L68Q cystatin C: Expression, cellular transport and turnover of the cystatin C variant forming amyloid in patients with Hereditary Cystatin C Amyloid Angiopaty (HCCAA)

Hereditary cystatin C amyloid angiopathy (HCCAA) is a disorder characterised by multiple strokes in young adults, resulting in paralysis and dementia. The disease is caused by a mutation in the gene coding for the peptidase inhibitor cystatin C. The mutation causes an amino acid substitution Leu68 -> Gln in cystatin C (L68Q cystatin C) which results in deposition of the protein in the blood vessels in the brain. The overall goal of this study was to elucidate the pathogenic mechanisms leading to amyloid deposition in patients suffering from HCCAA. This was addressed by studying factors involved in the regulation of cystatin C expression and the behaviour of the L68Q variant in different cellular systems and clinical samples. When studying factors involved in cystatin C gene regulation we found that the steroid dexamethasone causes a significant and dose-dependent increase in cystatin C secretion from cultivated HeLa cells, up to a maximal increase of 80% at biological levels of the steroid. When looking at monocytes isolated from HCCAA patients and transfected mouse and human neuroblastoma cells we saw, in all cells tested, that the mutated cystatin C was secreted, but at a significantly lower rate than the wildtype protein. An increased accumulation of cystatin C was seen in all transfected cells expressing the gene encoding L68Q cystatin C. The accumulated L68Q cystatin C is insoluble and located mainly in the endoplasmic reticulum. When studying human neuroblastoma cell-lines we saw that they expressed and secreted very high levels of cystatin C compared to other human cell-lines tested. The neuroblastoma cells were also different from other cell types in that they showed intracellular insoluble wildtype cystatin C. Finally we investigated the turnover of cystatin C in plasma and cerebrospinal fluid (CSF) from HCCAA patients. Both L68Q and wildtype cystatin C were found in plasma from these patients but only the wildtype cystatin C could be detected in CSF.