Periodic Reporting for period 4 - ZF-BrainReg (Regeneration of the adult zebrafish brain) Reporting period: 2020-07-01 to 2021-12-31 Summary of the context and overall objectives of the project Age-related cognitive decline and neurodegenerative diseases, like Alzheimer’s, Parkinson’s or Amyotrophic Lateral Sclerosis (ALS), are associated with neuronal cell loss and represent huge unsolved biomedical problems in western societies. Controlled brain regeneration, either by supplying programmed cells, or by activating endogenous progenitor cells, might provide a solution. The objective of this project were: (i) to unravel the underlying mechanisms for the ability of the adult zebrafish brain to regenerate itself after a severe lesion, and (ii) to compare these mechanisms with the mammalian brain, which is unable to regenerate. Overall, this project identified neuroepithelial cells as a second important stem cell type in the central nervous system of adult zebrafish and addressed the function of three identified genes during homeostasis and regeneration, in particular. Moreover, we identified acute inflammation as a common feature underlying successful neural regeneration, a topic that is now being pursued in large parts of the field. Finally, we tested two selected candidate genes that are critically important in the regeneration-competent zebrafish if they promote neurogenesis after lesion in the regeneration-incompetent mouse model. In conclusion, our work provided novel, fundamental insights into the cellular and molecular pathways underlying brain regeneration in vertebrates. The project also showed that the direct translation of regenerative cues gained from the adult zebrafish brain into the adult mouse brain is a big and challenging step indicating that intermediate steps testing the potential of candidate genes initially in vitro will be required prior to their analysis in vivo. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far One of the main objectives of this project was the identification of the cellular and molecular mechanisms allowing brain regeneration to occur in the regeneration-capable adult zebrafish. Towards this goal, we identified radial glia as well as neuroepithelial cells as the predominant source of new neurons in the zebrafish forebrain, cerebellum and retina. Publications: Kaslin et al., Development 2017; Kaslin and Brand, Essentials of Cerebellum and Cerebellar Disorders 2016; Lange et al., Development 2020. Moreover, we used gain- and loss-of-function studies to unravel in particular the function of Thyroglobulin, Lrrk2 and Hippo signaling during homeostasis and regeneration. Publications: Suzzi et al., BioRxiv 2017; Suzzi et al., Plos Genetics 2021. The project also identified acute inflammation as a central and common feature of regeneration across different neural tissues. Publication: Bosak et al., International Journal of Developmental Biology 2018. The second main objective of the project involved the translation of insights from the regeneration-competent zebrafish to the regeneration-incompetent mouse model. This undertaking involved a highly risky endeavor, in which we investigated the ability of the evolutionary conserved genes gata3 and cxcr5 to promote the production of new neurons directly in the lesioned, adult mouse brain. Independent of that, we established an adult mouse astrocyte in vitro cell culture system as an intermediate step to test the potential of candidate genes initially in vitro. This cell culture system shows all features of adult in vivo astrocytes and can be easily cultured as well as manipulated to allow subsequent screening of reprogramming factors. Finally, we set up a high throughput-screening platform to functionally screen for drugs affecting neural regeneration. Publications: Arulmozhivarman et al., Journal of Biomolecular Screening; Arulmozhivarman et al., Scientific Reports 2017. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The project built on the knowledge and tools that my group had assembled in the years before and all work packages have been successfully executed. Moreover, given the fast pace of regeneration research, we employed various novel methodologies that could not have been anticipated at the time of grant application. For instance, single cell RNA sequencing, a recent and highly dynamic field, allowed us to unravel the transcriptome of radial glia as well as their respective progeny and identified distinct transcriptional profiles in unprecedented depth. Moreover, based on the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) technology, we were able to generate tailor-made genomic modifications in the zebrafish genome and even implemented a novel conditional gene inactivation system that allows to not only describe the endogenous stem/progenitor cells but also to interrogate them functionally. As proposed, we also engaged into the translation of our findings into the mammalian context. However, we learned that the direct activation of regenerative cues, which promote regeneration in the adult zebrafish brain, do not improve neural regeneration in the adult mouse brain. Despite the setback in the in vivo reprogramming of mammalian stem cells, I conclude that all aims of the project have been achieved which is demonstrated by the manifold publications. Similarities and difference in inflammation in mammals and zebrafish following brain injury
