Beata Sikorska

Ultrastructural study of florid plaques in variant Creutzfeldt-Jakob disease: a comparison with amyloid plaques in kuru, sporadic Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker disease.

Background: Although the histological features of the amyloid plaques in variant Creutzfeldt–Jakob disease (vCJD) are distinct from those in other forms of prion disease [kuru, sporadic Creutzfeldt–Jakob disease (sCJD) and Gerstmann–Sträussler–Scheinker disease (GSS)], their ultrastructural features have only been described in a single case report. Aims: To study vCJD plaques systematically and compare them with plaques in kuru, sCJD, GSS and Alzheimer disease (AD). Methods: Amyloid plaques were studied by transmission electron microscopy and image analysis in five cases of vCJD, three cases of GSS, two cases of sCJD, one case of kuru and five cases of AD. Immunohistochemistry was performed on paraffin sections from one case of vCJD, two cases of GSS, one case of kuru and two cases of sCJD. Results: The florid plaques in vCJD were either compact or more diffuse; in both forms, the radiating fibrils were organized into thick ‘tongues’, in contrast to kuru plaques. Dystrophic neurites (DNs) containing lysosomal electron‐dense bodies or vesicles surrounded florid plaques. Microglial cells were found within florid plaques; occasional amyloid fibrils were identified in membrane‐bound pockets of microglial cells. In vCJD, there was significant tau immunoreactivity in DNs around florid plaques while, in sCJD, GSS and kuru, minimal tau immunoreactivity was observed around plaques. Conclusions: The ultrastructure of the florid plaques and DNs in vCJD is more reminiscent of neuritic plaques in AD than kuru or multicentric plaques. These findings may reflect differences both in the strains of the transmissible agents responsible for these disorders and in host factors.

Ultrastructural pathology of prion diseases revisited: brain biopsy studies.

We report here a detailed ultrastructural comparison of brain biopsies from 13 cases of Creutzfeldt‐Jakob disease (CJD) and from one case of fatal familial insomnia (FFI). The latter disease has not heretofore benefited from ultrastructural study. In particular, we searched for tubulovesicular structures (TVS), 35‐nm particles regarded as the only disease‐specific structures at the level of thin‐section electron microscopy. Our material consisted of brain biopsies obtained by open surgery from one FFI case from a new French family, one case of variant CJD (vCJD), nine cases of sporadic CJD (sCJD), two cases of iatrogenic (human growth hormone) CJD and one case of hereditary CJD (Val203Iso). The ultrastructural picture of the cerebral cortex of the FFI patient was virtually indistinguishable from that of CJD. TVS were found, albeit only after prolonged search. Typical spongiform change was observed, consisting of intracellular membrane‐bound vacuoles containing secondary chambers (vacuoles within vacuoles) and amorphous material. Neuronal degeneration was widespread: some processes contained degenerating mitochondria and lysosomal electron‐dense bodies and these met the criteria for neuroaxonal dystrophy. Other processes contained branching cisterns; still others were filled with electron‐dense masses and amorphous vesicles. The overall ultrastructural appearance of variant CJD was similar to that of FFI cerebral cortex, except for a much higher number of cellular processes containing TVS. We detected TVS in the majority of sCJD cases that, in addition to typical spongiform change and robust astrocytic reaction, showed widespread neuritic and synaptic degeneration and autophagic vacuoles. We conclude that TVS are readily found in FFI, vCJD and sCJD and that widespread neuritic degeneration is a part of ultrastructural pathology in prion diseases.

