Mutant prion protein‐mediated aggregation of normal prion protein in the endoplasmic reticulum: implications for prion propagation and neurotoxicity

Abstract

Familial prion disorders are believed to result from spontaneous conversion of mutant prion protein (PrP M ) to the pathogenic isoform (PrP Sc ). While most familial cases are heterozygous and thus express the normal (PrP C ) and mutant alleles of PrP, the role of PrP C in the pathogenic process is unclear. Plaques from affected cases reveal a heterogeneous picture; in some cases only PrP M is detected, whereas in others both PrP C and PrP M are transformed to PrP Sc . To understand if the coaggregation of PrP C is governed by PrP mutations or is a consequence of the cellular compartment of PrP M aggregation, we coexpressed PrP M and PrP C in neuroblastoma cells, the latter tagged with green fluorescent protein (PrP C-GFP ) for differentiation. Two PrP M forms (PrP 231T , PrP 217R/231T ) that aggregate spontaneously in the endoplasmic reticulum (ER) were generated for this analysis. We report that PrP C-GFP aggregates when coexpressed with PrP 231T or PrP 217R/231T , regardless of sequence homology between the interacting forms. Furthermore, intracellular aggregates of PrP 231T induce the accumulation of a C-terminal fragment of PrP, most likely derived from a potentially neurotoxic transmembrane form of PrP ( Ctm PrP) in the ER. These findings have implications for prion pathogenesis in familial prion disorders, especially in cases where transport of PrP M from the ER is blocked by the cellular quality control.

Trapping Prion Protein in the Endoplasmic Reticulum Impairs PrPC Maturation and Prevents PrPSc Accumulation

“…These biochemical characteristics are reminiscent of those described in cell models of genetic prion diseases (11,53,54) in which PrP C molecules carrying mutations are delayed in their maturation, accumulate in the ER, tend to misfold and aggregate (55,56). In our model we find that ER-trapped wild type PrP C can acquire the same properties of inherited prion mutants but in the absence of mutations.…”

Alpha‐ and beta‐ cleavages of the amino‐terminus of the cellular prion protein

“…1). Moreover, the N2 terminal fragment described here could be the complement of the faint C2 terminal fragment detected earlier in cells and brain extracts by us and others (Gu et al, 2003;Jimenez-Huete et al, 1998). Importantly, its size would indicate that cleavage could occur physiologically into the octapeptide repeat region.…”

Prion Protein Promotes Kidney Iron Uptake via Its Ferrireductase Activity

“…Immunostaining of endogenous mouse PrP C in non-transfected cells revealed a similar localization of PrP C , making it unlikely that intracellular localization is due to overexpression of transfected PrP (data not shown). The presence in vesicular structures was surprising as PrP C and PrP ⌬51-89 are expressed mainly on the plasma membrane of neuronal cells with some reactivity in the Golgi area (28,33). To confirm these observations, polarized PT cells expressing non-GFP-tagged PrP C and PrP ⌬51-89 were permeabilized with Triton-X and co-immunostained with antibody specific to human PrP C (green) and the tight junction protein ZO-1 (red) (Fig.…”

“…It was surprising to note that a significant amount of PrP C in PT cells was localized to recycling endosomes or late endosomes and lysosomes, a distribution markedly different from the mainly plasma membrane expression on neuronal cells (33,42,43). This can be explained by the large apical endocytosis activity of proximal tubule cells responsible in vivo for the uptake of a large daily load of small filtered proteins (6,8).…”

“…To evaluate whether exposure to hemin or FAC alters the detergent solubility of PrP C , hemin-and FAC-exposed cells were lysed in a buffer containing non-ionic detergents, and clarified extracts were centrifuged at high speed to separate the detergent-soluble (S) and insoluble (P) fractions (33,37). All fractions were resolved by SDS-PAGE, and transferred proteins were probed for PrP and ␤-actin (Fig.…”