Abnormally Upregulated αB-crystallin Was Highly Coincidental with the Astrogliosis in the Brains of Scrapie-Infected Hamsters and Human Patients with Prion Diseases

αB-crystallin is a member of the small heat shock protein family constitutively presenting in brains at a relatively low level. To address the alteration of αB-crystallin in prion disease, the αB-crystallin levels in the brains of scrapie agent 263 K-infected hamsters were analyzed. The levels of αB-crystallin were remarkably increased in the brains of 263 K-infected hamsters, showing a time-dependent manner along with incubation time. Immunohistochemical (IHC) and immunofluorescent (IFA) assays illustrated more αB-crystallin-positive signals in the regions of the cortex and thalamus containing severe astrogliosis. Double-stained IFA verified that the αB-crystallin signals colocalized with the enlarged glial fibrillary acidic protein-positive astrocytes, but not with neuronal nuclei-positive cells. IHC and IFA of the serial brain sections of infected hamsters showed no colocalization and correlation between PrP^Sc deposits and αB-crystallin increase. Moreover, increased αB-crystallin deposits were observed in the brain sections of parietal lobe of a sporadic Creutzfeldt–Jakob disease (sCJD) case, parietal lobe and thalamus of a G114V genetic CJD case, and thalamus of a fatal family insomnia (FFI) case, but not in a parietal lobe of FFI where only very mild astrogliosis was addressed. Additionally, the molecular interaction between αB-crystallin and PrP was only observed in the reactions of recombinant proteins purified from Escherichia coli, but not either in that of brain homogenates or in that of the cultured cell lysates expressing human PrP and αB-crystallin. Our data indicate that brain αB-crystallin is abnormally upregulated in various prion diseases, which is coincidental with astrogliosis. Direct interaction between αB-crystallin and PrP seems not to be essential during the pathogenesis of prion infection.     

Autopsied case of sporadic Creutzfeldt–Jakob disease classified as MM1+2C‐type

We encountered an autopsy case of sporadic Creutzfeldt‐Jakob disease (CJD) pathologically classified as MM1+2C‐type, where Western blot analysis of prion protein (PrP) mainly showed type‐1 scrapie PrP (PrP^Sc) but also, partially, mixed type‐2 PrP^Sc. A Japanese woman complained of visual disorder at the age of 86 years and then showed disorientation and memory disturbances. Magnetic resonance imaging (MRI) showed cerebral cortical hyperintensity on diffusion‐weighted images. The patient died 2 months after the onset of symptoms; her condition did not reach the akinetic mutism state and periodic sharp‐wave complexes on electroencephalography and myoclonus were not recognized. The brain weighed 1100 g and neuropathological examination showed extensive fine vacuole‐type spongiform changes in the cerebral cortex. In some cortical regions, large confluent vacuole‐type spongiform changes were also present. Gliosis and hypertrophic astrocytosis were generally mild, and tissue rarefaction of the neuropil and neuronal loss were not apparent. PrP immunostaining showed diffuse synaptic‐type PrP deposition in the cerebral gray matter, but some regions with large confluent vacuoles showed perivacuolar‐type deposition. We speculated, based on the clinicopathological findings and previous reports, that most MM1‐type sporadic CJD cases may be associated with type‐2 PrP^Sc, at least partially, within certain regions of the cerebrum.     

