Profiling of methylation and demethylation pathways during brain development and ageing.

Numerous signal pathways are epigenetically controlled during brain development and ageing. Thereby, both 5-methylcytosine (5mC) and the newly described 5-hydroxymethylcytosine (5hmC) are highly exhibited in the brain. As there is an uneven distribution of 5hmC in the brain depending on age and region, there is the need to investigate the underlying mechanisms being responsible for 5hmC generation and decline. The aim of this study was to quantify expression levels of genes that are associated with DNA methylation/demethylation in different brain regions and at different ages. Therefore, we investigated frontal cortex and cerebellum of 40 mice (strain C57BL/6), each eight mice sacrificed at day 0, 7, 15, 30 and 120 after birth. We performed expression profiling of methylation/demethylation genes depending on age and brain region. Interestingly, we see significant expression differences of genes being responsible for methylation/demethylation with a significant reduction of expression levels during ageing. Validating selected expression data on protein level using immunohistochemistry verified the expression data. In conclusion, our findings demonstrate that the regulation of methylation/demethylation pathways is highly controlled depending on brain region and age. Thus our data will help to better understand the complexity and plasticity of the brain epigenome.

Clinical findings and diagnosis in genetic prion diseases in Germany.

To describe the clinical syndrome and diagnostic tests in patients with genetic prion diseases (gPD) in Germany. Clinical features, MRI, EEG, and CSF markers were studied in 91 patients (28 D178N, 20 E200K, 17 inserts, 13 V210I, 8 P102L, 5 E196K). Dementia (35 %) and ataxia (29 %) were the most common initial symptoms and signs. A wide variety and high frequency of neurological/psychiatric symptoms and signs was found during disease course in all patients independently of the type of the mutation. Psychiatric manifestations were frequent (87 %). Neuropsychological abnormalities were observed in 67 %, and aphasia was the most common disturbance (45 %). In E200K, V210I and D178N patients, visual/oculomotor deficits were followed by ataxia early in the disease. Dementia followed by ataxia at onset was common in patients with insert and E196K mutation. P102L patients had isolated ataxia over a longer time period followed by pyramidal signs. Dementia was present only late in the disease course. All clinical routine tests such as MRI, EEG and CSF tests were less sensitive than in sporadic CJD. We provide the first detailed analysis of clinical signs and symptoms in a large group of patients with gPD. Frequency of clinical symptoms and signs was similar in different mutations in a later disease course, but the sequence of occurrence may be of great diagnostic importance. CSF markers were shown to be more sensitive than MRI and EEG.

The cellular prion protein mediates neurotoxic signalling of ß-sheet-rich conformers independent of prion replication.

Formation of aberrant protein conformers is a common pathological denominator of different neurodegenerative disorders, such as Alzheimer's disease or prion diseases. Moreover, increasing evidence indicates that soluble oligomers are associated with early pathological alterations and that oligomeric assemblies of different disease-associated proteins may share common structural features. Previous studies revealed that toxic effects of the scrapie prion protein (PrP(Sc)), a ß-sheet-rich isoform of the cellular PrP (PrP(C)), are dependent on neuronal expression of PrP(C). In this study, we demonstrate that PrP(C) has a more general effect in mediating neurotoxic signalling by sensitizing cells to toxic effects of various ß-sheet-rich (ß) conformers of completely different origins, formed by (i) heterologous PrP, (ii) amyloid ß-peptide, (iii) yeast prion proteins or (iv) designed ß-peptides. Toxic signalling via PrP(C) requires the intrinsically disordered N-terminal domain (N-PrP) and the GPI anchor of PrP. We found that the N-terminal domain is important for mediating the interaction of PrP(C) with ß-conformers. Interestingly, a secreted version of N-PrP associated with ß-conformers and antagonized their toxic signalling via PrP(C). Moreover, PrP(C)-mediated toxic signalling could be blocked by an NMDA receptor antagonist or an oligomer-specific antibody. Our study indicates that PrP(C) can mediate toxic signalling of various ß-sheet-rich conformers independent of infectious prion propagation, suggesting a pathophysiological role of the prion protein beyond of prion diseases.

