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. 2020 Mar 2;130(3):1350-1362.
doi: 10.1172/JCI131564.

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

Alejandro M Sevillano  1 , Patricia Aguilar-Calvo  1 , Timothy D Kurt  1 , Jessica A Lawrence  1 , Katrin Soldau  1 , Thu H Nam  1 , Taylor Schumann  1 , Donald P Pizzo  1 , Sofie Nyström  2 , Biswa Choudhury  3 , Hermann Altmeppen  4 , Jeffrey D Esko  3 , Markus Glatzel  4 , K Peter R Nilsson  2 , Christina J Sigurdson  1   5   6
Affiliations

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

Alejandro M Sevillano et al. J Clin Invest. .

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.

Keywords: Glycobiology; Infectious disease; Neurodegeneration; Neuroscience; Prions.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. PrP180Q/196Q traffics similarly to WT PrPC in primary neurons and in mice.
(A) Representative Western blot of PNGase-F–treated brain extracts from age-matched Prnp180Q/196Q and WT mice reveal similar PrPC expression levels (quantified in right panel) (100–250 day old mice); n = 4/group. (B) PrP immunocytochemistry shows that unglycosylated PrP180Q/196Q traffics to neuronal processes in primary cortical neurons, as does PrP in WT neurons; n = 3 experiments. Scale bars: 10 μm. (C) Representative Western blots of phospholipase C–cleaved (PIPLC-cleaved) PrP180Q/196Q and WT PrP from the surface of cortical neurons show that surface PrPC levels are similar (media); n = 3 experiments. The additional band in the media (~23 kDa) may be a cleaved form of PrP. (D) PrP180Q/196Q and WT PrPC, together with flotillin, localize to detergent-resistant membranes in the brain; n = 3/group. Unpaired, 2-tailed Student’s t test, no significant differences (A and C).
Figure 2
Figure 2. Survival and histopathologic lesions in prion-infected Prnp180Q/196Q and WT mice.
(A) For mCWD-infected mice, there was no difference in survival times. In contrast, 22L- and ME7-infected Prnp180Q/196Q mice showed prolonged survival compared with WT mice. By the second and third passage, ME7-infected Prnp180Q/196Q mice showed a 100% attack rate and similar survival times as the WT mice. (B) Brain sections stained with H&E or immunolabelled for PrP revealed severe spongiform degeneration and plaque-like deposits (RML) or plaques (22L, ME7, and mCWD, arrows) in prion-infected Prnp180Q/196Q mice. Less spongiform degeneration was noted in the mCWD-infected mice. Scale bar: 50 μm. (C) Lesion scores of spongiform change, gliosis, and PrPSc in 8 brain regions differed significantly in RML-, 22L-, and ME7-infected Prnp180Q/196Q mice compared with WT mice. Cerebellum was consistently less severely affected in the Prnp180Q/196Q mice. 1-dorsal medulla, 2-cerebellum, 3-hypothalamus, 4-medial thalamus, 5-hippocampus, 6-septum, 7-cerebral cortex, and 8-cerebral peduncle. (D) Right panel shows that PrPSc levels differed in the cerebellum (RML versus 22L; 0.2 ± 0.2 versus 1.2 ± 0.2, respectively) and hypothalamus (22L versus ME7; 1.8 ± 0.5 versus 2.3 ± 0.7, respectively) of the Prnp180Q/196Q mice. (E) ME7 and mCWD plaques bind Congo red in Prnp180Q/196Q brains. Scale bar: 50 μm. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; 1-way ANOVA with Tukey’s test (A), 2-way ANOVA with Bonferroni’s post hoc test (C and D). RML: n = 5–8 mice per group; ME7: n = 5–7 mice per group; 22L: n = 5–8 mice per group, mCWD: n = 5–9 mice per group, mock: n = 4–8 mice per group.
Figure 3
Figure 3. Comparison of PK resistance, solubility, and stability of RML, 22L, ME7, and mCWD prions in Prnp180Q/196Q and WT brain samples.
(A) Western blots of brain homogenates reveal PK-resistant unglycosylated PrPSc in RML-, 22L-, ME7-, and mCWD-infected Prnp180Q/196Q mice. (B) Western blots of soluble (S, supernatant) and insoluble (P, pellet) PrPSc show similar levels of insoluble PrPSc in Prnp180Q/196Q and WT brains on first passage, but significantly higher levels in ME7-infected Prnp180Q/196Q brains on further passage. The GPI-ME7 was included to compare samples with a known highly fibrillar prion; n = 2 (ME7, first passage only) and n = 3–7/strain (all other strains, including ME7, second and third passage). (C) Representative PrPSc stability curves from single mouse brain samples run in triplicate. The right graph shows the guanidine hydrochloride concentration at which half the PrPSc remains ([GndHCl]2) and reveals no significant differences in the stability of RML or 22L, but a significantly more stable ME7 prion after passage in Prnp180Q/196Q mice. Plotted (right graph) are the mean and SEM for n = 2 (ME7-infected Prnp180Q/196Q), n = 3 (WT), or n = 4 (RML- and 22L-infected Prnp180Q/196Q) mice per strain, each run in triplicate. **P ≤ 0.01, 1-way ANOVA with Tukey’s test (B).
