Enhancing predictability of IDH mutation status in glioma patients at initial diagnosis: a comparative analysis of radiomics from MRI, [18F]FET PET, and TSPO PET.

PURPOSE: According to the World Health Organization classification for tumors of the central nervous system, mutation status of the isocitrate dehydrogenase (IDH) genes has become a major diagnostic discriminator for gliomas. Therefore, imaging-based prediction of IDH mutation status is of high interest for individual patient management. We compared and evaluated the diagnostic value of radiomics derived from dual positron emission tomography (PET) and magnetic resonance imaging (MRI) data to predict the IDH mutation status non-invasively. METHODS: Eighty-seven glioma patients at initial diagnosis who underwent PET targeting the translocator protein (TSPO) using [18F]GE-180, dynamic amino acid PET using [18F]FET, and T1-/T2-weighted MRI scans were examined. In addition to calculating tumor-to-background ratio (TBR) images for all modalities, parametric images quantifying dynamic [18F]FET PET information were generated. Radiomic features were extracted from TBR and parametric images. The area under the receiver operating characteristic curve (AUC) was employed to assess the performance of logistic regression (LR) classifiers. To report robust estimates, nested cross-validation with five folds and 50 repeats was applied. RESULTS: TBRGE-180 features extracted from TSPO-positive volumes had the highest predictive power among TBR images (AUC 0.88, with age as co-factor 0.94). Dynamic [18F]FET PET reached a similarly high performance (0.94, with age 0.96). The highest LR coefficients in multimodal analyses included TBRGE-180 features, parameters from kinetic and early static [18F]FET PET images, age, and the features from TBRT2 images such as the kurtosis (0.97). CONCLUSION: The findings suggest that incorporating TBRGE-180 features along with kinetic information from dynamic [18F]FET PET, kurtosis from TBRT2, and age can yield very high predictability of IDH mutation status, thus potentially improving early patient management.

TSPO PET, tumour grading and molecular genetics in histologically verified glioma: a correlative 18F-GE-180 PET study.

BACKGROUND: The 18-kDa translocator protein (TSPO) is overexpressed in brain tumours and represents an interesting target for glioma imaging. 18F-GE-180, a novel TSPO ligand, has shown improved binding affinity and a high target-to-background contrast in patients with glioblastoma. However, the association of uptake characteristics on TSPO PET using 18F-GE-180 with the histological WHO grade and molecular genetic features so far remains unknown and was evaluated in the current study. METHODS: Fifty-eight patients with histologically validated glioma at initial diagnosis or recurrence were included. All patients underwent 18F-GE-180 PET, and the maximal and mean tumour-to-background ratios (TBRmax, TBRmean) as well as the PET volume were assessed. On MRI, presence/absence of contrast enhancement was evaluated. Imaging characteristics were correlated with neuropathological parameters (i.e. WHO grade, isocitrate dehydrogenase (IDH) mutation, O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and telomerase reverse transcriptase (TERT) promoter mutation). RESULTS: Six of 58 patients presented with WHO grade II, 16/58 grade III and 36/58 grade IV gliomas. An (IDH) mutation was found in 19/58 cases, and 39/58 were classified as IDH-wild type. High 18F-GE-180-uptake was observed in all but 4 cases (being WHO grade II glioma, IDH-mutant). A high association of 18F-GE-180-uptake and WHO grades was seen: WHO grade IV gliomas showed the highest uptake intensity compared with grades III and II gliomas (median TBRmax 5.15 (2.59-8.95) vs. 3.63 (1.85-7.64) vs. 1.63 (1.50-3.43), p < 0.001); this association with WHO grades persisted within the IDH-wild-type and IDH-mutant subgroup analyses (p < 0.05). Uptake intensity was also associated with the IDH mutational status with a trend towards higher 18F-GE-180-uptake in IDH-wild-type gliomas in the overall group (median TBRmax 4.67 (1.56-8.95) vs. 3.60 (1.50-7.64), p = 0.083); however, within each WHO grade, no differences were found (e.g. median TBRmax in WHO grade III glioma 4.05 (1.85-5.39) vs. 3.36 (2.32-7.64), p = 1.000). No association was found between uptake intensity and MGMT or TERT (p > 0.05 each). CONCLUSION: Uptake characteristics on 18F-GE-180 PET are highly associated with the histological WHO grades, with the highest 18F-GE-180 uptake in WHO grade IV glioblastomas and a PET-positive rate of 100% among the investigated high-grade gliomas. Conversely, all TSPO-negative cases were WHO grade II gliomas. The observed association of 18F-GE-180 uptake and the IDH mutational status seems to be related to the high inter-correlation of the IDH mutational status and the WHO grades.

