Distinct origins of dura mater graft-associated Creutzfeldt-Jakob disease: past and future problems

Dura mater graft-associated Creutzfeldt-Jakob disease (dCJD) can be divided into two subgroups that exhibit distinct clinical and neuropathological features, with the majority represented by a non-plaque-type of dCJD (np-dCJD) and the minority by a plaque-type of dCJD (p-dCJD). The two distinct phenotypes of dCJD had been considered to be unrelated to the genotype (methionine, M or valine, V) at polymorphic codon 129 of the PRNP gene or type (type 1 or type 2) of abnormal isoform of prion protein (PrPSc) in the brain, while these are major determinants of clinicopathological phenotypes of sporadic CJD (sCJD). The reason for the existence of two distinct subgroups in dCJD had remained elusive. Recent progress in research of the pathogenesis of dCJD has revealed that two distinct subgroups of dCJD are caused by infection with different PrPSc strains from sCJD, i.e., np-dCJD caused by infection with sCJD-MM1/MV1, and p-dCJD caused by infection with sCJD-VV2 or -MV2. These studies have also revealed previously unrecognized problems as follows: (i) the numbers of p-dCJD patients may increase in the future, (ii) the potential risks of secondary infection from dCJD, particularly from p-dCJD, may be considerable, and (iii) the effectiveness of the current PrPSc decontamination procedures against the PrPSc from p-dCJD is uncertain. To prevent secondary infection from p-dCJD, the establishment of effective decontamination procedures is an urgent issue. In this review, we summarize the past and future problems surrounding dCJD.


Introduction
Dura mater grafts used to repair the dural defects at neurosurgery can cause fatal disease years to decades later. The tragedy of dura mater graft-associated Creutzfeldt-Jakob disease (dCJD) was considered to be nearly over. However, recent progress in research of the pathogenesis of dCJD has revealed previously unrecognized problems. In this review, we summarize the past and future problems surrounding dCJD.
Creutzfeldt-Jakob disease (CJD) is a lethal transmissible neurodegenerative disease. The central event in the pathogenesis of CJD is a conformational change of the normal cellular isoform of prion protein (PrP C ) into an abnormal infectious isoform of prion protein (PrP Sc ) [1]. The conformational change of PrP C can occur due to either one of three causes: spontaneous conversion in sporadic CJD (sCJD), mutations in the PRNP gene in genetic CJD, or infection with PrP Sc in iatrogenic CJD and variant CJD.
One of the most frequent sources of iatrogenic PrP Sc infection is dura mater grafts obtained from human cadavers undiagnosed as CJD. The sum of dCJD (228 cases) and growth hormone-associated CJD (226 cases) accounts for 97% of total iatrogenic CJD cases [2]. A single brand of dura mater graft, Lyodura®, was used for all the dCJD cases in whom the brand name was identified. Although the causative dura mater grafts were manufactured by a German company, 62% (142 cases) of total dCJD cases have been found in Japan [2,3]. Persistent efforts of a Japanese CJD surveillance team have clarified the outline of dCJD outbreaks. The onset of Japanese dCJD patients peaked in the late 1990s, and most of the patients had received the grafts during 1983-1987, while as many as 100,000 persons received the Lyodura® grafts during this period [4,5]. In the process of conducting this elaborate survey, a puzzling mystery about dCJD emerged.
sCJD also shows wide phenotypic heterogeneity, and its clinicopathological phenotypes are determined by the genotype at polymorphic codon 129 of the PRNP gene and type of PrP Sc in the brain ( Figure 2) [17,18]. The codon 129 of the PRNP gene shows methionine (M)/valine (V) polymorphism. Two types of PrP Sc (type 1 and type 2) are distinguishable according to the size of the proteinase K-resistant core of unglycosylated PrP Sc (21 and 19 kDa, respectively), reflecting differences in the proteinase K-cleavage site (at residues 82 and 97, respectively) [19]. On the other hand, the two distinct phenotypes of dCJD had been considered to be unrelated to their PRNP genotype or type of PrP Sc in the brain [11]. In Japan, almost all dCJD patients had the same genotype, i.e., homozygous for methionine at codon 129 (129 M/M), except two heterozygotes [2], and the type of PrP Sc in their brains had been reported as type 1 [6,10,11]. The reason for the existence of two distinct subgroups in dCJD had remained elusive.

