6FCH). by 24 months. In addition, we observed that Fyn+/?3xTg+/? males were delayed in developing A pathology compared TOFA to females, and displayed better spatial learning overall performance at 18 months. Overall, these findings suggest that loss of Fyn at early stages of disease increases soluble A accumulation and worsens spatial learning Rabbit polyclonal to SP3 in the absence of changes in tau phosphorylation. models show that Fyn exacerbates A-induced neuronal and behavioral deficits, and these effects are blocked by the genetic ablation of Fyn (Chin et al., 2005; Chin et al., 2004). However, our group has found that Fyn causes decreased A production and Fyn knock-out mice have decreased -secretase APP products (Hoe et al., 2008). These findings suggest antagonistic functions for Fyn TOFA in increasing tau phosphorylation and A-mediated neurotoxicity and in decreasing A production, leading to the question of whether Fyn inhibition will ultimately show beneficial or detrimental for the treatment of AD. In order to investigate this question, we utilized a triple transgenic model of AD (3xTg) harboring mutations in human APP, presenilin-1, and tau, which recapitulates both the A and tau pathologies of AD, to determine the effect of Fyn on each (Oddo et al., 2003a; Oddo et al., 2003b). We employed a genetic approach by breeding 3xTg mice with either wild-type or Fyn knock-out mice to generate wild-type or heterozygous Fyn mice on a heterozygous 3xTg background (Fyn+/+3xTg+/? and Fyn+/?3xTg+/?). We found that knock-down of Fyn resulted in both increased A levels and TOFA decreased tau phosphorylation accompanied by deficits in spatial learning around the Morris water maze. These findings implicate a greater role for any, and not tau, pathology in mediating cognitive overall performance at early disease stages in the triple transgenic model of AD. Taken together, our study implicates a harmful effect of long-term reduction of Fyn kinase on A production and cognitive overall performance. 2. Materials and methods 2.1 Animals and breeding Fyn knock-out mice were obtained from Jackson Laboratories (Bar Harbor, ME) at 3 months of age for analysis of phospho-tau. Wild-type B6129SF2/J controls were also purchased from Jackson Laboratories. We crossed male Fyn knock-out and wild-type mice with female 3xTg AD mice originally generated by co-microinjection of human APP (K670M/N671L) and tau (P301L) transgenes under the control of the Thy 1.2 promoter into mutant PS-1 (M146V) knock-in mice (Oddo et al., 2003b). Female and male Fyn+/+3xTg+/? or Fyn+/?3xTg+/? mice were generated and euthanized by quick cervical dislocation (to eliminate anesthesia-mediated tau phosphorylation (Planel et al., 2007)) at 15, 18, 21, and 24 months of age for females and 18, 21, and 24 months of age for males. Brains were quickly isolated, and hemi-brains were either snap-frozen in dry ice for biochemical analyses or immersion fixed in 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4, for histochemical analyses. 2.2 Chemicals and antibodies We used antibody 6E10 (Signet, Dedham, MA) to detect A, antibodies AT8, AT180, and AT270 (Pierce, Rockford, IL) for phospho-tau epitopes, and Tau46 (Sigma, St. Louis, MO) for total tau. Polyclonal Fyn antibody was purchased from Millipore (Billerica, MA). Antibodies 1A10 (for A1C40), 1C3 (for A1C42), and 82E1 (human-specific A antibody) for any ELISAs were obtained from IBL (Gunma, Japan). 2.3 Tissue preparation Mouse brains were homogenized in a 10-fold volume of 50 mM TrisCHCl buffer, pH 7.6, containing 250 mM sucrose and protease inhibitor cocktail (Sigma). Soluble APP and A were extracted in 0.4% diethylamine (DEA), as previously explained (Nishitomi et al., 2006). Briefly, crude 10% brain homogenate was mixed with an equal volume of 0.4% DEA, sonicated, and ultracentrifuged for 1 hr at 100,000 g. TOFA The supernatant was collected and neutralized with 10% 0.5M Tris base, pH 6.8. The producing DEA portion was utilized for soluble A ELISA analyses. Insoluble A TOFA was extracted from your pellet after ultracentrifugation in formic acid (FA), sonicated, and ultracentrifuged for 1 hr at 100,000 g. The supernatant was collected and neutralized with 1M Tris base and 0.5M Na2HPO4, and the producing FA fraction was utilized for insoluble A ELISA analyses. 2.4 Western blot Proteins were extracted from brain homogenates with radioimmunoprecipitation assay (RIPA) buffer made up of 50mM Tris-HCl, pH 7.4, 150mM NaCl, 0.25% deoxycholic acid, 1% NP-40, and 1mM EDTA (Millipore), and probed for phosphorylated tau using antibodies AT8 (Ser202/Thr205), AT180 (Thr231), or AT270 (Thr181) (Hirata-Fukae et al., 2008). Total tau was detected with antibody Tau46 as a band at 60 kDa, and all phosphorylated tau values were normalized to total tau. Proteins were separated by SDS-PAGE on 10% polyacrylamide gels,.