Analysis of movement disorder-related genes following knockdowns of ANO10, WDR81, and VLDLR in zebrafish (DANIO RERIO)

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2018-06
Advisor
Adams, Michelle Marie
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Bilkent University
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English
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Abstract

Movement Disorders are the neurological symptoms that cause alterations in normal motility, posture and muscle tone. Certain brain areas, such as the cerebellum, mediate correct motor control and functioning. When defects or congenital lesions occur in the cerebellum, neural disruption in motor coordination causes the development of a particular movement disorder known as cerebellar ataxia. The focus of this study was to examine how three genes of interest (ano10, wdr81, vldlr), contributing to multiple varieties of cerebellar ataxias, influence one another and other genes that are associated with this disorder. Mutations in vldlr and wdr81 are associated with Cerebellar Ataxia Mental Retardation Disequilibrium Syndrome type 1 and type 2 (CAMRQ1 and CAMRQ2), respectively, whereas mutations in the ano10 gene is responsible for the development of Autosomal Recessive Cerebellar Ataxia Type 3 (ARCA3). In this work, five key scientific findings were reported. Firstly, in silico analysis predicted a common Ca2+ activated Casein Kinase 2 (CK2) domain in the protein sequences of the genes of interest and also predicted a common interacting UBC protein. These predicted interactions, a common CK2 domain and a UBC interacting protein may explain the observed neurodegenerative phenotype in cerebellar ataxia. Secondly, the transcript level analysis (qPCR and RNASeq) of ano10a, wdr81 and vldlr using zebrafish embryos collected from early embryonic and late larval stages showed that the three genes were expressed relatively higher at 1 hpf, 2 hpf and 5 hpf developmental stages than others and may suggest their importance in developmental processes. Additionally, the comparison of the expression patterns of ano10a, wdr81 and vldlr during early embryogenesis indicated that three targeted genes were co-localized at diencephalon, midbrain (optic tectum) and cerebellum. These spatiotemporal results may restrict the involvement of these three genes selectively in early neurodevelopmental processes. Thirdly, this study also examined the expression level analysis of three targeted genes in 12 different adult tissues in a sexually dimorphic manner. Findings showed that genes of interest were expressed significantly higher at the eyes, brain and gonads (p-values < 0.05). Moreover, the gender specific examination in the 12 adult tissues revealed that ano10a and wdr81 expression differed significantly at eyes, gills, liver and gonads (p-values < 0.05) whereas, vldlr gene expression was significantly different at swim bladder and gonads in male and female individuals (p-values < 0.05). Fourthly, the clustergram analysis indicated that three genes of interest were grouped within close families with each other and 9 additional cerebellar ataxia associated genes and may imply that targeted genes alter functions in the converging pathways. Finally, results from the clustergram analysis helped to design and carry out a study knocking down the expression of ano10a, wdr81 and vldlr separately with MO antisense technology to examine the effect of the silenced mRNA on the expression levels of each other and 9 other highly correlated cerebellar ataxia-related genes. Single MO injections caused the significant upregulation of all investigated genes especially at 72 hours after/post injection (hpi) (p-values < 0.05) when ano10a transcript was silenced suggesting either an activated compensatory mechanism or activated alternative disease specific cascade molecules in response to its absence. Taken together, the outcomes of functional knockdowns can pave the way for the development of novel therapeutic targets using inhibitors or antagonists of activated cellular pathway components or the enhancers of downregulated genes to prevent or at least slow down the progression of not only cerebellar ataxia but also several other neurodegenerative disorders.

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Zebrafish, Gene Expression, Movement Disorders, Cerebellar Ataxia, Ano10, Wdr81, Vldlr, Morpholino Injection, Gene Knockdown, Sexual Dimorphism, In Situ Hybridization, Clustergram Analysis, Bioinformatics Analysis And qPCR
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Published Version (Please cite this version)