ABalphaC, a major protein phosphatase 2A (PP2A) heterotrimeric enzyme, binds to and regulates the microtubule cytoskeleton and tau. We have shown that ABalphaC protein expression levels are selectively reduced in Alzheimer disease (AD). Notably, the carboxyl methylation of PP2A catalytic subunit (PP2A(C)) is critically required for ABalphaC holoenzyme assembly, and catalyzed by a specific methyltransferase (PPMT). Here, we provide the first analysis of human PPMT and methylated PP2A(C) in brain regions from AD, non-AD demented, and aged control autopsy cases. Immunoblotting analyses revealed that PPMT protein expression and PP2A(C) methylation levels were quantitatively decreased in AD-affected brain regions. Immunohistochemical studies showed that PPMT was abundant in neurons throughout the cortex in normal control and non-AD demented cases. However, in AD, there was a regional loss of PPMT immunoreactivity that closely paralleled the severity of tau pathology, but not amyloid plaque burden. We propose that the deregulation of PPMT and PP2A methylation/demethylation cycles contributes to AD pathogenesis, by inducing changes in PP2A heteromultimeric composition and substrate specificity. In turn, PP2A dysfunction compromises the mechanisms that control tau, neuronal plasticity, and survival.

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.

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.

The voltage-gated Ca 2؉ channels that effect tonic release of neurotransmitter from hair cells have unusual pharmacological properties: unlike most presynaptic Ca 2؉ channels, they are sensitive to dihydropyridines and therefore are L-type. To characterize these Ca 2؉ channels, we investigated the expression of L-type ␣ 1 subunits in hair cells of the chicken's cochlea. In PCRs with five different pairs of degenerate primers, we always obtained ␣ 1D products, but only once an ␣ 1C product and never an ␣ 1S product. A full-length ␣ 1D mRNA sequence was assembled from overlapping PCR products; the predicted amino acid sequence of the ␣ 1D subunit was about 90% identical to those of the mammalian ␣ 1D subunits. In situ hybridization confirmed that the ␣ 1D mRNA is present in hair cells. By using a quantitative PCR assay, we determined that the ␣ 1D mRNA is 100-500 times more abundant than the ␣ 1C mRNA. We conclude that most, if not all, voltage-gated Ca 2؉ channels in hair cells contain an ␣ 1D subunit. Furthermore, we propose that the ␣ 1D subunit plays a hitherto undocumented role at tonic synapses. At most synapses, transmitter release depends on N-type or P-type Ca 2ϩ channels, which are blocked by -conotoxin GIVA and -agatoxin IVA, respectively (reviewed in ref. 5). In hair cells, however, the Ca 2ϩ channels have different pharmacological properties: they are sensitive to dihydropyridines and therefore are L-type channels (6-9). In only a few other cell types, such as cultured sensory neurons (10) and retinal bipolar cells (11), have L-type channels been shown to effect neurotransmitter release.The drug sensitivity and permeability of a voltage-gated Ca 2ϩ channel depend on its type of ␣ 1 subunit (reviewed in ref.12). The pore-forming ␣ 1 protein is 160-240 kDa in size, with a cytoplasmic amino terminus, four homologous repeats (I-IV) of six transmembrane segments (S1-S6) each, and a cytoplasmic carboxyl terminus. L-type channels contain the product of the ␣ 1C (cardiac), the ␣ 1D (neuroendocrine), or the ␣ 1S (skeletal muscle) gene. To characterize the unusual L-type Ca 2ϩ channels that control synaptic transmission, we sought to determine which of these three ␣ 1 genes are expressed in hair cells of the chicken's cochlea. MATERIALS AND METHODSHistology. White Leghorn chickens (Gallus gallus) were asphyxiated with CO 2 and decapitated. The temporal bones with intact cochleae were excised and fixed overnight at 4°C with 0.75% (wt͞vol) paraformaldehyde and 2.5% (vol͞vol) glutaraldehyde in a buffer solution containing 70 mM sodium phosphate (pH 7.4), 75 mM sucrose, and 0.9 mM CaCl 2 . After two rinses in the buffer solution, the cochleae were carefully dissected from the bone, fixed with 1% (wt͞vol) OsO 4 in buffer solution, dehydrated successively with ethanol and propylene oxide, and embedded in epoxy resin consisting of EMbed 812, Araldite 6005, dodecenyl succinic anhydride, and 2,4,6-tris(dimethylaminomethyl)phenol (25:20:60:1 by volume; Electron Microscopy Sciences, Fort Washington, PA). Semithin ...