Recent advances in the histo‐molecular pathology of human prion disease

Creutzfeldt–Jakob disease and fatal insomnia

Secondary pathology in CJD and FI: tauopathy and microgliosis

Besides the primary tauopathies, in which the deposition of abnormal tau protein in the brain constitutes the dominant and often only pathology, several other neurodegenerative conditions with diverse etiologies demonstrate tau pathology. These conditions are known as secondary tauopathies, as other proteins play a central role in their pathogenesis. While GSS has been classified among secondary tauopathies for a long time 48, recently, a secondary tauopathy unrelated to AD pathology has also been documented in sCJD 85, 87, 140. Most frequently, tau deposits in CJD appear as either tiny rod‐ or stub‐like punctate inclusions in the neuropil or as thin neuritic threads 85, 87, 140. Notably, their morphology is also influenced by the pattern of PrP^Sc deposition, which ultimately depends on the disease subtype 87, 140. According to one study, the deposits of hyperphosphorylated tau (p‐tau) positively correlated with both the burden and the morphology of PrP^Sc aggregates 140. Intense PrP^Sc deposition and formation of small plaques elicited a stronger tau deposition, while perineuronal PrP^Sc aggregates were associated with a moderate density of tau particles. Furthermore, while synaptic PrP^Sc deposits correlated with tiny tau granules showing a homogeneous distribution, coarse granular PrP^Sc co‐localized with larger and well discernible tau granules 140. Notably, tau deposition in sCJD was not limited to the hippocampus and neocortical structures, but also involved the granular, molecular and Purkinje cell layers of the cerebellum 140. Although the prion‐specific secondary tauopathy may occur in all sCJD subtypes, it is especially seen in those linked to the V2 strain (ie, VV2 and MV2K subtypes) 87, in which PrP^Sc typically accumulates as kuru‐plaques and plaque‐like deposits. These findings and the relatively high prevalence of a secondary tauopathy in GSS and other PrP‐amyloidosis suggest a link between structural properties of PrP^Sc aggregates and tau hyperphosphorylation/deposition. In agreement with this conclusion, a large number of p‐tau immunoreactive neuritic profiles, often clustered around PrP‐amyloid deposits, have also been observed in vCJD in both the cerebral cortex and the cerebellum, in the absence of Aβ 49. In addition to sporadic and variant CJD, tau pathology has been observed in some phenotypes of gCJD, sometimes with atypical distribution and features [eg, neurofibrillary tangles occurring in brainstem nuclei of young patients carrying E200K 83, and granular p‐tau deposits in the medulla oblongata and pons in gCJD linked to the R208H substitution 93, 145]. Finally, tau deposition has also been described in the thalamus of a single case of FFI 67.

Inflammation is thought to be a major component of neurodegenerative diseases, although the precise role played by microglia, the key contributor to “neuroinflammation,” has yet to be defined. Experimental evidence supports the idea of a dual role of microglia in prion disease pathogenesis. On one hand, especially in the early disease stages, microglia would increase the phagocytosis of PrP^Sc aggregates, and therefore, have a protective role. On the other hand, during disease progression, both the accumulation of PrP^Sc and the neuronal damage would concur to switch microglia to a proinflammatory phenotype contributing to brain toxicity. This dual role of microglia appears to be regulated by multiple pathways, which polarize these cells into distinct phenotypes with characteristic functions 3. To date, studies investigating the microglial response in human prion disease support its role in disease pathogenesis. Microglial cell activation and proliferation occur early in CJD brains 42, 151, 157 where the microgliosis appears to be closely associated with PrP^Sc deposits 42, 52, 102, 107. Interestingly, besides the stage/severity of the disease, the molecular subtype significantly influences the degree of microglial reactivity 42, 154, 166. Other studies demonstrating that microglia markers, pro‐ and anti‐inflammatory cytokines, members of the complement system, regulatory proteins involved in microglial proliferation and key regulators of inflammation COX‐1/2 and PGE2, are all elevated in CJD brains 38, 50, 88 further speak for a crucial role of neuroinflammation in human prion disease.

