Lithium chloride alters Tau phosphorylation, kinase activity, and Rho GTPase signaling in cell models.

Alzheimer’s disease (AD) is a degenerative neurological disease that primarily affects the elderly. Drug therapy is the main strategy for AD treatment, but current treatments suffer from poor efficacy and a number of side effects. Non-drug therapy is attracting more attention and may be a better strategy for treatment of AD. Hypoxia is one of the important factors that contribute to the pathogenesis of AD. Multiple cellular processes synergistically promote hypoxia, including aging, hypertension, diabetes, hypoxia/obstructive sleep apnea, obesity, and traumatic brain injury. Increasing evidence has shown that hypoxia may affect multiple pathological aspects of AD, such as amyloid-beta metabolism, tau phosphorylation, autophagy, neuroinflammation, oxidative stress, endoplasmic reticulum stress, and mitochondrial and synaptic dysfunction. Treatments targeting hypoxia may delay or mitigate the progression of AD. Numerous studies have shown that oxygen therapy could improve the risk factors and clinical symptoms of AD. Increasing evidence also suggests that oxygen therapy may improve many pathological aspects of AD including amyloid-beta metabolism, tau phosphorylation, neuroinflammation, neuronal apoptosis, oxidative stress, neurotrophic factors, mitochondrial function, cerebral blood volume, and protein synthesis. In this review, we summarized the effects of oxygen therapy on AD pathogenesis and the mechanisms underlying these alterations. We expect that this review can benefit future clinical applications and therapy strategies on oxygen therapy for AD.

Most individuals with Down syndrome show early onset of Alzheimer disease (AD), resulting from the extra copy of chromosome 21. Located on this chromosome is a gene that encodes the dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). One of the pathological hallmarks in AD is the presence of neurofibrillary tangles (NFTs), which are insoluble deposits that consist of abnormally hyperphosphorylated Tau. Previously it was reported that Tau at the Thr-212 residue was phosphorylated by Dyrk1A in vitro. To determine the physiological significance of this phosphorylation, an analysis was made of the amount of phospho-Thr-212-Tau (pT212) in the brains of transgenic mice that overexpress the human DYRK1A protein (DYRK1A TG mice) that we recently generated. A significant increase in the amount of pT212 was found in the brains of DYRK1A transgenic mice when compared with age-matched littermate controls. We further examined whether Dyrk1A phosphorylates other Tau residues that are implicated in NFTs. We found that Dyrk1A also phosphorylates Tau at Ser-202 and Ser-404 in vitro. Phosphorylation by Dyrk1A strongly inhibited the ability of Tau to promote microtubule assembly. Following this, using mammalian cells and DYRK1A TG mouse brains, it was demonstrated that the amounts of phospho-Ser-202-Tau and phospho-Ser-404-Tau are enhanced when DYRK1A amounts are high. These results provide the first in vivo evidence for a physiological role of DYRK1A in the hyperphosphorylation of Tau and suggest that the extra copy of the DYRK1A gene contributes to the early onset of AD.

Hyperphosphorylated tau is the major component of neurofibrillary tangles in Alzheimer disease (AD), and the tangle distribution largely overlaps with zinc-containing glutamatergic neurons, suggesting that zinc released in synaptic terminals may play a role in tau phosphorylation. To explore this possibility, we treated cultured hippocampal slices or primary neurons with glutamate or Bic/4-AP to increase the synaptic activity with or without pretreatment of zinc chelators, and then detected the phosphorylation levels of tau. We found that glutamate or Bic/4-AP treatment caused tau hyperphosphorylation at multiple AD-related sites, including Ser-396, Ser-404, Thr-231, and Thr-205, while application of intracellular or extracellular zinc chelators, or blockade of zinc release by extracellular calcium omission almost abolished the synaptic activity-associated tau hyperphosphorylation. The zinc release and translocation of excitatory synapses in the hippocampus were detected, and zinc-induced tau hyperphosphorylation was also observed in cultured brain slices incubated with exogenously supplemented zinc. Tau hyperphosphorylation induced by synaptic activity was strongly associated with inactivation of protein phosphatase 2A (PP2A), and this inactivation can be reversed by pretreatment of zinc chelator. Together, these results suggest that synaptically released zinc promotes tau hyperphosphorylation through PP2A inhibition.

