Cecilia Bjorkdahl

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.

Mechanism of zinc-induced phosphorylation of p70 S6 kinase and glycogen synthase kinase 3β in SH-SY5Y neuroblastoma cells

We have previously reported an aberrant accumulation of activated protein kinase B (PKB), glycogen synthase kinase (GSK)‐3β, extracellular signal‐regulated kinase (ERK1/2), c‐Jun N‐terminal kinase (JNK), p38 and p70 S6 kinase (p70S6K) in neurons bearing neurofibrillary tangles (NFTs) in Alzheimers disease (AD). However, the mechanism by which these tau candidate kinases are involved in the regulation of p70S6K and GSK‐3β phosphorylation is unknown. In the current study, 100 μm zinc sulfate was used, and influences of various components of phosphatidylinositol 3‐kinase (PI3K) and mitogen‐activated protein kinase (MAPK) pathways on p70S6K and GSK‐3β phosphorylation have been investigated in serum‐deprived SH‐SY5Y neuroblastoma cells. We found that zinc could induce an increase of phosphorylated (p) p70S6K, p‐PKB, p‐GSK‐3β, p‐ERK1/2, p‐JNK and p‐p38, especially in long‐term treatment (4–8 h). Treatment with different inhibitors including rapamycin, wortmannin, LY294002, and U0126, and their combinations, indicated that phosphorylation of p70S6K and GSK‐3β is regulated by rapamycin‐dependent, PI3K and MAPK pathways. Furthermore, phosphorylation of p70S6K and GSK‐3β affected levels of tau unphosphorylated at the Tau‐1 site and phosphorylated at the PHF‐1 site, and p70S6K phosphorylation affected the total tau level. Thus, 100 μm zinc might activate PKB, GSK‐3β, ERK1/2, JNK, p38 and p70S6K, that are consequently involved in tau changes in SH‐SY5Y cells.

Tau hyperphosphorylation correlates with reduced methylation of protein phosphatase 2A.

The down-regulation of protein phosphatase 2A (PP2A) activity is thought to play an important role in the formation of tau hyperphosphorylation in the Alzheimers disease (AD) brain. Methylation of the PP2A catalytic subunit at the L309 site can potently activate PP2A for some substrates via the increasing recruitment of its regulatory subunits into the holoenzyme. Abeta is overproduced yet estrogen is deficient in the brains of the menopausal AD patients. Both Abeta and estrogen deficiency can interact with tau kinases such as protein kinase B and glycogen synthase kinase 3. In the current study, levels of demethylated (-m) PP2A (L309) were significantly increased, and methylated (+m) PP2A (L309) were significantly decreased, which corresponded with the increased tau phosphorylation at the Tau-1 and PHF-1 sites in both mouse N2a cells carrying the human APP with Swedish mutation (APPswe) and transgenic APPswe/presenilin (PS) 1 (A246E) mice. These findings were replicated in wild-type N2a cells treated with Abeta25-35, and to a relatively larger extent, in both wild-type N2a cells and APPswe treated by okadaic acid, as well as in the brains of estrogen receptor (ER) alpha-/- and ERbeta-/- mice that mimic the status of estrogen deficiency in menopausal AD patients. Together, these findings suggested that the increased demethylation of PP2A (L309) mediated by Abeta overproduction or estrogen deficiency (ERalpha-/- and ERbeta-/-) may contribute to the reduced PP2A activity observed in the AD brain, resulting in the compromised dephosphorylation of abnormally hyperphosphorylated tau.

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.

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.

Zinc induces neurofilament phosphorylation independent of p70 S6 kinase in N2a cells.

Hyperphosphorylated neurofilaments are a part of neurofibrillary tangles in Alzheimers disease brains. Zinc has been shown to be increased in the brain areas heavily affected by Alzheimer pathologies. Zinc could induce tau hyperphosphorylation in SH-SY5Y and N2a cells, and tau phosphorylation may be mediated by p70 S6 kinase activation. Many of the tau kinases can also phosphorylate neurofilaments, and in this study we wanted to see whether neurofilament phosphorylation is regulated by p70 S6 kinase in N2a cells. We found that zinc induces rapamycin-dependent p70 S6 kinase phosphorylation at Thr421/Ser424 and Thr389, and rapamycin-independent phosphorylation of neurofilaments at the SMI34 epitope. Although zinc could induce cell proliferation and cell growth, and increased phosphorylation of neurofilaments, only cell growth appeared to be related to p7056kinase activation.