Variably protease‐sensitive prionopathy

VPSPr is a recently recognized sporadic prion disease that owes its name to the peculiar properties of PrP^Sc aggregates 182. The estimated prevalence in the US, where most of ~40 cases reported to date worldwide have been recognized, is approximately 1%–2% of all prion diseases. Although VPSPr may affect subjects harboring each of the three PRNP codon 129 genotypes, it is most frequently linked to valine homozygosity 10, 45, 55, 56, 182. The clinical phenotype is more often consistent with an AD‐type or frontotemporal‐type of dementia, sometimes associated with atypical parkinsonism. However, atypical presentations may also occur as in two recently reported patients with autopsy‐confirmed VPSPr who received a clinical diagnosis of amyotrophic lateral sclerosis 167. Spongiform change is characterized by non‐confluent vacuoles of intermediate size [ie, larger than in sCJDMM(V)1, but smaller than in sCJDMM2C] and the lesion profile by a predominant involvement of cerebral cortices, striatum and thalamus, while the cerebellum is variably affected and the hippocampus relatively spared. PrP immunostaining reveals microplaques in the molecular layer of cerebellum and clusters of granular deposits often arranged in a target‐like pattern. Western blot analysis of PrP^Sc shows a ladder‐like profile of at least five mildly PK‐resistant fragments migrating in the 7–26 kDa range 182. The lack or minimal representation of diglycosylated PrP^Sc is another distinctive feature of VPSPr.

As per the disease name, both PrP^Sc amount and resistance to PK digestion are lower in VPSPr than in typical sCJDMM(V)1 152. However, PK‐resistance is also significantly influenced by the PRNP codon 129 genotype, being lowest in VPSPr‐129VV, highest in 129MM, and intermediate in 129MV. A recent study documented a regional variability of PrP^Sc properties in VPSPr involving both PK‐resistance and the presence of a CJD‐like fully glycosylated (ie, comprising the diglycosylated form) PrP^Sc form of 19 kDa 128. Further biochemical similarities with CJD were revealed by in vitro seeding assays, such as protein misfolding cyclic amplification (PMCA) and real time‐quaking induced conversion (RT‐QuIC), which showed striking similarities in the profile of newly converted PrP^Sc induced by both sCJDVV2 and VPSPr. Indeed, in VPSPr the converted PrP^Sc appears mainly determined by the 19 kDa CJD‐like fragment rather than by the GSS‐like ~7 kDa fragment 128.

Given the atypical clinical phenotype limiting the chance of a clinical diagnosis of suspected prion disease and the peculiar molecular and biochemical properties, requiring a modified western blot protocol for a consistent PrP^Sc detection, the prevalence of VPSPr is likely underestimated. Distinctive pathological features, which should direct to the correct diagnosis are the presence of cerebellar PrP^Sc microplaques and overall mild/moderate changes despite the relatively long disease duration (usually >20 months) (Figure 4).

Inherited PrP‐amyloidosis

Inherited PrP‐amyloidosis designates a group of genetic prion diseases characterized by the accumulation of an internal PrP^Sc fragment, truncated at both C‐ and N‐termini, mainly, although not exclusively, in the form of amyloid plaques or cerebral amyloid angiopathy. Depending on the causative mutation, the molecular mass of the abnormal PrP^Sc fragment may range from 6 to 11 kDa and can be occasionally associated with other PrP‐truncated species with higher molecular mass. The broad clinical and pathological heterogeneity observed in this group depends not only on the several disease‐associated PRNP mutations (ie, point and stop‐codon mutations, 7–12 extra octarepeat insertions, duplication in the hydrophobic domain), but also on the specific haplotype determined by the combination of PRNP codon 129 genotype/mutation, and other individual factors, exemplified by the significant intra‐familial phenotypic heterogeneity.

Iatrogenic CJD

A number of recent studies have suggested that the age‐adjusted prevalence of Aβ co‐pathology in iCJD is unexpectedly high (up to 40% in patients <55 years) 25, 57, 69, 142. A similar high prevalence of Aβ co‐pathology has been reported in both d‐iCJD and hGH‐iCJD. Overall, the reported burden of Aβ deposits in iCJD is significantly higher than in age‐matched sCJD controls but lower than in AD 25. Aβ accumulation in iCJD frequently involves the vessels in the form of CAA 25, 142. Moreover, diffuse parenchymal deposits extending to the subpial spaces are more frequent than amyloid core plaques 25, 142.