A traceback phenomenon can reveal the origin of prion infection

The transmission of prions to animals with incongruent prion protein (PrP) gene (referred to as cross‐sequence transmission) results in a relatively long incubation period and can generate a new prion strain with unique transmissibility designated as a traceback phenomenon. For example, cross‐sequence transmission of bovine spongiform encephalopathy (BSE) prions to human generated variant Creutzfeldt‐Jakob disease (vCJD) prions which retained the transmissibility to mice expressing bovine PrP. This finding suggests that traceback studies could enable us to identify the origin of prions. There are two distinct phenotypes in dura mater graft‐associated Creutzfeldt‐Jakob disease (dCJD), with the majority represented by a non‐plaque‐type of dCJD (np‐dCJD) and the minority by a plaque‐type of dCJD (p‐dCJD). To identify the origin of p‐dCJD, we performed a traceback study using mice expressing human PrP with methionine homozygosity (129M/M) or valine homozygosity (129V/V) at polymorphic codon 129. The characteristics of p‐dCJD such as the accumulation of abnormal isoform of PrP (PrP^Sc) intermediate in size between type 1 and type 2, and plaque‐type PrP deposition in the brain were maintained after transmission to the 129M/M mice. Furthermore, the 129V/V mice were more susceptible to p‐dCJD prions than the 129M/M mice and produced type 2 PrP^Sc that were identical in size to those from the 129V/V mice inoculated with sporadic CJD prions from a patient with 129V/V and type 2 PrP^Sc (sCJD‐VV2). In addition, we performed intracerebral transmission of sCJD‐VV2 prions to the 129M/M mice as an experimental model for p‐dCJD. These 129M/M mice showed the accumulation of the intermediate type PrP^Sc and plaque‐type PrP deposition in the brain. These results suggest that p‐dCJD could be caused by cross‐sequence transmission of sCJD‐VV2 prions to individuals with the 129M/M genotype.     

Neuropathological and biochemical criteria to identify acquired Creutzfeldt‐Jakob disease among presumed sporadic cases

As an experimental model of acquired Creutzfeldt‐Jakob disease (CJD), we performed transmission studies of sporadic CJD using knock‐in mice expressing human prion protein (PrP). In this model, the inoculation of the sporadic CJD strain V2 into animals homozygous for methionine at polymorphic codon 129 (129 M/M) of the PRNP gene produced quite distinctive neuropathological and biochemical features, that is, widespread kuru plaques and intermediate type abnormal PrP (PrP^Sc). Interestingly, this distinctive combination of molecular and pathological features has been, to date, observed in acquired CJD but not in sporadic CJD. Assuming that these distinctive phenotypic traits are specific for acquired CJD, we revisited the literature and found two cases showing widespread kuru plaques despite the 129 M/M genotype, in a neurosurgeon and in a patient with a medical history of neurosurgery without dura mater grafting. By Western blot analysis of brain homogenates, we revealed the intermediate type of PrP^Sc in both cases. Furthermore, transmission properties of brain extracts from these two cases were indistinguishable from those of a subgroup of dura mater graft‐associated iatrogenic CJD caused by infection with the sporadic CJD strain V2. These data strongly suggest that the two atypical CJD cases, previously thought to represent sporadic CJD, very likely acquired the disease through exposure to prion‐contaminated brain tissues. Thus, we propose that the distinctive combination of 129 M/M genotype, kuru plaques, and intermediate type PrP^Sc, represents a reliable criterion for the identification of acquired CJD cases among presumed sporadic cases.     

Virus-induced alterations of membrane lipids affect the incorporation of PrP Sc into cells

Prion diseases are fatal neurodegenerative disorders characterized by long incubation periods. To investigate whether concurrent diseases can modify the clinical outcome of prion‐affected subjects, we tested the effect of viral infection on the binding and internalization of PrP^Sc, essential steps of prion propagation. To this effect, we added scrapie brain homogenate or purified PrP^Sc to fibroblasts previously infected with minute virus of mice (MVM), a mouse parvovirus. We show here that the rate of incorporation of PrP^Sc into MVM‐infected cells was significantly higher than that observed for naïve cells. Immunostaining of cells and immunoblotting of subcellular fractions using antibodies recognizing PrP and LysoTracker, a lysosomal marker, revealed that in both control and MVM‐infected cells the incorporated PrP^Sc was associated mostly with lysosomes. Interestingly, floatation gradient analysis revealed that the majority of the PrP^Sc internalized into MVM‐infected cells shifted toward raft‐containing low‐density fractions. Concomitantly, the MVM‐infected cells demonstrated increased levels of the glycosphingolipid GM1 (an essential raft lipid component) throughout the gradient and a shift in caveolin 1 (a raft protein marker) toward lighter membrane fractions compared with noninfected cells. Our results suggest that the effect of viral infection on membrane lipid composition may promote the incorporation of exogenous PrP^Sc into rafts. Importantly, membrane rafts are believed to be the conversion site of PrP^C to PrP^Sc; therefore, the association of exogenous PrP^Sc with such membrane microdomains may facilitate prion infection. © 2008 Wiley‐Liss, Inc.     

