Defining the phenotypic spectrum of sporadic Creutzfeldt-Jakob disease MV2K: the kuru plaque type.

The current classification of sporadic Creutzfeldt-Jakob disease identifies six major subtypes mainly defined by the combination of the genotype at polymorphic codon 129 (methionine/M or valine/V) of the prion protein gene and the type (1 or 2) of misfolded prion protein accumulating in the brain (e.g. MM1, MM2, MV1, MV2, etc.). Here, we systematically characterized the clinical and histo-molecular features associated with the third prevalent subtype, the MV2 subtype with kuru plaques (MV2K), in the most extensive series collected to date. We evaluated neurological histories, cerebrospinal biomarkers, brain MRI and EEG results in 126 patients. The histo-molecular assessment included misfolded prion protein typing, standard histologic staining and immunohistochemistry for prion protein in several brain areas. We also investigated the prevalence and topographic extent of coexisting MV2-cortical features, the number of cerebellar kuru plaques and their effect on clinical phenotype. Systematic regional typing revealed a western blot profile of misfolded prion protein comprising a doublet of 19 and 20 kDa unglycosylated fragments, with the former more prominent in neocortices and the latter in the deep grey nuclei. The 20/19 kDa fragment ratio positively correlated with the number of cerebellar kuru plaques. The mean disease duration was exceedingly longer than in the typical MM1 subtype (18.0 versus 3.4 months). Disease duration correlated positively with the severity of pathologic change and the number of cerebellar kuru plaques. At the onset and early stages, patients manifested prominent, often mixed, cerebellar symptoms and memory loss, variably associated with behavioural/psychiatric and sleep disturbances. The cerebrospinal fluid prion real-time quaking-induced conversion assay was positive in 97.3% of cases, while 14-3-3 protein and total-tau positive tests were 52.6 and 75.9%. Brain diffusion-weighted MRI showed hyperintensity of the striatum, cerebral cortex and thalamus in 81.4, 49.3 and 33.8% of cases, and a typical profile in 92.2%. Mixed histotypes (MV2K + MV2-cortical) showed an abnormal cortical signal more frequently than the pure MV2K (64.7 versus 16.7%, P = 0.007). EEG revealed periodic sharp-wave complexes in only 8.7% of participants. These results further establish MV2K as the most common 'atypical' subtype of sporadic Creutzfeldt-Jakob disease, showing a clinical course that often challenges the early diagnosis. The plaque-type aggregation of the misfolded prion protein accounts for most of the atypical clinical features. Nonetheless, our data strongly suggest that the consistent use of the real-time quaking-induced conversion assay and brain diffusion-weighted MRI allows an accurate early clinical diagnosis in most patients.

From Kuru to Alzheimer: A personal outlook.

Seventy years ago, we learned from Chris Anfinsen that the stereochemical code necessary to fold a protein is embedded into its amino acid sequence. In water, protein morphogenesis is a spontaneous reversible process leading from an ensemble of disordered structures to the ordered functionally competent protein; conforming to Aristotle's definition of substance, the synolon of matter and form. The overall process of folding is generally consistent with a two state transition between the native and the denatured protein: not only the denatured state is an ensemble of several structures, but also the native protein populates distinct functionally relevant conformational (sub)states. This two-state view should be revised, given that any globular protein can populate a peculiar third state called amyloid, characterized by an overall architecture that at variance with the native state, is by-and-large independent of the primary structure. In a nut shell, we should accept that beside the folded and unfolded states, any protein can populate a third state called amyloid which gained center stage being the hallmark of incurable neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases as well as others. These fatal diseases are characterized by clear-cut clinical differences, yet display some commonalities such as the presence in the brain of amyloid deposits constituted by one misfolded protein specific for each disease. Some aspects of this complex problem are summarized here as an excursus from the prion's fibrils observed in the brain of aborigines who died of Kuru to the amyloid detectable in the cortex of Alzheimer's patients.

An autopsied case of MV2K + C-type sporadic Creutzfeldt-Jakob disease presenting with widespread cerebral cortical involvement and Kuru plaques.

