Drug combination of topical ripasudil and brimonidine enhances neuroprotection in a mouse model of optic nerve injury.

PURPOSE: To determine whether combination of topical ripasudil and brimonidine has more effective neuroprotection on retinal ganglion cells (RGCs) following injury to axons composing the optic nerve. METHODS: Topical ripasudil, brimonidine, or mixture of both drugs were administered to adult mice after optic nerve injury (ONI). The influence of drug conditions on RGC health were evaluated by the quantifications of surviving RGCs, phosphorylated p38 mitogen-activated protein kinase (phospho-p38), and expressions of trophic factors and proinflammatory mediators in the retina. RESULTS: Topical ripasudil and brimonidine suppressed ONI-induced RGC death respectively, and mixture of both drugs further stimulated RGC survival. Topical ripasudil and brimonidine suppressed ONI-induced phospho-p38 in the whole retina. In addition, topical ripasudil suppressed expression levels of TNFα, IL-1ß and monocyte chemotactic protein-1 (MCP-1), whereas topical brimonidine increased the expression level of basic fibroblast growth factor (bFGF). CONCLUSIONS: Combination of topical ripasudil and brimonidine may enhance RGC protection by modulating multiple signaling pathways in the retina.

Neuroprotection and axon regeneration by novel low-molecular-weight compounds through the modification of DOCK3 conformation.

Dedicator of cytokinesis 3 (DOCK3) is an atypical member of the guanine nucleotide exchange factors (GEFs) and plays important roles in neurite outgrowth. DOCK3 forms a complex with Engulfment and cell motility protein 1 (Elmo1) and effectively activates Rac1 and actin dynamics. In this study, we screened 462,169 low-molecular-weight compounds and identified the hit compounds that stimulate the interaction between DOCK3 and Elmo1, and neurite outgrowth in vitro. Some of the derivatives from the hit compound stimulated neuroprotection and axon regeneration in a mouse model of optic nerve injury. Our findings suggest that the low-molecular-weight DOCK3 activators could be a potential therapeutic candidate for treating axonal injury and neurodegenerative diseases including glaucoma.

Effects of constitutively active K-Ras on axon regeneration after optic nerve injury.

Visual disturbance after optic nerve injury is a serious problem. Attempts have been made to enhance the intrinsic ability of retinal ganglion cells (RGCs) to regenerate their axons, and the importance of PI3K/Akt and RAF/MEK/ERK signal activation has been suggested. Since these signals are shared with oncogenic signaling cascades, in this study, we focused on a constitutively active form of K-Ras, K-RasV12, to determine if overexpression of this molecule could stimulate axon regeneration. We confirmed that K-RasV12 phosphorylated Akt and ERK in vitro. Intravitreal delivery of AAV2-K-RasV12 increased the number of surviving RGCs and promoted 1.0 mm of axon regeneration one week after optic nerve injury without inducing abnormal proliferative effects in the RGCs. In addition, AAV2-K-RasV12 induced robust RGC axon regeneration, reaching as far as approximately 2.5 mm from the injury site, in eight weeks. Our findings suggest that AAV2-K-RasV12 could provide a good model for speedy and efficient analysis of the mechanism underlying axon regeneration in vivo.

Vision protection and robust axon regeneration in glaucoma models by membrane-associated Trk receptors.

Activation of neurotrophic factor signaling is a promising therapy for neurodegeneration. However, the transient nature of ligand-dependent activation limits its effectiveness. In this study, we solved this problem by inventing a system that forces membrane localization of the intracellular domain of tropomyosin receptor kinase B (iTrkB), which results in constitutive activation without ligands. Our system overcomes the small size limitation of the genome packaging in adeno-associated virus (AAV) and allows high expression of the transgene. Using AAV-mediated gene therapy in the eyes, we demonstrate that iTrkB expression enhances neuroprotection in mouse models of glaucoma and stimulates robust axon regeneration after optic nerve injury. In addition, iTrkB expression in the retina was also effective in an optic tract transection model, in which the injury site is near the superior colliculus. Regenerating axons successfully formed pathways to their brain targets, resulting in partial recovery of visual behavior. Our system may also be applicable to other trophic factor signaling pathways and lead to a significant advance in the field of gene therapy for neurotrauma and neurodegenerative disorders, including glaucoma.

