Topical Brimonidine or Intravitreal BDNF, CNTF, or bFGF Protect Cones Against Phototoxicity.

PURPOSE: To develop a focal photoreceptor degeneration model by blue light-emitting diode (LED)-induced phototoxicity (LIP) and investigate the protective effects of topical brimonidine (BMD) or intravitreal brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), or basic fibroblast growth factor (bFGF). METHODS: In anesthetized, dark-adapted, adult female Swiss mice, the left eye was dilated and exposed to blue light (10 seconds, 200 lux). After LIP, full-field electroretinograms (ERG) and spectral-domain optical coherence tomography (SD-OCT) were obtained longitudinally, and reactive-Iba-1+monocytic cells, TUNEL+ cells and S-opsin+ cone outer segments were examined up to 7 days. Left eyes were treated topically with BMD (1%) or vehicle, before or right after LIP, or intravitreally with BDNF (2.5 µg), CNTF (0.2 µg), bFGF (0.5 µg), or corresponding vehicle right after LIP. At 7 days, S-opsin+ cone outer segments were counted within predetermined fixed-size areas (PFA) centered on the lesion in both flattened retinas. RESULTS: SD-OCT showed a circular region in the superior-temporal left retina with progressive thinning (207.9 ± 5.6 µm to 160.7 ± 6.8 µm [7 days], n = 8), increasing TUNEL+ cells (peak at 3 days), decreasing S-opsin+ cone outer segments, and strong microglia activation. ERGs were normal by 3 days. Total S-opsin+ cones in the PFA for LIP-treated and fellow-retinas were 2330 ± 262 and 5601 ± 583 (n = 8), respectively. All neuroprotectants (n = 7-11), including topical BMD pre- or post-LIP, or intravitreal BDNF, CNTF, and bFGF, showed significantly greater S-opsin+ cone survival than their corresponding vehicle-treated groups. CONCLUSIONS: LIP is a reliable, quantifiable focal photoreceptor degeneration model. Topical BMD or intravitreal BDNF, CNTF, or bFGF protect against LIP-induced cone-photoreceptor loss. TRANSLATIONAL RELEVANCE: Topical BMD or intravitreal BDNF, CNTF, or bFGF protect cones against phototoxicity.

A novel in vivo model of focal light emitting diode-induced cone-photoreceptor phototoxicity: neuroprotection afforded by brimonidine, BDNF, PEDF or bFGF.

We have investigated the effects of light-emitting diode (LED)-induced phototoxicity (LIP) on cone-photoreceptors and their protection with brimonidine (BMD), brain-derived neurotrophic factor (BDNF), pigment epithelium-derived factor (PEDF), ciliary neurotrophic factor (CNTF) or basic fibroblast growth factor (bFGF). In anesthetized, dark adapted, adult albino rats a blue (400 nm) LED was placed perpendicular to the cornea (10 sec, 200 lux) and the effects were investigated using Spectral Domain Optical Coherence Tomography (SD-OCT) and/or analysing the retina in oriented cross-sections or wholemounts immune-labelled for L- and S-opsin and counterstained with the nuclear stain DAPI. The effects of topical BMD (1%) or, intravitreally injected BDNF (5 µg), PEDF (2 µg), CNTF (0.4 µg) or bFGF (1 µg) after LIP were examined on wholemounts at 7 days. SD-OCT showed damage in a circular region of the superotemporal retina, whose diameter varied from 1,842.4±84.5 µm (at 24 hours) to 1,407.7±52.8 µm (at 7 days). This region had a progressive thickness diminution from 183.4±5 µm (at 12 h) to 114.6±6 µm (at 7 d). Oriented cross-sections showed within the light-damaged region of the retina massive loss of rods and cone-photoreceptors. Wholemounts documented a circular region containing lower numbers of L- and S-cones. Within a circular area (1 mm or 1.3 mm radius, respectively) in the left and in its corresponding region of the contralateral-fellow-retina, total L- or S-cones were 7,118±842 or 661±125 for the LED exposed retinas (n = 7) and 14,040±1,860 or 2,255±193 for the fellow retinas (n = 7), respectively. BMD, BDNF, PEDF and bFGF but not CNTF showed significant neuroprotective effects on L- or S-cones. We conclude that LIP results in rod and cone-photoreceptor loss, and is a reliable, quantifiable model to study cone-photoreceptor degeneration. Intravitreal BDNF, PEDF or bFGF, or topical BMD afford significant cone neuroprotection in this model.

