Tear miRNA expression analysis reveals miR-203 as a potential regulator of corneal epithelial cells.

BACKGROUND: microRNAs (miRNAs) are small noncoding RNAs that negatively regulate gene expression. They are found within cells and in body fluids. Extracellular miRNAs have been shown to associate with the surrounding tissues. Therefore, we predicted that miRNAs in tears may contribute to regulate corneal epithelial cell function. However, information on the miRNA expression profile of tears is limited and the specific functions of tear miRNAs for corneal epithelial cells are still unknown. To study the role of tear miRNAs, we determined which miRNAs are highly expressed in tears and examined the involvement of miRNAs in corneal epithelial cell viability. METHODS: miRNAs extracted from monkey tears and sera were subjected to microarray analysis. miRNAs of which expression levels were higher in tears than in sera were selected, and their expression levels were quantified by quantitative polymerase chain reaction (qPCR). To examine miRNA function, mimics and inhibitors of miRNAs were transfected into human corneal epithelial (HCE-T) cells and incubated for 24 or 48 h. After transfection of miRNA mimics and inhibitors, the viability of HCE-T cells was measured using the water soluble tetrazolium salt (WST) assay, and microarray analysis and qPCR were performed using total RNA extracted from HCE-T cells. siRNAs of the candidate targets for miR-203 were transfected into HCE-T cells and the WST assay was performed. To determine a direct target gene for miR-203, a dual luciferase reporter assay was performed in HCE-T cells using a luciferase reporter plasmid containing 3'-UTR of human IGFBP5. RESULTS: Microarray and qPCR analyses showed that miR-184 and miR-203 were expressed significantly more highly in tears than in sera (165,542.8- and 567.8-fold, respectively, p < 0.05). Of these two miRNAs, transfection of a miR-203 mimic significantly reduced the viability of HCE-T cells (p < 0.05), while a miR-203 inhibitor significantly increased this viability (p < 0.05). miR-203 mimic downregulated insulin-like growth factor-binding protein 5 (IGFBP5) and nuclear casein kinase and cyclin-dependent kinase substrate 1 (NUCKS1), while miR-203 inhibitor upregulated these two genes. Transfection of IGFBP5-siRNA decreased the viability of HCE-T cells. miR-203 mimic significantly diminished the luciferase reporter activity. CONCLUSIONS: In this study, we identified miRNAs that are highly expressed in tears, and the inhibition of miR-203 increases the viability of corneal epithelial cells. Our results suggest that miR-203 contributes to regulating the homeostasis of corneal epithelial cells.

FK962 induces neurite outgrowth in cultured monkey trigeminal ganglion cells.

PURPOSE: Corneal sensation, cell proliferation, and wound healing all depend on adequate corneal innervation. Disruption of corneal innervation can lead to dry eye and delayed wound healing. Our studies in rats and rabbits show that the substituted fluorobenzamide drug FK962 accelerates the extension of neuronal processes and recovery of corneal sensitivity. The purpose of the present study was 1) to determine whether FK962 induces sprouting and elongation of neurites in cultured monkey trigeminal ganglion cells, and 2) to investigate the involvement of the neurotrophic peptide GDNF in FK962-induced neurite elongation. METHODS: Dissociated, cultured trigeminal ganglion cells, containing neuronal and Schwann cells were cultured for 48 h with or without FK962. Neuronal elongation was evaluated by immunostaining with a neurofilament-specific antibody. Culture with or without GDNF, or with antibody against GDNF, was used to determine the role of GDNF in FK962-induced neurite elongation. RESULTS: FK962 or GDNF were found to significantly induce neurite elongation. The GDNF antibody significantly inhibited elongation induced by FK962. CONCLUSION: GDNF was found to be a mediator of FK962-induced neurite elongation in a relevant primate model. FK962 may be a candidate drug for treatment of neurotrophic disorders in the human cornea.

Calpain inhibition reduces structural and functional impairment of retinal ganglion cells in experimental optic neuritis.