Reducing tau aggregates with anle138b delays disease progression in a mouse model of tauopathies.

Pathological tau aggregation leads to filamentous tau inclusions and characterizes neurodegenerative tauopathies such as Alzheimer's disease and frontotemporal dementia and parkinsonism linked to chromosome 17. Tau aggregation coincides with clinical symptoms and is thought to mediate neurodegeneration. Transgenic mice overexpressing mutant human P301S tau exhibit many neuropathological features of human tauopathies including behavioral deficits and increased mortality. Here, we show that the di-phenyl-pyrazole anle138b binds to aggregated tau and inhibits tau aggregation in vitro and in vivo. Furthermore, anle138b treatment effectively ameliorates disease symptoms, increases survival time and improves cognition of tau transgenic PS19 mice. In addition, we found decreased synapse and neuron loss accompanied by a decreased gliosis in the hippocampus. Our results suggest that reducing tau aggregates with anle138b may represent an effective and promising approach for the treatment of human tauopathies.

Loss of 5-hydroxymethylcytosine and intratumoral heterogeneity as an epigenomic hallmark of glioblastoma.

Glioblastoma (GBM) is the most malignant neoplasm with predominant astrocytic differentiation and the most frequent primary brain tumor of the adult. Here, we investigated 170 human GBM specimens deriving from 162 patients, as well as 66 healthy control tissue specimens deriving from 27 patients, and analyzed the amount of 5-hydroxymethylcytosine (5hmC) in GBMs compared to normal brain and tumor infiltration zones. Additionally, we correlated the amount of 5hmC with two different proliferation markers, Ki67 and H3S10p. Genetic characterization of GBMs enabled us to analyze the effect of isocitrate dehydrogenase 1 (IDH1) mutations, O6-methylguanin-DNA-methyltransferase (MGMT) promoter methylation, and loss of heterozygosity of chromosome 1p and 19q (LOH1p/19q) on 5hmC amount. We found that GBMs show a tremendous loss of 5hmC, and we observed that even the infiltration zones show reduced amounts of 5hmC. Interestingly, the amount of 5hmC was inversely proportional to the two investigated proliferation markers, Ki67 and H3S10p. Correlation of 5hmC amount and molecular genetic markers of GBMs showed that there are no correlations of 5hmC amount and IDH1 mutations, MGMT promoter methylation, and LOH1p/19q. Furthermore, we evaluated the intratumoral distribution of 5hmC in compact and infiltrating areas and found that the quantification of the 5hmC amount is a useful tool in evaluation of tumor infiltration. In summary, our data emphasize that GBMs show a disturbed hydroxymethylome that is disrupted by IDH1 independent pathways, and that loss of 5hmC shows astonishing intratumoral heterogeneity.

5-Hydroxymethylcytosine, the "Sixth Base", during brain development and ageing.

The epigenome is of fundamental importance for development and ageing. The discovery of 5-hydroxymethylcytosine (5hmC), a further base modification of cytosine beyond 5-methylcytosine, might be of high relevance in understanding the complexity of the human brain, as 5hmC is found in great extent in brain tissue. The aim of this study was to investigate the quantity of 5hmC containing nuclei by immunohistochemistry in human and murine brains at several developmental stages. We performed immunohistochemical stainings on frontal cortex, white matter and cerebellar cortex of 15 healthy controls. Three cases each were assigned to five age groups (foetus, adolescent, adult, elderly, aged). Additionally, cortex and cerebellum of 15 mice sacrificed between day 0 and 120 after birth were investigated. We found marked alterations of 5hmC amount during ageing. In human cortex there was an increase of 5hmC of 50%, in white matter we found an increase of even 200% during ageing. In the cerebellum both internal granular cell layer and molecular cell layer showed a significant increase of 5hmC till adulthood. Purkinje cell nuclei showed constantly positive signals for 5hmC. These data were paralleled in murine brains. Co-labelling of 5hmC and markers for mature and immature cells in murine cerebellar cortex at the age of 7 days revealed that 5hmC was found in mature but not in immature cells. In conclusion, the findings described in this study emphasise the importance of 5hmC in brain development and ageing and will help to better understand the complexity and plasticity of the brain.