ASK1 signaling regulates phase-specific glial interactions during neuroinflammation.

Neuroinflammation is well known to be associated with neurodegenerative diseases. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that has been implicated in neuroinflammation, but its precise cellular and molecular mechanisms remain unknown. In this study, we generated conditional knockout (CKO) mice that lack ASK1 in T cells, dendritic cells, microglia/macrophages, microglia, or astrocytes, to assess the roles of ASK1 during experimental autoimmune encephalomyelitis (EAE). We found that neuroinflammation was reduced in both the early and later stages of EAE in microglia/macrophage-specific ASK1 knockout mice, whereas only the later-stage neuroinflammation was ameliorated in astrocyte-specific ASK1 knockout mice. ASK1 deficiency in T cells and dendritic cells had no significant effects on EAE severity. Further, we found that ASK1 in microglia/macrophages induces a proinflammatory environment, which subsequently activates astrocytes to exacerbate neuroinflammation. Microglia-specific ASK1 deletion was achieved using a CX3CR1CreER system, and we found that ASK1 signaling in microglia played a major role in generating and maintaining disease. Activated astrocytes produce key inflammatory mediators, including CCL2, that further activated and recruited microglia/macrophages, in an astrocytic ASK1-dependent manner. Astrocyte-specific analysis revealed CCL2 expression was higher in the later stage compared with the early stage, suggesting a greater proinflammatory role of astrocytes in the later stage. Our findings demonstrate cell-type-specific roles of ASK1 and suggest phase-specific ASK1-dependent glial cell interactions in EAE pathophysiology. We propose glial ASK1 as a promising therapeutic target for reducing neuroinflammation.

Effects of lighting environment on the degeneration of retinal ganglion cells in glutamate/aspartate transporter deficient mice, a mouse model of normal tension glaucoma.

Lighting conditions may affect the development of retinal degenerative diseases such as macular degeneration. In this study, to determine whether the lighting environment affects the progression of degeneration of retinal ganglion cells (RGCs), we examined glutamate/aspartate transporter (GLAST) heterozygous (GLAST+/-) mice, a mouse model of normal tension glaucoma. GLAST+/- mice were reared under a 12-h light-dark cycle (Light/Dark) or complete darkness (Dark/Dark) condition after birth. The total RGC number in the Dark/Dark group was significantly decreased compared with the Light/Dark group at 3 weeks old, while the number of osteopontin-positive αRGCs were similar in both groups. At 6 and 12 weeks old, the total RGC number were not significantly different in both conditions. In addition, the retinal function examined by multifocal electroretinogram were similar at 12 weeks old. These results suggest that lighting conditions may regulate the progression of RGC degeneration in some types of glaucoma.

Suppression of Oxidative Stress as Potential Therapeutic Approach for Normal Tension Glaucoma.

Glaucoma is a neurodegenerative disease of the eye, which involves degeneration of retinal ganglion cells (RGCs): the output neurons of the retina to the brain, which with their axons comprise the optic nerve. Recent studies have shown the possible involvement of oxidative stress in the pathogenesis of glaucoma, especially in the subtype of normal tension glaucoma. Basic experiments utilizing rodent and primate models of glaucoma revealed that antioxidants protect RGCs under various pathological conditions including glutamate neurotoxicity and optic nerve injury. These results suggested that existing drugs and food factors may be useful for prevention and hence therapy of glaucoma. In this review, we highlight some therapeutic candidates, particularly those with antioxidant properties, and discuss the therapeutic potential of RGC protection by modulating gene expressions that prevent and ameliorate glaucoma.

