Search

The immunoproteasome is a central protease complex required for optimal antigen presentation. Immunoproteasome activity is also associated with facilitating degradation of misfolded and oxidized proteins, which prevents cellular stress. While extensively studied during diseases with increasing evidence suggesting a role for the immunoproteasome during pathological conditions including neurodegenerative diseases, this enzyme complex is believed to be mainly inactive in the healthy brain. Here, we show an age-dependent increase in polyubiquitination in the brain of wild-type mice, accompanied with induction of immunoproteasomes, which was most prominent in neurons and microglia. In contrast, mice completely lacking immunoproteasomes (triple-knockout (TKO) mice deficient for LMP2, LMP7 and MECL-1), displayed a strong increase in polyubiquitinated proteins already in the young brain and developed spontaneous epileptic seizures, beginning at the age of 6 months. Injections of kainic acid led to high epilepsy-related mortality of aged TKO mice, confirming increased pathological hyperexcitability states. Notably, the expression of the immunoproteasome was reduced in the brains of patients suffering from epilepsy. In addition, aged TKO mice showed increased anxiety, tau hyperphosphorylation and degeneration of Purkinje cell population with the resulting ataxic symptoms and locomotion alterations. Collectively, our study suggests a critical role for the immunoproteasome in the maintenance of a healthy brain during aging.

Background Antigen-specific neuroinflammation and neurodegeneration are characteristic for neuroimmunological diseases. In Parkinson’s disease (PD) pathogenesis, α-synuclein is a known culprit. Evidence for α-synuclein-specific T cell responses was recently obtained in PD. Still, a causative link between these α-synuclein responses and dopaminergic neurodegeneration had been lacking. We thus addressed the functional relevance of α-synuclein-specific immune responses in PD in a mouse model. Methods We utilized a mouse model of PD in which an Adeno-associated Vector 1/2 serotype (AAV1/2) expressing human mutated A53T-α-Synuclein was stereotactically injected into the substantia nigra (SN) of either wildtype C57BL/6 or Recombination-activating gene 1 (RAG1)\(^{-/-}\) mice. Brain, spleen, and lymph node tissues from different time points following injection were then analyzed via FACS, cytokine bead assay, immunohistochemistry and RNA-sequencing to determine the role of T cells and inflammation in this model. Bone marrow transfer from either CD4\(^{+}\)/CD8\(^{-}\), CD4\(^{-}\)/CD8\(^{+}\), or CD4\(^{+}\)/CD8\(^{+}\) (JHD\(^{-/-}\)) mice into the RAG-1\(^{-/-}\) mice was also employed. In addition to the in vivo studies, a newly developed A53T-α-synuclein-expressing neuronal cell culture/immune cell assay was utilized. Results AAV-based overexpression of pathogenic human A53T-α-synuclein in dopaminergic neurons of the SN stimulated T cell infiltration. RNA-sequencing of immune cells from PD mouse brains confirmed a pro-inflammatory gene profile. T cell responses were directed against A53T-α-synuclein-peptides in the vicinity of position 53 (68–78) and surrounding the pathogenically relevant S129 (120–134). T cells were required for α-synuclein-induced neurodegeneration in vivo and in vitro, while B cell deficiency did not protect from dopaminergic neurodegeneration. Conclusions Using T cell and/or B cell deficient mice and a newly developed A53T-α-synuclein-expressing neuronal cell culture/immune cell assay, we confirmed in vivo and in vitro that pathogenic α-synuclein peptide-specific T cell responses can cause dopaminergic neurodegeneration and thereby contribute to PD-like pathology.

The composition of the cell culture environment profoundly affects cultured cells. Standard cell culture equipment such as plastic and glass provide extremely stiff surfaces compared to physiological cell environments (i.e., tissue). A growing body of evidence documents the artificial behavior and morphology of cells cultured on supraphysiologically stiff surfaces, such as glass (elastic modulus ca. 70,000 MPA) or plastic (e.g., polystyrol ca. 3300 MPA). Therefore, polymer-based hydrogels are increasingly employed as more physiologically appropriate (100 kPA) supports for 2D or 3D culture. Since multiple properties that influence the cultured cells may be easily adjusted, hydrogels have become versatile tools for studying cells in a more native in vitro environment. Polyacrylamide-based hydrogels can be used as culture substrates for a broad variety of adherent cells and are easy to handle in most downstream biological assays, such as immunohistochemistry or molecular biology methods. We faced, however, serious difficulties with processing high stiffness polyacrylamide-based hydrogels for electron microscopy. To overcome this problem, we developed a simple protocol for embedding and processing cells grown on high stiffness polyacrylamide hydrogels that do not require modifications of routine embedding protocols. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Embedding of polyacrylamide-based hydrogels for transmission electron microscopy Alternate Protocol 1: Procedure for detached hydrogels Alternate Protocol 2: Procedure for attached hydrogels.

