Phospholemman, a Single-Span Membrane Protein, Is an Accessory Protein of Na,K-ATPase in Cerebellum and Choroid Plexus

Phospholemman (FXYD1) is a homolog of the Na,K-ATPase γ subunit (FXYD2), a small accessory protein that modulates ATPase activity. Here we show that phospholemman is highly expressed in selected structures in the CNS. It is most abundant in cerebellum, where it was detected in the molecular layer, in Purkinje neurons, and in axons traversing the granule cell layer. Phospholemman was particularly enriched in choroid plexus, the organ that secretes CSF in the ventricles, where it colocalized with Na,K-ATPase in the apical membrane. It was also enriched, with Na,K-ATPase, in certain tanycytes or ependymal cells of the ventricle wall. Two different experimental approaches demonstrated that phospholemman physically associated with the Na,K-ATPase in cerebellum and choroid plexus: the proteins copurified after detergent treatment and co-immunoprecipitated from solubilized crude membranes using either anti-phospholemman or anti-Na,K-ATPase antibodies. Phospholemman antibodies precipitated all three Na,K-ATPase α subunit isoforms (α1–α3) from cerebellum, indicating that the interaction is not specific to a particular α isoform and consistent with the presence of phospholemman in both neurons and glia. Antibodies against the C-terminal domain of phospholemman reduced Na,K-ATPase activityin vitro without effect on Na+affinity. At least two other FXYD family members have been detected in the CNS, suggesting that additional complexity of sodium pump regulation will be found

Histone deacetylase activity is necessary for left-right patterning during vertebrate development

<p>Abstract</p> <p>Background</p> <p>Consistent asymmetry of the left-right (LR) axis is a crucial aspect of vertebrate embryogenesis. Asymmetric gene expression of the TGFβ superfamily member <it>Nodal related 1 </it>(<it>Nr1) </it>in the left lateral mesoderm plate is a highly conserved step regulating the <it>situs </it>of the heart and viscera. In <it>Xenopus</it>, movement of maternal serotonin (5HT) through gap-junctional paths at cleavage stages dictates asymmetry upstream of <it>Nr1</it>. However, the mechanisms linking earlier biophysical asymmetries with this transcriptional control point are not known.</p> <p>Results</p> <p>To understand how an early physiological gradient is transduced into a late, stable pattern of <it>Nr1 </it>expression we investigated epigenetic regulation during LR patterning. Embryos injected with mRNA encoding a dominant-negative of Histone Deacetylase (HDAC) lacked <it>Nr1 </it>expression and exhibited randomized sidedness of the heart and viscera (heterotaxia) at stage 45. Timing analysis using pharmacological blockade of HDACs implicated cleavage stages as the active period. Inhibition during these early stages was correlated with an absence of <it>Nr1 </it>expression at stage 21, high levels of heterotaxia at stage 45, and the deposition of the epigenetic marker H3K4me2 on the <it>Nr1 </it>gene. To link the epigenetic machinery to the 5HT signaling pathway, we performed a high-throughput proteomic screen for novel cytoplasmic 5HT partners associated with the epigenetic machinery. The data identified the known HDAC partner protein Mad3 as a 5HT-binding regulator. While Mad3 overexpression led to an absence of <it>Nr1 </it>transcription and randomized the LR axis, a mutant form of Mad3 lacking 5HT binding sites was not able to induce heterotaxia, showing that Mad3's biological activity is dependent on 5HT binding.</p> <p>Conclusion</p> <p>HDAC activity is a new LR determinant controlling the epigenetic state of <it>Nr1 </it>from early developmental stages. The HDAC binding partner Mad3 may be a new serotonin-dependent regulator of asymmetry linking early physiological asymmetries to stable changes in gene expression during organogenesis.</p

Evaluation of an IGM-specific ELISA for early detection of bluetongue virus infections in domestic ruminants sera

International audienceCompetitive-ELISA (c-ELISA) is the most widely used serological test for the detection of Bluetongue virus (BTV) viral protein 7 (VP7) antibodies (Ab). However, these BTV c-ELISAs cannot to distinguish between IgG and IgM. IgM Ab are generated shortly after the primary immune response against an infectious agent, indicating a recent infection or exposure to antigens, such as after vaccination. Because the BTV genome or anti-VP7 Ab can be detected in ruminant blood months after infection, BTV diagnostic tools cannot discriminate between recent and old infections. In this study, we evaluated an IgM-capture ELISA prototype to detect ruminant anti-BTV VP7 IgM on 1,650 serum samples from cattle, sheep, or goats. Animals were BTV-naive, infected, or/and vaccinated with BTV-1, -2, -4, -8, -9, -16, or -27, and we also included 30 sera from cattle infected with the Epizootic haemorrhagic disease virus (EHDV) serotype 6. Results demonstrated that this ELISA kit is specific and can detect the presence of IgM with satisfactory diagnostic specificity and sensitivity from 1 to 5 weeks after BTV infection in domestic ruminants (for goats and cattle; for sheep, at least up to 24 days). The peak of anti-VP7 IgM was reached when the level of infectious viruses and BTV RNA in blood were the highest. The possibility of detecting BTV-RNA in IgM-positive sera allows the amplification and sequencing of the partial RNA segment 2 (encoding the serotype specific to VP2) to determine the causative BTV serotype/strain. Therefore, BTV IgM ELISA can detect the introduction of BTV (or EHDV) in an area with BTV-seropositive domestic animals regardless of their serological BTV status. This approach may also be of particular interest for retrospective epidemiological studies on frozen serum samples