Xin-Wen Zhou

Phosphorylated PP2A (tyrosine 307) is associated with Alzheimer neurofibrillary pathology.

Down‐regulation of protein phosphatase 2A (PP2A) is thought to play a critical role in tau hyperphosphorylation in Alzheimers disease (AD). In vitro phosphorylation of PP2A catalytic subunit at Y307 efficiently inactivates PP2A. A specific antibody against phosphorylated (p) PP2A (Y307) (PP2Ac‐Yp307) was used to investigate possible PP2A down‐regulation by known pathophysiological changes associated with AD, such as Aβ accumulation and oestrogen deficiency. Immunohistochemistry and immunofluorescence confocal microscopy showed an aberrant accumulation of PP2Ac‐Yp307 in neurons that bear pretangles or tangles in the susceptible brain regions, such as the entorhinal cortical cortex and the hippocampus. Experimentally, increased PP2Ac‐Yp307 was observed in mouse N2a neuroblastoma cells that stably express the human amyloid precursor protein with Swedish mutation (APPswe) compared with wild‐type, and in the brains of transgenic APPswe/ presenilin (PS1, A246E) mice, which corresponded to the increased tau phosphorylation. Treating N2a cells with Aβ25–35 mimicked the changes of PP2Ac‐Yp307 and tau phosphorylation in N2a APPswe cells. Knockout of oestrogen receptor (ER) α or ERβ gave similar changes of PP2Ac‐Yp307 level and tau phosphorylation in the mouse brain. Taken together, these findings suggest that increased PP2A phosphorylation (Y307) can be mediated by Aβ deposition or oestrogen deficiency in the AD brain, and consequently compromise dephosphorylation of abnormally hyperphosphorylated tau, and lead to neurofibrillary tangle formation.

P70 S6 kinase mediates tau phosphorylation and synthesis

Currently, we found that the 70‐kDa p70 S6 kinase (p70S6K) directly phosphorylates tau at S262, S214, and T212 sites in vitro. By immunoprecipitation, p‐p70S6K (T421/S424) showed a close association with p‐tau (S262 and S396/404). Zinc‐induced p70S6K activation could only upregulate translation of total S6 and tau but not global proteins in SH‐SY5Y cells. The requirement of p70S6K activation was confirmed in the SH‐SY5Y cells that overexpress wild‐type htau40. Level of p‐p70S6K (T421/S424) was only significantly correlated with p‐tau at S262, S214, and T212, but not T212/S214, in Alzheimers disease (AD) brains. These suggested that p70S6K might contribute to tau related pathologies in AD brains.

Small heat shock proteins Hsp27 or alphaB-crystallin and the protein components of neurofibrillary tangles: tau and neurofilaments.

The heat‐shock proteins (HSPs) Hsp27 and αB‐crystallin are up‐regulated in Alzheimers disease (AD), but the extent of this and the consequences are still largely unknown. The HSPs are involved in protein degradation and protection against protein aggregation, and they interact with several cytoskeletal components such as microtubules (MT) and neurofilaments (NF). AD pathology includes aggregated proteins (tau, NF), decreased protein degradation, and cytoskeletal disruption. It is thus of interest to investigate more closely the possible roles of the HSPs in AD pathology. The expressions of Hsp27 and αB‐crystallin in AD brain samples were significantly increased (by ∼20% and ∼30%, respectively) and correlated significantly with phosphorylated tau and NF proteins. To investigate the consequences of increased HSP levels on tau and NF regulation, N2a cells were transfected with Hsp27 or αB‐crystallin constructs, and overexpression of the HSPs was confirmed in the cells. Increased tau phosphorylation at the Ser262 site in the N2a cells was regulated by Hsp27 overexpression (possibly through p70S6k), whereas the overexpression of αB‐crystallin resulted in decreased levels of phosphorylated tau, NF, and GSK‐3β. It was also shown that overexpression of HSPs causes an increase in the percentage of cells present in the G1 phase. The results presented suggest that a cellular defense against dysregulated proteins, in the form of Hsp27 and αB‐crystallin, might contribute to the cell cycle reentry seen in AD cells. Furthermore, Hsp27 might also be involved in AD pathology by aggravating MT disruption by tau phosphorylation.

p70 S6 kinase and tau in Alzheimer's disease.

The 70-kDa S6 kinase (p70S6K) is a Ser/Thr (S/T)-directed kinase that plays a crucial role in cell growth, cell differentiation, and cell cycle control. This article presented evidence that supports both toxic and protective roles of p70S6K activity towards tau in Alzheimers disease (AD) brains. The p70S6K can phosphorylate tau at S262, S214, and T212 sites. Phosphorylation at these sites might release tau from microtubules, resulting in microtubule disruption. Evidence also suggests that p70S6K regulates the translation of tau mRNA by phosphorylating the 40S ribosomal protein S6. The extracellular amyloid-beta deposition in AD brains could be a causative factor that activates p70S6K. We hypothesized that amyloid-beta deposition activates p70S6K whose anti-apoptotic property subsequently keeps neurons from entering into the apoptotic process. This process provides the opportunity for the newly synthesized tau to be phosphorylated by p70S6K and by other tau kinases. This hyperphosphorylated tau then aggregates and is progressively deposited in neurons.

