Comparative analysis of Japanese and foreign L-type BSE prions期刊界 All Journals 搜尽天下杂志传播学术成果专业期刊搜索期刊信息化学术搜索

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Comparative analysis of Japanese and foreign L-type BSE prions

Authors:	Kentaro Masujin Ritsuko Miwa Hiroyuki Okada Shirou Mohri Takashi Yokoyama

Institution:	Prion Disease Research Center; National Institute of Animal Health; Tsukuba, Ibaraki Japan

Abstract:	L-type bovine spongiform encephalopathy (BSE) is an atypical form of BSE. To characterize the Japanese L-type BSE prion, we conducted a comparative study of the Japanese and foreign L-type BSE isolates. The L-type BSE isolates of Japan, Germany, France and Canada were intracerebrally inoculated into bovinized prion protein-overexpressing transgenic mice (TgBoPrP). All the examined L-type BSE isolates were transmitted to TgBoPrP mice, and no clear differences were observed in their biological and biochemical properties. Here, we present evidence that the Japanese and Canadian L-type BSE prions are identical to those from the European cases.Key words: prion, atypical BSE, L-type BSEBovine spongiform encephalopathy (BSE) is one of the transmissible spongiform encephalopathies (TSEs), or prion diseases, in cattle. TSE is characterized by spongiform changes in the central nervous system (CNS) and the accumulation of an abnormal prion protein (PrP^Sc) in the CNS.¹ PrP^Sc has been regarded as the major component of TSE pathogens.²BSE was detected in the UK in 1986,³ and subsequently spread to the other European countries, Japan and North America.⁴^–⁶ BSE is thought to be caused by a single prion strain, based on the analyses of its biological and biochemical characteristics.⁷ From 2003, however, several atypical neuropathological and molecular phenotypes of BSE (atypical BSE) have been detected in Japan, several European countries and North America.⁶^,⁸^–¹⁷ Currently, based on the molecular size of the proteinase-digested non-glycosylated form of PrP^Sc, atypical BSE is classified into two groups (L-type and H-type).¹⁴L-type BSE cases have been identified in the European countries, including Italy, France, Germany, Netherland, Poland and in Canada and Japan.⁸^–¹⁵ Two L-type BSE cases have been identified in Japan. One case was detected in a healthy 23-mo-old Holstein steer (BSE/JP8),⁸ and the other was detected in a 14-y-old black Japanese beef cattle (BSE/JP24).⁹ The latter case was successfully transmitted to bovinized transgenic mice and cattle, and the biological and biochemical properties differed from that of classical BSE (C-BSE).¹⁸^,¹⁹ However, it is unclear whether Japanese L-type BSE prion is identical to that of L-type BSE isolates from other countries. To characterize the Japanese L-type BSE isolate, we performed a comparative study of the Japanese and foreign L-type BSE isolates.A transmission study using experimental animals is a useful approach for prion characterization. Therefore, we performed a transmission study of the L-type BSE isolates in bovinized prion protein (PrP)-overexpressing transgenic mice (TgBoPrP).²⁰ Brain samples of L-type BSE-affected cattle from Japan (BSE/JP24),⁹ France,¹⁰ Germany¹¹ and Canada¹² were used in this study. The brain homogenates were intracerebrally inoculated into TgBoPrP using previously described methods in reference ¹⁸. All animal experiments were reviewed by the Committee of the Ethics on Animal Experiment of the National Institute of Animal Health.All the examined L-type BSE isolates were transmitted to TgBoPrP, and the affected mice developed progressive neurological diseases. Japanese L-type BSE isolate-affected TgBoPrP exhibited a unique clinical sign, the circling behavior. The same phenotype was observed when TgBoPrP were inoculated with German, French and Canadian L-type BSE isolates. On the other hand, in the first passage the incubation period for the Japanese L-type BSE isolate was significantly different from that of the other L-type BSE isolates (
	Incubation period (days)
	JPN	CAN	GER	FRA
First passage	197.7 (3.4)^†	172.8 (4.0)^*	173.3 (3.3)^*	175.7 (5.6)^*
	(10/10^·)	(12/12)	(12/12)	(10/10)
Second passage	152.0 (1.7)	145.7 (1.8)	143.1 (5.7)	143.1 (3.9)
	(24/24)	(23/23)	(18/18)	(18/18)
Third passage	145.1 (3.6)	143.7 (4.6)	145.3 (8.6)	141.6 (4.7)
	(21/21)	(25/25)	(12/12)	(20/20)

