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Simple Summary: Glioblastoma (GB) is the most common primary brain tumor in adults, but it remains incurable due to its high degree of therapy resistance. Glioblastoma stem-like cells (GSCs) are believed to drive the initiation, progression, and therapy resistance of GB, making them an ideal therapeutic target to improve patient outcomes. However, due to their heterogeneity, there are no universal markers to identify GSCs. We evaluated GARP as a novel marker for GSCs and found that GARP is more stably and uniformly expressed by human GSCs, across cellular states and disease stages, than the commonly used GSC marker, CD133. Additionally, we showed that GARP is intranuclearly localized in GSCs, and we are the first to show that nuclear GARP levels (GARPNU+) are associated with poor patient survival. Our findings indicate that GARP/GARPNU+ expression is an improved marker for GSCs and suggest a potential application of GARP as a prognostic biomarker for GB. Glioblastoma (GB) is notoriously resistant to therapy. GB genesis and progression are driven by glioblastoma stem-like cells (GSCs). One goal for improving treatment efficacy and patient outcomes is targeting GSCs. Currently, there are no universal markers for GSCs. Glycoprotein A repetitions predominant (GARP), an anti-inflammatory protein expressed by activated regulatory T cells, was identified as a possible marker for GSCs. This study evaluated GARP for the detection of human GSCs utilizing a multidimensional experimental design that replicated several features of GB: (1) intratumoral heterogeneity, (2) cellular hierarchy (GSCs with varied degrees of self-renewal and differentiation), and (3) longitudinal GSC evolution during GB recurrence (GSCs from patient-matched newly diagnosed and recurrent GB). Our results indicate that GARP is expressed by GSCs across various cellular states and disease stages. GSCs with an increased GARP expression had reduced self-renewal but no alterations in proliferative capacity or differentiation commitment. Rather, GARP correlated inversely with the expression of GFAP and PDGFR-α, markers of astrocyte or oligodendrocyte differentiation. GARP had an abnormal nuclear localization (GARPNU+) in GSCs and was negatively associated with patient survival. The uniformity of GARP/GARPNU+ expression across different types of GSCs suggests a potential use of GARP as a marker to identify GSCs. [ABSTRACT FROM AUTHOR]

Simple Summary: Molecular analyses discussed in Molecular Tumor Boards are expected to improve cancer treatment by identifying tumor-specific alterations. Here, we quantified the expression of the gangliosides GD2 and N-glycolyl GM3 in neuroblastoma and medulloblastoma, two aggressive pediatric tumors. Our data suggest that subtypes of both entities will benefit from an anti-GD2 directed therapy, and that the ganglioside signature can be helpful in the identification of GD2-positive samples. The integration of lipid analysis may help to identify patients who will benefit from personalized immunotherapy with monoclonal antibodies or CAR-T cells. Neuroblastoma (NBL) and medulloblastoma (MB) are aggressive pediatric cancers which can benefit from therapies targeting gangliosides. Therefore, we compared the ganglioside profile of 9 MB and 14 NBL samples by thin layer chromatography and mass spectrometry. NBL had the highest expression of GD2 (median 0.54 nmol GD2/mg protein), and also expressed complex gangliosides. GD2-low samples expressed GD1a and were more differentiated. MB mainly expressed GD2 (median 0.032 nmol GD2/mg protein) or GM3. Four sonic hedgehog-activated (SHH) as well as one group 4 and one group 3 MBs were GD2-positive. Two group 3 MB samples were GD2-negative but GM3-positive. N-glycolyl neuraminic acid-containing GM3 was neither detected in NBL nor MB by mass spectrometry. Furthermore, a GD2-phenotype predicting two-gene signature (ST8SIA1 and B4GALNT1) was applied to RNA-Seq datasets, including 86 MBs and validated by qRT-PCR. The signature values were decreased in group 3 and wingless-activated (WNT) compared to SHH and group 4 MBs. These results suggest that while NBL is GD2-positive, only some MB patients can benefit from a GD2-directed therapy. The expression of genes involved in the ganglioside synthesis may allow the identification of GD2-positive MBs. Finally, the ganglioside profile may reflect the differentiation status in NBL and could help to define MB subtypes. [ABSTRACT FROM AUTHOR]