Recruitment of septin cytoskeletal proteins by botulinum toxin A protease determines its remarkable stability.

Proteolytic cleavage of synaptosomal-associated protein 25 by the light chain of botulinum neurotoxin type A (LCA) results in a blockade of neurotransmitter release that persists for several months in motor neurons. The L428A/L429A mutation in LCA is known to significantly shorten both the proteolytic and neuroparalytic effects of the neurotoxin in mice. To elucidate the cellular mechanism for LCA longevity, we studied the effects of L428A/L429A mutation on the interactome, localization and stability of LCA expressed in cultured neuronal cells. Mass spectrometry analysis of the LCA interactome showed that the mutation prevented the interaction of LCA with septins. The wild-type LCA was concentrated in plasma-membrane-associated clusters, colocalizing with septins-2 and septin-7, which accumulated in these clusters only in the presence of LCA. The L428A/L429A mutation decreased co-clustering of LCA and septins and accelerated proteasomal and non-proteasomal degradation of LCA. Similarly, the impairment of septin oligomerization by forchlorfenuron or silencing of septin-2 prevented LCA interaction and clustering with septins and increased LCA degradation. Therefore, the dileucine-mediated LCA-septin co-clustering is crucial for the long-lasting stabilization of LCA-related proteolytic and presumably neuroparalytic activity.

Fusion of Golgi-derived vesicles mediated by SNAP-25 is essential for sympathetic neuron outgrowth but relatively insensitive to botulinum neurotoxins in vitro.

Sympathetic neurons ramify to innervate multiple cells in target tissues. In compartmentalized cultures of rat superior cervical ganglion neurons, cleavage of synaptosomal-associated protein of Mr  = 25 000 (SNAP-25) in neurites exposed to botulinum neurotoxin type A (BoNT/A) arrested their growth and collapsed interstitial branches, but this required large, nonclinical doses. A protease-inactive mutant proved ineffective, confirming involvement of SNAP-25 in neurite extension and arborization. BoNT/C1 acted like BoNT/A, but BoNT/E caused only mild inhibition, likely due to transient SNAP-25 proteolysis. Near-total lack of susceptibility to BoNT/B or BoNT/D revealed that vesicle-associated membrane protein (VAMPs) isoforms 1-3 are not essential. Neurite length was not reduced when either BoNT/A or BoNT/C1 was applied to the somata, with no detrimental effect on neuron viability being observed. Treatments that protect cells from deprivation of nerve growth factor failed to prevent the toxin-induced loss of neurites. Inactivation of SNAP-25 caused the accumulation at neurite branch sites of Golgi-derived organelles labelled with N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sphingosine conjugated to bovine serum albumin, prior to the collapse of arbors. Notably, neurite growth was ~ 1000-fold less susceptible to BoNT/A than cholinergic transmission in these neurons. Accordingly, a BoNT/A acceptor synaptic vesicle protein 2 (SV2) was found to be colocalized with VAMP 1-3, but not with VAMP 7, which is implicated in the growth of neurites. In conclusion, neurites depend on SNAP-25 for extension but this is quite resistant to BoNT/A, possibly, because of a low density of SV2 at growth sites that are distant from the highly susceptible regions of neurotransmitter release.

Mitochondrial permeability transition pore in inflammatory apoptosis of human conjunctival epithelial cells and T cells: effect of cyclosporin A.

PURPOSE: To investigate the role of mitochondrial permeability transition pore (MPTP) and effect of cyclosporin A (CsA) on inflammatory apoptosis of human conjunctival epithelial cells (IOBA-NHC) and T cells. METHODS: IOBA-NHC and Jurkat cells were stimulated with IFNγ, TNFα, αFas, or PMA/αCD3, in the presence or absence of CsA. MPTP was determined using the calcein-cobalt technique. Mitochondrial membrane potential (ΔΨm) was measured with JC-1. Apoptosis was quantified by Annexin V/PI staining. Apoptosis mediators were evaluated by flow cytometry or Western blot. RESULTS: In IOBA-NHC, TNFα, and IFNγ induced MPTP opening, ΔΨm loss, and increased cell apoptosis. This was accompanied by upregulation of Fas/FasL; Bax; and caspase-3, -8, and -9 activation. Addition of CsA prevented IOBA-NHC from cell death by blocking MPTP opening, ΔΨm loss, Fas/FasL, and caspase activation. In PMA/αCD3-activated Jurkat T cells, MPTP opening and ΔΨm loss were increased along with cell apoptosis and upregulated Fas/FasL/caspase expressions. CsA further promoted T-cell apoptosis, ΔΨm loss, and upregulation of Fas/FasL/caspase. CONCLUSIONS: Inflammation induces aberrant MPTP opening, resulting in an increased apoptosis in conjunctival epithelial cells. CsA protected IOBA-NHC from cell death by blocking both intrinsic and extrinsic apoptosis pathways. CsA promoted T-cell apoptosis via upregulating Fas/FasL and caspase activities with a minimal effect on MPTP. The findings suggest that the differential effect of CsA on T cells versus ocular surface resident epithelial cells may contribute to its therapeutic efficacy in treating ocular inflammation such as dry eye disease.

