Altered Expression Levels of the Protein Phosphatase 2A ABαC Enzyme Are Associated with Alzheimer Disease Pathology

The formation of amyloid-containing senile plaques and tau-rich neurofibrillary tangles are central events in Alzheimer disease (AD) pathogenesis. Significantly, ABαC, a major protein phosphatase 2A (PP2A) holoenzyme, specifically binds to and dephosphorylates tau. Deregulation of PP2A results in tau hyperphosphorylation in vivo. Here, we compared the expression levels and distribution of PP2A subunits in various brain regions from autopsy cases of AD and aged controls with or without histological evidence of age-related neurofibrillary degeneration. Immunoblotting analyses revealed that there was a significant reduction in the total amounts of ABαC in AD frontal and temporal cortices that matched the decrease in PP2A activity measured in the same brain homogenates. Immunohistochemical studies showed that neuronal ABαC expression levels were significantly and selectively decreased in AD-affected regions and in tangle-bearing neurons, but not in AD cerebellum and in non-AD dementias. Reduced neuronal ABαC immunoreactivity closely correlated with tangle load, but not plaque burden, suggesting that ABαC dysfunction contributes to AD tau pathology. Glial cells within senile plaques were also positive for ABαC. Increased glial PP2A immunoreactivity was observed in both AD and non-AD cases and may play a role in the brain's response to general inflammatory processes and amyloidogenesis.

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title: Tau Protein and the Neurofibrillary Pathology of Alzheimer's Disease
abstract: Abundant neurofibrillary tangles, neuropil threads and senile plaque neurites constitute the neurofibrillary pathology of Alzheimer's disease. They form in the nerve cells that undergo degeneration in the disease, in which their regional distribution correlates with the degree of dementia. Each lesion contains the paired helical filament (PHF) as its major component. PHFs are composed of the microtubule‐associated protein tau in a hyperphosphorylated state. PHF‐tau is hyperphosphorylated on all six adult brain isoforms. As a consequence, tau is unable to bind to microtubules and is believed to self‐assemble into the PHF. Several candidate protein kinases and protein phosphatases have been identified through in vitro experiments.

title: Phosphorylation of paired helical filament tau in Alzheimer's disease neurofibrillary lesions: focusing on phosphatases
abstract: The major brain abnormalities in patients with Alzheimer's disease (AD) include extracellular deposits of β‐amyloid, intraneuronal neurofibrillary lesions, and the massive loss of specific subsets of telencephalic neurons. Neurofibrillary lesions contain paired helical filaments (PHFs) that accumulate in the perikarya (e. g., neurofibrillary tangles) and processes (e. g., dystrophic neurites, neuropil threads) of selectively vulnerable telencephalic neurons. The subunits of PHFs are derivatized forms of central nervous system (CNS) tau proteins (CNS tau) known collectively as PHFtau. Until very recently it was thought that the aberrant phosphorylation of PHFtau was the most salient difference between normal human CNS tau and PHFtau. However, this view required modification when it was shown that fetal and biopsy‐derived human CNS tau proteins were phosphorylated at nearly all of the same sites found in PHFtau, albeit to a lesser extent. More significantly, these and other studies may be interpreted to suggest that the abnormal phosphorylation of PHFtau may result from the failure of protein phosphatases (i. e., PP2A and 2B) to dephosphorylate PHFtau. In this review, we summarize current understanding of the role of kinases and phosphatases in pathogenesis of PHFtau. We then consider how the formation PHFtau in neurons could disrupt the microtubule network, impair axonal transport, and compromise the viability of neurons, thereby contributing to the onset and progression of AD.—Trojanowski, J. Q., Lee, V. M.‐Y. Phosphorylation of paired helical filament tau in Alzheimer's disease neurofibrillary lesions: focusing on phosphatases.

title: Regulation of the Phosphorylation State and Microtubule-Binding Activity of Tau by Protein Phosphatase 2A
abstract: Recently, we reported that a pool of protein phosphatase 2A (PP2A) is associated with microtubules. Here, we demonstrate that specific isoforms of PP2A bind and dephosphorylate the neuronal microtubule-associated protein tau. Coexpression of tau and SV40 small t, a specific inhibitor of PP2A, in CV-1, NIH 3T3, or NT2 cells induced the phosphorylation of tau at multiple sites, including Ser-199, Ser-202, Thr-205, Ser-396, and Ser-404. Immunofluorescent and biochemical analyses revealed that hyperphosphorylation correlated with dissociation of tau from microtubules and a loss of tau-induced microtubule stabilization. Taken together, these results support the hypothesis that PP2A controls the phosphorylation state of tau in vivo.

