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Earlier we reported induction of neurotoxicity and neurodegeneration by tryptophan metabolites that link the metabolic alterations to Alzheimer’s disease (AD). Tryptophan is a product of the Shikimate pathway (SP). Human cells lack SP, which is found in human gut bacteria exclusively using SP to produce aromatic amino acids (AAA). This study is a first attempt toward gene-targeted analysis of human gut microbiota in AD fecal samples. The oligonucleotide primers newly-designed for this work target SP-AAA in environmental bacteria associated with human activity. Using polymerase chain reaction (PCR), we found unique gut bacterial sequence in most AD patients (18 of 20), albeit rarely in controls (1 of 13). Cloning and sequencing AD-associated PCR products (ADPP) enables identification of Na(+)-transporting NADH: Ubiquinone reductase (NQR) in Clostridium sp. The ADPP of unrelated AD patients possess near identical sequences. NQR substrate, ubiquinone is a SP product and human neuroprotectant. A defici in ubiquinone has been determined in a number of neuromuscular and neurodegenerative disorders. Antibacterial therapy prompted an ADPP reduction in an ADPP-positive control person who was later diagnosed with AD-dementia. We explored the gut microbiome databases and uncovered a sequence similarity (up to 97%) between ADPP and some healthy individuals from different geographical locations. Importantly, our main findin of the significan difference in the gut microbial genotypes between the AD and control human populations is a breakthrough.

Lacrimal gland inflammation triggers dry eye disease through impaired tear secretion by the epithelium. As aberrant inflammasome activation occurs in autoimmune disorders including Sjögren’s syndrome, we analyzed the inflammasome pathway during acute and chronic inflammation and investigated its potential regulators. Bacterial infection was mimicked by the intraglandular injection of lipopolysaccharide (LPS) and nigericin, known to activate the NLRP3 inflammasome. Acute injury of the lacrimal gland was induced by interleukin (IL)-1α injection. Chronic inflammation was studied using two Sjögren’s syndrome models: diseased NOD.H2b compared to healthy BALBc mice and Thrombospondin-1-null (TSP-1-/-) compared to TSP-1WTC57BL/6J mice. Inflammasome activation was investigated by immunostaining using the R26ASC-citrine reporter mouse, by Western blotting, and by RNAseq. LPS/Nigericin, IL-1α and chronic inflammation induced inflammasomes in lacrimal gland epithelial cells. Acute and chronic inflammation of the lacrimal gland upregulated multiple inflammasome sensors, caspases 1/4, and interleukins Il1b and Il18. We also found increased IL-1β maturation in Sjögren’s syndrome models compared with healthy control lacrimal glands. Using RNA-seq data of regenerating lacrimal glands, we found that lipogenic genes were upregulated during the resolution of inflammation following acute injury. In chronically inflamed NOD.H2b lacrimal glands, an altered lipid metabolism was associated with disease progression: genes for cholesterol metabolism were upregulated, while genes involved in mitochondrial metabolism and fatty acid synthesis were downregulated, including peroxisome proliferator-activated receptor alpha (PPARα)/sterol regulatory element-binding 1 (SREBP-1)-dependent signaling. We conclude that epithelial cells can promote immune responses by forming inflammasomes, and that sustained inflammasome activation, together with an altered lipid metabolism, are key players of Sjögren’s syndrome-like pathogenesis in the NOD.H2b mouse lacrimal gland by promoting epithelial dysfunction and inflammation.

Glaucoma is a multifactorial disease resulting in progressive vision loss due to retinal ganglion cell (RGC) dysfunction and death. Early events in the pathobiology of the disease include oxidative, metabolic, or mechanical stress that acts upon RGC, causing these to rapidly release danger signals, including extracellular ATP, resulting in micro- and macroglial activation and neuroinflammation. Danger signaling also leads to the formation of inflammasomes in the retina that enable maturation of proinflammatory cytokines such IL-1β and IL-18. Chronic neuroinflammation can have directly damaging effects on RGC, but it also creates a proinflammatory environment and compromises the immune privilege of the retina. In particular, continuous synthesis of proinflammatory mediators such as TNFα, IL-1β, and anaphylatoxins weakens the blood–retina barrier and recruits or activates T-cells. Recent data have demonstrated that adaptive immune responses strongly exacerbate RGC loss in animal models of the disease as T-cells appear to target heat shock proteins displayed on the surface of stressed RGC to cause their apoptotic death. It is possible that dysregulation of these immune responses contributes to the continued loss of RGC in some patients.

