Connexin30 and Connexin43 show a time-of-day dependent expression in the mouse suprachiasmatic nucleus and modulate rhythmic locomotor activity in the context of chronodisruption.

BACKGROUND: The astroglial connexins Cx30 and Cx43 contribute to many important CNS functions including cognitive behaviour, motoric capacity and regulation of the sleep-wake cycle. The sleep wake cycle, is controlled by the circadian system. The central circadian rhythm generator resides in the suprachiasmatic nucleus (SCN). SCN neurons are tightly coupled in order to generate a coherent circadian rhythm. The SCN receives excitatory glutamatergic input from the retina which mediates entrainment of the circadian system to the environmental light-dark cycle. Connexins play an important role in electric coupling of SCN neurons and astrocytic-neuronal signalling that regulates rhythmic SCN neuronal activity. However, little is known about the regulation of Cx30 and Cx43 expression in the SCN, and the role of these connexins in light entrainment of the circadian system and in circadian rhythm generation. METHODS: We analysed time-of-day dependent as well as circadian expression of Cx30 and Cx43 mRNA and protein in the mouse SCN by means of qPCR and immunohistochemistry. Moreover, we analysed rhythmic spontaneous locomotor activity in mice with a targeted deletion of Cx30 and astrocyte specific deletion of Cx43 (DKO) in different light regimes by means of on-cage infrared detectors. RESULTS: Fluctuation of Cx30 protein expression is strongly dependent on the light-dark cycle whereas fluctuation of Cx43 protein expression persisted in constant darkness. DKO mice entrained to the light-dark cycle. However, re-entrainment after a phase delay was slightly impaired in DKO mice. Surprisingly, DKO mice were more resilient to chronodisruption. CONCLUSION: Circadian fluctuation of Cx30 and Cx43 protein expression in the SCN is differently regulated. Cx30 and astroglial Cx43 play a role in rhythm stability and re-entrainment under challenging conditions.

Morphological study of a connexin 43-GFP reporter mouse highlights glial heterogeneity, amacrine cells, and olfactory ensheathing cells.

Connexin 43 (Cx43) is the main astrocytic connexin and forms the basis of the glial syncytium. The morphology of connexin-expressing cells can be best studied in transgenic mouse lines expressing cytoplasmic fluorescent reporters, since immunolabeling the plaques can obscure the shapes of the individual cells. The Cx43kiECFP mouse generated by Degen et al. (FASEBJ 26:4576, 2012) expresses cytosolic ECFP and has previously been used to establish that Cx43 may not be expressed by all astrocytes within a population, and this can vary in a region-dependent way. To establish this mouse line as a tool for future astrocyte and connexin research, we sought to consolidate reporter authenticity, studying cell types and within-region population heterogeneity. Applying anti-GFP, all cell types related to astroglia were positive-namely, protoplasmic astrocytes in the hippocampus, cortex, thalamus, spinal cord, olfactory bulb, cerebellum with Bergmann glia and astrocytes also in the molecular layer, and retinal Müller cells and astrocytes. Labeled cell types further comprise white matter astrocytes, olfactory ensheathing cells, radial glia-like stem cells, retinal pigment epithelium cells, ependymal cells, and meningeal cells. We furthermore describe a retinal Cx43-expressing amacrine cell morphologically reminiscent of ON-OFF wide-field amacrine cells, representing the first example of a mammalian CNS neuron-expressing Cx43 protein. In double staining with cell type-specific markers (GFAP, S100ß, glutamine synthetase), Cx43 reporter expression in the hippocampus and cortex was restricted to GFAP+ astrocytes. Altogether, this mouse line is a highly reliable tool for studies of Cx43-expressing CNS cells and astroglial cell morphology. © 2017 Wiley Periodicals, Inc.

Astrocyte uncoupling as a cause of human temporal lobe epilepsy.