Ependymoma with YAP1‐MAMLD1 fusion is a rare, recently described supratentorial neoplasm of childhood, with few cases published so far. We report on 15 pediatric patients with ependymomas carrying YAP1‐MAMLD1 fusions, with their characteristic histopathology, immunophenotype and molecular/cytogenetic, radiological and clinical features. The YAP1‐MAMLD1 fusion was documented by RT‐PCR/Sanger sequencing, and tumor genomes were studied by molecular inversion probe (MIP) analysis. Significant copy number alterations were identified by GISTIC (Genomic Identification of Significant Targets in Cancer) analysis. All cases showed similar histopathological features including areas of high cellularity, presence of perivascular pseudo‐rosettes, small to medium‐sized nuclei with characteristic granular chromatin and strikingly abundant cells with dot‐like cytoplasmic expression of epithelial membrane antigen. Eleven cases presented features of anaplasia, corresponding to WHO grade III. MRI showed large supratentorial multinodular tumors with cystic components, heterogeneous contrast enhancement, located in the ventricular or periventricular region. One of two variants of YAP1‐MAMLD1 fusions was detected in all cases. The MIP genome profiles showed balanced profiles, with focal alterations of the YAP1 locus at 11q22.1–11q21.2 (7/14), MAMLD1 locus (Xp28) (10/14) and losses of chromosome arm 22q (5/14). Most patients were female (13/15) and younger than 3 years at diagnosis (12/15; median age, 8.2 months). Apart from one patient who died during surgery, all patients are alive without evidence of disease progression after receiving different treatment protocols, three without postoperative further treatment (median follow‐up, 4.84 years). In this to date, largest series of ependymomas with YAP1‐MAMLD1 fusions we show that they harbor characteristic histopathological, cytogenetic and imaging features, occur mostly in young girls under 3 years and are associated with good outcome. Therefore, this genetically defined neoplasm should be considered a distinct disease entity. The diagnosis should be confirmed by demonstration of the specific fusion. Further studies on large collaborative series are warranted to confirm our findings. [ABSTRACT FROM AUTHOR]

Aims The antiphospholipid syndrome ( APS) is an autoimmune disease characterized by high titres of auto-antibodies ( aPL) leading to thrombosis and consequent infarcts. However, many affected patients develop neurological symptoms in the absence of stroke. Similarly, in a mouse model of this disease ( eAPS), animals consistently develop behavioural abnormalities despite lack of ischemic brain injury. Therefore, the present study was designed to identify structural alterations of hippocampal neurones underlying the neurological symptoms in eAPS. Methods Adult female Balb/C mice were subjected to either induction of eAPS by immunization with β2-Glycoprotein 1 or to a control group. After sixteen weeks animals underwent behavioural and cognitive testing using Staircase test (experiment 1 and 2) and Y-maze alternation test (experiment 1) and were tested for serum aPL levels (both experiments). Animals of experiment 1 ( n = 7/group) were used for hippocampal neurone analysis using Golgi- Cox staining. Animals of experiment 2 ( n = 7/group) were used to analyse molecular markers of total dendritic integrity ( MAP2), presynaptic plasticity (synaptobrevin 2/ VAMP2) and dendritic spines (synaptopodin) using immunohistochemistry. Results eAPS mice developed increased aPL titres and presented with abnormal behaviour and impaired short term memory. Further, they revealed a reduction of dendritic complexity of hippocampal CA1 neurones as reflected by decreased dendritic length, arborization and spine density, respectively. Additional decrease of the spine-associated protein expression of Synaptopodin points to dendritic spines as major targets in the pathological process. Conclusion Reduction of hippocampal dendritic complexity may represent the structural basis for the behavioural and cognitive abnormalities of eAPS mice. [ABSTRACT FROM AUTHOR]

The granulocyte colony-stimulating factor (G- CSF), being a member of the hematopoietic growth factor family, is also critically involved in controlling proliferation and differentiation of neural stem cells. Treatment with G- CSF has been shown to result in substantial neuroprotective and neuroregenerative effects in various experimental models of acute and chronic diseases of the central nervous system. Although G- CSF has been tested in a clinical study for treatment of acute ischemic stroke, there is only fragmentary data on the distribution of this cytokine and its receptor in the human brain. Therefore, the present study was focused on the immunohistochemical analysis of the protein expression of G- CSF and its receptor (G- CSF R) in the adult human brain. Since G- CSF has been shown not only to exert neuroprotective effects in animal models of Alzheimer's disease ( AD) but also to be a candidate for clinical treatment, we have also placed an emphasis on the regulation of these molecules in this neurodegenerative disease. One major finding is that both G- CSF and G- CSF R were ubiquitously but not uniformly expressed in neurons throughout the CNS. Protein expression of G- CSF and G- CSF R was not restricted to neurons but was also detectable in astrocytes, ependymal cells, and choroid plexus cells. However, the distribution of G- CSF and G- CSF R did not substantially differ between AD brains and control, even in the hippocampus, where early neurodegenerative changes typically occur. [ABSTRACT FROM AUTHOR]

