Published: July 16, 2020
AbstractPrion protein (PrP) mutations are linked to genetic prion diseases, a class of phenotypically heterogeneous neurodegenerative disorders with invariably fatal outcome. How mutant PrP triggers neurodegeneration is not known. Synaptic dysfunction precedes neuronal loss but it is not clear whether, and through which mechanisms, disruption of synaptic activity ultimately leads to neuronal death. Here we show that mutant PrP impairs the secretory trafficking of AMPA receptors (AMPARs). Specifically, intracellular retention of the GluA2 subunit results in synaptic exposure of GluA2-lacking, calcium-permeable AMPARs, leading to increased calcium permeability and enhanced sensitivity to excitotoxic cell death. Mutant PrPs linked to different genetic prion diseases affect AMPAR trafficking and function in different ways. Our findings identify AMPARs as pathogenic targets in genetic prion diseases, and support the involvement of excitotoxicity in neurodegeneration. They also suggest a mechanistic explanation for how different mutant PrPs may cause distinct disease phenotypes.
Genetic prion diseases are degenerative brain disorders caused by mutations in the gene encoding the prion protein (PrP). Different PrP mutations cause different diseases, including Creutzfeldt-Jakob disease, fatal familial insomnia and Gerstmann-Sträussler-Scheinker syndrome. How mutant PrP causes neuronal death and how different mutants encode distinct disease phenotypes is not known. Here we show that mutant PrP alters the subunit composition of glutamate AMPA receptors, promoting cell surface exposure of GluA2-lacking, calcium-permeable receptors, ultimately increasing neuronal vulnerability to excitotoxic cell death. We also demonstrate that the underlying molecular mechanism is the formation of a GluA2 subunit-PrP complex which is retained in the neuronal secretory pathway. PrP mutants associated with clinically different genetic prion diseases have distinct effects on GluA2 trafficking, depending on their tendency to misfold and aggregate in different intracellular organelles, indicating a possible contribution of this mechanism to the disease phenotype.
Citation: Ghirardini E, Restelli E, Morini R, Bertani I, Ortolan D, Perrucci F, et al. (2020) Mutant prion proteins increase calcium permeability of AMPA receptors, exacerbating excitotoxicity. PLoS Pathog 16(7):
https://doi.org/10.1371/journal.ppat.1008654Editor: Neil A. Mabbott, University of Edinburgh, UNITED KINGDOMReceived: January 1, 2020; Accepted: May 26, 2020; Published: July 16, 2020Copyright: © 2020 Ghirardini et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Data Availability: All relevant data are within the manuscript and its Supporting Information files.Funding: This work was supported by grants from Telethon Italy (GGP12115 to M.M. and R.C.), “Fondazione Cariplo” (2012-0560 to R.C. and M.M.), and the Italian Ministry of Health (RF- 2010-2314035 to R.C.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing interests: The authors have declared that no competing interests exist.
IntroductionSynaptic dysfunction is an early process in prion disease, preceding synapse loss and neuronal death. Understanding the mechanisms of primary changes in synaptic function that lead to irreversible neurodegeneration has important implications for therapy. We describe morphological and functional alterations in neurons expressing prion protein (PrP) mutations associated with genetic prion disease, indicating a neurotoxic mechanism involving α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs).
Genetic prion diseases are rare and currently untreatable neurodegenerative disorders linked to mutations in the PRNP gene, encoding PrP, on chromosome 20 . Approximately 70 pathogenic PRNP variants have been reported (see http://www.cureffi.org/2015/01/13/list-of-reportedly-pathogenic-prnp-variants/ for an up-to-date list), including missense mutations, expansions or deletions of a repeated sequence encoding an octapeptide motif in the N-terminal region, and stop codon mutations resulting in premature protein truncations.
One striking feature of genetic prion diseases is their phenotypic heterogeneity. Different PRNP mutations are associated with distinct disease subtypes, including genetic Creutzfeldt-Jakob disease (gCJD), fatal familial insomnia (FFI) and Gerstmann-Sträussler-Scheinker (GSS) syndrome . The disease presentation can be influenced by PRNP polymorphism at codon 129, where either methionine (M) or valine (V) may be present. A noteworthy example is prion disease linked to the substitution of asparagine (N) for aspartic acid (D) at codon 178 which, depending on the amino acid at codon 129 on the mutant allele, segregates with either FFI (D178N/M129), primarily characterized by severe sleep disorders and autonomic dysfunction, or CJD178 (D178N/V129), clinically identified by global cortical dementia and motor abnormalities .