Neurodevelopmental expression and localization of the cellular prion protein in the central nervous system of the mouse

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders caused by PrP^Sc, or prion, an abnormally folded form of the cellular prion protein (PrP^C). The abundant expression of PrP^C in the central nervous system (CNS) is a requirement for prion replication, yet despite years of intensive research the physiological function of PrP^C still remains unclear. Several routes of investigation point out a potential role for PrP^C in axon growth and neuronal development. Thus, we undertook a detailed analysis of the spatial and temporal expression of PrP^C during mouse CNS development. Our findings show regional differences of the expression of PrP, with some specific white matter structures showing the earliest and highest expression of PrP^C. Indeed, all these regions are part of the thalamolimbic neurocircuitry, suggesting a potential role of PrP^C in the development and functioning of this specific brain system. J. Comp. Neurol. 518:1879–1891, 2010. © 2010 Wiley‐Liss, Inc.     

Molecular biology and pathology of prion strains in sporadic human prion diseases

Prion diseases are believed to propagate by the mechanism involving self-perpetuating conformational conversion of the normal form of the prion protein, PrP^C, to the misfolded, pathogenic state, PrP^Sc. One of the most intriguing aspects of these disorders is the phenomenon of prion strains. It is believed that strain properties are fully encoded in distinct conformations of PrP^Sc. Strains are of practical relevance to human prion diseases as their diversity may explain the unusual heterogeneity of these disorders. The first insight into the molecular mechanisms underlying heterogeneity of human prion diseases was provided by the observation that two distinct disease phenotypes and their associated PrP^Sc conformers co-distribute with distinct PrP genotypes as determined by the methionine/valine polymorphism at codon 129 of the PrP gene. Subsequent studies identified six possible combinations of the three genotypes (determined by the polymorphic codon 129) and two common PrP^Sc conformers (named types 1 and 2) as the major determinants of the phenotype in sporadic human prion diseases. This scenario implies that each 129 genotype–PrP^Sc type combination would be associated with a distinct disease phenotype and prion strain. However, notable exceptions have been found. For example, two genotype–PrP^Sc type combinations are linked to the same phenotype, and conversely, the same combination was found to be associated with two distinct phenotypes. Furthermore, in some cases, PrP^Sc conformers naturally associated with distinct phenotypes appear, upon transmission, to lose their phenotype-determining strain characteristics. Currently it seems safe to assume that typical sporadic prion diseases are associated with at least six distinct prion strains. However, the intrinsic characteristics that distinguish at least four of these strains remain to be identified.     

Distribution of the cellular prion protein (PrPC) in brains of livestock and domesticated species

In transmissible spongiform encephalopathies (TSEs) the prion protein (PrP) plays a central role in pathogenesis. The PrP gene (Prnp) has been described in a number of mammalian and avian species and its expression product, the cellular prion protein (PrP^C), has been mapped in brains of different laboratory animals (rodent and non-human primates). However, mapping of PrP^C expression in mammalian species suffering from natural (bovine and ovine) and experimental (swine) TSE or in species in which prion disease has never been reported (equine and canine) deserves further attention. Thus, localising the cellular prion protein (PrP^C) distribution in brain may be noteworthy for the understanding of prion disease pathogenesis since lesions seem to be restricted to particular brain areas. In the present work, we analysed the distribution of PrP^C expression among several brain structures of the above species. Our results suggest that the expression of PrP^C, within the same species, differs depending on the brain structure studied, but no essential differences between the PrP^C distribution patterns among the studied species could be established. Positive immunoreaction was found mainly in the neuropil and to a lesser extent in neuronal bodies which occasionally appeared strongly stained in discrete regions. Overall, the expression of PrP^C in the brain was significantly higher in grey matter areas than in white matter, where accumulation of PrP^Sc is first observed in prion diseases. Therefore, other factors besides the level of expression of cellular PrP may account for the pathogenesis of TSEs     

