Simon J. O’Carroll

Connexin43 mimetic peptide is neuroprotective and improves function following spinal cord injury

Connexin43 (Cx43) is a gap junction protein up-regulated after spinal cord injury and is involved in the on-going spread of secondary tissue damage. To test whether a connexin43 mimetic peptide (Peptide5) reduces inflammation and tissue damage and improves function in an in vivo model of spinal cord injury, rats were subjected to a 10g, 12.5mm weight drop injury at the vertebral level T10 using a MASCIS impactor. Vehicle or connexin43 mimetic peptide was delivered directly to the lesion via intrathecal catheter and osmotic mini-pump for up to 24h after injury. Treatment with Peptide5 led to significant improvements in hindlimb function as assessed using the Basso-Beattie-Bresnahan scale. Peptide5 caused a reduction in Cx43 protein, increased Cx43 phosphorylation and decreased levels of TNF-α and IL-1β as assessed by Western blotting. Immunohistochemistry of tissue sections 5 weeks after injury showed reductions in astrocytosis and activated microglia as well as an increase in motor neuron survival. These results show that administration of a connexin mimetic peptide reduces secondary tissue damage after spinal cord injury by reducing gliosis and cytokine release and indicate the clinical potential for mimetic peptides in the treatment of spinal cord patients.

Pro-inflammatory TNFα and IL-1β differentially regulate the inflammatory phenotype of brain microvascular endothelial cells

BackgroundThe vasculature of the brain is composed of endothelial cells, pericytes and astrocytic processes. The endothelial cells are the critical interface between the blood and the CNS parenchyma and are a critical component of the blood-brain barrier (BBB). These cells are innately programmed to respond to a myriad of inflammatory cytokines or other danger signals. IL-1β and TNFα are well recognised pro-inflammatory mediators, and here, we provide compelling evidence that they regulate the function and immune response profile of human cerebral microvascular endothelial cells (hCMVECs) differentially.MethodsWe used xCELLigence biosensor technology, which revealed global differences in the endothelial response between IL-1β and TNFα. xCELLigence is a label-free impedance-based biosensor, which is ideal for acute or long-term comparison of drug effects on cell behaviour. In addition, flow cytometry and multiplex cytokine arrays were used to show differences in the inflammatory responses from the endothelial cells.ResultsExtensive cytokine-secretion profiling and cell-surface immune phenotyping confirmed that the immune response of the hCMVEC to IL-1β was different to that of TNFα. Interestingly, of the 38 cytokines, chemokines and growth factors measured by cytometric bead array, the endothelial cells secreted only 13. Of importance was the observation that the majority of these cytokines were differentially regulated by either IL-1β or TNFα. Cell-surface expression of ICAM-1 and VCAM-1 were also differentially regulated by IL-1β or TNFα, where TNFα induced a substantially higher level of expression of both key leukocyte-adhesion molecules. A range of other cell-surface cellular and junctional adhesion molecules were basally expressed by the hCMVEC but were unaffected by IL-1β or TNFα.ConclusionsTo our knowledge, this is the most comprehensive analysis of the immunological profile of brain endothelial cells and the first direct evidence that human brain endothelial cells are differentially regulated by these two key pro-inflammatory mediators.

Dose-dependent protective effect of connexin43 mimetic peptide against neurodegeneration in an ex vivo model of epileptiform lesion

Epileptic seizures typically result in delayed neuronal loss secondary to the initial damage and an up-regulation in connexin43 (Cx43). This study investigated the role of Cx43 gap junctions in lesion spread and cell loss following epileptiform activity. Epileptiform injury in hippocampal slice cultures was induced by 48 h exposure to 100 μM bicuculline methochloride (BMC). During the 24h recovery period following BMC treatment, lesion spread was observed in the CA1. A Cx43 mimetic peptide, applied during either the BMC treatment or recovery periods, produced concentration- and exposure time-dependent neuroprotection, as measured by propidium iodide uptake at the end of the recovery period. During the BMC period, peptide concentrations between 5 and 50 μM (sufficient to block hemichannels) had a protective effect while a substantial gap junction blockade with 500 μM peptide exacerbated the lesion. By contrast, all doses applied during the recovery period protected the CA1 region from further damage. The results indicate that while the slices are undergoing excessive neuronal firing and epileptic stress, gap junction communication appears to be essential for tissue survival but hemichannel opening may be damaging. Following epileptiform insult, however, gap junction communication plays a crucial role in the spread of neuronal damage. The findings from this study identify gap junction communication as a potential therapeutic target for epilepsy.

