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Gene silencing of cystatin B (CSTB) by RNAi : Implications for the altered JAK/STAT signaling pathway in Unverricht-Lundborg disease (EPM1)

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Title: Gene silencing of cystatin B (CSTB) by RNAi : Implications for the altered JAK/STAT signaling pathway in Unverricht-Lundborg disease (EPM1)
Author(s): Sandell, Katarin
Contributor: University of Helsinki, Faculty of Medicine, Institute of Biomedicine
Language: English
Acceptance year: 2013
Unverricht-Lundborg disease (EPM1/ULD, OMIM 254800) is an autosomal recessive inherited severe type of epilepsy with myoclonus and progressive neurological degeneration. The incidence of EPM1 in Finland is 1:20.000 births per year, and there are about 200 diagnosed cases. The age of disease onset is between 6 and 16 years. The symptoms start with epileptic seizures, stimulus sensitive myoclonus, and generalized tonic-clonic seizures, and progress within a few years to ataxia, incoordination, and dysarthria. Fourteen EPM1-associated loss-of-function mutations in the gene cystatin B (CSTB) have been described. CSTB is a ubiquitously expressed intracellular cysteine proteinase inhibitor, counteracting i.e. cathepsins B, L, and K. The expression levels of CSTB are higher in cerebellar Purkinje cells and in Bergmann glia of the adult central nervous system. There is a Cstb-deficient mouse model for EPM1, which shows progressive death of neurons and widespread gliosis. It has been earlier shown that Cstb knockdown sensitizes cerebellar granule neurons to cathepsin B mediated oxidative stress, resulting in cell death. This master's thesis is based on a previously done gene expression profiling of primary microglia of Cstb-/- mice, which revealed a downregulation of type I and type II interferon-regulated genes on the Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) –signaling pathway. The two aims of this study were to create an in vitro disease model for EPM1 in the human cervical adeno-carcinoma cell line HeLa, and in the murine leukemic cell line RAW264.7 by siRNA mediated RNA inhibition, and to study the effects of Cstb knockdown in selected interferon regulated genes of the JAK/STAT signaling pathway. Cystatin B was successfully knocked down in both cell lines HeLa and RAW264.7, and the obtained kinetics of Cstb knockdown in the cell line RAW264.7 provided with valuable information for the sec-ond part of the study. In the cell line HeLa, downregulation of CSTB did not change the expression lev-els of the genes of the JAK/STAT signaling pathway. In the cell line RAW264.7, CSTB knockdown on protein level was followed by a downregulation of the genes Stat1, Stat2, and Irf9. These results were in concordance with the results that had been obtained from the previously performed gene expression profiling of Cstb-/- microglia.

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