Topical ripasudil stimulates neuroprotection and axon regeneration in adult mice following optic nerve injury.

Optic nerve injury induces optic nerve degeneration and retinal ganglion cell (RGC) death that lead to visual disturbance. In this study, we examined if topical ripasudil has therapeutic potential in adult mice after optic nerve crush (ONC). Topical ripasudil suppressed ONC-induced phosphorylation of p38 mitogen-activated protein kinase and ameliorated RGC death. In addition, topical ripasudil significantly suppressed the phosphorylation of collapsin response mediator protein 2 and cofilin, and promoted optic nerve regeneration. These results suggest that topical ripasudil promotes RGC protection and optic nerve regeneration by modulating multiple signaling pathways associated with neural cell death, microtubule assembly and actin polymerization.

Roles of the DOCK-D family proteins in a mouse model of neuroinflammation.

The DOCK-D (dedicator of cytokinesis D) family proteins are atypical guanine nucleotide exchange factors that regulate Rho GTPase activity. The family consists of Zizimin1 (DOCK9), Zizimin2 (DOCK11), and Zizimin3 (DOCK10). Functions of the DOCK-D family proteins are presently not well-explored, and the role of the DOCK-D family in neuroinflammation is unknown. In this study, we generated three mouse lines in which DOCK9 (DOCK9-/-), DOCK10 (DOCK10-/-), or DOCK11 (DOCK11-/-) had been deleted and examined the phenotypic effects of these gene deletions in MOG35-55 peptide-induced experimental autoimmune encephalomyelitis, an animal model of the neuroinflammatory disorder multiple sclerosis. We found that all the gene knockout lines were healthy and viable. The only phenotype observed under normal conditions was a slightly smaller proportion of B cells in splenocytes in DOCK10-/- mice than in the other mouse lines. We also found that the migration ability of macrophages is impaired in DOCK10-/- and DOCK11-/- mice and that the severity of experimental autoimmune encephalomyelitis was ameliorated only in DOCK10-/- mice. No apparent phenotype was observed for DOCK9-/- mice. Further investigations indicated that lipopolysaccharide stimulation up-regulates DOCK10 expression in microglia and that microglial migration is decreased in DOCK10-/- mice. Up-regulation of C-C motif chemokine ligand 2 (CCL2) expression induced by activation of Toll-like receptor 4 or 9 signaling was reduced in DOCK10-/- astrocytes compared with WT astrocytes. Taken together, our findings suggest that DOCK10 plays a role in innate immunity and neuroinflammation and might represent a potential therapeutic target for managing multiple sclerosis.

Normal tension glaucoma-like degeneration of the visual system in aged marmosets.

The common marmoset (Callithrix jacchus) is a non-human primate that provides valuable models for neuroscience and aging research due to its anatomical similarities to humans and relatively short lifespan. This study was carried out to examine whether aged marmosets develop glaucoma, as seen in humans. We found that 11% of the aged marmosets presented with glaucoma-like characteristics; this incident rate is very similar to that in humans. Magnetic resonance imaging showed a significant volume loss in the visual cortex, and histological analyses confirmed the degeneration of the lateral geniculate nuclei and visual cortex in the affected marmosets. These marmosets did not have elevated intraocular pressure, but showed an increased oxidative stress level, low cerebrospinal fluid (CSF) pressure, and low brain-derived neurotrophic factor (BDNF) and TrkB expression in the retina, optic nerve head and CSF. Our findings suggest that marmosets have potential to provide useful information for the research of eye and the visual system.

Survival of Alpha and Intrinsically Photosensitive Retinal Ganglion Cells in NMDA-Induced Neurotoxicity and a Mouse Model of Normal Tension Glaucoma.

