MMP-3 deficiency alleviates endotoxin-induced acute inflammation in the posterior eye segment

Matrix metalloproteinase-3 (MMP-3) is known to mediate neuroinflammatory processes by activating microglia, disrupting blood-central nervous system barriers and supporting neutrophil influx into the brain. In addition, the posterior part of the eye, more specifically the retina, the retinal pigment epithelium (RPE) and the blood-retinal barrier, is affected upon neuroinflammation, but a role for MMP-3 during ocular inflammation remains elusive. We investigated whether MMP-3 contributes to acute inflammation in the eye using the endotoxin-induced uveitis (EIU) model. Systemic administration of lipopolysaccharide induced an increase in MMP-3 mRNA and protein expression level in the posterior part of the eye. MMP-3 deficiency or knockdown suppressed retinal leukocyte adhesion and leukocyte infiltration into the vitreous cavity in mice subjected to EIU. Moreover, retinal and RPE mRNA levels of intercellular adhesion molecule 1 (Icam1), interleukin 6 (Il6), cytokine-inducible nitrogen oxide synthase (Nos2) and tumor necrosis factor alpha (Tnf alpha), which are key molecules involved in EIU, were clearly reduced in MMP-3 deficient mice. In addition, loss of MMP-3 repressed the upregulation of the chemokines monocyte chemoattractant protein (MCP)-1 and (C-X-C motif) ligand 1 (CXCL1). These findings suggest a contribution of MMP-3 during EIU, and its potential use as a therapeutic drug target in reducing ocular inflammation

Selective PDE4 subtype inhibition provides new opportunities to intervene in neuroinflammatory versus myelin damaging hallmarks of multiple sclerosis.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterized by focal inflammatory lesions and prominent demyelination. Even though the currently available therapies are effective in treating the initial stages of disease, they are unable to halt or reverse disease progression into the chronic progressive stage. Thus far, no repair-inducing treatments are available for progressive MS patients. Hence, there is an urgent need for the development of new therapeutic strategies either targeting the destructive immunological demyelination or boosting endogenous repair mechanisms. Using in vitro, ex vivo, and in vivo models, we demonstrate that selective inhibition of phosphodiesterase 4 (PDE4), a family of enzymes that hydrolyzes and inactivates cyclic adenosine monophosphate (cAMP), reduces inflammation and promotes myelin repair. More specifically, we segregated the myelination-promoting and anti-inflammatory effects into a PDE4D- and PDE4B-dependent process respectively. We show that inhibition of PDE4D boosts oligodendrocyte progenitor cells (OPC) differentiation and enhances (re)myelination of both murine OPCs and human iPSC-derived OPCs. In addition, PDE4D inhibition promotes in vivo remyelination in the cuprizone model, which is accompanied by improved spatial memory and reduced visual evoked potential latency times. We further identified that PDE4B-specific inhibition exerts anti-inflammatory effects since it lowers in vitro monocytic nitric oxide (NO) production and improves in vivo neurological scores during the early phase of experimental autoimmune encephalomyelitis (EAE). In contrast to the pan PDE4 inhibitor roflumilast, the therapeutic dose of both the PDE4B-specific inhibitor A33 and the PDE4D-specific inhibitor Gebr32a did not trigger emesis-like side effects in rodents. Finally, we report distinct PDE4D isoform expression patterns in human area postrema neurons and human oligodendroglia lineage cells. Using the CRISPR-Cas9 system, we confirmed that pde4d1/2 and pde4d6 are the key targets to induce OPC differentiation. Collectively, these data demonstrate that gene specific PDE4 inhibitors have potential as novel therapeutic agents for targeting the distinct disease processes of MS

ROCK inhibition as a novel potential strategy for axonal regeneration in optic neuropathies

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Mitochondrial dysfunction underlying retinal diseases

Dysfunction of photoreceptors, retinal pigment epithelium (RPE) or both contribute to the initiation and progression of several outer retinal disorders. Disrupted Müller glia function might additionally subsidize to these diseases. Mitochondrial malfunctioning is importantly associated with outer retina pathologies, which can be classified as primary and secondary mitochondrial disorders. This review highlights the importance of oxidative stress and mitochondrial DNA damage, underlying outer retinal disorders. Indeed, the metabolically active photoreceptors/RPE are highly prone to these hallmarks of mitochondrial dysfunction, indicating that mitochondria represent a weak link in the antioxidant defenses of outer retinal cells.status: publishe

Inflammation as modulator of axonal regeneration: resolving the myeloid-neuroglial crosstalk

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Differential visual system organization and susceptibility to experimental models of optic neuropathies in three commonly used mouse strains

