Caspase-6-cleaved Tau fails to induce Tau hyperphosphorylation and aggregation, neurodegeneration, glial inflammation, and cognitive deficits.

Active Caspase-6 (Casp6) and Tau cleaved by Casp6 at amino acids 402 (Tau∆D402) and 421 (Tau∆D421) are present in early Alzheimer disease intraneuronal neurofibrillary tangles, which are made primarily of filamentous Tau aggregates. To assess whether Casp6 cleavage of Tau contributes to Tau pathology and Casp6-mediated age-dependent cognitive impairment, we generated transgenic knock-in mouse models that conditionally express full-length human Tau (hTau) 0N4R only (CTO) or together with human Casp6 (hCasp6) (CTC). Region-specific hippocampal and cortical hCasp6 and hTau expression were confirmed with western blot and immunohistochemistry in 2-25-month-old brains. Casp6 activity was confirmed with Tau∆D421 and Tubulin cleaved by Casp6 immunopositivity in 3-25-month-old CTC, but not in CTO, brains. Immunoprecipitated Tau∆D402 was detected in both CTC and CTO brains, but was more abundant in CTC brains. Intraneuronal hippocampal Tau hyperphosphorylation at S202/T205, S422, and T231, and Tau conformational change were absent in both CTC and CTO brains. A slight accumulation of Tau phosphorylated at S396/404 and S202 was observed in Cornu Ammonis 1 (CA1) hippocampal neuron soma of CTC compared to CTO brains. Eighteen-month-old CTC brains showed rare argentophilic deposits that increased by 25 months, whereas CTO brains only displayed them sparsely at 25 months. Tau microtubule binding was equivalent in CTC and CTO hippocampi. Episodic and spatial memory measured with novel object recognition and Barnes maze, respectively, remained normal in 3-25-month-old CTC and CTO mice, in contrast to previously observed impairments in ACL mice expressing equivalent levels of hCasp6 only. Consistently, the CTC and CTO hippocampal CA1 region displayed equivalent dendritic spine density and no glial inflammation. Together, these results reveal that active hCasp6 co-expression with hTau generates Tau cleavage and rare age-dependent argentophilic deposits but fails to induce cognitive deficits, neuroinflammation, and Tau pathology.

Purines and Pyrimidines: Metabolism, Function and Potential as Therapeutic Options in Neurodegenerative Diseases.

Various neurodegenerative disorders have various molecular origins but some common molecular mechanisms. In the current scenario, there are very few treatment regimens present for advanced neurodegenerative diseases. In this context, there is an urgent need for alternate options in the form of natural compounds with an ameliorating effect on patients. There have been individual scattered experiments trying to identify potential values of various intracellular metabolites. Purines and Pyrimidines, which are vital molecules governing various aspects of cellular biochemical reactions, have been long sought as crucial candidates for the same, but there are still many questions that go unanswered. Some critical functions of these molecules associated with neuromodulation activities have been identified. They are also known to play a role in foetal neurodevelopment, but there is a lacuna in understanding their mechanisms. In this review, we have tried to assemble and identify the importance of purines and pyrimidines, connecting them with the prevalence of neurodegenerative diseases. The leading cause of this class of diseases is protein misfolding and the formation of amyloids. A direct correlation between loss of balance in cellular homeostasis and amyloidosis is yet an unexplored area. This review aims at bringing the current literature available under one umbrella serving as a foundation for further extensive research in this field of drug development in neurodegenerative diseases.

S6 kinase phosphorylated at T229 is involved in tau and actin pathologies in Alzheimer's disease.

The 70-kDa ribosomal protein S6 kinase (S6K), a serine/threonine kinase that modulates the phosphorylation of the 40S ribosomal protein S6, regulates cell cycle progression and is known as a tau kinase in Alzheimer's disease (AD). In AD brains, neurofibrillary tangles (NFTs) have been shown to be positively stained with antibodies against S6K proteins phosphorylated at T389 (pT389-S6K) or T421/S424 (pT421/S424-S6K) by the mammalian target of rapamycin and mitogen-activated protein kinase pathways, respectively. However, there is little information available about S6K proteins directly phosphorylated at T229 (pT229-S6K) by the PI3K-PDK1 pathway. In the present study, we investigated the distribution of pT229-S6K in post mortem human brain tissues from elderly (control) and patients with AD using immnunoblotting and immunohistochemistry. pT229-S6K immunoreactivity was localized to small granular structures in neurons and endothelial cells in control and AD brains. In AD brains, intense pT229-S6K immunoreactivity was detected in 16.3% of AT8-positive NFTs, neuropil threads, and dystrophic neurites in the hippocampus and other vulnerable brain areas. In addition, Hirano bodies were also positive for pT229-S6K but were negative for pT389-S6K or pT421/S424-S6K. The present results indicate that S6K phosphorylation via the PI3K-PD1 pathway is involved in tau pathology in NFTs and abnormal neurites as well as actin pathology in Hirano bodies.

