Neuropeptide Y stimulates proliferation, migration and differentiation of neural precursors from the subventricular zone in adult mice

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Abstract

The neuropeptide Y (NPY) is widely expressed in the central nervous system and has been shown to stimulate neurogenesis in the hippocampus and the olfactory epithelium. Here, we demonstrate that intracerebroventricular injection of NPY stimulates proliferation of neural precursors in the mice subventricular zone (SVZ), one the most neurogenic areas of the brain. Newly generated neuroblasts migrate through the rostral migratory stream to the olfactory bulb and also directly to the striatum, as evidenced by BrdU labelling and cell phenotyping. Using knock-out mice, specific NPY receptor agonists and antagonists, we report that this neuroproliferative effect is mediated by the Y1 receptor subtype that we found to be highly expressed in the SVZ both at the mRNA and protein levels. Our data suggest that stimulating endogenous SVZ neural stem cells by NPY may be of a potential interest in cell replacement based therapies of neurodegenerative diseases affecting the striatum such as Huntington's disease.

Introduction

Over the last three decades, research exploring potential neuronal replacement therapies has focused on replacing lost neurons by transplanting cells into diseased regions of the brain (Bjorklund, 2003, Freeman, 2000, Gaillard, 2007). Among recent research avenues in the field of neurodegenerative diseases, a special interest has been gathered around promoting recruitment and differentiation of endogenous neural stem cells to replace lost cells (Romanko et al., 2004).

Neurogenesis persists in the adult mammalian brain in mainly two restricted areas, the subventricular zone (SVZ) of the lateral ventricles and the dentate gyrus of the hippocampus (Gage, 2000). Lining the lateral ventricles, the SVZ is the most neurogenic region in the adult brain. Newly generated neuroblasts from the SVZ migrate within the rostral migratory stream (RMS) to the olfactory bulb (OB) where they differentiate into interneurons and integrate the neuronal circuitry (Lois and Alvarez-Buylla, 1993, Lois, 1996). Neural stem cell proliferation has been shown to be regulated by endogenous factors such as neurotransmitters (dopamine, glutamate, GABA…), trophic factors such as brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), transforming growth factor α (TGFα), fibroblast growth factor-2 (FGF-2) and other molecules such as hormones and nitric oxide (Hagg, 2005, Ming and Song, 2005). Neurogenesis in the SVZ has been shown to be modulated in many physiopathological conditions (Curtis et al., 2007). For instance, inducing stroke in rats leads to a marked increase of cell proliferation in the SVZ; these cells migrate into the damaged area of the striatum where they express markers of striatal medium spiny neurons (Arvidsson et al., 2002). The production of progenitor cells from the SVZ was also shown to be increased in animal models of Huntington's disease (HD), where striatal degeneration occurs (Curtis et al., 2003). The SVZ of HD patients was found to be enriched in endogenous factors and receptors that actively regulate cell cycle and/or the differentiation of precursors. Identifying these factors is of key interest as they can be of potential help in brain repair. In this line, the SVZ of HD patients was found to be notably enriched in neuropeptide Y (NPY)-positive cells (Curtis et al., 2005).

NPY is a 36 amino acid peptide widely expressed in the central and peripheral nervous system during development and adulthood (Adrian, 1983, Allen, 1983, Tatemoto, 1982, Tatemoto, 1982) where is it described to be involved in many physiological and pathological conditions such as food intake, metabolic disorders, anxiety, seizures, memory, circadian rhythm, drug addiction and pain (Silva et al., 2002). The various effects of this peptide are mediated in rodents by the activation of at least five receptor subtypes belonging to the G-coupled receptor family designated as Y1, Y2, Y4, Y5 and y6 and that are broadly expressed in the developing and adult rodent brain (Kopp, 2002, Michel, 1998, Naveilhan, 1998, Stanic, 2006). Studies of the potential effects of NPY on stem cell proliferation were mainly performed in vitro (Hansel, 2001, Milenkovic, 2004) or on brain slice (Howell et al., 2005). In vivo, NPY knock-out mice show decreased proliferation of progenitor cells within the OB (Hansel et al., 2001). More recently, it has been shown that transgenic mice lacking NPY receptors have fewer proliferating cells, migratory neuroblasts and less interneurons within the OB compared to wild type mice, suggesting a role for NPY in regulating adult neurogenesis within the SVZ (Stanic et al., 2008). Moreover, a recent work has reported the pro-neurogenic effect of NPY in SVZ cultures (Agasse et al., 2008). However, no study thus far has reported the consequences of in vivo NPY administration on SVZ cell fate.

Here, we investigated the effects of intracerebroventricular (ICV) injection of NPY on SVZ cells in the adult mice. We demonstrate that NPY has a proliferative effect on neural precursor cells that results in cell migration of newly generated neurons to the striatum and the olfactory bulb. The neuroproliferative effect of NPY seems to be specifically mediated by the Y1 receptor which mRNA and protein are expressed in the SVZ. Our results may open new perspectives in cell replacement therapies in neurodegenerative disorders implicating the striatum such as Huntington's disease.

Section snippets

Animals

Experiments were carried out on adult C57BL/6 mice (4–5 month-old) supplied by R. Janvier (Le Genest-Saint Isles, France) and Y1 receptor knock-out mice (C57BL/6 background) kindly provided by Dr. T. Pedrazzini (Switzerland) (Pedrazzini et al., 1998). Animals were raised and housed under a 12 hour light/dark cycle with food and water ad libitum. All efforts were made to minimize animal number and suffering. All treatments conformed to the guidelines of the French Agriculture and Forestry

NPY stimulates cellular proliferation in the SVZ

We performed ICV injections of either saline, NPY (10 2 μg/μl), Y1 ([Leu31,Pro34], 10 2 μg/μl), Y2 (NPY3–36, 10 2 μg/μl) receptor agonists or NPY + Y1 receptor antagonist (BIBP3226, 10 μM) and evaluated after 48 h their effects on the proliferation of SVZ cells using single labelling with an antibody raised against BrdU (Figs. 1A–G). NPY induced a significant increase in the number of BrdU+ cells in the SVZ (305 ± 34) compared to saline group (166 ± 16) (P < 0.01). This effect seems to be mediated by

Discussion

Here we report that NPY promotes (i) significant cellular proliferation in the mouse SVZ, (ii) an increase in the number of cells found in the RMS, OB and striatum, and (iii) that these effects are mediated preferentially by the Y1 receptor. Proliferating cells were mainly neuroblasts, that were shown to express DCX and the transcription factor SOX2 which is known to maintain neuronal progenitors characteristics (Graham et al., 2003) and proliferative potential of stem cell (Episkopou, 2005).

Conclusion

The development of pharmacological drugs that would increase production of new neurons would be facilitated by identification of the endogenous or natural molecular regulators of adult neurogenesis in vivo. One of the residing main difficulties to the development of effective stem cell therapies is to direct the fate of the newly generated cells to specifically replace lost cells. Here, we demonstrate for the first time that NPY preferentially induces proliferation of neuronal cells in vivo and

Acknowledgments

We want to thank Bruno Merceron for his technical assistance, Eleni Paizanis for the help with BrdU staining protocol, and Frederic Gaillard for his help with the statistical analysis. Authors also thank Dr Philippe Naveilhan for generously providing us with NPY receptor plasmids and Dr Thierry Pedrazzini for providing us the transgenic Y1 knock-out mice. Authors certify that they have no conflict of interest. This work was supported by CNRS, Poitiers University, Fondation de France (2006),

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