Glial cells are now recognized as active communication partners in the central nervous system, and this new perspective has rekindled the question of their role in pathology. In the present study we analysed functional properties of astrocytes in hippocampal specimens from patients with mesial temporal lobe epilepsy without (n = 44) and with sclerosis (n = 75) combining patch clamp recording, K(+) concentration analysis, electroencephalography/video-monitoring, and fate mapping analysis. We found that the hippocampus of patients with mesial temporal lobe epilepsy with sclerosis is completely devoid of bona fide astrocytes and gap junction coupling, whereas coupled astrocytes were abundantly present in non-sclerotic specimens. To decide whether these glial changes represent cause or effect of mesial temporal lobe epilepsy with sclerosis, we developed a mouse model that reproduced key features of human mesial temporal lobe epilepsy with sclerosis. In this model, uncoupling impaired K(+) buffering and temporally preceded apoptotic neuronal death and the generation of spontaneous seizures. Uncoupling was induced through intraperitoneal injection of lipopolysaccharide, prevented in Toll-like receptor4 knockout mice and reproduced in situ through acute cytokine or lipopolysaccharide incubation. Fate mapping confirmed that in the course of mesial temporal lobe epilepsy with sclerosis, astrocytes acquire an atypical functional phenotype and lose coupling. These data suggest that astrocyte dysfunction might be a prime cause of mesial temporal lobe epilepsy with sclerosis and identify novel targets for anti-epileptogenic therapeutic intervention.

Characterization of Panglial Gap Junction Networks in the Thalamus, Neocortex, and Hippocampus Reveals a Unique Population of Glial Cells.

The thalamus plays important roles as a relay station for sensory information in the central nervous system (CNS). Although thalamic glial cells participate in this activity, little is known about their properties. In this study, we characterized the formation of coupled networks between astrocytes and oligodendrocytes in the murine ventrobasal thalamus and compared these properties with those in the hippocampus and cortex. Biocytin filling of individual astrocytes or oligodendrocytes revealed large panglial networks in all 3 gray matter regions. Combined analyses of mice with cell type-specific deletion of connexins (Cxs), semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and western blotting showed that Cx30 is the dominant astrocytic Cx in the thalamus. Many thalamic astrocytes even lack expression of Cx43, while in the hippocampus astrocytic coupling is dominated by Cx43. Deletion of Cx30 and Cx47 led to complete loss of panglial coupling, which was restored when one allele of either Cxs was present. Immunohistochemistry revealed a unique antigen profile of thalamic glia and identified an intermediate cell type expressing both Olig2 and Cx43. Our findings further the emerging concept of glial heterogeneity across brain regions.

Characterization of cytoplasmic polyadenylation element binding 2 protein expression and its RNA binding activity.

Cytoplasmic polyadenylation element binding (CPEB) proteins are translational regulators that are involved in the control of cellular senescence, synaptic plasticity, learning, and memory. We have previously found all four known CPEB family members to be transcribed in the mouse hippocampus. Aside from a brief description of CPEB2 in mouse brain, not much is known about its biological role. Hence, this study aims to investigate CPEB2 expression in mouse brain. With reverse transcription polymerase chain reaction (RT-PCR) of total mouse brain cDNA, we identified four distinct CPEB2 splice variants. Single-cell RT-PCR showed that CPEB2 is predominantly expressed in neurons of the juvenile and adult brain and that individual cells express different sets of splice variants. Staining of brain slices with a custom-made CPEB2 antibody revealed ubiquitous expression of the protein in many brain regions, including hippocampus, striatum, thalamus, cortex, and cerebellum. We also found differential expression of CPEB2 protein in excitatory, inhibitory, and dopaminergic neurons. In primary hippocampal cultures, the subcellular localization of CPEB2 in neurons and astrocytes resembled that of CPEB1. Electrophoretic mobility shift assay and RNA coimmunoprecipitation revealed CPEB2 interaction with ß-catenin and Ca(2+) /calmodulin-dependent protein kinase II (both established CPEB1 targets), indicating an overlap in RNA binding specificity between CPEB1 and CPEB2. Furthermore, we identified ephrin receptor A4 as a putative novel target of CPEB2. In conclusion, our study identifies CPEB2 splice variants to be differentially expressed among individual cells and across cell types of the mouse hippocampus, and reveals overlapping binding specificity between CPEB2 and CPEB1.