Protective effect of carnosic acid, a pro-electrophilic compound, in models of oxidative stress and light-induced retinal degeneration.

PURPOSE: The herb rosemary has been reported to have antioxidant and anti-inflammatory activity. We have previously shown that carnosic acid (CA), present in rosemary extract, crosses the blood-brain barrier to exert neuroprotective effects by upregulating endogenous antioxidant enzymes via the Nrf2 transcriptional pathway. Here we investigated the antioxidant and neuroprotective activity of CA in retinal cell lines exposed to oxidative stress and in a rat model of light-induced retinal degeneration (LIRD). METHODS: Retina-derived cell lines ARPE-19 and 661W treated with hydrogen peroxide were used as in vitro models for testing the protective activity of CA. For in vivo testing, dark-adapted rats were given intraperitoneal injections of CA prior to exposure to white light to assess protection of the photoreceptor cells. Retinal damage was assessed by measuring outer nuclear layer thickness and by electroretinogram (ERG). RESULTS: In vitro, CA significantly protected retina-derived cell lines (ARPE-19 and 661W) against H(2)O(2)-induced toxicity. CA induced antioxidant phase 2 enzymes and reduced formation of hyperoxidized peroxiredoxin (Prx)2. Similarly, we found that CA protected retinas in vivo from LIRD, producing significant improvement in outer nuclear layer thickness and ERG activity. CONCLUSIONS: These findings suggest that CA may potentially have clinical application to diseases affecting the outer retina, including age-related macular degeneration and retinitis pigmentosa, in which oxidative stress is thought to contribute to disease progression.

Novel chimeras of botulinum and tetanus neurotoxins yield insights into their distinct sites of neuroparalysis.

Botulinum neurotoxin (BoNT) A or E and tetanus toxin (TeTx) bind to motor-nerve endings and undergo distinct trafficking; their light-chain (LC) proteases cleave soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) peripherally or centrally and cause flaccid or spastic paralysis, respectively. To seek protein domains responsible for local blockade of transmitter release (BoNTs) rather than retroaxonal transport to spinal neurons (TeTx), their acceptor-binding moieties (H(C))--or in one case, heavy chain (HC)--were exchanged by gene recombination. Each chimera, expressed and purified from Escherichia coli, entered rat cerebellar neurons to cleave their substrates, blocked in vitro nerve-induced muscle contractions, and produced only flaccid paralysis in mice. Thus, the local cytosolic delivery of BoNT/A or BoNT/E proteases and the contrasting retrograde transport of TeTx are not specified solely by their HC or H(C); BoNT/A LC translocated locally irrespective of being targeted by either of the latter TeTx domains. In contrast, BoNT/E protease fused to a TeTx enzymatically inactive mutant (TeTIM) caused spastic paralysis and cleaved SNAP-25 in spinal cord but not the injected muscle. Apparently, TeTIM precludes cytosolic release of BoNT/E protease at motor nerve endings. It is deduced that the LCs of the toxins, acting in conjunction with HC domains, dictate their local or distant destinations.

Autoantibodies contribute to the immunopathogenesis of experimental dry eye disease.

