Diagnostic accuracy of cerebrospinal fluid biomarkers in genetic prion diseases.

Genetic prion diseases are a rare and diverse group of fatal neurodegenerative disorders caused by pathogenic sequence variations in the prion protein gene, PRNP. Data on CSF biomarkers in patients with genetic prion diseases are limited and conflicting results have been reported for unclear reasons. Here, we aimed to analyse the diagnostic accuracy of CSF biomarkers currently used in prion clinical diagnosis in 302 symptomatic genetic prion disease cases from 11 prion diagnostic centres, encompassing a total of 36 different pathogenic sequence variations within the open reading frame of PRNP. CSF samples were assessed for the surrogate markers of neurodegeneration, 14-3-3 protein (14-3-3), total-tau protein (t-tau) and α-synuclein and for prion seeding activity through the real-time quaking-induced conversion assay. Biomarker results were compared with those obtained in healthy and neurological controls. For the most prevalent PRNP pathogenic sequence variations, biomarker accuracy and associations between biomarkers, demographic and genetic determinants were assessed. Additionally, the prognostic value of biomarkers for predicting total disease duration from symptom onset to death was investigated. High sensitivity of the four biomarkers was detected for genetic Creutzfeldt-Jakob disease associated with the E200K and V210I mutations, but low sensitivity was observed for mutations associated with Gerstmann-Sträussler-Scheinker syndrome and fatal familial insomnia. All biomarkers showed good to excellent specificity using the standard cut-offs often used for sporadic Creutzfeldt-Jakob disease. In genetic prion diseases related to octapeptide repeat insertions, the biomarker sensitivity correlated with the number of repeats. New genetic prion disease-specific cut-offs for 14-3-3, t-tau and α-synuclein were calculated. Disease duration in genetic Creutzfeldt-Jakob disease-E200K, Gerstmann-Sträussler-Scheinker-P102L and fatal familial insomnia was highly dependent on PRNP codon 129 MV polymorphism and was significantly associated with biomarker levels. In a large cohort of genetic prion diseases, the simultaneous analysis of CSF prion disease biomarkers allowed the determination of new mutation-specific cut-offs improving the discrimination of genetic prion disease cases and unveiled genetic prion disease-specific associations with disease duration.

If Artificial In Vitro Microenvironment Can Influence Tumor Drug Resistance Network via Modulation of lncRNA Expression?-Comparative Analysis of Glioblastoma-Derived Cell Culture Models and Initial Tumors In Vivo.

The tumor resistance of glioblastoma cells in vivo is thought to be enhanced by their heterogeneity and plasticity, which are extremely difficult to curb in vitro. The external microenvironment shapes the molecular profile of tumor culture models, thus influencing potential therapy response. Our study examines the expression profile of selected lncRNAs involved in tumor resistance network in three different glioblastoma-derived models commonly utilized for testing drug response in vitro. Differential expression analysis revealed significant divergence in lncRNA profile between parental tumors and tumor-derived cell cultures in vitro, including the following particles: MALAT1, CASC2, H19, TUSC7, XIST, RP11-838N2.4, DLX6-AS1, GLIDR, MIR210HG, SOX2-OT. The examined lncRNAs influence the phenomenon of tumor resistance via their downstream target genes through a variety of processes: multi-drug resistance, epithelial-mesenchymal transition, autophagy, cell proliferation and viability, and DNA repair. A comparison of in vivo and in vitro expression identified differences in the levels of potential lncRNA targets, with the highest discrepancies detected for the MDR1, LRP1, BCRP and MRP1 genes. Co-expression analyses confirmed the following interrelations: MALAT1-TYMS, MALAT1-MRP5, H19-ZEB1, CASC2-VIM, CASC2-N-CAD; they additionally suggest the possibility of MALAT1-BCRP, MALAT1-mTOR and TUSC7-PTEN interconnections in glioblastoma. Although our results clearly demonstrate that the artificial ex vivo microenvironment changes the profile of lncRNAs related to tumor resistance, it is difficult to anticipate the final phenotypic effect, since this phenomenon is a complex one that involves a network of molecular interactions underlying a variety of cellular processes.

mRNA and miRNA Expression Analyses of the MYC/E2F/miR-17-92 Network in the Most Common Pediatric Brain Tumors.

