Abstract
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.
Highlights
We found that Hcy caused tau phosphorylation via activating glycogen synthase kinase 3β (GSK3β) and inactivating phosphatase 2A (PP2A), tau oligomerization, and tau cleavage via caspase 3 activation, and all of these modifications were associated with cell toxicity
Each blot was reprobed with anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody to confirm the same loading across the lanes (Figure 2A)
In cultures treated with 100 μM Hcy, the 45–62 kDa bands detected by Tau5 were increased by 157 ± 45% compared with the control
Summary
To test whether Hcy affects tau metabolism, tau expression was induced in M1C cells (by lowering tetracycline from 2 g/mL to 1 ng/mL) for 5 days, and on the 5th day, cells were exposed to L-Hcy (1, 10, 100 or 1000 μM) for 24 h. The elevation of Hcy is usually mild (>14 M) compared with in our cell-based studies (100 M). The supraphysiological concentration of Hcy was comparable with that in other in vitro studies Cell lysates were analysed by western blotting using Tau. Hcy treatment increased total tau protein (Tau5) in a dose-dependent fashion (Figure 2A,B). In cultures treated with 100 μM Hcy, the 45–62 kDa bands detected by Tau were increased by 157 ± 45% compared with the control. To establish that the increase in tau was not caused by the increase in tau mRNA expression, mRNA levels were examined. Hcy did not change tau mRNA levels as quantitated by qPCR (Figure 2C)
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