Mouse disease models have proven indispensable in glaucoma research, yet the complexity of the vast number of models and mouse strains has also led to confusing findings. In this study, we evaluated baseline intraocular pressure, retinal histology, and retinofugal projections in three mouse strains commonly used in glaucoma research, i.e. C57Bl/6, C57Bl/6-Tyr(c), and CD-1 mice. We found that the mouse strains under study do not only display moderate variations in their intraocular pressure, retinal architecture, and retinal ganglion cell density, also the retinofugal projections to the dorsal lateral geniculate nucleus and the superior colliculus revealed striking differences, potentially underlying diverging optokinetic tracking responses and visual acuity. Next, we reviewed the success rate of three models of (glaucomatous) optic neuropathies (intravitreal N-methyl-d-aspartic acid injection, optic nerve crush, and laser photocoagulation-induced ocular hypertension), looking for differences in disease susceptibility between these mouse strains. Different genetic backgrounds and albinism led to differential susceptibility to experimentally induced retinal ganglion cell death among these three mouse strains. Overall, CD-1 mice appeared to have the highest sensitivity to retinal ganglion cell damage, while the C57Bl/6 background was more resistant in the three models used.publisher: Elsevier articletitle: Differential visual system organization and susceptibility to experimental models of optic neuropathies in three commonly used mouse strains journaltitle: Experimental Eye Research articlelink: http://dx.doi.org/10.1016/j.exer.2016.01.006 content_type: article copyright: © 2016 Elsevier Ltd. All rights reserved.status: publishe

PDGF as an Important Initiator for Neurite Outgrowth Associated with Fibrovascular Membranes in Proliferative Diabetic Retinopathy

Purpose: The formation of fibrovascular membranes (FVMs) is a serious sight-threatening complication of proliferative diabetic retinopathy (PDR) that may result in retinal detachment and eventual blindness. During the formation of these membranes, neurite/process outgrowth occurs in retinal neurons and glial cells, which may both serve as a scaffold and have guiding or regulatory roles. To further understand this process, we investigated whether previously identified candidate proteins, from vitreous of PDR patients with FVMs, could induce neurite outgrowth in an experimental setting. Materials and methods: Retinal explants of C57BL6/N mouse pups on postnatal day 3 (P3) were cultured in poly-L-lysine- and laminin-coated dishes. Outgrowth stimulation experiments were performed with the addition of potential inducers of neurite outgrowth. Automated analysis of neurite outgrowth was performed by measuring β–tubulin-immunopositive neurites using Image J. Expression of PDGF receptors was quantified by RT-PCR in FVMs of PDR patients. Results: Platelet-derived growth factor (PDGF) induced neurite outgrowth in a concentration-dependent manner, whilst neuregulin 1 (NRG1) and connective tissue growth factor (CTGF) did not. When comparing three different PDGF dimers, treatment with PDGF-AB resulted in the highest neurite induction, followed by PDGF-AA and -BB. In addition, incubation of retinal explants with vitreous from PDR patients resulted in a significant induction of neurite outgrowth as compared to non-diabetic control vitreous from patients with macular holes, which could be prevented by addition of CP673451, a potent PDGF receptor (PDGFR) inhibitor. Abundant expression of PDGF receptors was detected in FVMs. Conclusion: Our findings suggest that PDGF may be involved in the retinal neurite outgrowth, which is associated with the formation of FVMs in PDR

AMA0428, a novel and potent ROCK inhibitor, as a neuroprotective and regenerative therapy for glaucoma

Despite successful IOP lowering therapies, developing alternative treatment strategies is still necessary, to prevent/repair glaucomatous damage to the retina and optic nerve. Neuroprotective approaches are warranted to protect RGCs and halt further axonal degeneration. In addition, strategies that stimulate injured RGCs to regenerate are necessary to repair structural and functional connectivity. Here, the potential role of Rho-associated kinase (ROCK) inhibitors was elucidated by using both ex vivo and in vivo models. More in detail, AMA0428, a novel and potent ROCK inhibitor, developed by Amakem Therapeutics, was investigated in comparison to commercially available ROCK inhibitors. The ex vivo neuroprotective effect of ROCK inhibitors was evaluated by using adult mouse retinal explants. The potential neuroprotective effect of AMA0428 was also studied in vivo, using the optic nerve crush (ONC) model. Second, the regenerative potential of the ROCK inhibitors was investigated ex vivo on postnatal day 3 (P3) retinal explants. Finally, experiments are ongoing to assess the regeneration-promoting effect of AMA0428 in vivo. Adult explants, treated with AMA0428, showed enhanced RGC survival compared to vehicle-treated explants. In contrast, preliminary in vivo data indicated no neuroprotective potential of AMA0428. Administration of AMA0428 to P3 retinal explants showed a significant induction of neurite outgrowth. Strikingly, even when dosed ten times lower, AMA0428 was more potent than the reference ROCK inhibitors. Evaluation of AMA0428 via ex vivo approaches, suggests that this novel ROCK inhibitor holds very promising neuroprotective and regeneration potential. Additional in vivo experiments are currently being performed to confirm these exciting ex vivo results.status: publishe