Zebrafish as a Model for Hereditary Spastic Paraplegia
by Vranda Garg
Date of Examination:2023-06-05
Date of issue:2023-06-28
Advisor:Dr. Bart Geurten
Referee:Dr. Bart Geurten
Referee:Prof. Dr. Ernst A. Wimmer
Persistent Address:
http://dx.doi.org/10.53846/goediss-9965
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Abstract
English
To investigate the mechanism of pathogenesis of the rare and complex neurodegenerative disorder Hereditary Spastic Paraplegia (HSP), which causes lower limb motility defects, it is crucial to establish simple and tractable animal models. We have utilised zebrafish as a model to emulate the neuroethological and physiological defects of HSP patients. In order to track the undulatory movement of adult zebrafish, we developed LACE (Limbless Animal traCkEr), a markerless pose estimator applicable to limbless animals. Our findings indicate that male zebrafish swim and turn more quickly than females due to a higher number of s-shaped thrust strokes produced within a given time period. We successfully established zebrafish as a model for HSP by developing the first stable CRISPRCas9 mutated zebrafish line for a known HSP gene, ZFYVE26 /SPASTIZIN, and identifying its novel roles in HSP pathogenesis. spastizin mutant fish swim slow as compared to their wild type counterparts and have defects in the functioning of the Mauthner cells. Mass spectrometry and electron microscopy revealed less cholesterol in the brain and demyelination in the large caliber spinal motor axons of spastizin mutants, respectively. We uncovered novel roles of Spastizin in regulating cholesterol metabolism and in maintaining the integrity of the myelin sheath and enhance the mechanistic understanding of the spastic paraplegia 15 (SPG15) pathogenesis. Moreover, we delineate the role of the outer mitochondrial membrane protein Tomm70 in HSP by demonstrating the behavioral and neurophysiological changes in tomm70 mutant fish. In this study, we found that tomm70 mutant fish also have defects in their normal and startle induced swimming and C-start escape behavior. At the cellular level, we found that the single point mutation in the tomm70 gene leads to the partial disruption in the interaction of Tomm70 with an ER protein called Lipid transfer at contact site 1 (Ltc1) or Lam6 which is mainly known for transporting sterols. As a consequence, less cholesterol was detected in the opaque eggs produced by the tomm70 mutants. We also observed defects in the transport of Tomm70 protein in the axons and dendrites of cultured brain neurons from tomm70 mutants, leading to signs of demyelination in large caliber axons of the spinal cord. In conclusion, we establish Tomm70 as a novel HSP gene, paving the way for further investigation into its role in this disorder and for potential therapeutic targets for the treatment of HSP patients.
Keywords: Hereditary Spastic Paraplegia; Zebrafish; Spastizin; Tomm70; Mauthner Cells; Demyelination; ER-mitochondria contact sites
