The band intensities recognized by anti-Tau PS422 antibody were not significantly altered by ethanol treatment (data not shown)

The band intensities recognized by anti-Tau PS422 antibody were not significantly altered by ethanol treatment (data not shown). is usually phosphorylated by GSK-3 and cleaved by caspase-3 during ethanol-induced neurodegeneration in the developing brain. strong class=”kwd-title” Keywords: ethanol, caspase-cleaved tau, glycogen synthase kinase-3, neurodegeneration, developing brain, microglia Introduction Ethanol triggers wide-spread apoptotic neurodegeneration in P7 mice, which are in the middle of the brain growth spurt and in a period of brain development corresponding to the human third trimester (Ikonomidou et al., 2000; Olney et al., 2002b). The P7 rodent model of fetal alcohol spectrum disorders has been widely used for elucidating mechanisms of ethanol-induced toxicity in the developing brain (Young et al., 2003; Carloni et al., 2004; Han et al., 2006). Ethanol-induced neurodegeneration in the P7 mouse brain is usually preceded by caspase-3 activation (Olney et al., 2002b; Saito et al., 2007) and by decreases in phosphorylation levels of Akt and GSK-3 (Chakraborty et al., 2008). GSK-3 and caspase-3 thus activated by ethanol may impact the downstream effector molecule tau. Phosphorylation of tau by GSK-3 decreases the microtubule-binding capacity of tau and disrupts microtubule stability (Utton Allopregnanolone et al., 1997; Sang et al., 2001). When the majority of tau is usually phosphorylated at Ser396/Ser404 (the PHF-1 antibody epitope) by GSK-3 activation, tau aggregation is usually favored (Chun et al., 2007). In addition to hyper-phosphorylation, tau can Rabbit Polyclonal to USP30 be cleaved at Asp421/422 by caspases-3, 6, 7, and 8 (Gamblin et al., 2003). The caspase-cleaved tau (C-tau) assembles more rapidly into filaments than full-length tau (Gamblin et al., 2003), and has been detected in Alzheimer brains (Rissman et al., 2004). C-tau is also detected in brains hurt by kainic acid (Zemlan et al., 2003) and trauma (Zemlan et al., 2002; Gabbita et al., 2005), and C-tau is usually pro-apoptotic in cultured neurons (Chung et al., 2001; Fasulo et al., 2005). Lithium, a GSK-3 inhibitor, which blocks ethanol-induced caspase-3 activation (Zhong et al., 2006; Chakraborty et al., 2008), has been shown to reduce phosphorylation and aggregation of tau in a mouse model of Alzheimers disease (Noble et al., 2005). From these studies, we hypothesized that ethanol-induced GSK-3 and caspase-3 activation change tau as a downstream target, which may result in neurodegeneration. We further hypothesized that lithium inhibits tau modification as well as subsequent neurodegeneration. Although recent studies have shown that ethanol induces tau accumulation in neuroblastoma cells (Gendron et al., 2008) and generates c-Tau in P7 mouse brains (Zhang et al., 2009), the effects of ethanol on tau are largely unknown. It is expected that the majority of tau proteins at P7 display a fetal form. This form is usually highly phosphorylated and recognized by PHF-1 antibody (Goedert and Jakes, 1990), although it is usually less phosphorylated compared to the paired helical filament tau observed in tauopathies and retains a low but significant level of Allopregnanolone activity for promoting tubulin assembly (Morishima-Kawashima et al., 1995). Here, we examined tau modifications during ethanol-induced neurodegeneration in the P7 mouse brain. Experimental Process Animals and treatment C57BL/6By mice were managed at the Animal Facility of the Nathan S. Kline Institute for Psychiatric Research. All procedures followed guidelines consistent with those developed by the National Institute of Health and the Institutional Animal Care and Use Committee of the Nathan S. Kline Institute. An ethanol treatment paradigm shown to induce strong neurodegeneration in P7 C57BL/6 mice (Olney et al., 2002b) was followed using P7 C57BL/6By mice as explained (Saito et al., 2007). Each mouse in a litter of more than 8 pups was assigned to a saline, a lithium, an ethanol, or an ethanol + lithium group, and the experiment was repeated 3 to 5 5 occasions using different litters. The mice were injected subcutaneously with saline or ethanol (2.5 g/kg, 20% solution in sterile saline) twice at 0 h and 2 h. Lithium chloride (LiCl, 0.6M, 10 l/g) or saline was injected intraperitoneally 15 min after the first ethanol injection as described previously (Zhong et al., 2006). Four to 48 h after the first ethanol injection, brains were removed and processed for immunoblotting and immunohistochemical staining. For experiments using a caspase 3/7 inhibitor, N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone (Z-DEVD-FMK) (BioVision, Mountain View, CA), the inhibitor was administered by intracerebroventricular (icv) injections as explained (Sadakata et al., 2007). Z-DEVD-FMK Allopregnanolone (1 g in 2 l of 1 1.5% DMSO in saline) or vehicle was given twice 1 h before and 4 h after the ethanol injection, which was administered once at a concentration of 5.0 g/kg. Ethanol administration once.