To evaluate this hypothesis, we immunoprecipitated Sp1 in lysates of NECs treated with or without PUGNAc and analyzed the O-GlcNAcylation. and streptozotocin, induced robust accumulation of O-GlcNAc in NECs and reduction of number of NECs. In O-GlcNAcase inhibitor-treated NECs, the Rasmitogen-activated protein kinase pathway and the phosphoinositide 3-kinase-Akt pathway, important for proliferation and survival, respectively, were intact, but caspase-3, an executioner for cell death, was activated. These results suggest the possibility that O-GlcNAc is involved in cell death signaling in NECs. Furthermore, for NECs, we identified an O-GlcNAc-modified protein, Sp1 transcription factor. Our study is the first to evaluate expression and functions of O-GlcNAc in NECs. Keywords:development, carbohydrate, signal transduction A neural stem cell (NSC) is an undifferentiated neural cell that is endowed with a high potential for proliferation and the capacity for self-renewal, with retention of multipotency to differentiate into neurons and glial cells (Weiss et al., 1996;McKay, 1997;Gage, 2000;Zhao et al., 2008). Because of their biological potential in neurogenesis and neural repair, there has been tremendous interest in the basic biology and clinical use of NSCs. The fate of NSCs is largely defined by extracellular cues, including cytokine signaling generated by the niche and intracellular programs such as epigenetic modifications (Fukuda and Taga, 2005;Zhao et al., 2008;Namihira et al., 2008). Not only cytokine signaling and epigenetic modifications but also glycosignaling mediated or modulated by carbohydrate antigens and glycoconjugates, however, is involved in NSC fate regulation (Yu and Yanagisawa, 2007). Glycoconjugates, including proteoglycans, glycoproteins, and glycolipids, are known as useful neural cell-lineage-specific markers, and emerging data indicate that glycoconjugates also mediate cell fate-regulating signals in NSCs (Yanagisawa and Yu, 2007). O-linked -N-acetylglucosamine (O-GlcNAc) is also expected to have important roles mediating glycosignaling in NSCs. O-GlcNAc is known to modify posttranslationally the serine and threonine residues of nuclear and cytoplasmic proteins (Wells et al., 2003;Kudlow, 2006;Zachara Uridine 5′-monophosphate and Hart, 2006;Hart et al., 2007;Rexach et al., Uridine 5′-monophosphate 2008); this modification is referred to as O-N-acetylglucosaminylation(O-GlcNAcylation). O-GlcNAcylation is catalyzed by O-linked N-acetylglucosamine transferase (O-GlcNAcT), which catalyzes the addition of a single O-GlcNAc residue from the donor UDP-GlcNAc to the carrier protein. Conversely, the O-GlcNAc is removed from the carrier protein by O-linked N-acetylglucosaminidase (O-GlcNAcase). O-GlcNAc and the enzymes regulating O-GlcNAcylation have been shown to play SGK2 various functional roles in cellular processes, including transcription, cell cycle regulation, signal transduction, stress response, apoptosis, glucose sensing, vesicular trafficking, Uridine 5′-monophosphate and proteasome degradation. Deletion of O-GlcNAcT in mouse embryonic stem cells is lethal, indicating the essential role of O-GlcNAc for cell integrity (Shafi et al., 2000). Because O-GlcNAcylation occurs in a manner similar to phosphorylation, and both appear at Uridine 5′-monophosphate the same or adjacent sites with serine/threonine residues, it has been proposed that O-GlcNAc has a reciprocal relationship with phosphorylation in modulating protein function. O-GlcNAcT and O-GlcNAcase were ubiquitously expressed but Uridine 5′-monophosphate most abundant in brain tissues (Kreppel et al., 1997;Gao et al., 2001), suggesting the importance of O-GlcNAc in brain tissues (Rexach et al., 2008). A number of reports have appeared on the occurrence of O-GlcNAc in brain tissues and cells (Griffith and Schmitz, 1999;Rex-Mathes et al., 2001;Khidekel et al., 2004;Khidekel et al., 2007;Rengifo et al., 2007). O-GlcNAc in brain tissues is found on proteins important for gene expression, neuronal signaling, and synaptic plasticity (Khidekel et al., 2004;Vosseller et al., 2006). Interestingly, it has been reported that O-GlcNAcylation is dynamically modulated by excitatory stimulation of the brain, suggesting the involvement of O-GlcNAcylation in neural circuitry (Khidekel et al., 2007). It has also been reported that neuron-specific deletion of the O-GlcNAcT gene in mice leads to abnormal development, locomotor defects, and postnatal death (O’Donnell et al., 2004). These reports clearly indicate that O-GlcNAc has significant roles in normal brain functions. In addition, O-GlcNAc is suggested to play roles in pathogenesis of Alzheimer’s disease (O’Donnell et al., 2004;Hart et al., 2007;Rexach et al., 2008) and aging (Flp et al., 2008). It is likely that such O-GlcNAcylation also has.