One of the hallmarks of stroke pathophysiology is the widespread death of many different types of brain cells. As our understanding of the complex disease that is stroke has grown, it is now generally accepted that various different mechanisms can result in cell damage and eventual death. A plethora of techniques is available to identify various pathological features of cell death in stroke; each has its own drawbacks and pitfalls, and most are unable to distinguish between different types of cell death, which partially explains the widespread misuse of many terms. The purpose of this review is to summarize the standard histopathological and immunohistochemical techniques used to identify various pathological features of stroke. We then discuss how these methods should be properly interpreted on the basis of what they are showing, as well as advantages and disadvantages that require consideration. As there is much interest in the visualization of stroke using noninvasive imaging strategies, we also specifically discuss how these techniques can be interpreted within the context of cell death. [ABSTRACT FROM AUTHOR]

1. The aim of the present study was to investigate the potential energy preserving effect of permanent bilateral common carotid artery occlusion (BCCAO) towards additional systemic hypotension of severe duration (30 min). In addition, the role of adenosine A1 receptors in cerebral ischaemic preconditioning was investigated in male Wistar rats. Thus, oligaemic rats were assigned randomly to continuous treatment with the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) or the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT), receiving daily intraperitoneal infusions of 0.1 mg/kg bodyweight CCPA or CPT or placebo (200 µL aqueous 2-hydropropyl-β-cyclodextrin) at a delivery rate of 0.5 µL/h over 14 days.2. Haemodynamic parameters and arterial blood gases were monitored. Rat cortical energy metabolites ATP, ADP, AMP, phosphocreatine and adenosine were measured using HPLC techniques. Adenosine A1 receptor expression was determined by immunhistochemistry and quantified by western blotting.3. Two weeks of permanent BCCAO induced an‘energy saving’ effect in rat cortical ATP concentrations. Under subchronic conditions, significant increases were detected in ADP and AMP concentrations after CCPA compared with placebo. Because similar changes were also seen after CPT, this adenosine A1 receptor-mediated effect does not seems to be specific. Furthermore, no differences in adenosine A1 receptor expression could be detected.4. Adenosine was not specifically involved in the‘preconditioning-like’ effect via the modulation of the adenosine A1 receptor in the present oligaemia model. Obviously, adenosine A1 receptor-specific effects after delayed cerebral ischaemic preconditioning do not seem to play an essential role if BCCAO is followed by a prolonged additional severe ischaemic event. [ABSTRACT FROM AUTHOR]

Kainic acid induces seizures with consecutive degeneration of highly vulnerable hippocampal CA3 neurons in adult rats. An abnormal influx of calcium through newly synthesized α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptors lacking the GluR2 subunit, which normally renders AMPA receptors calcium impermeable, is thought to play a pivotal role for postictal neuronal death (GluR2 hypothesis). Using a specific GluR2 antiserum, postictal hippocampal GluR2 protein expression was investigated and compared to GluR1 between 6 and 96 h after seizure induction. In addition, postictal protein expression of a recently cloned AMPA receptor binding protein (ABP), which anchors AMPA receptors in the plasma membrane was also analyzed, to address the question of whether its protein expression is associated with neuronal death or survival. At 6 h after seizure induction, GluR2 immunoreactivity (IR) in CA3 was more markedly reduced compared to GluR1, but at 24 h GluR2 IR reattained control levels. More importantly, GluR2 IR was also markedly, but transiently decreased between 6 and 48 h in hippocampal CA1 neurons, but no significant cell loss was observed. These findings modify the GluR2 hypothesis in so far as only a subset of, but not all, hippocampal CA1 and CA3 pyramidal neurons may die due to reduced GluR2 levels with consecutive calcium overload through calcium-permeable AMPA receptors. ABP was induced postictally in presumed CA2 and a subpopulation of CA3 neurons and seems not to be involved in mechanisms of delayed neuronal death. [ABSTRACT FROM AUTHOR]

