Pharmacological Inhibition of the Voltage-Gated Sodium Channel NaV1.7 Alleviates Chronic Visceral Pain in a Rodent Model of Irritable Bowel Syndrome.

The human nociceptor-specific voltage-gated sodium channel 1.7 (hNaV1.7) is critical for sensing various types of somatic pain, but it appears not to play a primary role in acute visceral pain. However, its role in chronic visceral pain remains to be determined. We used assay-guided fractionation to isolate a novel hNaV1.7 inhibitor, Tsp1a, from tarantula venom. Tsp1a is 28-residue peptide that potently inhibits hNaV1.7 (IC50 = 10 nM), with greater than 100-fold selectivity over hNaV1.3-hNaV1.6, 45-fold selectivity over hNaV1.1, and 24-fold selectivity over hNaV1.2. Tsp1a is a gating modifier that inhibits NaV1.7 by inducing a hyperpolarizing shift in the voltage-dependence of channel inactivation and slowing recovery from fast inactivation. NMR studies revealed that Tsp1a adopts a classical knottin fold, and like many knottin peptides, it is exceptionally stable in human serum. Remarkably, intracolonic administration of Tsp1a completely reversed chronic visceral hypersensitivity in a mouse model of irritable bowel syndrome. The ability of Tsp1a to reduce visceral hypersensitivity in a model of irritable bowel syndrome suggests that pharmacological inhibition of hNaV1.7 at peripheral sensory nerve endings might be a viable approach for eliciting analgesia in patients suffering from chronic visceral pain.

Venom Peptides with Dual Modulatory Activity on the Voltage-Gated Sodium Channel NaV1.1 Provide Novel Leads for Development of Antiepileptic Drugs.

Voltage-gated sodium (NaV) channels play a fundamental role in normal neurological function, especially via the initiation and propagation of action potentials. The NaV1.1 subtype is found in inhibitory interneurons of the brain and it is essential for maintaining a balance between excitation and inhibition in neuronal networks. Heterozygous loss-of-function mutations of SCN1A, the gene encoding NaV1.1, underlie Dravet syndrome (DS), a severe pediatric epilepsy. We recently demonstrated that selective inhibition of NaV1.1 inactivation prevents seizures and premature death in a mouse model of DS. Thus, selective modulators of NaV1.1 might be useful therapeutics for treatment of DS as they target the underlying molecular deficit. Numerous scorpion-venom peptides have been shown to modulate the activity of NaV channels, but little is known about their activity at NaV1.1. Here we report the isolation, sequence, three-dimensional structure, recombinant production, and functional characterization of two peptidic modulators of NaV1.1 from venom of the buthid scorpion Hottentotta jayakari. These peptides, Hj1a and Hj2a, are potent agonists of NaV1.1 (EC50 of 17 and 32 nM, respectively), and they present dual α/ß activity by modifying both the activation and inactivation properties of the channel. NMR studies of rHj1a indicate that it adopts a cystine-stabilized αß fold similar to known scorpion toxins. Although Hj1a and Hj2a have only limited selectivity for NaV1.1, their unusual dual mode of action provides an alternative approach to the development of selective NaV1.1 modulators for the treatment of DS.

Complement receptor C3aR1 controls neutrophil mobilization following spinal cord injury through physiological antagonism of CXCR2.

Traumatic spinal cord injury (SCI) triggers an acute-phase response that leads to systemic inflammation and rapid mobilization of bone marrow (BM) neutrophils into the blood. These mobilized neutrophils then accumulate in visceral organs and the injured spinal cord where they cause inflammatory tissue damage. The receptor for complement activation product 3a, C3aR1, has been implicated in negatively regulating the BM neutrophil response to tissue injury. However, the mechanism via which C3aR1 controls BM neutrophil mobilization, and also its influence over SCI outcomes, are unknown. Here, we show that the C3a/C3aR1 axis exerts neuroprotection in SCI by acting as a physiological antagonist against neutrophil chemotactic signals. We show that C3aR1 engages phosphatase and tensin homolog (PTEN), a negative regulator of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, to restrain C-X-C chemokine receptor type 2-driven BM neutrophil mobilization following trauma. These findings are of direct clinical significance as lower circulating neutrophil numbers at presentation were identified as a marker for improved recovery in human SCI. Our work thus identifies C3aR1 and its downstream intermediary, PTEN, as therapeutic targets to broadly inhibit neutrophil mobilization/recruitment following tissue injury and reduce inflammatory pathology.

