PKA modulates GSK-3β- and cdk5-catalyzed phosphorylation of tau in site- and kinase-specific manners

AbstractPhosphorylation of tau protein is regulated by several kinases, especially glycogen synthase kinase 3β (GSK-3β), cyclin-dependent protein kinase 5 (cdk5) and cAMP-dependent protein kinase (PKA). Phosphorylation of tau by PKA primes it for phosphorylation by GSK-3β, but the site-specific modulation of GSK-3β-catalyzed tau phosphorylation by the prephosphorylation has not been well investigated. Here, we found that prephosphorylation by PKA promotes GSK-3β-catalyzed tau phosphorylation at Thr181, Ser199, Ser202, Thr205, Thr217, Thr231, Ser396 and Ser422, but inhibits its phosphorylation at Thr212 and Ser404. In contrast, the prephosphorylation had no significant effect on its subsequent phosphorylation by cdk5 at Thr181, Ser199, Thr205, Thr231 and Ser422; inhibited it at Ser202, Thr212, Thr217 and Ser404; and slightly promoted it at Ser396. These studies reveal the nature of the inter-regulation of tau phosphorylation by the three major tau kinases

Long-Term Efficacy and Safety of Human Umbilical Cord Mesenchymal Stromal Cells in Rotenone-Induced Hemiparkinsonian Rats

Several studies have shown functional improvements, neuroprotective, and neuroregenerative effects after mesenchymal stem cells transplantation to parkinsonian animal models. However, questions remain about the safety, feasibility, and long-term efficacy of this approach. In this study, we investigated migration, therapeutic, tumorigenesis, and epileptogenic effects of human umbilical cord mesenchymal stromal cells (HUMSCs) 1 year after transplantation into rotenone-induced hemiparkinsonian rats. Our data indicated that DiI-labeled HUMSCs migrated in the lesioned hemisphere, from corpus striatum (CPu) to substantia nigra. By integrating with host cells and differentiating into NSE, GFAP, Nestin, and tyrosine hydroxylase-positive cells, HUMSCs prevented 48.4% dopamine neurons from degeneration and 56.9% dopamine terminals from loss, both correlating with improvement of apomorphine-induced rotations. The CD50 and CD97 value of pentylenetetrazol and semiquantitative immunohistochemical analysis of proliferating cell nuclear antigen (PCNA), β-catenin, C-myc, and NF-κB expression showed no significant difference between HUMSCs transplanted and untransplanted groups, whereas the expressions of Bcl-2 and P53 in the grafted CPu were upregulated by 281% and 200% compared to ungrafted CPu. The results of this long-term study suggest that HUMSCs transplantation, 1 of the most potential treatments for Parkinson's disease, is an effective and safe approach

Differential Effects of an O-GlcNAcase Inhibitor on Tau Phosphorylation

Abnormal hyperphosphorylation of microtubule-associated protein tau plays a crucial role in neurodegeneration in Alzheimer's disease (AD). The aggregation of hyperphosphorylated tau into neurofibrillary tangles is also a hallmark brain lesion of AD. Tau phosphorylation is regulated by tau kinases, tau phosphatases, and O-GlcNAcylation, a posttranslational modification of proteins on the serine or threonine residues with β-N-acetylglucosamine (GlcNAc). O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase, the enzyme catalyzing the transfer of GlcNAc to proteins, and N-acetylglucosaminidase (OGA), the enzyme catalyzing the removal of GlcNAc from proteins. Thiamet-G is a recently synthesized potent OGA inhibitor, and initial studies suggest it can influence O-GlcNAc levels in the brain, allowing OGA inhibition to be a potential route to altering disease progression in AD. In this study, we injected thiamet-G into the lateral ventricle of mice to increase O-GlcNAcylation of proteins and investigated the resulting effects on site-specific tau phosphorylation. We found that acute thiamet-G treatment led to a decrease in tau phosphorylation at Thr181, Thr212, Ser214, Ser262/Ser356, Ser404 and Ser409, and an increase in tau phosphorylation at Ser199, Ser202, Ser396 and Ser422 in the mouse brain. Investigation of the major tau kinases showed that acute delivery of a high dose of thiamet-G into the brain also led to a marked activation of glycogen synthase kinase-3β (GSK-3β), possibly as a consequence of down-regulation of its upstream regulating kinase, AKT. However, the elevation of tau phosphorylation at the sites above was not observed and GSK-3β was not activated in cultured adult hippocampal progenitor cells or in PC12 cells after thiamet-G treatment. These results suggest that acute high-dose thiamet-G injection can not only directly antagonize tau phosphorylation, but also stimulate GSK-3β activity, with the downstream consequence being site-specific, bi-directional regulation of tau phosphorylation in the mammalian brain