Panglial gap junctional communication is essential for maintenance of myelin in the CNS.

In this study, we have investigated the contribution of oligodendrocytic connexin47 (Cx47) and astrocytic Cx30 to panglial gap junctional networks as well as myelin maintenance and function by deletion of both connexin coding DNAs in mice. Biocytin injections revealed complete disruption of oligodendrocyte-to-astrocyte coupling in the white matter of 10- to 15-d-old Cx30/Cx47 double-deficient mice, while oligodendrocyte-to-oligodendrocyte coupling was maintained. There were no quantitative differences regarding cellular networks in acute brain slices obtained from Cx30/Cx47 double-null mice and control littermates, probably caused by the upregulation of oligodendrocytic Cx32 in Cx30/Cx47 double-deficient mice. We observed early onset myelin pathology, and ∼40% of Cx30/Cx47 double-deficient animals died within 42 to 90 d after birth, accompanied by severe motor impairments. Histological and ultrastructural analyses revealed severe vacuolization and myelination defects in all white matter tracts of the CNS. Furthermore, Cx30/Cx47 double-deficient mice exhibited a decreased number of oligodendrocytes, severe astrogliosis, and microglial activation in white matter tracts. Although less affected concerning motor impairment, surviving double-knock-out (KO) mice showed behavioral alterations in the open field and in the rotarod task. Vacuole formation and thinner myelin sheaths were evident also with adult surviving double-KO mice. Since interastrocytic coupling due to Cx43 expression and interoligodendrocytic coupling because of Cx32 expression are still maintained, Cx30/Cx47 double-deficient mice demonstrate the functional role of both connexins for interastrocytic, interoligodendrocytic, and panglial coupling, and show that both connexins are required for maintenance of myelin.

Functional redundancy and compensation among members of gap junction protein families?

Gap junctions are intercellular conduits for small molecules made up by protein subunits called connexins. A large number of connexin genes were found in mouse and man, and most cell types express several connexins, lending support to the view that redundancy and compensation among family members exist. This review gives an overview of the current knowledge on redundancy and functional compensation - or lack thereof. It takes into account the different properties of connexin subunits which comprise gap junctional intercellular channels, but also the compatibility of connexins in gap junctions. Most insight has been gained by the investigation of mice deficient for one or more connexins and transgenic mice with functional replacement of one connexin gene by another. Most single deficient mice show phenotypical alterations limited to critical developmental time points or to specific organs and tissues, while mice doubly deficient for connexins expressed in the same cell type usually show more severe phenotypical alterations. Replacement of a connexin by another connexin in some cases gave rise to rescue of phenotypical alterations of connexin deficiencies, which were restricted to specific tissues. In many tissues, connexin substitution did not restore phenotypical alterations of connexin deficiencies, indicating that connexins are specialized in function. In some cases, fatal consequences arose from the replacement. The current consensus gained from such studies is that redundancy and compensation among connexins exists at least to a limited extent. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

Dual reporter approaches for identification of Cre efficacy and astrocyte heterogeneity.

Gene inactivation reporters are powerful tools to circumvent limitations of the widely used Cre/loxP system of conditional mutagenesis. With new conditional transgenic mouse lines expressing the enhanced cyan fluorescent protein (ECFP) instead of connexin43 (Cx43) after Cre-mediated recombination, we demonstrate dual reporter approaches to simultaneously examine astrocyte subpopulations expressing different connexins, identify compensatory up-regulation within gene families, and quantify Cre-mediated deletion at the allelic level. Analysis of a newly generated Cx43 knock-in ECFP mouse revealed an unexpected heterogeneity of Cx43-expressing astrocytes across brain areas.

Gap junctions mediate intercellular spread of sodium between hippocampal astrocytes in situ.

