Analysis of zebrafish Lrrk2 loss-of-function during brain development and adult brain regeneration
Year: 2023
Type: dissertation
Abstract: The neurodegenerative disorder Parkinson's Disease (PD) represents both a major socioeco-nomic challenge and an individual burden for many patients. Despite major efforts, neither satisfactory explanations of the pathogenesis of PD, nor disease-modifying drugs have been developed to date. Mutations of the multidomain kinase LRRK2 represent the overall most common cause of he-reditary PD. Furthermore, LRRK2 mutations have been linked to dysregulations of the immune system such as inflammatory bowel disease, cancer, or the susceptibility towards mycobacte-rial infections. Several pathogenic point mutations have been identified that – directly or indi-rectly – lead to a pathological gain-of-function of the protein’s kinase domain. Despite recent advances, the physiological functions of LRRK2, as well as the underlying processes of LRRK2-mediated pathologies, remain largely unknown. Much research effort has aimed at generating reliable animal models for the study of LRRK2. Nevertheless, neither loss-of-function of the gene, nor overexpression of normal or mutant LRRK2, has yielded definitive results. Previous work from our research group on zebrafish (Danio rerio) has generated two genetic lrrk2 knock-out lines using different mutagenesis strat-egies: lrrk2TILLING and lrrk2CRISPR (Ahrendt, 2011; Suzzi, 2017). These studies’ results were par-tially contradictory, and the described phenotypes were not stable. Whilst this previous work investigated lrrk2 loss-of-function, so far, no genetic knock-in line carrying one of the multiple known pathogenic lrrk2 mutations, has been reported in zebrafish. Therefore, this work aimed to further investigate the effects of Lrrk2-deficiency in zebrafish and to establish a genetic knock-in of the common pathogenic G2019S substitution to model the genotype of PD patients more accurately. A variety of methods was applied to achieve these aims. Immunohistochemistry and conventional histology studies were performed on zebrafish brains and kidneys at different developmental stages, both under physiological conditions and following the induction of brain regeneration. Since the zebrafish’s neuroregenerative capabil-ity is closely linked to an initial neuroinflammation, and previous studies on lrrk2 knock-out zebrafish have suggested an impaired immune response and reduced brain regeneration, the neuroinflammation and neuroregeneration of adult lrrk2TILLING zebrafish were investigated by inducing a telencephalic stab-lesion and a subsequent BrdU-pulse-chase analysis. To investi-gate functional effects of the gene knock-out, a set of behavioral experiments was performed. Using CRISPR/Cas9 genome editing, the basis for a knock-in of the G2019S substitution was established. Immunohistochemistry analyses of larval and adult zebrafish brains were performed in a set of experiments. By quantifying all mitotic cells in larval brains at different time points, the basal brain proliferation levels during development were analyzed, as well as the levels of constitutive neurogenesis during adulthood. Under physiological conditions, basal brain prolif-eration was found unimpaired in Lrrk2-deficient zebrafish. Similarly, the number of microglia found in the telencephalon of Lrrk2-deficient zebrafish was not reduced under physiological conditions, although one experimental group showed signs of neuroinflammation. Upon induc-tion of a traumatic brain injury in adult fish, neither the trauma-induced proliferation of leuko-cytes, nor the number of regenerated neurons were altered in Lrrk2-deficient animals. A multi-dimensional behavioral analysis of Lrrk2-deficient zebrafish revealed no significant constraints. The total swimming distance, average velocity and ratio of mobility states were unimpaired upon lrrk2-knock-out, as was the fish’s exploratory behavior in an anxiety model using a light-dark-box. In a test for social preference, Lrrk2-deficient and wild-type zebrafish showed the same tendency to join a group of conspecific animals, suggesting no major deficits in overall social interaction. In contrast to these preserved functions, adult Lrrk2-deficient kidneys revealed a pronounced accumulation of vacuole-like particles in the proximal renal tubules, a finding that may indicate disruptions in the endolysosomal pathway and that is in line with phenotypes described in LRRK2-deficient rodents as well as with the side effects induced by pharmacological LRRK2 inhibitors. These findings represent a promising lead for future exploration. During this work a CRISPR/Cas9 target site with high cleavage efficiency was established within the Lrrk2 kinase domain of freshly spawned zebrafish eggs. In combination with recent advances in CRISPR methodology, these results provide an opportunity for the generation of a genetic Lrrk2-G2019S knock-in line in zebrafish. In summary, this work found Lrrk2-deficient zebrafish unimpaired regarding various physiolog-ical functions. While in line with previously reported results, a satisfactory explanation for Lrrk2-mediatied pathogenesis is still lacking. Morphological alterations of Lrrk2-deficient kidneys hint towards perturbations in the lysosomal homeostasis, and a promising target for future re-search. Modelling human LRRK2 genotypes more precisely will hopefully provide further in-sights into the enigma of LRRK2 and its link to neurodegeneration.
Author: Wirsching, Paul
Language: English
Subfield: Clinical Neurology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W7008066492