Comparison of 18F-GE-180 and dynamic 18F-FET PET in high grade glioma: a double-tracer pilot study.

BACKGROUND: PET represents a valuable tool for glioma imaging. In addition to amino acid tracers such as 18F-FET, PET targeting the 18-kDa mitochondrial translocator-protein (TSPO) is of high interest for high-grade glioma (HGG) imaging due to its upregulation in HGG cells. 18F-GE-180, a novel TSPO ligand, has shown a high target-to-background contrast in HGG. Therefore, we intra-individually compared its uptake characteristics to dynamic 18F-FET PET and contrast-enhanced MRI in patients with HGG. METHODS: Twenty HGG patients (nine IDH-wildtype, 11 IDH-mutant) at initial diagnosis (n = 8) or recurrence (n = 12) were consecutively included and underwent 18F-GE-180 PET, dynamic 18F-FET PET, and MRI. The maximal tumour-to-background ratios (TBRmax) and biological tumour volumes (BTV) were evaluated in 18F-GE-180 and 18F-FET PET. Dynamic 18F-FET PET analysis included the evaluation of minimal time-to-peak (TTPmin). In MRI, the volume of contrast-enhancement was delineated (VOLCE). Volumes were spatially correlated using the Sørensen-Dice coefficient. RESULTS: The median TBRmax tended to be higher in 18F-GE-180 PET compared to 18F-FET PET [4.58 (2.33-8.95) vs 3.89 (1.56-7.15); p = 0.062] in the overall group. In subgroup analyses, IDH-wildtype gliomas showed a significantly higher median TBRmax in 18F-GE-180 PET compared to 18F-FET PET [5.45 (2.56-8.95) vs 4.06 (1.56-4.48); p = 0.008]; by contrast, no significant difference was observed in IDH-mutant gliomas [3.97 (2.33-6.81) vs 3.79 (2.01-7.15) p = 1.000]. Only 5/20 cases showed higher TBRmax in 18F-FET PET compared to 18F-GE-180 PET, all of them being IDH-mutant gliomas. No parameter in 18F-GE-180 PET correlated with TTPmin (p > 0.05 each). There was a tendency towards higher median BTVGE-180 [32.1 (0.4-236.0) ml] compared to BTVFET [19.3 (0.7-150.2) ml; p = 0.062] with a moderate spatial overlap [median Sørensen-Dice coefficient 0.55 (0.07-0.85)]. In MRI, median VOLCE [9.7 (0.1-72.5) ml] was significantly smaller than both BTVFET and BTVGE180 (p < 0.001 each), leading to a poor spatial correlation with BTVGE-180 [0.29 (0.01-0.48)] and BTVFET [0.38 (0.01-0.68)]. CONCLUSION: PET with 18F-GE-180 and 18F-FET provides differing imaging information in HGG dependent on the IDH-mutational status, with diverging spatial overlap and vast exceedance of contrast-enhancement in MRI. Combined PET imaging might reveal new insights regarding non-invasive characterization of tumour heterogeneity and might influence patients' management.

Correction to: Biological tumour volumes of gliomas in early and standard 20-40 min 18F-FET PET images differ according to IDH mutation status.

The name of M. Unterrainer was inadvertently presented as M. Unterrrainer in the original article.

Biological tumour volumes of gliomas in early and standard 20-40 min 18F-FET PET images differ according to IDH mutation status.