The sheddase ADAM10 is a potent modulator of prion disease

The prion protein (PrPC) is highly expressed in the nervous system and critically involved in prion diseases where it misfolds into pathogenic PrPSc. Moreover, it has been suggested as a receptor mediating neurotoxicity in common neurodegenerative proteinopathies such as Alzheimers disease. PrPC is shed at the plasma membrane by the metalloprotease ADAM10, yet the impact of this on prion disease remains enigmatic. Employing conditional knockout mice, we show that depletion of ADAM10 in forebrain neurons leads to posttranslational increase of PrPC levels. Upon prion infection of these mice, clinical, biochemical, and morphological data reveal that lack of ADAM10 significantly reduces incubation times and increases PrPSc formation. In contrast, spatiotemporal analysis indicates that absence of shedding impairs spread of prion pathology. Our data support a dual role for ADAM10-mediated shedding and highlight the role of proteolytic processing in prion disease. DOI: http://dx.doi.org/10.7554/eLife.04260.001

High incidence of MGMT promoter methylation in primary glioblastomas without correlation with TP53 gene mutations

O(6)-methylguanine DNA methyltransferase (MGMT) reduces cytotoxicity of therapeutic or environmental alkylating agents. MGMT promoter methylation has been associated with TP53 G: C to A:T transition mutations in various types of cancers, and with poor prognosis in patients who did not receive chemotherapy. Mutations of TP53 are more frequent in secondary than in primary glioblastoma, thus the expected MGMT promoter methylation was low in primary glioblastoma. Glioblastoma patients with MGMT promoter methylation showed better response to chemotherapy based on alkylating agents and longer survival than patients without MGMT methylation. We examined 32 primary glioblastomas, treated with radiotherapy and surgery, for TP53 mutation by direct sequencing and MGMT promoter methylation by methylation-specific PCR. MGMT promoter methylation and TP53 mutations were detected in 72% and 31% of primary glioblastoma, respectively. Although not statistically significant, the frequency of TP53 G:C to A:T mutations were higher in cases with (26%) than without (11%) MGMT promoter methylation (p=0.376). MGMT promoter methylation had no impact on patient survival. Our data indicate that MGMT promoter methylation occurs frequently in primary glioblastoma, but does not lead to G:C to A:T TP53 mutations, has no independent prognostic value and is not a predictive marker unless glioblastoma patients are treated with chemotherapy.

Molecular heterogeneity of meningioma with INI1 mutation

Background: INI1 (hSNF5) mutations are linked to rhabdoid tumours, but mutations in meningiomas with hot spot mutations in position 377 have also been reported. Aims: To analyse the INI1 gene in meningioma. Methods: Exons 1, 4, 5, and 9 of the INI1 gene were analysed by the polymerase chain reaction and direct sequencing in 80 meningiomas. For all cases, western blotting of the INI1 protein was performed. Results: Only one of the 80 samples showed a cytosine insertion in codon 376. This mutation changed the open reading frame in almost the whole exon 9 and resulted in a longer hSNF5 protein. Complex analysis of the above described tumour sample by western blotting, DNA sequencing, and loss of heterozygosity (LOH) analysis showed that this particular meningioma consisted of heterogeneic cellular components. One of these components had a mutated INI1 gene, whereas in the other component INI1 was intact. Conclusions: INI1 mutation is a rare event in the molecular pathology of meningiomas. It is possible for the INI1 gene to be mutated in only a proportion of meningioma cells.

Boginia virus, a newfound hantavirus harbored by the Eurasian water shrew (Neomys fodiens) in Poland

BackgroundGuided by decades-old reports of hantaviral antigens in the Eurasian common shrew (Sorex araneus) and the Eurasian water shrew (Neomys fodiens) in European Russia, we employed RT-PCR to analyze lung tissues of soricine shrews, captured in Boginia, Huta Dłutowska and Kurowice in central Poland during September 2010, 2011 and 2012.FindingsIn addition to Seewis virus (SWSV), which had been previously found in Eurasian common shrews elsewhere in Europe, a genetically distinct hantavirus, designated Boginia virus (BOGV), was detected in Eurasian water shrews captured in each of the three villages. Phylogenetic analysis, using maximum likelihood and Bayesian methods, showed that BOGV formed a separate lineage distantly related to SWSV.ConclusionsAlthough the pathogenic potential of BOGV and other recently identified shrew-borne hantaviruses is still unknown, clinicians should be vigilant for unusual febrile diseases and clinical syndromes occurring among individuals reporting exposures to shrews.

Kuru: genes, cannibals and neuropathology.

