Extensive dysregulations of oligodendrocytic and astrocytic connexins are associated with disease progression in an amyotrophic lateral sclerosis mouse model.

BACKGROUND: Non-cell-autonomous motor neuronal death is suggested in a mutant Cu/Zn superoxide dismutase 1 (mSOD1)-mediated amyotrophic lateral sclerosis (ALS) model, in which glial cells play significant roles in disease progression. Connexins (Cxs) form homotypic or heterotypic gap junctions (GJs) and allow direct intercellular communications among nervous tissue cells. The role of Cxs in motor neuron disease has never been investigated; therefore, we aimed to evaluate alterations of Cxs in mSOD1-transgenic (mSOD1-Tg) mice in comparison with their non-transgenic (non-Tg) littermates at the same ages. METHODS: We pathologically evaluated temporal changes to astrocytic Cx43/Cx30 and oligodendrocytic Cx47/Cx32 immunoreactivities at presymptomatic, disease-progressive, and end stages, relative to aquaporin-4 (AQP4), glial fibrillary acidic protein (GFAP), excitatory amino acid transporter-2 (EAAT2), myelin-oligodendrocyte glycoprotein (MOG), and Nogo-A immunoreactivities, and observed neuronal loss by NeuN and neurofilament immunostaining, and microglial response by Iba-1 immunostaining. We also performed quantitative immunoblotting and real-time PCR analyses for Cxs. RESULTS: The mSOD1-Tg mice showed neuronal and axonal loss in the anterior horns of the lumbar spinal cord accompanied by increased activation of microglia compared with non-Tg mice at the disease-progressive and end stages. Expression patterns of Cxs were not different between mSOD1-Tg and non-Tg mice at the presymptomatic stage, but immunoreactivities for GFAP, Cx43, Cx30 and AQP4 were increased in the anterior horns of mSOD1-Tg mice at the disease-progressive and end stages. By contrast, Cx47 and Cx32 immunoreactivities were markedly diminished in Nogo-A-positive oligodendrocytes in the anterior horns of mSOD1-Tg mice at the disease-progressive and end stages, especially in oligodendrocytes showing SOD1 accumulation. EAAT2 immunoreactivity was also diminished in the anterior horns of mSOD1-Tg mice at the disease-progressive and end stages. Quantitative immunoblotting revealed a significant reduction in Cx47 and Cx32 protein levels in mSOD1-Tg mice at the disease-progressive and end stages. The levels of Cx47 and Cx32 mRNAs were also decreased at these stages. CONCLUSIONS: Our findings indicate that oligodendrocytic and astrocytic GJ proteins in the anterior horns of spinal cord in mSOD1-Tg mice are profoundly affected at the disease-progressive and end stages, where disruption of GJs among glial cells may exacerbate motor neuronal death.

Connexin 43 astrocytopathy linked to rapidly progressive multiple sclerosis and neuromyelitis optica.

BACKGROUND: Multiple sclerosis (MS) and neuromyelitis optica (NMO) occasionally have an extremely aggressive and debilitating disease course; however, its molecular basis is unknown. This study aimed to determine a relationship between connexin (Cx) pathology and disease aggressiveness in Asian patients with MS and NMO. METHODS/PRINCIPAL FINDINGS: Samples included 11 autopsied cases with NMO and NMO spectrum disorder (NMOSD), six with MS, and 20 with other neurological diseases (OND). Methods of analysis included immunohistochemical expression of astrocytic Cx43/Cx30, oligodendrocytic Cx47/Cx32 relative to AQP4 and other astrocytic and oligodendrocytic proteins, extent of demyelination, the vasculocentric deposition of complement and immunoglobulin, and lesion staging by CD68 staining for macrophages. Lesions were classified as actively demyelinating (n=59), chronic active (n=58) and chronic inactive (n=23). Sera from 120 subjects including 30 MS, 30 NMO, 40 OND and 20 healthy controls were examined for anti-Cx43 antibody by cell-based assay. Six NMO/NMOSD and three MS cases showed preferential loss of astrocytic Cx43 beyond the demyelinated areas in actively demyelinating and chronic active lesions, where heterotypic Cx43/Cx47 astrocyte oligodendrocyte gap junctions were extensively lost. Cx43 loss was significantly associated with a rapidly progressive disease course as six of nine cases with Cx43 loss, but none of eight cases without Cx43 loss regardless of disease phenotype, died within two years after disease onset (66.7% vs. 0%, P=0.0090). Overall, five of nine cases with Cx43 loss and none of eight cases without Cx43 loss had distal oligodendrogliopathy characterized by selective myelin associated glycoprotein loss (55.6% vs. 0.0%, P=0.0296). Loss of oligodendrocytic Cx32 and Cx47 expression was observed in most active and chronic lesions from all MS and NMO/NMOSD cases. Cx43-specific antibodies were absent in NMO/NMOSD and MS patients. CONCLUSIONS: These findings suggest that autoantibody-independent astrocytic Cx43 loss may relate to disease aggressiveness and distal oligodendrogliopathy in both MS and NMO.