title: Protein phosphatase 2A is the major enzyme in brain that dephosphorylates tau protein phosphorylated by proline-directed protein kinases or cyclic AMP-dependent protein kinase.
abstract: The paired helical filament (PHF), which makes up the major fibrous component of the neurofibrillary lesions of Alzheimer's disease, is composed of hyperphosphorylated and abnormally phosphorylated microtubule-associated protein tau. Previous studies have identified serine and threonine residues phosphorylated in PHF-tau and have shown that tau can be phosphorylated at several of these sites by proline-directed protein kinases and cyclic AMP-dependent protein kinase. Here we have investigated which protein phosphatase activities can dephosphorylate recombinant tau phosphorylated with mitogen-activated protein kinase, glycogen synthase kinase-3 beta, neuronal cdc2-like kinase, or cyclic AMP-dependent protein kinase. We show that protein phosphatase 2A is by far the major protein phosphatase activity in brain that dephosphorylates tau phosphorylated in this manner.

title: Paired helical filament-like phosphorylation of tau, deposition of β/A4-amyloid and memory impairment in rat induced by chronic inhibition of phosphatase 1 and 2A
abstract: Alzheimer's disease is histopathologically characterized by neurofibrillary tangles, formed by the abnormally high phosphorylated tau protein, and senile plaques which largely consist of the beta/A4-amyloid peptide. Metabolism of the amyloid precursor protein and its processing into beta/A4-amyloid is regulated by protein phosphorylation. Thus, an imbalance between protein phosphorylation and dephosphorylation might be crucial for the development of the molecular hallmarks of Alzheimer's disease. We report here that chronic infusion into rat brain ventricles of okadaic acid, a specific inhibitor of the serine/threonine protein phosphatases 1 and 2A, results in a severe memory impairment, accompanied by a paired helical filament-like phosphorylation of tau protein and the formation of beta/A4-amyloid containing plaque-like structures in gray and white matter areas.

title: Neurotoxic and Synaptic Effects of Okadaic Acid, an Inhibitor of Protein Phosphatases
abstract: Protein phosphorylation and dephosphorylation reactions, catalyzed by kinases and phosphatases, are involved in the regulation of a wide variety of physiological processes. In the nervous system, such reactions seem to modulate the function of several proteins crucial in synaptic transmission, including voltage-gated and ligand-gated channels, neurotransmitter release, and neurotransmitter transporters. On the other hand, hyperphosphorylation of certain cytoskeletal proteins or receptors may lead to neuronal death. In the present work we review the neurotoxic effect of okadaic acid (OKA), a potent and specific inhibitor of the serine/threonine protein phosphatases 1 and 2A, as well as its action on synaptic function. We analyze recent findings demonstrating that the microinjection of OKA in rat hippocampus induces neuronal stress, hyperexcitation and neurodegeneration, and discuss their possible relationships to alterations of protein phosphorylation-dephosphorylation observed in Alzheimer's disease brain. These results suggest that protein hyperphosphorylation due to inhibition of phosphatases in vivo induces neuronal stress and subsequent neurodegeneration.

title: Spatial memory deficit and neurodegeneration induced by the direct injection of okadaic acid into the hippocampus in rats
abstract: We investigated the effects of okadaic acid (OA), a specific inhibitor of protein phosphatases 1 and 2A, on spatial memory and neuronal survival in rats. Rats were initially trained on a spatial memory task in an eight arm radial maze. Spatial reference and working memory was impaired 1 day after the unilateral microinjection of OA into the dorsal hippocampus. The impairment was transient, and had disappeared by the following day. In contrast, neurodegeneration induced by OA was persistent and extended to the contralateral side 13 days after the injection. These results suggest that OA causes spatial memory impairment and neurodegeneration when injected directly into the hippocampus. Our findings also indicate dissociation between memory impairment and neurodegeneration induced by OA.

title: Reduced protein phosphatase 2A activity induces hyperphosphorylation and altered compartmentalization of tau in transgenic mice.
abstract: Hyperphosphorylated isoforms of the microtubule-associated protein tau are the major components of neurofibrillary lesions in Alzheimer's disease (AD). Protein phosphatase (PP) 2A is a major phosphatase implicated in tau dephosphorylation in vitro. Dephosphorylation of tau can be blocked in vivo by okadaic acid, a potent inhibitor of PP2A. Moreover, activity of PP2A is reduced in AD brains. To elucidate the role of PP2A in tau phosphorylation and pathogenesis, we expressed a dominant negative mutant form of the catalytic subunit Calpha of PP2A, L199P, in mice by using a neuron-specific promoter. We obtained mice with high expression levels of Calpha L199P in cortical, hippocampal, and cerebellar neurons. PP2A activity in brain homogenates of transgenic mice was reduced to 66%. Endogenous tau protein was hyperphosphorylated at distinct sites including the AT8 epitope Ser-202/Thr-205, a major AD-associated tau phosphoepitope. AT8-positive tau aggregates accumulated in the soma and dendrites of cortical pyramidal cells and cerebellar Purkinje cells and co-localized with ubiquitin. Our data establish that PP2A plays a crucial role in tau phosphorylation. Our results also show that reduced PP2A activity is associated with altered compartmentalization and ubiquitination of tau, resembling a key pathological finding in AD.