Made available in DSpace on 2018-12-11T17:21:24Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-08-01 FP7 Ideas: European Research Council Liver fibrosis is the final common pathway for almost all causes of chronic liver injury. This chronic disease is characterized by excessive deposition of extracellular matrix components mainly due to transdifferentiation of quiescent hepatic stellate cell into myofibroblasts-like cells, which in turn is driven by cell death and inflammation. In the last few years, paracrine signaling through pannexin1 channels has emerged as a key player in the latter processes. The current study was set up to investigate the role of pannexin1 signaling in liver fibrosis. Wild-type and whole body pannexin1 knock-out mice were treated with carbon tetrachloride or subjected to bile duct ligation. Evaluation of the effects of pannexin1 deletion was based on a number of clinically relevant read-outs, including markers of liver damage, histopathological analysis, oxidative stress, inflammation and regenerative capacity. In parallel, to elucidate the molecular pathways affected by pannexin1 deletion as well as to mechanistically anchor the clinical observations, whole transcriptome analysis of liver tissue was performed. While pannexin1 knock-out mice treated with carbon tetrachloride displayed reduced collagen content, hepatic stellate cell activation, inflammation and hepatic regeneration, bile duct ligated counterparts showed increased hepatocellular injury and antioxidant enzyme activity with a predominant immune response. Gene expression profiling revealed a downregulation of fibrotic and immune responses in pannexin1 knock-out mice treated with carbon tetrachloride, whereas bile duct ligated pannexin1-deficient animals showed a pronounced inflammatory profile. This study shows for the first time an etiology-dependent role for pannexin1 signaling in experimental liver fibrosis. Department of In Vitro Toxicology and Dermato-Cosmetology Vrije Universiteit Brussel Department of Pathology School of Veterinary Medicine and Animal Science University of São Paulo Bascom Palmer Eye Institute Department of Ophthalmology University of Miami Miller School of Medicine Department of Cell Biology and Anatomy University of Miami Miller School of Medicine Department of Food and Experimental Nutrition Faculty of Pharmaceutical Sciences University of São Paulo Department of Pathology Botucatu Medical School UNESP-São Paulo State University Department of Pathology Botucatu Medical School UNESP-São Paulo State University FP7 Ideas: European Research Council: 335476

Pannexins are transmembrane proteins that form communication channels connecting the cytosol of an individual cell with its extracellular environment. A number of studies have documented the presence of pannexin1 in liver as well as its involvement in inflammatory responses. In this study, it was investigated whether pannexin1 plays a role in acute liver failure and non-alcoholic steatohepatitis, being prototypical acute and chronic liver pathologies, respectively, both featured by liver damage, oxidative stress and inflammation. To this end, wild-type and pannexin1(−/−) mice were overdosed with acetaminophen for 1, 6, 24 or 48 hours or were fed a choline-deficient high-fat diet for 8 weeks. Evaluation of the effects of genetic pannexin1 deletion was based on a number of clinically relevant read-outs, including markers of liver damage, histopathological analysis, lipid accumulation, protein adduct formation, oxidative stress and inflammation. In parallel, in order to elucidate molecular pathways affected by pannexin1 deletion as well as to mechanistically anchor the clinical observations, whole transcriptome analysis of liver tissue was performed. The results of this study show that pannexin1(−/−) diseased mice present less liver damage and oxidative stress, while inflammation was only decreased in pannexin1(−/−) mice in which non-alcoholic steatohepatitis was induced. A multitude of genes related to inflammation, oxidative stress and xenobiotic metabolism were differentially modulated in both liver disease models in wild-type and in pannexin1(−/−) mice. Overall, the results of this study suggest that pannexin1 may play a role in the pathogenesis of liver disease.

Earlier we reported induction of neurotoxicity and neurodegeneration by tryptophan metabolites that link the metabolic alterations to Alzheimer's disease (AD). Tryptophan is a product of Shikimate pathway (SP). Human cells lack SP, which is found in human gut bacteria exclusively using SP to produce aromatic amino acids (AAA). This study is a first attempt toward gene-targeted analysis of human gut microbiota in AD fecal samples. The oligonucleotide primers newly-designed for this work target SP-AAA in environmental bacteria associated with human activity. Using polymerase chain reaction (PCR), we found unique gut bacterial sequence in most AD patients (18 of 20), albeit rarely in controls (1 of 13). Cloning and sequencing AD-associated PCR products (ADPP) enables identification of Na(+)-transporting NADH: Ubiquinone reductase (NQR) in Clostridium sp. The ADPP of unrelated AD patients possess near identical sequences. NQR substrate, ubiquinone is a SP product and human neuroprotectant. A deficit in ubiquinone has been determined in a number of neuromuscular and neurodegenerative disorders. Antibacterial therapy prompted an ADPP reduction in an ADPP-positive control person who was later diagnosed with AD-dementia. We explored the gut microbiome databases and uncovered a sequence similarity (up to 97%) between ADPP and some healthy individuals from different geographical locations. Importantly, our main finding of the significant difference in the gut microbial genotypes between the AD and control human populations is a breakthrough.

Pannexin 1 (Panx1) forms plasma membrane channels that release ATP, an important vascular tone regulator. However, despite the abundant expression of Panx1 in the vascular system, its effects on smooth muscle contraction are not evident in all arteries. In this study, we tested the hypothesis that the functional consequences of Panx1 deficiency can be masked by the augmented action of ATP secreted in a Panx1-independent way. Experiments were performed on small mesenteric arteries obtained from Panx1-knockout (Panx1–/–) and C57Bl/6 (Panx1+/+) male mice using wire myography of endothelium-denuded arterial preparations and reverse-transcription quantitative PCR techniques. Arterial contractile responses to phenylephrine did not differ in two experimental groups. Ecto-ATPase inhibitor ARL67156 (100 μM) potentiated the responses to phenylephrine in Panx1+/+ but not in Panx1–/–, while ARL67156 did not affect the contractile responses to the thromboxane A2 receptor agonist in any of the two groups. Contractile responses to exogenous ATP (10 μM) were smaller in Panx1+/+ than in Panx1–/– mice. By contrast, NTPDase1 mRNA content was higher in Panx1+/+ than in Panx1–/– mice. These results suggest that ATP released from smooth muscle cells through Panx1 channels can potentiate contractile responses of murine mesenteric arteries upon activation of α1-adrenoceptors. In Panx1–/– mice an increased arterial ATP sensitivity and diminished NTPDase1 activity may augment the contractile effects of Panx1-independent ATP.