Notably, evidence indicates that Aβ deposition in iCJD is an independent process rather than the result of a cross‐seeding mechanism with PrP^Sc. Indeed, in d‐iCJD, the regional distribution of Aβ aggregates is consistent with a spread from the graft (eg, subpial deposits) 54, 84. Most significantly, in a recent study, significant Aβ co‐pathology has also been observed in post‐mortem samples that received growth hormone treatment but did not develop CJD 142. Finally, another study suggested that neurosurgery with Aβ‐contaminated tools can transmit Aβ pathology and lead to intracerebral hemorrhage 57.

One study also documented Aβ contaminant in the hGH batches (old archived samples) and, after centrifugation, in the pellets, suggesting the presence of insoluble Aβ aggregates in the hormone preparation 39. Taken together these observations support the spreading of Aβ in a prion‐like manner through a peripheral route after subcutaneous hGH administration or centrally through use of dural mater grafts 54, 84, 142. This interpretation is in line with the results of transmission studies conducted in animal models showing that both intracerebral and intraperitoneal inoculation of Aβ induces CAA in APP‐transgenic mice 40, 99.

Given that AD pathology comprises a double proteinopathy, the above‐mentioned observations concerning Aβ raise the question about the role of tau. Two recent studies failed to demonstrate an increased prevalence of tau pathology in iCJD compared to sCJD 25, 142. Tau deposits, when present, were independent of Aβ deposits and compatible with primary age‐related tauopathy (PART). At variance, a very recent study in a French series of hGH‐iCJD brains 39 documented, in three cases, an atypical tau pathology characterized by positive staining of perikarya and neurites, and punctate deposits of a few microns in diameter in the hippocampus and temporal isocortex. Based on this finding and the detection of tau contaminant in hGH batches and pellets, a prion‐like transmission through the peripheral route was also suggested for tau.

Sporadic and genetic prion disease

Very few studies investigated the prevalence and type of neurodegenerative co‐pathologies in sporadic and genetic CJD. Moreover, they mainly focused on AD pathology while other reported associations were limited to case reports. Conflicting results also concern the frequency of the association between CJD and AD pathology, especially regarding the question of whether the co‐existence of the two diseases significantly exceeds that expected by chance based on their age‐related prevalence. A relatively old study that specifically addressed the prevalence of AD‐related pathology in sCJD 53 yielded a prevalence comparable to that observed in elderly non‐demented controls supporting the hypothesis of an age‐related unspecific co‐existence of the two pathologies. At variance, another study showed an inverse correlation between the total amount of accumulated Aβ and PrP^Sc in a subgroup of patients with the co‐occurrence of pathological changes typical for sCJD and AD, with patients with high Aβ load displaying significantly less PrP^Sc than those with low Aβ load. These results suggest that common pathways are involved in the generation of the two pathogenic proteins or their clearance or both 37.

Age‐related primary tauopathies, such as aging‐related tau astrogliopathy (ARTAG), argyrophilic grain disease (AGD) and PART have also been documented in sCJD, although in a minority of cases (5%–10%) 85, 87, 100. A predominantly gray matter type of ARTAG pathology characterized by granular/fuzzy astrocytes occurring in clusters, oligodendroglial coiled bodies, NFTs, pre‐tangles and neuropil threads with predominant localization in the limbic and frontotemporal cortices and, to a lesser extent, in thalamus and basal ganglia, has also been described in a patient carrying the V203I mutation 85.

Finally, in few case reports and small series, the CJD‐specific pathology was found to co‐exist with a synucleinopathy [sCJD+dementia with Lewy bodies (LBD) 41, 100, 168, 169, sCJD+preclinical multiple system atrophy 144] or a primary tauopathy [sCJD+progressive supranuclear palsy (PSP) 100]. Even more rarely, multiple proteinopathies have been documented in the same patient, namely sCJD+AD+LBD+AGD or CJD+AD+PSP+AGD 100. The very low prevalence of these cases suggests a by‐chance association rather than a common origin, although a possible cross‐seeding mechanism cannot be completely ruled out.