Numerous molecular factors disrupt the correctness of the cell cycle process leading to the development of cancer due to increased cell proliferation. Among known causative factors of such process is abnormal gene expression. Nowadays in the light of current knowledge such alterations are frequently considered in the context of mRNA-miRNA correlation. One of the molecular factors with potential value in tumorigenesis is the feedback loop between MYC and E2F genes in which miR-17-5p and miR-20a from the miR-17-92 cluster are involved. The current literature shows that overexpression of the members of the OncomiR-1 are involved in the development of many solid tumors. In the present work, we investigated the expression of components of the MYC/E2F/miR-17-92 network and their closely related elements including members of MYC and E2F families and miRNAs from two paralogs of miR-17-92: miR-106b-25 and miR-106a-363, in the most common brain tumors of childhood, pilocytic astrocytoma (PA), WHO grade 1; ependymoma (EP), WHO grade 2; and medulloblastoma (MB), WHO grade 4. We showed that the highest gene expression was observed in the MYC family for MYCN and in the E2F family for E2F2. Positive correlation was observed between the gene expression and tumor grade and type, with the highest expression being noted for medulloblastomas, followed by ependymomas, and the lowest for pilocytic astrocytomas. Most members of miR-17-92, miR-106a-363 and miR-106b-25 clusters were upregulated and the highest expression was noted for miR-18a and miR-18b. The rest of the miRNAs, including miR-19a, miR-92a, miR-106a, miR-93, or miR-25 also showed high values. miR-17-5p, miR-20a obtained a high level of expression in medulloblastomas and ependymomas, while close to the control in the pilocytic astrocytoma samples. miRNA expression also depended on tumor grade and histology.

Expression-based decision tree model reveals distinct microRNA expression pattern in pediatric neuronal and mixed neuronal-glial tumors.

BACKGROUND: The understanding of the molecular biology of pediatric neuronal and mixed neuronal-glial brain tumors is still insufficient due to low frequency and heterogeneity of those lesions which comprise several subtypes presenting neuronal and/or neuronal-glial differentiation. Important is that the most frequent ganglioglioma (GG) and dysembryoplastic neuroepithelial tumor (DNET) showed limited number of detectable molecular alterations. In such cases analyses of additional genomic mechanisms seem to be the most promising. The aim of the study was to evaluate microRNA (miRNA) profiles in GGs, DNETs and pilocytic asytrocytomas (PA) and test the hypothesis of plausible miRNA connection with histopathological subtypes of particular pediatric glial and mixed glioneronal tumors. METHODS: The study was designed as the two-stage analysis. Microarray testing was performed with the use of the miRCURY LNA microRNA Array technology in 51 cases. Validation set comprised 107 samples used during confirmation of the profiling results by qPCR bioinformatic analysis. RESULTS: Microarray data was compared between the groups using an analysis of variance with the Benjamini-Hochberg procedure used to estimate false discovery rates. After filtration 782 miRNAs were eligible for further analysis. Based on the results of 10 × 10-fold cross-validation J48 algorithm was identified as the most resilient to overfitting. Pairwise comparison showed the DNETs to be the most divergent with the largest number of miRNAs differing from either of the two comparative groups. Validation of array analysis was performed for miRNAs used in the classification model: miR-155-5p, miR-4754, miR-4530, miR-628-3p, let-7b-3p, miR-4758-3p, miRPlus-A1086 and miR-891a-5p. Model developed on their expression measured by qPCR showed weighted AUC of 0.97 (95% CI for all classes ranging from 0.91 to 1.00). A computational analysis was used to identify mRNA targets for final set of selected miRNAs using miRWalk database. Among genomic targets of selected molecules ZBTB20, LCOR, PFKFB2, SYNJ2BP and TPD52 genes were noted. CONCLUSIONS: Our data showed the existence of miRNAs which expression is specific for different histological types of tumors. miRNA expression analysis may be useful in in-depth molecular diagnostic process of the tumors and could elucidate their origins and molecular background.