Optic neuritis (ON), inflammation of the optic nerve, is strongly associated with multiple sclerosis. ON pathology is characterized by attack of autoreactive T cells against optic nerve antigens, resulting in demyelination, death of retinal ganglion cells, and cumulative visual impairment. A model of experimental autoimmune encephalomyelitis (EAE) was utilized to study the onset and progression of ON and the neuroprotective efficacy of oral treatment with the calpain inhibitor SNJ 1945. EAE was actively induced in B10.PL mice with myelin basic protein on Days 0 and 2, and mice received twice daily oral dosing of SNJ 1945 from Day 9 until sacrificing (Day 26). Visual function was determined by electroretinogram recordings and daily measurement of optokinetic responses (OKR) to a changing pattern stimulus. Optic nerve and retinal histopathology was investigated by immunohistochemical and luxol fast blue staining. EAE mice manifested losses in OKR thresholds, a measurement of visual acuity, which began early in the disease course. There was a significant bias toward unilateral OKR impairment among EAE-ON eyes. Treatment with SNJ 1945, initiated after the onset of OKR threshold decline, improved visual acuity, pattern electroretinogram amplitudes, and paralysis, with attenuation of retinal ganglion cell death. Furthermore, calpain inhibition spared oligodendrocytes, prevented degradation of axonal neurofilament protein, and attenuated reactive astrocytosis. The trend of early, unilateral visual impairment in EAE-ON parallels the clinical presentation of ON exacerbations associated with multiple sclerosis. Calpain inhibition may represent an ideal candidate therapy for the preservation of vision in clinical ON. As in multiple sclerosis (MS) patients, optic neuritis (ON) and early, primarily monocular loss in spatial acuity is observed in a rodent model (EAE, experimental autoimmune encephalomyelitis). Daily oral treatment with the calpain inhibitor SNJ 1945 preserves visual acuity and preserves retinal ganglion cells (Brn3a, brain-specific homeobox/POU domain protein 3A) and their axons (MOSP, myelin oligodendrocyte-specific protein). Calpain inhibition may represent a candidate therapy for the preservation of vision in ON.

Neuron-microglia interaction induced bi-directional cytotoxicity associated with calpain activation.

Activated microglia release pro-inflammatory factors and calpain into the extracellular milieu, damaging surrounding neurons. However, mechanistic links to progressive neurodegeneration in disease such as multiple sclerosis (MS) remain obscure. We hypothesize that persistent damaged/dying neurons may also release cytotoxic factors and calpain into the media, which then activate microglia again. Thus, inflammation, neuronal damage, and microglia activation, i.e., bi-directional interaction between neurons and microglia, may be involved in the progressive neurodegeneration. We tested this hypothesis using two in vitro models: (i) the effects of soluble factors from damaged primary cortical neurons upon primary rat neurons and microglia and (ii) soluble factors released from CD3/CD28 activated peripheral blood mononuclear cells of MS patients on primary human neurons and microglia. The first model indicated that neurons due to injury with pro-inflammatory agents (IFN-γ) release soluble neurotoxic factors, including COX-2, reactive oxygen species, and calpain, thus activating microglia, which in turn released neurotoxic factors as well. This repeated microglial activation leads to persistent inflammation and neurodegeneration. The released calpain from neurons and microglia was confirmed by the use of calpain inhibitor calpeptin or SNJ-1945 as well as µ- and m-calpain knock down using the small interfering RNA (siRNA) technology. Our second model using activated peripheral blood mononuclear cells, a source of pro-inflammatory Th1/Th17 cytokines and calpain released from auto-reactive T cells, corroborated similar results in human primary cell cultures and confirmed calpain to be involved in progressive MS. These insights into reciprocal paracrine regulation of cell injury and calpain activation in the progressive phase of MS, Parkinson's disease, and other neurodegenerative diseases suggest potentially beneficial preventive and therapeutic strategies, including calpain inhibition.

Galectin-3 enhances extracellular matrix associations and wound healing in monkey corneal epithelium.

Poor healing of epithelial wounds in cornea is a major clinical problem, leading to persistent epithelial defects and ulceration. The primary cause is poor cell migration over the wound. Carbohydrate-binding protein galectin-3 binds to extracellular matrixes (ECMs) and promotes lamellipodia formation by cross-linking to α3 integrin. Recombinant galectin-3 also facilitates wound healing in the rodent cornea. The purposes of the present experiments were to: (1) establish epithelial wound healing models in monkey corneal explant culture, the models more relevant to human, (2) evaluate the healing effect of galectin-3 in our models, and (3) determine if galectin-3 enhances cell adhesion by interacting with ECMs on corneal surface and their ligand integrins. Monkey corneas with central wounds produced by sodium hydroxide (NaOH) or n-heptanol were incubated with or without recombinant galectin-3. The defected area was stained with sodium fluorescein. Primary isolated corneal epithelial cells from monkey were cultured with or without galectin-3 on plates coated with ECMs or integrins, and the number of adhering cells was counted. Galectin-3 expression in various eye tissues was visualized by immunoblotting. NaOH caused loss of epithelial cells and basement membrane. n-Heptanol removed epithelial cells, but the basement membrane was retained. These corneal defects spontaneously became smaller in a time-dependent manner. Exogenous galectin-3 enhanced wound healing in both NaOH and n-heptanol models. Galectin-3 also enhanced cell adhesion onto the major ECMs found in the basement and Bowman's membranes and onto integrins. Relatively high levels of galectin-3 were detected in corneal and conjunctival epithelium, but tear fluid contained negligible galactin-3. These results suggested that the enhanced binding of epithelial cells to ECMs and integrins caused by galectin-3 might promote cell migration over wounded corneal surfaces. Since tear fluid contained relatively low levels of galectin-3, exogenous galectin-3 may be a beneficial drug to enhance re-epithelialization in human corneal diseases.