Abstract: Ischemic preconditioning has been established as a powerful experimental neuroprotective strategy, both after global and focal cerebral ischemia. Little is known, however, about the structural and functional long-term outcome. Therefore, our present study was designed to check for potential subtle alterations in the hippocampus after long-term survival. Gerbils were subjected either to short-term ischemia of 2.5 min duration usually used for ischemic preconditioning (n =8) or to sham operation (n =6) and allowed to survive for 6 weeks. Hippocampi with neuronal densities comparable to those of sham-operated control animals were analyzed for dendritic marker proteins MAP2, MAP1B and synaptopodin, respectively. Although MAP2 immunoreactivity was widely unchanged in all hippocampal subfields, both MAP1B and synaptopodin protein expression was decreased to about 80% to 90% of sham controls. A significant reduction, however, was only seen for synaptopodin immunoreactivity in stratum oriens and pyramidale of hippocampal CA1 subfield. In conclusion, our data indicate a dissociation of neuronal survival and dendritic integrity 6 weeks after short-term ischemia usually used for ischemic preconditioning. Analysis of postischemic synaptopodin protein expression is the most sensitive method to detect subtle dendritic changes compared to the classical dendritic marker molecules MAP2 and MAP1B. [Copyright &y& Elsevier]

Objective: Systemic PaO2 oscillations occur during cyclic recruitment and derecruitment of atelectasis in acute respiratory failure and might harm brain tissue integrity.Design: Controlled animal study.Setting: University research laboratory.Subjects: Adult anesthetized pigs.Interventions: Pigs were randomized to a control group (anesthesia and extracorporeal circulation for 20 hr with constant PaO2, n = 10) or an oscillation group (anesthesia and extracorporeal circulation for 20 hr with artificial PaO2 oscillations [3 cycles min⁻¹], n = 10). Five additional animals served as native group (n = 5).Measurements and Main Results: Outcome following exposure to artificial PaO2 oscillations compared with constant PaO2 levels was measured using 1) immunohistochemistry, 2) real-time polymerase chain reaction for inflammatory markers, 3) receptor autoradiography, and 4) transcriptome analysis in the hippocampus. Our study shows that PaO2 oscillations are transmitted to brain tissue as detected by novel ultrarapid oxygen sensing technology. PaO2 oscillations cause significant decrease in NISSL-stained neurons (p < 0.05) and induce inflammation (p < 0.05) in the hippocampus and a shift of the balance of hippocampal neurotransmitter receptor densities toward inhibition (p < 0.05). A pathway analysis suggests that cerebral immune and acute-phase response may play a role in mediating PaO2 oscillation-induced brain injury.Conclusions: Artificial PaO2 oscillations cause mild brain injury mediated by inflammatory pathways. Although artificial PaO2 oscillations and endogenous PaO2 oscillations in lung-diseased patients have different origins, it is likely that they share the same noxious effect on the brain. Therefore, PaO2 oscillations might represent a newly detected pathway potentially contributing to the crosstalk between acute lung and remote brain injury. [ABSTRACT FROM AUTHOR]

Abstract: Stroke outcome is determined by delayed neuronal cell death and edema formation. TWEAK, a cytokine of the TNF superfamily, and its membrane receptor Fn14 promote ischemia-induced neuronal apoptosis and leakage of the blood-brain barrier. Both TWEAK and Fn14 are upregulated in experimental stroke models. In this study, we investigated whether TWEAK and Fn14 are upregulated in stroke patients. We measured serum concentrations of TWEAK in stroke patients and matched control subjects by ELISA. Expression of Fn14 in the brain was evaluated by real-time RT-PCR and immunohistochemistry. TWEAK serum concentrations were elevated in stroke patients. In autopsy samples, we found elevated mRNA levels of the receptor Fn14 and a trend towards higher TWEAK mRNA levels. In the infarcted and peri-infarct tissue immunostaining for Fn14 was enhanced. These data show that the cytokine TWEAK and its membrane receptor Fn14 are upregulated in stroke and suggest that they contribute to stroke outcome. [Copyright &y& Elsevier]

Abstract: KCNQ-type potassium channels generate the so-called M-current regulating excitability in many neurons. Mutations in KCNQ2/KCNQ3 channels can cause benign familial neonatal convulsions (BFNC). We describe the immunohistochemical staining of adult and developing mouse brain using an antibody directed against the N-terminus of KCNQ3 channels (KCNQ3N). A widespread KCNQ3N immunoreactivity predominantly of neuropil but also of somata was detected in different regions of the adult mouse brain, in particular in the hippocampus, cortex, thalamus and cerebellum. This staining pattern appeared gradually and became more intense during development. In the pyramidal cell layer of the hippocampus, the immunoreactivity changed from a more somatic to a neuropil staining during development. These changes during maturation might be related to the age-dependent phenotype of BFNC. [Copyright &y& Elsevier]