Tania O. Alexson

Neural stem cells, which exhibit self-renewal and multipotentiality, are generated in early embryonic brains and maintained throughout the lifespan. The mechanisms of their generation and maintenance are largely unknown. Here, we show that neural stem cells are generated independent of RBP-Jkappa, a key molecule in Notch signaling, by using RBP-Jkappa(-/-) embryonic stem cells in an embryonic stem cell-derived neurosphere assay. However, Notch pathway molecules are essential for the maintenance of neural stem cells; they are depleted in the early embryonic brains of RBP-Jkappa(-/-) or Notch1(-/-) mice. Neural stem cells also are depleted in embryonic brains deficient for the presenilin1 (PS1) gene, a key regulator in Notch signaling, and are reduced in PS1(+/-) adult brains. Both neuronal and glial differentiation in vitro were enhanced by attenuation of Notch signaling and suppressed by expressing an active form of Notch1. These data are consistent with a role for Notch signaling in the maintenance of the neural stem cell, and inconsistent with a role in a neuronal/glial fate switch.

Basic fibroblast growth factor (FGF2)-responsive definitive neural stem cells first appear in embryonic day 8.5 (E8.5) mouse embryos, but not in earlier embryos, although neural tissue exists at E7.5. Here, we demonstrate that leukemia inhibitory factor-dependent (but not FGF2-dependent) sphere-forming cells are present in the earlier (E5.5-E7.5) mouse embryo. The resultant clonal sphere cells possess self-renewal capacity and neural multipotentiality, cardinal features of the neural stem cell. However, they also retain some nonneural properties, suggesting that they are the in vivo cells' equivalent of the primitive neural stem cells that form in vitro from embryonic stem cells. The generation of the in vivo primitive neural stem cell was independent of Notch signaling, but the activation of the Notch pathway was important for the transition from the primitive to full definitive neural stem cell properties and for the maintenance of the definitive neural stem cell state.

Recently, Notch signaling has been reported to underscore the ability of neural stem cells (NSCs) to self-renew. Utilizing mice deficient in presenilin-1(PS1), we asked whether the function of Notch signaling in NSC maintenance was conserved. At embryonic day 14.5, all NSCs--both similar (cortex-, ganglionic eminence- and hindbrain-derived) and distinct (retinal stem cell)--require Notch signaling in a gene-dosage-sensitive manner to undergo expansionary symmetric divisions, as assessed by the clonal, in vitro neurosphere assay. Within the adult, however, Notch signaling modulates cell cycle time in order to ensure brain-derived NSCs retain their self-renewal property. At face value, the effects in the embryo and adult appear different. We propose potential hypotheses, including the ability of cell cycle to modify the mode of division, in order to resolve this discrepancy. Regardless, these findings demonstrate that PS1, and presumably Notch signaling, is required to maintain all NSCs.

Nicotine, the primary psychoactive component of tobacco smoke, is known to possess potent rewarding and aversive stimulus properties. The mammalian ventral tegmental area (VTA) is involved importantly in the mediation of the motivational effects of nicotine. However, the neural outputs from the VTA that may be involved in the transmission of the rewarding and aversive motivational effects of nicotine are not well understood. We report that bilateral lesions of the tegmental pedunculopontine nucleus (TPP) double dissociate the rewarding and aversive motivational effects of nicotine. Using a conditioned place preference paradigm, bilateral TPP lesions blocked a nicotine reward signal and revealed the aversive motivational properties of intra-VTA nicotine. These same TPP lesions did not block an aversive nicotine signal, as measured in a conditioned taste aversion paradigm. TPP lesions also produce an attenuation in nicotine-induced locomotor activity; however, neither learning nor performance deficits can account for these observed effects, because TPP-lesioned animals still showed clear aversive nicotine conditioning in two separate behavioral paradigms. Our results suggest that the rewarding effects of nicotine in the VTA are dependent on a nondopaminergic, descending reward pathway to the brainstem TPP.

Here we show a novel function for Retinoblastoma family member, p107 in controlling stem cell expansion in the mammalian brain. Adult p107-null mice had elevated numbers of proliferating progenitor cells in their lateral ventricles. In vitro neurosphere assays revealed striking increases in the number of neurosphere forming cells from p107(-/-) brains that exhibited enhanced capacity for self-renewal. An expanded stem cell population in p107-deficient mice was shown in vivo by (a) increased numbers of slowly cycling cells in the lateral ventricles; and (b) accelerated rates of neural precursor repopulation after progenitor ablation. Notch1 was up-regulated in p107(-/-) neurospheres in vitro and brains in vivo. Chromatin immunoprecipitation and p107 overexpression suggest that p107 may modulate the Notch1 pathway. These results demonstrate a novel function for p107 that is distinct from Rb, which is to negatively regulate the number of neural stem cells in the developing and adult brain.