Sporadic fatal insomnia with spongiform degeneration in the thalamus and widespread PrPSc deposits in the brain

We report a case of human prion disease of 29 months duration in a 74‐year‐old Japanese man. The disease started with progressive sleeplessness and dementia. MRI showed gradually progressive cerebral atrophy. Neuronal loss, spongiform change and gliosis were evident in the thalamus and cerebral cortex, as well as in the striatum and amygdaloid nucleus. In the cerebellar cortex, mild‐to‐moderate depletion of Pukinje cells and spongiform change were observed. Mild neuronal loss in the inferior olivary nucleus was also seen. Immunohistochemistry revealed widespread perivacuolar deposits of abnormal prion protein (PrP^Sc) in the cerebral cortex, thalamus, basal ganglia, and brainstem, and minimal plaque‐like deposits of PrP^Sc in the cerebellar cortex. In the cerebellar plaque‐like deposits, the presence of amyloid fibrils was confirmed ultrastructurally. The entire pathology appeared to lie halfway between those of CJD and fatal insomnia, and further demonstrated the relationship between spongiform degeneration and PrP^Sc deposits, especially in the diseased thalamus. By immunoblotting, the thalamus was shown to contain the lowest amount of PrP^SC among the brain regions examined. The PrP^Sc of type 2, in which the ratio of the three glycoforms was compatible with that of sporadic fatal insomnia (MM2‐thalamic variant) reported previously, was also demonstrated. Analysis of the prion protein gene (PRNP) showed no mutation, and homozygosity for methionine at codon 129. In conclusion, we considered that this patient had been suffering from sporadic, pathologically atypical fatal insomnia.     

Quantification of surviving cerebellar granule neurones and abnormal prion protein (PrPSc) deposition in sporadic Creutzfeldt-Jakob disease supports a pathogenic role for small PrPSc deposits common to the various molecular subtypes

B. A. Faucheux, E. Morain, V. Diouron, J.‐P. Brandel, D. Salomon, V. Sazdovitch, N. Privat, J.‐L. Laplanche, J.‐J. Hauw and S. Haïk (2011) Neuropathology and Applied Neurobiology 37, 500–512 Quantification of surviving cerebellar granule neurones and abnormal prion protein (PrP^Sc) deposition in sporadic Creutzfeldt–Jakob disease supports a pathogenic role for small PrP^Sc deposits common to the various molecular subtypes Aims: Neuronal death is a major neuropathological hallmark in prion diseases. The association between the accumulation of the disease‐related prion protein (PrP^Sc) and neuronal loss varies within the wide spectrum of prion diseases and their experimental models. In this study, we investigated the relationships between neuronal loss and PrP^Sc deposition in the cerebellum from cases of the six subtypes of sporadic Creutzfeldt–Jakob disease (sCJD; n = 100) that can be determined according to the M129V polymorphism of the human prion protein gene (PRNP) and PrP^Sc molecular types. Methods: The numerical density of neurones was estimated with a computer‐assisted image analysis system and the accumulation of PrP^Sc deposits was scored. Results: The scores of PrP^Sc immunoreactive deposits of the punctate type (synaptic type) were correlated with neurone counts – the higher the score the higher the neuronal loss – in all sCJD subtypes. Large 5‐ to 50‐µm‐wide deposits (focal type) were found in sCJD‐MV2 and sCJD‐VV2 subtypes, and occasionally in a few cases of the other studied groups. By contrast, the highest scores for 5‐ to 50‐µm‐wide deposits observed in sCJD‐MV2 subtype were not associated with higher neuronal loss. In addition, these scores were inversely correlated with neuronal counts in the sCJD‐VV2 subtype. Conclusions: These results support a putative pathogenic role for small PrP^Sc deposits common to the various sCJD subtypes. Furthermore, the observation of a lower loss of neurones associated with PrP^Sc type‐2 large deposits is consistent with a possible ‘protective’ role of aggregated deposits in both sCJD‐MV2 and sCJD‐VV2 subtypes.     

