Shortening heparan sulfate chains prolongs survival and reduces parenchymal plaques in prion disease caused by mobile, ADAM10-cleaved prions

Abstract

Cofactors are essential for driving recombinant prion protein into pathogenic conformers. Polyanions promote prion aggregation in vitro, yet the cofactors that modulate prion assembly in vivo remain largely unknown. Here we report that the endogenous glycosaminoglycan, heparan sulfate (HS), impacts prion propagation kinetics and deposition sites in the brain. Exostosin-1 haploinsufficient (Ext1^+/-) mice, which produce short HS chains, show a prolonged survival and a redistribution of plaques from the parenchyma to vessels when infected with fibrillar prions, and a modest delay when infected with subfibrillar prions. Notably, the fibrillar, plaque-forming prions are composed of ADAM10-cleaved prion protein lacking a glycosylphosphatidylinositol anchor, indicating that these prions are mobile and assemble extracellularly. By analyzing the prion-bound HS using liquid chromatography-mass spectrometry (LC-MS), we identified the disaccharide signature of HS differentially bound to fibrillar compared to subfibrillar prions, and found approximately 20-fold more HS bound to the fibrils. Finally, LC-MS of prion-bound HS from human patients with familial and sporadic prion disease also showed distinct HS signatures and higher HS levels associated with fibrillar prions. This study provides the first in vivo evidence of an endogenous cofactor that accelerates prion disease progression and enhances parenchymal deposition of ADAM10-cleaved, mobile prions.

Keywords: ADAM10 cleavage; Amyloid; Glycosaminoglycans; Neurodegeneration.

Fig. 1

Ext1^+/+ and Ext1^+/− mice infected with subfibrillar prions show similar survival times, brain lesions, and biochemical properties. a A comparison of survival times revealed no differences in RML-infected Ext1^+/− mice and a modest delay in ME7-infected Ext1^+/− mice as compared to the Ext1^+/+ mice. b Brain sections immunolabelled for PrP and GFAP, or stained with hematoxylin and eosin (HE), show indistinguishable prion aggregate distribution and morphology (arrowheads), spongiform degeneration (arrows), and astrogliosis in Ext1^+/+ and Ext1^+/− brains. c Lesion profiles of RML- and ME7-infected Ext1^+/+ and Ext1^+/− mice (1-dorsal medulla, 2-cerebellum, 3-hypothalamus, 4-medial thalamus, 5-hippocampus, 6-septum, and 7-cerebral cortex) are almost superimposable. d Electrophoretic mobility and e glycoprofiles of RML and ME7 strains in Ext1^+/+ and Ext1^+/− mice. The RML and ME7 inocula were loaded for comparison (first lane). *P< 0.05, Log-rank (Mantel-Cox) test (panel a). Cerebral cortex (RML) and hippocampus (ME7) shown in panel b. Scale bar = 50 μm (panel b). RML: n=6-7 mice/group; ME7: n=4-5 mice/group.

