Abstract
The role of pro NGF, the precursor of Nerve Growth Factor (NGF), on the biology of adult neural stem cells (aNSCs) is still unclear. Here we analyzed adult hippocampal neurogenesis in AD11 transgenic mice, in which the constitutive expression of anti-NGF antibody leads to an imbalanceof proNGF over mature NGF. We found increased proliferation of progenitors but a reduced neurogenesis in the AD11 DG- hippocampus (HP-DG). Alsoin vitro, AD11 hippocampal neural stemcells (NSCs) proliferated more but were unable to differentiate into morphologically mature neurons. By treating wild-type (WT) hippocampal progenitors with the uncleavable form of pro NGF(proNGF-KR) we demonstrated that pro NGF acts as mitogen on a NSCs at low concentration. Themitogenic effect of proNGF was specifically addressed to the radial glia-like (RGL) stem cells through the induction of cyclin D1 expression. These cells express high level of p75NTR, as demonstrated by immunofluorescence analyses performed ex vivo on RGL cells isolated from freshly-dissociated HP-DG or selected in vitro from NSCs by LIF (leukemia inhibitory factor). Clonogenic assay performed in the absence of mitogens showed that RGLs respond to pro NGF-KR by reactivating their proliferation and thus leading to neurospheres formation. The mitogenic effect of pro NGF was further exploited in the expansion of mouse induced Neural Stem Cells (iNSCs).Chronic exposure of iNSCs to pro NGF-KR increased their proliferation. Altogether, we demonstrated that pro NGF acts as mitogen on hippocampal and induced neural stem cells.
This work identifies for the first time a new role of pro NGF as a specific mitogenic factor for the proliferation of adult hippocampal neural stem cells and in induced neural stem cells (iNS).The relevance of this finding is that it adds new knowledge to complex picture of neural stem cells biology, and indicates pro NGF/NGF balance critical for adult hippocampal neurogenesis. In addition, our results opens new perspectives in the view of developing future therapeutic approaches based on the stimulation of endogenous adult neurogenesis or on cell-reprogramming protocols.
Understanding the cell biology of the adult neural stem cells (aNSCs), in terms of their maintenance, activation, proliferation, differentiation, survival and maturation into newborn neurons represents a challenging open question in the field of adult neurogenesis [1]. All these different steps are tightly regulated by the convergence of many signals provided by the surrounding neurogenic niche [2,3]. Among these, signaling by members of the neurotrophin (NTs) family warrants to be investigated,due to their crucial role in development of the central nervous system [4]. The neurotrophin family includes the Nerve Growth Factor (NGF),the Brain-Derived Neurotrophic Factor (BDNF),the Neurotrophin-3 (NT3), Neurotrophin-4/5(NT4/5) [5–10]. Neurotrophins exert their bio-logical actions by binding to two different classes of transmembrane receptors (NTRs), theTrk family of receptors and the pan-Neurotrophin Receptor p75 (p75NTR). Both receptors can trigger downstream signaling pathways to drive the biological effects of neurotrophins. In the CNS, neurotrophins are present in equilibrium between their mature and larger precursor forms, the pro neurotrophins (proNGF, proBDNF, proNT-3, proNT-4/5). Pro-neurotrophins preferentially bind to p75NTR(at variance with the mature forms, that area preferential Trk binders), but not all p75NTR expressing cells are sensitive to pro neurotrophins; expression of the neurotensin receptor Sortilin is apparently needed for neurotrophins to induce biological effects via p75NTR [11]. At the functional level, pro neurotrophins play specific actions that are antagonist to those of the mature neurotrophins [12]. Interestingly,the antagonist effects of the proneurotrophins have pathophysiological relevance in human neurodegenerative diseases and brain injury [13–17]. Beyond their well-established essential role during embryogenesis [4], neurotrophins play critical roles also supporting mature neurons, after development, by maintaining neuronal morphologies and functions. In the adult nervous system NTs control the balance of survival/death response of mature cells, through the pro-survival TrkA and a pro apoptotic p75NTR signaling respectively [10,18–22]. Neurotrophins have been shown to regulate neurogenesis during embryonic development [23,24]. Instead, little is known approximately the relevance of NTs family members in regulating a NSCs biology [25–27], in which the cell-cycle progression or exit of the aNSCs must be tightly regulated. In addition tosurvival-inducing activity, NTs can be potent regulators of cellcycle [28–31], which could be potentially very relevant for their actions on aNSCs. However, the interplay between the immature or mature form of neurotrophins and cell cycle regulation has been less explored toward the understanding of a NSCs biology. Exogenous mature NGF positively affects adult hippocampal neural precursor survival and consequently hippocampal neurogenesis in physiological and neurodegenerative conditions [32,33]. A more direct positive effect of NGF on adult SVZ neurogenesis has been recently shown by our group,as we demonstrated that NGF neutralization impairs both proliferation and neuronal differentiation of SVZ progenitors [25].For what concerns pro neurotrophins, a role in cell cycle modulation has only recently being uncovered [34,35], but their function in this context needs further direct investigations.In this study we explored the role of NGF and pro NGF in regulating hippocampal dentate gyrus (HP-DG) adult neurogenesis, both in vivo and in vitro. In particular, by analyzing quiescent stem cells separately from neural progenitors, we were able to demonstrate a dual role of pro NGF in hippocampal neurogenesis, which is cell-stage specific and dependent on a fine balance with its mature form NGF.
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