Novel method for classification of prion diseases by detecting PrP^res signal patterns from formalin-fixed paraffin-embedded samples

Figures & data

Table 1. Case profiles.

Case No.	Diagnosis		Age at death (y)	Sex	Disease duration	Brain Weight (g)
sCJD 1	sCJD	MM1 + 2	77	F	4y 11 m	800
sCJD 2	sCJD	MM1	73	F	3 m	1210
sCJD 3	sCJD	MM1	72	F	3y 4 m	650
sCJD 4	sCJD	MM1 + 2C	73	M	3 m	1090
sCJD 5	sCJD	MM1 + 2C	72	F	3.5 m	1400
sCJD 6	sCJD	MV2(+1)	78	F	1y 7 m	1115
sCJD 7	sCJD	MM2	55	F	4 m	1040
GSS 1	GSS		79	F	5y	1200
GSS 2	GSS		78	F	13y	1020
GSS 3	GSS		65	M	5y	820
GPIALP 1	GPIALP		37	F	11y	1269
GPIALP 2	GPIALP		36	M	16y	1425
V180I CJD 1	V180I (PPS-treated)		70	F	5y 9 m	484
V180I CJD 2	V180I (PPS-untreated)		85	M	1y	1300
control 1	LC		79	M	-	1350
control 2	SCA6		93	M	-	1161
control 3	EDMD		65	M	-	1275
control 4	CPEO		84	F	-	958
control 5	MDEM		78	M	-	1186

sCJD, sporadic Creutzfeldt–Jakob disease; GSS, Gerstmann–Sträussler – Scheinker disease; GPIALP, glycosylphosphatidylinositol-anchorless prion disease; PPS, pentosan polysulfate; LC, liver cirrhosis; SCA6, spinocerebellar ataxia type 6; EDMD, Emery–Dreifuss muscular dystrophy; CPEO, chronic progressive external ophthalmoplegia; MDEM, multiphasic disseminated encephalomyelitis.sCJD, sporadic Creutzfeldt–Jakob disease; GSS, Gerstmann–Sträussler–Scheinker disease; GPIALP, glycosylphosphatidylinositol-anchorless prion disease; PrPres, proteinase K resistant prion protein; SD, standard deviation.

Figure 1. Workflow of protein extraction from the formalin-fixed paraffin-embedded (FFPE) samples. The FFPE samples are sectioned into two tubes, one for proteinase K (PK) treatment (d) and the other without PK treatment (e), and the proteins are then extracted.

Figure 2. Western blot analyses of the formalin-fixed paraffin-embedded (FFPE) samples with the 3F4 anti-prion protein (PrP) antibody. (a) in control cases, proteinase K-untreated (PK [−]) samples show strong PrP signals at approximately 25–40kDa. Proteinase K-treated (PK [+]) samples show no signal. (b, c, d) in the sporadic Creutzfeldt–Jakob disease (sCJD) cases, PK-resistant PrP (PrP^res) signals are observed with varied densities per case. In sCJD 1, 2, and 3, three PrP^res bands are detected at 21–30 kDa (white arrowheads). sCJD 4, 6 and 7 also show three PrP^res bands, but the unglycosylated PrP^res are slightly wider at 19–21 kDa (black arrowheads). In sCJD 5, faint PrP^res is detected at 21–30 kDa. In all sCJD cases, high molecular smear PrP^res signals are observed with varied densities per case. (e) PK digestion reveals three bands of PrP^res at 21–30 kDa (white arrowheads) in Gerstmann–Sträussler–Scheinker disease (GSS) 1 and 3, as well as low molecular weight bands at approximately 8 kDa (arrows) and strong high molecular weight smear bands. In GSS 2, a high molecular weight smear band and a band at approximately 8 kDa (arrow) are present. In glycosylphosphatidylinositol-anchorless prion disease (GPIALP) cases, PK digestion reveals smear bands from low to high molecular weight and low molecular bands at approximately 9 kDa (arrows). In V180I CJD cases, weak high molecular PrP^res smear signals are observed in V180I CJD 1, PrP^res signal are unclear in V180I CJD 2.

