Galina Dvoriantchikova

Transgenic Inhibition of Astroglial NF-κB Improves Functional Outcome in Experimental Autoimmune Encephalomyelitis by Suppressing Chronic Central Nervous System Inflammation

In the CNS, the transcription factor NF-κB is a key regulator of inflammation and secondary injury processes. Following trauma or disease, the expression of NF-κB-dependent genes is activated, leading to both protective and detrimental effects. In this study, we show that transgenic inactivation of astroglial NF-κB (glial fibrillary acidic protein-IκBα-dominant-negative mice) resulted in reduced disease severity and improved functional recovery following experimental autoimmune encephalomyelitis. At the chronic stage of the disease, transgenic mice exhibited an overall higher presence of leukocytes in spinal cord and brain, and a markedly higher percentage of CD8+CD122+ T regulatory cells compared with wild type, which correlated with the timing of clinical recovery. We also observed that expression of proinflammatory genes in both spinal cord and cerebellum was delayed and reduced, whereas the loss of neuronal-specific molecules essential for synaptic transmission was limited compared with wild-type mice. Furthermore, death of retinal ganglion cells in affected retinas was almost abolished, suggesting the activation of neuroprotective mechanisms. Our data indicate that inhibiting NF-κB in astrocytes results in neuroprotective effects following experimental autoimmune encephalomyelitis, directly implicating astrocytes in the pathophysiology of this disease.

Inactivation of astroglial NF‐κB promotes survival of retinal neurons following ischemic injury

Reactive astrocytes have been implicated in neuronal loss following ischemic stroke. However, the molecular mechanisms associated with this process are yet to be fully elucidated. In this work, we tested the hypothesis that astroglial NF‐κB, a key regulator of inflammatory responses, is a contributor to neuronal death following ischemic injury. We compared neuronal survival in the ganglion cell layer (GCL) after retinal ischemia‐reperfusion in wild‐type (WT) and in GFAP‐IκBα‐dn transgenic mice, where the NF‐κB classical pathway is suppressed specifically in astrocytes. The GFAP‐IκBα‐dn mice showed significantly increased survival of neurons in the GCL following ischemic injury as compared with WT littermates. Neuroprotection was associated with significantly reduced expression of pro‐inflammatory genes, encoding Tnf‐α, Ccl2 (Mcp1), Cxcl10 (IP10), Icam1, Vcam1, several subunits of NADPH oxidase and NO‐synthase in the retinas of GFAP‐IκBα‐dn mice. These data suggest that certain NF‐κB‐regulated pro‐inflammatory and redox‐active pathways are central to glial neurotoxicity induced by ischemic injury. The inhibition of these pathways in astrocytes may represent a feasible neuroprotective strategy for retinal ischemia and stroke.

Expression of pannexin family of proteins in the retina

Expression of the Panx1 and Panx2 members of the pannexin family of gap junction proteins was studied in the retina by in situ hybridization and qRT‐PCR. Both pannexins showed robust expression across the retina with predominant accumulation in the retinal ganglion cells (RGCs). In concordance, immunohistochemical analysis showed accumulation of the Panx1 protein in RGCs, amacrine, horizontal cells and their processes. Two Panx1 isoforms were detected: a ubiquitously expressed 58 kDa protein, and a 43 kDa isoform that specifically accumulated in the retina and brain. Our results indicated that Panx1 and Panx2 are abundantly expressed in the retina, and may therefore contribute to the electrical and metabolic coupling, or to signaling between retinal neurons via the secondary messengers.

Pannexin1 stabilizes synaptic plasticity and is needed for learning.