Inhibitory effects of an ellagic acid glucoside, okicamelliaside, on antigen-mediated degranulation in rat basophilic leukemia RBL-2H3 cells and passive cutaneous anaphylaxis reaction in mice.

Degranulation inhibitors in plants are widely used for prevention and treatment of immediate-type allergy. We previously isolated a new ellagic acid glucoside, okicamelliaside (OCS), from Camellia japonica leaves for use as a potent degranulation inhibitor. Crude extracts from leaves also suppressed allergic conjunctivitis in rats. In this study, we evaluated the in vivo effect of OCS using a pure sample and performed in vitro experiments to elucidate the mechanism underlying the extraordinary high potency of OCS and its aglycon. The IC(50) values for degranulation of rat basophilic leukemia cells (RBL-2H3) were 14 nM for OCS and 3 µM for aglycon, indicating that the two compounds were approximately 2 to 3 orders of magnitude more potent than the anti-allergic drugs ketotifen fumarate, DSCG, and tranilast (0.17, 3, and >0.3 mM, respectively). Antigen-induced calcium ion (Ca(2+)) elevation was significantly inhibited by OCS and aglycon at all concentrations tested (p<0.05). Upstream of the Ca(2+) elevation in the principle signaling pathway, phosphorylation of Syk (Tyr525/526) and PLCγ-1 (Tyr783 and Ser1248) were inhibited by OCS and aglycon. In DNA microarray-screening test, OCS inhibited expression of proinflammatory cytokines [interleukin (IL)-4 and IL-13], cytokine-producing signaling factors, and prostaglandin-endoperoxidase 2, indicating that OCS broadly inhibits allergic inflammation. During passive cutaneous anaphylaxis in mice, OCS significantly inhibited vascular hyperpermeability by two administration routes: a single intraperitoneal injection at 10 mg/kg and per os at 5 mg/kg for 7 days (p<0.05). These results suggest the potential for OCS to alleviate symptoms of immediate-type allergy.

PP2A inhibition assay using recombinant enzyme for rapid detection of okadaic acid and its analogs in shellfish.

Okadaic acid and its analogs (OAs) responsible for diarrhetic shellfish poisoning (DSP) strongly inhibit protein phosphatase 2A (PP2A) and thus are quantifiable by measuring the extent of the enzyme inhibition. In this study, we evaluated the suitability of the catalytic subunit of recombinant human PP2A (rhPP2Ac) for use in a microplate OA assay. OA, dinophysistoxin-1(DTX1), and hydrolyzate of 7-O-palmitoyl-OA strongly inhibited rhPP2Ac activity with IC(50) values of 0.095, 0.104, and 0.135 nM, respectively. The limits of detection and quantitation for OA in the digestive gland of scallops and mussels were 0.0348 µg/g and 0.0611 µg/g respectively, and, when converted to the whole meat basis, are well below the regulation level proposed by EU (0.16 µg/g whole meat). A good correlation with LC-MS data was demonstrated, the correlation coefficient being 0.996 with the regression slope of 1.097.

The chromosomal association of condensin II is regulated by a noncatalytic function of PP2A.

Mitotic chromosomal assembly in vertebrates is regulated by condensin I and condensin II, which work cooperatively but have different chromosomal localization profiles and make distinct mechanistic contributions to this process. We show here that protein phosphatase 2A (PP2A), which interacts with condensin II but not condensin I, plays an essential role in targeting condensin II to chromosomes. Unexpectedly, our data indicate that PP2A acts as a recruiter protein rather than a catalytic enzyme to target condensin II to chromosomes. This recruiting activity of PP2A was inhibited by okadaic acid, but not by fostriecin, even though both molecules strongly inhibited the catalytic activity of PP2A. Additionally, we found that the chromokinesin KIF4a is also targeted to chromosomes via the noncatalytic activity of PP2A. Thus, our studies reveal a previously unknown contribution of PP2A to chromosome assembly.

The effect of structural variation in 21 microcystins on their inhibition of PP2A and the effect of replacing cys269 with glycine.

Microcystins (MCs) are a group of cyclic heptapeptide hepatotoxins produced by Microcystis and several other genera of cyanobacteria. The representative MC, MC-LR, strongly inhibits protein phosphatase 2A (PP2A), while the inhibitory potencies of at least 60MC analogs characterized from bloom samples and cultured strains have not been fully elucidated. In this study, we determined the IC(50) values for 21MC analogs for inhibiting the recombinant PP2A catalytic subunit (rPP2Ac). Of the 21MC analogs, MC-LR was the strongest inhibitor of rPP2Ac. Comparison of the IC(50) values indicates that demethylation of the amino acids at positions 3 or 7 leads to a greater reduction in activity than the substitution of l-amino acids at positions 2 and 4. To obtain further insight into the MC-PP2A interaction, we substituted cysteine at position 269 in PP2Ac with glycine. The mutant PP2Ac (C269G) was comparable to the wild-type PP2Ac in the hydrolysis of p-NPP, but was more resistant to MCs as indicated by the greater IC(50) values. Our results indicate that cys269 in PP2Ac and N-methyldehydroalanine (Mdha) at position 7 in MCs play important roles in the enzyme-inhibitor interaction. We also determined the LC(50) values of the MCs for cytotoxicity assay. Our results indicate that there is a weak correlation between the cytotoxicity and PP2A inhibiting activities of the MCs. The MCs and rPP2Ac used in this study were of high purity and the IC(50) values were determined under the same experimental conditions, ensuring the quality of the data. The IC(50) values are of practical importance because they enable the precise conversion of the amounts of various MCs detected using instrumental methods to MC-LR equivalents.