Alzheimer disease (AD) is characterized by neurodegeneration marked by loss of synapses and spines associated with hyperphosphorylation of tau protein. Accumulating amyloid β peptide (Aβ) in brain is linked to neurofibrillary tangles composed of hyperphosphorylated tau in AD. Here, we identify β2-adrenergic receptor (β2AR) that mediates Aβ-induced tau pathology. In the prefrontal cortex (PFC) of 1-year-old transgenic mice with human familial mutant genes of presenilin 1 and amyloid precursor protein (PS1/APP), the phosphorylation of tau at Ser-214 Ser-262 and Thr-181, and the protein kinases including JNK, GSK3α/β, and Ca(2+)/calmodulin-dependent protein kinase II is increased significantly. Deletion of the β2AR gene in PS1/APP mice greatly decreases the phosphorylation of these proteins. Further analysis reveals that in primary PFC neurons, Aβ signals through a β2AR-PKA-JNK pathway, which is responsible for most of the phosphorylation of tau at Ser-214 and Ser-262 and a significant portion of phosphorylation at Thr-181. Aβ also induces a β2AR-dependent arrestin-ERK1/2 activity that does not participate in phosphorylation of tau. However, inhibition of the activity of MEK, an upstream enzyme of ERK1/2, partially blocks Aβ-induced tau phosphorylation at Thr-181. The density of dendritic spines and synapses is decreased in the deep layer of the PFC of 1-year-old PS1/APP mice, and the mice exhibit impairment of learning and memory in a novel object recognition paradigm. Deletion of the β2AR gene ameliorates pathological effects in these senile PS1/APP mice. The study indicates that β2AR may represent a potential therapeutic target for preventing the development of AD.

The microtubule-associated protein tau, which becomes hyperphosphorylated and pathologically aggregates in a number of these diseases, is extremely sensitive to manipulations of chaperone signaling. For example, Hsp90 inhibitors can reduce the levels of tau in transgenic mouse models of tauopathy. Because of this, we hypothesized that a number of Hsp90 accessory proteins, termed co-chaperones, could also affect tau stability. Perhaps by identifying these co-chaperones, new therapeutics could be designed to specifically target these proteins and facilitate tau clearance. Here, we report that the co-chaperone Cdc37 can regulate aspects of tau pathogenesis. We found that suppression of Cdc37 destabilized tau, leading to its clearance, whereas Cdc37 overexpression preserved tau. Cdc37 was found to co-localize with tau in neuronal cells and to physically interact with tau from human brain. Moreover, Cdc37 levels significantly increased with age. Cdc37 knockdown altered the phosphorylation profile of tau, an effect that was due in part to reduced tau kinase stability, specifically Cdk5 and Akt. Conversely, GSK3β and Mark2 were unaffected by Cdc37 modulation. Cdc37 overexpression prevented whereas Cdc37 suppression potentiated tau clearance following Hsp90 inhibition. Thus, Cdc37 can regulate tau in two ways: by directly stabilizing it via Hsp90 and by regulating the stability of distinct tau kinases. We propose that changes in the neuronal levels or activity of Cdc37 could dramatically alter the kinome, leading to profound changes in the tau phosphorylation signature, altering its proteotoxicity and stability.

Abnormally phosphorylated tau is the major component of paired helical filaments found in the brains of patients suffering from Alzheimer's disease. Therefore, the identification of kinases that phosphorylate tau is of considerable interest. A DEAE-Sepharose column resolved porcine brain extract into five tau kinase activity peaks. Among these peaks, two were completely inhibited by EGTA, indicating that these two activity peaks contained Ca2+-dependent tau kinases. One of the above two Ca2+-dependent tau kinase activity peaks also contained phosphorylase kinase activity. The tau kinase and phosphorylase kinase activities associated with this peak could not be separated from each other by Superose 12 gel filtration, hydroxylapatite, and calmodulin-agarose affinity chromatographies. Phosphorylase kinase, purified from rabbit skeletal muscle, phosphorylated tau to a stoichiometry of 2.1 mol of phosphate/mol of tau and converted tau to a species with a retarded mobility on SDS-polyacrylamide gel electrophoresis. The apparent Km and kcat values for tau phosphorylation by muscle phosphorylase kinase were 6.9 microM and 47.4 min-1, respectively. As a substrate of muscle phosphorylase kinase, phosphorylase was eight times better than tau. Sequence analyses of tryptic and thermolytic phosphopeptides derived from tau phosphorylated by muscle phosphorylase kinase revealed five phosphorylation sites, Ser237, Ser262, Ser285, Ser305, and Ser352. Among these sites, Ser262 was previously shown to be phosphorylated in human tau from fetal, adult, and Alzheimer's diseased brains (Seubert, P., Mawal-Dewan, M., Barbour, R., Jakes, R., Goedert, M., Johnson, G. V. W., Litersky, J. M., Schenk, D., Lieberburg, I., Trojanowski, J. Q., and Lee, V. M. Y. (1995) J. Biol. Chem. 270, 18917-18922); and its phosphorylation abolished tau's binding to microtubules (Drewes, G., Trinczek, B., Illenberger, S., Biernat, J., Schmitt-Ulms, G., Meyer, H. E., Mandelkow, E.-M., and Mandelkow, E. (1995) J. Biol. Chem. 270, 7679-7688). Slot-blot analysis using a monoclonal antibody against muscle phosphorylase kinase and an activity assay using phosphorylase revealed that phosphorylase kinase was present in microtubules extensively purified by repeated cycles of polymerization and depolymerization. Taken together, these results suggest that in neurons, phosphorylase kinase may be one of the kinases that participate in the phosphorylation of tau.