IDH2 R172 mutations occur in sinonasal undifferentiated carcinoma (SNUC), large-cell neuroendocrine carcinoma (LCNEC), sinonasal adenocarcinomas, and olfactory neuroblastoma (ONB). We performed a clinical, pathologic, and genetic/epigenetic analysis of a large IDH2-mutated sinonasal tumor cohort to explore their distinct features. A total 165 sinonasal/skull base tumors included 40 IDH2 mutants studied by light microscopy, immunohistochemistry, and genome-wide DNA methylation, and 125 IDH2 wild-type tumors used for comparison. Methylation profiles were analyzed by unsupervised hierarchical clustering, t-distributed stochastic neighbor embedding dimensionality reduction and assessed for copy number alterations (CNA). Thirty-nine histologically assessable cases included 25 (64.1%) SNUC, 8 (20.5%) LCNEC, 2 (5.1%) poorly differentiated adenocarcinomas, 1 (2.7%) ONB, and 3 (7.7%) IDH2-mutated tumors with ONB features. All cases were high-grade showing necrosis (82.4%), prominent nucleoli (88.9%), and median 21 mitoses/10 HPFs. AE1/AE3 and/or CAM 5.2 were positive in all and insulinoma-associated protein 1 (INSM1) in 80% cases. All IDH2 mutants formed one distinct group by t-distributed stochastic neighbor embedding dimensionality reduction separating from all IDH2 wild-type tumors. There was no correlation between methylation clusters and histopathologic diagnoses. Recurrent CNA included 1q gain (79.3%), 17p loss (75.9%), and 17q gain (58.6%). No CNA differences were observed between SNUC and LCNEC. IDH2 mutants showed better disease-specific survival than SMARCB1-deficient (P=0.027) and IDH2 wild-type carcinomas overall (P=0.042). IDH2-mutated sinonasal tumors are remarkably homogeneous at the molecular level and distinct from IDH2 wild-type sinonasal malignancies. Biology of IDH2-mutated sinonasal tumors might be primarily defined by their unique molecular fingerprint rather than by their respective histopathologic diagnoses.

The processes leading from disturbed B cell development to adult B cell progenitor acute lymphoblastic leukemia (BCP-ALL) are poorly understood. Here, we describe Irf4−/− mice as prone to developing BCP-ALL with age. Irf4−/− preB-I cells exhibited impaired differentiation but enhanced proliferation in response to IL-7, along with reduced retention in the IL-7 providing bone marrow niche due to decreased CXCL12 responsiveness. Thus selected, preB-I cells acquired Jak3 mutations, probably following irregular AID activity, resulting in malignant transformation. We demonstrate heightened IL-7 sensitivity due to Jak3 mutants, devise a model to explain it and describe structural and functional similarities to Jak2 mutations often occurring in human Ph-like ALL. Finally, targeting JAK signaling with Ruxolitinib in vivo prolonged survival of mice bearing established Irf4−/− leukemia. Intriguingly, organ infiltration including leukemic meningeosis was selectively reduced without affecting blood blast counts. In this work, we present spontaneous leukemogenesis following IRF4 deficiency with potential implications for high-risk BCP-ALL in adult humans.

Glioblastoma multiforme (GBM) is the most aggressive type of brain cancer with a median survival of only 15 months. To complement standard treatments including surgery, radiation and chemotherapy, it is essential to understand the contribution of the GBM tumor microenvironment. Brain macrophages and microglia particularly contribute to tumor angiogenesis, a major hallmark of GBM. ADAM8, a metalloprotease-disintegrin strongly expressed in tumor cells and associated immune cells of GBMs, is related to angiogenesis and correlates with poor clinical prognosis. However, the specific contribution of ADAM8 to GBM tumorigenesis remains elusive. Knockdown of ADAM8 in U87 glioma cells led to significantly decreased angiogenesis and tumor volumes of these cells after stereotactic injection into striate body of mice. We found that the angiogenic potential of ADAM8 in GBM cells and in primary macrophages is mediated by the regulation of osteopontin (OPN), an important inducer of tumor angiogenesis. By in vitro cell signaling analyses, we demonstrate that ADAM8 regulates OPN via JAK/STAT3 pathway in U87 cells and in primary macrophages. As ADAM8 is a dispensable protease for physiological homeostasis, we conclude that ADAM8 could be a tractable target to modulate angiogenesis in GBM with minor side-effects.