Figure 3. Western blot analyses of the formalin-fixed paraffin-embedded (FFPE) samples with the EP1802Y anti-prion protein (PrP) antibody. (a) in control cases, proteinase K-untreated (PK [−]) samples show PrP signals at 25–40 kDa. In proteinase K-treated (PK [+]) samples, there is no PrP^res signals. (b, c) the PK (+) samples of sporadic Creutzfeldt–Jakob disease (sCJD) 1–7 show unglycosylated PK-resistant PrP (PrP^res) signals at 21 kDa (case 1–5 and 7: white arrowheads) and about 19 kDa (case 6: arrowhead). High molecular smear PrP^res signals are observed in all sCJD cases (*). In addition, a low molecular weight band at approximately 16 kDa (arrows) are also found in sCJD cases 1–5 and 7. The 16 kDa PrP^res signal is not observed in sCJD 6. (D) in the Gerstmann–Sträussler–Scheinker disease (GSS) cases, unglycosylated PrP^res at 21 kDa (white arrowheads) and high molecular smear bands are present (*). The low molecular weight band at approximately 8 kDa is not detectable. In glycosylphosphatidylinositol-anchorless prion disease (GPIALP) cases, no PrP^res signal is detected. In the PK (+) sample of V180I CJD 1 (pentosan polysulfate [PPS]-treated case), a high molecular smear band (*) is visible, whereas the PrP^res signal is substantially weak in V180I CJD 2 (PPS-untreated case).

Table 2. Relative percentage of protease-resistant prion protein by molecular weight.

	sCJD	GSS	GPIALP	V180I CJD 1
3F4
High molecular PrPres: >30kDa (% ± SD)	48.8 ± 23.9	46.3 ± 23.0	71.4 ± 2.6	100
Three PrPres bands: 19 or 21 ~ 30kDa (% ± SD)	51.2 ± 23.9	32.7 ± 18.0		0
Low molecular PrPres: <19kDa (% ± SD)	0	21.0 ± 0.5	28.6 ± 1.8	0
Relative percentage of protease-resistant prion protein by molecular weight
EP1802Y
High molecular PrPres: >30kDa (% ± SD)	56.4 ± 2.4	79.0 ± 7.9	0	100
Three PrPres bands: 19 or 21 ~ 30kDa (% ± SD)	36.7 ± 1.8	21.9 ± 7.9		0
Low molecular PrPres: <19kDa (% ± SD)	7.0 ± 0.8	0	0	0

Figure 4. Western blotting analyses of the formalin-fixed paraffin-embedded (FFPE) samples with the Tohoku 1 (specific for type 1 scrapie prion protein [PrP^Sc]) and Tohoku 2 (specific for type 2 PrP^Sc) anti-proteinase K-resistant PrP (PrP^res) antibodies. (a, b) Sporadic Creutzfeldt–Jakob disease (sCJD) 1 and 3 show bands of PrP^res at 21–30 kDa and high molecular smears with Tohoku 1, whereas Tohoku 2 shows no signal. In sCJD 2, 4, and 5, PrP^res signals are observed both with Tohoku 1 and 2. sCJD 6 shows a weak signal with Tohoku 1 but a strong signal with Tohoku 2. (c, d) sCJD 7 shows a smear band with Tohoku 1 and a weak smear band at approximately 20 kDa with Tohoku 2.

