Prion disorders manifest when a normal cell surface glycoprotein, the prion protein (PrPC), undergoes a conformational change from an a-helical to a 13-sheet rich structure (PrPSc) that is pathogenic. Deposits of PrPSC in the brain parenchyma are considered the principal cause of neurotoxicity in prion disorders. In familial forms of these disorders, a point mutation in the prion protein gene (PRNP) is thought to mediate the conformational change of mutant PrP to PrPSc, resulting in pathogenicity. Despite the reported high co-relation between PrPSc deposits and clinical disease, neurodegeneration in prion disorders is often seen without detectable PrPS, indicating the presence of alternative mechanisms of cellular toxicity. Results from my laboratory show that in familial prion disorders, abnormal metabolism of the mutant prion protein leads to neuronal toxicity by distinct cellular pathways, unique to each mutation. The stop codon mutation at residue 145 (PrP145) leads to accumulation of PrP145 in the endoplasmic reticulum (ER) and in the nucleus, while the Q21 7R mutation (PrP217) causes accumulation of PrP217 in the ER and in the lysosomal compartment. The P102L mutation (PrP'?2), on the other hand, results in the accumulation of an amyloidogenic fragment of PrP'?2 on the cell surface. Based on these observations, we hypothesize that point mutations in different region(s) of PrP alter PrP metabolism and initiate neuronal death through distinct intracellular pathways unique to each mutation (or a group of mutations), and not by the common pathway of PrPSc accumulation. In this proposal, we will investigate this hypothesis by analyzing the biogenesis of mutant PrP with point mutations in three distinct regions of PrP: (1) the glycosylphosphatidyl inositol anchor signal peptide (GPI-SP) of PrP, (2) up to twenty-five amino acids upstream from the GPI addition site, and (3) in the vicinity of PrP glycosylation sites. We will use transfected human neuroblastoma cells to investigate the processing, transport, and turnover of wild type and mutant PrP using immunoprecipitation, Western blotting, and other cell and molecular biology techniques. Sub-cellular localization of PrP will be examined by confocal and electron microscopy. The results obtained will be confirmed in differentiated NT-2N cells and human embryonic neurons in culture. A C-terminal Flag-tagged PrP and a PrP-GFP construct developed in my laboratory will be used to complement the above methods. The Flag tag will allow specific identification of the GPI-SP, and the PrP-GFP construct will enable analysis of mutant PrP in living cells. This study will uncover alternative pathways of neuronal toxicity by mutant PrP besides conversion to PrPSc, and help in developing rational therapeutic strategies.