title: B56-Associated Protein Phosphatase 2A Is Required For Survival and Protects from Apoptosis in Drosophila melanogaster
abstract: Protein phosphorylation and specific protein kinases can initiate signal transduction pathways leading to programmed cell death. The specific protein phosphatases regulating apoptosis have been more elusive. Using double-stranded RNA-mediated interference (RNAi), the role of protein phosphatase 2A (PP2A) in cellular signaling was investigated. Knockdown of A or C subunits individually or of combined B subunits led to concurrent loss of nontargeted PP2A subunits, suggesting that PP2A is an obligate heterotrimer in vivo. Global knockdown of PP2A activity or specific loss of redundant B56 regulatory subunits caused cell death with the morphological and biochemical changes characteristic of apoptosis in cultured S2 cells. PP2A-regulated apoptosis required caspases and the upstream regulators dark, reaper, head involution defective, and dp53. In Drosophila embryos, knockdown of B56-regulated PP2A activity resulted in apoptosis and failure of gastrulation, an effect that was blocked by concurrent RNAi of the caspase Drice. B56-regulated PP2A activity appears to be required upstream of dp53 to maintain a critical proapoptotic substrate in a dephosphorylated, inactive state, thereby preventing apoptosis in Drosophila S2 cells.

title: Protein phosphatase 2A: the Trojan Horse of cellular signaling.
abstract: Dynamic phosphorylation and dephosphorylation of proteins are fundamental mechanisms utilized by cells to transduce signals. Whereas transduction by protein kinases has been a major focus of studies in the last decade, protein phosphatase 2A (PP2A) enzymes emerge in this millenium as the most fashionable players in cellular signaling. Viral proteins target specific PP2A enzymes in order to deregulate chosen cellular pathways in the host and promote viral progeny. The observation that a variety of viruses utilize PP2A to alienate cellular behavior emphasizes the fundamental importance of PP2A in signal transduction. This review will primarily focus on discussing the uniqueness of PP2A regulation and uncovering the critical role played by protein-protein interactions in the modulation of PP2A signaling. Moreover, the place of PP2A in signaling pathways and its functional significance for human diseases will be discussed.

title: Brain protein phosphatase 2A: Developmental regulation and distinct cellular and subcellular localization by B subunits
abstract: Protein phosphatase 2A (PP2A) is a heterotrimeric enzyme consisting of a catalytic subunit (C), a structural subunit (A), and a variable regulatory subunit (B). We have investigated the spatial and temporal expression patterns of three members of the B subunit family, Bα, Bβ, and Bγ, both at the message level by using ribonuclease protection analysis and at the protein level by using specific antibodies. Although A, Bα, and C protein are expressed in many tissues, Bβ and Bγ were detectable only in brain. Bα, Bβ, and Bγ are components of the brain PP2A heterotrimer, because they copurified with A and C subunits on immobilized microcystin. Whereas Bα and Bβ are mainly cytosolic, Bγ is enriched in the cytoskeletal fraction. In contrast to A, C, and Bα, which are expressed at constant levels, Bβ and Bγ RNA and protein are developmentally regulated, with Bβ levels decreasing and Bγ levels increasing sharply after birth. RNA and immunoblot analyses of subdissected brain regions as well as immunohistochemistry demonstrated that B subunits are expressed in distinct but overlapping neuronal populations and cellular domains. These data indicate that B subunits confer tissue and cell specificity, subcellular localization, and developmental regulation to the PP2A holoenzyme. The Bα‐containing heterotrimer may be important in general neuronal functions that involve its partially nuclear localization. Holoenzymes containing Bβ likely function in early brain development as well as in somata and processes of subsets of mature neurons. Bγ may target PP2A to cytoskeletal substrates that are important in the establishment and maintenance of neuronal connections.  1998.

