Adult male C57BL/6 mice (3-5 months old) were obtained from Orient Bio (Sungnam, Korea). For traumatic brain injury, metal probe (3-mm diameter) was pre-chilled in the liquid nitrogen, and placed on the skull (2 mm lateral and 1 mm anterior from the bregma) for 60 seconds. The cut skin was sutured, and the animals returned to home cage. All experimental procedures were approved by Animal Care and Use Committee of Korea University.

Adult mouse neural stem cells (NSCs) were obtained and maintained as previously reported [14]. Briefly, the subventricular zone (SVZ) tissues were isolated from adult male mice (4-8 weeks old C57BL/6) and incubated with a mix of 0.8% papain (Worthington, Lakewood, NJ, USA) and 0.08% dispase II (Roche Applied Science, USA) in HBSS. Dissociated cells were seeded and maintained as neurosphere in DMEM/F12 media containing 1% N2, 2% B27, penicillin-streptomycin (Gibco BRL, USA), basic fibroblast growth factor (20 ng/ml, Invitrogen, USA), epidermal growth factor (20 ng/ml, Invitrogen, USA), and L-ascorbic acid (20 ng/ml, Sigma-Aldrich, USA). Neurospheres were dissociated with Accutase (Millipore, USA) and seeded on the laminin-coated dish as a monolayer for the differentiation. The differentiation was induced by the deprivation of the EGF/bFGF for 6 days.

Total RNAs (2 µg) were isolated from cultured NSCs or adult (8 weeks old) hippocampal tissues using the RNeasy mini kit (Qiagen, Cat# 74104, Germany), and were reverse-transcribed with reverse transcriptase (Bioneer, Cat# E-3100) and oligo (dT) primer. An aliquot of RT products was then subjected to PCR amplification as follows; initial denaturation at 95℃ for 10 min and 40 cycles of 95℃ for 15 s, 60℃ for 30 s, and 72℃ for 30 s. with specific primers. Forward primer, 4F (cac cgt ggc aat tct tga gc) was designed in exon 4 (Genbank # NM_001177955.1). Two reverse primers, 8R (ata aac actg gca ctg cgg a) and 10R (agc ctg cat ttt gct tgc at), were designed in exon 8 and exon 10, respectively. The combination of 4F and 8R (4F8R) yields 260 bp PCR products, whereas 4F and 10R (4F10R) yields two different PCR products from isoform-1 (563 bp) and isoform-2 (654 bp). PCR-amplified cDNA fragments were then separated onto 3% agarose gel containing of etidium bromide.

Animals were cardiac-perfused with 4% paraformaldehyde and the brains were isolated. Brains were then post-fixed in the same fixative overnight, and transferred to 30% sucrose solution until sinking. Following freezing, brains were cut (40 µm) coronal on a cryostat, and stored at -20℃ in the storage solution (50% phosphate buffered saline (PBS) and 50% glycerol). Following blocking with 3% bovine serum albumin (BSA) and 0.1% Triton-X100 in the PBS, primary antibodies were applied to the sections for 24 hours at room temperature. Primary antibodies used in this study are as follows: anti-GPM6B (Abcam, Cat# ab92988, 1:200), anti-glial fibrillary acidic protein (GFAP: SantaCruz, 1: 1,000), anti-Nestin (Abcam, 1:200), and anti-doublecortin (DCX: SantaCruz, 1: 200). After washes, appropriate secondary antibodies were incubated with sections for 30 min at room temperature. The signals were then observed under the confocal microscope (Zeiss, LSM510, Goettingen, Germany).

We examined the distribution of M6B-IR in the adult brain and found that it was strongly localized in neurogenic regions, including the subventricular zone (SVZ; Fig. 1A~C) and rostral migratory stream (RMS; Fig. 1D~F). Double labeling with GFAP, an astrocyte marker, revealed that cells expressing M6B-IR in these regions were astrocytes. In addition to the astrocytes in the neurogenic regions, the astrocytes associated with ventricles or with white matter showed strong M6B-IR. Examples of M6B-IR in the third ventricle (V III; Fig. 1G~I) and corpus callosum (Corp Cal; Fig. 1J~L) are shown. On the other hand, astrocytes in the grey matter or associated with blood vessels did not exhibit substantial M6B-IR (Fig. 1M~O). Some NeuN-expressing neurons also exhibited M6B-IR, as reported previously (Fig. 1M~O, insets).

Because neural stem cells (NSCs) in the SVZ are known to express GFAP, we examined whether M6B-IR was found in NSCs both in vivo and in vitro (Figs. 2 and 3). GFAP-expressing cells in the SVZ of the lateral ventricle expressed M6B-IR (Fig. 2A~C); these cells also expressed the NSC marker, nestin (Fig. 2D~F). Although the majority of GFAP or nestin-expressing cells in the SVZ also exhibited M6B-IR, we found that a small subset (<5%) of nesting-expressing NSCs did not exhibit M6B-IR (Fig. 2D~F, arrows). On the other hand, neuroblasts expressing DCX in the SVZ did not exhibit noticeable M6B-IR (Fig. 2G~I), suggesting that GPM6B expression was rapidly down-regulated by the differentiation of NSCs into neuroblasts.

In cultured NSCs, M6B-IR was found in proliferating nestin-expressing cells (Fig. 3A~C). When growth factors were removed from the culture media, NSCs spontaneously differentiate into neurons and glial cells. After spontaneous differentiation we also found that M6B-IR was maintained in the GFAP-expressing astrocytes, as expected (Fig. 3D~F). On the other hand, neuron-like cells containing multiple processes also exhibited M6B-IR (arrows, Fig. 3D~F); this finding is not similar to our in vivo results. Multiple isoforms of GPM6B have been identified [11, 15]. Because the antibody we used recognize all isoforms of GPM6B, we further confirmed the expression of GPM6B isoforms in the cultured NSCs at mRNA level by RT-PCR (Fig. 3G). Both cDNAs from culture NSCs and adult hippocampal tissues yielded the same size of PCR products by 4F8R primer set which amplifies common region (exon4-exon8). Interestingly, 4F10R primer set demonstrated that isoform-2 is more abundant in the NSCs comparing to the adult hippocampus, which is consistent with the previous finding that the expression of isoform-2 is down-regulated during the development [15].

Traumatic brain injury is known to promote the activation of quiescent astrocytes, resulting in the proliferation and hypertrophy of astrocytes in the injury penumbra region. Therefore, we investigated whether reactive astrocytes also exhibit the enhanced M6B-IR (Fig. 4). Double labeling of GPM6B and nestin, which is known to be expressed in immature reactive astrocytes, revealed that a subset of reactive astrocytes expressed GPM6B on 3 (Fig. 4D~F) or 7 days (Fig. 4G~I) after traumatic brain injury. Double labeling with GFAP and GPM6B also indicated that these cells were astrocytes (insets, Fig. 4D~F).