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

Abstract

BackgroundPrevious reports identified proteins associated with ‘apoptosis’ following cross-linking PrPC with motif-specific anti-PrP antibodies in vivo and in vitro. The molecular mechanisms underlying this IgG-mediated neurotoxicity and the role of the activated proteins in the apoptotic pathways leading to neuronal death has not been properly defined. Previous reports implicated a number of proteins, including apolipoprotein E, cytoplasmic phospholipase A2, prostaglandin and calpain with anti-PrP antibody-mediated ‘apoptosis’, however, these proteins are also known to play an important role in allergy. In this study, we investigated whether cross-linking PrPC with anti-PrP antibodies stimulates a neuronal allergenic response.MethodsInitially, we predicted the allergenicity of the epitope sequences associated with ‘neurotoxic’ anti-PrP antibodies using allergenicity prediction servers. We then investigated whether anti-PrP antibody treatment of mouse primary neurons (MPN), neuroblastoma cells (N2a) and microglia (N11) cell lines lead to a neuronal allergenic response.ResultsIn-Silico studies showed that both tail- and globular-epitopes were allergenic. Specifically, binding regions that contain epitopes for previously reported ‘neurotoxic’ antibodies such as ICSM18 (146-159), ICSM35 (91-110), POM 1 (138-147) and POM 3 (95-100) lead to activation of allergenic related proteins. Following direct application of anti-PrPC antibodies on N2a cells, we identified 4 neuronal allergenic-related proteins when compared with untreated cells. Furthermore, we identified 8 neuronal allergenic-related proteins following treatment of N11 cells with anti-PrPC antibodies prior to co-culture with N2a cells when compared with untreated cells. Antibody treatment of MPN or MPN co-cultured with antibody-treated N11 led to identifying 10 and 7 allergenic-related proteins when compared with untreated cells. However, comparison with 3F4 antibody treatment revealed 5 and 4 allergenic-related proteins respectively. Of importance, we showed that the allergenic effects triggered by the anti-PrP antibodies were more potent when antibody-treated microglia were co-cultured with the neuroblastoma cell line. Finally, co-culture of N2a or MPN with N11-treated with anti-PrP antibodies resulted in significant accumulation of NO and IL6 but not TNF-α in the cell culture media supernatant.ConclusionsThis study showed for the first time that anti-PrP antibody binding to PrPC triggers a neuronal hypersensitivity response and highlights the important role of microglia in triggering an IgG-mediated neuronal hypersensitivity response. Moreover, this study provides an important impetus for including allergenic assessment of therapeutic antibodies for neurodegenerative disorders to derive safe and targeted biotherapeutics.

Therapeutic anti‐amyloid β antibodies cause neuronal disturbances

“…This confirms that the activation of neurons only or simultaneous activation of both neurons and microglia following treatment with anti-Aβ antibody leads to increased TNF-α expression while activation of microglia only following anti-Aβ antibody treatment before coculture with neurons leads to decreased TNF-α expression. Of interest, in a previous study, we did not find significant expression of TNF-α when we co-cultured the anti-PrP antibody-treated microglia with mouse primary neurons (Adhikari et al, 2021a). TNF-α is considered as a key mediator of the atopic allergy inflammation (Ying et al, 1991), human lung allergic reactions (Casale et al, 1996), and allergic rhinitis (Iwasaki et al, 2003;Mo et al, 2011) highlighting its association with hypersensitivity responses.…”

“…We have previously shown that treatment of neuronal cell lines with anti-PrP monoclonal antibodies leads to a hypersensitivity-like reaction (Adhikari et al, 2021a). We wanted to investigate whether the 18 common allergy related proteins were involved in a type 1 and/or 2 hypersensitivity reaction.…”

“…8 were upregulated and were downregulated amongst the 16 allergenic-related AllgDMTAβ17-23, when compared to untreated control (Table S10). Over 82% of the allergy related protein (n=14; TYK2, PCNA, CTSD, ANXA1, MYLK, DSG1, PPARD, PIK3C2A, PRDX1, PGK1, ALB, EEF1A1, HSP90B1, ACTB) were common to AllgDMTAβ1-6 and AllgDMTAβ17-23 (Table S6; Figure 3A); notably, 8 out 14 (MYLK, PGK1, PRDX1, TYK2, PCNA, ANXA1, PPARD, ALB) are involved in pro-inflammatory responses (Chu et al, 2016;Kullmann et al, 1996;Magzal et al, 2017;Moraes et al, 2018;Poelzl et al, 2021;Wang et al, 2015a;Youssef and Badr, 2004;Zhao et al, 2016) and 7 out of 14 (MYLK, PPARD, PRDX1, PGK1, ALB, ACTB, EEF1A1) have previously been shown to be involved in the pathogenesis of allergy (Adhikari et al, 2021a). Reactome pathway associated with allergenic responses such as IL-4 and IL-13 signalling (Gour and Wills-Karp, 2015) was found for AllgDATAβ17-23, but not for AllgDATAβ1-6 in overrepresentation test by PANTHER classification system (Table S11).…”

“…Moreover, 9 proteins (MYLK, ACTB, ANXA1, ITGB4, PPARD, PRDX1, PGK1, EEF1A1, PPIA) related to both AllgDMATAβ1-6 and AllgDMATAβ17-23 were previously shown to be involved in the pathogenesis of allergy (Adhikari et al, 2021a;Liu et al, 2018a;Ng et al, 2011). Reactome pathway associated with allergenic response such as IL-4 and IL-13 signaling (Gour and Wills-Karp, 2015) was found for AllgDMAT Aβ1-6, but not for AllgDMAT Aβ17-23 in overrepresentation test by PANTHER classification system (Table S18).…”

“…The protocol for direct antibody treatment (DAT) was adapted from the previously described methods with slight modifications (Adhikari et al, 2021a).…”