About 95% of Glioblastoma (GBM) patients experience tumor relapse as a consequence of resistance to the first-line standard chemotherapy using temozolomide (TMZ). Recent studies reported consistently elevated expression levels of carbonic anhydrase CA2 in recurrent glioblastoma and temozolomide-resistant glioblastoma stem-like cells (GSCs). Here we show that CA2 is preferentially expressed in GSCs and upregulated by TMZ treatment. When expressed in GBM cell lines, CA2 exerts significant metabolic changes reflected by enhanced oxygen consumption and increased extracellular acidification causing higher rates of cell invasion. Notably, GBM cells expressing CA2 respond to combined treatment with TMZ and brinzolamide (BRZ), a non-toxic and potent CA2 inhibitor. Interestingly, brinzolamide was more effective than the pan-CA inhibitor Acetazolamide (ACZ) to sensitize naïve GSCs and TMZ-resistant GSCs to TMZ induced cell death. Mechanistically, we demonstrated that the combined treatment of GBM stem cells with TMZ and BRZ caused autophagy of GBM cell lines and GSCs, reflected by enhanced LC3 cleavage (LC3-II) and p62 reduction. Our findings illustrate the potential of CA2 as a chemo-sensitizing drug target in recurrent GBM and propose a combined treatment of TMZ with CA2 inhibitor to tackle GBM chemoresistance and recurrence.

Increased stiffness of solid tissues has long been recognized as a diagnostic feature of several pathologies, most notably malignant diseases. In fact, it is now well established that elevated tissue rigidity enhances disease progression and aggressiveness and is associated with a poor prognosis in patients as documented, for instance, for lung fibrosis or the highly desmoplastic cancer of the pancreas. The underlying mechanisms of the interplay between physical properties and cellular behavior are, however, not very well understood. Here, we have found that switching culture conditions from soft to stiff substrates is sufficient to evoke (macro) autophagy in various fibroblast types. Mechanistically, this is brought about by stiffness-sensing through an Integrin αV–focal adhesion kinase module resulting in sequestration and posttranslational stabilization of the metabolic master regulator AMPKα at focal adhesions, leading to the subsequent induction of autophagy. Importantly, stiffness-induced autophagy in stromal cells such as fibroblasts and stellate cells critically supports growth of adjacent cancer cells in vitro and in vivo. This process is Integrin αV dependent, opening possibilities for targeting tumor-stroma crosstalk. Our data thus reveal that the mere change in mechanical tissue properties is sufficient to metabolically reprogram stromal cell populations, generating a tumor-supportive metabolic niche.

Cerebral cavernous malformations are slow-flow thrombi-containing vessels induced by two-step inactivation of the CCM1, CCM2 or CCM3 gene within endothelial cells. They predispose to intracerebral bleedings and focal neurological deficits. Our understanding of the cellular and molecular mechanisms that trigger endothelial dysfunction in cavernous malformations is still incomplete. To model both, hereditary and sporadic CCM disease, blood outgrowth endothelial cells (BOECs) with a heterozygous CCM1 germline mutation and immortalized wild-type human umbilical vein endothelial cells were subjected to CRISPR/Cas9-mediated CCM1 gene disruption. CCM1−/− BOECs demonstrated alterations in cell morphology, actin cytoskeleton dynamics, tube formation, and expression of the transcription factors KLF2 and KLF4. Furthermore, high VWF immunoreactivity was observed in CCM1−/− BOECs, in immortalized umbilical vein endothelial cells upon CRISPR/Cas9-induced inactivation of either CCM1, CCM2 or CCM3 as well as in CCM tissue samples of familial cases. Observer-independent high-content imaging revealed a striking reduction of perinuclear Weibel-Palade bodies in unstimulated CCM1−/− BOECs which was observed in CCM1+/− BOECs only after stimulation with PMA or histamine. Our results demonstrate that CRISPR/Cas9 genome editing is a powerful tool to model different aspects of CCM disease in vitro and that CCM1 inactivation induces high-level expression of VWF and redistribution of Weibel-Palade bodies within endothelial cells.