The Use of Connexin-Based Therapeutic Approaches to Target Inflammatory Diseases

Alterations in Connexin43 (Cx43) expression levels have been shown to play a role in inflammatory processes including skin wounding and neuroinflammation. Cx43 protein levels increase following a skin wound and can inhibit wound healing. Increased Cx43 has been observed following stroke, epilepsy, ischemia, optic nerve damage, and spinal cord injury with gap junctional communication and hemichannel opening leading to increased secondary damage via the inflammatory response. Connexin43 modulation has been identified as a potential target for protection and repair in neuroinflammation and skin wound repair. This review describes the use of a Cx43 specific antisense oligonucleotide (Cx43 AsODN) and peptide mimetics of the connexin extracellular loop domain to modulate Cx43 expression and/or function in inflammatory disorders of the skin and central nervous system. An overview of the role of connexin43 in inflammatory conditions, how antisense and peptide have allowed us to elucidate the role of Cx43 in these diseases, create models of diseases to test interventions and their potential for use clinically or in current clinical trials is presented. Antisense oligonucleotides are applied topically and have been used to improve wound healing following skin injury. They have also been used to develop ex vivo models of neuroinflammatory diseases that will allow testing of intervention strategies. The connexin mimetic peptides have shown potential in a number of neuroinflammatory disorders in ex vivo models as well as in vivo when delivered directly to the injury site or when delivered systemically.

Gap junction proteins and their role in spinal cord injury

Gap junctions are specialized intercellular communication channels that are formed by two hexameric connexin hemichannels, one provided by each of the two adjacent cells. Gap junctions and hemichannels play an important role in regulating cellular metabolism, signaling, and functions in both normal and pathological conditions. Following spinal cord injury (SCI), there is damage and disturbance to the neuronal elements of the spinal cord including severing of axon tracts and rapid cell death. The initial mechanical disruption is followed by multiple secondary cascades that cause further tissue loss and dysfunction. Recent studies have implicated connexin proteins as playing a critical role in the secondary phase of SCI by propagating death signals through extensive glial networks. In this review, we bring together past and current studies to outline the distribution, changes and roles of various connexins found in neurons and glial cells, before and in response to SCI. We discuss the contribution of pathologically activated connexin proteins, in particular connexin 43, to functional recovery and neuropathic pain, as well as providing an update on potential connexin specific pharmacological agents to treat SCI.

A model for ex vivo spinal cord segment culture - a tool for analysis of injury repair strategies

Most spinal cord injury research is undertaken using in vivo animal models but the extensive care associated with spinalized animals, inherent variability between animals, and complex surgeries makes alternative models especially valuable. Here we present a novel ex vivo model that enables culture of intact post-natal spinal cord segments for up to five days and the assessment of peripheral nerve grafting repair, enhanced with connexin43 antisense oligodeoxynucleotides (Cx43 AsODN), in this model. Down-regulating Cx43 expression with Cx43 AsODN in cultured spinal cord segments prevents cell death and inhibits inflammation spreading from the site of injury to neighbouring tissue, hence maintaining culture viability. Reduction in segment swelling and improvement in neuron survival were evident after Cx43 AsODN treatment. Furthermore, the combination of Cx43 AsODN with peripheral nerve graft implants into cultured spinal cords promoted axon sprouting from the spinal cord into the peripheral nerve graft. This ex vivo spinal cord segment culture model provides a valuable addition to tools currently available for spinal cord injury research.

Statins Inhibit Fibrillary β-Amyloid Induced Inflammation in a Model of the Human Blood Brain Barrier

Background Astrocytes and cerebral endothelial cells are important components of the blood-brain barrier (BBB). Disruption to this barrier through inflammation is a major contributor to Alzheimer’s disease (AD) pathology. The amyloid beta (Aβ) protein is known to exist in several forms and is a key modulator of AD that is known to cause inflammation and changes to BBB function. While one of these forms, fibrillary Aβ (fAβ), is known to cause endothelial cell death at the BBB, no studies have looked specifically at its role on inflammation in a model of the human BBB. Aims To determine if fAβ is inflammatory to the human BBB. As statins have been shown to be anti-inflammatory and protective in AD, we also tested if these could inhibit the inflammatory effect of fAβ. Methods Using cultured cerebral endothelial cells and astrocytes we determined changes in cytokine release, cell toxicity and barrier function in response to fibrillary β-amyloid1–42 (fAβ1–42) alone and in combination with statins. Results fAβ1–42 induced inflammatory cytokine release from endothelial cells in the absence of cell toxicity. It also induced astrocyte cytokine release and cell death and caused a loss of barrier integrity. Statin treatment inhibited all of these effects. Conclusions We conclude that fAβ1–42 has both inflammatory and cytotoxic effects on the BBB and the protective effect of statins in AD may in part be through inhibiting these effects.

