Self-cleaving-ribozyme-mediated reduction of betaAPP in human rhabdomyosarcoma cells.

A self-cleaving hammerhead ribozyme targeted to codon 47 in beta-amyloid precursor protein (betaAPP) mRNA was cloned as a eucaryotic transcription cassette into the 3' UTR of enhanced green fluorescence protein (EGFP) mRNA, producing a C-terminal fusion mRNA. CMV promotor-driven vectors bearing this construct or a mutationally inactive ribozyme construct were transiently transfected into human embryonic rhabdomyosarcoma (A-204) cells and their effects studied. Ribozyme self-cleavage in vivo was demonstrated by Northern blotting and the site of self-cleavage was delineated using site-specific deoxyoligonucleotide probes and primer extension arrest. Using this ribozyme reporter we demonstrated that ribozyme expression correlated with lower betaAPP levels in the transfected cells. Control studies with the inactive ribozyme construct showed that both ribozyme cleavage and antisense mechanisms combined to produce the observed effect. Furthermore, production of truncated EGFP mRNA via ribozyme self-cleavage reduced EGFP-reporter expression compared to full-length EGFP control mRNAs, indicating that truncation affects the translatability of the reporter. This occurred because of a slight decrease in the stability of the fusion mRNA. The results of these studies suggest that self-cleaving ribozyme vectors may be an effective means of delivering and visualizing the expression of small active ribozymes in vivo.

Cellular processing of the amyloidogenic cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type.

An important gap in our understanding of the pathogenesis of the amyloidoses is the identification of the cellular events that lead from synthesis of an amyloid precursor protein to its conversion to the amyloid fiber subunit. We address this question by characterizing the effects of an amyloidogenic mutation on the intracellular processing of its protein product. The protein, a mutant of the cysteine protease inhibitor cystatin C, is the amyloid precursor protein in Hereditary Cerebral Hemorrhage with Amyloidosis--Icelandic type (HCHWA-I). The amyloid fibers are composed of mutant cystatin C (L68Q) that lacks the first 10 amino acids. We have previously shown that processing of wild-type cystatin C entails formation of a transient intracellular dimer that dissociates prior to secretion, such that extracellular cystatin C is monomeric. We report here that the cystatin C mutation engenders several alterations in its intracellular trafficking. It forms a stable intracellular dimer that is partially retained in the endoplasmic reticulum and degraded. The bulk of mutant cystatin C that is secreted does not dissociate and is secreted as an inactive dimer. Thus, formation of the stable mutant cystatin C dimer is an early event in the pathogenesis of this disease.

Human cystatin C forms an inactive dimer during intracellular trafficking in transfected CHO cells.

To define the cellular processing of human cystatin C as well as to lay the groundwork for investigating its contribution to lcelandic Hereditary Cerebral Hemorrhage with Amyloidosis (HCHWA-I), we have characterized the trafficking, secretion, and extracellular fate of human cystatin C in transfected Chinese hamster ovary (CHO) cells. It is constitutively secreted with an intracellular half-life of 72 min. Gel filtration of cell lysates revealed the presence of three cystatin C immunoreactive species; an 11 kDa species corresponding to monomeric cystatin C, a 33 kDa complex that is most likely dimeric cystatin C and immunoreactive material, > or = 70 kDa, whose composition is unknown. Intracellular monomeric cystatin C is functionally active as a cysteine protease inhibitor, while the dimer is not. Medium from the transfected CHO cells contained only active monomeric cystatin C indicating that the cystatin C dimer, formed during intracellular trafficking, is converted to monomer at or before secretion. Cells in which exit from the endoplasmic reticulum (ER) was blocked with brefeldin A contained the 33 kDa species, indicating that cystatin C dimerization occurs in the ER. After removal of brefeldin A, there was a large increase in intracellular monomer suggesting that dimer dissociation occurs later in the secretion pathway, after exiting the ER but prior to release from the cell. Extracellular monomeric cystatin C was found to be internalized into lysosomes where it again dimerized, presumably as a consequence of the low pH of late endosome/lysosomes. As a dimer, cystatin C would be prevented from inhibiting the lysosomal cysteine proteases. These results reveal a novel mechanism, transient dimerization, by which cystatin C is inactivated during the early part of its trafficking through the secretory pathway and then reactivated prior to secretion. Similarly, its uptake by the cell also leads to its redimerization in the lysosomal pathway.

Isolation and characterization of macrophages from scrapie-infected mouse brain.

We have isolated and characterized a population of brain macrophages from normal and scrapie-infected mice. The cells are phagocytic, possess Fc-IgG receptors, Mac-1 surface antigen and proliferate in the presence of macrophage colony stimulating factor. They resemble microglia in that they have a plasmalemmal distribution of the enzyme nucleoside diphosphatase, a property tht is characteristic of microglia in situ. In two of the three combinations of scrapie agent and mouse strain examined, the number of brain macrophages was several fold higher than in normal control mice. The increase was not observed in mice infected intraperitoneally or in control mice inoculated with normal brain homogenate. The increase is detectable as early as 3-5 weeks postinoculation. The agent/host combination that failed to show an increase in brain macrophages is one that develops large numbers of amyloid plaques. These observations suggest that these cells are closely associated with the scrapie pathogenic process in the CNS. The failure of these cells to increase in the plaque forming model of scrapie disease also suggests that they play a role in the control of CNS amyloidogenesis.