Pannexin 1 (Panx1) represents a class of vertebrate membrane channels, bearing significant sequence homology with the invertebrate gap junction proteins, the innexins and more distant similarities in the membrane topologies and pharmacological sensitivities with gap junction proteins of the connexin family. In the nervous system, cooperation among pannexin channels, adenosine receptors, and KATP channels modulating neuronal excitability via ATP and adenosine has been recognized, but little is known about the significance in vivo. However, the localization of Panx1 at postsynaptic sites in hippocampal neurons and astrocytes in close proximity together with the fundamental role of ATP and adenosine for CNS metabolism and cell signaling underscore the potential relevance of this channel to synaptic plasticity and higher brain functions. Here, we report increased excitability and potently enhanced early and persistent LTP responses in the CA1 region of acute slice preparations from adult Panx1−/− mice. Adenosine application and N-methyl-D-aspartate receptor (NMDAR)-blocking normalized this phenotype, suggesting that absence of Panx1 causes chronic extracellular ATP/adenosine depletion, thus facilitating postsynaptic NMDAR activation. Compensatory transcriptional up-regulation of metabotropic glutamate receptor 4 (grm4) accompanies these adaptive changes. The physiological modification, promoted by loss of Panx1, led to distinct behavioral alterations, enhancing anxiety and impairing object recognition and spatial learning in Panx1−/− mice. We conclude that ATP release through Panx1 channels plays a critical role in maintaining synaptic strength and plasticity in CA1 neurons of the adult hippocampus. This result provides the rationale for in-depth analysis of Panx1 function and adenosine based therapies in CNS disorders.

Genetic Ablation of Pannexin1 Protects Retinal Neurons from Ischemic Injury

Pannexin1 (Panx1) forms large nonselective membrane channel that is implicated in paracrine and inflammatory signaling. In vitro experiments suggested that Panx1 could play a key role in ischemic death of hippocampal neurons. Since retinal ganglion cells (RGCs) express high levels of Panx1 and are susceptible to ischemic induced injury, we hypothesized that Panx1 contributes to rapid and selective loss of these neurons in ischemia. To test this hypothesis, we induced experimental retinal ischemia followed by reperfusion in live animals with the Panx1 channel genetically ablated either in the entire mouse (Panx1 KO), or only in neurons using the conditional knockout (Panx1 CKO) technology. Here we report that two distinct neurotoxic processes are induced in RGCs by ischemia in the wild type mice but are inactivated in Panx1KO and Panx1 CKO animals. First, the post-ischemic permeation of RGC plasma membranes is suppressed, as assessed by dye transfer and calcium imaging assays ex vivo and in vitro. Second, the inflammasome-mediated activation of caspase-1 and the production of interleukin-1β in the Panx1 KO retinas are inhibited. Our findings indicate that post-ischemic neurotoxicity in the retina is mediated by previously uncharacterized pathways, which involve neuronal Panx1 and are intrinsic to RGCs. Thus, our work presents the in vivo evidence for neurotoxicity elicited by neuronal Panx1, and identifies this channel as a new therapeutic target in ischemic pathologies.

Microarray analysis of gene expression in adult retinal ganglion cells

Retinal ganglion cells (RGCs) transfer visual information to the brain and are known to be susceptible to selective degeneration in various neuropathies such as glaucoma. This selective vulnerability suggests that these highly specialized neurons possess a distinct gene expression profile that becomes altered by neuropathy‐associated stresses, which lead to the RGC death. In this study, to identify genes expressed predominantly in adult RGCs, a global transcriptional profile of purified primary RGCs has been compared to that of the whole retina. To avoid alterations of the original gene expression profile by cell culture conditions, we isolated RNA directly from adult RGCs purified by immunopanning without prior sub‐cultivation. Genes expressed predominantly in RGCs included: Nrg1, Rgn, 14‐3‐3 family (Ywhah, Ywhaz, Ywhab), Nrn1, Gap43, Vsnl1, Rgs4. Some of these genes may serve as novel markers for these neurons. Our analysis revealed enrichment in genes controlling the pro‐survival pathways in RGCs as compared to other retinal cells.

Microarray analysis of fiber cell maturation in the lens

The mammalian lens consists of an aged core of quiescent cells enveloped by layers of mature fully elongated cells and younger, continuously elongating transcriptionally active cells. The fiber cell maturation is initiated when fiber cells cease to elongate. The process of maturation represents a radical switch from active elongation to a life‐long quiescence and has not been studied previously. It may also include critical stages of preparation for the organelle removal and denucleation. In the present study, we used laser capture microdisection (LCM) microdissection and RNA amplification to compare global gene expression profiles of young elongating and mature, non‐elongating fiber cells. Analysis of microarray data from three independent dye‐swap experiments identified 65 differentially expressed genes (FDR < 0.1) with greater than 2‐fold change in expression levels. Microarray array results for a group of randomly selected genes were confirmed by quantitative RT‐PCR. These microarray results provide clues to understanding the molecular pathways underlying lens development. The identified changes in the profile of gene expression reflected a shift in cell physiology characterizing the lens fiber maturation.