Abstract Kuru was the first human transmissible spongiform encephalopathy (TSE) or prion disease identified, occurring in the Fore linguistic group of Papua New Guinea. Kuru was a uniformly fatal cerebellar ataxic syndrome, usually followed by choreiform and athetoid movements. Kuru imposed a strong balancing selection on the Fore population, with individuals homozygous for the 129 Met allele of the gene (PRNP) encoding for prion protein (PrP) being the most susceptible. The decline in the incidence of kuru in the Fore has been attributed to the exhaustion of the susceptible genotype and ultimately by discontinuation of exposure via cannibalism. Neuropathologically, kuru-affected brains were characterized by widespread degeneration ofneurons, astroglial and microglial proliferation, and the presence of amyloid plaques. These early findings have been confirmed and extended by recent immunohistochemical studies for the detection of the TSE-specific PrP (PrPTSE). Confocal laser microscopy also showed theconcentration of glial fibrillary acidic protein–positive astrocytic processes at the plaque periphery. The fine structure of plaques corresponds to that described earlier by light microscopy. The successful experimental transmission of kuru led to the awareness of its similarityto Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker disease and formed a background against which the recent epidemics of iatrogenic and variant Creutzfeldt-Jakob disease could be studied.

Ultrastructural Characteristics (or Evaluation) of Creutzfeldt-Jakob Disease and Other Human Transmissible Spongiform Encephalopathies or Prion Diseases

The authors report on a large series of human prion diseases to establish ultrastructural characteristics that may be useful for their diagnosis. For Creutzfeldt-Jakob disease (CJD and its variant, vCJD) and fatal familial insomnia (FFI) only vacuolation (spongiform change) and the presence of tubulovesicular structures are consistent findings. Other changes, such as the presence of myelinated vacuoles, branching cisternae, neuroaxonal dystrophy, and autophagic vacuoles, were present in different proportions in either CJD or FFI, but they are nonspecific ultrastructural findings that can also occur in other neurodegenerative conditions. The hallmark of Gerstmann-Sträussler-Scheinker disease (GSS) and vCJD is the amyloid plaque, but plaques of GSS and kuru are different than those of vCJD. Whereas the former are typical unicentric kuru type or multicentric plaques, the latter are unicentric florid plaques. Also, kuru plaques are nonneuritic, whereas GSS florid plaques are usually neuritic; however, a proportion of plaques from GSS was also found to have nonneuritic characteristics. Thus, the presence or absence of dystrophic neurites is not a discriminatory factor for GSS and vCJD. Furthermore, plaques from GSS with different mutations were also slightly different. In GSS with mutations P102L, 232T, and A117V plaques were stellate while in 1 case with 144 base-pair insertion and in GSS-A117V, round plaques were also observed, and typical primitive neuritic plaques, i.e., composed of dystrophic neurites with little or no amyloid, were found only in a P102L case from the original Austrian family. In 2 cases of sporadic CJD, the kuru stellate plaque predominated.

Creutzfeldt-Jakob disease.

Creutzfeldt-Jakob disease (CJD), a neurodegenerative disorder that is the commonest form of human prion disease or transmissible spongiform encephalopathies (TSEs). Four types of CJD are known: Sporadic (sCJD), familial or genetic (gCJD); iatrogenic (iCJD) and variant CJD (vCJD). The latter results from transmission of bovine spongiform encephalopathy (BSE) from cattle to humans. The combination of PrP(Sc) peptide (either 21 kDa or 19 kDa) and the status of the codon 129 of the gene (PRNP) encoding for PrP (either Methionine or Valine) is used to classify sCJD into 6 types: MM1 and MV1, the most common; VV2; MV2 (Brownell/Oppenheimer syndrome); MM2; VV1 and sporadic fatal insomnia, in that order of prevalence. Genetic CJD is caused by diverse mutations in the PRNP gene. The neuropathology of CJD consists of spongiform change, astro- and microgliosis and poorly defined neuronal loss. In a proportion of cases, amyloid plaques, like those of kuru, are seen. PrP immunohistochemistry reveals different types of PrP(Sc) deposits - the most common is the synaptic-type, but perivacuolar, perineuronal and plaque-like deposits may be also detected.

