Several agents are currently being tested for their ability to inhibit PrP-scrapie (PrPSc) infection in experimental models, and the most promising drugs are undergoing clinical trials on patients with new variant Creutzfeldt–Jakob disease (CJD), a prion infection acquired from ingestion of contaminated beef products. Unfortunately, similar trials for the equally devastating familial prion disorders that result from point mutations in the prion protein gene have received less attention. A plausible explanation may relate to the lack of a clear understanding of the common pathway(s) of neurotoxicity in the various familial prion disorders, making it difficult to design an effective therapeutic strategy.
Previous studies aimed at elucidating the pathophysiology of familial prion disorders in cell and animal models suggest two basic pathogenic mechanisms: (1) abnormal metabolism of the mutant PrP, and/or (2) a spontaneous change in its conformation to the pathogenic conformer PrPSc. Diverse pathways of mutant PrP metabolism that might contribute to the observed neurotoxicity have been described, such as: (1) upregulation of a transmembrane form of PrP, (2) accumulation of mutant PrP in the cytosol, endoplasmic reticulum (ER), lysosomes, or in the cell nucleus, and (3) a conformational change of mutant PrP to a PrPSc-like form in an intracellular compartment or in lipid-rich membrane domains. Although these studies fall short of identifying a common pathway that can be targeted for therapeutic drug development, the phenomenon underlying most of the above observations is inability of the mutant PrP to fold into its mature conformation following synthesis in the ER. As a result, misfolded PrP molecules are subject to the cellular quality control mechanism(s) that may block their transport from the ER, mediate retro-translocation to the cytosol for proteasome-mediated turnover, or target these for lysosomal degradation. The ultimate fate probably depends on the extent of PrP misfolding. Cellular toxicity may therefore result from a loss of normal PrP function due to its degradation or sequestration in an abnormal cellular compartment, a gain of toxic function due to abnormal interaction with other proteins, or by conversion to the amyloidogenic PrPSc form that is relatively resistant to cellular proteases. Thus, agents that facilitate folding of mutant PrP in the ER in doses that can be tolerated over a prolonged period of time may be potentially useful in the prevention or treatment of certain familial prion disorders.
In this study, we report the effect of different drugs and protein-folding agents on the metabolism of mutant PrP H187R (PrP187R) in a cell model, using this as a prototype of familial CJD. In particular, we selected drugs that are safe for human use and have proved efficacious in clearing PrPSc from experimental models. To facilitate a quick and efficient evaluation of different agents on the transport and processing of mutant PrP187R, we expressed green fluorescent protein (GFP) tagged wild-type PrP (PrPC-GFP), or mutant PrP187R (PrP187R-GFP) in human neuroblastoma cells. Here we show that unlike PrPC-GFP that is mainly expressed on the cell surface, majority of PrP187R-GFP accumulates in lysosomes, and only minimal amounts are transported to the plasma membrane. Treatment of PrP187R-GFP cells with quinacrine or doxycycline, drugs that decrease PrPSc production in cell models and in vitro studies gave variable results. Quinacrine accentuated the lysosomal accumulation of PrP187R-GFP. In contrast, doxycycline decreased lysosomal accumulation and promoted transport of PrP187R-GFP to the cell surface. Similar results were obtained with agents that promote protein folding in the ER such as incubation at low temperature (28°C), and the chemical chaperones glycerol and dimethyl sulphoxide (DMSO). These data have significant implications for the development of effective therapeutic strategies for familial prion disorders where the underlying defect is misfolding of mutant PrP in the ER.
June 23, 2004