Fig 1.
(A) Workflow of brain PrP interactome discovery analysis. (B) CRISPR-Cas9-based knockout of PrP in multi-potent human neural stem cell line. (C) Validation of top-listed PrP interactor.
Fig 2.
PrP interactors in mouse brain.
Truncated list of proteins observed in eluate fractions in this study. The intensity of (i) gray shading reflects the sequence coverage, (ii) blue shading reflects the number of peptide-to-spectrum matches detected, and (iii) green shading reflects the relative enrichment in wild-type versus PrP knockout samples.
Fig 3.
Evidence for selective PrP co-enrichment of NKAs in mouse brain.
(A) Quantitative mass spectrometry sample processing workflow. (B-D) Side-by-side box plots depicting relative peptide enrichment levels in tryptic digests of PrP brain interactome eluates. (B) Selective enrichment of PrP-derived peptides in wild-type eluates (not in PrP ko eluates). (C) Exemplary PrP co-enrichment of the NKA alpha-1 subunit ATP1A1 (note that other NKA subunits were also observed to co-enrich with PrP; see Fig 2 for details). (D) Immunoglobulin levels were observed at similar abundance levels in all samples, including interactome eluates from PrP ko cells, consistent with the interpretation that levels of the bait antibody were balanced in all samples.
Fig 4.
Validation of NKA binding to PrP.
(A) Western blot-based validation of co-immunoprecipitation of NKA subunits with PrP. (B) Evidence of partial cellular co-localization of PrPC and ATP1A1 by co-immunocytochemical analysis of ReN VM cells, with Hoechst stain serving as the nuclear counter stain. Scale bar: 10 nm. (C) Chemical structure of the NKA inhibitor ouabain. (D) Parallel ouabain concentration-dependent effects on NKA and PrPC levels. Exposure of differentiated ReN VM cells to ouabain at concentrations up to 50 nM causes a biphasic effect on PrPC and ATP1A1 levels, with concentrations up to 30 nM leading to a ouabain concentration-dependent reduction in steady-state levels of both proteins, and exposure to 50 nM provoking a reproducible slowing of PrPC during SDS-PAGE separation that parallels an increase in steady-state ATP1A1 levels.
Fig 5.
PrP deficiency or prion infection alter 86Rb+ uptake activity of NKA.
(A) Design of gRNA targeting PRNP coding sequence. (B) CRISPR-Cas9-based knockout of PrP in ReN VM cells. (C) Validation of PrP knockout in ReN VM cells. Note that PrP deficiency has no effect on steady-state ATP1A1 levels in this model. (D) PrP knockout diminishes 86Rb+ uptake in ReN VM cells. Depicted are normalized mean plus standard deviation. (E) Increased steady-state ATP1A1 levels in RML-infected Neuro2a cells. (F) Similar to PrP knockout, RML-infection of Neuro2a cells compromises 86Rb+ uptake, albeit to a lesser extent. Statistical analyses in subpanels of this figure were based on the two-tailed t-test applied to three biological replicates.
Fig 6.
ReN VM cells respond to PrP-deficiency or cardiac glycoside exposure with an increase in the expression of a 60 kDa Coomassie-stained signal, originating from 5’-nucleotidase.
(A) Observation of Coomassie-stained protein band signal at 60 kDa that is conspicuously increased in the presence of 48 hour exposure to ouabain. The blot depicting levels of actin B (ACTB) serves as an additional loading control of samples that had been adjusted for total protein levels. (B) Scheme for the on-blot digestion and mass spectrometry-based identification of the 60 kDa band-of-interest. (C) MS/MS spectrum documenting identification of 5-nucleotidase (5’-NT) as the protein underlying this band. (D) Spectral count comparison of MS-based 5’-NT identification from Western blot bands observed in the absence or presence of ouabain. (E) Western blot validation of changes to 5’-NT expression upon prolonged exposure to low levels of ouabain. (F) CRISPR/Cas9-mediated knockout of PrP mimics low levels of ouabain in its effect on steady-state 5’-NT levels. Initially, the changes in 5’-NT expression were studied in pools of CRISPR-Cas9 gene engineered ReN VM PrP-/- cells generated with two distinct gRNAs. The pools differed in the percentages of cells depleted for PrP expression. Note that the 5’-NT signal levels correlate inversely with the degree of PrP knockout (i.e., the percentage of cells exhibiting the knockout). (G) Analysis of three separate ReN VM-derived PrP-/- clones corroborates changes in 5’-NT expression relative to wild-type Ren VM cells.
Fig 7.
Prolonged exposure of ReN cells to cardiac glycoside or prion infection causes cleavage of GFAP.
(A) Western blot documenting an increase in the intensity of GFAP antibody-reactive signals of 40–48 kDa in aged prion-infected mice that is not observed in vehicle-infected littermates. (B) Western blot documenting similar appearance of GFAP reactive bands in differentiated ReN cells exposed to low nanomolar concentrations of ouabain. (C) Calpain I inhibitor blocks ouabain-dependent formation of GFAP antibody reactive bands of 40–48 kDa. (D) Side-by-side western blot analysis of differentiated ReN cells, treated in the presence or absence of a calpain I inhibitor (A6185) next to brain lysates of RML-infected mice.
Fig 8.
The steady-state levels of isoforms of PrPC and NKA alpha-subunits are individually controlled by extracellular CGs and intracellular Ca2+ ions.
Exposure to 20 nM ouabain caused a reduction in the steady-state levels of PrPC, ATP1A1 and ATP1A2—but not ATP1A3—in a manner that cannot be reversed by concomitant incubation with YM24476. In contrast, co-incubation of cells with ouabain and A6185 rescued the reduction in ATP1A1 levels and caused the appearance of signals detected with the PrPC- and ATP1A2-directed antibodies that migrated with different apparent molecular weights than the dominant signals for these proteins observed in untreated cells. Finally, the intensity of relatively fast migrating signals detected with a GFAP-directed antibody increase under conditions that favor intracellular calcium-dependent calpain activity.
Fig 9.
Cartoons depicting how PrP-knockout or CG exposure may cause 5-NT overexpression, GFAP cleavage and NKA overexpression.
(A) Normal cell. (B) Cell exposed to low nanomolar CG levels or engineered to be PrP deficient. (C) Cell exposed to toxic high nanomolar CG levels. Note that the cartoon depicts a generic cell and omits subtleties related to the existence of NKA paralogs and isoforms.