Intracellular mechanisms mediating the neuronal death and astrogliosis induced by the prion protein fragment 106–126

Prion encephalopathies include fatal diseases of the central nervous system of men and animals characterized by nerve cell loss, glial proliferation and deposition of amyloid fibrils into the brain. During these diseases a cellular glycoprotein (the prion protein, PrP^C) is converted, through a not yet completely clear mechanism, in an altered isoform (the prion scrapie, PrP^Sc) that accumulates within the brain tissue by virtue of its resistance to the intracellular catabolism. PrP^Sc is believed to be responsible for the neuronal loss that is observed in the prion disease. The PrP 106–126, a synthetic peptide that has been obtained from the amyloidogenic portion of the prion protein, represents a suitable model for studying the pathogenic role of the PrP^Sc, retaining, in vitro, some characteristics of the entire protein, such as the capability to aggregate in fibrils, and the neurotoxicity. In this work we present the results we have recently obtained regarding the action of the PrP 106–126 in different cellular models. We report that the PrP 106–126 induces proliferation of cortical astrocytes, as well as degeneration of primary cultures of cortical neurons or of neuroectodermal stable cell lines (GH₃ cells). In particular, these two opposite effects are mediated by the same attitude of the peptide to interact with the L-type calcium channels: in the astrocytes, the activity of these channels seems to be activated by PrP 106–126, while, in the cortical neurons and in the GH₃ cells, the same treatment causes a blockade of these channels causing a toxic effect.     

Using Protein Misfolding Cyclic Amplification Generates a Highly Neurotoxic PrP Dimer Causing Neurodegeneration

Under the “protein-only” hypothesis, prion-based diseases are proposed to result from an infectious agent that is an abnormal isoform of the prion protein in the scrapie form, PrP^Sc. However, since PrP^Sc is highly insoluble and easily aggregates in vivo, this view appears to be overly simplistic, implying that the presence of PrP^Sc may indirectly cause neurodegeneration through its intermediate soluble form. We generated a neurotoxic PrP dimer with partial pathogenic characteristics of PrP^Sc by protein misfolding cyclic amplification in the presence of 1-palmitoyl-2-oleoylphosphatidylglycerol consisting of recombinant hamster PrP (23–231). After intracerebral injection of the PrP dimer, wild-type hamsters developed signs of neurodegeneration. Clinical symptoms, necropsy findings, and histopathological changes were very similar to those of transmissible spongiform encephalopathies. Additional investigation showed that the toxicity is primarily related to cellular apoptosis. All results suggested that we generated a new neurotoxic form of PrP, PrP dimer, which can cause neurodegeneration. Thus, our study introduces a useful model for investigating PrP-linked neurodegenerative mechanisms.     

Accumulation of prion protein in the vagus nerve in creutzfeldt–jakob disease

Disease‐associated proteins are thought to propagate along neuronal processes in neurodegenerative diseases. To detect disease‐associated prion protein (PrP^Sc) in the vagus nerve in different forms and molecular subtypes of Creutzfeldt–Jakob disease (CJD), we applied 3 different anti‐PrP antibodies. We screened the vagus nerve in 162 sporadic and 30 genetic CJD cases. Four of 31 VV‐2 type sporadic CJD and 7 of 30 genetic CJD cases showed vagal PrP^Sc immunodeposits with distinct morphology. Thus, PrP^Sc in CJD affects the vagus nerve analogously to α‐synuclein in Parkinson disease. The morphologically diverse deposition of PrP^Sc in genetic and sporadic CJD argues against uniform mechanisms of propagation of PrP^Sc. Ann Neurol 2019;85:782–787     

Novel Compounds Identified by Structure-Based Prion Disease Drug Discovery Using In Silico Screening Delay the Progression of an Illness in Prion-Infected Mice

