Screening en in vivo validatie van bioactieve peptiden op axonale uitgroei en regeneratie.

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 molecules that 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 to show that the observed diminished outgrowth in MMP-2 deficient explants can be rescued by administration 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 to study 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.