Regulation of human cerebro-microvascular endothelial baso-lateral adhesion and barrier function by S1P through dual involvement of S1P1 and S1P2 receptors

Herein we show that S1P rapidly and acutely reduces the focal adhesion strength and barrier tightness of brain endothelial cells. xCELLigence biosensor technology was used to measure focal adhesion, which was reduced by S1P acutely and this response was mediated through both S1P1 and S1P2 receptors. S1P increased secretion of several pro-inflammatory mediators from brain endothelial cells. However, the magnitude of this response was small in comparison to that mediated by TNFα or IL-1β. Furthermore, S1P did not significantly increase cell-surface expression of any key cell adhesion molecules involved in leukocyte recruitment, included ICAM-1 and VCAM-1. Finally, we reveal that S1P acutely and dynamically regulates microvascular endothelial barrier tightness in a manner consistent with regulated rapid opening followed by closing and strengthening of the barrier. We hypothesise that the role of the S1P receptors in this process is not to cause barrier dysfunction, but is related to controlled opening of the endothelial junctions. This was revealed using real-time measurement of barrier integrity using ECIS ZΘ TEER technology and endothelial viability using xCELLigence technology. Finally, we show that these responses do not occur simply though the pharmacology of a single S1P receptor but involves coordinated action of S1P1 and S1P2 receptors.

ECIS technology reveals that monocytes isolated by CD14+ve selection mediate greater loss of BBB integrity than untouched monocytes, which occurs to a greater extent with IL-1β activated endothelium in comparison to TNFα

Background We have previously shown that TNFα and IL-1β differentially regulate the inflammatory phenotype of human brain endothelial cells (hCMVECs). In this regard, IL-1β treatment was considerably more potent than TNFα at increasing expression of inflammatory chemokines and leukocyte adhesion molecules. We therefore hypothesised that interaction of the hCMVECs with human monocytes would also be dependent on the activation status of the endothelium. Therefore, the primary aim of this study was to assess whether brain endothelial cells activated by IL-1β or TNFα differed in their interaction with monocytes. Methods Monocyte interaction was measured using the real time, label-free impedance based ECIS technology, to evaluate endothelial barrier integrity during monocyte attachment and transendothelial migration. Results ECIS technology revealed that there was a greater loss of barrier integrity with IL-1β activation and this loss lasted for longer. This was expected and consistent with our hypothesis. However, more striking and concerning was the observation that the method of monocyte enrichment greatly influenced the extent of endothelial barrier compromise. Importantly, we observed that positively isolated monocytes (CD14+ve) caused greater reduction in barrier resistance, than the negatively selected monocytes (untouched). Analysis of the isolated monocyte populations revealed that the CD14+ve isolation consistently yields highly pure monocytes (>92%), whereas the untouched isolation was much more variable, yielding ~70% enrichment on average. These two enrichment methods were compared as it was thought that the presence of non-classical CD16hi monocytes in the untouched enrichment may mediate greater compromise than the classical CD14hi monocytes. This however, was not the case and these observations raise a number of important considerations pertaining to the enrichment strategy, which are essential for generating reliable and consistent data. Conclusions We conclude that IL-1β and TNFα differentially influence monocyte interaction with brain endothelial cells and moreover, the enrichment method also influences the monocyte response as revealed using ECIS technology.

Tonabersat Prevents Inflammatory Damage in the Central Nervous System by Blocking Connexin43 Hemichannels

The cis benzopyran compound tonabersat (SB-220453) has previously been reported to inhibit connexin26 expression in the brain by attenuating the p38-mitogen-activated protein kinase pathway. We show here that tonabersat directly inhibits connexin43 hemichannel opening. Connexin43 hemichannels have been called “pathological pores” based upon their role in secondary lesion spread, edema, inflammation, and neuronal loss following central nervous system injuries, as well as in chronic inflammatory disease. Both connexin43 hemichannels and pannexin channels released adenosine triphosphate (ATP) during ischemia in an in vitro ischemia model, but only connexin43 hemichannels contributed to ATP release during reperfusion. Tonabersat inhibited connexin43 hemichannel-mediated ATP release during both ischemia and reperfusion phases, with direct channel block confirmed using electrophysiology. Tonabersat also reduced connexin43 gap junction coupling in vitro, but only at higher concentrations, with junctional plaques internalized and degraded via the lysosomal pathway. Systemic delivery of tonabersat in a rat bright-light retinal damage model (a model for dry age-related macular degeneration) resulted in significantly improved functional outcomes assessed using electroretinography. Tonabersat also prevented thinning of the retina, especially the outer nuclear layer and choroid, assessed using optical coherence tomography. We conclude that tonabersat, already given orally to over 1000 humans in clinical trials (as a potential treatment for, and prophylactic treatment of, migraine because it was thought to inhibit cortical spreading depression), is a connexin hemichannel inhibitor and may have the potential to be a novel treatment of central nervous system injury and chronic neuroinflammatory disease.