title: Developmental expression and co-localization of cyclin G1 and the B' subunits of protein phosphatase 2a in neurons.
abstract: Cyclin G1 is a recently cloned transcriptional target of p53, it is located in neurons and ventricular ependymal cells and is elevated in neurons after axotomy and cerebral ischemia. The biological function for cyclin G1 in differentiated neurons has thus far not been elucidated. Recently, cyclin G1 has been shown to interact with the B' subunits of serine/threonine protein phosphatase 2A (PP2A) in a rat fibroblast cell line [K. Okamoto, C., Kamibayashi, M. Serrano, C. Prives, M.C. Mumby, D. Beach, p53-dependent association between cyclin G and the B' subunit of protein phosphatase 2A,  (1996) 6593-6602]. To further explore whether a similar interaction between cyclin G1 and PP2A B' subunits exists in the central nervous system, the present study compared the regional and developmental expression pattern, subcellular distribution and complex formation between cyclin G1 and the PP2A B' regulatory subunits in the rat brain. In situ hybridization of cyclin G1 and the B'alpha and B'beta subunits of PP2A showed an overlapping distribution in neurons of the cerebral cortex, hippocampus and thalamus at embryonic and early postnatal ages, but their developmental regulation differed. Whereas mRNA and protein levels of PP2A B' subunits were high in the cortical plate, subiculum, hippocampal areas and thalamus at E20 and decreased with age, those of cyclin G1 increased with age and were maximal in the adult cortex and hippocampus. In rat 14-day-old embryonic cortical cultures, cyclin G1 and PP2A B'alpha protein co-localized in nuclear and perinuclear areas of neurons, and both proteins were highly expressed in nuclei of cortical and hippocampal pyramidal cells and the mitral cell layer of the neonatal olfactory bulb. Both cyclin G1 and the PP2A regulatory B'alpha subunits were specifically expressed in neurons and not in glial cells. Antibodies raised against the B'alpha subunits of PP2A immunoprecipitated cyclin G1 in adult cortical lysates, indicating the presence of a complex involving cyclin G1 and the B'alpha subunits of PP2A. This study shows that the regional and subcellular localization of PP2A B' regulatory subunits and cyclin G1 are very similar at early postnatal stages. We discuss the possible functions of a cyclin G1-PP2A B'alpha complex in neurons.

title: Diversity, developmental regulation and distribution of murine PR55/B subunits of protein phosphatase 2A
abstract: Protein phosphatase (PP2A) 2A is a hetero‐trimeric holoenzyme that consists of a core dimer composed of a catalytic subunit that is tightly complexed with the scaffolding subunit PR65/A. This core dimer associates with variable regulatory subunits of the PR55/B, PR61/B′, PR72/B′′ and PR93/PR110/B′′′ families. As PP2A holoenzymes containing PR55/B have been shown to be involved in the pathogenesis of Alzheimer's disease, we characterized the PR55/B family with particular emphasis on its distribution and expression in the brain. We determined the genomic organization of all members of the PR55/B family and cloned their murine cDNAs. Thereby, two novel splice variants of PR55/Bβ were identified. In addition, Northern blot analysis revealed multiple transcripts for the different PR55 subunits, suggesting a higher variability within the PR55 family. In situ hybridization analysis revealed that all PR55/B subunits were widely expressed in the brain. PR55/Bα and Bβ protein expression varies significantly in areas of the brain affected by neurodegenerative diseases such as the hippocampus or cerebellum. At the cellular level, PR55/Bβ protein expression was confined to neurons, whereas PR55/Bα was also expressed in activated astrocytes indicating that the PR55 isoforms confer a different function to the holoenzyme complex. As PP2A dysfunction has been demonstrated to contribute to various human diseases, dissecting the PP2A holoenzyme and its particular function in different cell types will assist in the development of novel therapeutic strategies.

title: Characterization of polyclonal antibodies to brain protein phosphatase 2A and immunohistochemical localization of the enzyme in rat brain
abstract: Polyclonal antibodies to the catalytic subunit of protein phosphatase 2A (PrP-2A) from bovine brain were prepared by immunizing rabbits and then purified by antigen-affinity column chromatography. The purified antibodies recognized only PrP-2A among proteins examined, including calcineurin and PrP-1. The antibodies cross-reacted only with a protein in the crude homogenate from rat brain, which comigrated with the catalytic subunit of PrP-2A on SDS-PAGE. The antibodies completely inhibited the activity of PrP-2A, and immunoprecipitated the purified enzyme. Immunoblot analysis demonstrates that, among the subcellular fractions from rat brain, the cytosol fraction and the synaptosomal cytosol fraction show high immunoreactivities, and that any of examined regions of rat brain shows immunoreactivity more or less, in which the caudatoputamen was highest. Immunohistochemical studies demonstrate that the enzyme is distributed widely in various regions of rat brain and that the immunoreactivity is localized mainly in neurons. In general, immunostaining of neurons was strong in neurites as well as somata. It was noted that intracerebellar nuclei were strongly stained in both neuronal somata and dendrites. Amygdaloid complex, thalamus, neocortex, hippocampal formation, and caudatoputamen were moderately stained.

