Peptides and proteases as modulators of neurite outgrowth: insights from ex vivo and in vivo mouse models studying axonal regeneration of retinal ganglion cells

Due to the increasing life expectancy, CNS injury and neurodegenerative diseases, such as Alzheimer's and Parkinson’s disease, age-related macular degeneration and glaucoma, are more prevalent than ever before. The accompanying CNS damage leads to a severe deterioration of life quality. Current pharmacological therapies are able to attenuate the disease progression and reduce the symptoms, however restoration of neuronal function remains largely elusive, not least due to the lack of regenerative capacity in the adult mammalian CNS. A deeper understanding of underlying processes and identification of moleculesnbsp;are involved in CNS regeneration will lead the way to novel therapies to restore these CNS pathologies and improve the quality of life for many patients. Efforts to identify novel compounds for regenerative treatments are limited by a gap between initial results from high-throughput in vitro screening models and their (pre-)clinical validation. In contrast to cells, tissue explant cultures closely resemble the in vivo situation, making them ideal to study the effects of molecules in a neuro-glial network. We therefore developed a retinal explant model from neonatal mice to determine the effect of compounds on neurite outgrowth. During the development of this model, an automated analysis of neurite outgrowth was established and neurite outgrowth was shown to be independent from the region within the retina that the explant was originating from. In order to validate our ex vivo findings, an in vivo mouse model was established to study axonal regeneration of retinal ganglion cells after optic nerve injury. Within this regenerative optic nerve crush (RONC) model, microscopic visualization of regenerating axons was optimized. In search for new modulators of axonal outgrowth, we used the ex vivo retinal explant model to screen a peptide library, developed by the Functional Genomics and Proteomics research group. From the 26 peptides tested, we identified 3 axon-outgrowth stimulating and 3 inhibiting peptides. In a second phase, the most promising peptide, P318, was further characterized to unravel its mode of action. Our first findings suggest that P318 stimulates neurite outgrowth via glial cells Despite its ability to stimulate neurite outgrowth, we also found that administration of the peptide induces cell death in retinal explants. Further research is required to unravel the different underlying mechanisms. In a parallel study, we used the retinal explant model to study the effect of matrix metalloproteinases (MMPs) on neurite outgrowth. MMPs are zinc dependent endoproteinases that cleave extracellular matrix proteins and other signaling molecules. Previous studies of our lab have shown that both MMP-2 and MT1-MMP deficiency results in an impaired neurite outgrowth in neonatal mouse retinal explants. Using the retinal explant model, we were now able tonbsp;that the observed diminished outgrowth in MMP-2 deficient explants can be rescued bynbsp;of exogenous MMP-2. Furthermore, our studies revealed that MT1-MMP is implicated in neurite outgrowth via the activation of pro-MMP-2. Finally, we investigated the role of MMP-2 during axonal regeneration in the in vivo RONC model. In this pilot study, a reduced axonal regeneration was observed in MMP-2 deficient mice. Our preliminary results confirm the ex vivo data, namely that MMP-2 is involved in axonal regeneration. Within this study, the ex vivo retinal explant and in vivo RONC model were successfully established to study axonal outgrowth and regeneration. These models were used tonbsp;the role of the bio-active peptide P318 and of MMP-2/MT1-MMP during neurite outgrowth and/or axonal regeneration. These results could form a first step in the development of novel regenerative therapies in the adult CNS.status: publishe

Automated Analysis of Neurite Outgrowth in Mouse Retinal Explants

Despite intensive research efforts over the past years, regeneration of injured axons in the central nervous system remains elusive. In the quest for neurostimulatory agents that promote regeneration, well-defined models and analysis methods are required. Tissue explant cultures closely resemble the in vivo situation, making them ideal to study the effect of compounds on the neuro-glial network. This study reports the optimization of an explant culture technique using retinas of neonatal mice and the development of an analysis script that allows for rapid and automated analysis of neurite outgrowth from these explants. The key features of this script (i.e., local thresholding and form selection) allow for swift and unbiased detection of neurite outgrowth. The novel analysis method is compared with two commonly used manual methods and successfully validated by performing dose-response studies with molecules known to either inhibit (anti-β1-integrin antibody) or stimulate (brain-derived neurotrophic factor and ciliary neurotrophic factor) neurite outgrowth from retinal explants. Finally, the new analysis script is used to study whether retinal explant origin has any effect on neurite outgrowth.status: publishe