Immunohistochemical analysis of hippocampal butyrylcholinesterase: Implications for regional vulnerability in Alzheimer's disease.

Studies of acetylcholine degrading enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in Alzheimer's disease (AD) have suggested their potential role in the development of fibrillar amyloid-ß (Aß) plaques (amyloid plaques). A recent genome-wide association study analysis identified a novel association between genetic variations in the BCHE locus and amyloid burden. We studied BChE immunoreactivity in hippocampal tissue sections from AD and control cases, and examined its relationship with amyloid plaques, neurofibrillary tangles (NFT), dystrophic neurites (DN) and neuropil threads (NT). Compared to controls, AD cases had greater BChE immunoreactivity in hippocampal neurons and neuropils in CA2/3, but not in the CA1, CA4 and dentate gyrus. The majority of amyloid plaques (> 80%, using a pan-amyloid marker X-34) contained discrete neuritic clusters which were dual-labeled with antibodies against BChE and phosphorylated tau (clone AT8). There was no association between overall regional BChE immunoreaction intensity and amyloid plaque burden. In contrast to previous reports, BChE was localized in only a fraction (~10%) of classic NFT (positive for X-34). A similar proportion of BChE-immunoreactive pyramidal cells were AT8 immunoreactive. Greater NFT and DN loads were associated with greater BChE immunoreaction intensity in CA2/3, but not in CA1, CA4 and dentate gyrus. Our results demonstrate that in AD hippocampus, BChE accumulates in neurons and plaque-associated neuritic clusters, but only in a small proportion of NFT. The association between greater neurofibrillary pathology burden and markedly increased BChE immunoreactivity, observed selectively in CA2/3 region, could reflect a novel compensatory mechanism. Since CA2/3 is generally considered more resistant to AD pathology, BChE upregulation could impact the cholinergic modulation of glutamate neurotransmission to prevent/reduce neuronal excitotoxicity in AD hippocampus.

Genetic Variation in δ-Opioid Receptor Associates with Increased ß- and γ-Secretase Activity in the Late Stages of Alzheimer's Disease.

The agonist-induced activation of human δ-opioid receptor (δOR) has been shown to increase ß- (BACE1) and γ-secretase activities leading to increased production of amyloid-ß (Aß) peptide. We have recently shown that phenylalanine to cysteine substitution at amino acid 27 in δOR (δOR-Phe27Cys) increases amyloid-ß protein precursor processing through altered endocytic trafficking. Also, a genetic meta-analysis of the δOR-Phe27Cys variation (rs1042114) in two independent Alzheimer's disease (AD) patient cohorts indicated that the heterozygosity of δOR-Phe27Cys increases the risk of AD. Here, we investigated α-, ß-, and γ-secretase activities in human brain with respect to δOR-Phe27Cys variation in the temporal cortex of 71 subjects with varying degree of AD-related neurofibrillary pathology (Braak stages I-VI). As a result, a significant increase in ß- (p = 0.03) and γ- (p = 0.01), but not α-secretase, activities was observed in late stage AD samples (Braak stages V-VI), which were heterozygous for δOR-Phe27Cys as compared to the δOR-Phe27 and δOR-Cys27 homozygotes. The augmented ß-secretase activity was not associated with increased mRNA expression or protein levels of BACE1 in the late stage AD patients, who were heterozygous for the δOR-Phe27Cys variation. These findings suggest that δOR-Phe27Cys variation modulates ß- and γ-secretase activity in the late stages of AD likely via post-translational mechanisms other than alterations in the mRNA or protein levels of BACE1, or, in the expression of γ-secretase complex components.

Cleavage of tau by asparagine endopeptidase mediates the neurofibrillary pathology in Alzheimer's disease.