Solving the mystery
In 2003, an unusual p-dCJD case was reported [9]. This patient showed the accumulation of unusual PrP Sc with intermediate electrophoretic mobility between types 1 and 2 PrP Sc . Then, we reevaluated the biochemical properties of PrP Sc in the two subgroups of dCJD and found that the size of PrP Sc from p-dCJD was invariably smaller than that of type 1 PrP Sc from np-dCJD Figure 1 Clinicopathological features of the two subgroups of dCJD. The patients with dCJD can be divided into two subgroups, with the majority represented by a non-plaque-type of dCJD (np-dCJD) and the minority by a plaque-type of dCJD (p-dCJD) [11,12]. Neuropathological hallmark of p-dCJD is widespread PrP Sc amyloid plaques, while np-dCJD shows diffuse synaptic-type PrP Sc deposition.  To resolve the mystery of the existence of two distinct subgroups in dCJD, we hypothesized that they might be caused by infection with different PrP Sc strains from distinct sCJD subgroups. According to the PRNP genotype and type of PrP Sc in the brain, sCJD is classified into six subgroups (MM1, MV1, VV1, MM2, MV2, or VV2) [17]. MM1 and MV1, which are the predominant subgroups in sCJD, show the same clinicopathological features. Meanwhile, MM2 can be divided into three subgroups based on histopathological criteria (MM2T, thalamic form showing characteristic atrophy of thalamic and inferior olivary nuclei; MM2C, cortical form showing a predominant cortical pathology; or MM2T + C, mixed form) [17,21]. In addition, MV2 is also divided into three subgroups based on histopathological criteria (MV2K showing kuru type PrP Sc amyloid plaques, MV2C showing a predominant cortical pathology, or MV2K + C showing mixed histopathology) [17,18]. The clinicopathological features of np-dCJD, such as short duration of illness, PSWC on EEG, or diffuse synaptic-type PrP Sc deposition in the brain, are identical to those of sCJD-MM1/MV1. In contrast, the clinicopathological features of p-dCJD, such as ataxic gait as an initial symptom, slow progression of neurological symptoms, absence or late occurrence of PSWC on EEG, or formation of PrP Sc plaques in the brain, are similar to those of sCJD-VV2, −MV2K, or -MV2K + C. These similarities raised the possibility that np-dCJD might be caused by infection with sCJD-MM1/MV1, whereas p-dCJD might be caused by infection with sCJD-VV2, −MV2K, or -MV2K + C.