Purpose To characterize the ocular surface microbiome of healthy volunteers using a combination of microbial culture and high-throughput DNA sequencing techniques. Methods Conjunctival swab samples from 107 healthy volunteers were analyzed by bacterial culture, 16S rDNA gene deep sequencing (n = 89), and biome representational in silico karyotyping (BRiSK; n = 80). Swab samples of the facial skin (n = 42), buccal mucosa (n = 50), and environmental controls (n = 27) were processed in parallel. 16S rDNA gene quantitative PCR was used to calculate the bacterial load in each site. Bacteria were characterized by site using principal coordinate analysis of metagenomics data. BRiSK data were analyzed for presence of fungi and viruses. Results Corynebacteria, Propionibacteria, and coagulase-negative Staphylococci were the predominant organisms identified by all three techniques. Quantitative 16S PCR demonstrated approximately 0.1 bacterial 16S rDNA/human actin copy on the ocular surface compared with greater than 10 16S rDNA/human actin copy for facial skin or the buccal mucosa. The conjunctival bacterial community structure is distinct compared with the facial skin (R = 0.474, analysis of similarities P = 0.0001), the buccal mucosa (R = 0.893, P = 0.0001), and environmental control samples (R = 0.536, P = 0.0001). 16S metagenomics revealed substantially more bacterial diversity on the ocular surface than other techniques, which appears to be artifactual. BRiSK revealed presence of torque teno virus (TTV) on the healthy ocular surface, which was confirmed by direct PCR to be present in 65% of all conjunctiva samples tested. Conclusions Relative to adjacent skin or other mucosa, healthy ocular surface microbiome is paucibacterial. Its flora are distinct from adjacent skin. Torque teno virus is a frequent constituent of the ocular surface microbiome. (ClinicalTrials.gov number, NCT02298881.).

Pannexin1 (PanX1) are recently discovered proteins that can form large pore channels at the cell surface. They have been implicated in ATP-dependent cell-to-cell communication and in several pathophysiological processes such as inflammation, cell death and epilepsy. Using immunohistochemistry in the adult mouse, we describe the presence of PanX1 in the deep cerebellar nuclei, in large cells of the granular layer, presumably Golgi interneurones, in some Bergmann glia radial processes as well as in the soma and dendrites of Purkinje cells. In the latter, PanX1, like many other proteins, distribute heterogeneously. Only Zebrin II-positive Purkinje cells express PanX1, in accordance with the so-called “zebra-striped” modular architecture of the cerebellum. This distribution in zebra-stripes suggest that PanX1 may contribute to the control of ensemble activity within cerebellar microdomains or to the response of Purkinje cell to excitotoxicity and cell-death messages.

P2X7 receptors are present in presynaptic membranes of motor synapses, but their regulatory role in modulation of neurotransmitter release remains poorly understood. P2X7 receptors may interact with pannexin 1 channels to form a purinergic signaling unit. The potential mechanism of P2X7 receptor-dependent modulation of acetylcholine (ACh) release was investigated by recording miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) in neuromuscular junctions of wild-type (WT) and pannexin 1 knockout (Panx1(−/−)) mice. Modulation of P2X7 receptors with the selective inhibitor A740003 or the selective agonist BzATP did not alter the parameters of either spontaneous or evoked ACh release in WT mice. In Panx1(−/−) mice, BzATP-induced activation of P2X7 receptors resulted in a uniformly increased quantal content of EPPs during a short stimulation train. This effect was accompanied by an increase in the size of the readily releasable pool, while the release probability did not change. Inhibition of calmodulin by W-7 or of calcium/calmodulin-dependent kinase II (CaMKII) by KN-93 completely prevented the potentiating effect of BzATP on the EPP quantal content. The blockade of L-type calcium channels also prevented BzATP action on evoked synaptic activity. Thus, the activation of presynaptic P2X7 receptors in mice lacking pannexin 1 resulted in enhanced evoked ACh release. Such enhanced release was provoked by triggering the calmodulin- and CaMKII-dependent signaling pathway, followed by activation of presynaptic L-type calcium channels. We suggest that in WT mice, this pathway is downregulated due to pannexin 1-dependent tonic activation of inhibitory presynaptic purinergic receptors, which overcomes P2X7-mediated effects.

Pannexins (Panx) are integral membrane proteins, with Panx1 being the best-characterized member of the protein family. Panx1 is implicated in sensory processing, and knockout (KO) animal models have become the primary tool to investigate the role(s) of Panx1 in sensory systems. Extending previous work from our group on primary olfaction, the expression patterns of Panxs in the vomeronasal organ (VNO), an auxiliary olfactory sense organ with a role in reproduction and social behavior, were compared. Using qRT-PCR and Immunohistochemistry (IHC), we confirmed the loss of Panx1, found similar Panx2 expression levels in both models, and a significant upregulation of Panx3 in mice with a global ablation of Panx1. Specifically, Panx3 showed upregulated expression in nerve fibers of the non-sensory epithelial layer in juvenile and adult KO mice and in the sensory layer of adults, which overlaps with Panx1 expression areas in WT populations. Since both social behavior and evoked ATP release in the VNO was not compromised in KO animals, we hypothesized that Panx3 could compensate for the loss of Panx1. This led us to compare Panx1 and Panx3 channels in vitro, demonstrating similar dye uptake and ATP release properties. Outcomes of this study strongly suggest that Panx3 may functionally compensate for the loss of Panx1 in the VNO of the olfactory system, ensuring sustained chemosensory processing. This finding extends previous reports on the upregulation of Panx3 in arterial walls and the skin of Panx1 KO mice, suggesting that roles of Panx1 warrant uncharacterized safeguarding mechanisms involving Panx3.