Neurofibrillary tangles (NFTs), composed of truncated and hyperphosphorylated tau, are a common feature of numerous aging-related neurodegenerative diseases, including Alzheimer's disease (AD). However, the molecular mechanisms mediating tau truncation and aggregation during aging remain elusive. Here we show that asparagine endopeptidase (AEP), a lysosomal cysteine proteinase, is activated during aging and proteolytically degrades tau, abolishes its microtubule assembly function, induces tau aggregation and triggers neurodegeneration. AEP is upregulated and active during aging and is activated in human AD brain and tau P301S-transgenic mice with synaptic pathology and behavioral impairments, leading to tau truncation in NFTs. Tau P301S-transgenic mice with deletion of the gene encoding AEP show substantially reduced tau hyperphosphorylation, less synapse loss and rescue of impaired hippocampal synaptic function and cognitive deficits. Mice infected with adeno-associated virus encoding an uncleavable tau mutant showed attenuated pathological and behavioral defects compared to mice injected with adeno-associated virus encoding tau P301S. Together, these observations indicate that AEP acts as a crucial mediator of tau-related clinical and neuropathological changes. Inhibition of AEP may be therapeutically useful for treating tau-mediated neurodegenerative diseases.

Monoamine oxidase inhibitors: promising therapeutic agents for Alzheimer's disease (Review).

Activated monoamine oxidase (MAO) has a critical role in the pathogenesis of Alzheimer's disease (AD), including the formation of amyloid plaques from amyloid ß peptide (Aß) production and accumulation, formation of neurofibrillary tangles, and cognitive impairment via the destruction of cholinergic neurons and disorder of the cholinergic system. Several studies have indicated that MAO inhibitors improve cognitive deficits and reverse Aß pathology by modulating proteolytic cleavage of amyloid precursor protein and decreasing Aß protein fragments. Thus, MAO inhibitors may be considered as promising therapeutic agents for AD.

Protein disulfide isomerase P5-immunopositive inclusions in patients with Alzheimer's disease.

Amyloid plaques and neurofibrillary tangles (NFTs) are the major pathological characteristics of Alzheimer's disease (AD). NFTs are composed of tubular filaments and paired helical filaments containing polymerized hyperphosphorylated tau protein. Another feature of AD is excessive generation of nitric oxide (NO). Protein disulfide isomerase (PDI) is a chaperon protein located in the endoplasmic reticulum (ER). It was recently reported that NO-induced S-nitrosylation of PDI inhibits its enzymatic activity, leading to the accumulation of polyubiquitinated proteins, and activates the unfolded protein response. In addition, we previously reported the presence of PDI-immunopositive NFTs in AD. Here, we found that protein disulfide isomerase P5 (P5), which is a member of the PDI protein family, was co-localized with tau in NFTs. To our knowledge, this is the first report of P5-immunopositive inclusion in AD. Furthermore, we showed that S-nitrosylated P5 was present and the expression level of P5 was decreased in AD brains compared with that of control brains. We also demonstrated that the knock-down of PDI or P5 by siRNA could affect the viability of SH-SY5Y cells under ER stress. Previously, the observation of S-nitrosylated PDI in AD was reported. NO may inhibit P5 by inducing S-nitrosylation in the same manner as PDI, which inhibits its enzymatic activity allowing protein misfolding to occur in AD. The accumulation of misfolded proteins induces ER stress and may cause apoptosis of neuronal cells through S-nitrosylation and down-regulation of PDI and P5 in AD.

Caspase-6 as a novel early target in the treatment of Alzheimer's disease.

A recent paradigm shift appears to be underway on what scientists believe to be the cause of Alzheimer's disease (AD). The amyloid hypothesis has dominated the field of basic research for the last 25 years, and although these massive efforts have culminated in efficient removal of amyloid from the brains of patients, the absence of beneficial effects for the patient have been greatly disappointing. This has created a shift in the focus on amyloid to a much greater focus on Tau protein, in the hope that preventing tangle formation may inhibit or delay the progression of AD. Although there are promising developments in this area of research, diversifying our efforts to identify novel early targets by understanding the upstream molecular mechanisms that lead to, or occur with, neurofibrillary tangle and plaque formation may provide more efficient therapies against AD. Among many areas in development, an emphasis on the role of caspase-6 (Casp6) activity in early neurodegenerative mechanisms brings hope of a novel target against AD. Casp6 activity is intimately associated with the pathologies that define AD, correlates well with lower cognitive performance in aged individuals, and is involved in axonal degeneration in several cellular and in vivo animal models. This is a review of the evidence showing the relevance of Casp6 activation as an early event that could be inhibited to prevent the progression of AD.