Alzheimer’s Disease (AD) is the most common single cause of dementia in our ageing society. Traditionally thought of as an untreatable degenerative condition, recent advances in drug therapy have challenged this view. The disease is characterised by an insidious decline in cognitive and non-cognitive function. Classically, short and long-term memory is impaired while language skills, concentration and attention are often affected. This results in impaired ability to learn and retain new skills as well as the loss of existing ones. Non-cognitive function is the global term used to describe problems such as depression, agitation, personality changes, delusions and hallucinations. These factors have a significant impact on patient behaviour and a very real impact on the quality of life for both patients and caregivers. Diagnosis of AD is clinically based, and using the NINCDS-ADRDA criteria (Table 1) [1], a diagnosis of probable or possible AD can be made. Definitive diagnosis relies on pathological confirmation, which in the majority of cases is rarely completed. With the development of AD specific treatments, definition of AD from other types of dementia is very important. Table 1 NINCDS-ADRDA Criteria for clinical diagnosis of Alzheimer’s disease. Pathogenesis The pathogenesis of AD has not yet been elucidated. It is widely accepted that a combination of genetic susceptibility factors and environmental triggers are responsible for late onset sporadic AD, the most common form of the disease. An understanding of the disease mechanism remains elusive, and is the key to developing a disease modifying agent. Currently, it is proposed that beta amyloid protein, abnormal tau protein or possibly both play key factors in the development of disease. It has been widely postulated that oxidative damage and a slow inflammatory process are two possible mechanisms involved. As yet, no product with proven disease modifying properties is available, and current treatments offer symptomatic benefit only. The development of acetylcholinesterase (AChe) inhibitor drugs has followed the finding that cholinergic pathways in the cerebral cortex and basal forebrain are compromised in AD [2] and the resultant cholinergic deficit contributes to the cognitive impairment of these patients [3]. Although many believe this ‘cholinergic hypothesis’ to be important, others feel it represents a less significant component of the disease process [4]. Many other neurotransmitters are affected in AD, and the relative importance of each in relation to clinical findings has not been fully elucidated. Initial work focused on the use of acetylcholine precursors, using a similar rationale to dopamine therapy in Parkinson’s disease. A series of small trials using precursors such as choline and phosphatidylcholine showed no reliable improvement in cognitive function, with only 10 out of 43 trials reporting any positive effect [5]. There has been renewed interest in muscarinic agonists drugs, which when first introduced, had major problems with adverse cholinergic effects. Better understanding of the molecular pathology of muscarinic receptors and their subtypes has led to the development of more specific agonists. Drugs such as xanomeline, milameline, and civimeline have reached clinical trials, and the improvements seen in cognitive function are reviewed by Avery et al. [6]. There are also claims that these drugs have disease modifying properties, with effects on APP processing and tau phosphorylation. Muscarinic agonists remain in trial, but have yet to fulfil their potential in AD treatment. The only group of drugs currently licensed for AD treatment is the AChe inhibitors, which act through inhibition of the enzyme acetylcholinesterase (AChe), responsible for the breakdown of ACh in the neural synapse. A meta-analysis of the early AChe inhibitor treatments was encouraging [7] and these proceeded to larger placebo controlled double-blind trials.