Sensitive detection of FGFR1 N546K mosaic mutation in patient with encephalocraniocutaneous lipomatosis and pilocytic astrocytoma.

Encephalocraniocutaneous lipomatosis (ECCL) is a rare neurocutaneous disorder, with only about 100 cases reported worldwide. It is characterized by congenital lesions of the eye, skin, and central nervous system. Only recently, potential causative FGFR1 point mutations have been identified in brain tumors and cultured skin biopsies from patients with this condition. Here, we analyzed the molecular status of a patient with ECCL and a coexisting pilocytic astrocytoma with detected FGFR1 N546K mutation. The presence of the alteration in both affected and unaffected tissues has been evaluated using Sanger sequencing and droplet digital polymerase chain reaction (ddPCR) technique. The ddPCR analysis showed differential distribution of the alteration in all specimens, including unaffected and untreated samples. Therefore, we confirm that FGFR1 N546K is a plausible causative mutation of ECCL patients and could be associated with a risk of brain tumor development. We also show the usefulness of sensitive ddPCR method for detection of low levels of autosomal mosaic mutation in blood or swabs. We suggest that utilization of this method may improve the diagnostic process, especially when targeted therapies are considered.

The toxicity of antiprion antibodies is mediated by the flexible tail of the prion protein.

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. 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, monovalent Fab1 fragments and recombinant single-chain variable fragment miniantibodies. Similar to prion infections, 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, 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.

Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma.

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. 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) and DAXX (death-domain associated protein), encoding two subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres, 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.

Cerebrospinal fluid neurofilament light levels in neurodegenerative dementia: Evaluation of diagnostic accuracy in the differential diagnosis of prion diseases.

INTRODUCTION: Neurofilament light (NFL) levels in the cerebrospinal fluid are increased in several neurodegenerative dementias. However, their diagnostic accuracy in the differential diagnostic context is unknown. METHODS: Cerebrospinal fluid NFL levels were quantified in nonprimarily neurodegenerative neurological and psychiatric diseases (n = 122), mild cognitive impairment (n = 48), Alzheimer's disease (n = 108), dementia with Lewy bodies/Parkinson's disease dementia (n = 53), vascular dementia (n = 46), frontotemporal dementia (n = 41), sporadic Creutzfeldt-Jakob disease (sCJD, n = 132), and genetic prion diseases (n = 182). RESULTS: The highest NFL levels were detected in sCJD, followed by vascular dementia, frontotemporal dementia, dementia with Lewy bodies/Parkinson's disease dementia, Alzheimer's disease, and mild cognitive impairment. In sCJD, NFL levels correlated with cerebrospinal fluid tau and disease duration. NFL levels were able to differentiate sCJD from nonprimarily neurodegenerative neurological and psychiatric diseases (area under the curve = 0.99, 95% confidence interval: 0.99-1) and from the other diagnostic groups showing cognitive impairment/dementia of a non-CJD etiology (area under the curve = 0.90, 95% confidence interval: 0.87-0.92). Compared to nonprimarily neurodegenerative neurological and psychiatric diseases, NFL was also elevated in genetic prion diseases associated with the E200K, V210I, P102L, and D178N prion protein gene mutations. DISCUSSION: Increased NFL levels are a common feature in neurodegenerative dementias.

Cerebrospinal Fluid Biomarker-Based Diagnosis of Sporadic Creutzfeldt-Jakob Disease: A Validation Study for Previously Established Cutoffs.

