Prion protein glycans reduce intracerebral fibril formation and spongiosis in prion disease
Alejandro M. Sevillano, Patricia Aguilar-Calvo, Timothy D. Kurt, Jessica A. Lawrence, Katrin Soldau, Thu H. Nam, Taylor Schumann, Donald P. Pizzo, Sofie Nyström, Biswa Choudhury, Hermann Altmeppen, Jeffrey D. Esko, Markus Glatzel, K. Peter R. Nilsson, Christina J. Sigurdson
Alejandro M. Sevillano, Patricia Aguilar-Calvo, Timothy D. Kurt, Jessica A. Lawrence, Katrin Soldau, Thu H. Nam, Taylor Schumann, Donald P. Pizzo, Sofie Nyström, Biswa Choudhury, Hermann Altmeppen, Jeffrey D. Esko, Markus Glatzel, K. Peter R. Nilsson, Christina J. Sigurdson
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Research Article Infectious disease Neuroscience

Prion protein glycans reduce intracerebral fibril formation and spongiosis in prion disease

Abstract

Posttranslational modifications (PTMs) are common among proteins that aggregate in neurodegenerative disease, yet how PTMs impact the aggregate conformation and disease progression remains unclear. By engineering knockin mice expressing prion protein (PrP) lacking 2 N-linked glycans (Prnp180Q/196Q), we provide evidence that glycans reduce spongiform degeneration and hinder plaque formation in prion disease. Prnp180Q/196Q mice challenged with 2 subfibrillar, non–plaque-forming prion strains instead developed plaques highly enriched in ADAM10-cleaved PrP and heparan sulfate (HS). Intriguingly, a third strain composed of intact, glycophosphatidylinositol-anchored (GPI-anchored) PrP was relatively unchanged, forming diffuse, HS-deficient deposits in both the Prnp180Q/196Q and WT mice, underscoring the pivotal role of the GPI-anchor in driving the aggregate conformation and disease phenotype. Finally, knockin mice expressing triglycosylated PrP (Prnp187N) challenged with a plaque-forming prion strain showed a phenotype reversal, with a striking disease acceleration and switch from plaques to predominantly diffuse, subfibrillar deposits. Our findings suggest that the dominance of subfibrillar aggregates in prion disease is due to the replication of GPI-anchored prions, with fibrillar plaques forming from poorly glycosylated, GPI-anchorless prions that interact with extracellular HS. These studies provide insight into how PTMs impact PrP interactions with polyanionic cofactors, and highlight PTMs as a major force driving the prion disease phenotype.

Authors

Alejandro M. Sevillano, Patricia Aguilar-Calvo, Timothy D. Kurt, Jessica A. Lawrence, Katrin Soldau, Thu H. Nam, Taylor Schumann, Donald P. Pizzo, Sofie Nyström, Biswa Choudhury, Hermann Altmeppen, Jeffrey D. Esko, Markus Glatzel, K. Peter R. Nilsson, Christina J. Sigurdson

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Figure 6

PrP glycans hinder binding to HS.

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PrP glycans hinder binding to HS. (A) Immunoblots of heparin affinity ch...
(A) Immunoblots of heparin affinity chromatography experiments assessing variably glycosylated PrP. Relative levels of PrP from each elution are shown in the graphs. Differences in the binding affinity are most notable in the 0.5 M NaCl elution (red arrow). Asterisk color indicates the mutants with significant differences. The triglycosylated PrP isoform level was low in RK13 cells; n = 3–4 experiments. (B) Among the WT PrP glycoforms, diglycosylated PrP has a lower heparin affinity than mono- or unglycosylated PrP (unbound is PrP in the flow-through); n = 5 experiments, 4 also included in A. (C) Affinity chromatography of the soluble brain fraction reveals that total and ADAM10-cleaved PrP180Q/196Q have significantly higher heparin affinity than the corresponding WT PrP. PrP180Q/196Q shows a second band (blue arrow) that corresponds to ADAM10-cleaved PrPC. (D) Quantification of PrP shown in C; n = 3/strain. (E) ADAM10-cleaved PrP has a higher heparin-binding affinity than total PrP for both WT PrP and PrP180Q/196Q. (F) Immunolabelling reveals HS colocalizes to ME7 plaques in the Prnp180Q/196Q brain only and to mCWD plaques in both the WT and Prnp180Q/196Q brain; n = 4/strain. Scale bar: 50 μm. (G) LC-MS reveals approximately 6-fold more HS bound to unglycosylated ME7 PrPSc than to highly glycosylated ME7 PrPSc (WT); n = 3/strain. (H) Composition analysis of HS bound to purified PrPSc (ME7) reveals less N-sulfated (NS) and 6-O sulfated (6-O) HS bound to unglycosylated (Prnp180Q/196Q) as compared with glycosylated (WT) PrPSc; n = 3/group. (I) The overall HS composition in ME7-infected Prnp180Q/196 and WT whole-brain lysates are similar; n = 3/group. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001; 2-way ANOVA with Bonferroni’s post hoc test (A, D, E, and H). *P < 0.05; 1-way ANOVA with Tukey’s test (B). **P ≤ 0.01, unpaired, 2-tailed Student’s t test (G).