PTEN levels in Alzheimer's disease medial temporal cortex

Phosphatase and tensin homologue deleted from chromosome 10 (PTEN) is a dual (protein tyrosine and lipid) phosphatase one of the functions of which is to dephosphorylate phosphatidylinositol 3,4,5-trisphosphate to phosphatidylinositol-3,4-biphosphate thereby inhibiting phosphoinositide-dependent kinase activation of the cell survival kinase Akt. Akt activity is up regulated in Alzheimers disease (AD) brain in parallel to the progression of neurofibrillary pathology. The present study determined whether altered expression of PTEN occurs in Alzheimers disease brain. Western immunoblotting revealed no significant changes of PTEN protein levels in nuclear and membrane fractions of medial temporal cortex from a series of Alzheimers disease and control cases. Similarly, no changes in PTEN protein levels, as determined by dot-blotting, were seen in temporal cortex homogenates from a separate series of Alzheimers disease and control brains. A small but significant decrease in the levels of Ser(380) p-PTEN was seen in homogenates of Alzheimers disease temporal cortex. Immunohistochemistry revealed PTEN immunoreactivity in a number of brain structures including neurons, capillaries and structures resembling oligodendrocytes and astrocytes. The majority of temporal cortex pyramidal neurons (93-100%) were PTEN immunopositive. The Alzheimers disease cases had significantly lower numbers of total ( approximately 12% loss, P<0.02) and PTEN immunopositive ( approximately 15% loss, P<0.01) pyramidal neurons as compared to the control cases.

Assessments of the accumulation severities of amyloid β-protein and hyperphosphorylated tau in the medial temporal cortex of control and Alzheimer's brains

Alzheimers disease (AD) is characterized neuropathologically by neuritic plaques (NPs), and neurofibrillary tangles (NFTs). So far, the following key issues are not yet answered to the disease: (1) the accumulation degrees of three Abeta variants, and tau phosphorylation epitopes in AD as compared to control; (2) the correlation degrees of levels of three Abeta variants with different tau phosphorylation epitopes; (3) the correlation degrees of levels of three Abeta variants and different tau phosphorylation epitopes with Braak and CERAD staging systems. To address these issues, levels of Abeta40, Abeta42, and Abeta43, and phosphorylated tau were assessed by dot blots in homogenates of the medial temporal cortex from AD and control brains in the present study. These data implied different roles of tau phosphorylation epitopes in formation of NFTs, and in this process, Abeta might play a key role. Assessments of levels of these abnormal proteins by dot blots may serve as a useful complement to the morphological evaluations in diagnosis of AD.

Interactions Between Glycogen Synthase Kinase 3β, Protein Kinase B, and Protein Phosphatase 2A in Tau Phosphorylation in Mouse N2a Neuroblastoma Cells

In this study, we investigated how tau phosphorylation is regulated by protein kinase glycogen synthase kinase 3beta (GSK3 beta), protein kinase B (PKB), and protein phosphatase 2A (PP2A) in mouse N2a neuroblastoma cells. Results showed that GSK3 beta overexpression significantly increased PKB phosphorylation at the S473 site but not the T308 site. Neither GSK3 beta nor PKB overexpression could reduce the PP2AC phosphorylation at the Y307 site. In contrast, either PKB or GSK3 beta knockdown could increase PP2A phosphorylation at the Y307 site. PP2AC knockdown increased GSK3 beta phosphorylation at the S9 site but not at the Y216 site, and PKB phosphorylation at the T308 site but not at the S473 site. Tau phosphorylation at the S396 site was increased by GSK3 beta or PKB overexpression. Tau phosphorylation at the S214 site was only induced by PKB overexpression in the study. While GSK3 beta knockdown decreased tau phosphorylation at the S396 site, PKB knockdown increased tau phosphorylation at both the S396 and S214 sites. PP2AC knockdown decreased tau phosphorylation at the S396 and S214 sites. These findings suggest that tau phosphorylation at the S396 and S214 sites is differentially regulated by GSK3 beta, PKB, and PP2A in N2a cells. The final phosphorylation state of tau is possibly caused by the synergic action of the three enzymes.

Parallel increase in p70 kinase activation and tau phosphorylation (S262) with Aβ overproduction

This study set out to search for a link between overproduction of Aβ and p70S6 kinase (p70S6K) phosphorylation/activation. Results showed that levels of p‐p70S6K at T421/S424 and T389 are significantly increased in mouse N2a neuroblastoma cells carrying human APP with Swedish mutation (APPswe), and in transgenic APPswe/PS1 (A246E) mice as compared with respective controls, corresponding to the increase of tau phosphorylation at S262. This parallel increase in p70S6K activation and tau phosphorylation could be demonstrated by treating wild‐type N2a cells with Aβ25–35. Our results suggest that the Aβ deposition in senile plaques in Alzheimer disease brains might be a primary event that activates p70S6K and phosphorylates tau at S262, resulting in microtubule disruption.