Changes in cellular prion protein expression, processing and localisation during differentiation of the neuronal cell line CAD 5

Abstract

Background informationCellular prion protein (PrPC) is infamous for its role in prion diseases. The physiological function of PrPC remains enigmatic, but several studies point to its involvement in cell differentiation processes. To test this possibility, we monitored PrPC changes during the differentiation of prion‐susceptible CAD 5 cells, and then we analysed the effect of PrPC ablation on the differentiation process.ResultsNeuronal CAD 5 cells differentiate within 5 days of serum withdrawal, with the majority of the cells developing long neurites. This process is accompanied by an up to sixfold increase in PrPC expression and enhanced N‐terminal β‐cleavage of the protein, which suggests a role for the PrPC in the differentiation process. Moreover, the majority of PrPC in differentiated cells is inside the cell, and a large proportion of the protein does not associate with membrane lipid rafts. In contrast, PrPC in proliferating cells is found mostly on the cytoplasmic membrane and is predominantly associated with lipid rafts. To determine the importance of PrPC in cell differentiation, a CAD 5 PrP−/− cell line with ablated PrPC expression was created using the CRISPR/Cas9 system. We observed no considerable difference in morphology, proliferation rate or expression of molecular markers between CAD 5 and CAD 5 PrP−/− cells during the differentiation initiated by serum withdrawal.ConclusionsPrPC characteristics, such as cell localisation, level of expression and posttranslational modifications, change during CAD 5 cell differentiation, but PrPC ablation does not change the course of the differentiation process.SignificanceAblation of PrPC expression does not affect CAD 5 cell differentiation, although we observed many intriguing changes in PrPC features during the process. Our study does not support the concept that PrPC is important for neuronal cell differentiation, at least in simple in vitro conditions.

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“…Lipid rafts are also involved in the metal/copper-mediated endocytosis of prions via clathrin-coated pits or caveolae [ 370 , 371 , 372 ]. In proliferating neuronal CAD 5 cell lines, PrP C is predominantly associated with lipid rafts on cytoplasmic membranes [ 752 ] while in human dental pulp mesenchymal stem cells, the integrity of lipid rafts is essential for the preservation of recombinant prion protein (23–231) physiological activities affecting neuronal differentiation and signaling. The critical localization of PrP in lipid raft microdomains allows prions to recruit and interact with important biochemical signaling partners [ 753 , 754 ].…”

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“…Prion diseases, such as human prion diseases, are a group of progressive neurodegenerative disorders caused by conformational conversion of the α-helix-rich isoform of the prion protein (PrP C ), which is the normal form, into the β-sheet rich isoform (PrP Sc ), which is the disease-associated form [1,149,150]. Abnormal folding of the protein (PrP Sc ) leads to brain damage and causes high fatality rates in both humans and animals [151][152][153][154][155][156][157][158][159][160][161][162][163][164][165][166].…”

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“…As further detailed below, this domain also contains two potential sites for N-linked glycosylation that appear to underlie distinct biochemical properties associated with PrP Sc aggregates [67,68,74]. The primary function of PrP C is still unclear, albeit many potential biological functions including pro-apoptotic [75,76] and anti-apoptotic [77,78] roles, receptor for toxic amyloid-β (Aβ) oligomers [79][80][81][82], neuronal differentiation [83], and others have been described for it. Nevertheless, transgenic mice lacking this protein are viable, have a normal lifespan and do not show gross abnormalities [84].…”