The accumulation of abnormal prion protein (PrP^Sc) produced by the structure conversion of PrP (PrP^C) in the brain induces prion disease. Although the conversion process of the protein is still not fully elucidated, it has been known that the intramolecular chemical bridging in the most fragile pocket of PrP, known as the “hot spot,” stabilizes the structure of PrP^C and inhibits the conversion process. Using our original structure-based drug discovery algorithm, we identified the low molecular weight compounds that predicted binding to the hot spot. NPR-130 and NPR-162 strongly bound to recombinant PrP in vitro, and fragment molecular orbital (FMO) analysis indicated that the high affinity of those candidates to the PrP is largely dependent on nonpolar interactions, such as van der Waals interactions. Those NPRs showed not only significant reduction of the PrP^Sc levels but also remarkable decrease of the number of aggresomes in persistently prion-infected cells. Intriguingly, treatment with those candidate compounds significantly prolonged the survival period of prion-infected mice and suppressed prion disease-specific pathological damage, such as vacuole degeneration, PrP^Sc accumulation, microgliosis, and astrogliosis in the brain, suggesting their possible clinical use. Our results indicate that in silico drug discovery using NUDE/DEGIMA may be widely useful to identify candidate compounds that effectively stabilize the protein.Electronic supplementary materialThe online version of this article (10.1007/s13311-020-00903-9) contains supplementary material, which is available to authorized users.     

Creutzfeldt‐Jakob disease

This review will explore the clinical and pathological findings of the various forms of Creutzfeldt‐Jakob disease (CJD). Clinical findings of CJD are characterized by rapidly progressive cognitive dysfunction, diffusion‐weighted magnetic resonance imaging (DWI) hyperintensity, myoclonus, periodic sharp‐wave complexes on electroencephalogram and akinetic mutism state. Neuropathologic findings of CJD are characterized by spongiform changes in gray matter, gliosis—particularly hypertrophic astrocytosis—neuropil rarefaction, neuron loss and prion protein (PrP) deposition. The earliest pathological symptom observed by HE staining in the cerebral cortex is spongiform change. This spongiform change begins several months before clinical onset, and is followed by gliosis. Subsequently, neuropil rarefaction appears, followed by neuron loss. Regions showing fine vacuole‐type spongiform change reflect synaptic‐type PrP deposition and type 1 PrP^Sc deposition, whereas regions showing large confluent vacuole‐type spongiform changes reflect perivacuolar‐type PrP deposition and type 2 PrP^Sc deposition. Hyperintensities of the cerebral gray matter observed in DWI indicate the pathology of the spongiform change in CJD. The cerebral cortical lesions with large confluent vacuoles and type 2 PrP^Sc show higher brightness and more continuous hyperintensity on DWI than those with fine vacuoles and type 1 PrP^Sc. CJD cases showing diffuse myelin pallor of cerebral white matter have been described as panencephalopathic‐type, and this white matter pathology is mainly due to secondary degeneration caused by cerebral cortical involvement, particularly in regard to neuron loss. In conclusion, clinical and neuroimaging findings and neuropathologic observations are well matched in both typical and atypical cases in CJD. The clinical diagnosis of CJD is relatively easy for typical CJD cases such as the MM1‐type. However, even in atypical cases it seems that clinical findings can be used for an accurate diagnosis.     

Creutzfeldt-Jakob disease in a patient with an R208H mutation of the prion protein gene (PRNP) and a 17-kDa prion protein fragment

A case of Creutzfeldt-Jakob disease (CJD) with a rare mutation of the prion protein (PrP) gene (PRNP) at codon 208 (R208H) is described. By comparison with two preceding reports, the case described here displayed two distinct biochemical and neuropathological features. Western blot analysis of brain homogenates showed, in addition to the commonly observed three bands of abnormal protease-resistant PrP isoform (PrP^Sc), an additional band of about 17 kDa. Neuropathological examination of the post mortem brain revealed tau pathology in the hippocampus and entorhinal cortex, as well as ballooned neurons in the cortex, hippocampus and subcortical gray matter.     

