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 ...

The thymus is the primary organ responsible for the production of mature TCR α / β T cells. Quantification of a DNA excision circle that is produced during TCR rearrangement, termed a signal joint TCR rearrangement excision circle (sjTREC) can be used as a measure of thymic function. Here sjTREC measurement has been applied to two monkey species used as animal models of human disease, rhesus macaques (Asian origin) and sooty mangabeys (African origin). Initial PCR analysis determined that the TCR δRec‐ΨJα rearrangement leading to sjTREC formation occurs in both species. Primers to a DNA sequence conserved in macaques, mangabeys and humans were used in a quantitative competitive PCR assay to quantify sjTREC. We found that as in humans, sjTREC in these two monkey species decline with age. sjTREC are first generated in thymocytes during the early stages of TCR rearrangement. Lymph node CD4+ and CD8+ T cells contain more sjTREC than peripheral blood T cell populations, suggesting that recent thymic emigrants home to the lymphoid tissues. The sjTREC level is significantly higher within the peripheral blood CD4+ and CD8+ T cells of mangabeys compared to macaques. Removal of the thymus in four macaques led to a profound decrease in peripheral blood sjTREC level by 1 year post‐thymectomy, indicating the lack of a significant extra‐thymic source of peripheral naive T cells in macaques. Our results indicate that production, trafficking, and proliferation of recent thymic emigrants in these two monkey species represents a useful animal model system for understanding human immunological disorders.

one of the authors regrets that she inadvertently omitted references to the computer program and protein potential function that the authors used for their simulations of barnase cited above. The following sentence should have been the first sentence of the Methods section: Molecular dynamics simulations were performed with the program ENCAD (44) and the potential energy function of Levitt et al. (45).

The thymus is responsible for de novo production of CD4؉ and CD8 ؉ T cells and therefore is essential for T-cell renewal. The goal of this study was to assess the impact of simian immunodeficiency virus (SIV) infection on the production of T cells by the thymus. Levels of recent thymic emigrants within the peripheral blood were assessed through quantification of macaque T-cell receptor excision circles (TREC). Comparison of SIVinfected macaques (n ‫؍‬ 15) to uninfected macaques (n ‫؍‬ 23) revealed stable or increased TREC levels at 20 to 34 weeks postinfection. Further assessment of SIV-infected macaques (n ‫؍‬ 4) determined that TREC levels decreased between 24 and 48 weeks postinfection. Through the assessment of longitudinal time points in three additional SIVmac239-infected macaques, the SIV infection was divided into two distinct phases. During phase 1 (16 to 30 weeks), TREC levels remained stable or increased within both the CD4 and CD8 T-cell populations. During phase 2 (after 16 to 30 weeks), TREC levels declined in both T-cell populations. As has been described for human immunodeficiency virus (HIV)-infected patients, this decline in TREC levels did at times correlate with an increased level of T-cell proliferation (Ki67؉ cells). However, not all TREC decreases could be attributed to increased T-cell proliferation. Further evidence for thymic dysfunction was observed directly in a SIVmac239-infected macaque that succumbed to simian AIDS at 65 weeks postinfection. The thymus of this macaque contained an increased number of memory/effector CD8 ؉ T cells and an increased level of apoptotic cells. In summary, reduced levels of TREC can be observed beginning at 16 to 30 weeks post-SIV infection and correlate with changes indicative of dysfunction within the thymic tissue. SIV infection of macaques will be a useful model system to elucidate the mechanisms responsible for the thymic dysfunction observed in HIVinfected patients.

Abstract:The floristic composition and vegetation partitioning of the ephemeral wetlands of the Pilliga Outwash within the Pilliga National Park and Pilliga State Conservation Area (30˚30'S, 149˚22'E) on the North Western Plains of New South Wales are described. SPOT5 imagery was used to map 340 wetlands across the Pilliga Outwash. A total of 240 plots within 31 wetlands explored composition and species richness in relation to water depth and wetland size. The predominant community described is the species-rich herbfield of shallow basin wetlands, along with the structurally distinct but the less common sedgeland/herbfield of the deeper 'tank' wetlands and a single wetland with a floristically depauperate Diplachne fusca wet grassland. A total of 131 taxa were recorded including three species listed under the NSW Threatened Species Conservation Act (1995): Eriocaulon australasicum, Lepidium monoplocoides and Myriophyllum implicatum. New records for an additional six taxa were recorded for the North Western Plains. 11% of taxa were exotic in origin.