To test this possibility, we examined the transmission properties of the dCJD and sCJD subgroups using humanized mice carrying human PrP with either the 129 M/M or V/V genotype [20,22,23]. In these transmission experiments, p-dCJD and sCJD-VV2, −MV2K, or -MV2K + C were identical in the transmissibility to the PrP-humanized mice (Table 1, Figure 4a) and in the neuropathological and biochemical features in the inoculated mice (Figure 4b, c). By contrast, np-dCJD showed the same transmission properties as sCJD-MM1. In particular, the 129 M/M mice inoculated with sCJD-VV2, −MV2K, or -MV2K + C material showed widespread PrP Sc plaques and type i PrP Sc accumulation similar to the p-dCJD patients, whereas the 129 M/M mice inoculated with sCJD-MM1 material showed diffuse synaptic-type PrP Sc deposition and type 1 PrP Sc accumulation similar to the np-dCJD patients. Thus, these animal models support the hypothesis that the origin of np-dCJD is sCJD-MM1/MV1 and that of p-dCJD is  Table 1 Transmission of dCJD or sCJD to PrP-humanized mice p-dCJD (KR) 420 ± 10 (5/5) 685 ± 51 (5/5) 259 ± 6 (6/6)  sCJD-VV2, −MV2K, or -MV2K + C. Indeed, the incidence rate of p-dCJD (32%) among total dCJD is close to the sum total of the incidence of sCJD-VV2 (15%), −MV2K (8%), and -MV2K + C (3%) [18].   (Table 2). However, M2C PrP Sc lacks or has very low infectivity and does not affect the transmission properties of the coexisting PrP Sc [23]. Therefore, the transmission of sCJD-MV2K + C to the 129 M/M mice can also result in the generation of Mi PrP Sc (Figure 4c). Taken together, Mi PrP Sc in p-dCJD is generated by infection with Mi PrP Sc and/or V2 PrP Sc from sCJD-VV2, −MV2K, or -MV2K + C. It is noteworthy that Mi PrP Sc can be observed in the 129 M/M mice inoculated with V2 PrP Sc but not in sCJD patients with the 129 M/M genotype, suggesting that Mi PrP Sc in sCJD-MV2K or -MV2K + C is also generated by V2 PrP Sc seed-dependent conversion but not by spontaneous conversion of the 129 M PrP C . Therefore, the primary origin of Mi PrP Sc is V2 PrP Sc . This can account for the similarities in transmission properties between Mi PrP Sc and V2 PrP Sc . Thus, M1 PrP Sc in np-dCJD and Mi PrP Sc in p-dCJD are completely different with regard to the neuropathological phenotypes, biochemical features, and transmission properties, reflecting their distinct PrP Sc origins. In contrast to M1 PrP Sc , which is the most common PrP Sc observed in sCJD patients with the 129 M/M genotype, Mi PrP Sc has never been observed in sCJD patients with the 129 M/M genotype. Therefore, the detection of Mi PrP Sc can be sound evidence of iatrogenic infection in individuals with the 129 M/M genotype and would contribute to reliable surveillance of iatrogenic cases such as p-dCJD.
To verify experimentally that Mi PrP Sc originates from V2 PrP Sc and its transmission properties are identical to those of the parental V2 PrP Sc , we performed a modeling study using PrP-humanized mice (Figure 5a) [25]. As described above, the 129 M/M mice inoculated with V2 PrP Sc showed widespread PrP Sc plaques and Mi PrP Sc accumulation in the brain as an experimental model of p-dCJD. We then inoculated the Mi PrP Sc from these mice into other PrP-humanized mice with either the 129 M/M or V/V genotype. This secondary passage revealed that the transmission properties of the Mi PrP Sc , i.e., 129 M/M mouse-passaged sCJD-VV2, are identical to those of the parental V2 PrP Sc . In particular, although the incompatibility of the codon 129 genotypes between host and inoculum usually results in a prolonged incubation period [20], the 129 V/V mice inoculated with the Mi PrP Sc showed a shorter incubation period compared with the 129 M/M mice (Table 1). Moreover, the altered neuropathological phenotype and biochemical properties at the primary passage in the 129 M/M mice reverted to the original ones in the secondary passage in the 129 V/V mice (Figure 5b, c). Thus, this modeling study shows that (i) V2 PrP Sc infection in a host with the incompatible codon 129 M/M genotype generates an unusual PrP Sc with altered conformational properties, i.e., Mi PrP Sc , (ii) the emerging Mi PrP Sc retains the memory of the parental V2 PrP Sc within its conformational properties, and (iii) the parental V2 PrP Sc re-emerges and proliferates rapidly if the Mi PrP Sc is transmitted to the original host with the codon 129 V/V genotype. This phenomenon, designated as traceback, can be a useful tool to identify the origin of PrP Sc infection if atypical PrP Sc emerges in the future [20,22,26].