Stereotaxical Infusion of Rotenone: A Reliable Rodent Model for Parkinson's Disease

A clinically-related animal model of Parkinson's disease (PD) may enable the elucidation of the etiology of the disease and assist the development of medications. However, none of the current neurotoxin-based models recapitulates the main clinical features of the disease or the pathological hallmarks, such as dopamine (DA) neuron specificity of degeneration and Lewy body formation, which limits the use of these models in PD research. To overcome these limitations, we developed a rat model by stereotaxically (ST) infusing small doses of the mitochondrial complex-I inhibitor, rotenone, into two brain sites: the right ventral tegmental area and the substantia nigra. Four weeks after ST rotenone administration, tyrosine hydroxylase (TH) immunoreactivity in the infusion side decreased by 43.7%, in contrast to a 75.8% decrease observed in rats treated systemically with rotenone (SYS). The rotenone infusion also reduced the DA content, the glutathione and superoxide dismutase activities, and induced alpha-synuclein expression, when compared to the contralateral side. This ST model displays neither peripheral toxicity or mortality and has a high success rate. This rotenone-based ST model thus recapitulates the slow and specific loss of DA neurons and better mimics the clinical features of idiopathic PD, representing a reliable and more clinically-related model for PD research

Tau phosphorylation and μ-calpain activation mediate the dexamethasone-induced inhibition on the insulin-stimulated Akt phosphorylation.

Evidence has suggested that insulin resistance (IR) or high levels of glucocorticoids (GCs) may be linked with the pathogenesis and/or progression of Alzheimer's disease (AD). Although studies have shown that a high level of GCs results in IR, little is known about the molecular details that link GCs and IR in the context of AD. Abnormal phosphorylation of tau and activation of μ-calpain are two key events in the pathology of AD. Importantly, these two events are also related with GCs and IR. We therefore speculate that tau phosphorylation and μ-calpain activation may mediate the GCs-induced IR. Akt phosphorylation at Ser-473 (pAkt) is commonly used as a marker for assessing IR. We employed two cell lines, wild-type HEK293 cells and HEK293 cells stably expressing the longest human tau isoform (tau-441; HEK293/tau441 cells). We examined whether DEX, a synthetic GCs, induces tau phosphorylation and μ-calpain activation. If so, we examined whether the DEX-induced tau phosphorylation and μ-calpain activation mediate the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation. The results showed that DEX increased tau phosphorylation and induced tau-mediated μ-calpain activation. Furthermore, pre-treatment with LiCl prevented the effects of DEX on tau phosphorylation and μ-calpain activation. Finally, both LiCl pre-treatment and calpain inhibition prevented the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation. In conclusion, our study suggests that the tau phosphorylation and μ-calpain activation mediate the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation

Effects of dexamethasone on the insulin-stimulated Akt phosphorylation.

<p>A): Both wild-type HEK293 and HEK293/tau441 cells were treated with 1 µM dexamethasone (DEX) for 1–6 days and then stimulated with 100 nM insulin (i) for 15 min. Representative immunoblots from the results on the third day (D3) and the sixth day (D6) after DEX treatment were shown. Bars representing means ± SEM were shown below. Total amounts of Akt remained stable under the conditions. In HEK293/tau441 cells, DEX prevented the insulin-stimulated increases in pAkt on D3 and D6. In wild-type HEK293 cells, the inhibitory effect of DEX was evident on D6 but not on D3. Each experiment was repeated three times unless stated otherwise. ^*<i>P</i><0.05 versus control. B): Culture cells were pre-treated with mifepristone (RU; 20 µM, 30 min) and then treated with DEX for 3 days or 6 days. Representative immunoblots from wild-type HEK293 cells on D6 and those from HEK293/tau441 cells on D3 were shown, with bars representing means ± SEM below. Pre-treatment with RU prevented the inhibitory effects of DEX. ^*<i>P</i><0.05 versus DEX group.</p

Correction: A Chinese version of the Language Screening Test (CLAST) for early-stage stroke patients.