Pannexins belong to a family of ATP-release channels expressed in almost all cell types. An increasing body of literature on pannexins suggests that these channels play dual and sometimes contradictory roles, contributing to normal cell function, as well as to the pathological progression of disease. In this review, we summarize our understanding of pannexin "protective" and "harmful" functions in inflammation, regeneration and mechanical signaling. We also suggest a possible basis for pannexin's dual roles, related to extracellular ATP and K+ levels and the activation of various types of P2 receptors that are associated with pannexin. Finally, we speculate upon therapeutic strategies related to pannexin using eyes, lacrimal glands, and peripheral nerves as examples of interesting therapeutic targets.

Taste compounds detected by G protein-coupled receptors on the apical surface of Type 2 taste cells initiate an intracellular molecular cascade culminating in the release of ATP. It has been suggested that this ATP release is accomplished by pannexin 1 (PANX1). However, we report here that PANX1 knockout mice do not differ from wild-type controls in response to representative taste solutions, measured using 5-s brief-access tests or 48-h two-bottle choice tests. This implies that PANX1 is unnecessary for taste detection and consequently that ATP release from Type 2 taste cells does not require PANX1.

Pannexin 1 (Panx1) is involved in endothelium-dependent vasodilation in large arteries, but the exact mechanistic role remains poorly understood. We hypothesized that Panx1 facilitates large vessel relaxations regulating endothelium-derived hyperpolarization (EDH)-like mechanisms. The EDH-like component of acetylcholine-induced relaxation of saphenous arteries studied in isometric myograph after inhibition of NO-synthase and cyclooxygenase was significantly impaired in mice with genetically ablated Panx1 (KO) relative to that in the wild type (WT) mice. Application of P1-receptor antagonist and apyrase significantly reduced this component in WT, but not in KO mice, indicating participation of ATP released via Panx1 in the EDH-like relaxation.

Purpose Sjogren's syndrome is a systemic chronic autoimmune inflammatory disease that primarily targets the salivary and lacrimal glands (LGs). Currently there is no cure; therefore, cell-based regenerative therapy may be a viable option. LG inflammation is facilitated by extracellular ATP and mediated by the Pannexin-1 (Panx1) membrane channel glycoprotein. We propose that suppression of inflammation through manipulation of Panx1 activity can stimulate epithelial cell progenitor (EPCP) engraftment. Methods The expression of pannexins in the mouse and human LG was assayed by qRT-PCR and immunostaining. Acute LG inflammation was induced by interleukin-1α (IL1α) injection. Prior to EPCP transplantation, IL1α-injured or chronically inflamed LGs of thrombospondin-1-null mice (TSP-1-/-) were treated with the Panx1-specific blocking peptide (10panx) or the self-deliverable RNAi (sdRNAi). The efficacy of cell engraftment and the area of inflammation were analyzed by microscopy. Results Panx1 and Panx2 were detected in the mouse and human LGs. Panx1 and proinflammatory factors were upregulated during acute inflammation at days 1 to 3 after the IL1α injection. The analysis of EPCP engraftment demonstrated a significant and reproducible positive correlation between the 10panx peptide or Panx1 sdRNAi treatment and the number of engrafted cells. Similarly, treatment of the LG of the TSP-1-/- mouse (mouse model of chronic LG inflammation) by either Panx1 or Caspase-4 (also known as Casp11) sdRNAi showed a significant decrease in expression of proinflammatory markers and the lymphocyte infiltration. Conclusions Our results suggest that blocking Panx1 and/or Casp4 activities is a beneficial strategy to enhance donor cell engraftment and LG regeneration through the reduction of inflammation.

During brain homeostasis, both neurons and astroglia release ATP that is rapidly converted to adenosine in the extracellular space. Pannexin-1 (Panx1) hemichannels represent a major conduit of non-vesicular ATP release from brain cells. Previous studies have shown that Panx1−/− mice possess severe disruption of the sleep-wake cycle. Here, we review experimental data supporting the involvement of pannexins (Panx) in the coordination of fundamental sleep-associated brain processes, such as neuronal activity and regulation of cerebrovascular tone. Panx1 hemichannels are likely implicated in the regulation of the sleep-wake cycle via an indirect effect of released ATP on adenosine receptors and through interaction with other somnogens, such as IL-1β, TNFα and prostaglandin D2. In addition to the recently established role of Panx1 in the regulation of endothelium-dependent arterial dilation, similar signaling pathways are the major cellular component of neurovascular coupling. The new discovered role of Panx in sleep regulation may have broad implications in coordinating neuronal activity and homeostatic housekeeping processes during the sleep-wake cycle.

Pannexin 1 (Panx1) forms ATP-permeable membrane channels that play a key role in purinergic signaling in the nervous system in both normal and pathological conditions. In the retina, particularly high levels of Panx1 are found in retinal ganglion cells (RGCs), but the normal physiological function in these cells remains unclear. In this study, we used patch clamp recordings in the intact inner retina to show that evoked currents characteristic of Panx1 channel activity were detected only in RGCs, particularly in the OFF-type cells. The analysis of pattern electroretinogram (PERG) recordings indicated that Panx1 contributes to the electrical output of the retina. Consistently, PERG amplitudes were significantly impaired in the eyes with targeted ablation of the Panx1 gene in RGCs. Under ocular hypertension and ischemic conditions, however, high Panx1 activity permeated cell membranes and facilitated the selective loss of RGCs or stably transfected Neuro2A cells. Our results show that high expression of the Panx1 channel in RGCs is essential for visual function in the inner retina but makes these cells highly sensitive to mechanical and ischemic stresses. These findings are relevant to the pathophysiology of retinal disorders induced by increased intraocular pressure, such as glaucoma.

