Molecular biology of progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1)

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessively inherited disorder characterized by stimulus-sensitive myoclonus, tonic-clonic epileptic seizures, age of onset at 6-15 years and a progressive course. Mutations in the cystatin B (CSTB) gene underlie EPM1. The most common underlying mutation is a dodecamer repeat expansion in the putative promoter region of the gene. In addition, prior to this work, seven other EPM1associated CSTB mutations had been published. CSTB, a cysteine protease inhibitor, is a ubiquitously expressed tightly binding reversible inhibitor of cathepsins B, L, H and S, but its physiological function is unknown. In this study, we characterized the regulation and expression of the CSTB gene, determined the subcellular localization of the CSTB protein and elucidated the consequences of patient mutations on expression and subcellular localization of the CSTB gene and protein. Two novel EPM1-associated CSTB mutations, c.149G>A and c.168+1_18del were identified. Using promoter-luciferase reporter gene constructs, the basal CSTB promoter activity was located in the region from -670 to -1 bp from the translation initiation codon. The binding activity of transcription factors to five Sp1 and four AP1 sites as well as weak binding to an androgen response element (ARE) half site was demonstrated. The mouse Cstb transcript was present in all embryonic stages and adult tissues examined, the expression being highest at embryonic day 7. In adult tissues highest expression was detected in thymus. In sectioned E17 embryos highest expression was seen in the thymus, intestine and neopallial cortex. In postnatal brain Cstb expression was cell-specific, being highest in the cortex, caudate putamen, thalamus and hippocampus, as well as in the Purkinje cell layer of the cerebellum. Cystatin B expression thus seems to be tightly temporally and spatially regulated. Several alternatively spliced CSTB isoforms were identified, of these CSTB2 was widely expressed, also in brain, but with very low levels compared to CSTB expression. The other splice variants showed more restricted tissue expression. The endogenous CSTB protein localized to the nucleus, cytoplasm and lysosomes in COS-1 cells and human myoblasts. When myoblasts were differentiated into myotubes CSTB was excluded from the nucleus and lysosomes and merely Molecular biology of progressive myoclonus epilepsy of Unverrich-Lundborg type (EPM1) 11 localized to the cytoplasm, suggesting that the subcellular distribution of CSTB is dependent on the differentation status of the cells. Compatible with some 10-fold lowered in vitro transcriptional activity of the CSTB promoter containing a “premutation” length dodocamer repeat expansion, the CSTB expression was clearly decreased in individuals with “premutation” repeat expansions. The expression of CSTB mRNA and CSTB protein was markedly reduced in cells of EPM1 patients irrespective of the mutation type with the exception of one missense mutation. The proteins representing patient missense mutations were localized to cytoplasm and nucleus, but failed to associate with lysosomes, implying the importance of the lysosomal association for the proper physiological function of CSTB. By quantitative real-time PCR the splice variant CSTB2 was shown to be expressed at very low levels in all tissues. Whether CSTB2 protein is translated in vivo and its physiological relevance remain unknown. In patients the level of CSTB2 expression was reduced similarly to that of CSTB . We were unable to detect endogenous CSTB2 protein in cell lines with the antibodies available. The overexpressed wild-type and p.Gly4Arg mutant CSTB2 proteins localized evenly to cytoplasm and nucleus, but not to lysosomes. Instead, the p.Gly50Glu CSTB2 protein aggregated in cytoplasm and nucleus. The data produced in this study lay the basis for further understanding of the role of CSTB in health and disease.