Autopsy case of MV2K‐type sporadic Creutzfeldt‐Jakob disease with spongiform changes of the cerebral cortex

Comprehensive analysis is required for the accurate diagnosis of MV2‐type sporadic Creutzfeldt–Jakob disease (sCJD) because it shows a wide clinicopathological spectrum. Here, we describe the clinical findings and neuropathologic observations of an autopsy‐confirmed MV2K‐type sCJD case with extensive spongiform changes of the cerebral cortex. In the early disease stages, the patient exhibited gait disturbance with ataxia and gradually showed cognitive dysfunction. Diffusion‐weighted magnetic resonance images revealed hyperintense regions in the cerebral cortex, basal ganglia, and particularly in the thalamus. Prion protein (PrP) gene analysis revealed no mutations, and polymorphic codon 129 exhibited methionine and valine heterozygosity. During the course of the disease, a startle reaction was observed, whereas myoclonus was not observed. Electroencephalography showed no periodic sharp wave complexes. The patient died at age 61 years with 13 months total disease duration and did not reach the akinetic mutism state. Pathologic investigation revealed extensive fine vacuole‐type spongiform change in the cerebral cortex, and the appearance of vacuolation tended to be more pronounced in the deeper layers. Numerous kuru plaques were observed in the cerebellum. PrP immunostaining revealed extensive diffuse synaptic‐type PrP deposition in the cerebral cortex, and the finding was prominent in the deeper layer with perineuronal‐type PrP deposition. In the limbic system, basal ganglia, and thalamus, mixed small plaque‐type PrP with synaptic‐type PrP deposition was observed. In the cerebellar cortex, diffuse synaptic‐type PrP depositions were observed with numerous strongly immunopositive plaques. Western blot analysis of examined brain samples revealed mixed type 2 PrP^Sc (scrapie type) and intermediate‐type PrP^Sc.     

On the biology of prions

Summary Prions cause scrapie and Creutzfeldt-Jakob disease (CJD); these infectious pathogens are composed largely, if not entirely, of protein molecules. No prion-specific polynucleotide has been identified. Purified preparations of scrapie prions contain high titers (10^9.5 ID₅₀/ml), one protein (PrP 27-30) and amyloid rods (10–20 nm in diameter ×100–200 nm in length). Considerable evidence indicates that PrP 27-30 is required for and inseparable from scrapie infectivity. PrP 27-30 is encoded by a cellular gene and is derived from a larger protein, denoted PrP^Sc or PrP 33-35^Sc, by protease digestion. A cellular isoform, designated PrP^C or PrP 33-35^C, is encoded by the same gene as PrP^Sc and both proteins appear to be translated from the same 2.1 kb mRNA. Monoclonal antibodies to PrP 27-30, as well as antisera to PrP synthetic peptides, specifically react with both PrP^C and PrP^Sc, establishing their relatedness. PrP^C is digested by proteinase K, while PrP^Sc is converted to PrP 27-30 under the same conditions. Prion proteins are synthesized with signal peptides and are integrated into membranes. Detergent extraction of microsomal membranes isolated from scrapie-infected hamster brains solubilizes PrP^C but induces PrP^Sc to polymerize into amyloid rods. This procedure allows separation of the two prion protein isoforms and the demonstration that PrP^Sc accumulates during scrapie infection, while the level of PrP^C does not change. The prion amyloid rods generated by detergent extraction are identical morphologically, except for length, to extracellular collections of prion amyloid filaments which form plaques in scrapie- and CJD-infected brains. The prion amyloid plaques stain with antibodies to PrP 27-30 and PrP peptides. PrP 33-35^C does not accumulate in the extracellular space. Prion rods composed of PrP 27-30 can be dissociated into phospholipid vesicles with full retention of scrapie infectivity. The murine PrP gene (Prn-p) is linked to thePrn-i gene which controls the length of the scrapie incubation period. Prolonged incubation times are a cardinal feature of scrapie and CJD. While the central role of PrP^Sc in scrapie pathogenesis is well established, the chemical as well as conformational differences between PrP^C and PrP^Sc are unknown but probably arise from post-translational modifications.Supported by research grants from the National Institutes of Health (AG 02132 and NS 14069) and a Senator Jacob Javits Center of Excellence in Neuroscience (NS 22786) as well as by gifts from RJR-Nabisco, Inc. and Sherman Fairchild FoundationThis review is based upon a plenary lecture entitled Biology and Neuropathology of Prions presented at the Xth International Congress of Neuropathology, Stockholm, Sweden, September 11, 1986, and is dedicated to the memory of Peter Wilhelm Lampert (1929–1986)     