Neuroblastoma (NB), a pediatric cancer of the peripheral sympathetic nervous system, represents the most frequent solid malignancy in infants. Treatment of high-risk patients is still challenging and, depending on the genetic make-up and involved risk factors, the 5-year survival rate can drop to only 30%. Here, we found that the expression of the Dual Specificity Tyrosine Phosphorylation Regulated Kinase 3 (DYRK3) is increased in NB and is associated with decreased survival in NB patients. We further identified DYRK3 as a cytoplasmic kinase in NB cells and found that its levels are increased by hypoxic conditions. Further mechanistic studies revealed that DYRK3 acts as a negative regulator of HIF-driven transcriptional responses, suggesting that it functions in a negative feedback loop controlling the hypoxic response. Moreover, DYRK3 negatively impacted on NB cell differentiation, proposing an oncogenic role of this kinase in the etiology of NB. In summary, we describe novel functions of the DYRK3 kinase in NB, which will help to further improve the understanding of this disease eventually leading to the design of improved therapeutic concepts.

Significance Displaced myofibrils termed ring fibers have been reported for human congenital myopathies and for myopathies of unknown etiology, but their natural cause largely remained elusive. Cyclase-associated protein 2 (CAP2) is an actin regulator with unknown physiological function. We identified CAP2 as a regulator for the exchange of α-actin isoforms during myofibril differentiation. In CAP2 mutant mice, myofibrils remained in an undifferentiated stage at the onset of excessive voluntary muscle contractions, predisposing them to form ring fibers that compromise skeletal muscle function. Our data confirm that subtle cytoskeletal changes can cause ring fibers. Moreover, they led us to propose that CAP2 inactivation contributes to defects of human 6p22.3 deletion syndrome such as developmental delay and hypotonia., Actin filaments (F-actin) are key components of sarcomeres, the basic contractile units of skeletal muscle myofibrils. A crucial step during myofibril differentiation is the sequential exchange of α-actin isoforms from smooth muscle (α-SMA) and cardiac (α-CAA) to skeletal muscle α-actin (α-SKA) that, in mice, occurs during early postnatal life. This “α-actin switch” requires the coordinated activity of actin regulators because it is vital that sarcomere structure and function are maintained during differentiation. The molecular machinery that controls the α-actin switch, however, remains enigmatic. Cyclase-associated proteins (CAP) are a family of actin regulators with largely unknown physiological functions. We here report a function for CAP2 in regulating the α-actin exchange during myofibril differentiation. This α-actin switch was delayed in systemic CAP2 mutant mice, and myofibrils remained in an undifferentiated stage at the onset of the often excessive voluntary movements in postnatal mice. The delay in the α-actin switch coincided with the onset of motor function deficits and histopathological changes including a high frequency of type IIB ring fibers. Our data suggest that subtle disturbances of postnatal F-actin remodeling are sufficient for predisposing muscle fibers to form ring fibers. Cofilin2, a putative CAP2 interaction partner, has been recently implicated in myofibril actin cytoskeleton differentiation, and the myopathies in cofilin2 and CAP2 mutant mice showed striking similarities. We therefore propose a model in which CAP2 and cofilin2 cooperate in actin regulation during myofibril differentiation.

The impact of food antigens on intestinal homeostasis and immune function is poorly understood. Here, we explored the impact of dietary antigens on the phenotype and fate of intestinal T cells. Physiological uptake of dietary proteins generated a highly activated CD44(+)Helios(+)CD4(+) T cell population predominantly in Peyer patches. These cells are distinct from regulatory T cells and develop independently of the microbiota. Alimentation with a protein-free, elemental diet led to an atrophic small intestine with low numbers of activated T cells, including Tfh cells and decreased amounts of intestinal IgA and IL-10. Food-activated CD44(+)Helios(+)CD4(+) T cells in the Peyer patches are controlled by the immune checkpoint molecule PD-1. Blocking the PD-1 pathway rescued these T cells from apoptosis and triggered proinflammatory cytokine production, which in IL-10–deficient mice was associated with intestinal inflammation. In support of these findings, our study of patients with Crohn’s disease revealed significantly reduced frequencies of apoptotic CD4(+) T cells in Peyer patches as compared with healthy controls. These results suggest that apoptosis of diet-activated T cells is a hallmark of the healthy intestine.

