The role of mTor in the pathogenesis of tau-related pathologies in Alzheimer Disease

Sammanfattning: An important neurophathological hallmark of Alzheimer disease (AD) is the progressive formation of neurofibrillary tangles composed of aberrant hyperphosphorylated tau aggregates. Evidence from human postmortem AD brains and in vitro and in vivo rapamycin-treated drug models implicated an abnormal accumulation of the mammalian target of rapamycin (mTor) in AD brains. Previous evidence also indicated that the sequential molecular events, such as the formation and phosphorylation of tau, can be regulated by p70S6 kinase, the well characterized downstream target of mTor. Since mTor is a serine/threonine(S/T) kinase that plays a key role in the regulation of protein homeostasis and degradation, and in cellular functioning, including cell survival, cell growth and proliferation, we chose in our studies to investigate mTor’s involvement in the regulation of tau associated pathologies in AD. In Paper I we investigated whether or not mTor mediates tau protein homeostasis. By immunostaining we had found that the active form of mTor aggregated in tangle-bearing neurons in AD brains. Employing mass spectrometry and Western blotting, we identified that mTor directly phosphorylated tau protein at three phosphoepitopes (S214, S356 and T231). We have further developed a variety of stable cell lines with genetic modification of mTor activity using SH-SY5Y neuroblastoma cells as background, confirmed the tau phosphorylation sites found in vitro, and found that mTor mediates the synthesis and accumulation of tau, resulting in compromised microtubule stability. The altered mTor activity has been found to cause fluctuation of the level of a battery of tau kinases such as protein kinase A (PKA), v-Aktmurine thymoma viral oncogene homolog-1(Akt), glycogen synthase kinase 3?(GSK-3?), cyclin-dependent kinase 5 (cdk5), and tau protein phosphatase 2A(PP2A). In Paper II we investigated whether mTor mediatescell survival. We used mass spectrometry to identify specific protein expression changes associated with cell survival in SH-SY5Y cells expressing genetically modification of mTor. By inducing cell death in SH-SY5Y cells using moderate serum deprivation, we found that up-regulated mTor complex 2 (mTorC2) increases viable cells by flow cytometry. By employing a combination proteomic method and enrichment analysis we observed that mTor significantly altered expression of several proteins (Peroxiredoxin-5, Thioredoxin-dependent peroxide reductase, Cofilin 1 [non-muscle], Mortalin, Annexin A5, and 14-3-3 protein zeta/delta) involved in mitochondrial integrity, apoptosis, and pro-survival functions. In Paper III we investigated the influence of mTor in tau distribution and secretion. By immunostaining, we found that tau protein was localized within different organelles (autophagic vacuoles, endoplasmic reticulum, Golgi complexes, and mitochondria) in postmortem human AD brain. By employingSH-SY5Y cells stably carrying different genetic variants of mTor, we further found that mTor was responsible for the changed balance of phosphorylated (p-)/ non phosphorylated (Np-) tau in the cytoplasm and different cellular compartments. Up-regulated mTor activity resulted in a significant increase in the amount of cytosolic tau as well as its localization in exocytotic vesicles that were not associated with exosomes to release into extracellular space. In Paper IV we investigated the effects of mTorin relationship to cell growth and proliferation. We found that the suppression of mTor decreases the rate of cell proliferation assessed by WST-1(4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay. By using microarray, we exhibited that mTor knockdown up-regulated 27 genes and down-regulated 49 genes related to the regulation of cell growth and proliferation. Silenced mTor seems to inhibit cell growth and proliferation, by regulating not only the AKT-mTor-S6K signaling pathway, but also directly or indirectly affecting key regulator such as Bcl2, CDK4 inhibitor, various interleukins, or the TGF beta superfamily. These findings can provide a better understanding of the complex role of mTor involved in the biomechanics of different aspects of tau changes in AD and could contribute to developments of mTor as a novel therapeutic target in the future.