Phosphatidylinositol 4-phosphate (PI4P) is a low abundant phospholipid with important roles in lipid transport and membrane trafficking. However, little is known of its metabolism and function in neurons. Here, we investigate its subcellular distribution and functional roles in dendrites of rodent hippocampal neurons during resting state and long-term synaptic potentiation (LTP). We show that neural activity causes dynamic reversible changes in PI4P metabolism in dendrites. Upon LTP induction, PI4KIIIα, a type III phosphatidylinositol 4-kinase, localizes to the dendritic plasma membrane (PM) in a calcium-dependent manner and causes substantial increase in the levels of PI4P. Acute inhibition of PI4KIIIα activity abolishes trafficking of the AMPA-type glutamate receptor to the PM during LTP induction, and silencing of PI4KIIIα expression in the hippocampal CA1 region causes severe impairment of LTP and long-term memory. Collectively, our results identify an essential role for PI4KIIIα-dependent PI4P synthesis in synaptic plasticity of central nervous system neurons.

Aquaporins are integral proteins that facilitate the transmembrane transport of water and small solutes. In addition to enabling water flux, mammalian Aquaporin-1 (AQP1) channels activated by cyclic GMP can carry non-selective monovalent cation currents, selectively blocked by arylsulfonamide compounds AqB007 (IC50 170 μM) and AqB011 (IC50 14 μM). In silico models suggested that ligand docking might involve the cytoplasmic loop D (between AQP1 transmembrane domains 4 and 5), but the predicted site of interaction remained to be tested. Work here shows that mutagenesis of two conserved arginine residues in loop D slowed the activation of the AQP1 ion conductance and impaired the sensitivity of the channel to block by AqB011. Substitution of residues in loop D with proline showed effects on ion conductance amplitude that varied with position, suggesting that the structural conformation of loop D is important for AQP1 channel gating. Human AQP1 wild type, AQP1 mutant channels with alanines substituted for two arginines (R159A+R160A), and mutants with proline substituted for single residues threonine (T157P), aspartate (D158P), arginine (R159P, R160P), or glycine (G165P) were expressed in Xenopus laevis oocytes. Conductance responses were analyzed by two-electrode voltage clamp. Optical osmotic swelling assays and confocal microscopy were used to confirm mutant and wild type AQP1-expressing oocytes were expressed in the plasma membrane. After application of membrane-permeable cGMP, R159A+R160A channels had a significantly slower rate of activation as compared with wild type, consistent with impaired gating. AQP1 R159A+R160A channels showed no significant block by AqB011 at 50 μM, in contrast to the wild type channel which was blocked effectively. T157P, D158P, and R160P mutations had impaired activation compared to wild type; R159P showed no significant effect; and G165P appeared to augment the conductance amplitude. These findings provide evidence for the role of the loop D as a gating domain for AQP1 ion channels, and identify the likely site of interaction of AqB011 in the proximal loop D sequence.

Vesicular glutamate transporters (VGLUTs) fill synaptic vesicles with glutamate. VGLUTs were originally identified as sodium-dependent transporters of inorganic phosphate (Pi), but the physiological relevance of this activity remains unclear. Heterologous expression of all three VGLUTs greatly augments intracellular Pi levels. Using neuronal models, we show that translocation of VGLUTs to the plasma membrane during exocytosis results in highly increased Pi uptake. VGLUT-mediated Pi influx is counteracted by Pi efflux. Synaptosomes prepared from perinatal VGLUT2-/- mice that are virtually free of VGLUTs show drastically reduced cytosolic Pi levels and fail to import Pi. Glutamate partially competes with sodium (Na+)/Pi (NaPi)-uptake mediated by VGLUTs but does not appear to be transported. A nanobody that blocks glutamate transport by binding to the cytoplasmic domain of VGLUT1 abolishes Pi transport when co-expressed with VGLUT1. We conclude that VGLUTs have a dual function that is essential for both vesicular glutamate loading and Pi restoration in neurons.

Our lab previously established that metformin, a first-line type two diabetes treatment, activates the Nrf2 pathway and improves post-stroke recovery. Metformin's brain permeability value and potential interaction with blood-brain barrier (BBB) uptake and efflux transporters are currently unknown. Metformin has been shown to be a substrate of organic cationic transporters (Octs) in the liver and kidneys. Brain endothelial cells at the BBB have been shown to express Octs; thus, we hypothesize that metformin uses Octs for its transport across the BBB. We used a co-culture model of brain endothelial cells and primary astrocytes as an in vitro BBB model to conduct permeability studies during normoxia and hypoxia using oxygen-glucose deprivation (OGD) conditions. Metformin was quantified using a highly sensitive LC-MS/MS method. We further checked Octs protein expression using Western blot analysis. Lastly, we completed a plasma glycoprotein (P-GP) efflux assay. Our results showed that metformin is a highly permeable molecule, uses Oct1 for its transport, and does not interact with P-GP. During OGD, we found alterations in Oct1 expression and increased permeability for metformin. Additionally, we showed that selective transport is a key determinant of metformin's permeability during OGD, thus, providing a novel target for improving ischemic drug delivery.

