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Keywords:

  • Alzheimer’s;
  • disease;
  • hyperphosphorylation of tau and toll-like receptor 3;
  • innate immunity

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHOD
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES

Abstract  Neurofibrillary tangles of abnormally phosphorylated tau are one of the characteristic pathological hallmarks of Alzheimer's disease (AD). In addition, immunological and inflammatory changes including complements and activated microglia are also common phenomena in AD. However, these pathological changes are yet to be interlinked in a common explainable background. In this study, the relevant mechanism of phosphorylation of tau protein and an innate immune signal transduction system were investigated. Toll-like receptor 3 (TLR3) is a receptor working in the innate immune system and its expression in the brain has already been reported. Total RNA was isolated from SH-SY5Y cells and reverse transcriptase polymerase chain reaction was done to see endogenous expression of TLR3 in SH-SY5Y cells that was further confirmed at protein level by Western blot analysis. Cells were treated with 50 µg/mL of polyinosinic–polycytidylic acid (pIpC), a synthetic analog of dsRNA and the changes of phosphorylation of tau protein were investigated. Further the level of phosphorylation of tau protein was investigated after the cells had been previously treated with 10 ng/mL of lipopolysaccharide (LPS) for 6 h to induce over-expression of TLR3. Increased phosphorylation of tau protein at PHF-1 site (Ser396/404), activation of Jun N-terminal kinase and p38 MAPK were observed in cells treated with pIpC. These effects were enhanced when cells were pretreated with LPS, a known transducer of TLR3. These data suggest that toll-like receptor 3, an innate immune molecule, might be a potential link to mediate hyperphosphorylation of tau in neurodegenerative processes of AD.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHOD
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES

Neurofibrillary tangles (NFTs) are pathological hallmarks of Alzheimer's disease (AD) and abnormally hyperphosphorylated tau is the major protein component of NFTs.1–4 In addition, activation of immune cells such as activated microglia and accumulation of inflammation-associated proteins, including various cytokines and complements, are also a common phenomena associated with AD.5–7 The etiopathogenesis of hyperphosphorylation of tau has been studied for ages by number of investigators. Numerous protein kinases and protein phosphatases have been implicated in the pathogenesis of aberrant phosphorylation of tau protein in the AD brain.8–12 A recent study has suggested hyperphosphorylation of tau in cortical neurons to be mediated by interleukin-1 of activated microglia through p38-MAPK pathway thus linking activation of innate immune cells of CNS and hyperphosphorylation of tau.13

The evolutionarily ancient innate immune system provides the first line of host defense against a large variety of pathogens, tissue insults and also controls many aspects of the adaptive immune response.14 Cells of the innate immune system recognize invariant pathogens associated with molecular patterns through a series of genetically conserved and stable cell surface receptors related to the Drosophila gene toll that are therefore referred to as toll- like receptors (TLR).15 Broad expression of various TLRs (10 in number) has already been reported in the human brain.16 Moreover activation of innate immunity in CNS is found to trigger neurodegeneration through a toll-like receptor 4-dependant pathway.17

We focused on TLR3, which responds to its two known ligands: double stranded RNA (dsRNA, replication intermediary for many viruses)18 and endogenous mRNA (released from or associated with necrotic cells).19 Upon binding with its ligand, TLR3 activates a variety of signaling pathways including activation of p38 MAP kinase and Jun N-terminal kinase (JNK).18–20 Moreover subsclerosing pan encephalitis (SSPE), one of the known taupathies caused by the measles virus (RNA virus) has shown the evidence of neuronal loss and infiltration of inflammatory cells along with formation of NFTs.21 Taken together, we hypothesized whether the ligand-mediated activation of TLR3 can induce hyperphosphorylation of tau through the activation of MAP kinases or not.

In the present study we evaluated the expression level of TLR3 in human SH-SY5Y neuroblastoma cell line and determined ligand-induced activation of TLR3 to mediate hyperphosphorylation of tau.