title: Phosphatase Activity Toward Abnormally Phosphorylated τ: Decrease in Alzheimer Disease Brain
abstract: Microtubule‐associated protein τ is abnormally hyperphosphorylated and aggregated in affected neurons of Alzheimer disease brain. This hyperphosphorylated τ can be dephosphorylated at some of the abnormal phosphorylated sites by purified protein phosphatase‐1, 2A, and 2B in vitro. In the present study, we have developed an assay to measure protein phosphatase activity toward τ‐1 sites (Ser199/Ser202) using the hyperphosphorylated τ isolated from Alzheimer disease brain as substrate. Using this assay, we have identified that in normal brain, protein phosphatase‐2A and 2B and, to a lesser extent, 1 are involved in the dephosphorylation of τ. The Km values of dephosphorylation of the hyperphosphorylated τ by protein phosphatase‐2A and 2B are similar. The τ phosphatase activity is decreased by ∼30% in brain of Alzheimer disease patients compared with those of age‐matched controls. These findings suggest that a defect of protein phosphatase could be the cause of the abnormal hyperphosphorylation of τ in Alzheimer disease.

title: The effects of aging on gene expression in the hypothalamus and cortex of mice.
abstract: A better understanding of the molecular effects of aging in the brain may help to reveal important aspects of organismal aging, as well as processes that lead to age-related brain dysfunction. In this study, we have examined differences in gene expression in the hypothalamus and cortex of young and aged mice by using high-density oligonucleotide arrays. A number of key genes involved in neuronal structure and signaling are differentially expressed in both the aged hypothalamus and cortex, including synaptotagmin I, cAMP-dependent protein kinase C beta, apolipoprotein E, protein phosphatase 2A, and prostaglandin D. Misregulation of these proteins may contribute to age-related memory deficits and neurodegenerative diseases. In addition, many proteases that play essential roles in regulating neuropeptide metabolism, amyloid precursor protein processing, and neuronal apoptosis are up-regulated in the aged brain and likely contribute significantly to brain aging. Finally, a subset of these genes whose expression is affected by aging are oppositely affected by exposure of mice to an enriched environment, suggesting that these genes may play important roles in learning and memory.

title: A gene expression profile of Alzheimer's disease.
abstract: Postmortem analysis of brains of patients with Alzheimer's disease (AD) has led to diverse theories about the causes of the pathology, suggesting that this complex disease involves multiple physiological changes. In an effort to better understand the variety and integration of these changes, we generated a gene expression profile for AD brain. Comparing affected and unaffected brain regions in nine controls and six AD cases, we showed that 118 of the 7050 sequences on a broadly representative cDNA microarray were differentially expressed in the amygdala and cingulate cortex, two regions affected early in the disease. The identity of these genes suggests the most prominent upregulated physiological correlates of pathology involve chronic inflammation, cell adhesion, cell proliferation, and protein synthesis (31 upregulated genes). Conversely, downregulated correlates of pathology involve signal transduction, energy metabolism, stress response, synaptic vesicle synthesis and function, calcium binding, and cytoskeleton (87 downregulated genes). The results support several separate theories of the causes of AD pathology, as well as add to the list of genes associated with AD. In addition, approximately 10 genes of unknown function were found to correlate with the pathology.

title: PP2A mRNA Expression Is Quantitatively Decreased in Alzheimer's Disease Hippocampus
abstract: Since abnormal tau phosphorylation may play a role in neurofibrillary tangle (NFT) formation in aging and Alzheimer's disease (AD), we probed the distribution and abundance of protein phosphatase 2A (PP2A) catalytic (Calpha) and regulatory (PR55alpha and gamma, PR61varepsilon and delta) subunit mRNA in control and AD hippocampus using in situ hybridization. Quantitation of grain density per neuron area of PP2A subunits and beta-actin was determined for the CA3 region of hippocampus and cerebellum, while a qualitative assessment was performed for CA1, CA4, and dentate gyrus. All subunits are expressed in neurons, while PR55gamma and PR55alpha mRNA are also evident in glia. The expression levels of Calpha, all PP2A regulatory subunits studied, and beta-actin were similar in control and AD cerebellum. beta-Actin mRNA was, however, reduced in AD hippocampus. In addition to the generalized reduction of mRNA, as indicated by decreased beta-actin signal, there was a significant loss of Calpha, PR55gamma, and PR61epsilon mRNA in the CA3 hippocampus of AD. This study delineates the distribution of critical PP2A mRNAs and reveals a neuron- and subunit-specific reduction in PP2A catalytic and regulatory mRNA in AD hippocampus. This could result in decreased protein expression and phosphatase activity, leading to the hyperphosphorylation of tau and the formation of NFTs, as well as neuron degeneration in AD.

title: The Consortium to Establish a Registry for Alzheimer's Disease (CERAD)
abstract: The Neuropathology Task Force of the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) has developed a practical and standardized neuropathology protocol for the postmortem assessment of dementia and control subjects. The protocol provides neuropathologic definitions of such terms as “definite Alzheimer's disease” (AD), “probable AD,” “possible AD,” and “normal brain” to indicate levels of diagnostic certainty, reduce subjective interpretation, and assure common language. To pretest the protocol, neuropathologists from 15 participating centers entered information on autopsy brains from 142 demented patients clinically diagnosed as probable AD and on eight nondemented patients. Eighty-four percent of the dementia cases fulfilled CERAD neuropathologic criteria for definite AD. As increasingly large numbers of prospectively studied dementia and control subjects are autopsied, the CERAD neuropathology protocol will help to refine diagnostic criteria, assess overlapping pathology, and lead to a better understanding of early subclinical changes of AD and normal aging.