We characterized a novel GJB2 missense variant, c.133G>A, p.Gly45Arg, and compared it with the only other variant at the same amino acid position of the connexin 26 protein (Cx26) reported to date: c.134G>A, p.Gly45Glu. Whereas both variants are associated with hearing loss and are dominantly inherited, p.Gly45Glu has been implicated in the rare fatal keratitis-ichthyosis-deafness (KID) syndrome, which results in cutaneous infections and septicemia with premature demise in the first year of life. In contrast, p.Gly45Arg appears to be non-syndromic. Subcellular localization experiments in transiently co-transfected HeLa cells demonstrated that Cx26-WT (wild-type) and p.Gly45Arg form gap junctions, whereas Cx26-WT with p.Gly45Glu protein does not. The substitution of a nonpolar amino acid glycine in wildtype Cx26 at position 45 with a negatively charged glutamic acid (acidic) has previously been shown to interfere with Ca2+ regulation of hemichannel gating and to inhibit the formation of gap junctions, resulting in cell death. The novel variant p.Gly45Arg, however, changes this glycine to a positively charged arginine (basic), resulting in the formation of dysfunctional gap junctions that selectively affect the permeation of negatively charged inositol 1,4,5-trisphosphate (IP3) and contribute to hearing loss. Cx26 p.Gly45Arg transfected cells, unlike cells transfected with p.Gly45Glu, thrived at physiologic Ca2+ concentrations, suggesting that Ca2+ regulation of hemichannel gating is unaffected in Cx26 p.Gly45Arg transfected cells. Thus, the two oppositely charged amino acids that replace the highly conserved uncharged glycine in p.Gly45Glu and p.Gly45Arg, respectively, produce strikingly different effects on the structure and function of the Cx26 protein.

The interleukin-1 (IL-1) family cytokines are cytosolic proteins that exhibit inflammatory activity upon release into the extracellular space. These factors are released following various cell death processes, with pyroptosis being a common mechanism. Recently, it was recognized that phagocytes can achieve a state of hyperactivation, which is defined by their ability to secrete IL-1 while retaining viability, yet it is unclear how IL-1 can be secreted from living cells. Herein, we report that the pyroptosis regulator gasdermin D (GSDMD) was necessary for IL-1β secretion from living macrophages that have been exposed to inflammasome activators, such as bacteria and their products or host-derived oxidized lipids. Cell- and liposome-based assays demonstrated that GSDMD pores were required for IL-1β transport across an intact lipid bilayer. These findings identify a non-pyroptotic function for GSDMD, and raise the possibility that GSDMD pores represent conduits for the secretion of cytosolic cytokines under conditions of cell hyperactivation.

HIV replication is closely regulated by a complex pathway of host factors, many of them being determinants of cell tropism and host susceptibility to HIV infection. These host factors are known to exert a positive or negative influence on the replication of the two major types of HIV, HIV-1 and HIV-2, thereby modulating virus infectivity, host response to infection and ultimately disease progression profiles characteristic of these two types. Understanding the differential regulation of host cellular factors in response to HIV-1 and HIV-2 infections will help us to understand the apparent differences in rates of disease progression and pathogenesis. This knowledge would aid in the discovery of new biomarkers that may serve as novel targets for therapy and diagnosis. The objective of this study was to determine the differential expression of host genes in response to HIV-1/HIV-2 infection. To achieve this, we analyzed the effects of HIV-1 (MN) and HIV-2 (ROD) infection on the expression of host factors in PBMC at the RNA level using the Agilent Whole Human Genome Oligo Microarray. Differentially expressed genes were identified and their biological functions determined. Host gene expression profiles were significantly changed. Gene expression profiling analysis identified a subset of differentially expressed genes in HIV-1 and HIV-2 infected cells. Genes involved in cellular metabolism, apoptosis, immune cell proliferation and activation, cytokines, chemokines, and transcription factors were differentially expressed in HIV-1 infected cells. Relatively few genes were differentially expressed in cells infected with HIV-2.