Identification of new molecular targets for PET imaging of the microglial anti-inflammatory activation state.

Microglia are potential targets for therapeutic intervention in neurological and neurodegenerative diseases affecting the central nervous system. In order to assess the efficacy of therapies aimed to reduce the tissue damaging activities of microglia and/or to promote the protective potential of these cells, suitable pre-clinical and clinical tools for the in vivo analysis of microglia activities and dynamics are required. The aim of this work was to identify new translational markers of the anti-inflammatory / protective state of microglia for the development of novel PET tracers. Methods: New translational markers of the anti-inflammatory/protective activation state of microglia were selected by bioinformatic approaches and were in vitro and ex vivo validated by qPCR and immunohistochemistry in rodent and human samples. Once a viable marker was identified, a novel PET tracer was developed. This tracer was subsequently confirmed by autoradiography experiments in murine and human brain tissues. Results: Here we provide evidence that P2RY12 expression increases in murine and human microglia following exposure to anti-inflammatory stimuli, and that its expression is modulated in the reparative phase of experimental and clinical stroke. We then synthesized a novel carbon-11 labeled tracer targeting P2RY12, showing increased binding in brain sections of mice treated with IL4, and low binding to brain sections of a murine stroke model and of a stroke patient. Conclusion: This study provides new translational targets for PET tracers for the anti-inflammatory/protective activation state of microglia and shows the potential of a rationale-based approach. It therefore paves the way for the development of novel non-invasive methodologies aimed to monitor the success of therapeutic approaches in various neurological diseases.

Efficient Enzymatic Ligation of Inhibitor Cystine Knot Spider Venom Peptides: Using Sortase A To Form Double-Knottins That Probe Voltage-Gated Sodium Channel NaV1.7.

Gating modifier toxins from spider venom are disulfide-rich peptides that typically comprise a stabilizing inhibitor cystine knot (ICK). These knottin peptides are being pursued as therapeutic leads for a range of conditions linked to transmembrane proteins. Recently, double-knottin peptides discovered in spider venom and produced by recombinant expression have provided insights into the pharmacology of transmembrane channels. Here, we use chemoenzymatic ligation to produce double-knottins to probe the effect of bivalent modulation on the voltage-gated sodium channel subtype 1.7 (NaV1.7), which is implicated in pain signaling. Monovalent knottins were oxidatively folded and then biochemically conjugated using sortase A, to form double-knottins. The structural integrity of the peptides was confirmed using NMR, and fluorescence-based activity assays provided evidence suggesting that coincubated monovalent and bivalent knottins can cooperatively modulate NaV1.7. We anticipate that double-knottins will provide novel tools for enhancing our understanding of, and design strategies for, therapeutically relevant voltage-gated ion channels.

Potassium channel expression and function in microglia: Plasticity and possible species variations.

Potassium channels play important roles in microglia functions and thus constitute potential targets for the treatment of neurodegenerative diseases like Alzheimer, Parkinson and stroke. However, uncertainty still prevails as to which potassium channels are expressed and at what levels in different species, how the expression pattern changes upon activation with M1 or M2 polarizing stimuli compared with more complex exposure paradigms, and - most importantly - how these findings relate to the in vivo situation. In this mini-review we discuss the functional potassium channel expression pattern in cultured neonatal mouse microglia in the light of data obtained previously from animal disease models and immunohistochemical studies and compare it with a recent study of adult human microglia isolated from epilepsy patients. Overall, microglial potassium channel expression is very plastic and possibly shows species differences and therefore should be studied carefully in each disease setting and respective animal models.

Quantification of the functional expression of the Ca2+ -activated K+ channel KCa 3.1 on microglia from adult human neocortical tissue.

