Mutant prion protein enhances NMDA receptor activity, activates PKC, and triggers rapid excitotoxicity in mice
Joie Lin, … , John R. Yates III, Christina J. Sigurdson
Joie Lin, … , John R. Yates III, Christina J. Sigurdson
Published April 4, 2025
Citation Information: J Clin Invest. 2025;135(10):e186432. https://doi.org/10.1172/JCI186432.
View: Text | PDF
Research Article Aging Neuroscience

Mutant prion protein enhances NMDA receptor activity, activates PKC, and triggers rapid excitotoxicity in mice

Abstract

Neuronal hyperexcitability precedes synapse loss in certain neurodegenerative diseases, yet the synaptic membrane interactions and downstream signaling events remain unclear. The disordered amino terminus of the prion protein (PrPC) has been implicated in aberrant signaling in prion and Alzheimer’s disease. To disrupt neuronal interactions and signaling linked to the amino terminus, we CRISPR-engineered a knockin mouse expressing mutant PrPC (G92N), generating an N-linked glycosylation site between 2 functional motifs. Mice developed seizures and necrosis of hippocampal pyramidal neurons, similar to prion-infected mice and consistent with excitotoxicity. Phosphoproteomics analysis revealed phosphorylated glutamate receptors and calcium-sensitive kinases, including protein kinase C (PKC). Additionally, 92N-PrPC-expressing neurons showed persistent calcium influx as well as dendritic beading, which was rescued by an N-methyl-d-aspartate receptor (NMDAR) antagonist. Finally, survival of Prnp92N mice was prolonged by blocking active NMDAR channels. We propose that dysregulated PrPC-NMDAR–induced signaling can trigger an excitatory-inhibitory imbalance, spongiform degeneration, and neurotoxicity and that calcium dysregulation is central to PrPC-linked neurodegeneration.

Authors

Joie Lin, Julia A. Callender, Joshua E. Mayfield, Daniel B. McClatchy, Daniel Ojeda-Juárez, Mahsa Pourhamzeh, Katrin Soldau, Timothy D. Kurt, Garrett A. Danque, Helen Khuu, Josephina E. Ronson, Donald P. Pizzo, Yixing Du, Maxwell A. Gruber, Alejandro M. Sevillano, Jin Wang, Christina D. Orrú, Joy Chen, Gail Funk, Patricia Aguilar-Calvo, Brent D. Aulston, Subhojit Roy, Jong M. Rho, Jack D. Bui, Alexandra C. Newton, Stuart A. Lipton, Byron Caughey, Gentry N. Patrick, Kim Doré, John R. Yates III, Christina J. Sigurdson

×

Figure 5

PrPC and NMDAR localization and calcium response to NMDA in primary cortical neurons.

Options: View larger image (or click on image) Download as PowerPoint
PrPC and NMDAR localization and calcium response to NMDA in primary cort...
(A) Western blot of step gradient fractions from least to most dense (fraction 10) for GluN2B, PSD95, PrPC, as well as flotillin 1 (Flot-1) to identify fractions containing DRMs and the transferrin receptor (TfR) as a non-DRM protein control. Fraction 1 is denoted as the first fraction with a detectable amount of flotillin 1 (higher fractions are not shown). Graph x-axis labels for fractions 2–5 indicate the sum of the fraction blot intensity: fractions 2 and 3 (“2”), 4 and 5 (“3”), 6 and 7 (“4”), and 8 and 9 (“5”). n = 4 per genotype. (B) Primary rat neurons expressing mCherry-tagged WT-PrPC or 92N-PrPC immunolabeled for MAP2 (dendrites) and PSD95 (postsynaptic density) show newly expressed PrPC along dendrites. Straightened dendrites (right) show PSD95 and WT-PrPC or 92N-PrPC on spines. Scale bars: 20 μm (neurons) and 5 μm (dendrites). (C) Cortical neurons (DIV 14–16) loaded with Fura-2 AM were stimulated with low NMDA (5 μM) followed by saturating NMDA (100 μM). Graph (left) depicts the mean ± SEM for relative changes in cytosolic calcium concentration (normalized 340:380 nm ratio) over time. Quantifications show the AUC from 0–25 minutes (middle) or the mean 340:380 ratio ± SEM (right). n = 58 or 47 cells from 6 (Prnp92N) or 7 (PrnpWT) mice, respectively, from 4 (PrnpWT) to 5 (Prnp92N) separate experiments. *P < 0.05, ***P < 0.001, and ****P < 0.0001, by 2-way ANOVA with Tukey’s multiple-comparison test (A) and unpaired, 2-tailed t test with Welch’s correction (C).