Activation of glutamatergic synapses results in long-lasting sodium transients in astrocytes mediated mainly by sodium-dependent glutamate uptake. Sodium elevations activate Na(+) /K(+) -ATPase and glucose uptake by astrocytes, representing key signals for coupling glial metabolism to neuronal activity. Here, we analyzed the spread of sodium signals between astrocytes in hippocampal slice preparations. Stimulation of a single astrocyte resulted in an immediate sodium elevation that spread to neighboring astrocytes within a distance of ∼ 100 µm. Amplitude, slope, and propagation speed of sodium elevations in downstream cells decayed monotonically with increasing distance, indicative of a diffusion process. In contrast to sodium, calcium increases elicited by electrical stimulation were restricted to the stimulated cell and a few neighboring astrocytes. Pharmacological inhibition of mGluR1/5 slightly dampened the spread of sodium, whereas inhibition of glutamate uptake or purinergic receptors had no effect. Spread of sodium to neighboring cells was disturbed on pharmacological inhibition of gap junctions, reduced in animals at P4 and virtually omitted in Cx30/Cx43 double-deficient mice. In contrast to results obtained earlier in cultured astrocytes, our data thus indicate that calcium signaling and metabotropic glutamate receptors are supportive of, but not prerequisites for, the spread of sodium between hippocampal astrocytes in situ, whereas expression of Cx30 and Cx43 is essential. Cx30/Cx43-mediated sodium diffusion between astrocytes could represent a signal indicating increased metabolic needs, independent of concomitant calcium signaling. Spread of sodium might also serve a homeostatic function by supporting the re-establishment of steep sodium gradients and by lowering the metabolic burden imposed on single cells.

Connexin expression by radial glia-like cells is required for neurogenesis in the adult dentate gyrus.

In the adult dentate gyrus, radial glia-like cells represent putative stem cells generating neurons and glial cells. Here, we combined patch-clamp recordings, biocytin filling, immunohistochemistry, single-cell transcript analysis, and mouse transgenics to test for connexin expression and gap junctional coupling of radial glia-like cells and its impact on neurogenesis. Radial glia-like cells were identified in mice expressing EGFP under control of the nestin and gfap promoters. We show that a majority of Radial glia-like cells are coupled and express Cx43. Neuronal precursors were not coupled. Mice lacking Cx30 and Cx43 in GFAP-positive cells displayed almost complete inhibition of proliferation and a significant decline in numbers of radial glia-like cells and granule neurons. Inducible virus-mediated ablation of connexins in the adult hippocampus also reduced neurogenesis. These findings strongly suggest the requirement of connexin expression by radial glia-like cells for intact neurogenesis in the adult brain and point to possible communication pathways of these cells.

Role of astroglial connexin30 in hippocampal gap junction coupling.

The impact of connexin30 (Cx30) on interastrocytic gap junction coupling in the normal hippocampus is matter of debate; reporter gene analyses indicated a weak expression of Cx30 in the mouse hippocampus. In contrast, mice lacking connexin43 (Cx43) in astrocytes exhibited only 50% reduction in coupling. Complete uncoupling of hippocampal astrocytes in mice lacking both Cx30 and Cx43 suggested that Cx30 participates in interastrocytic gap junction coupling in the hippocampus. With comparative reporter gene assays, immunodetection, and cre/loxP-based reporter approaches we demonstrate that Cx30 is more abundant than previously thought. The specific role of Cx30 in interastrocytic coupling has never been investigated. Employing tracer coupling analyses in acute slices of Cx30 deficient mice here we show that Cx30 makes a substantial contribution to interastrocytic gap junctional communication in the mouse hippocampus.

Involvement of the cytoplasmic C-terminal domain of connexin43 in neuronal migration.

