Implication of ‘‘Down Syndrome Cell Adhesion Molecule’’ in the Hippocampal Neurogenesis of Ischemic Monkeys Tetsumori Yamashima, 1 * Boryana K. Popivanova, 1,2 Jianzhong Guo, 1,3 Susumu Kotani, 1 Tomohiko Wakayama, 3 Shoichi Iseki, 3 Kazunobu Sawamoto, 4,5 Hideyuki Okano, 5 Chifumi Fujii, 2 Naofumi Mukaida, 2 and Anton B. Tonchev 1,6 ABSTRACT: Molecular signals regulating adult neurogenesis in prima- tes are largely unknown. Here the authors used differential display to ana- lyze gene expression changes that occur in dentate gyrus of adult monkeys after transient global cerebral ischemia. Among 14 genes upregulated, the authors focused on Down syndrome cell adhesion molecule (DSCAM) known to play crucial role during neuronal development, and character- ized its expression pattern at the protein level. In contrast with approxi- mately threefold upregulation of Dscam gene on days 5 and 7, immuno- blotting and immunofluorescence analyses using specific antibodies showed a gradual decrease of DSCAM after ischemia until day 9 followed by recovery on day 15. In the control, immunofluorescence reactivity of DSCAM was detected in dentate gyrus granule cells and CA4 neurons but decreased after ischemia, being compatible with the immunoblotting data. However, in the subgranular zone, cerebral ischemia led to a marked increase of DSCAM-positive cells on days 9 and 15. DSCAM upregulation was seen in two cell types: one is immature neurons positive for polysialylated neural cell adhesion molecule or bIII-tubulin, while another is astrocytes positive for S100b. Young astrocytes were in inti- mate contact with newly generated neurons in the subgranular zone. These data suggest implication of DSCAM in the adult neurogenesis of pri- mate hippocampus upregulated after ischemia. V V C 2006 Wiley-Liss, Inc. KEY WORDS: primate; cerebral ischemia; subgranular zone; Down syndrome; neural progenitor INTRODUCTION Prominent in the embryonic brain, neurogenesis also continues in adulthood, but only in the restricted brain regions. For example, the hip- pocampal dentate gyrus (DG) retains multipotent pro- genitor cells residing in its subgranular zone (SGZ) (Gage et al., 1998). The existence of neuronal renewal in the adult DG under normal conditions has been shown from rodents (Altman and Das, 1965; Kaplan, 1977; Kempermann and Gage, 1999) to primates including monkeys (Gould et al., 1999; Kornack and Rakic, 1999) and humans (Eriksson et al., 1998; Roy et al., 2002). Various pathological conditions have been shown to affect DG neurogenesis. Among these, many rodent models have demonstrated that cerebral ische- mia increases the proliferation of SGZ progenitor cells with the consequent enhanced neuronal replacement in DG (Liu et al., 1998; Takagi et al., 1999; Jin et al., 2001; Kee et al., 2001; Yagita et al., 2001). Ischemic injury also increased DG progenitor cells in monkeys, but their proliferation and neuronal differentiation were much less compared with the rodent response (Tonchev et al., 2003a). This distinction appears to be serious for developing strategy of the brain restoration in humans. Recent research has identified signals affecting the postischemic progenitor proliferation in the rodent DG. These include basic fibroblast growth factor (Yoshimura et al., 2001), erythropoietin (Shingo et al., 2001), epidermal growth factor (Nakatomi et al., 2002), insulin-like growth factor-I, glial cell-derived neurotrophic factor (Dempsey et al., 2003), vascular endothelial growth factor (Sun et al., 2003), and hepa- rin-binding epidermal growth factor-like growth factor (Jin et al., 2004). However, the knowledge of the DG progenitor regulation in primates has been extremely limited until now, and for this reason the present study using macaque monkeys was undertaken. We con- structed a primate model of transient global cerebral is- chemia, which selectively destroys the hippocampal cornu Ammonis (CA)-1 neurons (Yamashima et al., 1996, 1998; Yamashima, 2000). In this study, we initially sought to investigate gene expression regarding SGZ progenitors. As gene expres- sion must precede the changes in stem cell behavior of day 9 (Tonchev et al., 2003a), we considered that post- ischemic day 5 and 7 time points are appropriate to investigate the gene expression changes regarding mon- key SGZ progenitor cell proliferation. In addition, to monitor more carefully dynamic changes of protein Departments of 1 Restorative Neurosurgery; 2 Molecular Bioregulation; 3 Histology and Embryology, Kanazawa University, Graduate School of Medical Science, Kanazawa, Japan; 4 Bridgestone Laboratory of Devel- opmental and Regenerative Neurobiology, Keio University School of Medicine, Tokyo, Japan; 5 Department of Physiology; 6 Division of Cell Biology, Department of Forensic Medicine, Medical University of Varna, Varna, Bulgaria Grant sponsor: Japanese Ministry of Education, Culture, Sports, Science and Technology; Grant numbers: Kiban-Kennkyu (B): 15390432 , 18390392. Abbreviations used: ABC, abidin–biotin peroxidase complex method; BLAST, basic local alignment search tool; CA, cornu Ammonis; DG, dentate gyrus; DSCAM, Down syndrome cell adhesion molecule; FDD, fluorescent differential display; GCL, granule cell layer; PSA-NCAM, polysialylated neural cell adhesion molecule; SGZ, subgranular zone. *Correspondence to: Tetsumori Yamashima, Department of Restorative Neurosurgery, Graduate School of Medical Science, Kanazawa Univer- sity, Takara-machi 13-1, Kanazawa 920-8641, Japan. E-mail: yamashim@med.kanazawa-u.ac.jp Accepted for publication 28 July 2006 DOI 10.1002/hipo.20223 Published online 18 September 2006 in Wiley InterScience (www.interscience. wiley.com). HIPPOCAMPUS 16:924–935 (2006) V V C 2006 WILEY-LISS, INC.