title: Making the diagnosis of Alzheimer's disease. A primer for practicing pathologists.
abstract: There is demand on community pathologists to perform autopsies to confirm the clinical diagnosis of Alzheimer's disease, the most common cause of dementia in our increasingly aging society. Yet many pathologists are reluctant to examine autopsy brains because they have little experience with neuropathology and with the common histopathologic staining methods needed to evaluate dementia cases. This article provides interested pathologists with a simple, practical protocol to use in meeting this demand. While there is no absolute diagnostic gold standard for Alzheimer's disease and the histopathologic diagnosis remains imperfect, the guidelines presented are adapted from those used by many neuropathologists at Alzheimer's disease research centers participating in CERAD, the Consortium to Establish a Registry for Alzheimer's Disease. Recipes for appropriate stains and specific case examples are provided for convenience.

title: Molecular Interactions among Protein Phosphatase 2A, Tau, and Microtubules
abstract: Hyperphosphorylated forms of the neuronal microtubule (MT)-associated protein tau are major components of Alzheimer’s disease paired helical filaments. Previously, we reported that ABαC, the dominant brain isoform of protein phosphatase 2A (PP2A), is localized on MTs, binds directly to tau, and is a major tau phosphatase in cells. We now describe direct interactions among tau, PP2A, and MTs at the submolecular level. Using tau deletion mutants, we found that ABαC binds a domain on tau that is indistinguishable from its MT-binding domain. ABαC binds directly to MTs through a site that encompasses its catalytic subunit and is distinct from its binding site for tau, and ABαC and tau bind to different domains on MTs. Specific PP2A isoforms bind to MTs with distinct affinities in vitro, and these interactions differentially inhibit the ability of PP2A to dephosphorylate various substrates, including tau and tubulin. Finally, tubulin assembly decreases PP2A activity in vitro, suggesting that PP2A activity can be modulated by MT dynamics in vivo. Taken together, these findings indicate how structural interactions among ABαC, tau, and MTs might control the phosphorylation state of tau. Disruption of these normal interactions could contribute significantly to development of tauopathies such as Alzheimer’s disease.

title: A novel pool of protein phosphatase 2A is associated with microtubules and is regulated during the cell cycle
abstract: Immunofluorescence microscopy revealed the presence of protein phosphatase 2A (PP2A) on microtubules in neuronal and nonneuronal cells. Interphase and mitotic spindle microtubules, as well as centrosomes, were all labeled with antibodies against individual PP2A subunits, showing that the AB alpha C holoenzyme is associated with microtubules. Biochemical analysis showed that PP2A could be reversibly bound to microtubules in vitro and that approximately 75% of the PP2A in cytosolic extracts could interact with microtubules. The activity of microtubule-associated PP2A was differentially regulated during the cell cycle. Enzymatic activity was high during S phase and intermediate during G1, while the activity in G2 and M was 20-fold lower than during S phase. The amount of microtubule-bound PP2A remained constant throughout the cell cycle, implying that cell cycle regulation of its enzymatic activity involves factors other than microtubules. These results raise the possibility that PP2A regulates cell cycle-dependent microtubule functions, such as karyokinesis and membrane transport.

title: Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex
abstract: Tight junctions (TJs) play a crucial role in the establishment of cell polarity and regulation of paracellular permeability in epithelia. Here, we show that upon calcium-induced junction biogenesis in Madin-Darby canine kidney cells, ABαC, a major protein phosphatase (PP)2A holoenzyme, is recruited to the apical membrane where it interacts with the TJ complex. Enhanced PP2A activity induces dephosphorylation of the TJ proteins, ZO-1, occludin, and claudin-1, and is associated with increased paracellular permeability. Expression of PP2A catalytic subunit severely prevents TJ assembly. Conversely, inhibition of PP2A by okadaic acid promotes the phosphorylation and recruitment of ZO-1, occludin, and claudin-1 to the TJ during junctional biogenesis. PP2A negatively regulates TJ assembly without appreciably affecting the organization of F-actin and E-cadherin. Significantly, inhibition of atypical PKC (aPKC) blocks the calcium- and serum-independent membrane redistribution of TJ proteins induced by okadaic acid. Indeed, PP2A associates with and critically regulates the activity and distribution of aPKC during TJ formation. Thus, we provide the first evidence for calcium-dependent targeting of PP2A in epithelial cells, we identify PP2A as the first serine/threonine phosphatase associated with the multiprotein TJ complex, and we unveil a novel role for PP2A in the regulation of epithelial aPKC and TJ assembly and function.

