Pineal-dependent increase of hypothalamic neurogenesis contributes to the timing of seasonal reproduction in sheep.

To survive in temperate latitudes, species rely on the photoperiod to synchronize their physiological functions, including reproduction, with the predictable changes in the environment. In sheep, exposure to decreasing day length reactivates the hypothalamo-pituitary-gonadal axis, while during increasing day length, animals enter a period of sexual rest. Neural stem cells have been detected in the sheep hypothalamus and hypothalamic neurogenesis was found to respond to the photoperiod. However, the physiological relevance of this seasonal adult neurogenesis is still unexplored. This longitudinal study, therefore aimed to thoroughly characterize photoperiod-stimulated neurogenesis and to investigate whether the hypothalamic adult born-cells were involved in the seasonal timing of reproduction. Results showed that time course of cell proliferation reached a peak in the middle of the period of sexual activity, corresponding to decreasing day length period. This enhancement was suppressed when animals were deprived of seasonal time cues by pinealectomy, suggesting a role of melatonin in the seasonal regulation of cell proliferation. Furthermore, when the mitotic blocker cytosine-b-D-arabinofuranoside was administered centrally, the timing of seasonal reproduction was affected. Overall, our findings link the cyclic increase in hypothalamic neurogenesis to seasonal reproduction and suggest that photoperiod-regulated hypothalamic neurogenesis plays a substantial role in seasonal reproductive physiology.

Leptin-dependent neurotoxicity via induction of apoptosis in adult rat neurogenic cells.

Adipocyte-derived hormone leptin has been recently implicated in the control of neuronal plasticity. To explore whether modulation of adult neurogenesis may contribute to leptin control of neuronal plasticity, we used the neurosphere assay of neural stem cells derived from the adult rat subventricular zone (SVZ). Endogenous expression of specific leptin receptor (ObRb) transcripts, as revealed by RT-PCR, is associated with activation of both ERK and STAT-3 pathways via phosphorylation of the critical ERK/STAT-3 amino acid residues upon addition of leptin to neurospheres. Furthermore, leptin triggered withdrawal of neural stem cells from the cell cycle as monitored by Ki67 labeling. This effect was blocked by pharmacological inhibition of ERK activation thus demonstrating that ERK mediates leptin effects on neural stem cell expansion. Leptin-dependent withdrawal of neural stem cells from the cell cycle was associated with increased apoptosis, as detected by TUNEL, which was preceded by cyclin D1 induction. Cyclin D1 was indeed extensively colocalized with TUNEL-positive, apoptotic nuclei. Cyclin-D1 silencing by specific shRNA prevented leptin-induced decrease of the cell number per neurosphere thus pointing to the causal relationship between leptin actions on apoptosis and cyclin D1 induction. Leptin target cells in SVZ neurospheres were identified by double TUNEL/phenotypic marker immunocytofluorescence as differentiating neurons mostly. The inhibition of neural stem cell expansion via ERK/cyclin D1-triggered apoptosis defines novel biological action of leptin which may be involved in adiposity-dependent neurotoxicity.

Adult-brain-derived neural stem cells grafting into a vein bridge increases postlesional recovery and regeneration in a peripheral nerve of adult pig.

We attempted transplantation of adult neural stem cells (ANSCs) inside an autologous venous graft following surgical transsection of nervis cruralis with 30 mm long gap in adult pig. The transplanted cell suspension was a primary culture of neurospheres from adult pig subventricular zone (SVZ) which had been labeled in vitro with BrdU or lentivirally transferred fluorescent protein. Lesion-induced loss of leg extension on the thigh became definitive in controls but was reversed by 45-90 days after neurosphere-filled vein grafting. Electromyography showed stimulodetection recovery in neurosphere-transplanted pigs but not in controls. Postmortem immunohistochemistry revealed neurosphere-derived cells that survived inside the venous graft from 10 to 240 post-lesion days and all displayed a neuronal phenotype. Newly formed neurons were distributed inside the venous graft along the severed nerve longitudinal axis. Moreover, ANSC transplantation increased CNPase expression, indicating activation of intrinsic Schwann cells. Thus ANSC transplantation inside an autologous venous graft provides an efficient repair strategy.

Effects of aging and caloric restriction on brainstem satiety center signals in rats.