During brain development, young neurons closely associate with radial glial while migrating from the ventricular zone (VZ) to the cortical plate (CP) of the neocortex. It has been shown previously that gap junctions are needed for this migration to occur properly, but the precise mechanism responsible is still in question. Here, we used Cre recombinase, driven by the nestin promoter, to conditionally knock-out a floxed coding DNA of the connexin43 (Cx43) gene in mice. Radial glia in the VZ normally express connexin43. They undergo divisions that produce neurons and astrocytes and serve as migratory guides for the daughter cells that they produce. Based on histological analysis, we suggest that removing Cx43 from radial glia alters the normal lamination of the mouse neocortex. To monitor newborn neurons during development, we introduced a plasmid containing green fluorescent protein driven by a neuronal (Talpha1 tubulin) promoter into the embryonic neocortex using in utero electroporation. The transfected migrating neurons remain in the VZ/intermediate zone (IZ) of the Cx43 conditional knock-out (Cx43cKO) animals, whereas in Cx43(fl/fl) mice, neurons migrate through the IZ into the CP, indicating that deletion of Cx43 from nestin-positive cells disrupts neuronal migration. We were able to rescue migration of Cx43cKO neurons by electroporating a cytomegalovirus-Cx43 expression plasmid into the embryonic cortex. In contrast, a C-terminal truncated form of Cx43 failed to rescue neuronal migration. In addition, Cx43(K258stop) mice, in which Cx43 lacks the last 125 amino acid residues of the cytoplasmic C-terminal domain, gave results similar to those seen with the Cx43cKO mice. This study illustrates that deletion of the C-terminal domain of Cx43 alters neuronal migration in the neocortex.

Cytoplasmic Polyadenylation Element Binding Proteins CPEB1 and CPEB3 Regulate the Translation of FosB and Are Required for Maintaining Addiction-Like Behaviors Induced by Cocaine.

A recurrent and devastating feature of addiction to a drug of abuse is its persistence, which is mediated by maladaptive long-term memories of the highly pleasurable experience initially associated with the consumption of the drug. We have recently found that members of the CPEB family of proteins (Cytoplasmic Polyadenylation Element-Binding Proteins) are involved in the maintenance of spatial memory. However, their possible role in the maintenance of memories that sustain addictive behavior has yet to be explored. Little is known about any of the mechanisms for maintaining memories for addictive behavior. To address the mechanisms whereby addictive behavior is maintained over time, we utilized a conditional transgenic mouse model expressing a dominant-negative version of CPEB1 that abolishes the activity in the forebrain of two of the four CPEB isoforms (CPEB1 and CPEB3). We found that, following cocaine administration, these dominant-negative (DN) CPEB mice showed a significant decrease, when compared to wild type (WT) mice, in both locomotor sensitizations and conditioned place preference (CPP), two indices of addictive behavior. Supporting these behavioral results, we also found a difference between WT and DN-CPEB1-3 mice in the cocaine-induced synaptic depression in the core of the Nucleus Accumbens (NAc). Finally, we found that (1) CPEB is reduced in transgenic mice following cocaine injections and that (2) FosB, known for its contribution to establishing the addictive phenotype, when its expression in the striatum is increased by drug administration, is a novel target of CPEBs molecules. Thus, our study highlights how CPEB1 and CPEB3 act on target mRNAs to build the neuroadaptative implicit memory responses that lead to the development of the cocaine addictive phenotypes in mammals.

The proapoptotic BCL-2 homology domain 3-only protein Bim is not critical for acute excitotoxic cell death.

Prolonged and repetitive epileptic activity is causally linked to neuronal cell death in the brain and is most marked in vulnerable subfields of the hippocampus. The Bcl-2 family protein Bim, a proapoptotic member of the BCL-2 homology domain 3-only subfamily, has been implicated as an important mediator of neuronal cell damage in various pathological conditions, although its role in epilepsy-associated cell death is not understood. We performed intrahippocampal stereotaxic injections of the glutamate analog kainic acid as an in vivo model of acute excitotoxicity to assess neuronal injury in Bim-deficient and control wild-type mice. A variety of cell death parameters including chromatin condensation, TdT-mediated dUTP nick end labeling, and caspase-3 activity was assessed. We found no differences in the extent of hippocampal neuronal death parameters between the 2 groups. Moreover, electroencephalographic recordings after kainic acid injection revealed indistinguishable patterns of seizure activity in Bim-deficient and wild-type animals. These in vivo and histological data suggest that Bim is not critically involved in excitotoxicity-induced acute neuronal cell injury.

Quality control of astrocyte-directed Cre transgenic mice: the benefits of a direct link between loss of gene expression and reporter activation.