A protein phosphatase 2A (PP2A) inhibition assay using a recombinant enzyme for rapid detection of microcystins.

Worldwide blooms of toxic cyanobacteria (blue-green algae) commonly occur in freshwater, often in drinking water sources, necessitating routine monitoring of water quality. Microcystin-LR and related cyanobacterial toxins strongly inhibit protein phosphatase 2A (PP2A) and are therefore assayable by measuring the extent of PP2A inhibition. In this study, we evaluated the suitability of the catalytic subunit of recombinant PP2A (rPP2Ac) expressed with a baculovirus system for use in a microplate microcystin assay. Five microcystin analogs, microcystin-LR, -RR, -YR, -LF, and -LW, and nodularin strongly inhibited rPP2Ac activity with IC(50) values of 0.048, 0.072, 0.147, 0.096, 0.114, and 0.54 nM, respectively. Microcystin-LR in a water sample could be assayed from 0.005 to 5 ng/ml. The assay could detect the toxin at a far lower level than required by the World Health Organization for regulation of microcystin-LR or its equivalent (1 microg/L). Pretreatment or concentration of water samples with low toxin concentrations was not necessary. The microplate assay using rPP2Ac was more sensitive than an enzyme-linked immunosorbent assay (ELISA) method and a cytotoxicity assay. The genetically engineered rPP2Ac was more stable than a commercially available dimeric enzyme, producing accurate and reproducible results. Our results confirm that the rPP2Ac we prepared is an excellent tool for detecting and quantifying microcystins in water.

Methylation of the C-terminal leucine residue of the PP2A catalytic subunit is unnecessary for the catalytic activity and the binding of regulatory subunit (PR55/B).

Protein phosphatase 2A (PP2A) is composed of structural (A), catalytic (C), and regulatory (B) subunits. The catalytic subunit (PP2A(C)) undergoes reversible carboxyl-methylation and -demethylation at its C-terminal leucine residue (Leu309), catalyzed by PP2A-methyltransferase (PMT) and PP2A methylesterase (PME-1), respectively. In this study, we observed that the activity of PP2A was largely unaffected by the addition of PME-1, and that the regulatory subunit (PR55/B) could bind demethylated PP2A(D). Furthermore, to study the precise effect of Leu309 demethylation on PP2A activity, we generated two His(8)-tagged mutant versions of PP2A(C) containing an alanine residue in place of Leu309, and a deletion of Leu309. Both recombinant mutants exhibited phosphatase activity. In addition, we demonstrated that both mutants could constitute a holoenzyme with the regulatory A and B subunits. Our collective results indicate that methylation of Leu309 of PP2A(C) is unnecessary for the PP2A activity and the binding of PR55/B.

Baculovirus expression, purification, and characterization of human protein phosphatase 2A catalytic subunits alpha and beta.

Protein phosphatase 2A (PP2A) contains a 36-kDa catalytic subunit (PP2Ac), a 65-kDa structural subunit (PR65/A), and a regulatory B subunit. The core enzyme consists of the structural and catalytic subunits. The catalytic subunit exists as two closely related isoforms, alpha and beta. Several natural toxins, including okadaic acid (OA) and microcystins, specifically inhibit PP2A. To obtain biologically active recombinant PP2A and to compare the properties of the PP2A catalytic subunit alpha and beta isoforms, we expressed human PP2Acalpha and cbeta in High Five insect cells. The recombinant PP2Acalpha and cbeta possess similar phosphatase activities using p-NPP and phosphopeptide as substrates and are strongly inhibited by OA and microcystin-LR to similar degrees. In addition, PP2Acalpha or cbeta was co-expressed with PR65/A and co-purified as a core dimer, PP2AD (Aalpha/calpha and Aalpha/cbeta) with PR65alpha/Aalpha. The recombinant PP2AD bound to the B subunit in vitro. These results show that the recombinant PP2Acalpha and cbeta are identical in their ability to associate with the A and B subunits, in their phosphatase activities, and in carboxyl-methylation. Furthermore, our results show that High Five insect cells can produce biologically active recombinant PP2A, which should be a valuable tool for detecting natural toxins and investigating the mechanism of PP2A catalysis and other protein interactions.