Nakamoto, Yasunari

AbstractBackgroundPathological hallmarks of Alzheimer's disease (AD) are senile plaques (SPs) and neurofibrillary tangles (NFTs), which are formed from highly phosphorylated tau. Lithium chloride (LiCl) has been shown to inhibit phosphorylated tau by inhibiting glycogen synthase kinase‐3β (GSK‐3β). In addition, autophagy lysosome system (Hamano et al., Eur J Neurosci 27; 1119, 2008) and proteasome (J Neurochem 2000) are known to play an important role as tau degradation pathways. We investigated the decrease of phosphorylated tau by LiCl by using cell culture model expressing wild‐type tau via TetOff induction system.MethodsTen to 50mM of LiCl treatment (24 hours) was performed to neuroblastoma cells (M1C) expressing wild‐type tau protein (4R0N) via TetOff induction. Total and phosphorylated tau (PHF‐1) levels and autophagy activation were examined by Western blot (WB) analysis.ResultsReduction of total tau (Tau5) and phosphorylated tau (PHF‐1, and CP13) with LiCl treatment was detected by WB analysis. C‐terminal truncated tau species detected by TauC3 was decreased by LiCl, too. In addition, increase of LC3Ⅱ/I, and decrease of P62 suggesting autophagy activation was observed. Major Tau kinase GSK3β was inactivated by LiCl.ConclusionsThese results imply that LiCl may decrease the phosphorylated tau through suppression of tau phosphokinase and activation of autophagy.

The neuropathological hallmarks of Alzheimer’s disease (AD) are senile plaques (SPs), which are composed of amyloid β protein (Aβ), and neurofibrillary tangles (NFTs), which consist of highly phosphorylated tau protein. As bio-metal imbalance may be involved in the formation of NFT and SPs, metal regulation may be a direction for AD treatment. Clioquinol (CQ) is a metal-protein attenuating compound with mild chelating effects for Zn2+ and Cu2+, and CQ can not only detach metals from SPs, but also decrease amyloid aggregation in the brain. Previous studies suggested that Cu2+ induces the hyperphosphorylation of tau. However, the effects of CQ on tau were not fully explored. To examine the effects of CQ on tau metabolism, we used a human neuroblastoma cell line, M1C cells, which express wild-type tau protein (4R0N) via tetracycline-off (TetOff) induction. In a morphological study and ATP assay, up to 10 μM CQ had no effect on cell viability; however, 100 μM CQ had cytotoxic effects. CQ decreased accumulation of Cu+ in the M1C cells (39.4% of the control), and both total and phosphorylated tau protein. It also decreased the activity of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) (37.3% and 60.7% levels of the control, respectively), which are tau kinases. Of note, activation of protein phosphatase 2A (PP2A), which is a tau phosphatase, was also observed after CQ treatment. Fractionation experiments demonstrated a reduction of oligomeric tau in the tris insoluble, sarkosyl soluble fraction by CQ treatment. CQ also decreased caspase-cleaved tau, which accelerated the aggregation of tau protein. CQ activated autophagy and proteasome pathways, which are considered important for the degradation of tau protein. Although further studies are needed to elucidate the mechanisms responsible for the effects of CQ on tau, CQ may shed light on possible AD therapeutics.

Neurofibrillary tangles, which consist of highly phosphorylated tau protein, and senile plaques (SPs) are pathological hallmarks of Alzheimer’s disease (AD). In swollen axons, many autophagic vacuoles are observed around SP in the AD brain. This suggests that autophagy function is disturbed in AD. We used a neuronal cellular model of tauopathy (M1C cells), which harbors wild type tau (4R0N), to assess the effects of the lysosomotrophic agent NH4Cl, and autophagy inhibitors chloroquine and 3 methyladenine (3MA). It was found that chloroquine, NH4Cl and 3MA markedly increased tau accumulation. Thus, autophagy lysosomal system disturbances disturbed the degradation mechanisms of tau protein. Other studies also revealed that tau protein, including aggregated tau, is degraded via the autophagy lysosome system. Phosphorylated and C terminal truncated tau were also reported to disturb autophagy function. As a therapeutic strategy, autophagy upregulation was suggested. Thus far, as autophagy modulators, rapamycin, mTOCR1 inhibitor and its analogues, lithium, metformin, clonidine, curcumin, nicotinamide, bexaroten, and torehalose have been proposed. As a therapeutic strategy, autophagic modulation may be the next target of AD therapeutics.