Cre recombinase activity for cell-type restricted deletion of floxed target genes (i.e., flanked by Cre recognition loxP-sites) is often measured by separate matings with recombination-activated reporter gene mice. Using a floxed Gja1 (Cx43) allele, we demonstrate the benefits of a direct link between reporter gene expression and target gene deletion to overcome critical limitations of the Cre/loxP system. The widely used human glial fibrillary acidic protein (hGFAP)-Cre transgene exhibits variable recombination activity and requires postexperimental validation. Such quality control is essential to correlate the extent of Cre-mediated Gja1 ablation with phenotypical alterations and to maintain the activity status of hGFAP-Cre in transgenic mouse colonies. We present several strategies to control for the fidelity of hGFAP-Cre mediated recombination. (c) 2008 Wiley-Liss, Inc.

Connexin43, but not connexin30, contributes to adult neurogenesis in the dentate gyrus.

The subgranular zone of the dentate gyrus represents a niche in which radial glia (RG)-like cells generate new neurons throughout postnatal life in the mammalian brain. Previous data showed that RG-like cells are coupled through gap junction channels, primarily formed by connexin43 (Cx43) and Cx30, and that the expression of these proteins is required for adult neurogenesis in the hippocampus. However, their individual function and underlying mechanisms remain unclear. Here we demonstrate that Cx43, but not Cx30, is crucial for adult neurogenesis. To assess whether Cx43-dependent intercellular coupling between RG-like cells or rather channel-independent interactions of the protein regulate neurogenesis, mice bearing a Cx43 point mutation (Cx43G138R) in RG-like cells and protoplasmic astrocytes cells were employed, which was expected to cause channel closure without affecting the trafficking of the protein to the membrane. We confirmed the disruption of coupling between RG-like cells and astrocytes in the hippocampus of Cx43G138R mice. Proliferative activity and neurogenesis in the DG were significantly decreased in the mutant mouse line, indicating that functional Cx43 channels are essential for proper adult neurogenesis. The fate of proliferating cells in the DG was not affected by Cx43 mutation as revealed by 5-bromo-2-deoxyuridine (BrdU) incorporation assays. Together, these findings suggest that adult neurogenesis in the hippocampus does not require Cx30 but channel-dependent functions of Cx43.

The impact of astrocytic gap junctional coupling on potassium buffering in the hippocampus.

Astrocytic gap junctions have been suggested to contribute to spatial buffering of potassium in the brain. Direct evidence has been difficult to gather because of the lack of astrocyte-specific gap junction blockers. We obtained mice with coupling-deficient astrocytes by crossing conditional connexin43-deficient mice with connexin30(-/-) mice. Similar to wild-type astrocytes, genetically uncoupled hippocampal astrocytes displayed negative resting membrane potentials, time- and voltage-independent whole-cell currents, and typical astrocyte morphologies. Astrocyte densities were also unchanged. Using potassium-selective microelectrodes, we assessed changes in potassium buffering in hippocampal slices of mice with coupling-deficient astrocytes. We demonstrate that astrocytic gap junctions accelerate potassium clearance, limit potassium accumulation during synchronized neuronal firing, and aid in radial potassium relocation in the stratum lacunosum moleculare. Furthermore, slices of mice with coupling-deficient astrocytes displayed a reduced threshold for the generation of epileptiform events. However, it was evident that radial relocation of potassium in the stratum radiatum was not dependent on gap junctional coupling. We suggest that the perpendicular array of individual astrocytes in the stratum radiatum makes these cells ideally suited for spatial buffering of potassium released by pyramidal cells, independent of gap junctions. In general, a surprisingly large capacity for K+ clearance was conserved in mice with coupling-deficient astrocytes, indicating that gap junction-dependent processes only partially account for K+ buffering in the hippocampus.

Emerging complexities in identity and function of glial connexins.