BACKGROUND: Several biomarkers have been proposed to discriminate sporadic Creutzfeldt-Jakob disease (sCJD) from other dementias and control cases. However, their clinical accuracy depends on the PRNP codon 129 genotype, leaving it unclear how well established markers behave in untested conditions. METHODS: We analyzed 14-3-3, tau, p-tau levels, and the p-tau/tau ratio in a population sample collected from Polish hospitals including nondementia, dementia, and definite sCJD cases and validated their parameters according to previously established cutoffs. Additionally, the correlation between biomarkers and disease duration as well as the influence of the PRNP129 polymorphism are reported. RESULTS: The tau levels and p-tau/tau ratios differed considerably between sCJD and clinically characterized non-CJD cases (p < 0.001). p-tau was only elevated in sCJD when compared to cases without dementia (p < 0.05). Tau and the p-tau/tau ratio showed a sensitivity of 95 and 100%, respectively, in detecting sCJD cases. A negative correlation between tau levels and disease duration, but not the timing of lumbar puncture was observed. CONCLUSION: The present findings confirmed the value of the p-tau/tau ratio as a robust sCJD biomarker and suggest a role for tau as prognostic marker.

Evaluation of α-synuclein as a novel cerebrospinal fluid biomarker in different forms of prion diseases.

INTRODUCTION: Accurate diagnosis of prion diseases and discrimination from alternative dementias gain importance in the clinical routine, but partial overlap in cerebrospinal fluid (CSF) biomarkers impedes absolute discrimination in the differential diagnostic context. METHODS: We established the clinical parameters for prion disease diagnosis for the quantification of CSF α-synuclein in patients with sporadic (n = 234) and genetic (n = 56) prion diseases, in cases with cognitive impairment/dementia or neurodegenerative disease (n = 278), and in the neurologic control group (n = 111). RESULTS: An optimal cutoff value of 680 pg/mL α-synuclein results in 94% sensitivity and 96% specificity when diagnosing sporadic Creutzfeldt-Jakob disease (CJD). Genetic CJD cases showed increased CSF α-synuclein values. No increased α-synuclein levels were detected in non-CJD cases with rapid progression course. DISCUSSION: Detection of α-synuclein in the CSF of patients with suspected CJD is a valuable diagnostic test reaching almost full discrimination from non-prion disease cases. These data highlight the utility of CSF α-synuclein quantification in front of classical CSF biomarkers in clinical routine.

Germline and somatic FGFR1 abnormalities in dysembryoplastic neuroepithelial tumors.

Dysembryoplastic neuroepithelial tumor (DNET) is a benign brain tumor associated with intractable drug-resistant epilepsy. In order to identify underlying genetic alterations and molecular mechanisms, we examined three family members affected by multinodular DNETs as well as 100 sporadic tumors from 96 patients, which had been referred to us as DNETs. We performed whole-exome sequencing on 46 tumors and targeted sequencing for hotspot FGFR1 mutations and BRAF p.V600E was used on the remaining samples. FISH, copy number variation assays and Sanger sequencing were used to validate the findings. By whole-exome sequencing of the familial cases, we identified a novel germline FGFR1 mutation, p.R661P. Somatic activating FGFR1 mutations (p.N546K or p.K656E) were observed in the tumor samples and further evidence for functional relevance was obtained by in silico modeling. The FGFR1 p.K656E mutation was confirmed to be in cis with the germline p.R661P variant. In 43 sporadic cases, in which the diagnosis of DNET could be confirmed on central blinded neuropathology review, FGFR1 alterations were also frequent and mainly comprised intragenic tyrosine kinase FGFR1 duplication and multiple mutants in cis (25/43; 58.1 %) while BRAF p.V600E alterations were absent (0/43). In contrast, in 53 cases, in which the diagnosis of DNET was not confirmed, FGFR1 alterations were less common (10/53; 19 %; p < 0.0001) and hotspot BRAF p.V600E (12/53; 22.6 %) (p < 0.001) prevailed. We observed overexpression of phospho-ERK in FGFR1 p.R661P and p.N546K mutant expressing HEK293 cells as well as FGFR1 mutated tumor samples, supporting enhanced MAP kinase pathway activation under these conditions. In conclusion, constitutional and somatic FGFR1 alterations and MAP kinase pathway activation are key events in the pathogenesis of DNET. These findings point the way towards existing targeted therapies.

Mitochondrial DNA differentiates Alzheimer's disease from Creutzfeldt-Jakob disease.

