Lack of adrenomedullin affects growth and differentation of adult neural stem/progenitor cells.

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.Peer Reviewe

L-DOPA-induced increase in TH-immunoreactive striatal neurons in parkinsonian mice: Insights into regulation and function

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.Peer Reviewe

Role of Nurr1 in the Generation and Differentiation of Dopaminergic Neurons from Stem Cells

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.Peer Reviewe

Nurr1 blocks the mitogenic effect of FGF‐2 and EGF, inducing olfactory bulb neural stem cells to adopt dopaminergic and dopaminergic‐GABAergic neuronal phenotypes

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.Contract grant sponsor: Spanish Ministerio de Economía yCompetitividad (MINECO: BFU2010-1963 and SAF2013-47596-R) (to C.V.-A.).Contract grant sponsor: Spanish Ministerio de Economía y Competitividad (BFU2010-20664 and SAF2013-48532-R) (to R.M.).Contract grant sponsor: Ministerio de Sanidad Política Social eIgualdad (Instituto de Salud Carlos III, ISCIII: CIBERNED CB06/05/065) (to C.V.-A.).Contract grant sponsor: Ministerio de Sanidad Polıtica Social eIgualdad (Instituto de Salud Carlos III, ISCIII: CIBERNED CB06/05/0055) (to R.M.).Contract grant sponsor: Comunidad de Madrid (CM: S2011/BMD-2336) (to R.M. and C.V.-A.).Contract grant sponsor: CM, CONACYT (Gobierno de Mex-ico), and MINECO (to H.R.M.-G., O.S., and E.R.-T.)