PURPOSE: For the clinical evaluation of O-(2-18F-fluoroethyl)-L-tyrosine (18F-FET) PET images, the use of standard summation images obtained 20-40 min after injection is recommended. However, early summation images obtained 5-15 min after injection have been reported to allow better differentiation between low-grade glioma (LGG) and high-grade glioma (HGG) by capturing the early 18F-FET uptake peak specific for HGG. We compared early and standard summation images with regard to delineation of the PET-derived biological tumour volume (BTV) in correlation with the molecular genetic profile according the updated 2016 WHO classification. METHODS: The analysis included 245 patients with newly diagnosed, histologically verified glioma and a positive 18F-FET PET scan prior to any further treatment. BTVs were delineated during the early 5-15 min and standard 20-40 min time frames using a threshold of 1.6 × background activity and were compared intraindividually. Volume differences between early and late summation images of >20% were considered significant and were correlated with WHO grade and the molecular genetic profile (IDH mutation and 1p/19q codeletion status). RESULTS: In 52.2% of the patients (128/245), a significant difference in BTV of >20% between early and standard summation images was found. While 44.3% of WHO grade II gliomas (31 of 70) showed a significantly smaller BTV in the early summation images, 35.0% of WHO grade III gliomas (28/80) and 37.9% of WHO grade IV gliomas (36/95) had a significantly larger BTVs. Among IDH-wildtype gliomas, an even higher portion (44.4%, 67/151) showed significantly larger BTVs in the early summation images, which was observed in 5.3% (5/94) of IDH-mutant gliomas only: most of the latter had significantly smaller BTVs in the early summation images, i.e. 51.2% of IDH-mutant gliomas without 1p/19q codeletion (21/41) and 39.6% with 1p/19q codeletion (21/53). CONCLUSION: BTVs delineated in early and standard summation images differed significantly in more than half of gliomas. While the standard summation images seem appropriate for delineation of LGG as well as IDH-mutant gliomas, a remarkably high percentage of HGG and, particularly, IDH-wildtype gliomas were depicted with significantly larger volumes in early summation images. This finding might be of interest for optimization of treatment planning (e.g. radiotherapy) in accordance with the individual IDH mutation status.

Loss of neuronal GSK3ß reduces dendritic spine stability and attenuates excitatory synaptic transmission via ß-catenin.

Central nervous glycogen synthase kinase 3ß (GSK3ß) is implicated in a number of neuropsychiatric diseases, such as bipolar disorder, depression, schizophrenia, fragile X syndrome or anxiety disorder. Many drugs employed to treat these conditions inhibit GSK3ß either directly or indirectly. We studied how conditional knockout of GSK3ß affected structural synaptic plasticity. Deletion of the GSK3ß gene in a subset of cortical and hippocampal neurons in adult mice led to reduced spine density. In vivo imaging revealed that this was caused by a loss of persistent spines, whereas stabilization of newly formed spines was reduced. In electrophysiological recordings, these structural alterations correlated with a considerable drop in the frequency and amplitude of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-dependent miniature excitatory postsynaptic currents. Expression of constitutively active ß-catenin caused reduction in spine density and electrophysiological alterations similar to GSK3ß knockout, suggesting that the effects of GSK3ß knockout were mediated by the accumulation of ß-catenin. In summary, changes of dendritic spines, both in quantity and in morphology, are correlates of experience-dependent synaptic plasticity; thus, these results may help explain the mechanism of action of psychotropic drugs inhibiting GSK3ß.

Amyloid-PET predicts inhibition of de novo plaque formation upon chronic γ-secretase modulator treatment.

In a positron-emission tomography (PET) study with the ß-amyloid (Aß) tracer [(18)F]-florbetaben, we previously showed that Aß deposition in transgenic mice expressing Swedish mutant APP (APP-Swe) mice can be tracked in vivo. γ-Secretase modulators (GSMs) are promising therapeutic agents by reducing generation of the aggregation prone Aß42 species without blocking general γ-secretase activity. We now aimed to investigate the effects of a novel GSM [8-(4-Fluoro-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-[1-(3-methyl-[1,2,4]thiadiazol-5-yl)-piperidin-4-yl]-amine (RO5506284) displaying high potency in vitro and in vivo on amyloid plaque burden and used longitudinal Aß-microPET to trace individual animals. Female transgenic (TG) APP-Swe mice aged 12 months (m) were assigned to vehicle (TG-VEH, n=12) and treatment groups (TG-GSM, n=12), which received daily RO5506284 (30 mg kg(-1)) treatment for 6 months. A total of 131 Aß-PET recordings were acquired at baseline (12 months), follow-up 1 (16 months) and follow-up 2 (18 months, termination scan), whereupon histological and biochemical analyses of Aß were performed. We analyzed the PET data as VOI-based cortical standard-uptake-value ratios (SUVR), using cerebellum as reference region. Individual plaque load assessed by PET remained nearly constant in the TG-GSM group during 6 months of RO5506284 treatment, whereas it increased progressively in the TG-VEH group. Baseline SUVR in TG-GSM mice correlated with Δ%-SUVR, indicating individual response prediction. Insoluble Aß42 was reduced by 56% in the TG-GSM versus the TG-VEH group relative to the individual baseline plaque load estimates. Furthermore, plaque size histograms showed differing distribution between groups of TG mice, with fewer small plaques in TG-GSM animals. Taken together, in the first Aß-PET study monitoring prolonged treatment with a potent GSM in an AD mouse model, we found clear attenuation of de novo amyloidogenesis. Moreover, longitudinal PET allows non-invasive assessment of individual plaque-load kinetics, thereby accommodating inter-animal variations.