[This corrects the article DOI: 10.1371/journal.pone.0196646.]

Overexpression of Dyrk1A contributes to neurofibrillary degeneration in Down syndrome

Adults with Down syndrome (DS) develop Alzheimer neurofibrillary degeneration in the brain, but the underlying molecular mechanism is unknown. Here, we report that the presence of an extra copy of the dual-specificity tyrosine-phosphorylated and regulated kinase 1A (Dyrk1A) gene due to trisomy 21 resulted in overexpression of Dyrk1A and elevated kinase activity in DS brain. Dyrk1A phosphorylated tau at several sites, and these sites were hyperphosphorylated in adult DS brains. Phosphorylation of tau by Dyrk1A primed its further phosphorylation by glycogen synthase kinase-3β (GSK-3β). Dyrk1A-induced tau phosphorylation inhibited tau’s biological activity and promoted its self-aggregation. In Ts65Dn mouse brain, an extra copy of the Dyrk1A gene caused increased expression and activity of Dyrk1A and resulted in increased tau phosphorylation. These findings strongly suggest a novel mechanism by which the overexpression of Dyrk1A in DS brain causes neurofibrillary degeneration via hyperphosphorylating tau. Liu, F., Liang, Z., Wegiel, J., Hwang, Y.-W., Iqbal, K., Grundke-Iqbal, I., Ramakrishna, N., Gong, C.-X. Overexpression of Dyrk1A contributes to neurofibrillary degeneration in Down syndrome

Involvement of μ-calpain in the inhibitory effect of DEX.

<p>A): Both wild-type HEK293 and HEK293/tau441 cells were pre-treated with LiCl (10 mM, 1 h) and then treated with DEX for 3 days (D3) or 6 days (D6). Activation of μ-calpain was determined by the ratios of the active/truncated calpain (78-kDa bands) and the inactive/full-length calpain (80-kDa bands). Bars representing means ± SEM. On D3 in HEK293/tau441 cells, DEX induced μ-calpain activation and pre-treatment with LiCl prevented the activation of μ-calpain. ^*<i>P</i><0.05 versus control. B): HEK293/tau441 cells were pre-treated with E-64d (30 µg/ml, 1 h) or LiCl (10 mM, 1 h), and then treated with DEX for 3 days. The level of tau phosphorylation was determined by the ratio of Tau-1/R134d. Bars representing means ± SEM. E-64d did not have obvious effect on the DEX-induced increase in tau phosphorylation. ^*<i>P</i><0.05 versus control, ^#<i>P</i><0.05 between indicated groups. C) Both wild-type HEK293 and HEK293/tau441 cells were pre-treated with E-64d (30 µg/ml, 1 h) and then treated with DEX for 3 days (D3) or 6 days (D6), followed by stimulation of insulin and Western analysis of pAkt and Akt. Bars representing means ± SEM. Pre-treatment with E-64d prevented the inhibitory effect of DEX in HEK293/tau441 cells on D3. ^*<i>P</i><0.05 versus control.</p

Effects of DEX and lithium chloride on tau phosphorylation.

<p>HEK293/tau441 cells were pre-treated with lithium chloride (LiCl; 10 mM, 1 h) and then treated with DEX for 3 days (D3) or 6 days (D6), followed by Western blotting analysis of tau phosphorylation with Tau-1 (against dephosphorylated tau within the epitope 189–207) and R134d (against total tau) antibodies. Total amounts of tau remained largely stable and the level of tau phosphorylation was determined by the Tau-1/R134d ratio. Bars representing means ± SEM. On D3, DEX induced an increase in tau phosphorylation, as evidenced by the decrease in the Tau-1/R134d ratio. Pre-treatment with LiCl prevented the effect of DEX on tau phosphorylation on D3. ^*<i>P</i><0.05 versus control, ^#<i>P</i><0.05 between indicated groups.</p