Identification of the Sites of Tau Hyperphosphorylation and Activation of Tau Kinases in Synucleinopathies and Alzheimer’s Diseases

Abstract

ObjectiveMost neurodegenerative diseases contain hyperphosphorylated Tau [p-Tau]. We examined for the first time epitopes at which Tau is hyperphosphorylated in Parkinson’s disease, dementia with Lewy bodies and Alzheimer’s disease, and also select Tau kinases.MethodsPostmortem frontal cortex from Parkinson’s disease, dementia with Lewy bodies, Alzheimer’s disease and striata from Parkinson’s disease, were analyzed by immunoblots using commercially available antibodies against 20 different phospho-epitopes of Tau. Major Tau kinases were also screened. Results in diseased tissues were compared to nondiseased controls.ResultsIn Alzheimer’s disease, Tau was hyperphosphorylated at all the 20 epitopes of p-Tau. In dementia with Lewy bodies, p-Tau formation occurred at 6 sites sharing 30% overlap with Alzheimer’s disease, while in Parkinson’s frontal cortex, an area which does not degenerate, Tau hyperphosphorylation was seen at just 3 epitopes, indicating 15% overlap with Alzheimer’s disease. In Parkinson’s disease striatum, an area which undergoes considerable neurodegeneration, Tau was hyperphosphorylated at 10 epitopes, sharing 50% overlap with Alzheimer’s disease. Between frontal cortex of Parkinson’s disease and dementia with Lewy bodies, there were only two p-Tau epitopes in common. In striata of Parkinson’s disease, there were 3 clusters of Tau hyperphosphorylated at 3 contiguous sites, while two such clusters were detected in dementia with Lewy bodies; such clusters disrupt axonal transport of mitochondria, cause microtubule remodeling and result in cell death. p-GSK-3β, a major Tau kinase, was activated in all brain regions examined, except in dementia with Lewy bodies. Activation of other Tau kinases was seen in all brain regions, with no clear pattern of activation.InterpretationOur studies suggest that the three neurodegenerative diseases each have a signature-specific profile of p-Tau formation which may be useful in understanding the genesis of the diseases and for the development of a panel of specific biomarkers.

GSK-3β dysregulation contributes to parkinson’s-like pathophysiology with associated region-specific phosphorylation and accumulation of tau and α-synuclein

“…As expected, elevated levels of α-Syn immunostaining were also seen in LBs of PD SN neurons ( Figure 2i, lower panel). Although we have analyzed total GSK-3β and p-GSK-3β-S9 in brains of PD patients by immunoblotting, 12,13 this is the first study to provide direct immunopositive evidence of increased p-GSK-3β-Y216 expression in DA-neurons of SN from PD patients.…”

“…In postmortem PD striatum, we have recently examined 20 different epitopes of p-Tau and found hyperphosphorylation to occur at 10 sites. 12 We, therefore, examined the pattern of Tau hyperphosphorylation by immunoblotting at these 10 epitopes in midbrain (Figures 3a and b), striatum (Figures 3c and d), and frontal cortex (Figures 3e and f) of 15-month-old TG and control mice. In midbrain, significant hyperphosphorylation was seen at 8 of the 10 epitopes examined (T205, T212, S235, S262, S356, S396/404, S409, and S422).…”

“…Tissues were prepared as previously described. 12,19,20 See supplementary material and methods for further details.…”

“…Western blot analyses of mouse tissue homogenates were performed as previously described. 12 See supplementary material and methods for further details.…”

“…12,13,16,19,20 In in vitro studies of MPTPtreated SH-SY5Y cells, blockade of GSK-3β with lithium, or with the highly selective non-ATP competitive inhibitor, TDZD--8, prevented the induction of p-GSK-3β-Y216, abolished p-Tau formation, and reversed the accumulation and aggregation of both p-Tau and α-Syn, averting cell death. 16 Other studies using Rotenone or MPTP/MPP+ in chemical PD models, have shown similar results of decreased neuronal viability during treatments accompanied by dose-and timedependent increases in GSK-3β activation, with decreased cytotoxicity detected when GSK-3β was inhibited or knockeddown through the use of GSK-3β-specific small molecule inhibitors or through RNAi.…”