Serum antibodies against ßH-crystallins in the American Cocker Spaniel.

OBJECTIVE: To detect antibodies for lens ßH-crystallins in the serum from the American Cocker Spaniel (ACS) presenting with and without cataracts and with and without uveitis. ANIMAL STUDIED: Seventy-three American Cocker Spaniels and six normal Beagles. PROCEDURES: Sera were collected from 73 ACSs, including those with normal lenses and those with cataracts, or uveitis. Fractionated, normal Beagle lens ßH-crystallins were separated by one- or two-dimensional electrophoresis. The separated lens ßH-crystallins were used on immunoblots as sentinel substrates against which the ACS sera were tested for the presence of antibodies against ßH-crystallins. RESULTS: Sera from approximately two-thirds of study animals contained antibodies to some ßH-crystallin polypeptides, but reactivity varied among patients. Contrary to some hypotheses, serum antibodies to groups of ßH-crystallins did not relate to the stages of cataract. However, detailed analysis by two-dimensional immunoblotting and mass spectrometry showed that three spots originating from ßA1-crystallin were detected only in sera from cataract patients. CONCLUSION: Serum antibodies to ßA1-crystallin may be associated with the development of cataract.

SNJ-1945, a calpain inhibitor, protects SH-SY5Y cells against MPP(+) and rotenone.

Complex pathophysiology of Parkinson's disease involves multiple CNS cell types. Degeneration in spinal cord neurons alongside brain has been shown to be involved in Parkinson's disease and evidenced in experimental parkinsonism. However, the mechanisms of these degenerative pathways are not well understood. To unravel these mechanisms SH-SY5Y neuroblastoma cells were differentiated into dopaminergic and cholinergic phenotypes, respectively, and used as cell culture model following exposure to two parkinsonian neurotoxicants MPP(+) and rotenone. SNJ-1945, a cell-permeable calpain inhibitor was tested for its neuroprotective efficacy. MPP(+) and rotenone dose-dependently elevated the levels of intracellular free Ca(2+) and induced a concomitant rise in the levels of active calpain. SNJ-1945 pre-treatment significantly protected cell viability and preserved cellular morphology following MPP(+) and rotenone exposure. The neurotoxicants elevated the levels of reactive oxygen species more profoundly in SH-SY5Y cells differentiated into dopaminergic phenotype, and this effect could be attenuated with SNJ-1945 pre-treatment. In contrast, significant levels of inflammatory mediators cyclooxygenase-2 (Cox-2 and cleaved p10 fragment of caspase-1) were up-regulated in the cholinergic phenotype, which could be dose-dependently attenuated by the calpain inhibitor. Overall, SNJ-1945 was efficacious against MPP(+) or rotenone-induced reactive oxygen species generation, inflammatory mediators, and proteolysis. A post-treatment regimen of SNJ-1945 was also examined in cells and partial protection was attained with calpain inhibitor administration 1-3 h after exposure to MPP(+) or rotenone. Taken together, these results indicate that calpain inhibition is a valid target for protection against parkinsonian neurotoxicants, and SNJ-1945 is an efficacious calpain inhibitor in this context. SH-SY5Y cells, differentiated as dopaminergic (TH positive) and cholinergic (ChAT positive), were used as in vitro models for Parkinson's disease. MPP+ and rotenone induced up-regulation of calpain, expression, and activity as a common mechanism of neurodegeneration. SNJ-1945, a novel calpain inhibitor, protected both the cell phenotypes against MPP+ and rotenone.

Effects of a novel orally administered calpain inhibitor SNJ-1945 on immunomodulation and neurodegeneration in a murine model of multiple sclerosis.