title: Subcellular distribution of protein phosphatases and abnormally phosphorylated τ in the temporal cortex from Alzheimer's disease and control brains
abstract: Microtubule-associated protein tau is abnormally hyperphosphorylated in the brain of patients with Alzheimer's disease (AD). In vitro studies have shown that protein phosphatases PP-2A and PP-2B can convert Alzheimer like tau to its normal state and that the activities of PP-1, PP-2A, and phosphotyrosyl-protein phosphatase (PTP) are reduced in AD brain. However, to have a direct effect on the regulation of phosphorylation on tau, these enzymes have to exist in neurons. Using specific polyclonal antibodies the levels of protein phosphatases PP-1, PP-2A, and PP-2B were determined by indirect ELISA in superior temporal cortical gray matter of AD and control brains. The protein levels of PP-2A and PP-2B were significantly increased in postsynaptosomal supernatant 2 (S2) of the AD group, and this alteration showed a significant linear correlation with levels of hyperphosphorylated tau. PP-1 and PTP-1B levels were not significantly changed in any of the AD fractions. Because of the large variation from case to case, the activity levels of none of the phosphatases investigated were significantly different between the AD and control groups. However, the PP-2B specific activity (activity/protein) showed a significant linear inverse correlation with hyperphosphorylated tau. These studies suggest that any attempt by the AD brain to compensate for the decreased tau phosphatase activity remains unsuccessful and that the decrease in phosphatase activity might contribute to increased levels of abnormally phosphorylated tau.

title: Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen.
abstract: Binding of different regulatory subunits and methylation of the catalytic (C) subunit carboxy-terminal leucine 309 are two important mechanisms by which protein phosphatase 2A (PP2A) can be regulated. In this study, both genetic and biochemical approaches were used to investigate regulation of regulatory subunit binding by C subunit methylation. Monoclonal antibodies selectively recognizing unmethylated C subunit were used to quantitate the methylation status of wild-type and mutant C subunits. Analysis of 13 C subunit mutants showed that both carboxy-terminal and active site residues are important for maintaining methylation in vivo. Severe impairment of methylation invariably led to a dramatic decrease in Balpha subunit binding but not of striatin, SG2NA, or polyomavirus middle tumor antigen (MT) binding. In fact, most unmethylated C subunit mutants showed enhanced binding to striatin and SG2NA. Certain carboxy-terminal mutations decreased Balpha subunit binding without greatly affecting methylation, indicating that Balpha subunit binding is not required for a high steady-state level of C subunit methylation. Demethylation of PP2A in cell lysates with recombinant PP2A methylesterase greatly decreased the amount of C subunit that could be coimmunoprecipitated via the Balpha subunit but not the amount that could be coimmunoprecipitated with Aalpha subunit or MT. When C subunit methylation levels were greatly reduced in vivo, Balpha subunits were found complexed exclusively to methylated C subunits, whereas striatin and SG2NA in the same cells bound both methylated and unmethylated C subunits. Thus, C subunit methylation is critical for assembly of PP2A heterotrimers containing Balpha subunit but not for formation of heterotrimers containing MT, striatin, or SG2NA. These findings suggest that methylation may be able to selectively regulate the association of certain regulatory subunits with the A/C heterodimer.

title: Expression of protein phosphatases (PP-1, PP-2A, PP-2B and PTP-1B) and protein kinases (MAP kinase and P34cdc2) in the hippocampus of patients with Alzheimer disease and normal aged individuals
abstract: Microtubule-associated protein tau is abnormally hyperphosphorylated in the brain of patients with Alzheimer disease (AD). Previous studies have shown (i) that in vitro tau can be phosphorylated to an Alzheimer abnormally phosphorylated state-like protein by proline-directed protein kinases MAP kinase and p34cdc2, and (ii) that the AD abnormally phosphorylated tau can be in vitro dephosphorylated by protein phosphatases PP-2B, PP-2A and PP-1 and not by PP-2C. However, to have a direct effect on the regulation of phosphorylation of tau, these enzymes should be present in the affected neurons. In the present study immunocytochemical localization of protein phosphatases PP-1, PP-2A, PP-2B and PTP, and protein kinases MAP kinase and p34cdc2 were studied in the hippocampal formation of AD and as a control in non-demented elderly patients. All the protein phosphatases and protein kinases studied were localized to both granular and pyramidal neurons. In the pyramidal neurons, the enzymes staining was observed in neuronal soma and neurites. PTP-1B, PP-1 and PP-2A were also highly expressed in microglia. The topographical distributions of all the enzymes studied were similar, i.e. the intensity of immunostaining in hippocampus in end-plate (CA3 and CA4) > prosubiculum, subiculum > entorhinal cortex > dentate gyrus > CA2 > CA1. Furthermore, the expression of all the enzymes was also observed in the tangle-bearing neurons. The PP-2B staining of the tangle-bearing neurons was weaker than the unaffected neurons in the same tissue section field in AD cases.