Correlation of two-photon in vivo imaging and FIB/SEM microscopy.

Advances in the understanding of brain functions are closely linked to the technical developments in microscopy. In this study, we describe a correlative microscopy technique that offers a possibility of combining two-photon in vivo imaging with focus ion beam/scanning electron microscope (FIB/SEM) techniques. Long-term two-photon in vivo imaging allows the visualization of functional interactions within the brain of a living organism over the time, and therefore, is emerging as a new tool for studying the dynamics of neurodegenerative diseases, such as Alzheimer's disease. However, light microscopy has important limitations in revealing alterations occurring at the synaptic level and when this is required, electron microscopy is mandatory. FIB/SEM microscopy is a novel tool for three-dimensional high-resolution reconstructions, since it acquires automated serial images at ultrastructural level. Using FIB/SEM imaging, we observed, at 10 nm isotropic resolution, the same dendrites that were imaged in vivo over 9 days. Thus, we analyzed their ultrastructure and monitored the dynamics of the neuropil around them. We found that stable spines (present during the 9 days of imaging) formed typical asymmetric contacts with axons, whereas transient spines (present only during one day of imaging) did not form a synaptic contact. Our data suggest that the morphological classification that was assigned to a dendritic spine according to the in vivo images did not fit with its ultrastructural morphology. The correlative technique described herein is likely to open opportunities for unravelling the earlier unrecognized complexity of the nervous system.

Mutational signature of extracranial meningioma metastases and their respective primary tumors.

Extracranial metastases of intracranial meningiomas are rare. Little is known about the mutational pattern of these tumors and their metastatic seeding. Here, we retrospectively explored the molecular alterations of these metastatic lesions and their respective intracranial tumor manifestations.Histology and genome sequencing were performed in intracranial meningiomas and their extracranial metastatic lesions operated upon between 2002 and 2021. Next-generation DNA/RNA sequencing (NGS) and methylome analysis were performed to determine molecular alterations.We analyzed the tumors of five patients with clinically suspected metastases of a meningioma using methylome analysis and next generation panel sequencing of the primary tumors as well as the metastatic lesions. Metastases were found in the spinal cord and one in the lung. In four of these patients, molecular analyses confirmed metastatic disease, while the fifth patient was found to harbor two molecularly distinct meningiomas. On pathological assessment, the primary lesions ranged from CNS WHO grades 1 to 3 (integrated molecular-morphologic meningioma classification scores 2 to 6). Of the four true metastatic cases, three out of the four metastasizing tumors harbored alterations in the BAP1 gene, comprising a stop-mutation combined with copy-number loss (WHO grade 1), copy number loss (WHO grade 3) and a frameshift mutation (WHO grade 2). Furthermore, the latter was confirmed to harbor a BAP1 tumor predisposition syndrome. The fourth metastasizing tumor had copy-number losses in NF2 and PTEN. Only one of four showed CDKN2A homozygous deletion; none showed TERT promotor mutation.Our results molecularly confirm true metastatic disease in four meningioma patients. BAP1 gene alterations were the most frequent. Larger cohorts, most likely from multicenter studies are necessary to evaluate the role of BAP-1 alterations to further understand the metastatic spread in meningiomas. for metastatic spread and might indicate patients at risk for metastatic spread. Further explorations within larger cohorts are necessary to validate these findings which might influence the clinical management in the future.