INTRODUCTION: Low content of cell-free mitochondrial DNA (mtDNA) in cerebrospinal fluid (CSF) is a biomarker of early stage Alzheimer's disease (AD), but whether mtDNA is altered in a rapid neurodegenerative dementia such as Creutzfeldt-Jakob disease is unknown. METHODS: CSF mtDNA was measured using digital polymerase chain reaction (dPCR) in two independent cohorts comprising a total of 112 patients diagnosed with sporadic Creutzfeldt-Jakob disease (sCJD), probable AD, or non-Alzheimer's type dementia. RESULTS: Patients with AD exhibit low mtDNA content in CSF compared with patients diagnosed with sCJD or with non-Alzheimer's type dementias. The CSF concentration of mtDNA does not correlate with Aß, t-tau, p-tau, and 14-3-3 protein levels in CSF. DISCUSSION: Low-CSF mtDNA is not a consequence of brain damage and allows the differential diagnosis of AD from sCJD and other dementias. These results support the hypothesis that mtDNA in CSF is a pathophysiological biomarker of AD.

The molecular pattern of histopathological progression to anaplastic meningioma - A case report.

Meningiomas (MGs) are the most frequent primary tumours of the central nervous system (CNS) and exhibit a large spectrum of histological types and clinical phenotypes. The WHO classification of CNS tumours established strict diagnostic criteria of the benign (Grade 1), atypical (Grade 2) and anaplastic (Grade 3) subtypes. Combined with the resection rate, WHO grading has the most crucial role as the prognostic factor. Additionally, such biomarkers as Ki-67/MIB-1, progesterone receptors and phosphor-histone H3 were correlated with MG progression. Recently, it was suggested that the aggressive behaviour of some MGs is attributed to molecular alterations, regardless of their histopathology. The analysis of loss of heterozygosity (LOH) at chromosomes 1, 9, 10, 14 and 22 was performed. The presented case of WHO Grade 2 MG initially exhibited LOH at chromosomes 10, 14 and 22. In the first recurrence, the tumour genetic profiling revealed additional LOH at chromosome 1p and atypical histopathology. During the second recurrence, an aggressive phenotype was observed and tumour progressed to an anaplastic form. Considering the appearance of the tumour relapses, the set of molecular changes overtook the histopathological progression. The genetic and histopathological imbalance in the tumour progression in secondary anaplastic MGs has not been previously described. The evolution of genetic and histopathological changes was presented in the same patient. In the future, the individualised therapy of potentially more aggressive forms of MGs could be based on certain chromosome aberrations.

Recurrence-associated chromosomal anomalies in meningiomas: Single-institution study and a systematic review with meta-analysis.

Complete removal of a meningioma (MG) does not guarantee relapse-free survival. Alterations on several chromosomes responsible for MG recurrence were suggested, although their role was not validated by a systematic review. Following the analysis of own 161 cases, all previously published data has been collected for evidence synthesis. Based on own series, WHO grade >I (odds ratio (OR)=92.0; 95%CI: 19.1-443.5) and a combination of loss of heterozygosity (LOH) on 1p and 14q (OR=10.2; 95%CI: 19-55.7) were the independent recurrence-specific prognosticators. The deleterious role of LOH on 1p/14q was demonstrated in a subset of parasagittal and falcine MGs. A total of 742 cases and 10 studies were pooled for the Individual Patient Data and Aggregate Data models of meta-analysis, respectively. The prognostic role of WHO classification (OR=90.4) and anomaly of chromosome 14 (OR=3.5) was confirmed. LOH on 14 showed lesser impact on recurrence than suggested by the WHO grading (area under the curve 0.65 for LOH vs. 0.74 for WHO). Fixed effect model of meta-analysis provided high summarized OR values for 1p (OR=5.4; 95%CI: 3.6-8.1) and 14q (OR=7.6; 95%CI: 4.3-13.6), and low for chromosome 22 (OR=1.6; 95%CI: 1.1-2.4). Final appraisal of recurrence-associated chromosomal alterations indicated that arms 1p and 14q deserve attention while predicting MG recurrence.

