The current study reports that endogenous expression of TLR3 in SH-SY5Y cells and ligand-induced TLR3-mediated hyperphosphorylation of tau can be further enhanced by LPS pretreatment. To trace TLR3-induced signal transduction pathway mediating hyperphosphorylation of tau we evaluated two kinases: p38 MAPK and JNK. We observed that these two kinases are at least involved in ligand (pIpC)-induced TLR3-mediated hyperphosphorylation of tau.
Amyloid plaques and NTFs are two well-known pathological hallmarks of AD.22,23 Moreover, a prominent innate immune response has been observed in association with pathological lesions of AD that includes activation of microglia, activation of complement, secretion of proinflammatory kinase such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α; expression of the chemokines such as MIP-1α, MIP-1β and MCP-1 and the secretion of nitric oxide.24,25 Recently, studies have shown that IL-1 released from activated microglia mediates hyperphosphorylation of tau in cortical neurons through the p38 MAPK pathway.13,26 Moreover activated microglia has been correlated with neurofibrillary pathology,27 including intracellular tau accumulation.28,29 These investigations therefore indicate the involvement of innate immune activation in the pathway of aberrant phosphorylation of tau. We focused on an innate immune receptor, TLR3, that is reported to be expressed in macrophage, astrocyte and oligodentrocyte in the human brain.16 The current study reports the endogenous expression of TLR3 in SH-SY5Y cells. We also showed that ligand-induced TLR3 mediated hyperphosphorylation of tau in the SH-SY5Y cell line, which highlights the involvement of TLR3, an innate immune molecule in the pathogenesis of tau hyperphosphorylation. To date, two ligands have been reported to bind with TLR3. One is dsRNA18 associated with viral infection and another is endogenous mRNA. Kariko et al. first reported that mRNA escaping from damaged tissue or contained within endocytosed cells is a potent host-derived ligand of TLR3.19 Moreover RNA sequestration to NFTs and amyloid plaques in AD and other neurodegenerative diseases has been demonstrated by Ginsberg et al.30–32 In addition, Marcinkiewicz et al.33 had shown that immature plaques and dystrophic dendrites are capable of concentrating specific mRNA. Although the mechanism(s) by which RNA become sequestered to NFTs in vivo remains unknown, in vitro evidence suggests that RNA may act as a pathological chaperone to accelerate the aggregation of tau proteins into insoluble paired helical filaments.34 However, the molecular mechanism of sequestration of this RNA and their role in the onset and progression of human neurodegenerative diseases are still not known. Given these precedents, it is reasonable to speculate that RNA released by necrotic cells or through phagocytes of necrotic cells in neurodegenerative and inflammatory process of AD could conceivably act as ligand to stimulate TLR3 signaling pathways, which may mediate hyperphosphorylation of tau and NFT formation. In our study, we used commercially available synthetic analog of dsRNA/pIpC as ligand of TLR3, considering the endogenous ligand (mRNA) would have mediated the same response. Moreover, one of the RNA groups of viruses (measles virus) causing SSPE characteristically had shown hyperphosphorylation of tau with NFT formation in association with neuronal loss, and infiltration of inflammatory cells.35–37 Ultrastructurally, these NFTs are made of PHF identical to those seen in AD. However, the reason behind this association of virus infection with NFTs formation in SSPE still remains unclear. Taken together, it can be speculated that a common phenomena might be involved to mediate hyperphosphorylation of tau and NFT formation in these two diseases, with much clinical diversity but showing identical pathological features. Our in vitro study of ligand-induced TLR-3-mediated hyperphosphorylation of tau thus helps to shed some light on the role of an innate immune receptor, which might play a common role in the pathogenesis of NFT formation in various neurodegenerative taupathies such as AD and SSPE.
The pIpC-induced TLR3-mediated signal transduction pathway has been studied by various investigators however there remains more to investigate.38,39 In brief, stimulation of TLR3 by a specific ligand induces nuclear transport of NF-kB and the activation of a set of Mitogen-activated protein kinases (MAPkinases) (extracellular signal regulated kinases (ERKs), JNK, and p38MAPK) through multiple signaling components or adaptor molecule. Since increased expression of active kinases, including JNK and p38 MAPK has been found in association with all the taupathies,40 we sought whether these two kinases were also involved in the TLR3-mediated hyperphosphorylation of tau. In our study we observed pIpC induced activation of p38 MAPK and JNK. These activations were further enhanced when TLR3 were overexpressed by LPS pretreatment, thus indicating the activation of these two kinases in the pathway of TLR3-mediated hyperphosphorylation of tau by pIpC. However, we did not check other upstream signaling components such as TRAF/TAK/MKK, which would have shown more detail of the TLR3-mediated activation of JNK/p38MAPK to mediate hyperphosphorylation of tau in SH-SY5Y cells.
In conclusion, this study showed evidence that TLR3 might be a potential mechanistic link between innate immunity and hyperphosphorylation of tau. A better understanding of how innate immunity affects hyperphosphorylation of tau and neurodegeneration will help to develop a new diagnostic and therapeutic approach.