Alzheimer's disease (AD) neuropathology is characterized by the accumulation of phosphorylated tau and amyloid-␤ peptides derived from the amyloid precursor protein (APP). Elevated blood levels of homocysteine are a significant risk factor for many age-related diseases, including AD. Impaired homocysteine metabolism favors the formation of S-adenosylhomocysteine, leading to inhibition of methyltransferase-dependent reactions. Here, we show that incubation of neuroblastoma cells with S-adenosylhomocysteine results in reduced methylation of protein phosphatase 2A (PP2A), a major brain Ser/Thr phosphatase, most likely by inhibiting PP2A methyltransferase (PPMT). PP2A methylation levels are also decreased after ectopic expression of PP2A methylesterase in Neuro-2a (N2a) cells. Reduced PP2A methylation promotes the downregulation of B␣-containing holoenzymes, thereby affecting PP2A substrate specificity. It is associated with the accumulation of both phosphorylated tau and APP isoforms and increased secretion of ␤-secretase-cleaved APP fragments and amyloid-␤ peptides. Conversely, incubation of N2a cells with S-adenosylmethionine and expression of PPMT enhance PP2A methylation. This leads to the accumulation of dephosphorylated tau and APP species and increased secretion of neuroprotective ␣-secretase-cleaved APP fragments. Remarkably, hyperhomocysteinemia induced in wild-type and cystathionine-␤-synthase ϩ/Ϫ mice by feeding a high-methionine, low-folate diet is associated with increased brain S-adenosylhomocysteine levels, PPMT downregulation, reduced PP2A methylation levels, and tau and APP phosphorylation. We reported previously that downregulation of neuronal PPMT and PP2A methylation occur in affected brain regions from AD patients. The link between homocysteine, PPMT, PP2A methylation, and key CNS proteins involved in AD pathogenesis provides new mechanistic insights into this disorder.

Dynamin is a microtubule-binding protein with a microtubule-activated GTPase activity. The gene encoding dynamin is mutated in shibire, a Drosophila mutant defective in endocytosis in nerve terminals and other cells. These observations place dynamin into two distinct functional contexts, suggesting roles in microtubule-based motility or in endocytosis. We report here that dynamin is identical to the neuronal phosphoprotein dephosphin (P96), originally identified by its stimulus-dependent dephosphorylation in nerve terminals. Dynamin is a protein doublet of M(r) 94 and 96K arising by alternative splicing of its primary transcript. In the nerve terminal, both forms of dynamin are phosphorylated by protein kinase C (PKC) and are quantitatively dephosphorylated on excitation. In vitro, dynamin is also phosphorylated by casein kinase II which inhibits PKC phosphorylation. Phosphorylation by PKC but not by casein kinase II enhances the GTPase activity of dynamin 12-fold. The dynamins are therefore a group of nerve terminal phosphoproteins whose GTPase is regulated by phosphorylation in parallel with synaptic vesicle recycling. The regulation of dynamin GTPase could serve as the trigger for the rapid endocytosis of synaptic vesicles after exocytosis.

Protein phosphatase 2A (PP2A) is a large family of enzymes that account for the majority of brain Ser/Thr phosphatase activity. While PP2A enzymes collectively modulate most cellular processes, sophisticated regulatory mechanisms are ultimately responsible for ensuring isoform-specific substrate specificity. Of particular interest to the Alzheimer’s disease (AD) field, alterations in PP2A regulators and PP2A catalytic activity, subunit expression, methylation and/or phosphorylation, have been reported in AD-affected brain regions. “PP2A” dysfunction has been linked to tau hyperphosphorylation, amyloidogenesis and synaptic deficits that are pathological hallmarks of this neurodegenerative disorder. Deregulation of PP2A enzymes also affects the activity of many Ser/Thr protein kinases implicated in AD. This review will more specifically discuss the role of the PP2A/Bα holoenzyme and PP2A methylation in AD pathogenesis. The PP2A/Bα isoform binds to tau and is the primary tau phosphatase. Its deregulation correlates with increased tau phosphorylation in vivo and in AD. Disruption of PP2A/Bα-tau protein interactions likely contribute to tau deregulation in AD. Significantly, alterations in one-carbon metabolism that impair PP2A methylation are associated with increased risk for sporadic AD, and enhanced AD-like pathology in animal models. Experimental studies have linked deregulation of PP2A methylation with down-regulation of PP2A/Bα, enhanced phosphorylation of tau and amyloid precursor protein, tau mislocalization, microtubule destabilization and neuritic defects. While it remains unclear what are the primary events that underlie “PP2A” dysfunction in AD, deregulation of PP2A enzymes definitely affects key players in the pathogenic process. As such, there is growing interest in developing PP2A-centric therapies for AD, but this may be a daunting task without a better understanding of the regulation and function of specific PP2A enzymes.