Cross‐seeding between PrP and other amyloidogenic proteins

Evidence supporting shared pathogenic mechanisms among neurodegenerative disorders caused by protein misfolding raised the question of the possible cross‐seeding between amyloidogenic proteins, an issue which is also relevant for the interpretation of the neuropathological studies described above on the prevalence of mixed neurodegenerative pathologies in autopsy series.

Current evidence suggesting a cross‐seeding role of PrP is limited to Aβ. However, the possibility of synergistic effect between prion and Aβ pathologies remains controversial. For example, published data indicated that PrP^Sc can exacerbate Aβ deposition at a time point where plaques are already present 106, with both pathologies working synergistically, but also that misfolded Aβ can interfere with prion pathogenesis 150 or do not affect prion pathology at all 139. Further work is needed to clarify this issue and also to understand the possible effect of the strain and the various routes of infection on PrP^Sc‐Aβ cross‐seeding.

Sporadic prion disease

The results of the studies conducted to date with sCJD prions indicate that five of the sCJD subtypes defined by the current classification behave as different prion strains 17, 68, 76, 79, 82, 104, 110, 124, 174, 175 (Figure 6). As the only exception, prions associated with VV2 and MV2K shared transmission properties despite the different phenotypic features in the natural host, suggesting a host genotype effect related to PRNP codon 129. Specifically, sCJD MM1 and MV1 isolates showed identical transmission properties (M1 strain), which significantly differed from those of sCJD VV2 or MV2K (V2 strain) 17, 124. Furthermore, sCJD MM2C, MM2T and VV1 isolates generated distinctive transmission properties, suggesting they are caused by specific strains too 17, 68, 80, 82, 91, 104, 114, 174, 175.

In two recent studies, the transmission of brain inocula from either sCJD MM1+2C or atypical p‐MM1 gave results indistinguishable from those obtained with M1 prions 80, 146. Despite the analogy, the results might have different meanings. The most likely explanation of the former result is that the much higher susceptibility of PrP‐humanized knock‐in mice (as humans and non‐human primates) to M1 prions than to M2C prions prevented the appearance of histopathological or biochemical features related to M2C prions in the animal recipients. Serial passages of sCJDMM1+2C inocula in animals that are susceptible to M2C prions but not to M1 prions will be needed to further clarify the issue. At variance, the latter result [ie, sCJD p‐MM1 transmission] more likely suggests a host‐derived effect (ie, genetic predisposition to amyloid plaque formation in white matter). Indeed, to consider an alternative possibility, one would postulate the unlikely scenario of the co‐occurrence in sCJD p‐MM1 of a distinct prion strain besides sCJD M1, not inducing a distinctive cerebral grey matter pathology, not affecting PrP^Sc properties, and not transmissible to bank voles 146.

Genetic CJD and FFI

Genetic CJD has been linked to the same pool of strains isolated from sCJD, although, to date, transmission studies have examined only part of the phenotypic variants or PRNP haplotype combinations that characterize the disease. When propagated in transgenic mice, non‐human primates or bank voles, CJD inocula from carriers of E200K‐129M and V210I‐129M haplotypes and accumulating PrP^Sc type 1 in the brain showed similar transmission properties to M1 prions 92, 110, 124. Similar results were obtained after transmission of brain homogenates from a subject carrying a 6‐OPRI‐129M allele into transgenic mice expressing human PrP^C and wild‐type FVB mice 96.

Interestingly, unlike with the E200K‐129M, a single E200K‐129V inoculum with PrP^Sc type 2 transmitted with prolonged incubation time and induced plaque‐like focal deposits in the affected mice 7, a result which parallels the findings obtained with sCJD MM1 and VV2 inocula in non‐human primates and transgenic mice 17, 124. On the same line, FFI and sFI inocula shared transmission properties when propagated into transgenic mice 91, 104.