Multiple sclerosis (MS) pathology is marked by the massive infiltration of myelin-specific T cells into the CNS. Hallmarks of T helper (Th) cells during active disease are pro-inflammatory Th1/Th17 cells that predominate over immunoregulatory Th2/Treg cells. Neurodegeneration, a major factor in progressive MS, is often overlooked when considering drug prescription. Here, we show that oral dosing with SNJ-1945, a novel water-soluble calpain inhibitor, reduces experimental autoimmune encephalomyelitis clinical scores in vivo and has a two pronged effect via anti-inflammation and protection against neurodegeneration. We also show that SNJ-1945 treatment down-regulates Th1/Th17 inflammatory responses, and promotes regulatory T cells (Tregs) and myeloid-derived suppressor cells in vivo, which are known to have the capacity to suppress helper as well as cytotoxic T cell functions. Through analysis of spinal cord samples, we show a reduction in calpain expression, decreased infiltration of inflammatory cells, and signs of inhibition of neurodegeneration. We also show a marked reduction in neuronal cell death in spinal cord (SC) sections. These results suggest that calpain inhibition attenuates experimental autoimmune encephalomyelitis pathology by reducing both inflammation and neurodegeneration, and could be used in clinical settings to augment the efficacy of standard immunomodulatory agents used to treat MS. Multiple sclerosis (MS) pathology is marked by inflammation and infiltration of myelin-specific T cells into the central nervous system. Inflammation leads to neurodegeneration in progressive MS which also leads to epitope spreading, feedback looping to more inflammation. Calpain can play a role in both arms of the disease. Here, oral dosing with SNJ-1945, a novel water-soluble calpain inhibitor, reduces experimental autoimmune encephalomyelitis clinical scores in vivo and has a two-pronged effect via anti-inflammation and protection against neurodegeneration.

Mechanism for laser-induced neovascularization in rat choroid: accumulation of integrin α chain-positive cells and their ligands.

PURPOSE: Inhibitors binding to integrins α5 and αv are antiangiogenic in models of choroidal neovascularization (CNV). However, a comprehensive understanding of the accumulation of integrin α isoform-positive cells, their ligands, and associations is limited. The purpose of the present study was to examine the localization of integrin α chain-positive cells and their extracellular matrix (ECM) ligands in the RPE/choroid after laser injury. METHODS: CNV, observed with fluorescein isothiocyanate (FITC)-labeled isolectin, was produced in Brown Norway rats with a 532 nm green laser. Localization of α5 and αv integrins and their ligands was performed with immunohistochemistry in consecutive cryosections. To test the binding specificity between the integrin α chains and ECM ligands, an in vitro cell adhesion assay was performed using retinal endothelial cells and specific antibodies. RESULTS: Angiogenesis was observed on day 7 after laser injury in choroidal flat mounts and cryosections. The number of integrin α5- and αv-positive cells markedly increased at day 3 and then gradually decreased, but was still elevated on day 14. One day after laser treatment, α integrin ligands fibronectin (FN) and vitronectin (VN) were markedly increased, and localized closely to integrins in the laser-injured regions. FN decreased on day 7, but was still retained until 14 days. In contrast, VN disappeared. Cell adhesion assays showed specific association of integrin α5 to FN, and integrin αv to VN. CONCLUSIONS: Laser-induced choroidal injury increased FN and VN, followed by accumulation of integrin α5- and αv-positive cells. The interaction between integrin α chain-positive cells and their specific ligands FN and VN may be important steps leading to CNV.

Calpain-specific breakdown fragment in human drusen.

PURPOSE: With aging and age-related macular dystrophy (AMD), proteolytic fragments are deposited in extracellular drusen located between the RPE and Bruch's membrane. Localized hypoxia may be a risk factor for AMD. Our hypothesis is that following hypoxia, activation of proteolytic enzymes called calpains may cause proteolysis/degeneration of retinal cells and RPE. No direct evidence has yet demonstrated activation of calpains in AMD. The purpose of the present study was to identify calpain-cleaved proteins in drusen. METHODS: Seventy-six (76) drusen were analyzed in human eye sections from six normal and twelve AMD human donor eyes. The sections were subjected to immunofluorescence for the calpain-specific 150 kDa breakdown product from α-spectrin, SBDP150 - a marker for calpain activation, and for recoverin - a marker for photoreceptor cells. RESULTS: Among 29 nodular drusen, 80% from normal eyes and 90% from AMD eyes stained positive for SBDP150. Among 47 soft drusen, mostly from AMD eyes, 72% stained positive for SBDP150. Thus, the majority of both soft and nodular drusen from AMD donors contained SBDP150. CONCLUSIONS: SBDP150 was detected for the first time in soft and nodular drusen from human donors. Our results suggest that calpain-induced proteolysis participates in the degeneration of photoreceptors and/or RPE cells during aging and AMD. Calpain inhibitors may ameliorate AMD progression.