The KCa 3.1 channel (KCNN4) is an important modulator of microglia responses in rodents, but no information exists on functional expression on microglia from human adults. We isolated and cultured microglia (max 1% astrocytes, no neurons or oligodendrocytes) from neocortex surgically removed from epilepsy patients and employed electrophysiological whole-cell measurements and selective pharmacological tools to elucidate functional expression of KCa 3.1. The channel expression was demonstrated as a significant increase in the voltage-independent current by NS309, a KCa 3.1/KCa 2 activator, followed by full inhibition upon co-application with NS6180, a highly selective KCa 3.1 inhibitor. A major fraction (79%) of unstimulated human microglia expressed KCa 3.1, and the difference in current between full activation and inhibition (ΔKCa 3.1) was estimated at 292 ± 48 pA at -40 mV (n = 75), which equals at least 585 channels per cell. Serial KCa 3.1 activation/inhibition significantly hyperpolarized/depolarized the membrane potential. The isolated human microglia were potently activated by lipopolysaccharide (LPS) shown as a prominent increase in TNF-α production. However, incubation with LPS neither changed the KCa 3.1 current nor the fraction of KCa 3.1 expressing cells. In contrast, the anti-inflammatory cytokine IL-4 slightly increased the KCa 3.1 current per cell, but as the membrane area also increased, there was no significant change in channel density. A large fraction of the microglia also expressed a voltage-dependent current sensitive to the KCa 1.1 modulators NS1619 and Paxilline and an inward-rectifying current with the characteristics of a Kir channel. The high functional expression of KCa 3.1 in microglia from epilepsy patients accentuates the need for further investigations of its role in neuropathological processes. GLIA 2016;64:2065-2078.

Mobilisation of the splenic monocyte reservoir and peripheral CX3CR1 deficiency adversely affects recovery from spinal cord injury.

Macrophages in the injured spinal cord originate from resident microglia and blood monocytes. Whether this diversity in origins contributes to their seemingly dual role in immunopathology and repair processes has remained poorly understood. Here we took advantage of Cx3cr1(gfp) mice to visualise monocyte-derived macrophages in the injured spinal cord via adoptive cell transfer and bone marrow (BM) chimera approaches. We show that the majority of infiltrating monocytes at 7 days post-injury originate from the spleen and only to a lesser extent from the BM. Prevention of early monocyte infiltration via splenectomy was associated with improved recovery at 42 days post-SCI. In addition, an increased early presence of infiltrating monocytes/macrophages, as a result of CX3CR1 deficiency within the peripheral immune compartment, correlated with worsened injury outcomes. Adoptive transfer of identified Cx3cr1(gfp/+) monocytes confirmed peak infiltration at 7 days post-injury, with inflammatory (Ly6C(high)) monocytes being most efficiently recruited. Focal SCI also changed the composition of the two major monocyte subsets in the blood, with more Ly6C(high) cells present during peak recruitment. Adoptive transfer experiments further suggested high turnover of inflammatory monocytes in the spinal cord at 7 days post-injury. Consistent with this, only a small proportion of infiltrating cells unequivocally expressed polarisation markers for pro-inflammatory (M1) or alternatively activated (M2) macrophages at this time point. Our findings offer new insights into the origins of monocyte-derived macrophages after SCI and their contribution to functional recovery, providing a basis for further scrutiny and selective targeting of Ly6C(high) monocytes to improve outcomes from neurotraumatic events.

Detection of endogenous iron deposits in the injured mouse spinal cord through high-resolution ex vivo and in vivo MRI.

The main aim of this study was to employ high-resolution MRI to investigate the spatiotemporal development of pathological features associated with contusive spinal cord injury (SCI) in mice. Experimental mice were subjected to either sham surgery or moderate contusive SCI. A 16.4-T small-animal MR system was employed for nondestructive imaging of post-mortem, fixed spinal cord specimens at the subacute (7 days) and more chronic (28-35 days) stages post-injury. Routine histological techniques were used for subsequent investigation of the observed neuropathology at the microscopic level. The central core of the lesion appeared as a dark hypo-intense area on MR images at all time points investigated. Small focal hypo-intense spots were also observed spreading through the dorsal funiculi proximal and distal to the site of impact, an area that is known to undergo gliosis and Wallerian degeneration in response to injury. Histological examination revealed these hypo-intense spots to be high in iron content as determined by Prussian blue staining. Quantitative image analysis confirmed the increased presence of iron deposits at all post-injury time points investigated (p<0.05). Distant iron deposits were also detectable through live imaging without the use of contrast-enhancing agents, enabling the longitudinal investigation of this pathology in individual animals. Further immunohistochemical evaluation showed that intracellular iron deposits localised to macrophages/microglia, astrocytes and oligodendrocytes in the subacute phase of SCI, but predominantly to glial fibrillary acidic protein-positive, CC-1-positive astrocytes at later stages of recovery. Progressive, widespread intracellular iron accumulation is thus a normal feature of SCI in mice, and high-resolution MRI can be effectively used to detect and monitor these neuropathological changes with time.

