AktSer473 and P-AktThr308, 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, This work was funded by the grants from the MEC (Spain) BFU 2004-2352 to F.d.P., SAF2004-05798 to C.V.-A. and BMC 2003-07751 to E.J.d.l.R., and from the Fundación "la Caixa" NE03/72-02 to C.V.-A. G.O., M.J.Y.-B, E.V.-V. and H.R.M.-G. were supported by doctoral fellowships either from the MEC or the "Comunidad Autónoma de Madrid"

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 10days 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. © 2012 Elsevier Inc.

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 doparninergic neurons.