Open in a separate window^†Mean (standard deviation)^·Number of affected mice/number of inoculated mice^*p < 0.05 for Japanese L-type BSE isolate vs. other L-type BSE isolates in the first passage (Student''s t-test)Neuropathological examination of the L-type BSE isolate-affected TgBoPrP were performed using previously described methods.¹⁸ Lesion profile analysis revealed that the degree of brain vacuolation due to the Japanese L-type BSE isolate was similar to that caused by the other L-type BSE isolates (Fig. 1A). All the L-type BSE isolates caused severe spongiform changes in the hippocampus, septal nuclei of the paraterminal body and cerebral cortex. We next examined the PrP^Sc deposition pattern in the brain using paraffin-embedded tissue (PET) blot, as described previously in reference ¹⁸. The distributions of PrP^Sc deposits in Japanese L-type BSE isolate-inoculated mice were similar to that of mice inoculated with the other L-type BSE isolates; fine punctate and fine granular PrP^Sc were predominantly and uniformly distributed in the pons, cerebellar medulla, midbrain, thalamus and corpus callosum (Fig. 1B). Furthermore, similar PrP^Sc deposits and distribution patterns were observed in the brain in the first and subsequent passages of all the L-type BSE isolates (data not shown).Open in a separate window Figure 1Neuropathological analysis of L-type BSE isolate-affected TgBoPrP. (A) Lesion profile in the first passage. The vacuolation in the following brain regions was scored on a scale of 0–5 (mean values): 1, dorsal medulla; 2, cerebellar cortex; 3, superior cortex; 4, hypothalamus; 5, thalamus; 6, hippocampus; 7, septal nuclei of the paraterminal body; 8, cerebral cortex at the levels of the hypothalamus and thalamus; and 9, cerebral cortex at the level of the septal nuclei of the paraterminal body. The data are presented as mean ± standard deviation (n = 5). ◆, Japanese L-type BSE (JPN); □, Canadian L-type BSE (CAN); ▲, German L-type BSE (GER); ○, French L-type BSE (FRA). (B) The neuroanatomical distribution of PrP^Sc in the brain of TgBoPrP mice infected with Canadian (CAN), German (GER), French (FRA) and Japanese (JPN) L-type BSE isolate by PET-blot analysis. The PET-blot analysis reveals preferential and intense PrP^Sc immunolabeling along with periventricular areas, corpus callosum and cerebellar gray matter. Widespread PrP^Sc immunolabeling is also detected in the thalamic and brainstem nuclei, while PrP^Sc immunostaining in the cerebral and cerebellar cortices and basal ganglia is less conspicuous. Dewaxed membranes were treated with PK (80 µg/mL), followed by denaturation with 3 M guanidine thiocyanate. The monoclonal antibody (mAb) SAF84 was used. Blots corresponding to the brain areas at the level of frontal lob, septal nuclei, hippocampus, midbrain and medulla and cerebellum. FC, frontal cortex; OV, olfactory ventricle; LO, lateral orbital cortex; CC, cingulated cortex; CP, caudate putamen; PB, paraterminal body; CP, caudate putamen; PC, parietal cortex; TC, temporal cortex; H, hippocampus; T, thalamus; HT, hypothalamus; AM, amygdala; SSC, stratum moleculare of the cerebellum; NCG, nucleus corporis geniculati; NR, nucleus rubber; SN, substantia nigra; NM, nucleus mammillaris; NC, deep nuclei of the cerebellum.We further examined the biochemical properties of PrP^Sc, such as the glycoform ratio and molecular mass of proteinase K (PK)-digested PrP^Sc (PrPcore). PrP^Sc were extracted from the brain of L-type BSE isolate-affected TgBoPrP using previously described methods in reference ¹⁸. Western blotting analysis revealed that the glycoform patterns and molecular mass of the PrPcore of the Japanese L-type BSE isolate resembled that of the other L-type BSE isolates. In contrast, clear differences were observed between C-BSE and L-type BSE isolates (Fig. 2A and B). Next, we examined the relative PK resistance of PrP^Sc from L-type BSE isolate-affected TgBoPrP, as described previously in reference ¹⁸. The PrP concentration of the sample was adjusted using the signal intensity of western blot. The PK resistance of PrP^Sc from the Japanese L-type BSE was similar to that of the foreign L-type BSE isolates. The PrP^Sc of C-BSE-affected TgBoPrP was resistant to digestion with 1,000 µg/ml of PK. In contrast to C-BSE, the PrP^Sc signal from the L-type BSE isolates decreased when digested with 500 µg/ml of PK (Fig. 2C).Open in a separate window Figure 2Western blot analysis of proteinase K (PK)-digested prion protein (PrPcore) from the brain of L-type BSE isolate-affected TgBoPrP. (A) Lane 1, Classical-BSE; Lane 2, Japanese L-type BSE; Lane 3, Canadian L-type BSE; Lane 4, French L-type BSE; Lane 5, German L-type BSE. All the samples were digested with 50 µg/ml PK at 37°C for 1 h (upper part), and digested aliquots were treated with N-glycosidase F (PNGaseF), according to the manufacturer''s instructions (bottom part). PrPcore was detected with mAb 6H4. Molecular markers are shown on the left (kDa). (B) The relative amounts of the diglycosylated (solid black bar), monoglycosylated (gray bar), and unglycosylated (clear bar) forms in the PrPcore from the brain of L-type BSE isolate-affected TgBoPrP. The lane numbers are as listed in (A). The results are presented as mean ± standard deviation from 5 experiments. (C) Relative PK resistance of PrP^Sc from L-type BSE isolate-affected TgBoPrP. The PrP^Sc concentration of the sample was adjusted using the western blot signal intensity. The samples were treated with various concentrations of PK (100–1,000 µg/mL). The results are presented as mean ± standard deviation from 3 experiments. PrP^Sc was detected with mAb 6H4. ◆, Japanese L-type BSE (JPN); □, Canadian L-type BSE (CAN); ▲, German L-type BSE (GER); ○, French L-type BSE (FRA); ●, Classical-BSE (C-BSE).The analyses of L-type BSE cases have been performed using different bovinized PrP-overexpressing transgenic mice, such as TgBoPrP,¹⁸ Tgbov XV¹¹^,²¹ and Tg540.²² Thus, it has been impossible to compare the properties of L-type BSE isolates in detail. In this study, therefore, we performed a transmission study of the L-type BSE isolates using identical bovinized PrP-overexpressing transgenic mice to further characterize the Japanese L-type BSE prion. All the L-type BSE isolates transmitted to TgBoPrP, and their incubation periods converged at approximately 145 d following serial passages (Fig. 1A and B). Besides the biological characteristics, no differences were observed in the biochemical characteristics of PrP^Sc from the L-type BSE isolates (Fig. 2A–C). These findings suggest that the examined L-type BSE cases were caused by prions with identical characteristics.Italian L-type BSE cases are called bovine amyloid spongiform encephalopathy (BASE). We could not compare the characteristics of the Japanese L-type BSE with those of the Italian isolates. In a transmission study using transgenic mice, the French L-type BSE isolate and BASE exhibit similar biological characteristics.²² Our data indicated that the properties of the Japanese L-type BSE prion are identical to those of the French L-type BSE isolate. It has also been reported that the characteristics of Japanese L-type BSE isolate closely resemble those of BASE in an experimental transmission study in cattle.¹⁹The origin of L-type BSE prion is unknown. The present study showed that the Japanese and Canadian L-type BSE prions are identical to those from the European cases. The fact that identical L-type BSE prions exhibit a worldwide distribution is important insight for devising atypical BSE control measures.

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