Neurodegenerative tauopathies, including Alzheimer disease, are characterized by abnormal hyperphosphorylation of the microtubule-associated protein Tau. One group of tauopathies, known as frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), is directly associated with mutations of the gene tau. However, it is unknown why mutant Tau is highly phosphorylated in the patient brain. In contrast to in vivo high phosphorylation, FTDP-17 Tau is phosphorylated less than wild-type Tau in vitro. Because phosphorylation is a balance between kinase and phosphatase activities, we investigated dephosphorylation of mutant Tau proteins, P301L and R406W. Tau phosphorylated by Cdk5-p25 was dephosphorylated by protein phosphatases in rat brain extracts. Compared with wild-type Tau, R406W was dephosphorylated faster and P301L slower. The two-dimensional phosphopeptide map analysis suggested that faster dephosphorylation of R406W was due to a lack of phosphorylation at Ser-404, which is relatively resistant to dephosphorylation. We studied the effect of the peptidyl-prolyl isomerase Pin1 or microtubule binding on dephosphorylation of wild-type Tau, P301L, and R406W in vitro. Pin1 catalyzes the cis/trans isomerization of phospho-Ser/Thr-Pro sequences in a subset of proteins. Dephosphorylation of wild-type Tau was reduced in brain extracts of Pin1-knockout mice, and this reduction was not observed with P301L and R406W. On the other hand, binding to microtubules almost abolished dephosphorylation of wild-type and mutant Tau proteins. These results demonstrate that mutation of Tau and its association with microtubules may change the conformation of Tau, thereby suppressing dephosphorylation and potentially contributing to the etiology of tauopathies.

Extracellular amyloid plaques, intracellular neurofibrillary tangles, and loss of basal forebrain cholinergic neurons in the brains of Alzheimer's disease (AD) patients may be the end result of abnormalities in lipid metabolism and peroxidation that may be caused, or exacerbated, by beta-amyloid peptide (Abeta). Apolipoprotein E (apoE) is a major apolipoprotein in the brain, mediating the transport and clearance of lipids and Abeta. ApoE-dependent dendritic and synaptic regeneration may be less efficient with apoE4, and this may result in, or unmask, age-related neurodegenerative changes. The increased risk of AD associated with apoE4 may be modulated by diet, vascular risk factors, and genetic polymorphisms that affect the function of other transporter proteins and enzymes involved in brain lipid homeostasis. Diet and apoE lipoproteins influence membrane lipid raft composition and the properties of enzymes, transporter proteins, and receptors mediating Abeta production and degradation, tau phosphorylation, glutamate and glucose uptake, and neuronal signal transduction. The level and isoform of apoE may influence whether Abeta is likely to be metabolized or deposited. This review examines the current evidence for diet, lipid homeostasis, and apoE in the pathogenesis of AD. Effects on the cholinergic system and response to cholinesterase inhibitors by APOE allele carrier status are discussed briefly.

Amyloid Activates GSK-3β to Aggravate Neuronal Tauopathy in Bigenic Mice

Intraneuronal accumulation of hyperphosphorylated protein tau in paired helical filaments together with amyloid-beta peptide (Abeta) deposits confirm the clinical diagnosis of Alzheimer disease. A common cellular mechanism leading to the production of these potent toxins remains elusive. Here we show that, in cultured neurons, membrane depolarization induced a calcium-mediated transient phosphorylation of both microtubule-associated protein tau and amyloid precursor protein (APP), followed by a dephosphorylation of these proteins. Phosphorylation was mediated by glycogen synthase kinase 3 and cyclin-dependent kinase 5 protein kinases, while calcineurin was responsible for dephosphorylation. Following the transient phosphorylation of APP, intraneuronal Abeta accumulated and induced neurotoxicity. Phosphorylation of APP on Thr-668 was indispensable for intraneuronal accumulation of Abeta. Our data demonstrate that an increase in cytosolic calcium concentration induces modifications of neuronal metabolism of APP and tau, similar to those found in Alzheimer disease.

Insulin deficiency results in reversible protein kinase A activation and tau phosphorylation

