Characterization of spontaneously generated prion-like conformers in cultured cells

Abstract

A distinct conformational transition from the α-helix-rich cellular prion protein (PrPC) into its β-sheet-rich pathological isoform (PrPSc) is the hallmark of prion diseases, a group of fatal transmissible encephalopathies that includes spontaneous and acquired forms. Recently, a PrPSc-like intermediate form characterized by the formation of insoluble aggregates and protease-resistant PrP species termed insoluble PrPC (iPrPC) has been identified in uninfected mammalian brains and cultured neuronal cells, providing new insights into the molecular mechanism(s) of these diseases. Here, we explore the molecular characteristics of the spontaneously formed iPrPC in cultured neuroblastoma cells expressing wild-type or mutant human PrP linked to two familial prion diseases. We observed that although PrP mutation at either residue 183 from Thr to Ala (PrPT183A) or at residue 198 from Phe to Ser (PrPF198S) affects glycosylation at both N-linked glycosylation sites, the T183A mutation that results in intracellular retention significantly increased the formation of iPrPC. Moreover, while autophagy is increased in F198S cells, it was significantly decreased in T183A cells. Our results indicate that iPrPC may be formed more readily in an intracellular compartment and that a significant increase in PrPT183A aggregation may be attributable to the inhibition of autophagy.

Glycoform-Selective Prion Formation in Sporadic and Familial Forms of Prion Disease

“…In combination these data suggests that mono181 is not converted into PrP res in the two diseases and that mono181 of VPSPr and fCJD V180I is different from that of sCJD. Notably, mono197 from all conditions migrated more slowly than did mono181 at ∼0.5–1 kDa, consistent with previous observations [11], [12]. Moreover, the ratio of mono197 intensity to unglycosylated PrP was significantly greater than that of mono181 to unglycosylated PrP in all three diseases (sCJD: 0.92 ± 0.02 vs. 0.43 ± 0.08, p < 0.001; VPSPr: 0.81 ± 0.…”

“…In contrast with PK-treated PrP, mono181 or mono197 was detected in both VPSPr and fCJD V180I in addition to C2 and C1, two fragments identified previously in human brains [11], [15], similar to those detected in the sCJD control (Fig. 2C).…”

“…S1 and S2) [11]–[14]. On the blot probed with the Bar209 antibody that specifically detects mono197, both mono197 and unglycosylated PrP migrating at ∼26–28 kDa and ∼19–20 kDa, respectively, were detected in all samples treated with PK alone except for fCJD T183A that had a significantly under-represented unglycosylated PrP (Fig.…”

“…On the blot probed with the V14 antibody that specifically detects mono181 [11], [12], the PrP band migrating at ∼24–26 kDa corresponding to mono181 was detected in sCJD in addition to the band migrating at ∼19–20 kDa corresponding to the unglycosylated PrP (Fig. 2B).…”

“…This is because the T183A PrP mutation completely abolishes the first N-linked glycosylation site at residue 181 (N181) [9]–[11] and the detected diglycosylated PrP is derived only from wild-type PrP (3, the current study). In contrast, the PrP glycoforms in VPSPr appear typical prior to PK-treatment; however, there is no detectable diglycosylated PrP Sc after PK-treatment.…”