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Biochemical and structural investigation of a model for prion-induced neurodegeneration—antibody binding to PrP C —reveals the role of the PrP flexible tail and reactive oxygen species in mediating toxicity. Prion tail linked to neurotoxicity The cellular prion protein contains a globular domain and a flexible tail. Here it is shown that antibodies against the globular domain cause neurotoxicity in mice and cerebellar cultured slices. This neurodegeneration is accompanied by a burst of reactive oxygen species production, and is suppressed by antioxidants, and toxicity is dependent on the presence of the superoxide-producing enzyme NOX2. Octapeptide repeats within the flexible-tail domain seem also to be required for toxicity, and antibodies against these repeats are able to prolong life in in mice expressing a toxic deletion mutant PrP C protein. These novel neurotoxicity mechanisms may be relevant to the pathogenesis of neurodegenerative disease due to prions and other agencies. Prion infections cause lethal neurodegeneration. This process requires the cellular prion protein (PrP C ; ref. 1 ), which contains a globular domain hinged to a long amino-proximal flexible tail 2 . Here we describe rapid neurotoxicity in mice and cerebellar organotypic cultured slices exposed to ligands targeting the α1 and α3 helices of the PrP C globular domain. Ligands included seven distinct monoclonal antibodies 3 , monovalent Fab 1 fragments and recombinant single-chain variable fragment miniantibodies. Similar to prion infections 4 , 5 , 6 , the toxicity of globular domain ligands required neuronal PrP C , was exacerbated by PrP C overexpression, was associated with calpain activation and was antagonized by calpain inhibitors. Neurodegeneration was accompanied by a burst of reactive oxygen species, and was suppressed by antioxidants. Furthermore, genetic ablation of the superoxide-producing enzyme NOX2 (also known as CYBB) protected mice from globular domain ligand toxicity. We also found that neurotoxicity was prevented by deletions of the octapeptide repeats within the flexible tail. These deletions did not appreciably compromise globular domain antibody binding, suggesting that the flexible tail is required to transmit toxic signals that originate from the globular domain and trigger oxidative stress and calpain activation. Supporting this view, various octapeptide ligands were not only innocuous to both cerebellar organotypic cultured slices and mice, but also prevented the toxicity of globular domain ligands while not interfering with their binding. We conclude that PrP C consists of two functionally distinct modules, with the globular domain and the flexible tail exerting regulatory and executive functions, respectively. Octapeptide ligands also prolonged the life of mice expressing the toxic PrP C mutant 7 , PrP(Δ94–134), indicating that the flexible tail mediates toxicity in two distinct PrP C -related conditions. Flexible tail-mediated toxicity may conceivably play a role in further prion pathologies, such as familial Creutzfeldt-Jakob disease in humans bearing supernumerary octapeptides.

Recurrent mutations affecting the histone H3.3 residues Lys27 or indirectly Lys36 are frequent drivers of pediatric high-grade gliomas (over 30 % of HGGs). To identify additional driver mutations in HGGs, we investigated a cohort of 60 pediatric HGGs using whole-exome sequencing (WES) and compared them to 543 exomes from non-cancer control samples. We identified mutations in SETD2 , a H3K36 trimethyltransferase, in 15 % of pediatric HGGs, a result that was genome-wide significant (FDR = 0.029). Most SETD2 alterations were truncating mutations. Sequencing the gene in this cohort and another validation cohort (123 gliomas from all ages and grades) showed SETD2 mutations to be specific to high-grade tumors affecting 15 % of pediatric HGGs (11/73) and 8 % of adult HGGs (5/65) while no SETD2 mutations were identified in low-grade diffuse gliomas (0/45). Furthermore, SETD2 mutations were mutually exclusive with H3F3A mutations in HGGs ( P  = 0.0492) while they partly overlapped with IDH1 mutations (4/14), and SETD2 -mutant tumors were found exclusively in the cerebral hemispheres ( P  = 0.0055). SETD2 is the only H3K36 trimethyltransferase in humans, and SETD2 -mutant tumors showed a substantial decrease in H3K36me3 levels ( P  < 0.001), indicating that the mutations are loss-of-function. These data suggest that loss-of-function SETD2 mutations occur in older children and young adults and are specific to HGG of the cerebral cortex, similar to the H3.3 G34R/V and IDH mutations. Taken together, our results suggest that mutations disrupting the histone code at H3K36, including H3.3 G34R/V, IDH1 and/or SETD2 mutations, are central to the genesis of hemispheric HGGs in older children and young adults.