Age-related increases of body weight and adiposity, indicating dysregulation of food intake/energy expenditure, can be prevented in rodents by long-term 40% caloric restriction. The dorsal vagal complex (DVC), the brainstem center mediating the satiety reflex, has recently emerged as a determinant effector of long-term feeding adaptation. To study the effects of aging and caloric restriction on satiety circuits, leptin and brain-derived neurotrophic factor (BDNF) signaling systems were studied in 2- and 19-month-old ad libitum-fed (AL) and 19-month-old calorie-restricted (CR) rats. Age-induced hyperleptinemia in AL rats was correlated with elevated DVC BDNF immunoreactive concentrations and satiety threshold stability, suggesting functional desensitization of the DVC to these signals. To better understand this phenomenon, mRNA levels of receptor and post-receptor signaling effectors were measured by real-time RT-PCR. Aging selectively increased BDNF receptors and suppressor of cytokine signaling-3 (SOCS-3) mRNA levels. Caloric restriction prevented age-related increases of serum leptin, DVC BDNF and SOCS-3 mRNA levels, but not those of BDNF receptors. In CR rats, prevention of leptin resistance-promoting SOCS-3 induction was also observed at the protein level. This study suggests that leptin post-receptor targets and BDNF signaling play a role in the establishment of age-related DVC dysfunction.

Emerging new sites for adult neurogenesis in the mammalian brain: a comparative study between the hypothalamus and the classical neurogenic zones.

In adult mammalian brain, two main germinative regions located in the subventricular zone of the lateral ventricle and in the subgranular cell layer of the hippocampal dentate gyrus have been considerably documented and are still under intense scrutiny. However, new neuron formation has recently been reported in various other brain areas including the hypothalamus. This central structure, responsible for the control of many major neuroendocrine functions such as reproduction, expresses high levels of PSA-NCAM and nestin, both proteins being involved in structural and morphological plasticity mechanisms. Cell proliferation and new neuron production have been demonstrated in the adult hypothalamus of numerous species, although not hitherto described in non-human primates and humans. Similarly to the subventricular zone and in the subgranular cell layer, the adult hypothalamic neurogenesis process is subject to dynamic regulation by various physiological and pharmacological signals. Several pieces of evidence support the hypothesis that a stem cell niche-like architecture exist in the hypothalamus region lining the third ventricle thereby enabling adult neural stem cells to continuously generate neurons in vivo throughout life. Furthermore, recent data indicating that new hypothalamic neurons may become functionally implicated in sensory information processing endorse the assumption that the hypothalamus might be a neurogenic region.

In vitro isolation of neural precursor cells from the adult pig subventricular zone.

In order to improve cell therapy techniques, we have characterized a multipotent neural precursor cell isolation technique from the subventricular zone of adult pig brain. The pig is a non-primate species that is immunologically closest to human. The proliferative zone of this neurogenic structure was first localized in situ in the pig brain by Ki-67 immunohistochemistry, as a ventral subfield of the Nissl-stained subventricular zone. For in vitro cultures, the striatal forebrain was sampled from deeply anaesthetized adult pigs and SVZ tissue explants were immediately microdissected out and dissociated in the appropriate medium. Primary cell culture in the presence of EGF and bFGF allowed growth of spherical masses that exhibited sustained growth and self-renewal capacity through six subsequent passages. Molecular characterization using reverse transcription-polymerase chain reaction (RT-PCR) showed that expanded pro-differentiating neurospheres expressed markers of proliferation, neural stem cells, and committed neural progenitors. After growth factor removal, the spheres became adherent and were shown to contain the three neural cell lineages by triple immunocytofluorescence and confocal microscopy. The present protocol therefore allowed for in vitro expansion of pig brain primary cells that display capacities for proliferation, self-renewal, and multipotency, i.e., the cardinal features of multipotent neural precursor cells.

Microenvironmental determinants of adult neural stem cell proliferation and lineage commitment in the healthy and injured central nervous system.

The discovery of neural stem cells (NSC) which ensure continuous neurogenesis in the adult mammalian brain, has led to a conceptual revolution in basic neuroscience and to high hopes for clinical nervous tissue repair. However, several research issues remain to address before neural stem cells can be harnessed for regenerative therapies. The presence of NSC in a nervous structure is demonstrated in vitro by primary culture of dissociated adult nervous tissue in the presence of the specific mitogens EGF and bFGF. This leads to spherical masses of proliferating cells endowed with capacities for self-renewal and, after growth factor removal, differentiation into the three characteristic cell types of nervous tissue (neurons, astrocytes, oligodendrocytes). In vivo, neurogenesis per se, i.e. production of new neurons, occurs only in a small subset of NSC-endowed structures. The production of oligodendrocytes, i.e. myelinating glial cells, is similarly restricted. Such in vivo restrictions were formally demonstrated to arise from the tissular microenvironnement, which led to the emerging concept of "neurogenic niche". In this context, major challenges now consist in identifying the nature of tissue-specific extracellular signals that determine lineage commitment of NSC progeny, understanding why NSCs display weak in vivo reactivity to lesions compared to other stem cell types in adults, and identifying the factors behind the very high resistance to tumorigenesis displayed by NSCs. Altogether, the current data offer hope for the future use of adult NSCs in regenerative therapies, provided that tissue-specific signals are identified in view of counteracting the intrinsic repression of new cell genesis and/or stimulating endogenous NSC recruitment to lesion sites.