As adenosine 5'-triphosphate (ATP) released from astrocytes can modulate many neural signaling systems, the triggers and pathways for this ATP release are important. Here, the ability of mechanical strain to trigger ATP release through pannexin channels and the effects of sustained strain on pannexin expression were examined in rat optic nerve head astrocytes. Astrocytes released ATP when subjected to 5% of equibiaxial strain or to hypotonic swelling. Although astrocytes expressed mRNA for pannexins 1-3, connexin 43, and VNUT, pharmacological analysis suggested a predominant role for pannexins in mechanosensitive ATP release, with Rho kinase contribution. Astrocytes from panx1(-/-) mice had reduced baseline and stimulated levels of extracellular ATP, confirming the role for pannexins. Swelling astrocytes triggered a regulatory volume decrease that was inhibited by apyrase or probenecid. The swelling-induced rise in calcium was inhibited by P2X7 receptor antagonists A438079 and AZ10606120, in addition to apyrase and carbenoxolone. Extended stretch of astrocytes in vitro upregulated expression of panx1 and panx2 mRNA. A similar upregulation was observed in vivo in optic nerve head tissue from the Tg-MYOC(Y437H) mouse model of chronic glaucoma; genes for panx1, panx2, and panx3 were increased, whereas immunohistochemistry confirmed increased expression of pannexin 1 protein. In summary, astrocytes released ATP in response to mechanical strain, with pannexin 1 the predominant efflux pathway. Sustained strain upregulated pannexins in vitro and in vivo. Together, these findings provide a mechanism by which extracellular ATP remains elevated under chronic mechanical strain, as found in the optic nerve head of patients with glaucoma.

Many blinding diseases of the inner retina are associated with degeneration and loss of retinal ganglion cells (RGCs). Recent evidence implicates several new signaling mechanisms as causal agents associated with RGC injury and remodeling of the optic nerve head. Ion channels such as Transient receptor potential vanilloid isoform 4 (TRPV4), pannexin-1 (Panx1) and P2X7 receptor are localized to RGCs and act as potential sensors and effectors of mechanical strain, ischemia and inflammatory responses. Under normal conditions, TRPV4 may function as an osmosensor and a polymodal molecular integrator of diverse mechanical and chemical stimuli, whereas P2X7R and Panx1 respond to stretch- and/or swelling-induced adenosine triphosphate release from neurons and glia. Ca(2+) influx, induced by stimulation of mechanosensitive ion channels in glaucoma, is proposed to influence dendritic and axonal remodeling that may lead to RGC death while (at least initially) sparing other classes of retinal neuron. The secondary phase of the retinal glaucoma response is associated with microglial activation and an inflammatory response involving Toll-like receptors (TLRs), cluster of differentiation 3 (CD3) immune recognition molecules associated with the T-cell antigen receptor, complement molecules and cell type-specific release of neuroactive cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). The retinal response to mechanical stress thus involves a diversity of signaling pathways that sense and transduce mechanical strain and orchestrate both protective and destructive secondary responses.Mechanistic understanding of the interaction between pressure-dependent and independent pathways is only beginning to emerge. This review focuses on the molecular basis of mechanical strain transduction as a primary mechanism that can damage RGCs. The damage occurs through Ca(2+)-dependent cellular remodeling and is associated with parallel activation of secondary ischemic and inflammatory signaling pathways. Molecules that mediate these mechanosensory and immune responses represent plausible targets for protecting ganglion cells in glaucoma, optic neuritis and retinal ischemia.

Background Retinal ganglion cell (RGC) soma death is a consequence of optic nerve damage, including in optic neuropathies like glaucoma. The activation of the innate immune network in the retina after nerve damage has been linked to RGC pathology. Since the eye is immune privileged, innate immune functions are the responsibility of the glia, specifically the microglia, astrocytes, and Müller cells that populate the retina. Glial activation, leading to the production of inflammatory cytokines, is a hallmark feature of retinal injury resulting from optic nerve damage and purported to elicit secondary degeneration of RGC somas. Methods A mouse model of optic nerve crush (ONC) was used to study retinal glial activation responses. RGC apoptosis was blocked using Bax-deficient mice. Glial activation responses were monitored by quantitative PCR and immunofluorescent labeling in retinal sections of activation markers. ATP signaling pathways were interrogated using P2X receptor agonists and antagonists and Pannexin 1 (Panx1)-deficient mice with RGC-specific deletion. Results ONC induced activation of both macroglia and microglia in the retina, and both these responses were dramatically muted if RGC death was blocked by deletion of the Bax gene. Macroglial, but not microglial, activation was modulated by purinergic receptor activation. Release of ATP after optic nerve damage was not mediated by PANX1 channels in RGCs. Conclusions RGC death in response to ONC plays a principal stimulatory role in the retinal glial activation response. Electronic supplementary material The online version of this article (doi:10.1186/s12974-016-0558-y) contains supplementary material, which is available to authorized users.