Localization of Nav 1.7 in the normal and injured rodent olfactory system indicates a critical role in olfaction, pheromone sensing and immune function.

Loss-of-function mutations in the pore-forming α subunit of the voltage-gated sodium channel 1.7 (Nav 1.7) cause congenital indifference to pain and anosmia. We used immunohistochemical techniques to study Nav 1.7 localization in the rat olfactory system in order to better understand its role in olfaction. We confirm that Nav 1.7 is expressed on olfactory sensory axons and report its presence on vomeronasal axons, indicating an important role for Nav 1.7 in transmission of pheromonal cues. Following neuroepithelial injury, Nav 1.7 was transiently expressed by cells of monocytic lineage. These findings support an emerging role for Nav 1.7 in immune function. This sodium channel may provide an important pharmacological target for treatment of inflammatory injury and inflammatory pain syndromes.

CX3CR1 deficiency exacerbates neuronal loss and impairs early regenerative responses in the target-ablated olfactory epithelium.

The olfactory epithelium is a site of sustained adult neurogenesis where olfactory sensory neurons are continuously replaced from endogenous stem/progenitor cells. Epithelial macrophages have been implicated in the phagocytosis of degenerating cells but the molecular mechanisms allowing for their recruitment and activation while maintaining a neurogenic microenvironment are poorly understood. We have previously shown that the chemokine fractalkine (CX3CL1) is expressed by olfactory sensory neurons and ensheathing cells in the olfactory epithelium. In turn, the fractalkine receptor, CX3CR1, is expressed on macrophages and dendritic cells within the olfactory epithelium. We report that a selective cell death of olfactory sensory neurons in the epithelium of CX3CR1-deficient mice via target ablation (i.e. olfactory bulbectomy) results in an exacerbated loss of olfactory sensory neurons compared to wild-type mice. In addition, reduced proliferation of intraepithelial stem/progenitor cells was observed in lesioned CX3CR1-deficient mice, suggesting an impaired regenerative response. Importantly, a lack of CX3CL1-signaling caused increased recruitment of macrophages into the olfactory epithelium, which in turn contained higher levels of pro-inflammatory cytokines (e.g. TNF-α and IL-6) as determined by qPCR. We also present novel data showing that, relative to wild-type, CX3CR1-deficient macrophages have diminished phagocytic activity following stimulation with CX3CL1. Collectively, our data indicate that signaling through the CX3CR1 receptor modulates macrophage activity, resulting in an environment conducive to olfactory sensory neuron clearance and targeted replacement from endogenous stem/progenitor cells.

Bone marrow chimeric mice reveal a role for CX3CR1 in maintenance of the monocyte-derived cell population in the olfactory neuroepithelium.

Macrophages in the olfactory neuroepithelium are thought to play major roles in tissue homeostasis and repair. However, little information is available at present about possible heterogeneity of these monocyte-derived cells, their turnover rates, and the role of chemokine receptors in this process. To start addressing these issues, this study used Cx3cr1(gfp) mice, in which the gene sequence for eGFP was knocked into the CX3CR1 gene locus in the mutant allele. Using neuroepithelial whole-mounts from Cx3cr1(gfp/+) mice, we show that eGFP(+) cells of monocytic origin are distributed in a loose network throughout this tissue and can be subdivided further into two immunophenotypically distinct subsets based on MHC-II glycoprotein expression. BM chimeric mice were created using Cx3cr1(gfp/+) donors to investigate turnover of macrophages (and other monocyte-derived cells) in the olfactory neuroepithelium. Our data indicate that the monocyte-derived cell population in the olfactory neuroepithelium is actively replenished by circulating monocytes and under the experimental conditions, completely turned over within 6 months. Transplantation of Cx3cr1(gfp/gfp) (i.e., CX3CR1-deficient) BM partially impaired the replenishment process and resulted in an overall decline of the total monocyte-derived cell number in the olfactory epithelium. Interestingly, replenishment of the CD68(low)MHC-II(+) subset appeared minimally affected by CX3CR1 deficiency. Taken together, the established baseline data about heterogeneity of monocyte-derived cells, their replenishment rates, and the role of CX3CR1 provide a solid basis to further examine the importance of different monocyte subsets for neuroregeneration at this unique frontier with the external environment.