Long non-coding RNA normalisers in human brain tissue.

The family of long non-coding RNA (lncRNA) is of increasing scientific interest as there is emerging evidence, that lncRNAs are of essential importance for transcriptional and translational control, genomic imprinting and regulation of normal development as well as neuronal plasticity. As the generation of reliable expression profiles requires adequate normalisers, it is of fundamental importance to determine suitable references for lncRNA studies. However, to date no systematic analysis of potential lncRNA normalisers has been performed on human postmortem brain tissue samples. In this study, we investigated three different brain regions (cortex, white matter, and cerebellum) of human postmortem tissue and analysed the expression stability of 90 lncRNAs. Bioinformatical analysis was performed to identify stably expressed lncRNAs. Subsequently, lncRNAs were classified according to their stability values using the NormFinder algorithm. We identified 30 suitable normalisers in cortex, 22 in white matter, and 41 in cerebellum. In addition, there were 13 suitable normalisers for studies comparing cortex and white matter, 25 for studies comparing cortex and cerebellum and 7 for studies comparing white matter and cerebellum. 5 lncRNAs (LUST, IGF2AS (family), 7SK, HOXA6as, NDM29) showed stable expression in all investigated brain regions. A subsequent analysis of the influence of postmortem intervals (PMI) on expression of lncRNAs revealed that expression levels of the newly identified 5 universal lncRNA normalisers are stable within PMI of up to 27 h. Thus, these 5 lncRNAs may be applicable as references for accurate normalisation of lncRNA profiling in multiple brain regions during long PMI, enabling the generation of highly reproducible datasets in lncRNA studies of the human brain.

Three novel presenilin 1 mutations marking the wide spectrum of age at onset and clinical patterns in familial Alzheimer's disease.

Presenilin 1 (PSEN1) mutations are the major cause of autosomal dominant Alzheimer's disease (ADAD). Here we report three novel PSEN1 mutations: Ile238_Lys239insIle, Ala246Pro and Ala164Val from patients who manifested in their twenties, forties and seventies, respectively, with variant clinical presentations of dementia. These cases exemplify the tremendous heterogeneity of clinical phenotypes and age of onset associated with PSEN1 mutations. The possibility of ADAD--not previously suspected in two of our patients--should always be considered in neurodegenerative conditions albeit they might neither exhibit the typical clinical picture of Alzheimer's disease nor early onset dementia, which is regarded the primary clinical sign of hereditary neurodegeneration.

Age-dependent levels of 5-methyl-, 5-hydroxymethyl-, and 5-formylcytosine in human and mouse brain tissues.

The absolute levels of 5-hydroxymethylcytosine (hmC) and 5-methylcytosine (mC) in human brain tissues at various ages were determined. Additionally, absolute levels of 5-formylcytosine (fC) in adult individuals and cytosine modification levels in sorted neurons were quantified. These data were compared with age-related fC, hmC, and mC levels in mouse brain samples. For hmC, an initial steady increase is observed, which levels off with age to a final steady-state value of 1.2 % in human brain tissue. This level is nearly twice as high as in mouse cerebral cortex. In contrast, fC declines rapidly with age during early developmental stages, thus suggesting that while hmC is a stable epigenetic mark, fC is more likely an intermediate of active DNA demethylation during early brain development. The trends in global cytosine modification dynamics during the lifespan of an organism are conserved between humans and mice and show similar patterns in different organs.

Constitutive activation of ß-catenin in neural progenitors results in disrupted proliferation and migration of neurons within the central nervous system.