Purpose: We assess if α retinal ganglion cells (αRGCs) and intrinsically photosensitive retinal ganglion cells (ipRGCs) survive in mouse models of glaucoma. Methods: Two microliters of N-methyl-D-aspartate (NMDA; 1 mM) or PBS were injected intraocularly 7 days before sacrifice. Immunohistochemical analyses of the retina were performed using antibodies against RNA-binding protein with multiple splicing (RBPMS), osteopontin, and melanopsin. Immunohistochemical analyses also were performed in adult mice with glutamate/aspartate transporter (GLAST) deletion (GLAST knockout [KO] mice), a mouse model of normal tension glaucoma. Results: NMDA-induced loss of RBPMS-positive total RGCs was 58.4% ± 0.4% compared to PBS-treated controls, whereas the loss of osteopontin-positive αRGCs was 5.0% ± 0.6% and that of melanopsin-positive ipRGCs was 7.6% ± 1.6%. In GLAST KO mice, the loss of total RGCs was 48.4% ± 0.9% compared to wild-type mice, whereas the loss of αRGCs and ipRGCs was 3.9% ± 0.4% and 9.3% ± 0.5%, respectively. The distribution of survived total RGCs, αRGCs, and ipRGCs was similar regardless of the location of the retina. Conclusions: These results suggest that αRGC and ipRGC are highly tolerant to NMDA-induced neurotoxicity and NTG-like neurodegeneration in GLAST KO mice.

DOCK8 is expressed in microglia, and it regulates microglial activity during neurodegeneration in murine disease models.

Dedicator of cytokinesis 8 (DOCK8) is a guanine nucleotide exchange factor whose loss of function results in immunodeficiency, but its role in the central nervous system (CNS) has been unclear. Microglia are the resident immune cells of the CNS and are implicated in the pathogenesis of various neurodegenerative diseases, including multiple sclerosis (MS) and glaucoma, which affects the visual system. However, the exact roles of microglia in these diseases remain unknown. Herein, we report that DOCK8 is expressed in microglia but not in neurons or astrocytes and that its expression is increased during neuroinflammation. To define the role of DOCK8 in microglial activity, we focused on the retina, a tissue devoid of infiltrating T cells. The retina is divided into distinct layers, and in a disease model of MS/optic neuritis, DOCK8-deficient mice exhibited a clear reduction in microglial migration through these layers. Moreover, neuroinflammation severity, indicated by clinical scores, visual function, and retinal ganglion cell (RGC) death, was reduced in the DOCK8-deficient mice. Furthermore, using a glaucoma disease model, we observed impaired microglial phagocytosis of RGCs in DOCK8-deficient mice. Our data demonstrate that DOCK8 is expressed in microglia and regulates microglial activity in disease states. These findings contribute to a better understanding of the molecular pathways involved in microglial activation and implicate a role of DOCK8 in several neurological diseases.

Differential effects of N-acetylcysteine on retinal degeneration in two mouse models of normal tension glaucoma.

N-acetylcysteine (NAC) is widely used as a mucolytic agent and as an antidote to paracetamol overdose. NAC serves as a precursor of cysteine and stimulates the synthesis of glutathione in neural cells. Suppressing oxidative stress in the retina may be an effective therapeutic strategy for glaucoma, a chronic neurodegenerative disease of the retinal ganglion cells (RGCs) and optic nerves. Here we examined the therapeutic potential of NAC in two mouse models of normal tension glaucoma, in which excitatory amino-acid carrier 1 (EAAC1) or glutamate/aspartate transporter (GLAST) gene was deleted. EAAC1 is expressed in retinal neurons including RGCs, whereas GLAST is mainly expressed in Müller glial cells. Intraperitoneal administration of NAC prevented RGC degeneration and visual impairment in EAAC1-deficient (knockout; KO) mice, but not in GLAST KO mice. In EAAC1 KO mice, oxidative stress and autophagy were suppressed with increased glutathione levels by NAC treatment. Our findings suggest a possibility that systemic administration of NAC may be available for some types of glaucoma patients.

Recent advances in genetically modified animal models of glaucoma and their roles in drug repositioning.