MATERIALS AND METHOD

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHOD
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES

Reagents

Polyinosinic–polycytidylic acid (pIpC) and lipopolysaccharide (LPS) from Escherichia coli 055:B5 were purchased from Sigma-Aldrich, Tokyo, Japan. PHF-1 antibody that can detect phosphorylated tau at Ser396/404 was a gift from Dr P. Davies (Albert Einstein University, New York, NY, USA). Anti-JNK/SAPK, anti phospho JNK/SAPK (Thr183/Tyr185), antip38/MAPK and antiphospho p38/MAPK (Thr180/Tyr182) were purchased from Cell Signaling Technology (MA, USA). Anti-TLR3 (rabbit polyclonal igG), Anti-TLR3 (mouse monoclonal igG), were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA and CALBIOCHEM, San Diego, CA, USA, respectively. Antibodies against C-terminal and N-terminal of human TLR3 were purchased from ABGENT, San Diego, CA, USA.

Cell line and culture condition

SH-SY5Y human neuroblastoma cells were cultured in DMEM/F12 (Invitrogen, Carlsbad, CA, USA) supplemented with 5% fetal calf-serum (FCS) and were maintained at 37°C in a humid atmosphere containing 95% air/5% CO2.

Cell treatment

Cells were treated with 50 µg/mL of pIpC for a different duration of time with or without the LPS (10 µg/mL) pretreatment for 6 h.

Rt-pcr

Total RNA was extracted from cultured cells using RNeasy® kit (Qiagen, Tokyo, Japan) according to the manufacturer's instruction. Total RNA (4 µg) was reversed transcribed using oligo (dT) primers with Thermo ScriptTM RT-PCR system (Invitrogen). To compare the mRNA levels among different RNA samples, RT was performed simultaneously using the reagents from a single master mix. Transcribed cDNA was used as a template for PCR amplification in a 50 µL reaction volume with Platinum® Taq DNA Polymerase (Invitrogen) for 35 cycles at 95°C for 45 s, 62°C for 40 s and 72°C for 1 min followed by a final extension for 10 min at 72°C. GAPDH was used as an internal control. PCR products were visualized on a 2% agarose gel by ethidium bromide staining. The TLR3 and GAPDH cDNA were amplified with the following primers: 5-TCCGTTGAGAAGAAGGTTTTCGGG-3 and 5-ATATCCTCCAGCCCTCCAAGTGGA-3 for TLR3, 5-CACAGTCCATGCCATCACTG-3 and 5-TACTCCTTGGAGGCCATGTG-3 for GAPDH.

Western blot

Cells were lyzed in 100 mM PIPES pH 6.8, 2 mM MgCl2, 0.1 mM EDTA, 1 mM PMSF, 5 µg/mL aprotinine, 5 µg/mL leupeptine, 25 mM NaF, 1 mM Na3 VO4, 0.1% Triton-X100 on ice, and centrifuged at 200 000 × g for 30 min.

Supernatants were employed for Western blot analysis. Aliquots (50 µg) of the supernatants were separated by SDS-PAGE and transferred to PVDF membrane. As secondary antibodies, peroxides labeled antirabbit IgG were used and the membranes were developed by ECL (Amersham Bioscience, Buckinghamshire, UK).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHOD
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES

We observed endogenous expression of TLR3 mRNA in SH-SY5Y cells by means of RT-PCR (Fig. 1a). Furthermore, expression of TLR3 at the protein level was confirmed by Western blot analysis (Fig. 1b). Lipopolysaccharide (LPS), which is known to induce over-expression of TLR3 in many other tissues18 was also found to upregulate TLR3 expression in SH-SY5Y cells (Fig. 1c). As is shown, LPS pretreatment for 6 h markedly increases the expression of TLR3 by pIpC at 0 h (Fig. 1c, lane 3) compared to pIpC treatment only (Fig. 1c, lane 1).

image

Figure 1. (A) Endogenous expression of TLR3 mRNA in SH-SY5Y cell line as evidenced by RT-PCR. Total RNA isolated from cultured SH-SY5Y cells were subjected to RT-PCR with specific primer for TLR3. (B) Endogenous expression of TLR3 protein in SH-SY5Y cell line as evidenced by Western blot analysis. (C) LPS induced over-expression of endogenous TLR3 in SH-SY5Y cells. Cells either pretreated with 10 ng/mL of LPS or without pretreatment (change of medium only) for 6 h, were treated with pIpC 50 µg/mL for the indicated number of hours. The level of expression of TLR3 protein was observed by Western blot using anti-TLR3 antibody. Data are representative of at least three separate experiments.