Remaining problems
Our transmission studies resolved the complicated pathogenesis of dCJD. However, they have also revealed several issues surrounding dCJD that need to be addressed in the future. First, the numbers of p-dCJD patients may increase in the future. The experimental p-dCJD model, i.e., the 129 M/M mice inoculated with Mi PrP Sc and/or V2 PrP Sc from sCJD-VV2, −MV2K, or -MV2K + C, showed a longer incubation period compared with the np-dCJD model, i.e., the 129 M/M mice inoculated with M1 PrP Sc from sCJD-MM1 (Table 1). This raises the concern that additional p-dCJD patients, who are presenting still at the subclinical stage, may emerge after a longer incubation period in the future. Although the numbers of patients with newly developed dCJD have dropped off, continuous surveillance will be required to find remaining p-dCJD cases.
Second, the potential risks of secondary infection from dCJD, particularly from p-dCJD, may be considerable. As described above, the transmission studies raise a concern about the existence of subclinical p-dCJD patients. dCJD patients may undergo more than one neurosurgical operation due to their underlying diseases (the primary disease for which the neurosurgery was performed) [4]. In addition, p-dCJD patients may be more frequently autopsied because the clinical features of p-dCJD are atypical compared with those of classical sCJD [11]. These facts suggest that there may be considerable risk of secondary infection from p-dCJD patients. Individuals with the 129 V/V genotype may be more vulnerable to the infection with Mi PrP Sc from p-dCJD, as suggested by the fact that the 129 V/V mice were highly susceptible to Mi PrP Sc in the transmission study (Table 1). Additionally, 129 M/M individuals may be also affected after a prolonged incubation period, as suggested by the high attack rate (100%) of the 129 M/M mice inoculated with Mi PrP Sc . Therefore, secondary infection from p-dCJD can occur regardless of the codon 129 genotype. Comprehensive analysis of the distribution of PrP Sc in the peripheral tissues of p-dCJD patients will be also required to assess the potential risks of secondary infection.
Finally, the efficacy of the current PrP Sc decontamination procedures against Mi PrP Sc needs to be tested in the future. Mi PrP Sc in p-dCJD and M1 PrP Sc in np-dCJD differ in the sizes of the proteinase K-resistant core, suggesting their conformational differences. Moreover, their parental PrP Sc strains are also different. Different PrP Sc strains can show different thermostability [27,28] and different susceptibility to the decontamination procedures [29]. To prevent the spread of secondary infection from dCJD patients to medical workers or other patients, adequate decontamination and disinfection of the instruments used for neurosurgery or autopsy are essential. However, the current PrP Sc decontamination procedures were developed using scrapie isolates and tested using CJD isolates other than p-dCJD [30][31][32]. Therefore, further studies using Mi PrP Sc will be needed to assess the effectiveness of the current procedures. For this purpose, sensitive detection systems for Mi PrP Sc are also prerequisite to evaluating quantitatively the reduction of infectivity after the decontamination procedures. Real-time quaking-induced conversion [33,34], protein misfolding cyclic amplification [35][36][37][38][39], or transgenic mice overexpressing human PrP with the 129 V genotype [20] might be useful to detect the reduced infectivity of Mi PrP Sc at high sensitivity. Using such sensitive detection systems, effective decontamination procedures for Mi PrP Sc can be established in the future.

Concluding remarks
Recent progress in the study of the pathogenesis of dCJD has revealed that the two distinct subgroups of dCJD are caused by infection with different PrP Sc strains of sCJD, i.e., np-dCJD caused by M1 PrP Sc from sCJD-MM1/MV1 and p-dCJD caused by Mi PrP Sc and/or V2 PrP Sc from sCJD-VV2, −MV2K, or -MV2K + C. Studies have also revealed previously unrecognized problems such as the considerable risks of secondary infection from dCJD, particularly from p-dCJD. To prevent secondary infection from p-dCJD, the effectiveness of the current decontamination procedures should be tested urgently using sensitive Mi PrP Sc detection systems.