Abnormal Activation of Microglia Accompanied with Disrupted CX3CR1/CX3CL1 Pathway in the Brains of the Hamsters Infected with Scrapie Agent 263K

Microglial cells are resident mononuclear phagocytes of the central nervous system (CNS). Active proliferation of microglia in the brain has been identified in neurodegenerative disorders, including some kinds of prion disease. However, the detailed regional distribution between microglia and PrP^Sc deposition has not been presented, and investigation of fractalkine signaling which is involved in the regulation of activation of microglia in prion disease is not well documented. In this study, the disease phenomenon of microglial accumulation in the CNS was thoroughly analyzed using a scrapie-infected experimental model. Western blots of microglia-specific markers Iba1 and CD68, immunohistochemical and immunofluorescent assays demonstrated obviously activation of microglia in almost whole brain regions in the infected animals. Under the dynamic analysis on hallmarks of activation of microglia, a time-dependent increase of Iba1 and CD68 was detected, accompanied by accumulation of PrP^Sc and progression of neurodegenerative symptoms. With serial brain sections and double staining of Iba1 and PrP^Sc, we observed that the microglia distributed around PrP^Sc deposits in 263K-infected hamsters’ brains, proposing PrP^Sc phagocytosis. Flow cytometry assays with the single-cell suspensions prepared from the cortical region of the infected brains verified an activation of microglial population. ELISA assays of the cytokines in brain homogenates revealed significant upregulations of interleukin (IL)-1β, IL-6 and TNF-α when infected. Evaluation of fractalkine signaling in the infected hamsters’ brains showed progressively downregulation of CX3CL1 during the incubation. Prion peptide PrP106-126 also disrupted fractalkine and evoked microglial activation in rat primary neuron–glia mixed cultures. Our data here demonstrate an activated status of microglia in CNS tissues of infectious prion disease, possibly through fractalkine signaling deficiency.     

An autopsied case of MM1 + MM2‐cortical with thalamic‐type sporadic Creutzfeldt‐Jakob disease presenting with hyperintensities on diffusion‐weighted MRI before clinical onset

A 78‐year‐old Japanese man presented with rapidly progressive dementia and gait disturbances. Eight months before the onset of clinical symptoms, diffusion‐weighted magnetic resonance imaging (DWI) demonstrated hyperintensities in the right temporal, right parietal and left medial occipital cortices. Two weeks after symptom onset, DWI showed extensive hyperintensity in the bilateral cerebral cortex, with regions of higher brightness that existed prior to symptom onset still present. Four weeks after clinical onset, periodic sharp wave complexes were identified on an electroencephalogram. Myoclonus was observed 8 weeks after clinical onset. The patient reached an akinetic mutism state and died 5 months after onset. Neuropathological examination showed widespread cerebral neocortical involvement of fine vacuole‐type spongiform changes with large confluent vacuole‐type spongiform changes. Spongiform degeneration with neuron loss and hypertrophic astrocytosis was also observed in the striatum and medial thalamus. The inferior olivary nucleus showed severe neuron loss with hypertrophic astrocytosis. Prion protein (PrP) immunostaining showed widespread synaptic‐type PrP deposition with perivacuolar‐type PrP deposition in the cerebral neocortex. Mild to moderate PrP deposition was also observed extensively in the basal ganglia, thalamus, cerebellum and brainstem, but it was not apparent in the inferior olivary nucleus. PrP gene analysis showed no mutations, and polymorphic codon 129 showed methionine homozygosity. Western blot analysis of protease‐resistant PrP showed both type 1 scrapie type PrP (PrP^Sc) and type 2 PrP^Sc. Based on the relationship between the neuroimaging and pathological findings, we speculated that cerebral cortical lesions with large confluent vacuoles and type 2 PrP^Sc would show higher brightness and continuous hyperintensity on DWI than those with fine vacuoles and type 1 PrP^Sc. We believe the present patient had a combined form of MM1 + MM2‐cortical with thalamic‐type sporadic Creutzfeldt‐Jakob disease (sCJD), which suggests a broader spectrum of sCJD clinicopathological findings.