Wnt signaling is known to play crucial roles in the development of multiple organs as well as in cancer. In particular, constitutive activation of Wnt/ß-Catenin signaling in distinct populations of forebrain or brainstem precursor cells has previously been shown to result in dramatic brain enlargement during embryonic stages of development as well as in the formation of medulloblastoma, a malignant brain tumor in childhood. In order to extend this knowledge to postnatal stages of both cerebral and cerebellar cortex development, we conditionally activated Wnt signaling by introducing a dominant active form of ß-catenin in hGFAP-positive neural precursors. Such mutant mice survived up to 21 days postnatally. While the mice revealed enlarged ventricles and an initial expansion of the Pax6-positive ventricular zone, Pax6 expression and proliferative activity in the ventricular zone was virtually lost by embryonic day 16.5. Loss of Pax6 expression was not followed by expression of the subventricular zone marker Tbr2, indicating insufficient neuronal differentiation. In support of this finding, cortical thickness was severely diminished in all analyzed stages from embryonic day 14.5 to postnatal day 12, and appropriate layering was not detectable. Similarly, cerebella of hGFAP-cre::Ctnnb1(ex3)(Fl/+) mice were hypoplastic and displayed severe lamination defects. Constitutively active ß-Catenin induced inappropriate proliferation of granule neurons and inadequate development of Bergmann glia, thereby preventing regular migration of granule cells and normal cortical layering. We conclude that Wnt signaling has divergent roles in the central nervous system and that Wnt needs to be tightly controlled in a time- and cell type-specific manner.

Analyses of protease resistance and aggregation state of abnormal prion protein across the spectrum of human prions.

Prion diseases are characterized by tissue accumulation of a misfolded, ß-sheet-enriched isoform (scrapie prion protein (PrP(Sc))) of the cellular prion protein (PrP(C)). At variance with PrP(C), PrP(Sc) shows a partial resistance to protease digestion and forms highly aggregated and detergent-insoluble polymers, two properties that have been consistently used to distinguish the two proteins. In recent years, however, the idea that PrP(Sc) itself comprises heterogeneous species has grown. Most importantly, a putative proteinase K (PK)-sensitive form of PrP(Sc) (sPrP(Sc)) is being increasingly investigated for its possible role in prion infectivity, neurotoxicity, and strain variability. The study of sPrP(Sc), however, remains technically challenging because of the need of separating it from PrP(C) without using proteases. In this study, we have systematically analyzed both PK resistance and the aggregation state of purified PrP(Sc) across the whole spectrum of the currently characterized human prion strains. The results show that PrP(Sc) isolates manifest significant strain-specific differences in their PK digestion profile that are only partially explained by differences in the size of aggregates, suggesting that other factors, likely acting on PrP(Sc) aggregate stability, determine its resistance to proteolysis. Fully protease-sensitive low molecular weight aggregates were detected in all isolates but in a limited proportion of the overall PrP(Sc) (i.e. <10%), arguing against a significant role of slowly sedimenting PK-sensitive PrP(Sc) in the biogenesis of prion strains. Finally, we highlight the limitations of current operational definitions of sPrP(Sc) and of the quantitative analytical measurements that are not based on the isolation of a fully PK-sensitive PrP(Sc) form.

Piperazine derivatives inhibit PrP/PrP(res) propagation in vitro and in vivo.

Prion diseases are fatal neurodegenerative disorders, which are not curable and no effective treatment exists so far. The major neuropathological change in diseased brains is the conversion of the normal cellular form of the prion protein PrPc(C) into a disease-associated isoform PrP(Sc). PrP(Sc) accumulates into multimeres and fibrillar aggregates, which leads to the formation of amyloid plaques. Increasing evidence indicates a fundamental role of PrP(Sc) species and its aggregation in the pathogenesis of prion diseases, which initiates the pathological cascade and leads to neurodegeneration accompanied by spongiform changes. In search of compounds that have the potential to interfere with PrP(Sc) formation and propagation, we used a cell based assay for the screening of potential aggregation inhibitors. The assay deals with a permanently prion infected cell line that was adapted for a high-throughput screening of a compound library composed of 10,000 compounds (DIVERset 2, ChemBridge). We could detect six different classes of highly potent inhibitors of PrP(Sc) propagation in vitro and identified piperazine derivatives as a new inhibitory lead structure, which increased incubation time of scrapie infected mice.

Common pathobiochemical hallmarks of progranulin-associated frontotemporal lobar degeneration and neuronal ceroid lipofuscinosis.