We demonstrated previously that Pannexin 1 (Panx1), an ion and metabolite channel, promotes the growth and proliferation of ventricular zone (VZ) neural precursor cells (NPCs)in vitro. To investigate its rolein vivo, we used floxed Panx1 mice in combination with viruses to delete Panx1 in VZ NPCs and to track numbers of Panx1-null and Panx1-expressing VZ NPCs over time. Two days after virus injection, Panx1-null cells were less abundant than Panx1-expressing cells, suggesting that Panx1 is required for the maintenance of VZ NPCs. We also investigated the effect of Panx1 deletion in VZ NPCs after focal cortical stroke via photothrombosis. Panx1 is essential for maintaining elevated VZ NPC numbers after stroke. In contrast, Panx1-null NPCs were more abundant than Panx1-expressing NPCs in the peri-infarct cortex. Together, these findings suggest that Panx1 plays an important role in NPC maintenance in the VZ niche in the naive and stroke brain and could be a key target for improving NPC survival in the peri-infarct cortex.SIGNIFICANCE STATEMENTHere, we demonstrate that Pannexin 1 (Panx1) maintains a consistent population size of neural precursor cells in the ventricular zone, both in the healthy brain and in the context of stroke. In contrast, Panx1 appears to be detrimental to the survival of neural precursor cells that surround damaged cortical tissue in the stroke brain. This suggests that targeting Panx1 in the peri-infarct cortex, in combination with other therapies, could improve cell survival around the injury site.

Astrocytes undergo rapid activation after injury, which is mediated in part by the transcription factor nuclear factor-kappaB (NF-κB). Consequently, activated astrocytes have been shown to induce the NF-κB regulated phagocyte NADPH oxidase (PHOX), resulting in elevated production of reactive oxygen species. We investigated the regulatory mechanisms of PHOX-induced oxidative stress in astrocytes and its non-cell-autonomous effects on retinal ganglion cell loss following retinal ischemia-reperfusion (IR) injury. To study PHOX activity and neurotoxicity mediated by glial NF-κB, we employed GFAP-IκBα-dn transgenic mice, where the NF-κB canonical pathway is suppressed specifically in astrocytes. Our analysis showed that NF-κB activation in astrocytes correlated with an increased expression of PHOX and reactive oxygen species production in primary cells and whole retinas subjected to oxygen-glucose deprivation or IR injury. Selective blockade of NF-κB in astrocytes or application of NADPH oxidase inhibitors suppressed retinal ganglion cell loss in co-cultures with astroglia challenged by oxygen-glucose deprivation. Furthermore, genetic suppression of astroglial NF-κB reduced oxidative stress in ganglion layer neurons in vivo in retinal IR. Collectively, our results suggest that astroglial NF-κB-regulated PHOX activity is a crucial toxicity pathway in the pathogenesis of retinal IR injury.

Quantitative proteomic analysis was pursued of retinal ganglion cells (RGCs) from rats with unilateral experimental glaucoma. RGCs were isolated from 22 animals by immunopanning after 8 weeks of sustained elevated intraocular pressure. Proteins were quantified by LC MS/MS iTRAQ technology. Of the 268 proteins quantified, approximately 8% appeared elevated and approximately 13% decreased in glaucomatous RGCs. Voltage-dependent anion channel protein 2, aldose reductase, and ubiquitin were among the significantly elevated proteins while prothymosin was among the significantly decreased. The results demonstrate the feasibility of identifying global proteomic differences in protein expression between purified glaucomatous and control in vivo RGCs.

We investigated the systemic effect of liposomes bearing apoptotic signals on the level of inflammation and neuronal death induced by ischemia–reperfusion (IR). Using a model of retinal ischemia, we showed that treatment with phosphatidylserine (PS) and phosphatidylcholine (PC) liposomes significantly reduced the expression of proinflammatory genes, including that of Il1b, Il6, Ccl2, Ccl5, Cxcl10, and Icam1, 24 h after reperfusion. Phosphatidylserine liposome treatment was the most efficient and correlated with significantly reduced neuronal death in the retina 7 days after reperfusion. The results of our study indicate that therapeutic strategy based on mimicking a systemic increase in apoptotic signaling can significantly reduce central nervous system damage induced by IR and improve neurologic outcome.

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-kappaB, 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-IkappaBalpha-dn transgenic mice, where the NF-kappaB classical pathway is suppressed specifically in astrocytes. The GFAP-IkappaBalpha-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-alpha, Ccl2 (Mcp1), Cxcl10 (IP10), Icam1, Vcam1, several subunits of NADPH oxidase and NO-synthase in the retinas of GFAP-IkappaBalpha-dn mice. These data suggest that certain NF-kappaB-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.

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.

Different sub-populations of retinal ganglion cells (RGCs) vary in their sensitivity to pathological conditions such as retinal ischemia, diabetic retinopathy and glaucoma. Comparative transcriptomic analysis of such groups will likely reveal molecular determinants of differential sensitivity to stress. However, gene expression profiling of primary neuronal sub-populations represent a challenge due to the cellular heterogeneity of retinal tissue. In this manuscript, we report the use of a fluorescent neural tracer to specifically label and selectively isolate RGCs with different soma sizes by fluorescence-activated cell sorting (FACS) for the purpose of differential gene expression profiling. We identified 145 genes that were more active in the large RGCs and 312 genes in the small RGCs. Differential data were validated by quantitative RT-PCR, several corresponding proteins were confirmed by immunohistochemistry. Functional characterization revealed differential activity of genes implicated in synaptic transmission, neurotransmitter secretion, axon guidance, chemotaxis, ion transport and tolerance to stress. An in silico reconstruction of cellular networks suggested that differences in pathway activity between the two sub-populations of RGCs are controlled by networks interconnected by SP-1, Erk2 (MAPK1), Egr1, Egr2 and, potentially, regulated via transcription factors C/EBPbeta, HSF1, STAT1- and c-Myc. The results show that FACS-aided purification of retrogradely labeled cells can be effectively utilized for transcriptional profiling of adult retinal neurons.