Quantitative assessment of neurite outgrowth in mouse retinal explants

Despite intensive research efforts over the past years, regeneration of injured axons in the central nervous system (CNS) remains elusive. The discovery of novel neuro-stimulatory agents that promote regeneration is hampered by a gap between high content analysis platforms using neuronal cells and time-consuming preclinical animal models. In this regard, tissue explant cultures, which are easily manageable and more closely resemble the in vivo situation, form an ideal model system to study the effect of compounds on the neuroglial network. Retinal explants have proven to be a useful tool to investigate the effect of molecules on neuronal survival and regeneration. In this chapter, we report a detailed description of how to isolate and culture retinal explants and how to immunolabel the outgrowing neurites. Furthermore, we describe different analysis tools, both manual and automated, to quantify neurite outgrowth from retinal explants.status: publishe

MMP-2 positively affects axonal regeneration of retinal ganglion cells upon optic nerve crush in adult mice

Purpose: We investigated the involvement of matrix metalloproteinase-2 (MMP-2) in axonal regeneration of retinal ganglion cells (RGC) after optic nerve (ON) injury. Methods: MMP-2 deficient mice and their corresponding WT were subjected to an experimentally induced optic nerve crush (ONC) characterized by partial regeneration of RGC axons (RONC model) via intravitreal injection of Zymosan. RGC axonal regeneration was evaluated by anterograde labeling with CTB on ON cryosections at 14 and 42 dpi. RGC survival was determined by Brn3a IHC on whole mount retinas. Retinal and ON MMP-2 expression was evaluated at different time points after RONC. Leukocyte infiltration after RONC was determinated by IHC for CD45. Results: Axon outgrowth was significantly reduced near the crush site in MMP-2 deficient as compared to WT mice, both at 14 and 42 dpi. Also the number of regenerating axons was dramatically reduced at various distances from the crush site upon MMP-2 deficiency. No difference in the number of Brn3a+ surviving RGCs was observed between both genotypes at 14 dpi. Moreover MMP-2 expression was highly increased in the processes of Müller glia and in the ON at 7 and 14 dpi respectively. Strikingly MMP-2 was found to be expressed in CD-45(+) infiltrating cells in the vitreous after RONC. Conclusions: MMP-2 expressed by infiltrating cells could contribute to the induction of axonal regeneration in the RONC model. MMP-2 seems to play a beneficial role in RGC axonal regeneration after ONC without affecting RGC survival. Our data suggest a pleotropic role for MMP-2 in RGC regeneration.status: publishe

An aberrant cerebellar development in mice lacking matrix metalloproteinase-3

Cell-cell and cell-matrix interactions are necessary for neuronal patterning and brain wiring during development. Matrix metalloproteinases (MMPs) are proteolytic enzymes capable of remodelling the pericellular environment and regulating signaling pathways through cleavage of a large degradome. MMPs have been suggested to affect cerebellar development, but the specific role of different MMPs in cerebellar morphogenesis remains unclear. Here, we report a role for MMP-3 in the histogenesis of the mouse cerebellar cortex. MMP-3 expression peaks during the second week of postnatal cerebellar development and is most prominently observed in Purkinje cells (PCs). In MMP-3 deficient (MMP-3(-/-)) mice, a protracted granule cell (GC) tangential migration and a delayed GC radial migration results in a thicker and persistent external granular layer, a retarded arrival of GCs in the inner granular layer, and a delayed GABAergic interneuron migration. Importantly, these neuronal migration anomalies, as well as the consequent disturbed synaptogenesis on PCs, seem to be caused by an abnormal PC dendritogenesis, which results in reduced PC dendritic trees in the adult cerebellum. Of note, these developmental and adult cerebellar defects might contribute to the aberrant motor phenotype observed in MMP-3(-/-) mice and suggest an involvement of MMP-3 in mouse cerebellar development.status: publishe

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