Heterozygous loss-of-function mutations in the progranulin (GRN) gene and the resulting reduction of GRN levels is a common genetic cause for frontotemporal lobar degeneration (FTLD) with accumulation of TAR DNA-binding protein (TDP)-43. Recently, it has been shown that a complete GRN deficiency due to a homozygous GRN loss-of-function mutation causes neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disorder. These findings suggest that lysosomal dysfunction may also contribute to some extent to FTLD. Indeed, Grn(-/-) mice recapitulate not only pathobiochemical features of GRN-associated FTLD-TDP (FTLD-TDP/GRN), but also those which are characteristic for NCL and lysosomal impairment. In Grn(-/-) mice the lysosomal proteins cathepsin D (CTSD), LAMP (lysosomal-associated membrane protein) 1 and the NCL storage components saposin D and subunit c of mitochondrial ATP synthase (SCMAS) were all found to be elevated. Moreover, these mice display increased levels of transmembrane protein (TMEM) 106B, a lysosomal protein known as a risk factor for FTLD-TDP pathology. In line with a potential pathological overlap of FTLD and NCL, Ctsd(-/-) mice, a model for NCL, show elevated levels of the FTLD-associated proteins GRN and TMEM106B. In addition, pathologically phosphorylated TDP-43 occurs in Ctsd(-/-) mice to a similar extent as in Grn(-/-) mice. Consistent with these findings, some NCL patients accumulate pathologically phosphorylated TDP-43 within their brains. Based on these observations, we searched for pathological marker proteins, which are characteristic for NCL or lysosomal impairment in brains of FTLD-TDP/GRN patients. Strikingly, saposin D, SCMAS as well as the lysosomal proteins CTSD and LAMP1/2 are all elevated in patients with FTLD-TDP/GRN. Thus, our findings suggest that lysosomal storage disorders and GRN-associated FTLD may share common features.

Molecular stereotactic biopsy technique improves diagnostic accuracy and enables personalized treatment strategies in glioma patients.

BACKGROUND: In gliomas molecular biomarkers are increasingly gaining diagnostic, prognostic and predictive significance. Determination of biomarker status after biopsy is important as not all patients are eligible for open tumor resection. We developed and validated prospectively (6/10-12/11) a protocol allowing for both reliable determination of multiple biomarkers and representative histological diagnoses from small-sized biopsies. METHODS: All molecular stereotactic biopsies were performed according to a detailed workflow. The selection of specimens best suited for molecular analyses was intra-operatively guided by the attending neuropathologist. Postoperative screening was done by methylation specific PCR using two distinct cryopreserved specimens to test for reproducibility of the findings and to rule out contamination. The DNA of a single best-suited specimen (1 mm(3)) was subjected to detailed molecular analysis (MGMT promoter methylation, IDH1/2 mutational status, LOH 1p and/or 19q). RESULTS: 159 consecutively enrolled untreated gliomas were analyzed (94 glioblastomas, 2 gliosarcomas, 24 anaplastic astrocytomas, 10 oligo-tumors grade II/III, 20 grade II astrocytomas and 9 pilocytic astrocytomas). Transient morbidity was 2 %. Overall, the drop-out rate due to tissue contamination was 0.4 %. Median time from biopsy to histological and molecular genetic analyses was 3 and 5 days, respectively. Distributions of the respective biomarker status for tumor subgroups were consistent with the literature. The final histological diagnosis was changed/modified in 5/159 patients according to molecular findings. Treatment after molecular biopsy was highly personalized. CONCLUSIONS: Molecular stereotactic biopsy is feasible and safe, can be implemented in daily clinical practice, improves diagnostic precision and enables personalized treatment.

Dipeptide repeat protein pathology in C9ORF72 mutation cases: clinico-pathological correlations.

