The toxicity of antiprion antibodies is mediated by the flexible tail of the prion protein

Abstract

Prion infections cause lethal neurodegeneration. This process requires the cellular prion protein (PrP(C); ref. 1), which contains a globular domain hinged to a long amino-proximal flexible tail. Here we describe rapid neurotoxicity in mice and cerebellar organotypic cultured slices exposed to ligands targeting the 1 and 3 helices of the PrP(C) globular domain. Ligands included seven distinct monoclonal antibodies, monovalent Fab1 fragments and recombinant single-chain variable fragment miniantibodies. Similar to prion infections, the toxicity of globular domain ligands required neuronal PrP(C), was exacerbated by PrP(C) overexpression, was associated with calpain activation and was antagonized by calpain inhibitors. Neurodegeneration was accompanied by a burst of reactive oxygen species, and was suppressed by antioxidants. Furthermore, genetic ablation of the superoxide-producing enzyme NOX2 (also known as CYBB) protected mice from globular domain ligand toxicity. We also found that neurotoxicity was prevented by deletions of the octapeptide repeats within the flexible tail. These deletions did not appreciably compromise globular domain antibody binding, suggesting that the flexible tail is required to transmit toxic signals that originate from the globular domain and trigger oxidative stress and calpain activation. Supporting this view, various octapeptide ligands were not only innocuous to both cerebellar organotypic cultured slices and mice, but also prevented the toxicity of globular domain ligands while not interfering with their binding. We conclude that PrP(C) consists of two functionally distinct modules, with the globular domain and the flexible tail exerting regulatory and executive functions, respectively. Octapeptide ligands also prolonged the life of mice expressing the toxic PrP(C) mutant, PrP(Δ94-134), indicating that the flexible tail mediates toxicity in two distinct PrP(C)-related conditions. Flexible tail-mediated toxicity may conceivably play a role in further prion pathologies, such as familial Creutzfeldt-Jakob disease in humans bearing supernumerary octapeptides. (Fig. 1b). None of three high-affinity antibodies to the octapeptide repeats (OR, residues 50-90 embedded within the FT) were neurotoxic (Fig. 1b). Antibodies POM3 and D13, which bind the "charged cluster-2" 11 (CC2, residues 95-110), were innocuous at 67 nM but neurotoxic at 200 nM (Fig. 1b). None of the tested antibodies were toxic to Prnp o/o COCS ( Supplementary Fig. 2a). The identity of the targeted epitopes appeared to be a better predictor of PrP C antibody toxicity than their affinity to PrP C , suggesting that neurotoxicity resulted from the interaction of antibodies with specific PrP C domains (Supplementary Table 2).The mechanisms of neurotoxicity were further explored using POM1, a highly toxic antibody targeting the GD. Wild-type (wt) and tga20 COCS lost most granule cells (CGC) within 28 and 14 days post-exposure (dpe) to POM1, respectively (Fig. 2a-c). Controls included POM1-treated Prnp o/o COCS 12 , t...

A neuroprotective role for microglia in prion diseases

“…Not only did we confirm our previous study that depletion of microglia in COCS leads to enhanced PrP Sc deposition, but we also detected a dramatic acceleration in prion-induced neurodegeneration of the cerebellar granule layer. Notably, microglia ablation does not affect antiprion antibody-induced neurotoxicity (Sonati et al, 2013), suggesting that microglia exert neuroprotection by reducing the prion load rather than by acting on downstream events shared by prion infections and antiprion antibody treatment (Herrmann et al, 2015). The notion that microglia protects the brain against prion-induced neurotoxicity was confirmed in vivo using CD11b-HSV TK transgenic mice.…”

In Vivo Longitudinal 1H MRS Study of Transgenic Mouse Models of Prion Disease in the Hippocampus and Cerebellum at 14.1 T

“…The PrP D32-121 mice suffer from ataxia and seem to have different pathology from prion infections characterized by astrogliosis and cerebellar granule cell loss mainly in the cerebellum [5], even thought our measurements showed modifications which are very similar to the human prion disease (decrease NAA in both hippocampus and cerebellum and increased Ins in cerebellum). In addition, some recent transcriptomic analyses showed that there is considerable overlap between the mice used in our study, the exposure to antibodies targeting the cellular prion protein and the pathologies related to prions, namely prion infections [43,44].…”

PrPSc-specific antibodies do not induce prion disease or misfolding of PrPCin highly susceptible Tga20 mice

“…A recent extensive study found that monoclonal antibodies binding to the globular domain of PrP C elicit cytotoxic signaling mediated by the N-terminal octapeptide repeat region. 13 The antibody that caused the most dramatic neuronal loss ex vivo and when injected into the brains of tga20 mice was one that binds a region of native PrP C comprising residues 138-147 and 204, 208, and 212. 13 The SN6b antigenic region we targeted is distinct from this sequence (159-174, by the same numbering), comprising a region that is buried in PrP C .…”

“…13 The antibody that caused the most dramatic neuronal loss ex vivo and when injected into the brains of tga20 mice was one that binds a region of native PrP C comprising residues 138-147 and 204, 208, and 212. 13 The SN6b antigenic region we targeted is distinct from this sequence (159-174, by the same numbering), comprising a region that is buried in PrP C . These recent results highlight the importance of directing antibodies specifically against misfolded PrP Sc , ELISa titers are expressed as the reciprocal of the highest serum dilution resulting in a reading exceeding 2 standard deviations above the negative control (pre-immune).…”

“…Circulating antibodies against PrP C may trigger complement-dependent lysis of cells or induce autoimmune disease by breaking tolerance to this molecule. Alternatively, antibodies against PrP C may impair or alter the function of this normal cellular protein by triggering apoptosis in neurons, 12 superoxide mediated cytotoxicity, 13 or activation of inappropriate cell signaling cascades.…”