Most transmissible spongiform encephalopathies arise either spontaneously or by infection. Mutations of PRNP, which encodes the prion protein, PrP, segregate with phenotypically similar diseases. Here we report that moderate overexpression in transgenic mice of mPrP(170N, 174T), a mouse PrP with two point mutations that subtly affect the structure of its globular domain, causes a fully penetrant lethal spongiform encephalopathy with cerebral PrP plaques. This genetic disease was reproduced with 100% attack rate by intracerebral inoculation of brain homogenate to tga20 mice overexpressing WT PrP, and from the latter to WT mice, but not to PrP-deficient mice. Upon successive transmissions, the incubation periods decreased and PrP became more protease-resistant, indicating the presence of a strain barrier that was gradually overcome by repeated passaging. This shows that expression of a subtly altered prion protein, with known 3D structure, efficiently generates a prion disease.

Prions cause neurodegeneration in vivo, yet prion-infected cultured cells do not show cytotoxicity. This has hampered mechanistic studies of prion-induced neurodegeneration. Here we report that prion-infected cultured organotypic cerebellar slices (COCS) experienced progressive spongiform neurodegeneration closely reproducing prion disease, with three different prion strains giving rise to three distinct patterns of prion protein deposition. Neurodegeneration did not occur when PrP was genetically removed from neurons, and a comprehensive pharmacological screen indicated that neurodegeneration was abrogated by compounds known to antagonize prion replication. Prion infection of COCS and mice led to enhanced fodrin cleavage, suggesting the involvement of calpains or caspases in pathogenesis. Accordingly, neurotoxicity and fodrin cleavage were prevented by calpain inhibitors but not by caspase inhibitors, whereas prion replication proceeded unimpeded. Hence calpain inhibition can uncouple prion replication from its neurotoxic sequelae. These data validate COCS as a powerful model system that faithfully reproduces most morphological hallmarks of prion infections. The exquisite accessibility of COCS to pharmacological manipulations was instrumental in recognizing the role of calpains in neurotoxicity, and significantly extends the collection of tools necessary for rigorously dissecting prion pathogenesis.

Genetically distinct hantaviruses have been identified in five species of fossorial moles (order Eulipotyphla, family Talpidae) from Eurasia and North America. Here, we report the isolation and partial characterization of a highly divergent hantavirus, named Nova virus (NVAV), from lung tissue of a European mole ( Talpa europaea ), captured in central Poland in August 2013. Typical hantavirus-like particles, measuring 80–120 nm in diameter, were found in NVAV-infected Vero E6 cells by transmission electron microscopy. Whole-genome sequences of the isolate, designated NVAV strain Te34, were identical to that amplified from the original lung tissue and phylogenetic analysis of the full-length L, M and S segments, using maximum-likelihood and Bayesian methods, showed that NVAV was most closely related to hantaviruses harbored by insectivorous bats, consistent with an ancient evolutionary origin. Infant Swiss Webster mice, inoculated with NVAV by the intraperitoneal route, developed weight loss and hyperactivity, beginning at 16 days, followed by hind-limb paralysis and death. High NVAV RNA copies were detected in lung, liver, kidney, spleen and brain by quantitative real-time RT-PCR. Neuropathological examination showed astrocytic and microglial activation and neuronal loss. The first mole-borne hantavirus isolate will facilitate long-overdue studies on its infectivity and pathogenic potential in humans.