Figure 4
Figure 4. Fluorescence lifetime (FLIM) decay of h-FTAA bound to prion aggregates.
(A) Fluorescence lifetime images and (B and C) intensity-weighted mean lifetime (ti) distributions of h-FTAA–stained RML, 22L, and ME7 prion plaques in Prnp180Q/196Q (red) or WT (blue) brain show that the FLIM decay curves in Prnp180Q/196Q brain differ from those in WT brain and reveal 3 different aggregate conformers in prion-infected Prnp180Q/196Q mice. The decay data were collected with the excitation wavelength set at 490 nm. The color bar represents lifetimes from 200 ps (orange) to 1000 ps (blue) and the images are color coded according to the representative lifetime. Decay curves were collected from 5–10 prion deposits from a minimum of n = 3 mice/strain. Scale bar: 20 µm.
Figure 5
Figure 5. Increased ADAM10-cleaved PrPSc in prion-infected Prnp180Q/196Q mice.
(A) Western blots reveal 22L- and ME7-infected Prnp180Q/196Q mice harbor higher levels of ADAM10-cleaved PrPSc in the brain compared with WT mice; n = 3/group. Note that the PK-digested ADAM10-cleaved PrP runs at a lower molecular weight due to lack of the GPI-anchor and terminal 3 amino acids. *P ≤ 0.05, unpaired, 2-tailed Student’s t test. (B) Western blots show that uninfected Prnp180Q/196Q and WT mice have similar levels of ADAM10-cleaved/total PrPC in the brain; n = 5 WT and 11 Prnp180Q/196Q mice. (C) Brain sections immunolabeled for PrP with SAF84 (labelled PrP) or with sPrPG228 antibody (labelled ADAM10-cleaved PrP) reveal ADAM10-cleaved PrP localizes to plaque-like and plaque deposits in ME7-infected Prnp180Q/196Q, mCWD-infected Prnp180Q/196Q, and WT brain sections. Note that the diffuse aggregates are not labelled by the sPrPG228 antibody. Cortex (ME7-infected WT), thalamus (ME7-infected Prnp180Q/196Q), hippocampus (mCWD-infected Prnp180Q/196Q), and corpus callosum (WT-infected mCWD) are shown. Scale bar: 50 μm.
Figure 6
Figure 6. PrP glycans hinder binding to HS.
(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).
Figure 7
Figure 7. Knockin mice expressing triglycosylated PrP show a reversal in the clinical, histologic, and biochemical phenotype when infected with the plaque-forming prion strain, mCWD.
(A) Survival curves of Prnp187N mice inoculated with mCWD prions show a profound decrease in the survival time as compared with WT mice; n = 5–9/group. (B) The spongiform degeneration is similar in Prnp187N mice as compared with WT mice; however, PrP deposits switched from the typical large, dense plaques in WT mice to primarily diffuse aggregates, with occasional rare small plaques in the corpus callosum (lower panel). (C) HS immunolabelling of the diffuse aggregates or plaque-like structures was not evident in the Prnp187N brain. (D) Western blot reveals that the PrPSc in the Prnp187N brain is no longer composed of ADAM10-cleaved PrPSc that predominated in the original mCWD strain in tga20 mice (overexpressed WT mouse PrP); n = 3–5/mouse strain. (E) Western blot of soluble versus insoluble PK-resistant PrPSc reveals a decrease in insoluble PrPSc in the Prnp187N mice; n = 4–7 WT or Prnp187N mice and n = 2 Tg (GPI-PrP) positive control brain samples. (F) Representative PrPSc stability assay and quantification show that the resistance to unfolding in chaotropes is similar for PrPSc aggregates in the mCWD-infected Prnp187N and WT brain; n = 2/group. (G) Western blot of Prnp187N mice inoculated intratongue (IT) or intracerebrally (IC) with mCWD-passaged Prnp187N brain homogenate shows that all clinically terminal mice (5/5 mice) harbored PrPSc in the brain. *A longer exposure of one sample is shown on the right. **P ≤ 0.01, ***P ≤ 0.001; 1-way ANOVA with Tukey’s test (A and E), unpaired, 2-tailed Student’s t test (D). Scale bar: 100 μm (B and C).
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