How disturbances of B cell development provoke adult B acute lymphoblastic leukemia (B-ALL) remains poorly understood. Here we describe Irf4–/– mice as prone to developing B-ALL with age. Irf4–/– pro/preB cells exhibited impaired differentiative but enhanced proliferative potential in vitro and accumulated in spleens of healthy Irf4–/– mice, suggesting reduced adherence to the IL-7 providing bone marrow niche. Thus selected, pro/preB cells transformed acquiring proliferative IL-7 independency through Jak3 gain-of-function mutations. Targeting JAK signaling with Ruxolitinib in vivo prolonged survival of mice bearing established Irf4–/– leukemia. Intriguingly, organ infiltration including leukemic meningeosis was selectively reduced without affecting blood blast counts. As low IRF4 expression and JAK3 mutations also characterize a subpopulation of Ph-like B-ALL in adult humans, our results imply Irf4–/– mice as a suitable model for investigating preleukemic conditions in adults. Using this model, we identified an unexpected effect of Ruxolitinib treatment in B-ALL.

Characterization of brain tumours requires neuropathological expertise and is generally performed by histological evaluation and molecular analysis. One emerging technique to assist pathologists in future tumour diagnostics is multimodal optical spectroscopy. In the current clinical routine, tissue preprocessing with formalin is widely established and suitable for spectroscopic investigations since degradation processes impede the measurement of native tissue. However, formalin fixation results in alterations of the tissue chemistry and morphology for example by protein cross-linking. As optical spectroscopy is sensitive to these variations, we evaluate the effects of formalin fixation on multimodal brain tumour data in this proof-of-concept study. Nonfixed and formalin-fixed cross sections of different common human brain tumours were subjected to analysis of chemical variations using ultraviolet and Fourier-transform infrared microspectroscopy. Morphological changes were assessed by elastic light scattering microspectroscopy in the visible wavelength range. Data were analysed with multivariate data analysis and compared with histopathology. Tissue type classifications deduced by optical spectroscopy are highly comparable and independent from the preparation and the fixation protocol. However, formalin fixation leads to slightly better classification models due to improved stability of the tissue. As a consequence, spectroscopic methods represent an appropriate additional contrast for chemical and morphological information in neuropathological diagnosis and should be investigated to a greater extent. Furthermore, they can be included in the clinical workflow even after formalin fixation.

Tumor-cell infiltration is a major obstacle to successful therapy for brain tumors. Membrane-type matrix metalloproteinases (MT-MMPs), a metzincin subfamily of six proteases, are important mediators of infiltration. The cellular source of MT-MMPs and their role in glioma biology, however, remain controversial. Thus, we comprehensively analyzed the expression of MT-MMPs in primary brain tumors. All MT-MMPs were differentially expressed in primary brain tumors. In diffuse gliomas, MT-MMP1, -3, and -4 were predominantly expressed by IDH1mutated tumor cells, while macrophages/microglia contributed significantly less to MT-MMP expression. For functional analyses, individual MT-MMPs were expressed in primary mouse p53&minus, /&minus, astrocytes. Invasion and migration potential of MT-MMP-transduced astrocytes was determined via scratch, matrigel invasion, and novel organotypic porcine spinal slice migration (OPoSSM) and invasion assays. Overall, MT-MMP-transduced astrocytes showed enhanced migration compared to controls. MMP14 was the strongest mediator of migration in scratch assays. However, in the OPoSSM assays, the glycosylphosphatidylinositol (GPI)-anchored MT-MMPs MMP17 and MMP25, not MMP14, mediated the highest infiltration rates of astrocytes. Our data unequivocally demonstrate for the first time that glioma cells, not microglia, are the predominant producers of MT-MMPs in glioma and can act as potent mediators of tumor-cell infiltration into CNS tissue. These proteases are therefore promising targets for therapeutic interventions.