The correlation of clinical and chromosomal alterations of benign meningiomas and their recurrences.

Meningiomas (MGs) are the frequent benign intracranial tumors. Their complete removal does not always guarantee relapse-free survival. Recurrence-associated chromosomal anomalies in MGs haves been proposed as prognostic factors in addition to the World Health Organisation (WHO) grading, tumor size and resection rate. The aim of this study was to evaluate the frequency of deletions on chromosomes in sporadic MGs and to correlate them with the clinical findings and tumor behaviour. Along with survival, the tumor recurrence was the main endpoint. Chromosomal loss of heterozygosity (LOH) was studied. 46 benign MGs were subjected to the analysis, complete tumor resection was intended and no early mortalities were observed. Incomplete removal was related to parasagittal location and psammomatous hisptopathology (p<0.01). Chromosomal alterations were present in 82.6% of cases; LOH at 22q (67.4%) and 1p (34.8%) were the most frequent and associated with male sex (p=0.04). Molecular findings were not specific for any of the histopathologic grade. Tumor recurrence (14 of 46) correlated with tumor size (≥35mm), LOH at 1p, 14q, coexistence of LOH at 1p/14q, 10q/14q, 'complex karyotype' status (≥2 LOHs excluding 22q), patient age (younger <35), and Simpson grading of resection rate (≥3 of worse prognosis). The last 3 variables were independent significant prognostic factors in multivariate analysis and of the same importance in recurrence prediction (Receiver Operating Characteristic curves comparison p>0.05). Among the cases of recurrence, tumor progression was observed in 3 of 14. In 2 cases, LOH on 1p and/or coexistence of LOH 1p/14q correlated with anaplastic transformation.

De novo prion aggregates trigger autophagy in skeletal muscle.

In certain sporadic, familial, and infectious prion diseases, the prion protein misfolds and aggregates in skeletal muscle in addition to the brain and spinal cord. In myocytes, prion aggregates accumulate intracellularly, yet little is known about clearance pathways. Here we investigated the clearance of prion aggregates in muscle of transgenic mice that develop prion disease de novo. In addition to neurodegeneration, aged mice developed a degenerative myopathy, with scattered myocytes containing ubiquitinated, intracellular prion inclusions that were adjacent to myocytes lacking inclusions. Myocytes also showed elevated levels of the endoplasmic reticulum chaperone Grp78/BiP, suggestive of impaired protein degradation and endoplasmic reticulum stress. Additionally, autophagy was induced, as indicated by increased levels of beclin-1 and LC3-II. In C2C12 myoblasts, inhibition of autophagosome maturation or lysosomal degradation led to enhanced prion aggregation, consistent with a role for autophagy in prion aggregate clearance. Taken together, these findings suggest that the induction of autophagy may be a central strategy for prion aggregate clearance in myocytes. IMPORTANCE In prion diseases, the prion protein misfolds and aggregates in the central nervous system and sometimes in other organs, including muscle, yet the cellular pathways of prion aggregate clearance are unclear. Here we investigated the clearance of prion aggregates in the muscle of a transgenic mouse model that develops profound muscle degeneration. We found that endoplasmic reticulum stress pathways were activated and that autophagy was induced. Blocking of autophagic degradation in cell culture models led to an accumulation of aggregated prion protein. Collectively, these findings suggest that autophagy has an instrumental role in prion protein clearance.

Transcriptional profiles of pilocytic astrocytoma are related to their three different locations, but not to radiological tumor features.

