Reelin function in neural stem cell biology

Abstract

In the adult brain, neural stem cells (NSC) must migrate to express their neuroplastic potential. The addition of recombinant reelin to human NSC (HNSC) cultures facilitates neuronal retraction in the neurospheroid. Because we detected reelin, alpha3-integrin receptor subunits, and disabled-1 immunoreactivity in HNSC cultures, it is possible that integrin-mediated reelin signal transduction is operative in these cultures. To investigate whether reelin is important in the regulation of NSC migration, we injected HNSCs into the lateral ventricle of null reeler and wild-type mice. Four weeks after transplantation, we detected symmetrical migration and extensive neuronal and glial differentiation of transplanted HNSCs in wild-type, but not in reeler mice. In reeler mice, most of the injected HNSCs failed to migrate or to display the typical differentiation pattern. However, a subpopulation of transplanted HNSCs expressing reelin did show a pattern of chain migration in the reeler mouse cortex. We also analyzed the endogenous NSC population in the reeler mouse using bromodeoxyuridine injections. In reeler mice, the endogenous NSC population in the hippocampus and olfactory bulb was significantly reduced compared with wild-type mice; in contrast, endogenous NSCs expressed in the subventricular zonewere preserved. Hence, it seems likely that the lack of endogenous reelin may have disrupted the migration of the NSCs that had proliferated in the SVZ. We suggest that a possible inhibition of NSC migration in psychiatric patients with a reelin deficit may be a potential problem in successful NSC transplantation in these patients.

Figure 1

Effect of recombinant reelin on HNSCs differentiated without serum. (a) βIII-tubulin (green) and GFAP (red) at time 0 (×400). (b) Typical time-lapse image of HNSC movements in the presence and absence of recombinant reelin (×50). quicktime movie files of these time-lapse images are published as Movies 1 and 2 as supporting information on the PNAS web site, www.pnas.org.

Figure 2

Expression of reelin and α3-integrin receptor subunits in cultures of HNSCs. (a) Reelin immunoreactivity (green) expressed by neuronal-like phenotypes and not by GFAP-positive cells (red) (×400). (b) Electron-microscopic image (×17,500) of α3−integrin receptor subunit immunoreactivity located on HNSC plasma membrane (arrows). (c) α3-Integrin receptor subunit immunoreactivity (green) expressed specifically by neuron-like phenotypes (×400). Nuclei were counterstained with DAPI (blue).

Figure 3

Western blot analysis of reelin (a), α3-integrin (b), and Dab-1 protein (c) extracted from HNSCs by immunoprecipitation with anti-reelin 142 antibody after nonserum (basal) or serum differentiation.

Figure 4

Immunohistochemistry of cerebral cortex in a wild-type mouse 4 weeks after HNSC transplantation into the brain. (a) HNSCs migrated into the cortex and differentiated into neuron-like phenotypes expressing βIII-tubulin immunoreactivity (green) (×100). (b) Pyramidal neuron-like phenotypes, with the presence of apical dendrites (green) expressing BrdUrd-positive nuclei (red) (×400). (c) Note a layer of GFAP-positive HNSCs (brown) (×100). (d) Double GFAP and βIII-tubulin immunostaining revealing a layer of GFAP-positive (red) and a layer of βIII-tubulin-positive (green) cells (×100). Nuclei were counterstained with DAPI (blue).

Figure 5

Immunohistochemistry of a reeler mouse brain 4 weeks after HNSC transplantation. (a) Low magnification (×100) of the cortex stained with anti-βIII-tubulin antibody and counterstained with DAPI to reveal nuclei. Note that βIII-tubulin-positive (green) cells are virtually undetectable. (b) Groups of BrdUrd-positive cells near the injection site (×400). Note that the morphology of these cells differs from that of cells expressed in the cortex of wild-type mice (see Fig. 4). (c) Chain migration of transplanted cells expressing reelin (green) in the cortex (×400). (d) Few GFAP-positive HNSCs (brown) detected in the cortex (×100). (e) Differentiation of HNSCs into βIII-tubulin-positive cells (green) near the injection site (×400). Nuclei were counterstained with DAPI (blue).

Figure 6

Comparison of endogenous NSC populations in reeler and wild-type mice (×100). BrdUrd incorporated into the nuclei of proliferating cells was detected by fluorescent immunohistochemistry (red), and all of the nuclei were counterstained with DAPI (blue). (Upper) Dramatically decreased population of stem cells in the hippocampus of homozygous (a) and heterozygous (b) reeler mice compared with wild-type mice (c). (Lower) Similar population of stem cells in the SVZ of homozygous reeler (d) and wild-type mice (e). Note the unorganized structure of the dentate gyrus in homozygous reeler mice (a). Nuclei were counterstained with DAPI (blue).

Figure 7

Number of BrdUrd-positive cells in heterozygous reeler (+/−), homozygous reeler (−/−), and wild-type mice in the hippocampus, olfactory bulb, and SVZ. Significant reduction of the stem cell population was observed in the hippocampus (P < 0.001) and olfactory bulb (P < 0.01) but not in the SVZ of reeler (+/−) and reeler (−/−) mice compared with wild-type mice by Fisher's Protected LSD post hoc analysis after ANOVA.