Gap junctions (GJs) are composed of proteins that form a channel connecting the cytoplasm of adjacent cells. Connexins were initially considered to be the only proteins capable of GJ formation. Another family of GJ proteins (innexins) were first found in invertebrates and were proposed to be renamed pannexins after their orthologs were discovered in vertebrates. The lack of both connexins and pannexins in the genomes of some metazoans suggests that other, still undiscovered GJ proteins exist. In vertebrates, connexins and pannexins co-exist. Here we discuss whether vertebrate pannexins have a nonredundant role in animal physiology. Pannexin channels appear to be suited for ATP and calcium signaling and play a role in the maintenance of calcium homeostasis by mechanisms implicating both GJ and nonjunctional function. Suggested roles in the ischemic death of neurons, schizophrenia, inflammation and tumor suppression have drawn much attention to exploring the molecular properties and cellular functions of pannexins.

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 58kDa protein, and a 43kDa 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.

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.

SummaryIn rod photoreceptors, arrestin localizes to the outer segment (OS) in the light and to the inner segment (IS) in the dark. Here, we demonstrate that redistribution of arrestin between these compartments can proceed in ATP-depleted photoreceptors. Translocation of transducin from the IS to the OS also does not require energy, but depletion of ATP or GTP inhibits its reverse movement. A sustained presence of activated rhodopsin is required for sequestering arrestin in the OS, and the rate of arrestin relocalization to the OS is determined by the amount and the phosphorylation status of photolyzed rhodopsin. Interaction of arrestin with microtubules is increased in the dark. Mutations that enhance arrestin-microtubule binding attenuate arrestin translocation to the OS. These results indicate that the distribution of arrestin in rods is controlled by its dynamic interactions with rhodopsin in the OS and microtubules in the IS and that its movement occurs by simple diffusion.

The mammalian lens consists of an aged core of quiescent cells enveloped by a layer of synthetically active cells. Abundant gap junctions within and between these cell populations ensure that the lens functions as an electrical syncytium and facilitates the exchange of small molecules between surface and core cells. In the present study, we utilized an in vivo mouse model to characterize the properties of an additional pathway, permeable to macromolecules, which co-exists with gap-junction-mediated communication in the lens core. The TgN(GFPU)5Nagy strain of mice carries a green fluorescent protein (GFP) transgene. In the lenses of hemizyous animals, GFP was expressed in a variegated fashion, allowing diffusion of GFP to be visualized directly. Early in development, GFP expression in scattered fiber cells resulted in a checkerboard fluorescence pattern in the lens. However, at E15 and later, the centrally located fiber cells became uniformly fluorescent. In the adult lens, a superficial layer of cells, approximately 100 μm thick, retained the original mosaic fluorescence pattern, but the remainder, and majority, of the tissue was uniformly fluorescent. We reasoned that at the border between the two distinct labeling patterns, a macromolecule-permeable intercellular pathway was established. To test this hypothesis, we microinjected 10 kDa fluorescent dextran into individual fiber cells and followed its diffusion by time-lapse microscopy. Injections at depths of >100 μm resulted in intercellular diffusion of dextran from injected cells. By contrast, when injections were made into superficial fiber cells, the injected cell invariably retained the dextran. Together, these data suggest that, in addition to being coupled by gap junctions, cells in the lens core are interconnected by a macromolecule-permeable pathway. At all ages examined, a significant proportion of the nucleated fiber cell population of the lens was located within this region of the lens.

Connexin alpha1Cx43 has previously been shown to bind to the PDZ domain-containing protein ZO-1. The similarity of the carboxyl termini of this connexin and the lens fiber connexins alpha3Cx46 and alpha8Cx50 suggested that these connexins may also interact with ZO-1. ZO-1 was shown to be highly expressed in mouse lenses. Colocalization of ZO-1 with alpha3Cx46 and alpha8Cx50 connexins in fiber cells was demonstrated by immunofluorescence and by fracture-labeling electron microscopy but showed regional variations throughout the lens. ZO-1 was found to coimmunoprecipitate with alpha3Cx46 and alpha8Cx50, and pull-down experiments showed that the second PDZ domain of ZO-1 was involved in this interaction. Transiently expressed alpha3Cx46 and alpha8Cx50 connexins lacking the COOH-terminal residues did not bind to the second PDZ domain but still formed structures resembling gap junctions by immunofluorescence. These results indicate that ZO-1 interacts with lens fiber connexins alpha3Cx46 and alpha8Cx50 in a manner similar to that previously described for alpha1Cx43. The spatial variation in the interaction of ZO-1 with lens gap junctions is intriguing and is suggestive of multiple dynamic roles for this association.