Glaucoma is one of the leading causes of vision loss in the world. Currently, pharmacological intervention for glaucoma therapy is limited to eye drops that reduce intraocular pressure (IOP). Recent studies have shown that various factors as well as IOP are involved in the pathogenesis of glaucoma, especially in the subtype of normal tension glaucoma. To date, various animal models of glaucoma have been established, including glutamate/aspartate transporter knockout (KO) mice, excitatory amino acid carrier 1 KO mice, optineurin E50K knock-in mice, DBA/2J mice and experimentally induced models. These animal models are very useful for elucidating the pathogenesis of glaucoma and for identifying potential therapeutic targets. However, each model represents only some aspects of glaucoma, never the whole disease. This review will summarise the benefits and limitations of using disease models of glaucoma and recent basic research in retinal protection using existing drugs.

Role of neuritin in retinal ganglion cell death in adult mice following optic nerve injury.

Neuritin is a small extracellular protein that plays important roles in the process of neural development, synaptic plasticity, and neural cell survival. Here we investigated the function of neuritin in a mouse model of optic nerve injury (ONI). ONI induced upregulation of neuritin mRNA in the retina of WT mice. The retinal structure and the number of retinal ganglion cells (RGCs) were normal in adult neuritin knockout (KO) mice. In vivo retinal imaging and histopathological analyses demonstrated that RGC death and inner retinal degeneration following ONI were more severe in neuritin KO mice. Immunoblot analyses revealed that ONI-induced phosphorylation of Akt and ERK were suppressed in neuritin KO mice. Our findings suggest that neuritin has neuroprotective effects following ONI and may be useful for treatment of posttraumatic complication.

Topical Ripasudil Suppresses Retinal Ganglion Cell Death in a Mouse Model of Normal Tension Glaucoma.

Purpose: To assess if ripasudil has a neuroprotective effect using mice with excitatory amino acid carrier 1 (EAAC1) deletion (EAAC1 knockout [KO] mice), a mouse model of normal tension glaucoma. Methods: Topical administration (5 µL/day) of two different concentrations of ripasudil (0.4% and 2%) were applied to EAAC1 KO mice from 5 to 12 weeks old. Optical coherence tomography, multifocal electroretinograms, the measurement of intraocular pressure (IOP), and histopathology analyses were performed at 5, 8, and 12 weeks old. Retrograde labeling of retinal ganglion cells (RGCs), immunoblot, and immunohistochemical analyses of phosphorylated p38 mitogen-activated protein kinase (MAPK) in the retina were performed at 8 weeks old. Results: Topical ripasudil ameliorated retinal degeneration and improved visual function in EAAC1 KO mice at both 8 and 12 weeks old. Ripasudil reduced IOP and strongly suppressed the phosphorylation of p38 MAPK that stimulates RGC death in EAAC1 KO mice. Conclusions: These results suggest that, in addition to IOP reduction, ripasudil prevents glaucomatous retinal degeneration by neuroprotection, which is achieved by suppressing cell-death signaling pathways.

Edaravone Prevents Retinal Degeneration in Adult Mice Following Optic Nerve Injury.

Purpose: To assess the therapeutic potential of edaravone, a free radical scavenger that is used for the treatment of acute brain infarction and amyotrophic lateral sclerosis, in a mouse model of optic nerve injury (ONI). Methods: Two microliters of edaravone (7.2 mM) or vehicle were injected intraocularly 3 minutes after ONI. Optical coherence tomography, retrograde labeling of retinal ganglion cells (RGCs), histopathology, and immunohistochemical analyses of phosphorylated apoptosis signal-regulating kinase-1 (ASK1) and p38 mitogen-activated protein kinase (MAPK) in the retina were performed after ONI. Reactive oxygen species (ROS) levels were assessed with a CellROX Green Reagent. Results: Edaravone ameliorated ONI-induced ROS production, RGC death, and inner retinal degeneration. Also, activation of the ASK1-p38 MAPK pathway that induces RGC death following ONI was suppressed with edaravone treatment. Conclusions: The results of this study suggest that intraocular administration of edaravone may be a useful treatment for posttraumatic complications.