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 Alzheimer's 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. Prion proteins are anchored to the surface of brain cells called neurons. Normally, prion proteins are folded into a specific three-dimensional shape that enables them to carry out their normal roles in the brain. However, they can be misfolded into a different shape known as PrPSc, which can cause Creutzfeldt-Jakob disease and other serious conditions that affect brain function and ultimately lead to death. The PrPSc proteins can force normal prion proteins to change into the PrPSc form, so that over time this form accumulates in the brain. They are essential components of infectious particles termed ‘prions’ and this is why prion diseases are infectious: if prions from one individual enter the brain of another individual they can cause disease in the recipient. The UK outbreak of variant Creutzfeldt-Jakob disease in humans in the 1990s is thought to be due to the consumption of meat from cattle with a prion disease known as mad cow disease. An enzyme called ADAM10 can cut normal prion proteins from the surface of neurons. However, it is not clear whether ADAM10 can also target the PrPSc proteins and what impact this may have on the development of prion diseases. Here, Altmeppen et al. studied mutant mice that were missing ADAM10 in neurons in the front portion of their brain. These mice had a higher number of normal prion proteins on the surface of their neurons than normal mice did. When mice missing ADAM10 were infected with prions, more PrPSc accumulated in their brain and disease symptoms developed sooner than when normal mice were infected. This supports the view that mice with higher numbers of prion proteins are more vulnerable to prion disease. However, disease symptoms did not spread as quickly to other parts of the brain in the mice missing ADAM10. This suggests that by releasing prion proteins from the surface of neurons, ADAM10 helps PrPSc proteins to spread around the brain. Recently, it has been suggested that prion proteins may also play a role in Alzheimer's disease and other neurodegenerative conditions. Therefore, Altmeppen et al.'s findings may help to develop new therapies for other forms of dementia. The next challenge is to understand the precise details of how ADAM10 works.

The in vitro environment can influence not only the molecular background of glioblastoma drug-resistance and treatment efficiency, but also the mechanisms and pathways of cell death. Both crucial molecular pathways and the deregulation of miRNAs are thought to participate in tumor therapy-resistance. The aim of our study is to examine the potential influence of ex vivo conditions on the expression of miRNAs engaged in the machinery of tumor-drug resistance, since in vitro models are commonly used for testing new therapeutics. Glioblastoma-derived cells, cultured under three different sets of conditions, were used as experimental models in vitro. The expression of 84 miRNAs relevant to brain tumorigenesis was evaluated by multi-miRNA profiling for initial tumors and their corresponding cultures. Finally, the expression of selected miRNAs related to temozolomide-resistance (miR-125b, miR-130a, miR-21, miR-221, miR-222, miR-31, miR-149, miR-210, miR-181a) was assessed by real-time PCR for each tumor and neoplastic cells in cultures. Our results demonstrate significant discrepancies in the expression of several miRNAs between tumor cells in vivo and in vitro, with miR-130a, miR-221, miR-31, miR-21, miR-222, miR-210 being the most marked. Also differences were observed between particular models in vitro. The results of computational analysis revealed the interplay between examined miRNAs and their targets involved in processes of glioblastoma chemosensitivity, including the genes relevant to temozolomide response ( , , , , ). The artificial environment may influence the selective proliferation of cell populations carrying specific patterns of miRNAs and/or the phenotype of neoplastic cells (eg differentiation) by the action of molecular events including miRNAs. These phenomena may influence the tumor-responsiveness to particular drugs, disturbing the evaluation of their efficacy in vitro, with unpredictable results caused by the interdependency of molecular pathways.