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We then sought to elucidate whether LPS pretreated over-expressed TLR3 mediated enhanced activation of p38-MAPK and JNK by pIpC (Fig. 2). Only pIpC treatment increased activation of p38-MAPK (Fig. 2, upper panel lanes 1–3) and JNK (Fig. 2, lower panel, lanes 1–3) progressively at 1 h and 2 h. We used Phospho p38 and Phospho JNK antibody to detect activation of p38 MAPK and JNK, respectively. Moreover this activation was further enhanced when LPS pretreatment was done for 6 h followed by pIpC treatment (lanes 4–6). However, total p38-MAPK (Fig. 2, lanes 7, 8) and total JNK (Fig. 2, lanes 7, 8) did not show any difference even after LPS pretreatment.

image

Figure 2. LPS-induced TLR3-mediated enhanced activation of p38-MAPK and JNK by pIpC. Cells either pretreated with 10 ng/mL of LPS or without pretreatment (change of medium only) for 6 h were treated with pIpC 50 µg/mL for the indicated number of hours. The level of phosphorylation of p38-MAPK was observed and compared with unphosphorylated total-p38 MAPK (upper panel) by Western blot analysis. Further level of phosphorylation of JNK was observed and compared with unphosphorylated total-JNK by Western blot analysis (lower panel). Data are representative of at least three separate experiments.

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Activation of p38 MAPK and JNK has been associated with stress response and more specifically with hyperphosphorylation of tau in AD. Since an elevation of phosphorylated p38 MAPK and phosphorylated JNK were observed in our experiment through pIpC-induced activation of TLR3 (Fig. 2), we tested whether activation of these kinases participate in the TLR3-mediated hyperphosphorylation of tau or not. Cells were either pretreated with LPS for 6 h to induce over-expression of TLR3 or with the change of medium followed by pIpC treatment. Hyperphosphorylation of tau was detected employing the PHF-1 antibody, which specifically detects ser396/404 phospho epitope of tau protein. We could see pIpC-induced TLR3-mediated hyperphosphorylation of tau (Fig. 3, lanes 1–2) which was further enhanced by LPS pretreatment (Fig. 3, lanes 3–4), indicating that ligand (pIpC)-mediated activation of TLR3 can mediate hyperphosphorylation of tau (Fig. 3).

image

Figure 3. LPS-induced TLR3-mediated enhanced phosphorylation of tau by pIpC. Cells either pretreated with 10 ng/mL of LPS or without pretreatment (change of medium only) for 6 h, were treated with pIpC 50 µg/mL for the indicated number of hours. The level of phosphorylation of tau at ser396/404 was observed using PHF-1 antibody by Western blot analysis. Data are representative of at least three separate experiments.

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The pIpC-induced TLR3-mediated signaling pathways was studied mainly in peripheral macrophages and had shown activation of TRAF-6 (TNF receptor-activated factor 6) through multiple adaptor molecules. When TRAF-6 is activated, it transfers the downstream signal to TAK-1 (TGF β activated kinase 1). Activated TAK-1 then leads to phosphorylation and activation of MAPKKs (MAP kinase kinases) family of MKK3/6 and MKK4, which in turn activate the p38 and JNK pathway, respectively. Thus, on the basis of the known signaling pathway of TLR3 in peripheral macrophages, we speculated the same loop of pathway to be working in human SH-SY5Y cell line to activate JNK and p38 MAPK to mediate hyperphosphorylation of tau (Fig. 4).

image

Figure 4. Schematic demonstration of intracellular signal transduction pathway of toll-like receptor-3 to mediated hyperphosphorylation of tau. Upon binding of dsRNA (ligand) to Trans membrane receptor, TLR3, multiple adaptor molecule are recruited to the receptor to form a complex to activate TRAF-6 (TNF receptor activated factor 6). Activated TRAF-6 transfers the signal to TAK-1 (TGF β activated kinase 1). Thus TAK-1 can activate MAPKKs (MAP kinase kinase), family of MKK3/6 and MKK4, which in turn activate the p38 and JNK pathway, respectively.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHOD
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES

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.

ACKNOWLEDGMENT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHOD
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES

This work was supported by grant 16591136 from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHOD
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES
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