Acquired CJD

Experimental evidence indicates that iCJD MMiK in Japan originated from an incomplete adaptation of the V2 strain to the PRNP codon 129 MM genotype 77, 78. Indeed, both transmission studies in mice 75, 76 and in vitro experiments using PMCA 143, 161 convincingly demonstrated that (i) the transmission of the V2 strain to recipients carrying 129MM results in an unusual PrP^Sc with altered conformational properties (PrP^Sc type “i”); (ii) the V2 strain rapidly re‐emerges when the PrP^Sc type “i” is transmitted to a host expressing 129VV. Notably, like iCJD MMiK, experimentally transmitted kuru also demonstrated the transmission properties of V2 prions, which led to the conclusion that it originated from an individual affected by sCJD VV2 or MV2K 124.

In contrast to prions from kuru and iCJD, vCJD prions showed transmission properties that were strikingly distinct from sCJD prions 22, 59. The vCJD prions transmitted the disease to wild‐type mice far more efficiently than any other form of human prion disease 22, 59, 172 and the faithful propagation of the vCJD phenotype in transgenic mice was dependent upon the homozygous expression of M at PRNP codon 129 6, 14, 16, 171. Transgenic mice homozygous for human PrP 129V showed a pronounced transmission barrier to vCJD prions and propagated a distinct clinicopathological phenotype 6, 16, 59, 160, 171. The latter results raised the possibility that the BSE‐vCJD strain may be associated with other human pathological phenotypes besides that observed in subjects carrying MM at codon 129.

Interestingly, given that at variance with sporadic and inherited prion disease, acquired prion diseases have all been linked to M1, V2 or BSE‐derived prions, it appears that the definition of “infectious” prions (ie, causing a disease that has propagated from human‐to‐human) may only apply to these three human strains. Indeed, strictly speaking, all other human disease variants would better fit the definition of prion‐like disorders (or prionoids), the terms currently used for transmissible but not‐infectious neurodegenerative disorders characterized by protein misfolding, aggregation and seeded polymerization (eg, synucleinopathies, tauopathies) 153.

GSS

With the significant exception of the GSS P102L associated with spongiform change and a PrP^Sc type 1‐like, which showed CJD‐like transmission properties in non‐human primates and wild‐type mice 90, 162, the other GSS variants have been more difficult to transmit to animals in comparison to CJD and FI. This led to the suggestion that these GSS phenotypes (ie, those lacking spongiform change) are not bona‐fide transmissible spongiform encephalopathies (TSE), and would be better designated as non‐transmissible prion disorders. However, more recently, the use of transgenic mice carrying GSS mutations, such as A117V or the mouse equivalent of P102L, led to successful transmissions, although with some significant differences between the two models 8, 9. More specifically, the inoculation of brain extracts from a GSS P102L patient with no spongiform change caused almost no clinical disease but induced striking PrP‐amyloid deposition in brains of several recipient mice; the extracts of those brains failed to transmit neurological disease on further passage but again induced PrP‐amyloid plaques in recipient mice 9. At variance, the transmission of A117V prions induced a typical TSE phenotype with spongiform change and tissue deposition of PrP27‐30 8.

In a recent study, brain homogenates from patients carrying the PRNP mutations G131V, Y226X and Q227X were injected intra‐cerebrally into transgenic mice overexpressing human PrP 138. Transmissions were successful with the former two patients but not with the one carrying Q227X. In detail, half of the animals injected with Y226X brain homogenate had PrP^Sc detectable in the brain by immunostaining, immunoblot, and PrP amyloid seeding activity assayed by RT‐QuIC, while G131V‐injected mice showed PrP^Sc deposition in the brain, although none were positive by immunoblot or RT‐QuIC assay. In contrast, none of the mice injected with Q227X brain had PrP^Sc or PrP‐amyloid seeding activity detectable by these methods 138. Notably, Y226X is the only known truncating PRNP mutation associated with a familial disease that has been shown to be transmissible.

Recently, homogenates from GSS brains expressing the F198S and A117V mutations and showing the typical 8 kDa fragment as the main form of PrP^Sc after PK digestion were successfully transmitted to bank voles, where they induced a classic spongiform encephalopathy 135.