MRI-based contrast clearance analysis shows high differentiation accuracy between radiation-induced reactions and progressive disease after cranial radiotherapy.

BACKGROUND: Pseudoprogression (PsP) or radiation necrosis (RN) may frequently occur after cranial radiotherapy and show a similar imaging pattern compared with progressive disease (PD). We aimed to evaluate the diagnostic accuracy of magnetic resonance imaging-based contrast clearance analysis (CCA) in this clinical setting. PATIENTS AND METHODS: Patients with equivocal imaging findings after cranial radiotherapy were consecutively included into this monocentric prospective study. CCA was carried out by software-based automated subtraction of imaging features in late versus early T1-weighted sequences after contrast agent application. Two experienced neuroradiologists evaluated CCA with respect to PsP/RN and PD being blinded for histological findings. The radiological assessment was compared with the histopathological results, and its accuracy was calculated statistically. RESULTS: A total of 33 patients were included; 16 (48.5%) were treated because of a primary brain tumor (BT), and 17 (51.1%) because of a secondary BT. In one patient, CCA was technically infeasible. The accuracy of CCA in predicting the histological result was 0.84 [95% confidence interval (CI) 0.67-0.95; one-sided P = 0.051; n = 32]. Sensitivity and specificity of CCA were 0.93 (95% CI 0.66-1.00) and 0.78 (95% CI 0.52-0.94), respectively. The accuracy in patients with secondary BTs was 0.94 (95% CI 0.71-1.00) and nonsignificantly higher compared with patients with primary BT with an accuracy of 0.73 (95% CI 0.45-0.92), P = 0.16. CONCLUSIONS: In this study, CCA was a highly accurate, easy, and helpful method for distinguishing PsP or RN from PD after cranial radiotherapy, especially in patients with secondary tumors after radiosurgical treatment.

Long-term dynamics of aberrant neuronal activity in awake Alzheimer's disease transgenic mice.

Alzheimer's disease (AD) is associated with aberrant neuronal activity, which is believed to critically determine disease symptoms. How these activity alterations emerge, how stable they are over time, and whether cellular activity dynamics are affected by the amyloid plaque pathology remains incompletely understood. We here repeatedly recorded the activity from identified neurons in cortex of awake APPPS1 transgenic mice over four weeks during the early phase of plaque deposition using in vivo two-photon calcium imaging. We found that aberrant activity during this stage largely persisted over the observation time. Novel highly active neurons slowly emerged from former intermediately active neurons. Furthermore, activity fluctuations were independent of plaque proximity, but aberrant activity was more likely to persist close to plaques. These results support the notion that neuronal network pathology observed in models of cerebral amyloidosis is the consequence of persistent single cell aberrant neuronal activity, a finding of potential diagnostic and therapeutic relevance for AD.

Serial stereotactic biopsy of brainstem lesions in adults improves diagnostic accuracy compared with MRI only.

OBJECTIVE: The aim of the current prospective study was to analyse the validity of MRI based diagnosis of brainstem gliomas which was verified by stereotactic biopsy and follow-up evaluation as well as to assess prognostic factors and risk profile. METHODS: Between 1998 and 2007, all consecutive adult patients with radiologically suspected brainstem glioma were included. The MRI based diagnosis of the lesions was made independently by an experienced neuroradiologist. Histopathological evaluation was performed in all patients from paraffin embedded specimens obtained by multimodal image guided stereotactic serial biopsy technique. Histopathological results were compared with prior radiological assessment. Length of survival was estimated with the Kaplan-Meier method and prognostic factors were calculated using the Cox model. RESULTS: 46 adult patients were included. Histological evaluation revealed pilocytic astrocytoma (n = 2), WHO grade II glioma (n = 14), malignant glioma (n = 12), metastasis (n = 7), lymphoma (n = 5), cavernoma (n = 1), inflammatory disease (n = 2) or no tumour/gliosis (n = 3). Perioperative morbidity was 2.5% (n = 1). There was no permanent morbidity and no mortality. All patients with "no tumour" or "inflammatory disease" survived. Patients with low grade glioma and malignant glioma showed a 1 year survival rate of 75% and 25%, respectively; the 1 year survival rate for patients with lymphoma or metastasis was 30%. In the subgroup with a verified brainstem glioma, negative predictors for length of survival were higher tumour grade (p = 0.002) and Karnofsky performance score < or =70 (p = 0.004). CONCLUSION: Intra-axial brainstem lesions with a radiological pattern of glioma represent a very heterogeneous tumour group with completely different outcomes. Radiological features alone are not reliable for diagnostic classification. Stereotactic biopsy is a safe method to obtain a valid tissue diagnosis, which is indispensible for treatment decision.