Recent research results indicate that glial gap-junction communication is much more complex and widespread than originally thought, and has diverse roles in brain homeostasis and the response of the brain to injury. The situation is far from clear, however. Pharmacological agents that block gap junctions can abolish neuron-glia long-range signaling and can alleviate neuronal damage whereas, intriguingly, opposite effects are observed in mice lacking connexin43, a major gap-junction subunit protein in astrocytes. How can the apparently contradictory results be explained, and how is specificity achieved within the glial gap-junction system? Another key issue in understanding glial connexin function is that oligodendrocytes and astrocytes, each of which express distinct connexin isotypes, are thought to participate in brain homeostasis by forming a panglial syncytium. Molecular analysis has revealed a surprising diversity of connexin expression and function, and this has led to new hypotheses regarding their roles in the brain, which could be tested using new approaches.

Connexin43 Containing Gap Junction Channels Facilitate HIV Bystander Toxicity: Implications in NeuroHIV.

Human immunodeficiency virus-1 (HIV-1) infection compromises the central nervous system (CNS) in a significant number of infected individuals, resulting in neurological dysfunction that ranges from minor cognitive deficits to frank dementia. While macrophages/microglia are the predominant CNS cells infected by HIV, our laboratory and others have shown that HIV-infected astrocytes, although present in relatively low numbers with minimal to undetectable viral replication, play key role in NeuroAIDS pathogenesis. Our laboratory has identified that HIV "hijacks" connexin (Cx) containing channels, such as gap junctions (GJs) and hemichannels (HCs), to spread toxicity and apoptosis to uninfected cells even in the absence of active viral replication. In this study, using a murine model with an astrocyte-directed deletion of Cx43 gene (hGFAP-cre Cx43fl/fl) and control Cx43fl/fl mice, we examined whether few HIV-infected human astrocytoma cells (U87-CD4-CCR5), microinjected into the mouse cortex, can spread toxicity and apoptosis through GJ-mediated mechanisms, into the mouse cells, which are resistant to HIV infection. In the control Cx43fl/fl mice, microinjection of HIV-infected U87-CD4-CCR5 cells led to apoptosis in 84.28 ± 6.38% of mouse brain cells around the site of microinjection, whereas hGFAP-cre Cx43fl/fl mice exhibited minimal apoptosis (2.78 ± 1.55%). However, simultaneous injection of GJ blocker, 18α-glycyrrhetinic acid, and Cx43 blocking peptide along with microinjection of HIV-infected cells prevented apoptosis in Cx43fl/fl mice, demonstrating the Cx43 is essential for HIV-induced bystander toxicity. In conclusion, our findings demonstrate that Cx43 expression, and formation of GJs is essential for bystander apoptosis during HIV infection. These findings reveal novel potential therapeutic targets to reduce astrocyte-mediated bystander toxicity in HIV-infected individuals because despite low to undetectable viral replication in the CNS, Cx channels hijacked by HIV amplify viral neuropathogenesis.

Altered splicing leads to reduced activation of CPEB3 in high-grade gliomas.

Cytoplasmic polyadenylation element binding proteins (CPEBs) are auxiliary translational factors that associate with consensus sequences present in 3'UTRs of mRNAs, thereby activating or repressing their translation. Knowing that CPEBs are players in cell cycle regulation and cellular senescence prompted us to investigate their contribution to the molecular pathology of gliomas-most frequent of intracranial tumors found in humans. To this end, we performed methylation analyses in the promoter regions of CPEB1-4 and identified the CPEB1 gene to be hypermethylated in tumor samples. Decreased expression of CPEB1 protein in gliomas correlated with the rising grade of tumor malignancy. Abundant expression of CPEBs2-4 was observed in several glioma specimens. Interestingly, expression of CPEB3 positively correlated with tumor progression and malignancy but negatively correlated with protein phosphorylation in the alternatively spliced region. Our data suggest that loss of CPEB3 activity in high-grade gliomas is caused by expression of alternatively spliced variants lacking the B-region that overlaps with the kinase recognition site. We conclude that deregulation of CPEB proteins may be a frequent phenomenon in gliomas and occurs on the level of transcription involving epigenetic mechanism as well as on the level of mRNA splicing, which generates isoforms with compromised biological properties.