The pathological features of Alzheimer's disease (AD) include extracellular neuritic plaques containing β-amyloid (Aβ) peptide, a cleaved fragment of amyloid precursor protein (APP) via β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) and intracellular neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau. Cyclin-dependent kinase 5 (Cdk5) is increasingly thought to play a pivotal role in the pathogenesis of AD, both as a regulator of the production of Aβ and through its well-established role as a tau kinase. Fuzhisan (FZS), a Chinese herbal complex prescription, has been used for the treatment AD for over 20years, and is known to enhance the cognitive ability in AD patients as well as in AD model rats. To investigate mechanisms of AD and the potential therapy of FZS in AD, we treated senescence-accelerated mouse SAMP8 mice, a useful model of AD-related memory impairment, with FZS by intragastrical administration for 8weeks and Donepizel was used as a positive control. The results showed that FZS (0.3, 0.6, and 1.2g/kg/day) improved impaired cognitive ability of aged SAMP8 mice in a dose-dependent manner. FZS robustly decreased Aβ level and phosphorylation of tau. This was accompanied by a significant decrease in the BACE1 level and phosphorylated APP (Thr668). Futhermore, The p25/Cdk5 pathway was markedly down-regulated by FZS treatment. These results indicated that the memory ameliorating effect of FZS may be, in part, by regulation the p25/Cdk5 pathway which may contribute to down-regulation of Aβ and tau hyperphosphorylation.

Tau is an important microtubule-stabilizing protein in neurons. In its hyperphosphorylated form, Tau protein loses its ability to bind to microtubules and then accumulates and is part of pathological lesions characterizing tauopathies, e.g. Alzheimer disease. Glycogen synthase kinase-3beta (GSK-3beta), antagonized by protein phosphatase 2A (PP2A), regulates Tau phosphorylation at many sites. Diabetes mellitus is linked to an increased risk of developing Alzheimer disease. This could be partially caused by dysregulated GSK-3beta. In a long term experiment (-16 h) using primary murine neuron cultures, we interfered in the insulin/phosphoinositide 3-kinase (PI3K) (LY294002 treatment and insulin boost) and mammalian target of rapamycin (mTor) (AICAR and rapamycin treatment) signaling pathways and examined consequent changes in the activities of PP2A, GSK-3beta, and Tau phosphorylation. We found that the coupling of PI3K with mTor signaling, in conjunction with a regulatory interaction between PP2A and GSK-3beta, changed activities of both enzymes always in the same direction. These balanced responses seem to ensure the steady Tau phosphorylation at GSK/PP2A-dependent sites observed over a long period of time (>/=6 h). This may help in preventing severe changes in Tau phosphorylation under conditions when neurons undergo transient fluctuations either in insulin or nutrient supply. On the other hand, the investigation of Tau protein at Ser-262 showed that interference in the insulin/PI3K and mTor signaling potentially influenced the Tau phosphorylation status at sites where only one of two enzymes (in this case PP2A) is involved in the regulation.

The microtubule-associated protein tau is hyperphosphorylated and forms neurofibrillary tangles in Alzheimer disease. Additionally caspase-cleaved tau is present in Alzheimer disease brains co-localized with fibrillar tau pathologies. To further understand the role of site-specific phosphorylation and caspase cleavage of tau in regulating its function, constructs of full-length tau (T4) or tau truncated at Asp421 (T4C3) to mimic caspase-3 cleavage with and without site-directed mutations that mimic phosphorylation at Thr231/Ser235, Ser396/Ser404, or at all four sites (Thr231/Ser235/Ser396/Ser404) were made and expressed in cells. Pseudophosphorylation of T4, but not T4C3, at either Thr231/Ser235 or Ser396/Ser404 increased its phosphorylation at Ser262 and Ser199. Pseudophosphorylation at Thr231/Ser235 impaired the microtubule binding of both T4 and T4C3. In contrast, pseudophosphorylation at Ser396/Ser404 only affected microtubule binding of T4C3 but did make T4 less soluble and more aggregated, which is consistent with the previous finding (Abraha, A., Ghoshal, N., Gamblin, T. C., Cryns, V., Berry, R. W., Kuret, J., and Binder, L. I. (2000) J. Cell Sci. 113, 3737-3745) that pseudophosphorylation at Ser396/Ser404 enhances tau polymerization in vitro. In situ T4C3 was more prevalent in the cytoskeletal and microtubule-associated fractions compared with T4, whereas purified recombinant T4 bound microtubules with higher affinity than did T4C3 in an in vitro assay. These data indicate the importance of cellular factors in regulating tau-microtubule interactions and that, in the cells, phosphorylation of T4 might impair its microtubule binding ability more than caspase cleavage. Treatment of cells with nocodazole revealed that pseudophosphorylation of T4 at both Thr231/Ser235 and Ser396/Ser404 diminished the ability of tau to protect against microtubule depolymerization, whereas with T4C3 only pseudophosphorylation at Ser396/Ser404 attenuated the ability of tau to stabilize the microtubules. These results show that site-specific phosphorylation and caspase cleavage of tau differentially affect the ability of tau to bind and stabilize microtubules and facilitate tau self-association.