Fig. 2

mCWD-infected tga20^+/−Ext1^+/− mice show prolonged survival times and altered plaque distribution. a mCWD-infected tga20^+/−Ext1^+/− (“Ext1^+/−”) mice show a significant delay in survival time. b PrP immunolabelled brain sections show mCWD prion plaques in the corpus callosum (CC) of Ext1^+/+ mice, whereas in Ext1^+/− mice, plaques are present in other brain regions including thalamus (TH), and velum interpositum (VI). The plaque morphology was unchanged. c The distribution of mCWD plaques varied between the Ext1^+/+ and Ext1^+/− mice, as fewer Ext1^+/− mice developed plaques in the corpus callosum (CC), whereas more Ext1^+/− mice developed plaques in the basal ganglia (BG) and thalamus (TH) (HP: hippocampus, HT: hypothalamus, CX: cerebral cortex, and CB: cerebellum). d Ext1^+/− mice show fewer plaques in the corpus callosum and more plaques in the velum interpositum and cerebellum. e Lesion profiles comparing spongiform degeneration, astrogliosis, and PrP^Sc deposition are similar in Ext1^+/+ and Ext1^+/− mice (1-medulla, 2-cerebellum, 3-hypothalamus, 4-medial thalamus, 5-hippocampus, 6-septum, 7-cerebral cortex, 8-cerebral peduncle and 9-cerebellar peduncle). f mCWD plaques are congophilic in Ext1^+/+ and Ext1^+/− mice [shown is cerebellum (Ext1^+/+) and hippocampus (Ext1^+/−)]. g Dual immunostaining for PrP and endothelial cells (CD31) shows typical non-vascular plaques in the corpus callosum (upper panels), perivascular plaques in the basal ganglia, and periventricular plaques adjacent to the lateral ventricle (middle panels, white arrowheads show blood vessel; bottom panels, V=ventricle). Ext1^+/− mice show fewer parenchymal plaques in the corpus callosum and more vascular plaques in the velum interpositum and cerebellum. h Iba1 immunolabelling of activated microglia shows similar clustering of activated microglia around mCWD plaques in Ext1^+/+ and Ext1^+/− brain sections (arrowheads). Quantification of activated microglia shown in upper panel (for corpus callosum Ext1^+/+: n= 5 and Ext1^+/−: n= 3; for cerebellum Ext1^+/+: n= 3 and Ext1^+/−: n= 4). Scale bars = 500 μm (left) and 50 μm (right) (panel b), 50 μm (panels f and g), and 1 mm (panel h). *P< 0.05, Log-rank (Mantel-Cox) test (panel a). *P< 0.05, **P< 0.01, ***P< 0.001, Fisher's exact test (panel c), two-way ANOVA with Bonferroni’s post test (panels d and g), unpaired, 2-tailed Student’s t test (panel e). Ext1^+/+: n= 11 mice; Ext1^+/−: n=16 mice.

Fig. 3

mCWD prion conformation is similar in mice having long or short HS chains. a h-FTAA fluorescence life-time decay of mCWD prion plaques in tga20^+/−Ext1^+/+ (“Ext1^+/+”) and tga20^+/−Ext1^+/− (“Ext1^+/−”) brain sections are similar. b mCWD electrophoretic mobility and glycoprofile are also similar in Ext1^+/+ and Ext1^+/− mice. c Representative example of PrP^Sc aggregate stability as measured by GdnHCl denaturation in mCWD-infected Ext1^+/+ and Ext1^+/− mice. [GdnHCl]_1/2 values shown for mCWD in Ext1^+/+ and Ext1^+/− brain (n=4 mice/strain; each run in triplicate).

Fig. 4

ADAM10-cleaved and full length GPI-anchorless prions bind HS. a Schematic representation of ADAM10 cleavage at mouse PrP residue 228 shows the release of shed PrP lacking the GPI-anchor and three C-terminal amino acid residues (RRS). b Immunoblots of brain homogenate from prion-infected Ext1^+/+ mice using POM19 antibody (PrP) and sPrP^G228 antibody (ADAM10-cleaved PrP). c Ratios of ADAM10-cleaved PrP^Sc relative to total PrP^Sc reveal significantly higher levels of ADAM10-cleaved PrP in mCWD as compared to the RML and ME7 strains. d Brain immunolabelled for PrP with SAF84 (amino acids 163–169 of mouse PrP) and sPrP^G228 antibodies reveals all mCWD plaques, but few diffuse RML aggregates, are labelled by sPrP^G228. e Quantification of HS bound to GPI-anchored and –anchorless RML and 22L prions by LC-MS, and f a grouped comparison of HS bound to GPI-anchored prions (RML and 22L) versus GPI-anchorless prions (GPI⁻ RML, GPI⁻ 22L) and ADAM10-cleaved mCWD shows that full length, GPI-anchorless prions and ADAM10-cleaved prions (mCWD) bind more HS than their GPI-anchored counterparts. g Dual immunostaining of mCWD-infected brain sections for PrP and HS shows parenchymal prion plaques in the corpus callosum label strongly for HS. Pre-treating brain sections with heparinases abolished HS labelling of plaques. Scale bars = 200 μm and 500 μm for upper and lower panel (panel d) and 25 μm (panel g). *P< 0.05, **P< 0.01 and ***P< 0.001, Wilcoxon rank sum test (panel c) and one-way ANOVA with Tukey’s post test (panel f).