PURPOSE: The purpose of this study was to determine if autoantibodies play a role in the immunopathogenesis of experimental dry eye disease. METHODS: Dry eye was induced by exposing female C57BL/6 wild-type mice or hen egg lysozyme B-cell receptor transgenic mice to desiccating stress (subcutaneous scopolamine [0.5 mg/0.2 mL] 3 times a day, humidity < 40%, and sustained airflow) for 3 weeks, allowing sufficient time for a humoral immune response. Serum or purified IgG isolated from dry-eye mice or untreated controls was passively transferred to nude recipient mice, which were evaluated for ocular surface inflammation 3 days after transfer. To determine if complement activation contributed to serum-induced dry eye disease, cobra venom factor was used to deplete complement activity. RESULTS: Autoantibodies against kallikrein 13 were identified in serum from dry-eye mice, but were undetectable in untreated controls. Autoantibody-containing serum or purified IgG from dry-eye mice was sufficient to mediate complement-dependent ocular surface inflammation. Serum or purified IgG caused marked inflammatory burden and tissue damage within the ocular surface tissues, including elevated Gr1+ neutrophil infiltration and proinflammatory cytokines/chemokines associated with goblet cell loss. Moreover, complement C3b deposition was found within the ocular surface tissues of mice receiving dry-eye serum, but not in recipients of control serum. Functionally, complement depletion attenuated the ability to transfer dry-eye-specific serum or IgG-mediated disease. CONCLUSIONS: These data demonstrate for the first time a complement-dependent pathogenic role of dry-eye-specific autoantibodies, and suggest autoantibody deposition within the ocular surface tissues contributes to the predominantly T-cell-mediated immunopathogenesis of dry eye disease.

Comparison of human ocular distribution of bimatoprost and latanoprost.

PURPOSE: This study investigated the ocular distribution of bimatoprost, latanoprost, and their acid hydrolysis products in the aqueous humor, cornea, sclera, iris, and ciliary body of patients treated with a single topical dose of 0.03% bimatoprost or 0.005% latanoprost for understanding concentration-activity relationships. METHODS: Thirty-one patients undergoing enucleation for an intraocular tumor not affecting the anterior part of the globe were randomized to treatment with bimatoprost or latanoprost at 1, 3, 6 or 12 h prior to surgery. Concentrations of bimatoprost, bimatoprost acid, latanoprost, and latanoprost acid in the human aqueous and ocular tissues were measured using liquid chromatography tandem mass spectrometry. RESULTS: Following topical administration, intact bimatoprost was distributed in human eyes with a rank order of cornea/sclera >iris/ciliary body >aqueous humor. Bimatoprost acid was also detected in these tissues, where its low levels in the cornea relative to that of latanoprost acid indicated that bimatoprost hydrolysis was limited. Latanoprost behaved as a prodrug that entered eyes predominantly via the corneal route. Levels of latanoprost acid were distributed as cornea >>aqueous humor>iris>sclera>ciliary body. CONCLUSIONS: Our study provided experimental evidence that levels of bimatoprost in relevant ocular tissues, and not only aqueous humor, are needed to understand the mechanisms by which bimatoprost lowers intraocular pressure (IOP) in human subjects. The data suggest that bimatoprost reached the target tissues favoring the conjunctival/scleral absorption route. Findings of intact bimatoprost in the target ciliary body indicated its direct involvement in reducing IOP. However, bimatoprost acid may have only a limited contribution on the basis that bimatoprost has greater/similar IOP-lowering efficacy than latanoprost, yet bimatoprost acid levels were a fraction of latanoprost acid levels in the aqueous humor and cornea and only sporadically detectable in the ciliary body. In this report, human ocular tissues were examined concurrently with aqueous humor for the in vivo distribution of bimatoprost, bimatoprost acid, latanoprost, and latanoprost acid.

C6orf176: a novel possible regulator of cAMP-mediated gene expression.

cAMP mediates diverse cellular signals including prostaglandin (PG) E(2)-mediated intraocular pressure (IOP)-lowering activity in human ocular ciliary smooth muscle cells (hCSM). We have identified gene regulatory networks and key genes upon activation of the cAMP pathway in hCSM, using novel agonists highly selective for PGE(2) receptor subtypes EP2 or EP4, which are G protein-coupled receptors well known to activate cAMP signaling. Here we describe a novel, EP2/EP4-induced, primate-specific gene of hitherto unknown function, also known as C6orf176 (chromosome 6 open reading frame 176) and recently reclassified as noncoding RNA in NCBI's database. Its expression, as determined by quantitative real-time RT-PCR (qRT-PCR), is dramatically upregulated (>2,000-fold) subsequent to transduction of EP2/EP4/Gs/cAMP signaling not only in hCSM, but also in HEK cells overexpressing the recombinant receptors. Moreover, activation of other IOP lowering, Gs-coupled prostanoid receptors, such as DP1 and IP, as well as a direct activator of adenylyl cyclase, forskolin, also substantially upregulated C6orf176 in hCSM, while FP and TP, which are Gq-coupled prostanoid receptor subtypes, did not. Novel transcript variants carrying open reading frames, derived from an at least 67 kb genomic locus on chromosome 6q27 with putative alternative transcription start sites, were identified. Transcriptional upregulation of transcript variants as well as of two genes expressed in antisense orientation that partially overlap the transcribed C6orf176 region was observed, to varying degrees, subsequent to induction of cAMP signaling using various agonists. Small interfering RNA-mediated C6orf176 gene silencing experiments showed modulation of several cAMP-responsive genes. These transcriptional activities identify C6orf176 as a potential biomarker and/or therapeutic target in context with diseases linked to deregulated cAMP signaling. Also, the cAMP-inducible C6orf176 gene locus could be useful as a model system for studying transcriptional regulation by chromatin and RNA polymerase II.