MV2-type sporadic Creutzfeldt-Jakob disease (sCJD), which was previously called "Kuru-plaque variant", was gradually revealed to have a wide spectrum and has been classified into three pathological subtypes: MV2K, MV2C and MV2K + C. We herein describe the detailed clinical findings and neuropathologic observations from an autopsied MV2K + C-type Japanese sCJD case with widespread cerebral cortical pathology and Kuru plaques. In the early stages of the disease, the patient exhibited gait disturbance with ataxia and dysarthria as well as gradual appearance of cognitive dysfunction. Diffusion-weighted images (DWI) on MRI revealed extensive cerebral cortical hyperintensity. Pathologic investigation revealed extensive spongiform change in the cerebral cortex, particularly in the deeper layers. Vacuole size varied, and some were confluent. Prion protein (PrP) immunostaining revealed extensive PrP deposition in the cerebral cortex, basal ganglia, thalamus, cerebellum, brainstem and spinal cord. In the cerebral cortex, synaptic-type, Kuru plaque-like, and coarse plaque-type PrP depositions were mainly observed, along with some perivacuolar-type PrP depositions. Kuru plaques and coarse plaque-type PrP depositions also were observed in the cerebellar cortex. PrP gene analysis revealed no mutations, and polymorphic codon 129 exhibited Met/Val heterozygosity. Western blot analysis revealed a mixture of intermediate-type PrPSc and type 2 PrPSc . Based on previous reports regarding MV2-type sCJD and the clinicopathologic findings of the present case, we speculated that it may be possible to clinically distinguish each MV2 subtype. Clinical presentation of the MV2K + C subtype includes predominant cerebral cortical involvement signs with ataxia and DWI hyperintensity of the cerebral cortex on MRI.

Human prion diseases: from Kuru to variant Creutzfeldt-Jakob disease.

Transmissible spongiform encephalopathies (TSEs) or prion diseases are the names given to the group of fatal neurodegenerative disorders that includes kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker disease (GSS), fatal and sporadic familial insomnia and the novel prion disease variable protease-sensitive prionopathy (PSPr) in humans. Kuru was restricted to natives of the Foré linguistic group in Papua New Guinea and spread by ritualistic endocannibalism. CJD appears as sporadic, familial (genetic or hereditary) and infectious (iatrogenic) forms. Variant CJD is a zoonotic CJD type and of major public health importance, which resulted from transmission from bovine spongiform encephalopathy (BSE) through ingestion of contaminated meat products. GSS is a slowly progressive hereditary autosomal dominant disease and the first human TSE in which a mutation in a gene encoding for prion protein (PrP) was discovered. The rarest human prion disease is fatal insomnia, which may occur, in genetic and sporadic form. More recently a novel prion disease variable protease-sensitive prionopathy (PSPr) was described in humans.TSEs are caused by a still incompletely defined infectious agent known as a "prion" which is widely regarded to be an aggregate of a misfolded isoform (PrP(Sc)) of a normal cellular glycoprotein (PrP(c)). The conversion mechanism of PrP(c) into PrP(Sc) is still not certain.

Immunohistochemical investigations of the prion protein accumulation in human spongiform encephalopathies. Special report II.

Creutzfeldt-Jakob disease (CJD) in a proportion of cases may have nonspecific clinical signs and symptoms and no characteristic neuroimaging and EEG picture. Thus, neuropathological studies are mandatory for a diagnosis. However, spongiform change, neuronal loss and astrocyte proliferation--the hallmarks of prion diseases, may also be absent or variable. In such cases, the diagnosis should be supported by the detection of prion protein (PrP) by Western blotting or immunohistochemistry (ICC). PrP may not be visualised under "regular" conditions, but it is unmasked following pretreatment procedures: incubation in formic acid or guanidine thiocyanate, microwave treatment, and hydrated or hydrolytic autoclaving, and these methods were included in standard diagnostic procedures in several different protocols. The aim of this study was to compare the effectiveness of these pretreatment methods and to introduce an optimal protocol for our laboratory. For this purpose, we used brain sections of 11 cases of CJD, 1 case of Gerstmann-Sträussler-Scheinker syndrome (GSS), 1 case of kuru and 3 control brains. For pretreatment we used the hydrated and hydrolytic autoclaving and incubation with formic acid. Immunostaining was performed with monoclonal 3F4 antibody against PrP. The best results were achieved with hydrolytic autoclaving. By this procedure we were able to detect the "synaptic" type of PrP accumulation in all CJD cases, as well as in GSS and kuru, while with other two methods the signal was weaker or even absent.

Pathology and immunocytochemistry of a kuru brain.

