Identification of proteins underlying a reduced regenerative potential in the ageing zebrafish.

Brain injury and neurodegenerative disorders such as Alzheimer's and Parkinson's disease, multiple sclerosis or glaucoma represent a growing social and economic problem and affect an increasing number of people in our aging society. Neurotrauma and degeneration drastically diminish life quality and lead to severe impairments, largely because the central nervous system (CNS) of adult mammals has only a limited capacity to replace or repair damaged neurons and subsequent axonal regeneration – the latter being the focus of this study.

Following CNS injury, resident microglia and infiltrating myeloid cells dominate within the pathological environment and exert diverse effects, which depend on the specific context of the injury and can be both beneficial or detrimental to the regenerative outcome. Recent innovative insights into the dichotomous role of neuroinflammation have sprouted the idea that directing and instructing the inflammatory machinery may be a better therapeutic objective than suppressing it. Yet, little is known about how inflammation contributes to successful axonal regeneration in the adult CNS, and also the molecules and pathways connecting the two processes remain largely elusive.

In sharp contrast to mammals, zebrafish retain robust regenerative capacities into adulthood, including in the CNS. Importantly, despite these apparent differences in the regenerative ability between mammals and fish, the molecular mechanisms underlying CNS regeneration show remarkable phylogenetic conservation. Hence, zebrafish form a powerful model for comparative studies that aim at the identification of novel pro-regenerative molecules. In both mammals and zebrafish, the retinofugal system is one of the most studied CNS regeneration model systems. It forms an integral part of the CNS, encompassing the neural retina, optic nerve and visual target areas in the brain, and offers the advantage of its unique accessibility and well-described morphology and function, which are highly conserved across vertebrates. Furthermore, the axons of the optic nerve originate from a single neuronal cell population, the retinal ganglion cells (RGCs).

In this PhD dissertation, we exploited the robust regenerative capacity of the zebrafish optic nerve to investigate the mechanisms that underlie successful axonal regeneration, thereby focusing on the role of acute neuroinflammation.

Firstly, we aimed at the detailed characterization of the inflammatory response upon optic nerve crush (ONC) in zebrafish. We showed that optic nerve damage induces a timed induction and resolution of inflammatory cells throughout the retinofugal system. In contrast to mammals, the inflammatory reaction is rather moderate in zebrafish, and does not become chronic. Additionally, we revealed a restricted and transient reactivation of retinal Müller glia during optic nerve regeneration.

Subsequently, we sought to investigate the contribution of acute neuroinflammation to the regenerative process, by modulating the immune response during optic nerve regeneration. In a first approach, we studied the effects of inflammatory stimulation. We showed that robust ocular inflammation in zebrafish can be achieved via intravitreal injection of the yeast cell wall extract zymosan, while the lipopeptide Pam3Cys has only a modest effect. In addition, we demonstrated that zymosan, but not Pam3Cys, elevates the retinal cytokine expression, indicative of the increased inflammatory response. More importantly, inflammatory stimulation was found to accelerate optic nerve regeneration, reminiscent of previous findings in rodent models. Systemic immunosuppression on the other hand, achieved by administration of dex to the fish’ water, significantly impeded the regenerative response upon ONC. Additionally, we aimed to obtain local immunosuppression via intravitreal injection of dexamethasone (dex) or clodronate liposomes. Although this confined treatment resulted in an efficient depletion of microglia/macrophages in the uninjured retina, we were unable to achieve continued immunosuppression after ONC. Instead, our treatment paradigm provoked inflammatory stimulation, which was confirmed by increased retinal cytokine expression. Most likely, exposure to the immunosuppressive drugs caused sensitization of microglia/macrophages, thereby triggering an exaggerated inflammatory reaction upon ONC. Accordingly, we observed an accelerated regenerative response. Taken together, our data clearly point towards a beneficial role of acute inflammation during zebrafish optic nerve regeneration.

Furthermore, we disclosed that inflammatory stimulation induces proliferative gliosis of Müller glia in the zebrafish retina, a finding that has not been described before. Our data are suggestive for crosstalk between innate immune cells and Müller glia, and it is conceivable that zebrafish Müller glia mediate an important part of the beneficial effect of inflammatory stimulation on optic nerve regeneration, similar to their mammalian counterparts. Yet, further study is needed to elucidate the underlying molecular cues and signaling pathways, as well as the respective contributions of inflammatory cells and Müller glia to the spontaneous regenerative process in teleost fish.

Altogether, this dissertation shed new light on the role of acute neuroinflammatory responses in CNS axonal regrowth, and advanced our understanding of the mechanisms that underlie successful optic nerve regeneration in zebrafish. As such, we hope that this work may generate new conceptual insights that contribute to the development of novel therapeutics that can reverse neurodegeneration in the mammalian CNS.