Ocular surface APCs are necessary for autoreactive T cell-mediated experimental autoimmune lacrimal keratoconjunctivitis.

As specialized sentinels between the innate and adaptive immune response, APCs are essential for activation of Ag-specific lymphocytes, pathogen clearance, and generation of immunological memory. The process is tightly regulated; however, excessive or atypical stimuli may ignite activation of APCs in a way that allows self-Ag presentation to autoreactive T cells in the context of the necessary costimulatory signals, ultimately resulting in autoimmunity. Studies in both animal models and patients suggest that dry eye is a chronic CD4(+) T cell-mediated ocular surface autoimmune-based inflammatory disease. Using a desiccating stress-induced mouse model of dry eye, we establish the fundamental role of APCs for both the generation and maintenance of ocular-specific autoreactive CD4(+) T cells. Subconjunctival administration of liposome-encapsulated clodronate efficiently diminished resident ocular surface APCs, inhibited the generation of autoreactive CD4(+) T cells, and blocked their ability to cause disease. APC-dependent CD4(+) T cell activation required intact draining cervical lymph nodes, as cervical lymphadenectomy also inhibited CD4(+) T cell-mediated dry eye disease. In addition, local depletion of peripheral conjunctival APCs blocked the ability of dry eye-specific CD4(+) T cells to accumulate within the ocular surface tissues, suggesting that fully primed and targeted dry eye-specific CD4(+) T cells require secondary activation by resident ocular surface APCs for maintenance and effector function. These data demonstrate that APCs are necessary for the initiation and development of experimental dry eye and support the standing hypothesis that dry eye is a self-Ag-driven autoimmune disease.

Treatment of experimental anterior and intermediate uveitis by a dexamethasone intravitreal implant.

PURPOSE: To assess the efficacy of a dexamethasone (DEX) intravitreal implant in a rabbit model of anterior and intermediate uveitis. METHODS: Experimental anterior and intermediate uveitis was induced by a unilateral intracameral injection of Mycobacterium tuberculosis H37Ra antigen in preimmunized rabbits. Four days after uveitis induction, rabbits received DEX implant or underwent a sham procedure (no implant). Clinical and histopathologic signs of uveitis were assessed for 13 days, and levels of inflammatory markers in the iris/ciliary body were measured after 21 days. RESULTS: All signs of anterior and intermediate uveitis were reduced by the DEX implant compared with sham procedure. At day 13, mean anterior chamber cell scores ± SD for the DEX implant versus the sham procedure were, respectively, 1.9 ± 1.3 versus 4.0 ± 0.0 (P = 0.04), and mean total histologic inflammatory scores were 3.9 ± 2.5 versus 15.4 ± 6.0 (P = 0.026). Similarly, at day 13, mean vitreous haze severity scores (SD) for the DEX implant versus the sham procedure were, respectively, 0.1 ± 0.2 versus 2.7 ± 1.5 (P = 0.026), and mean vitreous inflammatory cell infiltration scores were 0.0 ± 0.0 versus 1.5 ± 1.3. Treatment with the DEX intravitreal implant also significantly reduced the proinflammatory immune response, as measured by cytokine levels in iris/ciliary body. CONCLUSIONS: A single administration of DEX implant significantly reduced inflammation in an animal model of anterior and intermediate uveitis.

Brimonidine prevents axonal and somatic degeneration of retinal ganglion cell neurons.