Pituitary adenylate cyclase-activating peptide induces neurite outgrowth in cultured monkey trigeminal ganglion cells: involvement of receptor PAC1.

PURPOSE: Our previous studies in the rabbit trigeminal nerve (TgN) showed that pituitary adenylate cyclase-activating peptide (PACAP) accelerated the extension of neuronal processes and recovery of corneal sensitivity. The purposes of the present study were 1) develop a procedure to culture trigeminal nerve (TgN) cells from monkeys, 2) test whether PACAP induces sprouting and elongation of axons in our culture system, 3) investigate the signaling mechanisms producing axon elongation induced by PACAP, and 4) test the action of PACAP on tear protein secretion by monkey lacrimal acinar cells. METHODS: Primary cultures of TgN cells were established from rhesus monkeys. Cellular distribution of the PACAP receptor, PAC1, was determined with immunostaining. Axonal length in cultured TgN ganglion cells was evaluated with staining by antibody for neurofilament. mRNA expression was determined with quantitative real-time polymerase chain reaction (qPCR). Secretion of tear protein from cultured acinar cells was measured with immunoblotting. RESULTS: Our results showed that dissociated, cultured TgN cells contained neuronal ganglion and Schwann cells, and the PAC1 receptor was expressed in both cell types. PACAP-27 significantly induced neurite outgrowth, which was inhibited by PACAP 6-27. Inhibitors for adenylate cyclase and phospholipase C also inhibited neurite outgrowth. Follistatin was upregulated by PACAP-27 during the culture period. PACAP enhanced secretion of tear proteins. CONCLUSIONS: Our data suggested PAC1 activation is involved in TgN neurite outgrowth.

Concerted inhibition of HIF-1α and -2α expression markedly suppresses angiogenesis in cultured RPE cells.

HIF-1α is known to play an important role in the induction of VEGF by hypoxia in retinal pigment epithelial (RPE) cells. However, the involvement of the other isoform, HIF-2α, in RPE cells remains unclear. Thus, the purpose of present study was to clarify the role of HIF-2α during induction of angiogenic genes in hypoxic RPE cells. When human RPE cells (ARPE-19) were cultured under hypoxic conditions, HIF-1α and HIF-2α proteins increased. This induced an increase in mRNA for VEGF, causing secretion of VEGF protein into the medium. This conditioned medium induced tube formation in human vascular endothelial cells (HUVEC). The increased expression of mRNA for VEGF in hypoxic RPE cells was partially inhibited by HIF-1α siRNA, but not by HIF-2α siRNA. However, co-transfection of HIF-1α siRNA and HIF-2α siRNA augmented downregulation of VEGF mRNA and protein in hypoxic RPE cells and inhibited formation of tube-like structures in HUVEC. GeneChip and PCR array analyses revealed that not only VEGF, but also expression of other angiogenic genes were synergistically downregulated by co-transfection of hypoxic RPE cells with HIF-1α and HIF-2α siRNAs. These findings suggest an important compensatory role for the HIF-2α isoform in the regulation of angiogenic gene expression. Thus, suppression of angiogenic genes for HIF-1α and HIF-2α may be a possible therapeutic strategy against retinal angiogenesis in Age-related macular degeneration (ARMD).

GCL loss in BRAO.

