Epitope-specific anti-prion antibodies upregulate apolipoprotein E and disrupt membrane cholesterol homeostasis

Abstract

The mechanisms of neuronal degeneration induced by the transformation of normal cellular prion protein (PrP C ) into disease-associated PrP Sc are not fully understood. Previous reports have demonstrated that cross-linking cellular prion protein by anti-PrP C antibodies can promote neuronal apoptosis. In this report, we now show that treatment of neuronal cells with anti-prion antibodies leads to sequestration of free cholesterol in cell membranes, significant overexpression of apolipoprotein E, and to cytoplasmic phospholipase A2 activation as well as to production of prostaglandin. These results confirm the in vivo toxic effects and indicate that anti-prion antibody treatment of neurons lead to deleterious effects. Finally, great caution should be exerted when adopting antibody-based therapy for prion diseases. INTRODUCTIONTransmissible spongiform encephalopathies or prion diseases are invariably fatal neurodegenerative diseases (Prusiner, 1994) thought to occur through the post-translational conversion of a normal cell membrane sialoglycoprotein PrP C , composed of primarily a-helical structure, into a disease-specific isoform, PrP Sc that is rich in b-sheet and partially proteinase-resistant (Prusiner, 1998).Molecular mechanisms underlying prion infection and the resulting replication/accumulation and neuronal death remain unknown (Chiesa & Harris, 2001). However, cellular prion protein, PrP C , plays an important role in these processes as PrP Sc itself is not capable of initiating the neurotoxic effects associated with prion diseases in the absence of PrP C expression, indicating that PrP C is necessary for neurodegeneration to occur (Brandner et al., 1996; Mallucci et al., 2003). Furthermore, cross-linking PrP C with specific anti-prion antibodies led to neuronal apoptosis in vivo (Jones et al., 2010;Solforosi et al., 2004) and to PrP C -dependent Fyn activation (Mouillet-Richard et al., 2000) and also led to modulation of calciumdependent protein kinase C in mice (Mazzoni et al., 2005), suggesting a role in signal transduction.Apolipoprotein E (ApoE) has been associated with the pathogenesis of Alzheimer's disease and has also been recognized in kuru plaques (Van Everbroeck et al., 2001). ApoE is a glycoprotein that plays an important role in cellular lipid transport and lipoprotein metabolism. High levels of ApoE are expressed in astrocytes, macrophages and hepatocytes, but are also seen in most peripheral tissues (Xu et al., 2006). Furthermore, increased plasma cholesterol levels have been shown to augment the progression of a number of neurodegenerative diseases including Alzheimer's disease (Puglielli et al., 2003), Parkinson's disease (Michel & Bakovic, 2007) and prion diseases (Mok et al., 2006; Kempster et al., 2007).Several studies have established the crucial role of ApoE in the transport of cholesterol (Kesäniemi et al., 1987;Mahley & Huang, 1999) and its genetic association with phospholipase A 2 (PLA 2 ) (Lung et al., 2006). The amount of free cholesterol within cell membranes is normally tig...

Cross-Linking Cellular Prion Protein Induces Neuronal Type 2-Like Hypersensitivity

“…Furthermore, inhibition of mGluRs, known to regulate histamine (34), abolished the anti-PrP antibody toxic effects (28). Taken together and in addition to the allergenic-related proteins associated with ICSM35 treatment reported by Tayebi and colleagues (21), this provides sufficient evidence to investigate the allergenic pathways potentially induced by treatment with anti-PrP antibodies which we refer to as "IgG-Mediated Neuronal Hypersensitivity". To that end, we treated mouse primary neurons (MPN), mouse neuroblastoma (N2a) and microglia (N11) cell lines with anti-PrP antibodies then assessed the neuronal allergenic proteome following mass spectrometry analysis as well as expression of neuronal FceR1a.…”

“…Furthermore, experimental treatment with anti-PrP antibodies directed against PrP C led to neuronal apoptosis based on microscopic assessments (23,24,26). Upon further analysis, some of these studies also revealed that anti-PrP antibodies induced activation of allergenic-related proteins identified by the AllerGAtlas database (21,27,28). We therefore sought molecular confirmation of a neuronal type 2-like hypersensitivity process associated with anti-PrP antibody treatment of neurons and microglia in vitro.…”

“…Prion diseases immunotherapeutics that directly target PrP Sc elimination and transient inhibition of PrP C have been efficacious in rodent models (16)(17)(18)(19)(20). However, several reports highlighted potential side-effects caused by anti-PrP C antibody treatment in vitro and in vivo (12,15,(21)(22)(23)(24)(25). Of note, the antibody-mediated 'neurotoxic' effects reported previously were made on the basis of microscopic assessments following TUNEL and/or standard histological staining but have not been characterized at a molecular level (23,24,26), with the exception of reports by Tayebi et al, Sonati et al and Goniotaki et al that confirmed the association of apolipoprotein E (APOE), cytoplasmic phospholipase A2 (cPLA2), prostaglandin (PG), calpain (CAPN) and group-I metabotropic glutamate receptors (mGluR1 and mGluR5) with anti-PrP mediated 'neurotoxicity' (21,27,28).…”

Treatment of Microglia with Anti-PrP Antibodies Induces Neuronal Allergenicity

“…Furthermore, inhibition of mGluRs, known to regulate histamine [34], abolished the anti-PrP antibody toxic effects [28]. Taken together and in addition to the allergenic-related proteins associated with ICSM35 treatment reported by Tayebi and colleagues [21], this provides su cient evidence to investigate the allergenic pathways potentially induced by treatment with anti-PrP antibodies which we refer to as "IgG-Mediated Neuronal Allergenic Toxicity".…”

“…Furthermore, experimental treatment with anti-PrP antibodies directed against PrP C led to neuronal apoptosis based on non-molecular microscopic assessments [23,24,26]. Upon further analysis, some of these studies also revealed that anti-PrP antibodies induced activation of allergenic-related proteins identi ed by the AllerGAtlas database [21,27,28]. We therefore sought molecular con rmation of a neuronal hypersensitivity/allergenic process associated with anti-PrP antibody treatment of neuroblastoma and microglia.…”