AbstractBackgroundThe pathological features of AD are neurofibrillary tangles (NFTs) composed of highly phosphorylated tau and senile plaques (SPs) composed of amyloid β protein (Aβ). It was suggested that Cu2 + induced hyperphosphorylation of tau and Zn2 + deposits on SPs. Clioquinol (CQ) has a mild chelating effect on Zn2 + and Cu2 +. In addition, CQ has been shown to reduce Aβ aggregation by removing Zn2+ from SPs. However, the effect of CQ on tau aggregation is not fully understood. The purpose of our study is to examine the effect of CQ on tau metabolism by using a cell culture model of tauopathy.MethodsHuman neuroblastoma cell line M1C cells that express wild‐type tau protein (4R0N) via tetracycline (Tet) off induction was used. M1C cells were exposed to 0.1‐10 μM CQ on day 4 of Tet‐Off induction, and harvested at the end of Day 5. Total tau detected by Tau5, and phosphorylated tau detected by PS199/202, AT180, AT270, CP13, and PHF‐1 antibodies, were examined using Western blot analysis. Immunocytochemical studies, fractional analysis and immunoprecipitation of ubiquitinated tau were also performed. Tau kinases including c‐Jun N‐terminal kinase (JNK), and P38 MAPK and tau phosphatase, protein phosphatase 2A (PP2A) were also examined. C‐terminal truncated pathological tau was detected by using the antibody TauC3. In addition, autophagy and proteasome activity were also examined.ResultPhosphorylated tau levels were reduced by 1‐10 μM CQ treatment. Tau kinases, JNK and p38 MAPK were inactivated by CQ treatment. Interestingly, activation of the tau phosphatase PP2A was also observed with CQ treatment. Fractionation studies showed that CQ treatment reduced AT180 and AT270 positive high molecular weight tau in the Tris‐insoluble, sarkosyl‐soluble fraction. CQ also reduced tau oligomer specific antibody TOC1‐positive oligomeric tau. CQ reduced caspase‐cleaved tau detected by antibody TauC3. P62 levels were reduced by CQ treatment, suggesting that CQ activates autophagy. In addition, CQ slightly reduced high molecular weight ubiquitinated tau. This implies activation of the ubiquitin‐proteasome by CQ.ConclusionCQ could provide beneficial effects against pathogenic forms of tau via multiple mechanisms and may represent a viable therapeutic approach for AD and other tauopathies.

AbstractBackgroundNeurofibrillary tangle (NFT), composed of highly phosphorylated tau is one of the pathological hallmarks of Alzheimer’s disease (AD). Spleen tyrosine kinase (Syk) is non receptor type tyrosine kinase, and has various physiological functions, including histamine release in mast cells, phagocytosis in macrophage, and B cell differentiation. The possibility of Syk inhibitor for clinical application was proposed in allergic rhinitis, rheumatoid arthritis, and immunologic thrombocytopenia. The possible relationship between Syk and AD has been reported recently. We have examined the effects of Syk specific inhibitor BAY61‐3606 on phosphorylation levels of tau protein and oligomeric tau by using cell culture model of tauopathy, M1C cells.MethodsWe utilized human neuroblastoma cell line M1C cells, which expresses wild type tau protein (4R0N) via tetracycline off induction (Tet Off induction). Reduction of phosphorylated tau protein and oligomeric tau protein by Syk inhibitor was examined by using various phospho‐tau antibodies, and tau oligomer specific antibody. Influences of activity of autophagy by Syk inhibitor was also examined by western blot analysis.ResultsIn this cell line, 72kDa of Syk protein was identified by Western blot analysis. Phosphorylated tau detected by various phospho‐tau antibodies, including PHF‐1, CP13, and AT270 was decreased by 1, 10 μM of Syk inhibitor. One of the patholoigical form of tau, caspase cleaved tau detected by TauC3 was also decreased by Syk inhibitor. Oligomeric tau, which detected by tau oligomer specific antibody, TOC1 was also decreased by Syk inhibitor. Syk inhibitor also inactivated glycogen synthase kinase 3β (GSK3β). Syk inhibitor upregulated autophagy detected by increase of LC3‐II, and reduction of P62. In summary, Syk inhibitor decreased phosphorylated tau as well as total tau by upregulating autophagy, and inactivating tau kinases, including GSK3β. Finally, oligomeric tau was reduced by Syk inhibitor.ConclusionSyk inhibitor may have promising for the treatment of tauopathy, including AD.

Increased plasma homocysteinemia is considered a risk factor of dementia, including Alzheimer’s disease (AD) and vascular dementia. However, the reason elevated plasma homocysteinemia increases the risk of dementia remains unknown. A pathological hallmark of AD is neurofibrillary tangles (NFTs) that consist of pathologically phosphorylated tau proteins. The effect of homocysteine (Hcy) on tau aggregation was explored using human neuroblastoma M1C cells that constitutively express human wild-type tau (4R0N) under the control of a tetracycline off system, primary mouse cultured neurons, and by inducing hyperhomocysteinemia in a mouse model of tauopathy (HHCy mice). A wide range of Hcy concentrations (10–1000 µM) increased total tau and phosphorylated tau protein levels. Hcy activated glycogen synthase kinase 3, and cyclin dependent kinase 5, major tau phosphokinases, and inactivated protein phosphatase 2A, a main tau phosphatase. Hcy exhibited cytotoxic effects associated with enhanced activation of caspase. Truncation of tau in the C-terminus, the cleavage site of caspase 3 (i.e., D421, detected by the TauC3 antibody) was also increased. Total tau, phosphorylated tau, as well as C-terminal cleaved tau were increased in the sarkosyl insoluble tau fraction. Hcy also increased the level of tau oligomers, as indicated by the tau oligomer complex 1 (TOC1) antibody that specifically identifies oligomeric tau species, in the tris insoluble, sarkosyl soluble fraction. The levels of TOC1-positive oligomeric tau were increased in brain lysates from HHCy mice, and treating HHCy mice with S-adenosylmethionine, an intermediate of Hcy, reduced the levels of oligomeric tau to control levels. These observations suggest that Hcy increases the levels of phosphorylated tau as well as truncated tau species via caspase 3 activation, and enhanced tau oligomerization and aggregation.