Hexanucleotide repeat expansion in C9ORF72 is the most common genetic cause of frontotemporal dementia and motor neuron disease. Recently, unconventional non-ATG translation of the expanded hexanucleotide repeat, resulting in the production and aggregation of dipeptide repeat (DPR) proteins (poly-GA, -GR and GP), was identified as a potential pathomechanism of C9ORF72 mutations. Besides accumulation of DPR proteins, the second neuropathological hallmark lesion in C9ORF72 mutation cases is the accumulation of TDP-43. In this study, we characterized novel monoclonal antibodies against poly-GA and performed a detailed analysis of the neuroanatomical distribution of DPR and TDP-43 pathology in a cohort of 35 cases with the C9ORF72 mutation that included a broad spectrum of clinical phenotypes. We found the pattern of DPR pathology to be highly consistent among cases regardless of the phenotype with high DPR load in the cerebellum, all neocortical regions (frontal, motor cortex and occipital) and hippocampus, moderate pathology in subcortical areas and minimal pathology in lower motor neurons. No correlation between DPR pathology and the degree of neurodegeneration was observed, while a good association between TDP-43 pathology with clinical phenotype and degeneration in key anatomical regions was present. Our data confirm that the presence of DPR pathology is intimately related to C9ORF72 mutations. The observed dissociation between DPR inclusion body load and neurodegeneration might suggest inclusion body formation as a potentially protective response to cope with soluble toxic DPR species. Moreover, our data imply that alterations due to the C9ORF72 mutation resulting in TDP-43 accumulation and dysmetabolism as secondary downstream effects likely play a central role in the neurodegenerative process in C9ORF72 pathogenesis.

Bidirectional transcripts of the expanded C9orf72 hexanucleotide repeat are translated into aggregating dipeptide repeat proteins.

Massive GGGGCC repeat expansion in the first intron of the gene C9orf72 is the most common known cause of familial frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Despite its intronic localization and lack of an ATG start codon, the repeat region is translated in all three reading frames into aggregating dipeptide-repeat (DPR) proteins, poly-(Gly-Ala), poly-(Gly-Pro) and poly-(Gly-Arg). We took an antibody-based approach to further validate the translation of DPR proteins. To test whether the antisense repeat RNA transcript is also translated, we raised antibodies against the predicted products, poly-(Ala-Pro) and poly-(Pro-Arg). Both antibodies stained p62-positive neuronal cytoplasmic inclusions throughout the cerebellum and hippocampus indicating that not only sense but also antisense strand repeats are translated into DPR proteins in the absence of ATG start codons. Protein products of both strands co-aggregate suggesting concurrent translation of both strands. Moreover, an antibody targeting the putative carboxyl terminus of DPR proteins can detect inclusion pathology in C9orf72 repeat expansion carriers suggesting that the non-ATG translation continues through the entire repeat and beyond. A highly sensitive monoclonal antibody against poly-(Gly-Arg), visualized abundant inclusion pathology in all cortical regions and some inclusions also in motoneurons. Together, our data show that the GGGGCC repeat is bidirectionally translated into five distinct DPR proteins that co-aggregate in the characteristic p62-positive TDP-43 negative inclusions found in FTLD/ALS cases with C9orf72 repeat expansion. Novel monoclonal antibodies against poly-(Gly-Arg) will facilitate pathological diagnosis of C9orf72 FTLD/ALS.

Atypical parkinsonism due to a D202N Gerstmann-Sträussler-Scheinker prion protein mutation: first in vivo diagnosed case.

BACKGROUND: Parkinsonism with dopa-sensitivity and a correlating DaTSCAN turned out to be due to a D202N mutation which is associated with the Gerstmann-Sträussler-Scheinker (GSS) disease. METHODS/RESULTS: We report a 51-year old female who presented with left-dominant parkinsonism and a positive DaTSCAN. She was diagnosed with idiopathic Parkinson's syndrome. Dopaminergic medication reduced her symptoms. In addition, punding-like behavior, deficits in organizing daily life and abnormal sleep behavior were reported. Neuropsychological testing, EEG, polysomnography as well as PET imaging with fluorodexyglucose (FDG), [F-18]-desmethoxyfallypride (DMFP), and [C-11]-6-OH-BTA-1 (PIB) were not diagnostic. Cerebral spinal fluid analysis revealed no 14-3-3 protein, but elevated neuron-specific enolase (NSE) and S100-beta and a very low phospho-tau/total-tau ratio. Analysis of the prion gene disclosed the rare D202N mutation. CONCLUSIONS: The D202N prion mutation has been associated with GSS pathology and up to now was only reported post mortem. Our patient is the very first case diagnosed in vivo.