Pannexin 1 (Panx1), the most extensively investigated member of a channel-forming protein family, is able to form pores conducting molecules up to 1.5 kDa, like ATP, upon activation. In the olfactory epithelium (OE), ATP modulates olfactory responsiveness and plays a role in proliferation and differentiation of olfactory sensory neurons (OSNs). This process continuously takes place in the OE, as neurons are replaced throughout the whole lifespan. The recent discovery of Panx1 expression in the OE raises the question whether Panx1 mediates ATP release responsible for modulating chemosensory function. In this study, we analyzed pannexin expression in the OE and a possible role of Panx1 in olfactory function using a Panx1(-/-) mouse line with a global ablation of Panx1. This mouse model has been previously used to investigate Panx1 functions in the retina and adult hippocampus. Here, qPCR, in-situ hybridization, and immunohistochemistry (IHC) demonstrated that Panx1 is expressed in axon bundles deriving from sensory neurons of the OE. The localization, distribution, and expression of major olfactory signal transduction proteins were not significantly altered in Panx1(-/-) mice. Further, functional analysis of Panx1(-/-) animals does not reveal any major impairment in odor perception, indicated by electroolfactogram (EOG) measurements and behavioral testing. However, ATP release evoked by potassium gluconate application was reduced in Panx1(-/-) mice. This result is consistent with previous reports on ATP release in isolated erythrocytes and spinal or lumbar cord preparations from Panx1(-/-) mice, suggesting that Panx1 is one of several alternative pathways to release ATP in the olfactory system.

Tissue injury involves coordinated systemic responses including inflammatory response, targeted cell migration, cell-cell communication, stem cell activation and proliferation, and tissue inflammation and regeneration. The inflammatory response is an important prerequisite for regeneration. Multiple studies suggest that extensive cell-cell communication during tissue regeneration is coordinated by purinergic signaling via extracellular adenosine triphosphate (ATP). Most recent data indicates that ATP release for such communication is mediated by hemichannels formed by connexins and pannexins. The Pannexin family consists of three vertebrate proteins (Panx 1, 2, and 3) that have low sequence homology with other gap junction proteins and were shown to form predominantly non-junctional plasma membrane hemichannels. Pannexin-1 (Panx1) channels function as an integral component of the P2X/P2Y purinergic signaling pathway and is arguably the major contributor to pathophysiological ATP release. Panx1 is expressed in many tissues, with highest levels detected in developing brain, retina and skeletal muscles. Panx1 channel expression and activity is reported to increase significantly following injury/inflammation and during regeneration and differentiation. Recent studies also report that pharmacological blockade of the Panx1 channel or genetic ablation of the Panx1 gene cause significant disruption of progenitor cell migration, proliferation, and tissue regeneration. These findings suggest that pannexins play important roles in activation of both post-injury inflammatory response and the subsequent process of tissue regeneration. Due to wide expression in multiple tissues and involvement in diverse signaling pathways, pannexins and connexins are currently being considered as therapeutic targets for traumatic brain or spinal cord injuries, ischemic stroke and cancer. The precise role of pannexins and connexins in the balance between tissue inflammation and regeneration needs to be further understood.

Purpose: To advance our understanding how the outer eye interacts with its environment, we asked which cellular receptors are expressed in the cornea, focusing on G protein-coupled receptors. Methods: Total RNA from the mouse cornea was subjected to next-generation sequencing using the Illumina platform. The data was analyzed with TopHat and CuffLinks software packages. Expression of a representative group of genes detected by RNA-seq was further analyzed by RT-PCR and in situ hybridization using RNAscope technology and fluorescent microscopy. Results: We generated more than 46 million pair-end reads from mouse corneal RNA. Bioinformatics analysis revealed that the mouse corneal transcriptome reconstructed from these reads represents over 10,000 gene transcripts. We identified 194 GPCR transcripts, of which 96 were putative olfactory receptors. RT-PCR analysis confirmed the presence of several olfactory receptors and related genes, including olfactory marker protein and the G protein associated with olfaction, Gaolf. In situ hybridization showed that mRNA for olfactory marker protein, Gaolf and possibly some olfactory receptors were found in the corneal epithelial cells. In addition to the corneal epithelium, Gaolf was present in the ganglionic and inner nuclear layers of the retina. One of the olfactory receptors, Olfr558, was present primarily in vessels of the eye co-stained with antibodies against alpha-smooth muscle actin, indicating expression in arterioles. Conclusions: Several species of mRNA encoding putative olfactory receptors and related genes are expressed in the mouse cornea and other parts of the eye indicating they may play a role in sensing chemicals in the ocular environment.

Pannexin1 (Panx1) forms non-selective membrane channels, structurally similar to gap junction hemichannels, and are permeable to ions, nucleotides, and other small molecules below 900 Da. Panx1 activity has been implicated in paracrine signaling and inflammasome regulation. Recent studies in different animal models showed that overactivation of Panx1 correlates with a selective demise of several types of neurons, including retinal ganglion cells, brain pyramidal, and enteric neurons. The list of Panx1 activators includes extracellular ATP, glutamate, high K(+), Zn(2+), fibroblast growth factors (FGFs),pro-inflammatory cytokines, and elevation of intracellular Ca(2+). Most of these molecules are released following mechanical, ischemic, or inflammatory injury of the CNS, and rapidly activate the Panx1 channel. Prolonged opening of Panx1 channel induced by these "danger signals" triggers a cascade of neurotoxic events capable of killing cells. The most vulnerable cell type are neurons that express high levels of Panx1. Experimental evidence suggests that Panx1 channels mediate at least two distinct neurotoxic processes: increased permeability of the plasma membrane and activation of the inflammasome in neurons and glia. Importantly, both pharmacological and genetic inactivation of Panx1 suppresses both these processes, providing a marked protection in several disease and injury models. These findings indicate that external danger signals generated after diverse types of injuries converge to activate Panx1. In this review we discuss molecular mechanisms associated with Panx1 toxicity and the crosstalk between different pathways.