Fyn, a potential target for Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia characterized by the presence of amyloid-ß (Aß) plaques and neurofibrillary tangles. The mechanisms leading to AD are not completely understood; however, recent evidence suggests that alterations in Fyn, a Src family kinase, might contribute to AD pathogenesis. A number of studies have demonstrated that Fyn is involved in synaptic plasticity, a cellular mechanism for learning and memory. In addition, Fyn plays a role in the regulation of Aß production and mediates Aß-induced synaptic deficits and neurotoxicity. Fyn also induces tyrosine phosphorylation of tau. Although many studies have implicated a role for Fyn in AD, the precise cellular and molecular mechanisms require further investigation. Novel insights into the role of Fyn in AD may help identify alternative pharmacological approaches for the treatment of AD.

Role of protein phosphatase 2A in Alzheimer's disease.

Alzheimer disease (AD) is the most common cause of dementia in adults. Aberrant hyperphosphorylation of microtubule associated protein Tau and neurofilament-M/H is one of the pathological hallmarks of AD. Most of the therapeutic strategies for treating AD are based on the inhibition of protein kinases such as glycogen synthase kinase-3ß, cyclin-dependent kinase 5, and other Tau kinases. Here, we focus on protein phosphatase 2A (PP2A) as a key player in AD. PP2A expression and activity are downregulated in AD brain, contributing to the aberrant phosphorylation of Tau and NF proteins in AD. Recent data published from our lab as well as others on PP2A deregulation in AD is reviewed. The role of peptidyl prolyl isomerase Pin1 in regulation of PP2A mediated neurodegeneration is further analyzed. Development of drugs for AD could be based on restoration of PP2A activity or targeting Pin1.

Link between DYRK1A overexpression and several-fold enhancement of neurofibrillary degeneration with 3-repeat tau protein in Down syndrome.

Triplication of chromosome 21 in Down syndrome (DS) results in overexpression of the minibrain kinase/dual-specificity tyrosine phosphorylated and regulated kinase 1A gene (DYRK1A). DYRK1A phosphorylates cytoplasmic tau protein and appears in intraneuronal neurofibrillary tangles (NFTs). We have previously shown significantly more DYRK1A-positive NFTs in DS brains than in sporadic Alzheimer disease (AD) brains. This study demonstrates a gene dosage-proportional increase in the level of DYRK1A in DS in the cytoplasm and the cell nucleus, and enhanced cytoplasmic and nuclear immunoreactivity of DYRK1A in DS. The results suggest that overexpressed DYRK1A may alter both phosphorylation of tau and alternative splicing factor (ASF). Two-dimensional electrophoresis revealed modification of ASF phosphorylation in DS/AD and AD in comparison to controls. Altered phosphorylation of ASF by overexpressed nuclear DYRK1A may contribute to the alternative splicing of the tau gene and an increase by 2.68 × of the 3R/4R ratio in DS/AD, and a several-fold increase in the number of 3R tau-positive NFTs in DS/AD subjects compared with that in sporadic AD subjects. These data support the hypothesis that phosphorylation of ASF by overexpressed DYRK1A may contribute to alternative splicing of exon 10, increased expression of 3R tau, and early onset of neurofibrillary degeneration in DS.

[GSK-3beta: a central kinase for neurodegenerative diseases?]. / GSK-3bêta :une kinase au coeur des maladies neuro-dégénératives?

Neurodegenerative diseases are more and more prevalent in our aging societies. There is strong evidence that glycogen synthase kinase (GSK)-3b plays a crucial role in Alzheimer's disease (AD). Indeed, it is involved in the regulation of the two major neuropathological hallmarks present in the brains of AD patients. Interestingly, the kinase has been implicated in multiple cellular processes and linked with the pathogenesis and neuronal loss in several neurodegenerative diseases, including Parkinson's and Huntington's diseases, in which abnormally elevated levels of GSK-3b activity have been reported. In this review, we will provide an overview of the current data pointing out the convergent role of GSK-3b in the neuropathological pathways of these diseases. We will also discuss the rationale for the development of specific inhibitors with therapeutic potentials for such devastating human diseases.