How mutant PrP causes neuronal death and how sequence variants of PRNP encode the information to specify distinct disease phenotypes is a central question in prion biology. PrP is a cell membrane glycoprotein highly expressed by neurons in the CNS. PrP is located at both pre- and post-synaptic sites, and there is ample evidence indicating a modulatory role in synaptic transmission which may be negatively affected by pathogenic mutations [3,4]. We previously found that PrP interacts physically with the α2δ-1 subunit of voltage-gated calcium channels (VGCCs) which govern depolarization-induced neurotransmitter release . Mutant PrP misfolding and intracellular retention of α2δ-1 impaired synaptic delivery of VGCCs, and glutamatergic neurotransmission is disrupted in transgenic (Tg) mouse models of GSS and CJD178 .
PrP engages functional interactions with glutamate receptors, including AMPARs [6,7]. AMPARs are tetrameric, cation-permeable ionotropic receptors, which mediate the largest part of fast excitatory neurotransmission in the brain. Upon binding of glutamate, the pore opening allows the influx of Na+ ions and the efflux of K+ ions to depolarize the postsynaptic compartment. Depending on the subunit composition, AMPARs also allow influx of Ca2+. In the adult brain, the majority of GluA2-containing AMPARs are largely Ca2+-impermeable, due to a RNA editing that replaces a glutamine with a positively charged arginine in the pore-forming region of the assembled channel, thus preventing Ca2+ influx . In contrast, GluA2-lacking AMPARs are Ca2+-permeable, and have higher single-channel conductance . The Ca2+ permeability of AMPARs is thought to have important consequences for plasticity as well as cell viability (reviewed in ).
We explored the contribution of AMPAR dysfunction in genetic prion diseases by morphological and functional analyses in primary neurons from Tg mice expressing mouse homologs of the CJD178 and FFI mutations (moPrP D177N/V128 and moPrP D177N/M128), and of a nine-octapeptide repeat insertion (moPrP PG14) associated with GSS [11–14]. Membrane delivery of the GluA2 subunit of AMPARs was impaired in a PrP mutation-specific manner, with alterations in the structure, function and plasticity of the excitatory synapses. In addition, intracellular retention of GluA2 modified the subunit composition of AMPARs in the mutant neurons, increasing the number of GluA2-lacking, calcium-permeable AMPARs and resulting in greater calcium permeability and more vulnerability to excitotoxic cell death. These results cast fresh light on the mechanisms of neurodegeneration and phenotypic variability in genetic prion diseases.
Synaptic structure and function are altered in hippocampal neurons expressing the FFI and CJD178 mutations
Dendritic spine loss has been described in prion-infected mice and organotypic cerebellar cultures, and in cultured hippocampal neurons exposed to PrPSc, the infectious isoform of PrP [15–18]. We investigated whether synaptic alterations were also detectable in neurons expressing FFI or CJD178 PrP. We analyzed the morphology of excitatory synapses in primary cultures of hippocampal neurons from wild-type (WT), FFI and CJD mice at 13–15 days in vitro (DIV) after transfection with a plasmid encoding EGFP to allow visualization of dendritic spines. Both FFI and CJD neurons had less spine density than WT cells (Fig 1A and 1C). Consistent with this, the co-localization between the pre- and post-synaptic markers Bassoon and Shank2 was significantly lower in the mutant neurons (Fig 1B and 1D). The number of Bassoon puncta per μm was not altered in the mutant neurons, indicating that the defect involves mainly the post-synaptic compartment (Fig 1E and 1F).
Fig 1. Synaptic structure and activity are altered in FFI and CJD hippocampal neurons.(A) Confocal representative images of WT, CJD and FFI neurons transfected with EGFP and (C) quantification of total spine density; 46–62 dendrites from 25–30 neurons for each condition. Kruskal-Wallis test followed by Dunn’s multiple comparison test: **p Read More