Fig. 5

GPI-anchored mCWD prions do not form plaques and bind low levels of HS. a Schematic representation of mCWD inoculated into tga20 and GPI-anchorless PrP^C expressing mice [tg(GPI-PrP)] with corresponding prion plaque morphology, Congo red, and Alcian blue staining of brain sections (corpus callosum). Note that the new mCWD prions do not bind Congo red or Alcian blue. b Western blots of PK-treated PrP labelled with anti-PrP POM19 (total PrP) or sPrP^G228 (ADAM10-cleaved PrP) antibodies reveal ADAM10-cleaved PrP in the original mCWD but not the GPI-anchorless mCWD or the new mCWD–infected brain. c Ratios of ADAM10-cleaved : total PrP^Sc are higher in the original mCWD than in the GPI-anchorless mCWD or in the new mCWD-infected brains (original mCWD results are also shown in Fig. 4c). d The new mCWD binds less HS than the original mCWD or GPI-anchorless mCWD (original mCWD results are also shown in Fig. 4f). e A grouped comparison shows HS bound to GPI-anchorless prions (mCWD and GPI⁻ mCWD, black bar) versus the new GPI-anchored mCWD (pink bar). f The HS bound to GPI-anchorless mCWD is more sulfated than the HS associated with ADAM10-cleaved mCWD and the new GPI-anchored mCWD, as it contains less unsulfated N-acetylated (NAc) HS and higher level of N-sulfated (NS) and 2-O sulfated (2-O) HS. g HS does not co-localize to the new mCWD aggregates (bottom panel). Scale bars = 100 μm for original and GPI⁻ mCWD and 200 μm for new mCWD (panel a) and 25 μm (panel g). *P< 0.05, **P< 0.01 and ***P< 0.001, Wilcoxon rank sum test (panels c and e), one-way ANOVA with Tukey’s post test (panel d), and two-way ANOVA with Bonferroni’s post test (panel f).

Fig. 6

Abundant HS is associated with prion plaques in human brain. a Immunoblots of PK-digested PrP purified from GSS and sCJD brain samples show differences in electrophoretic mobility and glycoprofile. Note that PrP from the GSS-P102L brain shows a PK core size of 21 kD and a different glycoprofile than PrP from the sCJD MM1 and MV1 brain samples. b Fibrillar prion plaques from GSS-F198S patients immunolabelled for PrP. Plaques also bind Alcian blue. c Mass spectrometry of HS from purified prion preparations derived from the GSS and sCJD patient brain samples reveal significantly higher HS levels associated with the GSS-F198S purified prions. d The HS bound to GSS-F198S prions shows lower levels of N-acetylated (NAc) and 6-O (6-O) sulfated disaccharides than the HS bound to sCJD. e Cerebellar GSS-F198S plaques show intense HS immunolabelling, primarily in the plaque core. Plaque cores in GSS-F198S affected brains are Congo red positive (shown is a representative example). Cerebellum (GSS-F198S) and thalamus (sCJD MM1) are shown in panel b. Scale bars = 50 μm (sCJD) and 100 μm GSS-F198S (panel b), 25 μm (dual IF, panel e) and 250 μm (Congo red, panel e). ***P< 0.001, One-way ANOVA with Tukey’s post test (panels c). *P< 0.05, Two-way ANOVA with Bonferroni’s post test (panel d).