BACKGROUND: Brimonidine is a common drug for lowering ocular pressure and may directly protect retinal ganglion cells in glaucoma. The disease involves early loss of retinal ganglion cell transport to brain targets followed by axonal and somatic degeneration. We examined whether brimonidine preserves ganglion cell axonal transport and abates degeneration in rats with elevated ocular pressure induced by laser cauterization of the episcleral veins. RESULTS: Ocular pressure was elevated unilaterally by 90% for a period of 8 weeks post- cauterization. During this time, brimonidine (1mg/kg/day) or vehicle (phosphate-buffered saline) was delivered systemically and continuously via subcutaneous pump. Animals received bilateral intravitreal injections of fluorescent cholera toxin subunit ß (CTB) two days before sacrifice to assess anterograde transport. In retinas from the vehicle group, elevated pressure induced a 44% decrease in the fraction of ganglion cells with intact uptake of CTB and a 14-42% reduction in the number of immuno-labelled ganglion cell bodies, with the worst loss occurring nasally. Elevated pressure also caused a 33% loss of ganglion cell axons in vehicle optic nerves and a 70% decrease in CTB transport to the superior colliculus. Each of these components of ganglion cell degeneration was either prevented or significantly reduced in the brimonidine treatment group. CONCLUSIONS: Continuous and systemic treatment with brimonidine by subcutaneous injection significantly improved retinal ganglion cell survival with exposure to elevated ocular pressure. This effect was most striking in the nasal region of the retina. Brimonidine treatment also preserved ganglion cell axon morphology, sampling density and total number in the optic nerve with elevated pressure. Consistent with improved outcome in the optic projection, brimonidine also significantly reduced the deficits in axonal transport to the superior colliculus associated with elevated ocular pressure. As transport deficits to and from retinal ganglion cell projection targets in the brain are relevant to the progression of glaucoma, the ability of brimonidine to preserve optic nerve axons and active transport suggests its neuroprotective effects are relevant not only at the cell body, but throughout the entire optic projection.

A dileucine in the protease of botulinum toxin A underlies its long-lived neuroparalysis: transfer of longevity to a novel potential therapeutic.

Blockade of neurotransmitter release by botulinum neurotoxin type A (BoNT(A)) underlies the severe neuroparalytic symptoms of human botulism, which can last a few years. The structural basis for this remarkable persistence remains unclear. Herein, recombinant BoNT(A) was found to match the neurotoxicity of that from Clostridium botulinum, producing persistent cleavage of synaptosomal-associated protein of 25 kDa (SNAP-25) and neuromuscular paralysis. When two leucines near the C terminus of the protease light chain of A (LC(A)) were mutated, its inhibition of exocytosis was followed by fast recovery of intact SNAP-25 in cerebellar neurons and neuromuscular transmission in vivo. Deletion of 6-7 N terminus residues diminished BoNT(A) activity but did not alter the longevity of its SNAP-25 cleavage and neuromuscular paralysis. Furthermore, genetically fusing LC(E) to a BoNT(A) enzymically inactive mutant (BoTIM(A)) yielded a novel LC(E)-BoTIM(A) protein that targets neurons, and the BoTIM(A) moiety also delivers and stabilizes the inhibitory LC(E), giving a potent and persistent cleavage of SNAP-25 with associated neuromuscular paralysis. Moreover, its neurotropism was extended to sensory neurons normally insensitive to BoNT(E). LC(E-)BoTIM(A)(AA) with the above-identified dileucine mutated gave transient neuromuscular paralysis similar to BoNT(E), reaffirming that these residues are critical for the persistent action of LC(E)-BoTIM(A) as well as BoNT(A). LC(E)-BoTIM(A) inhibited release of calcitonin gene-related peptide from sensory neurons mediated by transient receptor potential vanilloid type 1 and attenuated capsaicin-evoked nociceptive behavior in rats, following intraplantar injection. Thus, a long acting, versatile composite toxin has been developed with therapeutic potential for pain and conditions caused by overactive cholinergic nerves.

Comparison of prostaglandin E2 receptor subtype 4 agonist and sulfasalazine in mouse colitis prevention and treatment.

