Fig 1.
Cross-seeding of PrPC misfolding and replication in dgPMCAb.
a Analysis of the glycoform ratios of PrPC from Syrian hamster normal brain homogenate (HaNBH) before and after treatment with PNGase F. Black and white triangles mark di- and monoglycosylated glycoforms, respectively, whereas arrows mark the unglycosylated form. b and c Serial dgPMCAb reactions were seeded as labeled with (i) lysates of HeLa cells expressing WT α-synuclein (HeLa WT) or A30P mutant (HeLa A30P) and cultured under two conditions (#1, #2) as described in Methods; (ii) amyloid fibrils produced in vitro from recombinant α-synuclein from two sources (syn fibrils #1, #2 as described in Methods); (iii) mouse rPrP amyloid fibrils produced in vitro in 0, 0.1, 0.5, or 2.5M GdnHCl (Mo rPrP fibrils #1, #2, #3, #4, respectively); (iv) non-fibrillar α-synuclein (non-fibr); or (v) Aβ fibrils produced in vitro using six different protocols as described in Methods (Aβ fibrils, #1–#6). As a control for cross-contamination, non-seeded dgPMCAb reactions were conducted in parallel (non-seeded). Seven serial dgPMCAb rounds with 10-fold dilutions between rounds were conducted and the products of the seventh round were treated with PK and analyzed by Western blots using SAF-84 antibodies. As references, dgPMCAb-derived PrPres formed in serial dgPMCAb seeded with hamster rPrP fibrils produced in vitro in 0.5 M GdnHCl (Ha rPrP fibrils, panel b) or brain- derived PrPres from animals inoculated with hamster rPrP fibrils are provided (brain PrPres, panel c) [26]. d Analysis of PrPres dynamics in serial dgPMCAb reactions seeded with α-synuclein WT fibrils #1, lysates of HeLa cells expressing α-synuclein A30P variant and cultured under condition #2, or hamster rPrP fibrils.
Table 1.
Bioassay in Golden Syrian hamsters.
Fig 2.
Bioassay of dgPMCAb-derived PrPres in Syrian hamsters.
a Western blot analysis of brain material from hamsters inoculated IC with (i) fibrillar α-synuclein, (ii, iii) dgPMCAb-derived PrPres induced by WT α-synuclein fibrils or lysates of HeLa cells expressing the A30P variant, (iv) non-fibrillar α-synuclein, or (v) non-seeded dgPMCAb-derived material and stained with SAF-84 (top panels) or 3F4 antibody (bottom panels). Non-inoculated, age-matched animals were examined as a negative control. Brain materials marked by asterisks were used for the second passage. b Left panel shows Western blot analysis of dgPMCAb-derived PrPres used for inoculation and resulting brain-derived PrPres and PrPSc from animals of the 1st and 2nd passages. To match the amounts of dgPMCAb- and brain-derived PrPres on a blot, all brain materials were diluted 250-fold after PK treatment. Western blots were stained with SAF-84 antibody. On the right panel is a schematic representation of the PK resistant profile showing overlap between the three glycoforms of PrPres (gray boxes) and the three glycoforms of PrPSc (black boxes). c Western blot analysis of brain material from hamsters inoculated with the second passage of dgPMCAb-derived PrPres induced by α-synuclein fibrils; lysates of HeLa cells expressing the A30P mutant; or non-seeded dgPMCAb-derived material. Lanes marked by asterisks show brain material from animals from the first passages used for serial transmission. Western blots were stained with SAF-84 (top panels) or 3F4 antibody (bottom panels).
Fig 3.
Histopathological analysis of brains from the 1st passage of dgPMCAb-derived PrPres.
Representative images of the frontal cortex (a, b, c, d) and hippocampus (e, f, g, h) of animals inoculated with dgPMCAb products seeded with fibrillar WT α-synuclein (a,b,e,f) or dgPMCAb products seeded with lysates of HeLa cells expressing A30P α-synuclein (c,d,g,h). Note the lack of spongiform change in the sections stained with hematoxylin and eosin (a, c, e, g) and patchy reactive astrogliosis (immunostaining for GFAP are in the insets in panels a and c). Immunostaining for PrP using SAF-84 (b, d, f, h) revealed diffuse/synaptic and granular deposits. Scale bar in a = 50 μm.
Fig 4.
Histopathological analysis of brains from the 2nd passage of PrPres produced in dgPMCAb reactions seeded with fibrillar WT α-synuclein.
Representative images of caudate putamen (a) and cerebellum (b) stained with hematoxylin and eosin, hippocampus stained with anti-PrP 3F4 (c), anti-GFAP (d) or anti-Iba1 antibody (e), or subventricular zones stained with anti-PrP 3F4 (f), anti-GFAP (g) or anti-Iba1 antibody (h). Subventricular deposits are indicated by arrows and the ventricular surface of ependymal cells is indicated by an arrowhead. S-O, stratum oriens; S-R, stratum radiatum; S-L, stratum lacunosum-moleculare; d, dentate gyrus, LV, lateral ventricle. Scale bars: in a, b, f, g, h = 100 μm, c, d, e = 500 μm.
Fig 5.
Histopathological analysis of brains from the 2nd passage of PrPres produced in dgPMCAb reactions seeded with fibrillar WT α-synuclein.
Subcallosal (a), perineuronal (b, indicated by arrows) and perivascular (c, indicated by arrows) PrP immunoreactivity as stained with anti-PrP 3F4 antibody. Ctx, cortex; cc, corpus callosum; Hp, hippocampus; LV, lateral ventricle. Scale bars: in a, c = 100 μm, b = 50 μm.