Anticancer Activity and Mechanisms of Action of MAPK pathway inhibitors


I. reduced in comparison to wildtype hTau. In contrast, release of phosphomimetic hTauE14 was increased upon activation. We found that released hTau was phosphorylated in its proline-rich and C-terminal domains using phosphorylation site-specific tau antibodies (retinal; BL, blue light; CSF, cerebrospinal fluid; ChR2, channelrhodopsin 2; DIV, days (14) showed for the first time that tau can Rabbit Polyclonal to FZD10 be released from rat cortical neurons in culture by glutamate agonists. Activity-dependent tau release was also observed (15). A recent study (16) used a sophisticated optogenetics approach to specifically activate a subset of neurons, which subsequently released tau. Neuronal activity appears to be an important factor regulating tau release, but its role in tau pathology is unknown. In addition, the mechanism of activity-dependent tau release is still elusive in terms of its secretion pathways (pathological). Tau undergoes several post-translational modifications, and phosphorylation is the most commonly observed (17, 18, 19). More than 40 phosphorylation sites (pSites) of tau are thought to be involved in AD pathogenesis (2, 4, 19). Beta-mangostin Phosphorylation of specific tau pSites is important for its association with microtubules and normal function, but its hyperphosphorylation is a common feature of tauopathies (17, 20). Indeed, some of these tau pSites are known to be abnormally phosphorylated in paired helical filaments (PHFs) and neurofibrillary tangles during progression of AD but are not phosphorylated in healthy brains (3). Tau phosphorylation is also known to increase its release from neurons (21, 22). Phosphorylated tau proteins at specific Beta-mangostin sites (neurons in primary culture expressing human tau (hTau) and a variety of genetic approaches such as a binary UAS/Gal4 system (23) and optogenetics (24). We studied how tau phosphorylation contributes to its activity-dependent release using two tau mutant lines: phosphomimetic tauE14 (25) and phosphoresistant tauS11A (26). Finally, a selected group of pSite-specific tau antibodies (neuronal culture In order to study activity-dependent release of hTau, we used primary neuronal culture expressing hTau. Using Gal4? UAS binary system (23), the fly line was made by crossing the cholinergic driver Cha-Gal4 with UAS-hTau2N4R line. GFP transgene (UAS-GFP) was added to mark cholinergic neurons coexpressing hTau. The majority of GFP(+) neurons in ChaGFP-Gal4? UAS-hTau2N4R cultures at 9?days (DIV) was stained with antitotal hTau HT7 antibody (Fig.?1and Table?1). These data suggest that expression of hTau in fly neuronal culture results in neurodegeneration and intracellular tau aggregation. Open in a separate window Figure?1 Human tau (hTau) expressed in (DIV) neurons were stained. test. n?= number of neurites used for analysis. Results were from three independent experiments. aprimary neuronal culture (Fig.?1and Beta-mangostin (DIV) culture (ChaGFP-Gal4? hTau2N4R) was used. hTau bands quantified in CM and lysate were between 55 and 100?kDa. test. PHF, paired helical filament. Lysate tau bands are observed in the range of 40 to 250?kDa. In contrast, released tau proteins in the IP-CM were observed in the 55 to 100?kDa range, regardless of which tau antibody was used (and and endogenous fly tau (Fig.?2pSites, including phospho-specific AT8, AT100, AT180, and PHF1 epitopes (26). These genetic tools allowed a direct comparison of the effect of phosphorylation or dephosphorylation in the proline-rich domain Beta-mangostin (PRD) and C-terminal domain of hTau upon its depolarization-induced release. ChaGFP-Gal4 was used to drive the expression of UAS-hTauE14, UAS-hTauS11A, wildtype hTau 0N4R (UAS-hTau0N4R) and hTau 2N4R isoforms (UAS-hTau2N4R), respectively. E14 tau was mutated from 0N4R wildtype tau isoform, whereas S11A was from 2N4R. Therefore, we used 0N4R and 2N4R as controls for E14 and S11A, respectively. When hTauE14 was expressed, depolarization-dependent release was significantly increased when compared with neurons expressing hTau0N4R (Fig.?3, and and and test. hTau release was increased by optogenetic stimulation As our results suggested that hTau release is regulated by neuronal depolarization in neurons, we were prompted to confirm the depolarization-induced hTau release an optogenetic approach, which provides a highly controlled and precise method of inducing cellular depolarization. Optogenetic stimulation is physiologically relevant and.