Protein disulfide isomerase-immunopositive inclusions in patients with Alzheimer disease.

Alzheimer disease (AD) is the most common neurodegenerative disease, but there is currently no effective treatment available because the etiology or mechanism of AD is still unclear. Many neurodegenerative diseases feature inclusions, which contain accumulations of misfolded, aggregated proteins. Amyloid plaques and neurofibrillary tangles (NFTs) are the major pathological hallmarks of AD. NFTs are composed of tubular filaments, and paired helical filaments containing polymerized hyperphosphorylated tau protein. Another feature is excessive generation of nitric oxide synthetase, reactive nitrogen species, and reactive oxygen species. Protein disulfide isomerase (PDI) is a member of the thioredoxin (TX) superfamily and is believed to accelerate the folding of disulfide-bonded proteins by catalyzing the disulfide interchange reaction, which is the rate-limiting step during protein folding in the luminal space of the endoplasmic reticulum (ER). Nitric oxide (NO)-induced S-nitrosylation of PDI inhibits its enzymatic activity, leading to the accumulation of polyubiquitinated proteins, and activates the unfolded protein response in neurodegenerative diseases. In this study, we found NFTs positive for anti-PDI-antibody in the brain of patients with AD. As far as we know, this is the first report of anti-PDI-antibody-immunopositive inclusions in AD. In AD, NO may inhibit PDI by inducing S-nitrosylation, which inhibits its enzymatic activity and thus allows protein misfolding to occur. Consequently, the accumulation of misfolded proteins induces ER stress. The ER stress can cause apoptosis of neuronal cells. These results suggest that PDI could be a therapeutic target to prevent ER stress in neuronal cells in AD.

Caspase activation precedes and leads to tangles.

Studies of post-mortem tissue have shown that the location of fibrillar tau deposits, called neurofibrillary tangles (NFT), matches closely with regions of massive neuronal death, severe cytological abnormalities, and markers of caspase activation and apoptosis, leading to the idea that tangles cause neurodegeneration in Alzheimer's disease and tau-related frontotemporal dementia. However, using in vivo multiphoton imaging to observe tangles and activation of executioner caspases in living tau transgenic mice (Tg4510 strain), we find the opposite: caspase activation occurs first, and precedes tangle formation by hours to days. New tangles form within a day. After a new tangle forms, the neuron remains alive and caspase activity seems to be suppressed. Similarly, introduction of wild-type 4-repeat tau (tau-4R) into wild-type animals triggered caspase activation, tau truncation and tau aggregation. Adeno-associated virus-mediated expression of a construct mimicking caspase-cleaved tau into wild-type mice led to the appearance of intracellular aggregates, tangle-related conformational- and phospho-epitopes, and the recruitment of full-length endogenous tau to the aggregates. On the basis of these data, we propose a new model in which caspase activation cleaves tau to initiate tangle formation, then truncated tau recruits normal tau to misfold and form tangles. Because tangle-bearing neurons are long-lived, we suggest that tangles are 'off pathway' to acute neuronal death. Soluble tau species, rather than fibrillar tau, may be the critical toxic moiety underlying neurodegeneration.

Neurochemical basis for symptomatic treatment of Alzheimer's disease.

Neuron and synapse loss together with neurotransmitter dysfunction have, along with Abeta deposition and neurofibrillary tangles, been recognized as hallmarks of Alzheimer's disease (AD). Furthermore, clinical and preclinical studies point to neuronal loss and associated neurochemical alterations of several transmitter systems as a main factor underlying both cognitive and neuropsychiatric symptoms. Treatment for the cognitive decline in AD, based on early findings of a cholinergic deficit, has been in the clinic for more than a decade but provides only modest benefit in most patients. Therefore there is still considerable scope for new treatments that demonstrate greater efficacy against cognitive dysfunction in spite of the fact that the mainstays of current treatments, the cholinesterase inhibitors Aricept, Exelon and Reminyl (Razadyne) will become generic over the next few years. However, the most important area for drug development is for the treatment of behavioural disturbance in AD since many existing treatments have limited efficacy and have potentially life-threatening side effects. This review examines the neurochemical underpinning of both cognitive and neuropsychiatric symptoms in dementia and provides some basis for rational drug development.

A peptidyl-prolyl isomerase, FKBP12, accumulates in Alzheimer neurofibrillary tangles.