Retinal ganglion cell (RGC) programmed necrosis contributes to ischemia-reperfusion-induced retinal damage

Retinal ischemia-reperfusion (IR) injury remains a common cause of blindness and has a final pathway of retinal ganglion cell (RGC) death by apoptosis and necrosis. RGC apoptosis was intensively studied in IR injury, while RGC necrosis did not receive nearly enough consideration since it was viewed as an accidental and unregulated cellular event. However, there is evidence that necrosis, like apoptosis, can be implemented by a programmed mechanism. In this study, we tested the role of RGC programmed necrosis (necroptosis) in IR-induced retinal injury. We employed the mouse model of retinal IR injury for in vivo experiments. The oxygen and glucose deprivation (OGD) model was used as an IR model in vitro. Primary RGCs were isolated by an immunopanning technique. Necrostatin 1 (Nec1) was used to inhibit necroptosis in in vitro and in vivo experiments. The changes in gene expression were assessed by quantitative RT-PCR. The distribution of proteins in the retina and in RGC cultures was evaluated by immunohistochemistry and immunocytochemistry, respectively. Our data suggest that proteins (Ripk1 and Ripk3), which initiate necroptosis, were present in normal and ischemic RGCs. Treatment with Nec1 significantly reduced retinal damage after IR. Increased RGC survival and reduced RGC necrosis following OGD were observed in Nec1-treated cultures. We found significantly reduced expression of genes coding pro-inflammatory markers Il1b, Ccl5, Cxcl10, Nos2 and Cybb in Nec1-treated ischemic retinas. Thus, our findings suggest that RGC necroptosis contributes to retinal damage after IR through direct loss of cells and induction of associated inflammatory responses.

Neuronal NAD(P)H Oxidases Contribute to ROS Production and Mediate RGC Death after Ischemia

PURPOSE To study the role of neuronal nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase-dependent reactive oxygen species (ROS) production in retinal ganglion cell (RGC) death after ischemia. METHODS Ischemic injury was induced by unilateral elevation of intraocular pressure via direct corneal cannulation. For in vitro experiments, RGCs isolated by immunopanning from retinas were exposed to oxygen and glucose deprivation (OGD). The expression levels of NAD(P)H oxidase subunits were evaluated by quantitative PCR, immunocytochemistry, and immunohistochemistry. The level of ROS generated was assayed by dihydroethidium. The NAD(P)H oxidase inhibitors were then tested to determine if inhibition of NAD(P)H oxidase altered the production of ROS within the RGCs and promoted cell survival. RESULTS It was reported that RGCs express catalytic Nox1, Nox2, Nox4, Duox1, as well as regulatory Ncf1/p47phox, Ncf2/p67phox, Cyba/p22phox, Noxo1, and Noxa1 subunits of NAD(P)H oxidases under normal conditions and after ischemia. However, whereas RGCs express only low levels of catalytic Nox2, Nox4, and Duox1, and regulatory Ncf1/p47, Ncf2/p67 subunits, they exhibit significantly higher levels of catalytic subunit Nox1 and the subunits required for optimal activity of Nox1. It was observed that the nonselective NAD(P)H oxidase inhibitors VAS-2870, AEBSF, and the Nox1 NAD(P)H oxidase-specific inhibitor ML-090 decreased the ROS burst stimulated by OGD, which was associated with a decreased level of RGC death. CONCLUSIONS The findings suggest that NAD(P)H oxidase activity in RGCs renders them vulnerable to ischemic death. Importantly, high levels of Nox1 NAD(P)H oxidase subunits in RGCs suggest that this enzyme could be a major source of ROS in RGCs produced by NAD(P)H oxidases.

Astroglial NF-κB mediates oxidative stress by regulation of NADPH oxidase in a model of retinal ischemia reperfusion injury.

J. Neurochem. (2012) 120, 586–597.