Synucleinopathy with features of both multiple system atrophy and dementia with Lewy bodies.

Alpha-synuclein is a main component of neuronal inclusions in Parkinson’s disease, dementia with Lewy bodies (DLB) and glial and neuronal inclusions in multiple system atrophy (MSA) – as a group often referred to as alpha-synucleinopathies [1,2]. Dementia with Lewy bodies is the second most frequent neurodegenerative dementing disorder in the elderly after Alzheimer’s disease. Clinically, it is associated with fluctuating cognition and hallucinations in association with neurological features of Parkinsonism. DLB is neuropathologically characterized by the presence of Lewy bodies (LBs) in the brain [3]. Classically, LBs appear as one or more intraneuronal eosinophilic spherical structures with a dense core. They are often surrounded by a halo, although in the neocortex they usually lack the halo and may be inconspicuous in routine staining. Multiple system atrophy is a sporadic neurodegenerative disease clinically characterized by varying degrees of Parkinsonism, cerebellar ataxia and/or features of autonomic failure. The term MSA was coined to unify three separate entities: striatonigral degeneration, olivopontocerebellar atrophy and the Shy–Drager syndrome, which may overlap clinically and pathologically [4]. However, it was not until 1989, when Papp and Lantos described filamentous inclusions in the cytoplasm of oligodendroglial cells in the brains of patients with MSA, irrespective of clinical symptoms, that the existence of MSA as a clinicopathological entity was confirmed [5]. Glial cytoplasmic inclusions (GCIs), also called Papp–Lantos bodies, were more recently shown also to be composed mainly of alpha-synuclein [1,6]. Similar inclusions were later identified in neuronal cytoplasm (neuronal cytoplasmic inclusions, or NCIs) and in the nuclei of neurones and oligodendrocytes [7,8]. The clinical phenotype depends on the neuronal systems predominantly involved; hence, the two clinical presentations are now designated MSAcerebellar type and MSA-Parkinsonism. We report here an archival case of a 51-year-old woman with progressive cognitive deficits, psychotic symptoms (hallucinations) and muscle rigidity, who died of unrelated gastric haemorrhage a year after the onset of neurological and psychiatric signs. According to the archival files, the patient presented on admission with delirium superposed on progressive dementia. Neuroleptic sensitivity reaction (extrapyramidal signs) occurred during the course of the disease. Owing to the lack of detailed clinical data, thorough analysis of the clinical phenotype of this patient could not be performed in this retrospective study. Brain autopsy was performed after 14 days of fixation in 4% formaldehyde. Recently, the archival paraffin blocks were re-examined. Fivemicrometre-thick sections were obtained from each tissue block for routine stainings, conventional immunohistochemistry and double immunofluorescent labelling. Immunohistochemistry was performed using a panel of antibodies (Table 1) according to the producers’ protocols. As a secondary system we used the ChemMateTM detection kit Dako, Glostrup, Denmark. For immunofluorescent labelling and multichannel confocal microscopy we used mouse anti-microtubuleassociated protein (MAP)-2 monoclonal antibody (Chemicon International, Inc. Temecula, CA, USA, dilution 1:250) and rat anti-a-synuclein monoclonal antibody (Alexis Bichemicals, Lausen, Switzerland, dilution 1:100). The fluorescent-labelled secondary antibody for the antia-synuclein antibody was Alexa Fluor 488 rabbit anti-rat IgG (Molecular Probes, Eugene, USA, 1:200) and for antiMAP-2 Alexa Fluor 546 goat anti-mouse IgG (Molecular Probes, 1:200). To visualize cell nuclei, Sytox orange stain was used (Dako, Glostrup, Denmark). Immunofluorescence labelling was evaluated using a Zeiss LSM 510 laser scanning confocal microscope. Material for electron microscopy was retrieved from formalin-fixed, paraffin-embedded sections and reprocessed for electron microscopy. On gross neuropathological examination, moderate and diffuse brain atrophy was observed, along with slight depigmentation of the substantia nigra. Microscopic examin-