ASK1 in neurodegeneration.

Neurodegenerative diseases (NDDs) such as glaucoma, multiple sclerosis (MS), Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD) are characterized by the progressive loss of neurons, causing irreversible damage to patients. Longer lifespans may be leading to an increase in the number of people affected by NDDs worldwide. Among the pathways strongly impacting the pathogenesis of NDDs, oxidative stress, a condition that occurs because of an imbalance in oxidant and antioxidant levels, has been known to play a vital role in the pathophysiology of NDDs. One of the molecules activated by oxidative stress is apoptosis signal-regulating kinase 1 (ASK1), which has been shown to play a role in NDDs. ASK1 activation is regulated by multiple steps, including oligomerization, phosphorylation, and protein-protein interactions. In the oxidative stress state, reactive oxygen species (ROS) induce the dissociation of thioredoxin, a protein regulating cellular reduction and oxidation (redox), from the N-terminal region of ASK1, and ASK1 is subsequently activated by the oligomerization and phosphorylation of a critical threonine residue, leading to cell death. Here, we review experimental evidence that links ASK1 signaling with the pathogenesis of several NDDs. We propose that ASK1 may be a new point of therapeutic intervention to prevent or treat NDDs.

The Renin-Angiotensin System Regulates Neurodegeneration in a Mouse Model of Optic Neuritis.

The major role of the renin-angiotensin system (RAS), including that of angiotensin II (Ang II), the principal effector molecule, in the cardiovascular system is well known. Increasing evidence suggests that the RAS also plays a role in the development of autoimmune diseases. Optic neuritis (ie, inflammation of the optic nerve, with retinal ganglion cell loss) is strongly associated with multiple sclerosis. We investigated the effects of candesartan, an Ang II receptor antagonist, on optic neuritis in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. The Ang II concentration was increased in the early phase of EAE. Oral administration of candesartan markedly attenuated demyelination of the optic nerve and spinal cord and reduced retinal ganglion cell loss and visual impairment in mice with EAE. In vitro analyses revealed that Ang II up-regulated the expression of Toll-like receptor (TLR)-4 in astrocytes via the NF-κB pathway. In addition, Ang II treatment enhanced lipopolysaccharide-induced production of monocyte chemoattractant protein 1 in astrocytes, and pretreatment with candesartan or SN50, an NF-κB inhibitor, suppressed the effects of Ang II. The novel pathway of RAS-NF-κB-TLR4 in glial cells identified in the present study may be a valid therapeutic target for neurodegeneration in neuroinflammatory diseases.

Edaravone suppresses retinal ganglion cell death in a mouse model of normal tension glaucoma.

Glaucoma, one of the leading causes of irreversible blindness, is characterized by progressive degeneration of optic nerves and retinal ganglion cells (RGCs). In the mammalian retina, excitatory amino-acid carrier 1 (EAAC1) is expressed in neural cells, including RGCs. Loss of EAAC1 leads to RGC degeneration without elevated intraocular pressure (IOP) and exhibits glaucomatous pathology including glutamate neurotoxicity and oxidative stress. In the present study, we found that edaravone, a free radical scavenger that is used for treatment of acute brain infarction and amyotrophic lateral sclerosis (ALS), reduces oxidative stress and prevents RGC death and thinning of the inner retinal layer in EAAC1-deficient (KO) mice. In addition, in vivo electrophysiological analyses demonstrated that visual impairment in EAAC1 KO mice was ameliorated with edaravone treatment, clearly establishing that edaravone beneficially affects both histological and functional aspects of the glaucomatous retina. Our findings raise intriguing possibilities for the management of glaucoma by utilizing a widely prescribed drug for the treatment of acute brain infarction and ALS, edaravone, in combination with conventional treatments to lower IOP.