We investigated a possible role in Alzheimer's disease (AD) for FKBP12, a peptidyl-prolyl cis-trans isomerase known to be important in protein assembly, folding and transportation by using Western blotting and microscopic analyses in postmortem brain tissues from elderly controls and the patients with AD. FKBP12 was enriched and localized to neuronal cell bodies and neurites in control brains. Intense immunoreactivity was found in large neurons such as pyramidal cells. Many FKBP12 positive granules were located in the cytoplasm and the proximal portion of dendrites and axons, and in the nuclei. By contrast, the expression of FKBP12 in AD brains was lower than in control brains. Furthermore, numerous intracellular neurofibrillary tangles (NFTs) were stained for FKBP12 in the hippocampal CA1 subfield, subiculum, entorhinal cortex and angular gyrus. Neuritic pathology such as neuropil threads and dystrophic neurites (DNs) within senile plaques (SPs) and some reactive astrocytes were also immunolabeled for FKBP12 in AD. Double immunofluorescence staining showed dual labeling of intracellular NFTs for FKBP12 and tau. Similar results were obtained in reactive astrocytes for the combination of FKBP12 and glial fibrillary acidic protein (GFAP). Labeling for FKBP12 was dense in axons stained for highly phosphorylated neurofilament protein. Thus our results suggest that FKBP12 may be involved in neuronal or astrocytic cytoskeletal organization and in the abnormal metabolism of tau protein in AD damaged neurons.

Association of glucocerebrosidase mutations with dementia with lewy bodies.

BACKGROUND: Mutations in the glucocerebrosidase (GBA) gene are associated with Lewy body (LB) disorders. OBJECTIVE: To determine the relationship of GBA mutations and APOE4 genotype to LB and Alzheimer disease (AD) pathological findings. DESIGN: Case-control study. SETTING: Academic research. PARTICIPANTS: The 187 subjects included patients with primary neuropathological diagnoses of LB disorders with or without AD changes (95 cases), randomly selected patients with AD (without significant LB pathological findings; 60 cases), and controls with neither LB nor AD pathological findings (32 cases). MAIN OUTCOME MEASURES: GBA mutation status, APOE4 genotype, LB pathological findings (assessed according to the third report of the Dementia With Lewy Body Consortium), and Alzheimer plaque and tangle pathological findings (rated by criteria of Braak and Braak, the Consortium to Establish a Registry for Alzheimer Disease, and the National Institute on Aging-Reagan Institute). RESULTS: GBA mutations were found in 18% (34 of 187) of all subjects, including 28% (27 of 95) of those with primary LB pathological findings compared with 10% (6 of 60) of those with AD pathological findings and 3% (1 of 32) of those without AD or LB pathological findings (P=.001). GBA mutation status was significantly associated with the presence of cortical LBs (odds ratio, 6.48; 95% confidence interval, 2.45-17.16; P<.001), after adjusting for sex, age at death, and presence of APOE4. GBA mutation carriers were significantly less likely to meet AD pathological diagnostic (National Institute on Aging-Reagan Institute intermediate or high likelihood) criteria (odds ratio, 0.35; 95% confidence interval, 0.15-0.79; P=.01) after adjustment for sex, age at death, and APOE4. CONCLUSION: GBA mutations may be associated with pathologically "purer" LB disorders, characterized by more extensive (cortical) LB, and less severe AD pathological findings and may be a useful marker for LB disorders.

Substrate-specific reduction of PP2A activity exaggerates tau pathology.

Phosphorylation of the microtubule-associated protein tau is regulated by the balanced interplay of kinases and phosphatases. Disturbance of this balance causes hyperphosphorylation of tau and neurofibrillary tangle formation in Alzheimer's disease brain. Here, we crossed Dom5 mice that express a substrate-specific dominant negative mutant form, L309A Calpha, of protein phosphatase 2A (PP2A) with neurofibrillary-tangle-forming P301L mutant tau transgenic pR5 mice. This exacerbated the tau pathology of pR5 mice significantly. Double-transgenic Dom5/pR5 mice showed 7-fold increased numbers of hippocampal neurons that specifically phosphorylated the pathological S422 epitope of tau. They showed 8-fold increased numbers of tangles compared to pR5 mice, in agreement with our previous finding that tangle formation is correlated with and preceded by phosphorylation of tau at the S422 epitope. This suggests that, in addition to kinases, PP2A and its regulatory subunits may be a therapeutic target for Alzheimer's disease.