We report here results of modern staining techniques including anti-prion protein (PrP) immunocytochemistry to a set of archival brain specimens of a 16 year-old male who died from kuru in 1967. Brain suspensions transmitted disease to chimpanzees and New World monkeys. The PrP gene is homozygous for valine at the polymorphic codon 129. Histology shows neuronal loss, spongiform change, and astrogliosis. Lesions are maximal in parasagittal and interhemispheric areas of frontal, central and parietal cortex, cingulate cortex, striatum, and thalamus, and are accentuated in middle and deep cerebral cortical layers. PrP accumulates as diffuse synaptic type deposits and mostly unicentric plaques. PrP deposition is maximal in parasagittal and interhemispheric areas of frontal, central and parietal cortex, cingulate cortex, basal ganglia, and cerebellar cortex. Plaques are prominent in the striatum, thalamus, and granular layer of cerebellar cortex. Meticulous examination reveals only rare "florid" plaques with surrounding vacuolation. We conclude that 1) pathology including immunomorphology of PrP deposition in this kuru brain is within the lesion spectrum of Creutzfeldt-Jakob disease although plaques are unusually prominent and widespread; 2) kuru does not share the neuropathological hallmarks of the new Creutzfeldt-Jakob disease variant recently reported in the UK and France; 3) topographic prominence of PrP deposition parallels that of spongiform change and/or astrogliosis.

Is the prion structure solved?

We report here on the current knowledge on the nature of the scrapie agent or prion. Several lines of evidence suggest that the abnormal isoform of prion protein (PrP) is crucial for scrapie infectivity while evidence that PrP is also a part of the entire particle of the scrapie agent (prion) is much weaker. There is no doubt, however, that conformational changes (transitions from alpha-helical into beta-pleated structures) of PrP underlay scrapie pathogenesis. In view of the notorious puzzling nature of the scrapie agent, the electron microscopic search for the ultrastructural correlate of it is still warranted. Thus, we discuss the nature of tubulovesicular structures (TVS), the only diseases-specific particles known so far and the association between TVS and PrP fibrils which was recently discovered.

Relationship of microglia and scrapie amyloid-immunoreactive plaques in kuru, Creutzfeldt-Jakob disease and Gerstmann-Sträussler syndrome.

Kuru, Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler syndrome (GSS) are transmissible dementias affecting humans characterized neuropathologically by intraneuronal vacuolation, spongiform change, astrocytic hypertrophy and hyperplasia and the variable presence of amyloid plaques. It has been suggested that microglia are amyloid-forming cells, which play an essential role in amyloid plaque formation. To study the relationship between microglia and amyloid plaques in kuru, CJD and GSS, cerebellar tissues were examined by the double-immunostaining technique using anti-ferritin antibodies as the microglial marker and anti-scrapie amyloid antibody as plaque marker. Ferritin-immunoreactive microglia were observed interdigitating with and among unicentric, multicentric and diffuse types of scrapie amyloid-immunoreactive plaques and were found to a lesser extent in the neuropil. In kuru and CJD, scrapie amyloid-immunoreactive plaques were predominantly unicentric and were observed in the granular layer. In kuru, 53% of the amyloid plaques were associated with microglia, whereas only 30% of plaques in CJD were. In contrast, scrapie-amyloid-immunoreactive plaques in GSS were of the multicentric type, predominantly observed in the molecular layer, and 90% of these plaques were associated with microglia. Our data indicate that microglia are frequently associated with scrapie amyloid-immunoreactive plaques in GSS, less commonly in kuru and to a much lesser extent in CJD, suggesting that microglia may play a variable but important role in the formation of plaques in the transmissible spongiform encephalopathies.

Alzheimer's amyloid precursor protein-positive degenerative neurites exist even within kuru plaques not specific to Alzheimer's disease.

To clarify the relationship between amyloid formation and amyloid precursor protein (APP), the brain sections from eight patients with Alzheimer's disease (AD) and four with Gerstmann-Sträussler Syndrome (GSS) were investigated immunohistochemically by the double-immunostaining method. In AD, most APP-positive senile plaques belong to classical plaques or primitive plaques, whereas in diffuse plaques, APP-positive neuritic components are rarely observed. The authors documented that anti-APP-labeled degenerative neurites surrounding kuru plaques in all four GSS patients. These kuru plaques were verified by double immunostaining using anti-prion protein and anti-APP. The APP-positive structures in kuru plaques were almost identical with those seen in the classical plaques in AD. The authors concluded that APP-positive degenerative neurites are not an early event in the amyloid formation of senile plaques. It is therefore postulated that depositions of beta/A4 and prion proteins are primary events that may involve the surrounding microenvironment and result in the secondary formation of APP-positive degenerative neurites, not specific to AD.

[Molecular biology of subacute spongiform encephalitis]. / Biologie moléculaire des encéphalites spongiformes subaiguës.

Subacute spongiform encephalitis is a pathology that is common to 4 human and 4 animal diseases. These diseases are characterized by the neurological lesions they share and by the fact that they can be transmitted to animals. An abnormal isoform of an endogenous central nervous system protein has been identified. It might be the sole pathogenic agent, but it is certain that it plays a major role in the expressivity of the disease.

A comparative immunohistochemical study of Kuru and senile plaques with a special reference to glial reactions at various stages of amyloid plaque formation.