PURPOSE: Our recent publication used optical coherence tomography (OCT) to follow thinning of the retinal ganglion cell layer (GCL) in central retinal artery occlusion (CRAO). Thinning of the inner layers also occurs in patients with branch retinal artery occlusion (BRAO). The mechanism for such thinning may be partially due to proteolysis by a calcium-activated protease called calpain. Calpain inhibitor SNJ-1945 ameliorated the proteolysis in a past series of model experiments. The purposes of the present retrospective study were to: 1) use segmentation analysis of OCT images to follow the loss of retinal layers in BRAO compared to CRAO patients, and 2) predict the number of patients and days of observation needed for a clinical trial of a calpain inhibitor against BRAO. METHODS: A retrospective, case control study was conducted by computer-aided search in a medical records database for BRAO (ICD10 Code H34.239) with at least one OCT procedure (CPT: 92134). Non-proliferative, co-morbid eye diseases were allowed in the patient data base, and manual correction of auto-segmentation errors was performed. GCL thickness changes were followed over time and Cohen-d/sample size statistics were used to predict minimal patients needed for drug trials. RESULTS: The thickness of the GCL layer in BRAO decreased rapidly with time as in CRAO, but in more limited quadrants. The data, as fit to a single-phase decay curve, showed that GCL thickness could be used to provide sample size statistics in a clinical trial to test a calpain inhibitor. For example, a 60-day trial with a 60% effective inhibitor would need a minimum of 29 patients. CONCLUSIONS: Using thickness changes in the GCL layer to monitor the efficacy of potential inhibitors against BRAO and CRAO is practical in human trials requiring a reasonable number of patients and relatively short trial period. TRANSLATIONAL RELEVANCE: Measurement of GCL thickness would be a useful indicator of amelioration of BRAO and CRAO progression in a clinical trial of a putative inhibitor.

NFE2L2 activator RS9 protects against corneal epithelial cell damage in dry eye models.

Oxidative stress may cause ocular surface damage during the development of dry eye. Mammalian cells have defense systems against oxidative stress. A central regulator of the stress response is nuclear factor-erythroid 2-related factor 2 (NFE2L2). NFE2L2 is activated by the novel triterpenoid RS9 (a biotransformation compound of RTA 402). The purpose of this study was to assess the efficacy of RS9 against dry eye using in vitro and in vivo models. Bioactivity was estimated by the induction of mRNAs for two NFE2L2-targeted genes: NQO1 (prevents radical species) and GCLC (glutathione synthesis), using a corneal epithelial cell line (HCE-T). Protection against oxidation and cell damage was tested in vitro by culturing cells under hyperosmotic stress or by the addition of menadione, a generator of reactive oxygen species (ROS). Dry eye in vivo was induced by the injection of scopolamine into rats. Then, 930 nM of RS9 was applied to both eyes for 2 weeks. Oxidative stress was measured by the accumulation of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Corneal wound healing was measured by scoring for superficial punctate keratitis (SPK). Corneal epithelial cell densities were evaluated histologically. RS9 and RTA 402 induced the expression of NQO1 and GCLC mRNAs in HCE-T cells. And both compounds suppressed hyperosmotic-ROS generation and menadione induced cellular damage. However RS9 had a stronger protective effect than RTA 402. Ocular instillation of RS9 also significantly upregulated the expression of Nqo1 mRNA in the corneal epithelium. Accumulation of 8-OHdG, increase of SPK scores and decrement of basal cell density were observed in corneal epithelium from scopolamine-injected rats. These changes were significantly ameliorated by the topical administration of RS9. RS9 induced Nfe2l2 activation and Nfe2l2-targeted genes, reduced oxidation, and ameliorated symptoms of dry eye using in vitro and in vivo models. Thus, RS9 might be a potent candidate agent against dry eye disease.

Segmenting OCT for detecting drug efficacy in CRAO.

PURPOSE: Thinning of the inner layers of the retina occurs in patients with central retinal artery occlusion (CRAO). The mechanism for such thinning may be partially due to proteolysis by a calcium-activated protease called calpain. Calpain inhibitor SNJ-1945 ameliorated the proteolysis in a past series of model experiments. The purposes of the present retrospective study were to: 1) use segmentation analysis of optical coherence tomography (OCT) images to mathematically model the loss of specific retinal layers in CRAO patients, and 2) predict the number of patients and days of observation needed for clinical trials of inhibitors against CRAO. METHODS: A retrospective case control study was conducted by computer-aided search for CRAO (ICD10 H43.1) with at least one OCT procedure (CPT: 92134) in the OHSU Epic patient data base. RESULTS: After initial swelling, thinning of the inner retinal layers, especially the ganglion cell (GCL) layer followed exponential decay curves. Using sample size statistics and GCL thickness as a marker in a 30-day clinical trial, 19 eyes/group could theoretically detect a 20% beneficial effect of an inhibitor against CRAO. Other markers, such as the whole retinal thickness and combined inner layers could also be used as less-specific markers. CONCLUSIONS: Using thickness changes in the GCL layer to monitor the efficacy of potential inhibitors against CRAO is practical in human trials requiring a reasonable number of patients and relatively short trial period. TRANSLATIONAL RELEVANCE: Measurement of GCL thickness would be a useful indicator of CRAO progression in a clinical trial of putative inhibitors.

