Research Article: Familial CJD Associated PrP Mutants within Transmembrane Region Induced Ctm-PrP Retention in ER and Triggered Apoptosis by ER Stress in SH-SY5Y Cells

Date Published: January 27, 2011

Publisher: Public Library of Science

Author(s): Xin Wang, Qi Shi, Kun Xu, Chen Gao, Cao Chen, Xiao-Li Li, Gui-Rong Wang, Chan Tian, Jun Han, Xiao-Ping Dong, Colin Combs.

Abstract: Genetic prion diseases are linked to point and inserted mutations in the prion protein (PrP) gene that are presumed to favor conversion of the cellular isoform of PrP (PrPC) to the pathogenic one (PrPSc). The pathogenic mechanisms and the subcellular sites of the conversion are not completely understood. Here we introduce several PRNP gene mutations (such as, PrP-KDEL, PrP-3AV, PrP-A117V, PrP-G114V, PrP-P102L and PrP-E200K) into the cultured cells in order to explore the pathogenic mechanism of familial prion disease.

To address the roles of aberrant retention of PrP in endoplasmic reticulum (ER), the recombinant plasmids expressing full-length human PrP tailed with an ER signal peptide at the COOH-terminal (PrP-KDEL) and PrP with three amino acids exchange in transmembrane region (PrP-3AV) were constructed. In the preparations of transient transfections, 18-kD COOH-terminal proteolytic resistant fragments (Ctm-PrP) were detected in the cells expressing PrP-KDEL and PrP-3AV. Analyses of the cell viabilities in the presences of tunicamycin and brefeldin A revealed that expressions of PrP-KDEL and PrP-3AV sensitized the transfected cells to ER stress stimuli. Western blots and RT-PCR identified the clear alternations of ER stress associated events in the cells expressing PrP-KDEL and PrP-3AV that induced ER mediated apoptosis by CHOP and capase-12 apoptosis pathway. Moreover, several familial CJD related PrP mutants were transiently introduced into the cultured cells. Only the mutants within the transmembrane region (G114V and A117V) induced the formation of Ctm-PrP and caused the ER stress, while the mutants outside the transmembrane region (P102L and E200K) failed.

The data indicate that the retention of PrP in ER through formation of Ctm-PrP results in ER stress and cell apoptosis. The cytopathic activities caused by different familial CJD associated PrP mutants may vary, among them the mutants within the transmembrane region undergo an ER-stress mediated cell apoptosis.

Partial Text: Prion diseases, or transmissible spongiform encephalopathies (TSE), are a group of transmissible neurodegenerative disorders that afflict both humans and animals, including Creutzfeldt-Jakob disease (CJD) in human, scrapie in sheep and goat, bovine spongiform encephalopathy (BSE) in cattle[1]. The underlying cause of prion disease is the conversion of a host-derived cellular prion protein (PrPC) to the infectious scrapie prion protein (PrPSc), a misfolded and proteinase K (PK)-resistant isoform, which is the main component of infectious prion[2], [3]. Propagation and accumulation of infectious PrP have always been thought to be tightly linked to the pathogenesis of prion disease. However, transgenic mouse models have revealed that misfolded or mistargeted PrPC can also induce neuronal cell death in the absence of infectious prion[4]. The observation of substantial neurodegeneration in the absence of PrPSc accumulation in some cases of natural and experimental prion disease argues against its accumulation as the sole cause of pathology[5], [6], highlighting that other aspects, e.g. PrP expressing, folding, locating and trafficking, may feature in the pathophysiological mechanisms that ultimately cause disease.

In this study, we have provided the data that a novel transmembrane form of PrP tagged with an ER signal peptide (PrP-KDEL) induces the similar biological effectiveness, e.g. formation of Ctm-PrP, ER stress and cell apoptosis, as the previously reported recombinant PrP mutant (PrP-3AV)[9]. We also confirm that retention of full-length presenilin 1 tagged with ER signal peptide (PS1-KDEL) does not induce the ER stress. It supplies the direct evidence that simply retention of PrP in ER will cause subsequent ER stress and cell apoptosis.