of several cell types by SV40, especially when assayed Alphonse Garcia 3 in growth-arrested cells (Martin et al., 1979). Small t also Small t also activates AP-1 in microinjected CV-1 cells in a MAP kinase-dependent fashion (Frost et al., 1994). We have reported that inhibition of protein phosphatHowever, the mechanisms supporting the role of small t ase 2A (PP2A) by expression of SV40 small t stimulates during SV40 infection are far from being completely the mitogenic MAP kinase cascade. Here, we show that understood. SV40 small t can substitute for tumor necrosis factor-αLike PP2A, the atypical calcium-independent protein (TNF-α) or serum and stimulate atypical protein kinase kinase C ζ isoform (PKC ζ) has been involved in the C ζ (PKC ζ) activity, resulting in MEK activation, cell control of mitogenic signal transduction and survival proliferation and NF-κB-dependent gene transcrip- (Berra et al., 1993(Berra et al., , 1995Gomez et al., 1995; Diaz-Meco tional activation in CV-1 and NIH 3T3 cells. Kieser et al., 1996;Powell et al., 1996). PKC effects were abrogated by co-expression of kinase-ζ also plays a pivotal role in tumor necrosis factor-α deficient PKC ζ and inhibition of phosphatidylinositol (TNF-α) activation of NF-κB (Diaz-Meco et al., 1993, 3-kinase p85α-p110 by wortmannin, LY294002 and 1994; Dominguez et al., 1993;Lozano et al., 1994), an a dominant-negative mutant of p85α. In contrast, inducible transcriptional activator that participates in the expression of kinase-inactive ERK2 inhibited small control of cell proliferation and survival, as well as in t-dependent cell growth but was unable to abolish inflammatory response and viral gene expression small t-induced NF-κB transactivation. Our results (reviewed in Bauerle and Baltimore, 1996). NF-κB has provide the first in vivo evidence for a critical regulatory been especially implicated in the transcriptional activation role of PP2A in bifunctional PKC ζ signaling pathways of the human immunodeficiency virus type 1 (HIV-1) long controlled by phosphatidylinositol 3-kinase. Constituterminal repeat (LTR) (reviewed in Gaynor, 1992). NF-κB tive activation of PKC ζ and NF-κB following inhibition is the prototype of a family of heterodimeric transcription of PP2A supports new mechanisms by which SV40 factors composed of monomers that bind to DNA and the small t promotes cell growth and transformation. By I-κB inhibitors (reviewed in Baldwin, 1996). NF-κB is establishing PP2A as a key player in the response of retained in the cytoplasm in its inactive form by the I-κB cells to growth factors and stress signals like TNF-α, α inhibitor. Upon stimulation of cells, I-κB α becomes our findings could explain why PP2A is a primary phosphorylated and dissociates from NF-κB, resulting in target utilized during SV40 infection to alter cellular the translocation of NF-κB to the nucleus, where it carries behavior.out its transactivation function. The rapid phosphorylation Keywords: NF-κB/PI 3-kinase/PKC ζ/PP2A/SV40 of I-κB α represents a possible signal for its proteo...

