Prion Protein Deficiency Causes Diverse Proteome Shifts in Cell Models That Escape Detection in Brain Tissue

Abstract

A popular method for studying the function of a given protein is to generate and characterize a suitable model deficient for its expression. For the prion protein (PrP), best known for its role in several invariably fatal neurodegenerative diseases, a natural choice, therefore, would be to undertake such studies with brain samples. We recently documented the surprising observation that PrP deficiency caused a loss or enhancement of NCAM1 polysialylation, dependent on the cell model used. To identify possible causes for this disparity, we set out to systematically investigate the consequence of PrP deficiency on the global proteome in brain tissue and in four distinct cell models. Here we report that PrP deficiency causes robust but surprisingly divergent changes to the global proteomes of cell models but has no discernible impact on the global brain proteome. Amongst >1,500 proteins whose levels were compared in wild-type and PrP-deficient models, members of the MARCKS protein family exhibited pronounced, yet cell model-dependent changes to their steady-state levels. Follow-up experiments revealed that PrP collaborates with members of the MARCKS protein family in its control of NCAM1 polysialylation. We conclude that the physiological function of PrP may be masked in analyses of complex brain samples but its cell-type specific influence on a lipid raft-based NCAM1-related cell biology comes to the fore in investigations of specific cell types.

The prion protein is embedded in a molecular environment that modulates transforming growth factor β and integrin signaling

“…The study made use of four mouse cell lines from which we had previously derived PrP knockout clones by CRISPR-Cas9 technology (Fig. 1a ) 18 . The parental wild-type cell lines are familiar to the prion research community due to their distinct properties with regard to PrP: (1) NMuMG cells exhibit a more than five-fold increase in their PrP protein levels when EMT was induced by the addition of Tgfb1 (attempts to infect these cells with prions have been unsuccessful but were also not exhaustive) 19 ; (2) C2C12 cells are the only muscle cell model currently known to be susceptible to prion infection 20 , 21 ; (3) N2a neuroblastoma cells may be the most often used cell model in prion research and can readily be infected with mouse-adapted Rocky Mountain Laboratory (RML) prions; and (4) CAD5 catecholaminergic cells exhibit susceptibility to infection with several prion strains 22 .…”

“…We recently documented that the expression levels of the aforementioned ZIPs, PrP, and Ncam1 are several-fold upregulated during EMT in mammalian cells 17 , consistent with the interpretation that the interactions these proteins engage in change over time and depend on the cell lineage characteristics of the model. A hint that there may be additional differences in PrP interactions when comparisons are done across models came from an observation of model-dependent proteome shifts in PrP-deficient cells 18 . Not only did PrP-deficiency in distinct cell models cause the levels of members of the Marcks protein family to shift in opposite ways but it also prevented Ncam1 polysialylation in one cell model, yet caused a robust increase of this specific posttranslational modification (PTM) in another model 17 .…”

Physiological Functions of the Cellular Prion Protein

“…Indeed, such a role may not be restricted to the nervous system, since there is evidence that PrP C can influence some of the earliest differentiation processes that occur during embryogenesis. For example, PrP C may indirectly modulate NCAM1 polysialylation in order to regulate the epithelial-to-mesenchymal transition (Mehrabian et al, 2015, 2016), a vital developmental process that alters cell adhesion and enables cell migration. Ectodermal cells in the developing embryo must undergo epithelial-to-mesenchymal transition as part of gastrulation, which may explain why knockdown of PrP1 expression in zebrafish embryos has been shown to prevent this process (Malaga-Trillo et al, 2009).…”

“…Several proteomic studies have identified such changes in cell lines in which PrP C was knocked down or overexpressed (Provansal et al, 2010; Weiss et al, 2010; Mehrabian et al, 2014) and also in PrP C -null liver tissues compared with wild type controls (Arora et al, 2013). Analyses of brain tissues have been less successful at detecting differential expression of cytoskeletal proteins (or of any proteins for that matter), although this could be because cell type-specific effects of PrP C are averaged out over an entire tissue (Crecelius et al, 2008; Mehrabian et al, 2016). Another common feature of differentiation is a change of cell cycle progression.…”

NCAM1 Polysialylation

“…Closer investigations revealed that PrP C controls NCAM1 polysialylation in NMuMG cells by acting on St8sia2 gene transcription. The precise steps involved in the PrP-dependent signaling that controls transcription of the St8sia2 gene are not yet understood but may involve β-catenin (Mehrabian et al, 2015) as well as members of the MARCKS protein family (Mehrabian et al, 2016). Morevoer, we observed that distinct cell models exhibit a PrP-dependent upregulation or downregulation of St8sia2 gene transcription, suggesting that input from other signaling pathways must exist (Mehrabian et al, 2015).…”