Circumventricular organs: A novel site of neural stem cells in the adult brain

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Abstract

Neurogenesis in the adult mammalian nervous system is now well established in the subventricular zone of the anterolateral ventricle and subgranular zone of the hippocampus. In these regions, neurons are thought to arise from neural stem cells, identified by their expression of specific intermediate filament proteins (nestin, vimentin, GFAP) and transcription factors (Sox2). In the present study, we show that in adult rat and mouse, the circumventricular organs (CVOs) are rich in nestin+, GFAP+, vimentin+ cells which express Sox2 and the cell cycle-regulating protein Ki67. In culture, these cells proliferate as neurospheres and express neuronal (doublecortin+, β-tubulin III+) and glial (S100β+, GFAP+, RIP+) phenotypic traits. Further, our in vivo studies using bromodeoxyuridine show that CVO cells proliferate and undergo constitutive neurogenesis and gliogenesis. These findings suggest that CVOs may constitute a heretofore unknown source of stem/progenitor cells, capable of giving rise to new neurons and/or glia in the adult brain.

Introduction

In recent years, it has become increasingly clear that the adult mammalian brain, once thought to be a static structure, in fact retains the capacity to generate new neurons and glia throughout the life of the organism (Alvarez-Buylla and Lim, 2004, Doetsch et al., 1999, Doetsch, 2003b, Doetsch, 2003a). In particular, adult neurogenesis has been found in two discrete regions of the brain: the subventricular zone (SVZ) of the anterolateral ventricular wall and the subgranular zone (SGZ) in the dentate gyrus of the hippocampus (Lie et al., 2004). The neurons produced in these regions are thought to arise from neural stem cells (NSCs) found in highly regulated stem cell niches wherein self-renewal and differentiation of progenitors toward the neural cell fate is determined by local environmental and intrinsic cellular cues (Temple, 2001, Shen et al., 2004, Barkho et al., 2006). In the adult, NSCs are generated for the continuous replacement of specific classes of brain neurons (i.e. periglomerular and granule cells in the olfactory bulb and granule cells of the dentate gyrus) but also in response to neurodegenerative disease, traumatic injury, stroke, etc. (Schouten et al., 2004, Sundholm-Peters et al., 2005, Park et al., 2006).

It has been postulated that NSCs in the SVZ and SGZ derive from a unique cell type, the germinal astrocyte, which is characterized by the presence of specific intermediate filament proteins (i.e. nestin, glial fibrillary acidic protein [GFAP], vimentin) (Doetsch et al., 1999, Doetsch, 2003b, Doetsch, 2003a, Lendahl et al., 1990, Wei et al., 2002, Wiese et al., 2004) and nuclear factors (i.e. Sox1, Sox2, Musashi-1) (Komitova and Eriksson, 2004, Cai et al., 2006, Shin et al., 2007). Still others maintain that additional cells types, such as multiciliated ependymal cells (Johansson et al., 1999) and tanycytes (non-ciliated cells of the ependyma) (Xu et al., 2005) also serve as NSCs in the adult brain and spinal cord (Bruni, 1998, Liu et al., 2002, Mothe and Tator, 2005). Finally, the presence of dividing progenitors has also been reported in a number of discrete regions of the adult brain, such as the cortex (Magavi et al., 2000, Jiang et al., 2001), amygdala (Park et al., 2006), striatum (Benraiss et al., 2001, Pencea et al., 2001, Teramoto et al., 2003, Chmielnicki et al., 2004, Collin et al., 2005, Mohapel et al., 2005), and substantia nigra (Zhao et al., 2003, Van Kampen and Robertson, 2005).

In the present study, we will show that proliferating cells found at specific sites along the third and fourth ventricles express proteins normally associated with germinal astrocytes and their derivatives (transit amplifying cells) of the SVZ and SGZ. We will demonstrate that these cells give rise to neurons and glia both in culture and in vivo. Historically, the midline structures where these cells reside have together been termed circumventricular organs (CVOs) and include the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), median eminence (ME), pineal gland (PG), subcommissural organ (SCO), area postrema (AP) and the choroid plexus. CVOs are unique in structure and function. Their ventricular surfaces are lined with specialized ependymal cells called tanycytes, which act as a partial CSF barrier. In addition, CVOs lack a classic blood-brain-barrier. With the exception of the SCO, all other CVOs contain permeable fenestrated capillaries and are often referred to as the “windows of the brain” (Johnson and Gross, 1993). The CVOs are critically involved in the maintenance of a wide variety of sensory homeostatic and inflammatory pathways in the brain (Moyse et al., 2006). These unique properties indicate that CVOs may represent novel sites for the continued generation of neurons and/or glia in the adult brain.

Section snippets

CVOs express neural stem cell markers

To establish that CVO cells express proteins that identify neural stem cells, we examined CVO regions from both the adult nestin-GFP mouse and adult rat. Using double label immunocytochemistry on nestin-GFP mouse brain sections, we first confirmed in the traditional and most active niche of adult neural stem cells (SVZ shown in panels A–C) that GFP fluorescent cells are nestin-immunoreactive. Likewise, the CVO regions of the nestin-GFP mouse brain (ME in panels E–G and AP in panels H–J of Fig. 1

Discussion

The present findings demonstrate that specific sites along the third and fourth ventricles in the adult brain, regions historically known as the CVOs, contain cells that express intermediate filament proteins (nestin, vimentin, GFAP) and transcription factors (Sox2), markers that typically characterize self-renewing multipotent stem/progenitor cells of the embryonic neuroepithelium (Cai et al., 2006, Shin et al., 2007) and adult SVZ and SGZ (Mignone et al., 2004). Grown in culture these cells

Animals and treatments

All experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and under approval of Thomas Jefferson University's Institutional Animal Care and Use Committee.

BrdU administration

Nestin-GFP male and female mice (8–14 weeks old) were administered BrdU (Fisher Scientific, Fair Lawn, NJ; 1 mg/ml) in their drinking water for one (proliferation study) or four weeks (differentiation studies), then immediately perfused and brains processed for tissue

Acknowledgments

This work was supported by NIH NS32519, NS43309 and the Hassel Foundation. This project is funded, in part, under a grant with the Pennsylvania Department of Health: SAP4100026302 C.U.R.E. The Department specifically disclaims responsibility for analyses, interpretations or conclusions. Lori Bennett was supported in part by a Measey MD/PhD Student Fellowship from Thomas Jefferson University. The authors thank Xiaotao Wei, Dr. Rupal Mehta, and Steve Borson for their assistance.

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