Activation of Cytosolic Calpain, Not Caspase, Is Underlying Mechanism for Hypoxic RGC Damage in Human Retinal Explants.

Purpose: Activation of proteolytic enzymes, calpains and caspases, have been observed in many models of retinal disease. We previously demonstrated calpain activation in monkey retinal explants cultured under hypoxia. However, cellular responses are often species-specific. The purpose of the present study was to determine whether calpains or caspase-3 was involved in retinal ganglion cell (RGC) damage caused by hypoxia/reoxygenation in human retinal explants. The explant model was improved by use of an oxygen-controlled chamber. Methods: Human and monkey retinal explants were cultured under hypoxic conditions in an oxygen-controlled chamber and then reoxygenated. Calpain inhibitor SNJ-1945 was maintained throughout the culture period. Immunohistochemistry and immunoblotting were performed for calpains 1 and 2, calpastatin, α-spectrin, calpain-specific α-spectrin breakdown product at 150 kDa (SBDP150), caspase-3, and apoptosis-inducing factor (AIF). Propidium iodide (PI) staining measured membrane disruption, and TUNEL staining detected DNA fragmentation. Results: Activation of calpains in nerve fibers and increases of PI-positive RGCs were observed in retinal explants incubated for 16-hour hypoxia/8-hour reoxygenation. Except for autolysis of calpain 2, SNJ-1945 ameliorated these changes. In longer incubations under 24-hour hypoxia/16-hour reoxygenation, TUNEL-positive cells appeared, although activated caspase-3 and truncated AIF were not observed. DNA fragmentation was inhibited by SNJ-1945. Conclusions: An improved human retinal explant model showed that calpains, not caspase-3, were involved in cell damage induced by hypoxia/reoxygenation. This finding could be relevant for patient treatment with a calpain inhibitor if calpain activation is documented in human retinal ischemic diseases.

Cell-Permeable Calpain Inhibitor SJA6017 Provides Functional Protection to Spinal Motoneurons Exposed to MPP.

Extra-nigral central nervous system sites have been found to be affected in Parkinson's disease (PD). In addition to substantia nigra, degeneration of spinal cord motor neurons may play a role in the motor symptoms of PD. To this end, hybrid rodent VSC 4.1 cells differentiated into motoneurons were used as a cell culture model following exposure to Parkinsonian neurotoxicant MPP+. SJA6017, a cell-permeable calpain inhibitor, was tested for its neuroprotective efficacy against the neurotoxicant. SJA6017 attenuated MPP+-induced rise in intracellular free Ca2+ and concomitant increases in the active form of calpain. It also significantly prevented increased levels of proteases and their activities, as shown by reduced levels of 145 kDa calpain-specific and 120 kDa caspase-3-specific spectrin breakdown products. Exposure to MPP+ elevated the levels of reactive oxygen species in VSC 4.1 motoneurons; this was significantly diminished with SJA6017. The motor proteins in spinal motoneurons, i.e., dynein and kinesin, were also impaired following exposure to MPP+ through calpain-mediated mechanisms; this process was partially ameliorated by SJA6017 pretreatment. Cytoprotection provided by SJA6017 against MPP+-induced damage to VSC 4.1 motoneurons was confirmed by restoration of membrane potential via whole-cell patch-clamp assay. This study demonstrates that calpain inhibition is a prospective route for neuroprotection in experimental PD; moreover, calpain inhibitor SJA6017 appears to be an effective neuroprotective agent against MPP+-induced damage in spinal motoneurons.

Inhibition of calpain attenuates encephalitogenicity of MBP-specific T cells.

Multiple sclerosis (MS) is a T-cell mediated autoimmune disease of the CNS, possessing both immune and neurodegenerative events that lead to disability. Adoptive transfer (AT) of myelin basic protein (MBP)-specific T cells into naïve female SJL/J mice results in a relapsing-remitting (RR) form of experimental autoimmune encephalomyelitis (EAE). Blocking the mechanisms by which MBP-specific T cells are activated before AT may help characterize the immune arm of MS and offer novel targets for therapy. One such target is calpain, which is involved in activation of T cells, migration of immune cells into the CNS, degradation of axonal and myelin proteins, and neuronal apoptosis. Thus, the hypothesis that inhibiting calpain in MBP-specific T cells would diminish their encephalitogenicity in RR-EAE mice was tested. Incubating MBP-specific T cells with the calpain inhibitor SJA6017 before AT markedly suppressed the ability of these T cells to induce clinical symptoms of RR-EAE. These reductions correlated with decreases in demyelination, inflammation, axonal damage, and loss of oligodendrocytes and neurons. Also, calpain : calpastatin ratio, production of truncated Bid, and Bax : Bcl-2 ratio, and activities of calpain and caspases, and internucleosomal DNA fragmentation were attenuated. Thus, these data suggest calpain as a promising target for treating EAE and MS.