title: Elevated protein levels of protein phosphatases PP-2A and PP-2B in astrocytes of Alzheimer's disease temporal cortex
abstract: SummaryPrevious studies have shown that activities of the protein phosphatases PP-2A and PP-2B towards the microtubule associated protein tau are reduced in Alzheimer's disease (AD) frontal cortex (Gong et al., 1993, 1995), suggesting that PP-2A and PP-2B are involved in the hyper-phosphorylation of tau in AD. Most recently, we found that protein levels of PP-2A and PP-2B are elevated in postsynaptic supernatant (S2) fractions prepared from AD temporal cortex, and that the activities of these enzymes were not significantly different between AD and control cases (Pei et al., in press). In the present study, we found that astroglia positive for PP-2A and PP-2B immunoreactivities were greater in numbers in AD medial temporal cortex, compared to controls. GFAP levels, as determined by indirect ELISA, were approximately 1.5 times greater in the P1 (500 × g) fraction from AD temporal cortex, compared to controls. GFAP levels in the P1 fraction showed significant correlations with PP-2A and PP-2B levels in the postsynaptic S2 (20,000 × g) fraction from the same brains. These results suggest that astrogliosis probably accounts for the increased levels of PP-2A and PP-2B in the S2 fraction in AD brain and that the levels of these enzymes per neuron are likely to be decreased.

title: Tangle and neuron numbers, but not amyloid load, predict cognitive status in Alzheimer’s disease
abstract: To examine the relationship between stereologic estimates of AD-related pathology and severity of cognitive deficits in brain aging.  Previous studies reported substantial contributions of neurofibrillary tangles (NFT), amyloid deposits, and neuronal loss to the development of dementia. However, the prediction of cognitive status based on nonstereologic quantification of these measures has led to conflicting results. Such studies have measured densities, rather than absolute numbers, and most do not take into account the potential interaction between the above pathologic hallmarks in a global multivariate analysis.  Clinicopathologic study in 22 elderly cases. Cognitive status assessed prospectively using the Mini-Mental State Examination (MMSE); stereologic assessment of NFT, unaffected neurons, and total amyloid volume in the CA1 field of the hippocampus, entorhinal cortex, and area 9. Statistical analysis was performed using both univariate and multivariate linear regression models.  High total NFT counts but not amyloid volume were strongly associated with a lower number of unaffected neurons in all areas studied. A high proportion of variability in MMSE scores was explained by NFT and neuronal counts in the CA1 field (83% and 85.4%), entorhinal cortex (87.8% and 83.7%), and area 9 (87% and 79%); amyloid volume in the entorhinal cortex, but not in the CA1 field and area 9, accounted for 58.5% of MMSE variability. Multivariate analyses showed that total NFT counts in the entorhinal cortex and area 9 as well as neuron numbers in the CA1 field were the best predictors of MMSE score.  These new stereologic data indicate that neuronal pathology in hippocampal formation and frontal cortex closely reflects the progression of cognitive deficits in brain aging and AD. They also demonstrate that amyloid volume has no additional predictive value, in terms of clinicopathologic correlations, beyond its interaction with NFT.

title: Glia-related pathomechanisms in Alzheimer's disease: a therapeutic target?
abstract: Reactive glial cell properties could contribute to pathomechanisms underlying Alzheimer's disease by favoring oxidative neuronal damage and beta-amyloid toxicity. A critical step is apparently reached when pathological glia activation is no longer restricted to microglia and includes astrocytes. By giving up their differentiated state, astrocytes may lose their physiological negative feed-back control on microglial NO production and even contribute to neurotoxic peroxynitrate formation. Another consequence is the impairment of the astrocyte-maintained extracellular ion homeostasis favoring excitotoxic damage. By the production of apolipoprotein-E, triggered by the microglial cytokine interleukine-1beta, reactive astrocytes could promote the transformation of beta-amyloid into the toxic form. A pharmacologically reinforced cAMP signaling in rat glial cell cultures depressed oxygen radical formation in microglia and their release of TNF-alpha and interleukine-1beta, feed-forward signals which mediate oxidative damage and secondary astrocyte activation. Cyclic AMP also favored differentiation and expression of a mature ion channel pattern in astrocytes improving their glutamate buffering. A deficient cholinergic signaling that increases the risk of pathological APP processing was compensated by an adenosine-mediated reinforcement of the second messenger calcium. A combination therapy with acetylcholine-esterase inhibitors together with adenosine raising pharmaca, therefore, may be used to treat cholinergic deficiency in Alzheimer's disease.