Prodrugs of 5-aminosalicylic acid (5-ASA), such as sulfasalazine, have been the mainstay for the treatment and maintenance of inflammatory bowel disease (IBD) for decades, which is attributable to their antiadaptive immune activity. However, 5-ASA compromises regeneration of intestinal epithelia and induces apoptosis. The majority of patients eventually undergo colectomy. Agonists for the prostaglandin E(2) subtype 4 (EP4) receptor have been shown to protect epithelial barrier against colitis-inducing agents and could be valuable alternatives for sulfasalazine. Here, we compared sulfasalazine and a novel EP4 agonist for their abilities to prevent colitis induction and relieve symptoms of established colitis in a dextran sulfate sodium-indomethacin mouse model. The EP4 agonist dose-dependently alleviated weight loss in colitis mice. Compared with sulfasalazine at 100 mg/kg on the colitis induction model, the EP4 agonist at 0.2 mg/kg was superior in reducing colitis symptoms, preventing increase of innate immune cells, and ameliorating inflammation in colon. In mice with established colitis, sulfasalazine quickly reversed weight loss but with fading efficacy. The EP4 agonist, in contrast, had slow but sustained effects on body weight gain and was more efficacious in epithelial regeneration. Such temporal differences between sulfasalazine and the EP4 agonist actions seemingly led to no additive effect in combination therapy. In conclusion, the EP4 agonist would be more efficacious in the maintenance of remission because of both anti-innate immune responses and epithelial regeneration activity, whereas sulfasalazine would be more suitable for induction of remission because of its rapid onset of antiadaptive inflammation action.

Prostaglandin E2 receptor subtype EP2- and EP4-regulated gene expression profiling in human ciliary smooth muscle cells.

Prostanoids are an important class of intraocular pressure (IOP)-lowering antiglaucoma agents that act primarily via increased uveo-scleral aqueous humor outflow through the ciliary body. We have developed two novel PGE(2) analogs that are specific agonists for the PGE(2) receptor subtypes EP2 and EP4, respectively. To identify gene regulatory networks and key players that mediate the physiological effects observed in vivo, we performed genomewide expression studies using human ciliary smooth muscle cells. Quantitative real-time RT-PCR confirmed a largely overlapping gene expression profile subsequent to EP2 and EP4 agonist treatment, with 65 significantly regulated genes identified overall, 5 being specific for the EP2 agonist and 6 specific for the EP4 agonist. We found predicted functional cAMP-response elements in promoter regions of a large fraction of the predominantly upregulated genes, which suggests that the cAMP signaling pathway is the most important intracellular signaling pathway for these agonists in these cells. Several target genes were identified that, as part of complex regulatory networks, are implicated in tissue remodeling processes and osmoregulation (e.g., AREG, LOXL3, BMP2, AQP3) and thus may help elucidate the mechanism of action of these IOP-lowering drugs involving the uveo-scleral outflow path.

Nimodipine enhancement of alpha2 adrenergic modulation of NMDA receptor via a mechanism independent of Ca2+ channel blocking.

PURPOSE: To further understand alpha2 receptor signaling in the retina and the mechanisms that mediate ocular beneficial effects of brimonidine (an alpha2 agonist) and nimodipine (an L-type Ca(2+) channel blocker). METHODS: The authors used in situ retinal ganglion cells (RGCs) in the isolated rat retina to characterize alpha2 modulation of NMDA receptor function and a rabbit retinal NMDA excitotoxicity model to verify in vitro findings under in vivo conditions. Electrophysiological (whole-cell patch clamp) recordings and Ca(2+) imaging were used to characterize NMDA receptor function and to verify the effect of various Ca(2+) channel blockers. In vivo drug application in rabbits was achieved by intravitreal injections. RESULTS: Application of NMDA elicited a robust whole-cell inward current in individual in situ RGCs voltage clamped at -70 mV. Pretreatment with brimonidine significantly reduced NMDA-elicited currents in RGCs. This suppressive effect of brimonidine was substantially enhanced by background addition of nimodipine or isradipine, but not by diltiazem, verapamil, or cadmium. This effect of nimodipine was blocked by either a selective alpha2 antagonist, a cyclic adenosine monophosphate (cAMP) analogue, or an adenylate cyclase activator, indicating that nimodipine acts through the alpha2 receptor-G(alphai)-coupled pathway. Brimonidine protects RGCs in the rabbit excitotoxicity model. This brimonidine protection is also enhanced significantly by application of nimodipine but not of diltiazem. CONCLUSIONS: These in vitro and in vivo findings demonstrate a novel neural mechanism involving nimodipine enhancement of alpha2 signaling in RGCs. This nimodipine effect appears to be independent of its classic L-type Ca(2+) channel-blocking action.

EP4 agonist alleviates indomethacin-induced gastric lesions and promotes chronic gastric ulcer healing.