The aim of this study was to assess the presence of DYT6 mutations in Polish patients with isolated dystonia and to characterize their phenotype. We sequenced THAP1 exons 1, 2 and 3 including exon-intron boundaries and 5'UTR fragment in 96 non-DYT1 dystonia patients. In four individuals single nucleotide variations were identified. The coding substitutions were: c. 238A>G (p.Ile80Val), found in two patients, and c.167A>G (p.Glu56Gly), found in one patient. The same variations were present also in the patients' symptomatic as well as asymptomatic relatives. Mutation penetration in the analyzed families was 50-66.7%. In the fourth patient, a novel c.-249C>A substitution in the promoter region was identified. The patient, initially suspected of idiopathic isolated dystonia, finally presented with pantothenate kinase 2-associated neurodegeneration phenotype and was a carrier of two PANK2 mutations. This is the first identified NBIA1 case carrying mutations in both PANK2 and THAP1 genes. In all symptomatic THAP1 mutation carriers (four probands and their three affected relatives) the first signs of dystonia occurred before the age of 23. A primary localization typical for DYT6 dystonia was observed in six individuals. Five subjects developed the first signs of dystonia in the upper limb. In one patient the disease began from laryngeal involvement. An uncommon primary involvement of lower limb was noted in the THAP1 and PANK2 mutations carrier. Neither of these THAP1 substitutions were found in 150 unrelated healthy controls. To the contrary, we identified a heterozygous C/T genotype of c.57C>T single nucleotide variation (p.Pro19Pro, rs146087734) in one healthy control, but in none of the patients. Therefore, a previously proposed association between this substitution and DYT6 dystonia seems unlikely. We found also no significant difference between cases and controls in genotypes distribution of the two-nucleotide -237-236 GA>TT (rs370983900 & rs1844977763) polymorphism.

Recurrent histone mutations are linked to paediatric glioblastoma multiforme, an aggressive type of brain tumour. Drug targets in glioblastoma Exome sequencing of 48 childhood glioblastoma samples has identified somatic mutations in the H3.3-ATRX-DAXX chromatin remodelling pathway in 44% of tumours. Mutations in H3F3A, which encodes the replication-independent histone 3 variant H3.3, were found in 31% of tumours, the first indication that this gene is linked to tumorigenesis. These mutations are shown to be specific to glioblastoma and are highly prevalent in children and young adults, suggesting that defects of the chromatin architecture may underlie the condition. Childhood glioblastoma is fatal and resistant to therapy, so the discovery of potentially disease-relevant drug targets is an important advance. Glioblastoma multiforme (GBM) is a lethal brain tumour in adults and children. However, DNA copy number and gene expression signatures indicate differences between adult and paediatric cases 1 , 2 , 3 , 4 . To explore the genetic events underlying this distinction, we sequenced the exomes of 48 paediatric GBM samples. Somatic mutations in the H3.3-ATRX-DAXX chromatin remodelling pathway were identified in 44% of tumours (21/48). Recurrent mutations in H3F3A , which encodes the replication-independent histone 3 variant H3.3, were observed in 31% of tumours, and led to amino acid substitutions at two critical positions within the histone tail (K27M, G34R/G34V) involved in key regulatory post-translational modifications. Mutations in ATRX (α-thalassaemia/mental retardation syndrome X-linked) 5 and DAXX (death-domain associated protein), encoding two subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres 6 , 7 , were identified in 31% of samples overall, and in 100% of tumours harbouring a G34R or G34V H3.3 mutation. Somatic TP53 mutations were identified in 54% of all cases, and in 86% of samples with H3F3A and/or ATRX mutations. Screening of a large cohort of gliomas of various grades and histologies ( n = 784) showed H3F3A mutations to be specific to GBM and highly prevalent in children and young adults. Furthermore, the presence of H3F3A/ATRX-DAXX/TP53 mutations was strongly associated with alternative lengthening of telomeres and specific gene expression profiles. This is, to our knowledge, the first report to highlight recurrent mutations in a regulatory histone in humans, and our data suggest that defects of the chromatin architecture underlie paediatric and young adult GBM pathogenesis.

Kuru, the first human prion disease was transmitted to chimpanzees by D. Carleton Gajdusek (1923–2008). In this review, we summarize the history of this seminal discovery, its anthropological background, epidemiology, clinical picture, neuropathology, and molecular genetics. We provide descriptions of electron microscopy and confocal microscopy of kuru amyloid plaques retrieved from a paraffin-embedded block of an old kuru case, named Kupenota. The discovery of kuru opened new vistas of human medicine and was pivotal in the subsequent transmission of Creutzfeldt–Jakob disease, as well as the relevance that bovine spongiform encephalopathy had for transmission to humans. The transmission of kuru was one of the greatest contributions to biomedical sciences of the 20th century.