Altered folate homeostasis is associated with many clinical and pathological manifestations in the CNS. Notably, folate-mediated onecarbon metabolism is essential for methyltransferase-dependent cellular methylation reactions. Biogenesis of protein phosphatase 2A (PP2A) holoenzyme containing the regulatory B␣ subunit, a major brain tau phosphatase, is controlled by methylation. Here, we show that folate deprivation in neuroblastoma cells induces downregulation of PP2A leucine carboxyl methyltransferase-1 (LCMT-1) expression, resulting in progressive accumulation of newly synthesized demethylated PP2A pools, concomitant loss of B␣, and ultimately cell death. These effects are further accentuated by overexpression of PP2A methylesterase (PME-1) but cannot be rescued by PME-1 knockdown. Overexpression of either LCMT-1 or B␣ is sufficient to protect cells against the accumulation of demethylated PP2A, increased tau phosphorylation, and cell death induced by folate starvation. Conversely, knockdown of either protein accelerates folate deficiencyevoked cell toxicity. Significantly, mice maintained for 2 months on low-folate or folate-deficient diets have brain-region-specific alterations in metabolites of the methylation pathway. Those are associated with downregulation of LCMT-1, methylated PP2A, and B␣ expression and enhanced tau phosphorylation in susceptible brain regions. Our studies provide novel mechanistic insights into the regulation of PP2A methylation and tau. They establish LCMT-1-and B␣-containing PP2A holoenzymes as key mediators of the role of folate in the brain. Our results suggest that counteracting the neuronal loss of LCMT-1 and B␣ could be beneficial for all tauopathies and folate-dependent disorders of the CNS.

Background:The microtubule-associated proteins tau and MAP2 are dephosphorylated by PP2A, a major brain Ser/Thr phosphatase. Results: Identification of a common PP2A-binding motif in tau and MAP2. Conclusion: Soluble tau and MAP2 can compete for binding to and dephosphorylation by PP2A/B␣. Significance: Tau and MAP2 regulate neuronal plasticity and deregulation of tau is a hallmark of tauopathies.

Actin, one of the major filamentous cytoskeletal molecules, is involved in a variety of cellular functions. Whereas an association between muscle actin mutations and skeletal and cardiac myopathies has been well documented, reports of human disease arising from mutations of nonmuscle actin genes have been rare. We have identified a missense point mutation in the gene coding for beta -actin that results in an arginine-to-tryptophan substitution at position 183. The disease phenotype includes developmental midline malformations, sensory hearing loss, and a delayed-onset generalized dystonia syndrome in monozygotic twins. Cellular studies of a lymphoblastoid cell line obtained from an affected patient demonstrated morphological abnormalities of the actin cytoskeleton and altered actin depolymerization dynamics in response to latrunculin A, an actin monomer-sequestering drug. Resistance to latrunculin A was also observed in NIH 3T3 cells expressing the mutant actin. These findings suggest that mutations in nonmuscle actins may be associated with a broad spectrum of developmental malformations and/or neurological abnormalities such as dystonia.

Carboxymethylation and phosphorylation of protein phosphatase 2A (PP2A) catalytic C subunit are evolutionary conserved mechanisms that critically control PP2A holoenzyme assembly and substrate specificity. Down-regulation of PP2A methylation and PP2A enzymes containing the Ba regulatory subunit occur in Alzheimer's disease. In this study, we show that expressed wild-type and methylation-(L309D) and phosphorylation-(T304D, T304A, Y307F, and Y307E) site mutants of PP2A C subunit differentially bind to B, B¢, and B¢¢-type regulatory subunits in NIH 3T3 fibroblasts and neuro2a (N2a) neuroblastoma cells. They also display distinct binding affinity for microtubules (MTs). Relative to controls, expression of the wild-type, T304A and Y307F C subunits in N2a cells promotes the accumulation of acetylated and detyrosinated MTs. However, expression of the Y307E, L309D, and T304D mutants, which are impaired in their ability to associate with the Ba subunit, induces their loss. Silencing of Ba subunit in N2a and NIH 3T3 cells is sufficient to induce a similar breakdown of acetylated and detyrosinated MTs. It also confers increased sensitivity to nocodazole-induced MT depolymerization. Our findings suggest that changes in intracellular PP2A subunit composition can modulate MT dynamics. They support the hypothesis that reduced amounts of neuronal Ba-containing PP2A heterotrimers contribute to MT destabilization in Alzheimer's disease.