Anaerobic training in hypoxia: A new approach to stimulate the rating of effort perception.

This study compared subjective effort perception with objective physiological measures during high-intensive intermittent exercise performed in normoxia, moderate hypoxia (FiO2: 16.5%) and severe hypoxia (FiO2: 13.5%). Sixteen physically active subjects performed an equal training session on three different days. Training consisted of 6 "all-out" series of continuous jumps lasting for 15s each. Average power output during the jumps was similar in all three conditions (~3200W). Greater hypoxemia was observed in hypoxia as compared to normoxia. Likewise, a significantly higher value in perceived effort was observed after hypoxia training as compared to normoxia training (p<0.05). Whereas blood lactate concentrations immediately after training were not different between normoxia and hypoxia, creatine kinase increased in moderate (p=0.02) and severe (p<0.01) hypoxia compared to normoxia 24h after the training. Perceived fatigue was also significantly elevated 24h after hypoxic exercise only. Heart rate variability pre and 24h after exercise showed a tendency to sympathetic predominance in severe hypoxia as compared to moderate hypoxia and normoxia. In conclusion, a single session of anaerobic exercise can be executed at the same intensity in moderate/severe hypoxia as in normoxia. This type of hypoxic training may be considered as a method potentially to improve the ability tolerating discomfort and consequently also exercise performance.

The cerebellum-specific Munc13 isoform Munc13-3 regulates cerebellar synaptic transmission and motor learning in mice.

Munc13 proteins form a family of three, primarily brain-specific phorbol ester receptors (Munc13-1/2/3) in mammals. Munc13-1 is a component of presynaptic active zones in which it acts as an essential synaptic vesicle priming protein. In contrast to Munc13-1, which is present in most neurons throughout the rat and mouse CNS, Munc13-3 is almost exclusively expressed in the cerebellum. Munc13-3 mRNA is present in granule and Purkinje cells but absent from glia cells. Munc13-3 protein is localized to the synaptic neuropil of the cerebellar molecular layer but is not found in Purkinje cell dendrites, suggesting that Munc13-3, like Munc13-1, is a presynaptic protein at parallel fiber-Purkinje cell synapses. To examine the role of Munc13-3 in cerebellar physiology, we generated Munc13-3-deficient mutant mice. Munc13-3 deletion mutants exhibit increased paired-pulse facilitation at parallel fiber-Purkinje cell synapses. In addition, mutant mice display normal spontaneous motor activity but have an impaired ability to learn complex motor tasks. Our data demonstrate that Munc13-3 regulates synaptic transmission at parallel fiber-Purkinje cell synapses. We propose that Munc13-3 acts at a similar step of the synaptic vesicle cycle as does Munc13-1, albeit with less efficiency. In view of the present data and the well established vesicle priming function of Munc13-1, it is likely that Munc13-3-loss leads to a reduction in release probability at parallel fiber-Purkinje cell synapses by interfering with vesicle priming. This, in turn, would lead to increases in paired-pulse facilitation and could contribute to the observed deficit in motor learning.

Evidence of presynaptic location and function of the prion protein.

The prion protein (PrP(C)) is a copper-binding protein of unknown function that plays an important role in the etiology of transmissible spongiform encephalopathies. Using morphological techniques and synaptosomal fractionation methods, we show that PrP(C) is predominantly localized to synaptic membranes. Atomic absorption spectroscopy was used to identify PrP(C)-related changes in the synaptosomal copper concentration in transgenic mouse lines. The synaptic transmission in the presence of H(2)O(2), which is known to be decomposed to highly reactive hydroxyl radicals in the presence of iron or copper and to alter synaptic activity, was studied in these animals. The response of synaptic activity to H(2)O(2) was found to correlate with the amount of PrP(C) expression in the presynaptic neuron in cerebellar slice preparations from wild-type, Prnp(0/0), and PrP gene-reconstituted transgenic mice. Thus, our data gives strong evidence for the predominantly synaptic location of PrP(C), its involvement in the regulation of the presynaptic copper concentration, and synaptic activity in defined conditions.

