DNA methylation of the promoter region at the CREB1 binding site is a mechanism for the epigenetic regulation of brain-specific PKMζ.

Protein kinase M zeta, PKMζ, is a brain enriched kinase with a well characterized role in Long-Term Potentiation (LTP), the activity-dependent strengthening of synapses involved in long-term memory formation. However, little is known about the molecular mechanisms that maintain the tissue specificity of this kinase. Here, we characterized the epigenetic factors, mainly DNA methylation, regulating PKMζ expression in the human brain. The PRKCZ gene has an upstream promoter regulating Protein kinase C ζ (PKCζ), and an internal promoter driving PKMζ expression. A demethylated region, including a canonical CREB binding site, situated at the internal promoter was only observed in human CNS tissues. The induction of site-specific hypermethylation of this region resulted in decreased CREB1 binding and downregulation of PKMζ expression. Noteworthy, CREB binding sites were absent in the upstream promoter of PRKCZ locus, suggesting a specific mechanism for regulating PKMζ expression. These observations were validated using a system of human neuronal differentiation from induced pluripotent stem cells (iPSCs). CREB1 binding at the internal promoter was detected only in differentiated neurons, where PKMζ is expressed. The same epigenetic mechanism in the context of CREB binding site was identified in other genes involved in neuronal differentiation and LTP. Additionally, aberrant DNA hypermethylation at the internal promoter was observed in cases of Alzheimer's disease, correlating with decreased expression of PKMζ in patient brains. Altogether, we present a conserved epigenetic mechanism regulating PKMζ expression and other genes enhanced in the CNS with possible implications in neuronal differentiation and Alzheimer's disease.

Gas6 drives Zika virus-induced neurological complications in humans and congenital syndrome in immunocompetent mice.

Zika virus (ZIKV) has the ability to cross placental and brain barriers, causing congenital malformations in neonates and neurological disorders in adults. However, the pathogenic mechanisms of ZIKV-induced neurological complications in adults and congenital malformations are still not fully understood. Gas6 is a soluble TAM receptor ligand able to promote flavivirus internalization and downregulation of immune responses. Here we demonstrate that there is a correlation between ZIKV neurological complications with higher Gas6 levels and the downregulation of genes associated with anti-viral response, as type I IFN due to Socs1 upregulation. Also, Gas6 gamma-carboxylation is essential for ZIKV invasion and replication in monocytes, the main source of this protein, which was inhibited by warfarin. Conversely, Gas6 facilitates ZIKV replication in adult immunocompetent mice and enabled susceptibility to transplacental infection. Our data indicate that ZIKV promotes the upregulation of its ligand Gas6, which contributes to viral infectivity and drives the development of severe adverse outcomes during ZIKV infection.

GAS6 signaling tempers Th17 development in patients with multiple sclerosis and helminth infection.

Multiple sclerosis (MS) is a highly disabling neurodegenerative autoimmune condition in which an unbalanced immune response plays a critical role. Although the mechanisms remain poorly defined, helminth infections are known to modulate the severity and progression of chronic inflammatory diseases. The tyrosine kinase receptors TYRO3, AXL, and MERTK (TAM) have been described as inhibitors of the immune response in various inflammatory settings. We show here that patients with concurrent natural helminth infections and MS condition (HIMS) had an increased expression of the negative regulatory TAM receptors in antigen-presenting cells and their agonist GAS6 in circulating CD11bhigh and CD4+ T cells compared to patients with only MS. The Th17 subset was reduced in patients with HIMS with a subsequent downregulation of its pathogenic genetic program. Moreover, these CD4+ T cells promoted lower levels of the co-stimulatory molecules CD80, CD86, and CD40 on dendritic cells compared with CD4+ T cells from patients with MS, an effect that was GAS6-dependent. IL-10+ cells from patients with HIMS showed higher GAS6 expression levels than Th17 cells, and inhibition of phosphatidylserine/GAS6 binding led to an expansion of Th17 effector genes. The addition of GAS6 on activated CD4+ T cells from patients with MS restrains the Th17 gene expression signature. This cohort of patients with HIMS unravels a promising regulatory mechanism to dampen the Th17 inflammatory response in autoimmunity.

Effects of quinine, quinidine, and chloroquine on alpha9alpha10 nicotinic cholinergic receptors.

In this study, we report the effects of the quinoline derivatives quinine, its optical isomer quinidine, and chloroquine on alpha9alpha10-containing nicotinic acetylcholine receptors (nAChRs). The compounds blocked acetylcholine (ACh)-evoked responses in alpha9alpha10-injected Xenopus laevis oocytes in a concentration-dependent manner, with a rank order of potency of chloroquine (IC50 = 0.39 microM) > quinine (IC50 = 0.97 microM) approximately quinidine (IC50= 1.37 microM). Moreover, chloroquine blocked ACh-evoked responses on rat cochlear inner hair cells with an IC50 value of 0.13 microM, which is within the same range as that observed for recombinant receptors. Block by chloroquine was purely competitive, whereas quinine inhibited ACh currents in a mixed competitive and noncompetitive manner. The competitive nature of the blockage produced by the three compounds was confirmed by equilibrium binding experiments using [3H]methyllycaconitine. Binding affinities (Ki values) were 2.3, 5.5, and 13.0 microM for chloroquine, quinine, and quinidine, respectively. Block by quinine was found to be only slightly voltage-dependent, thus precluding open-channel block as the main mechanism of interaction of quinine with alpha9alpha10 nAChRs. The present results add to the pharmacological characterization of alpha9alpha10-containing nicotinic receptors and indicate that the efferent olivocochlear system that innervates the cochlear hair cells is a target of these ototoxic antimalarial compounds.

Direct interaction of serotonin type 3 receptor ligands with recombinant and native alpha 9 alpha 10-containing nicotinic cholinergic receptors.

In the present work, we characterized the effects of serotonin type 3 receptor ligands on recombinant and native alpha 9 alpha 10-containing nicotinic acetylcholine receptors (nAChRs). Our results indicate that the recombinant alpha 9 alpha 10 nAChR shares striking pharmacological properties with 5-HT(3) ligand-gated ion channels. Thus, 5-HT(3) receptor antagonists block ACh-evoked currents in alpha 9 alpha 10-injected Xenopus laevis oocytes with a rank order of potency of tropisetron (IC(50), 70.1 +/- 0.9 nM) > ondansetron (IC(50), 0.6 +/- 0.1 microM) = MDL 72222 (IC(50), 0.7 +/- 0.1 microM). Although serotonin does not elicit responses in alpha 9 alpha 10-injected oocytes, it blocks recombinant alpha 9 alpha 10 receptors in a noncompetitive and voltage-dependent manner (IC(50), 5.4 +/- 0.6 microM). On the other hand, we demonstrate an in vivo correlate of these properties of the recombinant receptor, with those of the alpha 9 alpha 10-containing nAChR of frog saccular hair cells. The possibility that the biogenic amine serotonin might act as a neuromodulator of the cholinergic efferent transmission in the vestibular apparatus and in the organ of Corti is discussed.