The prion protein (PrP) binds copper and has antioxidant activity enhancing the survival of neurones in culture. The ability of the PrP to bind other cations was tested and it was found that only manganese could substitute for copper. Although initially manganeseloaded PrP exhibited similar structure and activity to copper-loaded PrP, after aging, manganese-loaded PrP became proteinase resistant and lost function. It was also found that manganese could be incorporated into PrP expressed by astrocytes and that this PrP was partially proteinase resistant. These results show that it is possible to generate proteinase-resistant PrP from cells and suggest a possible mechanism for the formation of the scrapie isoform of the PrP as generated in sporadic prion disease.

We show here that mouse prion protein (PrP(C)) either as recombinant protein or immunoprecipitated from brain tissue has superoxide dismutase (SOD) activity. SOD activity was also associated with recombinant chicken PrP(C) confirming the evolutionary conserved phenotype suggested by sequence similarity. Acquisition of copper by PrP(C) during protein folding endowed SOD activity on the protein but the addition of copper following refolding did not. PrP(C) dependent SOD activity was abolished by deletion of the octapeptide-repeat region involved in copper binding. These results describe an enzymic function for PrP(C) consistent with its cellular distribution and suggest it has a direct role in cellular resistance to oxidative stress.

We show here that mouse prion protein (PrP(C)) either as recombinant protein or immunoprecipitated from brain tissue has superoxide dismutase (SOD) activity. SOD activity was also associated with recombinant chicken PrP(C) confirming the evolutionary conserved phenotype suggested by sequence similarity. Acquisition of copper by PrP(C) during protein folding endowed SOD activity on the protein but the addition of copper following refolding did not. PrP(C) dependent SOD activity was abolished by deletion of the octapeptide-repeat region involved in copper binding. These results describe an enzymic function for PrP(C) consistent with its cellular distribution and suggest it has a direct role in cellular resistance to oxidative stress.

Accumulation of misfolded proteins and protein assemblies is associated with neuronal dysfunction and death in several neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's disease (HD). It is therefore critical to understand the molecular mechanisms of drugs that act on pathways that modulate misfolding and/or aggregation. It is noteworthy that the mammalian target of rapamycin inhibitor rapamycin or its analogs have been proposed as promising therapeutic compounds clearing toxic protein assemblies in these diseases via activation of autophagy. However, using a cellular model of HD, we found that rapamycin significantly decreased aggregation-prone polyglutamine (polyQ) and expanded huntingtin and its inclusion bodies (IB) in both autophagy-proficient and autophagy-deficient cells (by genetic knockout of the atg5 gene in mouse embryonic fibroblasts). This result suggests that rapamycin modulates the levels of misfolded polyQ proteins via pathways other than autophagy. We show that rapamycin reduces the amount of soluble polyQ protein via a modest inhibition of protein synthesis that in turn significantly reduces the formation of insoluble polyQ protein and IB formation. Hence, a modest reduction in huntingtin synthesis by rapamycin may lead to a substantial decrease in the probability of reaching the critical concentration required for a nucleation event and subsequent toxic polyQ aggregation. Thus, in addition to its beneficial effect proposed previously of reducing polyQ aggregation/toxicity via autophagic pathways, rapamycin may alleviate polyQ disease pathology via its effect on global protein synthesis. This finding may have important therapeutic implications.

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The prion protein is a glycoprotein expressed by neurones and other cells. In its holo-form it binds copper and exhibits superoxide dismutase activity. Studies in mice have led to the description of two distinct alleles. Differences in these alleles are linked to long and short incubation times following infection with scrapie. We studied recombinant mouse protein corresponding to the products of either allele and two intermediates carrying single amino-acid residue substitutions. The different forms of the prion protein exhibited differences in superoxide dismutase (SOD) activity and conformation. Intermediates with single substitutions were less stable than either allelic product. The findings provide insight into the differences between the two alleles and might have consequences for understanding differences in susceptibility to prion disease.

Proteins containing extended polyglutamine repeats cause at least nine neurodegenerative disorders, but the mechanisms of diseaserelated neuronal death remain uncertain. We show that sympathetic neurons containing cytoplasmic inclusions formed by 97 glutamines expressed within human huntingtin exon1-enhanced green fluorescent protein (Q97) undergo a protracted form of nonapoptotic death that is insensitive to Bax deletion or caspase inhibition but is characterized by mitochondrial dysfunction. By treating the neurons with combined cytosine arabinoside and NGF withdrawal, we demonstrate that Q97 confers a powerful resistance to apoptosis at multiple levels: despite normal proapoptotic signaling (elevation of P-ser15-p53 and BimEL), there is no increase of Puma mRNA or Bax activation, both necessary for apoptosis. Even restoration of Bax translocation with overexpressed Puma does not activate apoptosis. We demonstrate that this robust inhibition of apoptosis is caused by Q97-mediated accumulation of Hsp70, which occurs through inhibition of proteasomal activity. Thus, apoptosis is reinstated by short hairpin RNA-mediated knockdown of Hsp70. These findings explain the rarity of apoptotic death in Q97-expressing neurons. Given the proteasomal blockade, we test whether enhancing lysosomal-mediated degradation with rapamycin reduces Q97 accumulation. Rapamycin reduces the amount of nonpathological Q25 by 70% over 3 d, but Q97 accumulation is unaffected. Interestingly, Q47 inclusions form more slowly as a result of constitutive lysosomal degradation, but fasterforming Q97 inclusions escape lysosomal control. Thus, cytoplasmic Q97 inclusions are refractory to clearance by proteasomal and lysosomal systems, leading to a toxicity that dominates over neuroprotective Hsp70. Our findings may explain the rarity of apoptosis but the inevitable cell death associated with polyQ inclusion diseases.

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