Figure 5. Histological findings of the prion disease cases. (A, C, E, G, I, K, M, O, Q, S, U, W: Hematoxylin and eosin staining (HE), B, D, F, H, J, L, N, P, R, T, V, X: Immunohistochemistry (IHC) with 3F4), (A, B: sporadic Creutzfeldt–Jakob disease case 1 (sCJD 1), C, D: sCJD 2, E, F: sCJD 3, G, H: sCJD 4, I, J: sCJD 5, K, L: sCJD 6, M, N: sCJD 7, O, P: Gerstmann–Sträussler–Scheinker disease case 1 (GSS 1), Q, R: glycosylphosphatidylinositol-anchorless prion disease case 1 (GPIALP 1), S, T: V180I CJD 1 (treated with pentosan polysulfate (PPS)), U, V: V180I CJD 2 without PPS, W, X: non prion case: control 3). (A, B: sCJD 1) HE staining indicating neuronal loss, gliosis and spongiform changes (a). IHC with 3F4 displaying synaptic prion protein (PrP) deposits in the cerebral cortex (b). (C, D: sCJD 2) HE staining shows spongiform changes and some large confluent vacuoles (inset) (c). 3F4 reveals synaptic PrP and perivacuolar PrP deposits (inset) (d). (E, F: sCJD 3) HE staining indicating neuronal loss, gliosis and spongiform changes (e). 3F4 displaying synaptic PrP deposits in the cerebral cortex (f). (G, H: sCJD 4) HE staining shows spongiform changes and some large confluent vacuoles (g). 3F4 reveals synaptic PrP deposits, perivacuolar PrP deposits and perineuronal PrP deposits (inset) (h). (I, J: sCJD 5) HE staining shows spongiform changes and some large confluent vacuoles (I). 3F4 reveals synaptic PrP and perivacuolar PrP deposits (j). (K, L: sCJD 6) HE staining shows spongiform changes (k). 3F4 reveals synaptic, perineuronal (inset), and plaque-type deposits (inset) (l). (M, N: sCJD 7) HE staining shows spongiform changes, gliosis, and large confluent vacuoles (m). 3F4 reveals PrP perivacuolar and diffuse synaptic deposits (n). (O, P: GSS 1) HE staining shows spongiform changes and PrP plaques (inset) (o). 3F4 reveals numerous PrP plaques (inset) and synaptic deposits (p). (Q, R: GPIALP 1) HE staining shows minimal spongiform changes and only mild neuronal loss (q). 3F4 reveals numerous PrP coarse granular deposits in the neuropil and around the vessels (r). (S, T: V180I CJD 1 treated with PPS) HE staining shows spongiform changes and gliosis (s). 3F4 reveals synaptic deposits and many PrP coarse deposits (t). (U, V: V180I CJD 2 without PPS) HE staining shows extensive and severe spongiform changes (u). 3F4 shows virtually no abnormal PrP deposits (v). (W, X: non prion case: control 3) HE shows that the neurons and neuropil are preserved, and there is no obvious neurodegenerative pathology (w). 3F4 shows a weak positive PrP staining in the cortex (x). (Scale bars: A-V: 50 µm, inset: C, D, H, O, P: 25 µm, L: 20 µm).

Figure 6. Schema for classing prion diseases using western blotting (WB) from formalin-fixed paraffin-embedded (FFPE) samples. In proteinase K-treated FFPE samples by western blotting with 3F4 antibody, the presence of 21–30 kDa three PrP^res bands, high molecular smear PrPres signals, or low molecular PrP^res signals strongly suggests prion disease. In 3F4, three PrP^res bands + moderate high molecular smear PrP^res signals suggest sCJD, strong high molecular smear PrP^res signals + moderate three PrP^res bands + moderate low molecular PrP^res signals suggests GSS or GPIALP. In V180I CJD, it is difficult to detect, and only V180I CJD 1 PPS-treated case show weak high molecular smear PrP^res signals. In sCJD cases, western blotting using Tohoku 1 (TH1) and Tohoku 2 (TH2) antibodies specific for type 1 and type 2 PrP^res, respectively, can also distinguish between type 1 and type 2 PrP^res with this method. In the case of V180I CJD, western blotting with EP1802Y antibody enhanced the high molecular smear PrP^res signal, which was weak in 3F4. However, this was also only in PPS-treated case (V180I CJD 1), and it was difficult to identify the PrP^res signal in untreated V180I CJD (V180I CJD 2).

Figure 7. Representative band patterns of western blotting (WB) of formalin-fixed paraffin-embedded (FFPE) samples using multiple antibodies specific for different epitopes. Band patterns for the conventional method using frozen samples are displayed at the bottom of the table.

Data availability statement

Data supporting the findings of this study are available from the corresponding author upon reasonable request.