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Axonal regeneration in the central nervous system (CNS) is an extremely complex process which, although possible in principle, does not take place under natural conditions. Axotomized retinal ganglion cells (RGCs) from adult rats normally undergo a progressive degeneration process, but regenerate partially under experimental conditions following a conditioned pre-treatment. Therefore the aim of neuroscience research is to delay neuronal degeneration and stimulate axonal regeneration by targeted modulation or application of growth-promoting substances. The aim of this study was to examine regeneration-associated differences in protein expression in order to unravel the molecular mechanisms of degeneration and to develop strategies that stimulate axonal regeneration.
To begin with, investigations were carried out on the previously established regeneration model of the adult rat retinal ganglion cells, which display a strong regeneration after a combined pre-treatment involving an optic nerve crush and simultaneous lens injury. Morphological, biochemical and immunohistochemical analysis showed that the lens injury mediates its regeneration-promoting effects by different signalling pathways than those following the crush injury. Previous studies postulated an activation of macrophages as mediators of regeneration, and this study showed that the neurotrophic factor bFGF and the alpha-, beta- and gamma-crystallins could be additional mediators of the regenerative effect.
In addition to the rat retina model, the monkey retina was found to be capable of sponataneous regeneration, and was established and characterised. The monkey retina exhibited a higher substrate specificity, which enabled age-dependent regeneration processes to be studied.
Biochemical and immunohistochemical studies revealed inter-species proteome variations due to axonal regeneration. Thus, both models showed an enhanced expression of a GAP-43-like protein following regeneration. Simultaneously, the enhanced inter-species up-regulation of GFAP, demonstrates that glia are involved in, but do not contradict the process of regeneration. Furthermore, the crystallins changed their expression and localization in both models, thus demonstrating their fundamental role in regeneration.
To analyse the differential protein expression the 2D-PAGE method was established and the high-resolution representation of the retinal proteome was optimized. In combination with MALDI-MS the retinal proteome of the rat and monkey was mapped. Thirty-two (rat retina) and 23 (monkey retina) proteins having a different function and localization were identified, respectively and proteome-maps of both species were generated, providing the basis for further investigations of other retinal diseases.
The proteomic analysis of regenerating and non-regenerating retinas revealed that some proteins are differentially regulated. Two of the proteins, up-regulated under non-regenerating conditions in the rat retina, were identified as synaptotagmin I and high mobility group protein 1 (HMG-1). Comparative analysis of the 2D-gels of the monkey retina showed age- and regeneration-dependent expression of some proteins, identified as calmodulin, alpha A-crystallin, fatty acid binding protein (FABP) and cellular retinoic acid binding protein (CRABP). Based on their function these proteins seem to be interesting candidates for further investigations.
This study showed that numerous factors and their corresponding signalling pathways are associated with the regeneration process. Functional testing of these candidate proteins, especially the crystallins, has to be performed in order to elucidate their role in regeneration. The generated rat and monkey proteome maps provide a basis for future, differential analysis of variable aspects. The newly established monkey retina regeneration model and the 2D-PAGE offer excellent potential to gain a deeper insight into regeneration-associated processes and to develop strategies that stimulate them.