Report about four novel mutations in the prion protein gene.

BACKGROUND/AIMS: Since detection of the prion protein gene (PRNP) more than 30 mutations have been discovered. Some have only been found in single case reports without known intrafamilial accumulation or neuropathological proof so that the causal connection between mutation and disease could not be proved. Those patients often present atypical clinical phenotypes, and it is not unusual that they are classified as diseases other than Creutzfeldt-Jakob disease (CJD). METHODS: Cases of suspected CJD have been reported to the national reference center for prion diseases. Clinical and diagnostic data were collected, and a classification of definite, possible or probable prion disease was made. Molecular analysis of PRNP was performed by capillary sequencing. RESULTS: We have described 4 cases with atypical clinical and diagnostic findings and unknown mutations in PRNP so far. CONCLUSION: Three patients fulfilled the criteria of probable CJD, and 1 patient fulfilled the criteria of possible CJD but the clinical picture in none of the patients was typical CJD; hence, it remained questionable whether the mutations were causal of the disease.

Proper cerebellar development requires expression of ß1-integrin in Bergmann glia, but not in granule neurons.

ß1-class integrins play essential roles both in developmental biology as well as in cancer. Particularly, a Nestin-driven deletion of ß1-integrin receptors results in severe abnormalities of brain development including a laminar disorganization of cerebellar granule neurons. However, since Nestin is expressed in all kinds of neural precursors, these data do not allow conclusions to be drawn about the role of ß1-integrins in distinct neuronal and glial cell types. By generating conditional knockout mice using granule cell-specific Math1-promoter sequences, we show here that the expression of ß1-integrins in granule neurons is dispensable for the development of the cerebellum. Also, deletion of ß1-integrin from tumors that arise in a mouse model of granule cell precursor-derived medulloblastoma did not result in a significant survival benefit. Last, expression levels of ß1-integrin in human medulloblastoma samples did not predict patient's outcome. However, a ß1-integrin knockout using hGFAP-promoter sequences led to cerebellar hypoplasia, inappropriate positioning of Bergmann glia cells in the molecular layer, undirected outgrowth of radial glia fibers, and granule cell ectopia. We therefore conclude that ß1-integrin expression in cerebellar granule neurons is not essential during normal development or medulloblastoma formation. In fact, it is the expression of ß1-integrin in glia that is crucial for the proper development of the cerebellar cortex.

Low values of 5-hydroxymethylcytosine (5hmC), the "sixth base," are associated with anaplasia in human brain tumors.

5-Methylcytosine (5 mC) in genomic DNA has important epigenetic functions in embryonic development and tumor biology. 5-Hydroxymethylcytosine (5 hmC) is generated from 5 mC by the action of the TET (Ten-Eleven-Translocation) enzymes and may be an intermediate to further oxidation and finally demethylation of 5 mC. We have used immunohistochemistry (IHC) and isotope-based liquid chromatography mass spectrometry (LC-MS) to investigate the presence and distribution of 5 hmC in human brain and brain tumors. In the normal adult brain, IHC identified 61.5% 5 hmC positive cells in the cortex and 32.4% 5 hmC in white matter (WM) areas. In tumors, positive staining of cells ranged from 1.1% in glioblastomas (GBMs) (WHO Grade IV) to 8.9% in Grade I gliomas (pilocytic astrocytomas). In the normal adult human brain, LC-MS also showed highest values in cortical areas (1.17% 5 hmC/dG [deoxyguanosine]), in the cerebral WM we measured around 0.70% 5 hmC/dG. levels were related to tumor differentiation, ranging from lowest values of 0.078% 5 hmC/dG in GBMs (WHO Grade IV) to 0.24% 5 hmC/dG in WHO Grade II diffuse astrocytomas. 5 hmC measurements were unrelated to 5 mC values. We find that the number of 5 hmC positive cells and the amount of 5 hmC/dG in the genome that has been proposed to be related to pluripotency and lineage commitment in embryonic stem cells is also associated with brain tumor differentiation and anaplasia.