Mice with combined gene knock-outs reveal essential and partially redundant functions of amyloid precursor protein family members.

The amyloid precursor protein (APP) involved in Alzheimer's disease is a member of a larger gene family including amyloid precursor-like proteins APLP1 and APLP2. We generated and examined the phenotypes of mice lacking individual or all possible combinations of APP family members to assess potential functional redundancies within the gene family. Mice deficient for the nervous system-specific APLP1 protein showed a postnatal growth deficit as the only obvious abnormality. In contrast to this minor phenotype, APLP2(-/-)/APLP1(-/-) and APLP2(-/-)/APP(-/-) mice proved lethal early postnatally. Surprisingly, APLP1(-/-)/APP(-/-) mice were viable, apparently normal, and showed no compensatory upregulation of APLP2 expression. These data indicate redundancy between APLP2 and both other family members and corroborate a key physiological role for APLP2. This view gains further support by the observation that APLP1(-/-)/APP(-/-)/APLP2(+/-) mice display postnatal lethality. In addition, they provide genetic evidence for at least some distinct physiological roles of APP and APLP2 by demonstrating that combinations of single knock-outs with the APLP1 mutation resulted in double mutants of clearly different phenotypes, being either lethal, or viable. None of the lethal double mutants displayed, however, obvious histopathological abnormalities in the brain or any other organ examined. Moreover, cortical neurons from single or combined mutant mice showed unaltered survival rates under basal culture conditions and unaltered susceptibility to glutamate excitotoxicity in vitro.

Popularity of hypoxic training methods for endurance-based professional and amateur athletes.

Scientific debate continues into whether hypoxic training has any performance benefit for athletes, and although this type of training seems popular, to our knowledge little empirical evidence on its popularity with endurance-based athletes exists. To quantify the usage of hypoxic training in endurance-based athletes we asked 203 athletes (amateur = 108, professional = 95) to complete a 17-question survey during 2013-2014 season. Compared to amateurs, professional athletes were 4.5 times (3.0-6.8, odds ratio, 95% confidence limits) more likely to undertake hypoxic training. Live-high train-low was the most popular hypoxic training protocol for athletes (52% professional and 80% amateur) with live-high train-high also used (38% professional, 20% amateur). Compared to amateurs, professional athletes tended to use evidence-based hypoxic training methods, seek advice on hypoxic training from reliable sources and were generally more realistic about the potential performance gains as a result of hypoxic training. Almost one third (25-30%) of all athletes suffered illness during their hypoxic training. Compared to amateurs, professional athletes are more likely to undertake hypoxic training and tend to follow current scientific guidelines. Attenuation of the ill effects that occur during hypoxic training may be accomplished if athletes give more attention to monitoring stress and training levels.

Clonal analysis in glioblastoma with epithelial differentiation.

Epithelial differentiation in glioblastomas (GBM) may be associated with circumscribed growth and focal keratin expression resembling carcinoma metastasis. Therefore these rare lesions can pose a diagnostic problem suggesting coincidental occurrence of two separate neoplasms. However molecular analysis should succeed in establishing a common origin of seemingly unrelated tumor samples. Five GBMs exhibiting epithelial differentiation were microdissected and analyzed for mutations in the TP53 gene. SSCP analysis of exons 5-8 was followed by direct sequencing of aberrantly migrating fragments. TP53 mutations were identified in tumors from two of five patients. A G-->T transversion in codon 176 was detected in a tumor, initially diagnosed as metastases of unknown origin, however, a later autopsy revealed GBM. In this lesion, the mutation was observed in both, areas of astrocytic differentiation and areas of epithelial differentiation. One tumor diagnosed as GBM with epithelial differentiation carried C-->T transition in codon 211 in both, areas of astrocytic and epithelial differentiation. Thus, molecular analysis proved clonality in two GBMs with epithelial differentiation, thereby excluding a collision tumor. The present data support the concept of clonal origin of these morphologically heterogeneous lesions.