The authors examined 10 patients with Gerstmann-Sträussler syndrome or Creutzfeldt-Jakob disease and 10 with Alzheimer's disease (AD). Immunohistochemistry using anti-prion protein (PrP) and anti-beta/A4 protein (beta/A4) coupled with formic acid pretreatment could detect Congophilic and non-Congophilic deposits. Prion protein deposits were classified into five types and compared with types of beta/A4 deposits. Kuru plaques with multicentric cores and fine granular deposits were a characteristic feature of PrP deposits. Some types of PrP or beta/A4 deposits depend on the anatomic sites. To clarify the relationship of microglia and astrocytes to PrP or beta/A4 deposits, double-immunostaining method was performed. In both kuru and senile plaques, microglia were closely linked to the Congophilic plaques. Astrocytes, however, extended their processes toward the plaques even in the non-Congophilic plaques. These observations strongly suggest that similar glial association with plaque formation may be involved in both kuru and senile plaques, although the amyloid core proteins differ.

Molecular biology and pathology of scrapie and the prion diseases of humans.

Scrapie and bovine spongiform encephalopathy of animals and Creutzfeldt-Jakob and Gerstmann-Sträussler-Scheinker diseases of humans are transmissible and genetic neurodegenerative diseases caused by prions. Infectious prion particles are composed largely, if not entirely, of an abnormal isoform of the prion protein which is encoded by a chromosomal gene. An as yet unidentified post-translational process converts the cellular prion protein into an abnormal isoform. Scrapie neuropathology, incubation times, and prion synthesis in transgenic mice are controlled by the prion protein gene. Point mutations in the prion protein genes of animals and humans are genetically linked to development of neurodegeneration. Transgenic mice expressing mutant prion proteins spontaneously develop neurologic dysfunction and spongiform neuropathology. Studies of prion diseases may advance investigations of other neurodegenerative disorders and of how neurons differentiate, function for decades and grow senescent.

Apolipoprotein E immunoreactivity in cerebral amyloid deposits and neurofibrillary tangles in Alzheimer's disease and kuru plaque amyloid in Creutzfeldt-Jakob disease.

During the course of our immunohistochemical studies on the change of lipids in Alzheimer's disease brains by using antibody to apolipoprotein E, a protein having a special relevance to nervous tissue, we unexpectedly found that apo E immunoreactivity was associated with amyloid in both senile plaques and cerebral vessels and neurofibrillary tangles. The immunoreactivity was also found in amyloid of kuru plaques in Creutzfeldt-Jakob disease. Pretreatment of the sections with formic acid greatly enhanced immunoreactivity of senile and kuru plaques to antibody to apo E.

Immunohistochemical localization of prion protein in spongiform encephalopathies and normal brain tissue.

We used polyclonal antibodies raised against hamster and mouse PrP27-30 as immunologic probes to study the localization of intracellular and extracellular deposits of prion protein in normal and scrapie-infected mouse and hamster brains and in Creutzfeldt-Jakob disease (CJD)-infected mouse brains. In addition, we examined normal human brain and brain tissues from patients with CJD, kuru, Alzheimer's disease, and idiopathic chronic encephalitis. There was positive staining in the cytoplasm of neurons of normal and scrapie- and CJD-infected mice, and in the neurons of normal and scrapie-infected hamsters. The staining pattern suggests the localization of PrP in an intracellular membrane compartment, most likely the rough endoplasmic reticulum or Golgi apparatus. Antibodies raised against a 15-amino-acid synthetic peptide of the N-terminal of hamster PrP27-30 displayed a similar pattern of staining in mouse brain sections. We observed no intracellular staining in human brain sections obtained at autopsy. Antibodies prepared against mouse and hamster PrP27-30 reacted with amyloid plaques in scrapie-infected mouse and kuru- and CJD-infected human brain sections but not with amyloid plaques in the brain of a patient with Alzheimer's disease.

The presence of complements in amyloid plaques of Creutzfeldt-Jakob disease and Gerstmann-Straussler-Scheinker disease.

The presence of complements Clq, C4, C3, C3b, C3c and C3d, as well as amyloid P component, in the amyloid plaques of Creutzfeldt-Jakob disease (CJD) and Gerstmann-Straussler-Scheinker disease (GSS) brains was demonstrated by means of immunofluorescent and immunoperoxidase techniques. Positive reaction was not observed in other tissue elements, including the blood vessels. These findings may not be due to an adsorption, but to the immunological binding of complements to the amyloid. Proteins such as scrapie associated fibrils or prion in the brain of patients with "unconventional' slow virus diseases are related to the amyloid plaques. It is conceivable that the complements in amyloid are related to these proteins.