Alzheimer's disease (AD) is characterized neuropathologically by intracellular neurofibrillary tangles (NFTs) formed of taubased paired helical filaments (PHFs) and extracellular b-amyloid plaques. The degree of Alzheimer dementia correlates with the severity of PHFs and NFTs. As an intraneuronal accumulation of oxidatively damaged proteins has been found in the brains of patients with AD, a dysfunction of the proteasomal system, which degrades damaged proteins, has been assumed to cause protein aggregation and therefore neurodegeneration in AD. In this study, we revealed that such proteasome dysfunction in AD brain results from the inhibitory binding of PHF-tau to proteasomes. We analysed the proteasome activity in brains from patients with AD and agematched controls, and observed a significant decrease to 56% of the control level in the straight gyrus of patients with AD. This loss of activity was not associated with a decrease in the proteasome protein. PHF-tau co-precipitated during proteasome immunoprecipitation and proteasome subunits could be co-isolated during isolation of PHFs from AD brain. Furthermore, the proteasome activity in human brains strongly correlated with the amount of co-precipitated PHF-tau during immunoprecipitation of proteasome. Incubation of isolated proteasomes with PHF-tau isolated from AD brain, and with PHFs after in vitro assembly from human recombinant tau protein, resulted in a distinct inhibition of proteasome activity by PHF-tau. As this inhibition of proteasome activity was sufficient to induce neuronal degeneration and death, we suggest that PHF-tau is able directly to induce neuronal damage in the AD brain.

Hyperphosphorylated tau proteins accumulate in the paired helical filaments of neurofibrillary tangles seen in such tauopathies as Alzheimer's disease. In the present paper we show that tau turnover is dependent on degradation by the proteasome (inhibited by MG132) in HT22 neuronal cells. Recombinant human tau was rapidly degraded by the 20 S proteasome in vitro, but tau phosphorylation by GSK3beta (glycogen synthase kinase 3beta) significantly inhibited proteolysis. Tau phosphorylation was increased in HT22 cells by OA [okadaic acid; which inhibits PP (protein phosphatase) 1 and PP2A] or CsA [cyclosporin A; which inhibits PP2B (calcineurin)], and in PC12 cells by induction of a tet-off dependent RCAN1 transgene (which also inhibits PP2B). Inhibition of PP1/PP2A by OA was the most effective of these treatments, and tau hyperphosphorylation induced by OA almost completely blocked tau degradation in HT22 cells (and in cell lysates to which purified proteasome was added) even though proteasome activity actually increased. Many tauopathies involve both tau hyperphosphorylation and the oxidative stress of chronic inflammation. We tested the effects of both cellular oxidative stress, and direct tau oxidative modification in vitro, on tau proteolysis. In HT22 cells, oxidative stress alone caused no increase in tau phosphorylation, but did subtly change the pattern of tau phosphorylation. Tau was actually less susceptible to direct oxidative modification than most cell proteins, and oxidized tau was degraded no better than untreated tau. The combination of oxidative stress plus OA treatment caused extensive tau phosphorylation and significant inhibition of tau degradation. HT22 cells transfected with tau-CFP (cyan fluorescent protein)/tau-GFP (green fluorescent protein) constructs exhibited significant toxicity following tau hyperphosphorylation and oxidative stress, with loss of fibrillar tau structure throughout the cytoplasm. We suggest that the combination of tau phosphorylation and tau oxidation, which also occurs in tauopathies, may be directly responsible for the accumulation of tau aggregates.

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