Taken together the data gathered to date indicate that GSS is a truly transmissible disease. However, with the exception of the P102L‐129M phenotype linked to PrP^Sc type 1‐like and spongiform change, it seems that the disease mostly behaves in humans as a prion‐like disorder capable of prion protein amyloid seeding without generating infectious prions. The development of a spongiform encephalopathy rather than prion‐related amyloidosis in bank voles underlines the importance of the recipient host in determining the degree of transmissibility and the associated neurodegenerative changes induced by a given PrP^Sc isolate.

Transmissions to assess the possible zoonotic origin of human prions

Given the lack of a definite answer about the origin of sCJD, the notion that classic‐BSE prions are zoonotic agents causing vCJD in humans keeps alive the possibility that certain sCJD cases may also originate from animal prion disease cross‐transmission. Some concern in this regard came from the observation that a BSE strain variant, designated L‐BSE, responsible for an atypical phenotype of prion disease in aged cattle, propagated more easily than classical BSE in transgenic mice expressing human PrP^C (tgHu) and shared biochemical PrP^Sc properties with sCJD M2C prions 13. However, a recent comparison of the transmission properties in tgHu mice between L‐BSE isolates and a series sCJD cases representative of the human prion strain diversity disclosed no etiological link between L‐BSE and sCJD M2C prions 68. Specifically, although the two isolates showed similar immunoblot profiles and conformational stabilities of PrP^Sc, a shared limited trophism for lymphoid tissue, and a comparable regional profile of PrP^Sc deposition, they significantly differed in the disease incubation time over serial passages and in the resistance to PK digestion of PrP^Sc aggregates.

Concern about the zoonotic potential of prion disease in ruminants was also kept alive by another transmission study in tgHu mice expressing human PrP codon 129 variants 30. In the latter, different scrapie isolates transmitted the disease to several tgHu mice models with an efficiency comparable to that of cattle BSE and led to the propagation of prions that were phenotypically identical to those causing sCJD in humans. If confirmed, these results will significantly strengthen the notion that scrapie prions have zoonotic potential and will generate new questions about the possible link between animal and human prions.

Conclusive remarks

There has been substantial progress on several aspects of human prion disease in recent years. The phenotypic spectrum of disease has been expanded with the addition or a better characterization of rare phenotypic variants such as sCJD p‐MM1, MM2T, VPSPr, iCJD MMiK, vCJD in codon 129 MV recipients, gCJD linked to E200G‐129V and A224V‐129V, GSS associated with P105T‐129V and, above all, PrP‐SA in patients carrying PRNP truncating mutations. Based on the finding of a specific form of tau deposition correlating with both amount and morphology of PrP^Sc aggregates, CJD has been added to the list of secondary tauopathies. Furthermore, recent studies on iCJD have raised concerns that Aβ may be transmissible from human‐to‐human through the injection of contaminated tissues, as it is the case for PrP^Sc. On a related issue, the increasing evidence supporting shared pathogenic mechanisms among neurodegenerative disorders caused by protein misfolding has also raised the question of the possible cross‐seeding between PrP^Sc and other amyloidogenic proteins such as Aβ and tau. However, despite the several studies, results remain controversial on this specific aspect and await further studies on both animal models and human brains.

Significant advances have also been made on the characterization of human prion strains and their molecular basis. The VPSPr, several GSS variants, and PrP‐CAA linked to the truncating mutation Y226X have been added to the list of prion diseases successfully transmitted to recipient animals. However, at variance with the CJD‐FI spectrum, in which current transmission studies indicate the existence of at least six well‐characterized strains, the above‐mentioned transmissions have not yet provided a conclusive picture regarding the extent of strain variation in GSS or VPSPr. Similarly, the critical question of whether or not the pathogenic mutations linked to genetic disease, which are thought to affect PrP conformation, are able to generate prion strains differing from those isolated from the sporadic disease remains unanswered. Finally, concern about the zoonotic potential of prion disease in ruminants remains alive based on the results of at least one study demonstrating the possible convergence of scrapie and sCJD prions into tgHu mice, and the occurrence of sCJD subtypes such as VV1 and MM2T in young adults with a mean age at onset incompatible with the hypothesis of a spontaneous etiology related to aging.