BACKGROUND: Pilocytic astrocytoma is the most common type of brain tumor in the pediatric population, with a generally favorable prognosis, although recurrences or leptomeningeal dissemination are sometimes also observed. For tumors originating in the supra-or infratentorial location, a different molecular background was suggested, but plausible correlations between the transcriptional profile and radiological features and/or clinical course are still undefined. The purpose of this study was to identify gene expression profiles related to the most frequent locations of this tumor, subtypes based on various radiological features, and the clinical pattern of the disease. METHODS: Eighty six children (55 males and 31 females) with histologically verified pilocytic astrocytoma were included in this study. Their age at the time of diagnosis ranged from fourteen months to seventeen years, with a mean age of seven years. There were 40 cerebellar, 23 optic tract/hypothalamic, 21 cerebral hemispheric, and two brainstem tumors. According to the radiological features presented on MRI, all cases were divided into four subtypes: cystic tumor with a non-enhancing cyst wall; cystic tumor with an enhancing cyst wall; solid tumor with central necrosis; and solid or mainly solid tumor. In 81 cases primary surgical resection was the only and curative treatment, and in five cases progression of the disease was observed. In 47 cases the analysis was done by using high density oligonucleotide microarrays (Affymetrix HG-U133 Plus 2.0) with subsequent bioinformatic analyses and confirmation of the results by independent RT-qPCR (on 39 samples). RESULTS: Bioinformatic analyses showed that the gene expression profile of pilocytic astrocytoma is highly dependent on the tumor location. The most prominent differences were noted for IRX2, PAX3, CXCL14, LHX2, SIX6, CNTN1 and SIX1 genes expression even within different compartments of the supratentorial region. Analysis of the genes potentially associated with radiological features showed much weaker transcriptome differences. Single genes showed association with the tendency to progression. CONCLUSIONS: Here we have shown that pilocytic astrocytomas of three different locations can be precisely differentiated on the basis of their gene expression level, but their transcriptional profiles does not strongly reflect the radiological appearance of the tumor or the course of the disease.

High variability of clinical symptoms in a Polish family with a novel THAP1 mutation.

BACKGROUND: Mutations in the THAP1 gene are associated with a broad spectrum of dystonia including focal and generalized forms. Missense, nonsense and frameshift mutations, including small insertions/deletions within the THAP1 gene, have been reported and majority of them cause autosomal dominant disease with limited penetrance of approximately 60%. Here, we describe a novel THAP1 mutation. MATERIALS AND METHODS: Blood samples were collected from consenting family members for extraction of genomic DNA. As controls, we analyzed 150 individuals without neurological disorders. THAP1 coding sequences were amplified with PCR and sequenced. RESULTS: We describe a Polish family with a novel heterozygous substitution: c.167A>G (p.Glu56Gly) in THAP1 exon 2. This is the largest reported family with the mutation in THAP1 exon 2. The mutation was found in four of five genetically studied family members, including two clinically affected male individuals and two asymptomatic carriers (male and female). Data on one deceased male symptomatic subject were available and two assumed carriers were identified. The substitution was not present in any of the analyzed healthy controls. The high variability of phenotype included age of onset, localization of the initial symptom as well as the rate and degree of generalization. CONCLUSIONS: Our findings strongly suggest the role of other genetic factors or environmental triggers in the pathogenesis of dystonia related to mutations in THAP1 gene.

Prion pathogenesis is faithfully reproduced in cerebellar organotypic slice cultures.

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.

Biochemical properties of highly neuroinvasive prion strains.

Infectious prions propagate from peripheral entry sites into the central nervous system (CNS), where they cause progressive neurodegeneration that ultimately leads to death. Yet the pathogenesis of prion disease can vary dramatically depending on the strain, or conformational variant of the aberrantly folded and aggregated protein, PrP(Sc). Although most prion strains invade the CNS, some prion strains cannot gain entry and do not cause clinical signs of disease. The conformational basis for this remarkable variation in the pathogenesis among strains is unclear. Using mouse-adapted prion strains, here we show that highly neuroinvasive prion strains primarily form diffuse aggregates in brain and are noncongophilic, conformationally unstable in denaturing conditions, and lead to rapidly lethal disease. These neuroinvasive strains efficiently generate PrP(Sc) over short incubation periods. In contrast, the weakly neuroinvasive prion strains form large fibrillary plaques and are stable, congophilic, and inefficiently generate PrP(Sc) over long incubation periods. Overall, these results indicate that the most neuroinvasive prion strains are also the least stable, and support the concept that the efficient replication and unstable nature of the most rapidly converting prions may be a feature linked to their efficient spread into the CNS.