AIM: To investigate EP4-selective agonist effect on indomethacin-induced gastric lesions and on the spontaneous healing of chronic gastric ulcers. METHODS: In a mouse model of gastric bleeding with high dose of indomethacin (20 mg/kg), an EP4-selective agonist was administered orally. Stomach lesions and gastric mucous regeneration were monitored. In a mouse model of chronic gastric ulcer induced by acetic acid, EP4 agonist effect on the healing of chronic gastric ulcer was evaluated in the presence or absence of low dose indomethacin (3 mg/kg). In cultured human gastric mucous cells, EP4 agonist effect on indomethacin-induced apoptosis was assessed by flow cytometry. RESULTS: The EP4-selective agonist reduced high dose indomethacin-induced acute hemorrhagic damage and promoted mucous epithelial regeneration. Low-dose indomethacin aggravated ulcer bleeding and inflammation, and delayed the healing of the established chronic gastric ulcer. The EP4 agonist, when applied locally, not only offset indomethacin-induced gastric bleeding and inflammation, but also accelerated ulcer healing. In the absence of indomethacin, the EP4 agonist even accelerated chronic gastric ulcer healing and suppressed inflammatory cell infiltration in the granulation tissue. In vitro, the EP4 agonist protected human gastric mucous cells from indomethacin-induced apoptosis. CONCLUSION: EP4-selective agonist may prevent indomethacin-induced gastric lesions and promote healing of existing and indomethacin-aggravated gastric ulcers, via promoting proliferation and survival of mucous epithelial cells.

Bimatoprost-induced calcium signaling in human T-cells does not involve prostanoid FP or TP receptors.

PURPOSE: The prostamide bimatoprost and prostanoid FP receptor agonists are highly efficacious drugs for glaucoma treatment. The presence of both prostamide and prostanoid FP receptors in bimatoprost-sensitive preparations has made prostamide receptor classification difficult. This study investigated a novel bimatoprost-sensitive preparation. METHODS: Human peripheral blood T lymphoblasts (Molt-3) and human osteoblasts (hFOB) were cultured for intracellular calcium signaling studies and quantitative real-time PCR analysis of RNA. RESULTS: Bimatoprost stimulated concentration-related increases in [Ca(2 +)](i) in a human T-cell line that does not express human FP receptor/variants, according to PCR analysis. The calcium signal induced by bimatoprost was not antagonized by prostanoid FP receptor antagonist/partial agonist AL-8810 or selective TP receptor antagonist SQ 29548. Conversely, bimatoprost did not elevate [Ca(2 +)](i) in human osteoblasts, which were confirmed to contain RNA of human FP receptor/variants. CONCLUSIONS: Molt-3 cells have been identified as a bimatoprost-sensitive preparation in which the activity of bimatoprost is independent of prostanoid FP receptors.

Experimental glutamatergic excitotoxicity in rabbit retinal ganglion cells: block by memantine.

PURPOSE: Excessive activity of NMDA-type (N-methyl-d-aspartate) glutamatergic channels has been implicated as a mechanism for neuronal injury in neurologic disorders, including glaucoma, and retinal disease. This study was designed to characterize the retinal response to experimental manipulations that mimic features of glutamatergic excitotoxic insult and also to determine whether memantine, an NMDA-type glutamatergic channel blocker, is effective in reversing experimental excitotoxicity. METHODS: Recordings of the electroretinogram (ERG) and spiking activity of single retinal ganglion cells (RGCs) were made from rabbit retinas. Excitotoxic insult was induced by either (1) application of NMDA, a selective NMDA receptor agonist; (2) application of TBOA (dl-threo-beta-benzyloxyaspartic acid), a selective inhibitor of glutamate transporters, or (3) perfusion with magnesium-free medium. For each condition, memantine was coapplied to determine its efficacy for reversal of experimental excitotoxicity. Memantine was also applied in isolation to characterize any effect on retinal responses to light stimuli. RESULTS: All three experimental manipulations were associated with an increase in the tonic level of RGC spiking activity, a reduction in RGC spike amplitude, and, in some cells, block of spike generation. Experimental excitotoxicity had little or no effect on ERG responses. Coapplication of memantine was associated with recovery of RGC tonic spiking activity and spike amplitude toward control levels. Application of memantine in isolation was associated with a dose-dependent effect on the timing of ERG and RGC-OFF responses. CONCLUSIONS: Memantine was effective in reversing acute experimental excitotoxicity at concentrations that have little effect on retinal light signaling.