PACAP induces neurite outgrowth in cultured trigeminal ganglion cells and recovery of corneal sensitivity after flap surgery in rabbits.

PURPOSE: To evaluate the ability of pituitary adenylate cyclase-activating polypeptide (PACAP) to induce growth of neuronal processes in cultured trigeminal ganglion cells, and to accelerate neurite outgrowth and recovery of corneal sensitivity after creation of a corneal flap in a rabbit model of laser-assisted in situ keratomileusis (LASIK) surgery. DESIGN: Animal study. METHODS: The cDNA of rabbit PACAP was sequenced, and the expression of PACAP receptors in the trigeminal ganglia from rabbits was quantified by quantitative real-time polymerase chain reaction. Trigeminal ganglion cells were isolated from rabbits and cultured for 48 hours with or without PACAP27 (bioactive N-terminal peptide from PACAP). Cells were stained with antibody against neurofilaments, and neurite outgrowth was quantified by cell counting. In the rabbit LASIK model, a corneal flap with a planned thickness of 130 microm and 8.5 mm diameter was created with a microkeratome. The rabbits then received eyedrops containing PACAP27 four times a day for eight weeks, and corneal sensitivity was measured. Neurite outgrowth was assessed by staining histologic sections of the flap area for cholinesterase. RESULTS: The deduced amino acid sequence of PACAP in rabbit was identical to that of human. PACAP receptor, PAC1, was highly expressed in trigeminal ganglia from newborn and adult rabbits. PACAP27 at 1 microM induced growth of neuronal processes in cultured primary trigeminal ganglion cells. In the LASIK model, extensions of neuronal processes from amputated nerve trunks in cornea were observed after administration of eyedrops containing 1 or 10 microM PACAP27. The 10 microM PACAP27 treatment also greatly accelerated recovery of corneal sensitivity. CONCLUSIONS: PACAP may be a candidate drug for ameliorating dry eye after LASIK surgery.

Contribution of calpain to cellular damage in human retinal pigment epithelial cells cultured with zinc chelator.

PURPOSE: We previously showed involvement of calpains in neural retina degeneration induced by hypoxia and ischemia-reperfusion. Age-related macular degeneration (AMD) is one of the leading causes for loss of vision. AMD showed degeneration of neural retina due to dysfunction and degeneration of the retinal pigment epithelium (RPE). RPE performs critical functions in neural retina, such as phagocytosis of shed rod outer segments. The purpose of the current study was to determine the contribution of calpain-induced proteolysis to damage in cultured human RPE cells. Zinc chelator TPEN was used to induce cellular damage as zinc deficiency is a suspected risk factor for AMD. METHODS: In RPE/choroid preparations from normal and AMD patients, calpain mRNAs were measured by qPCR, and calpain activity was assessed by casein zymography. Third- to fifth-passage cells from human RPE cells were cultured with TPEN. Cell damage was morphologically assessed under the phase-contrast microscope, and TUNEL staining was performed to detect apoptosis. Leakage of lactate dehydrogenase (LDH) into the medium was measured as a marker of RPE cell damage. Activation of calpains and proteolysis of the known calpain substrate alpha -spectrin were assessed by immunoblotting. To further confirm calpain-induced proteolysis, calpain in homogenized RPE was also activated directly by addition of calcium. RESULTS: RPE/choroid from normal patients expressed mRNAs for calpain 1, calpain 2, and calpastatin moderately, and calpain 2 activity tended to be lower in AMD patients. TPEN caused RPE cell damage with positive TUNEL staining. TPEN also caused leakage of LDH into the medium from RPE cells, and calpain inhibitor SJA6017 inhibited the leakage. Caspase-3 inhibitors z-VAD and z-DEVD also showed inhibitory effects. Immunoblotting for calpain and alpha -spectrin showed activation of calpain in RPE cells cultured with TPEN. Proteolysis by activated calpain was confirmed by addition of calcium to homogenized RPE. CONCLUSIONS: These results suggested that activation of calpain contributed to cellular damage induced by TPEN in cultured human RPE cells.