Simple Summary Most primary brain tumors infiltrate the surrounding brain even before the time of diagnosis and therefore cannot be removed completely. Matrix metalloproteases can degrade the extracellular proteins of the brain and thereby allow for the brain infiltration of glioma cells. Here, we demonstrate that tumor cells are the major source of several metalloproteases and as such responsible for the malignant behavior of gliomas. Our findings suggest that, controlling metalloproteases might be a promising therapeutic avenue in the treatment of glioma. Abstract Tumor-cell infiltration is a major obstacle to successful therapy for brain tumors. Membrane-type matrix metalloproteinases (MT-MMPs), a metzincin subfamily of six proteases, are important mediators of infiltration. The cellular source of MT-MMPs and their role in glioma biology, however, remain controversial. Thus, we comprehensively analyzed the expression of MT-MMPs in primary brain tumors. All MT-MMPs were differentially expressed in primary brain tumors. In diffuse gliomas, MT-MMP1, -3, and -4 were predominantly expressed by IDH1mutated tumor cells, while macrophages/microglia contributed significantly less to MT-MMP expression. For functional analyses, individual MT-MMPs were expressed in primary mouse p53−/− astrocytes. Invasion and migration potential of MT-MMP-transduced astrocytes was determined via scratch, matrigel invasion, and novel organotypic porcine spinal slice migration (OPoSSM) and invasion assays. Overall, MT-MMP-transduced astrocytes showed enhanced migration compared to controls. MMP14 was the strongest mediator of migration in scratch assays. However, in the OPoSSM assays, the glycosylphosphatidylinositol (GPI)-anchored MT-MMPs MMP17 and MMP25, not MMP14, mediated the highest infiltration rates of astrocytes. Our data unequivocally demonstrate for the first time that glioma cells, not microglia, are the predominant producers of MT-MMPs in glioma and can act as potent mediators of tumor-cell infiltration into CNS tissue. These proteases are therefore promising targets for therapeutic interventions.

Metastatic breast cancer affects long-term survival and is a major cause of cancer death for women worldwide. The Metalloprotease-Disintegrin ADAM8 promotes breast cancer development and brain metastasis in a mouse breast cancer model. Here, abundant ADAM8 expression was detected in primary human breast tumors and associated brain metastases. To investigate the function of ADAM8 in metastasis, MB-231 breast cancer cells with ADAM8 knockdown (MB-231_shA8) and scramble control cells (MB-231_shCtrl) were analyzed for their capability to develop metastases. In vitro, formation of metastatic complexes in hanging drops is dependent on ADAM8 and blocked by ADAM8 inhibition. MB-231_shA8 in contrast to MB-231_shCtrl cells were impaired in transmigration through an endothelial and a reconstituted blood-brain barrier. Out of 23 MMP and 22 ADAM genes, only the MMP-9 gene was affected by ADAM8 knockdown in MB-231_shA8 cells. Following re-expression of wild-type ADAM8 in contrast to ADAM8 lacking the cytoplasmic domain in MB-231_shA8 cells caused increased levels of activated pERK1/2 and pCREB (S133) that were associated with elevated MMP-9 transcription. Application of ADAM8 and MMP-9 antibodies reduced transmigration of MB-231 cells suggesting that ADAM8 affects transmigration of breast cancer cells by MMP-9 regulation. ADAM8-dependent transmigration was confirmed in Hs578t cells overexpressing ADAM8. Moreover, transmigration of MB-231 and Hs578t cells was significantly reduced for cells treated with an antibody directed against P-selectin glycoprotein ligand (PSGL-1), a substrate of ADAM8. From these data we conclude that ADAM8 promotes early metastatic processes such as transendothelial migration by upregulation of MMP-9 and shedding of PSGL-1 from breast cancer cells.

Purpose Epilepsy surgery is an evidence-based treatment for drug-refractory focal epilepsy. We aimed to evaluate how well preoperative outcome estimates of epilepsy surgery in clinical practice correlated with postoperative outcome and to compare prediction by the clinical team with available scores (m-SFS, ESN). Method Retrospective cohort study including patients with drug-refractory focal epilepsy who underwent resective epilepsy surgery at Epilepsy Center Hessen, Marburg, between 1998-2016. Patients were categorized into four groups based on their estimated chance of postoperative seizure freedom documented in preoperative medical records. Variables required for calculation of m-SFS and ESN were also extracted from presurgical medical records. Seizure outcome using Engel/ILAE classifications was extracted from postoperative medical records. Results 148 patients were included and 98 had follow-up at 5 years. 69 (70%) had Engel I and 50 (51%) ILAE 1 outcome. Observed 5-year outcome for very good candidates was 20/22 (91%) Engel I and 14/22 (64%) ILAE 1, for good candidates 29/40 (73%) Engel I and 21/40 (53%) ILAE 1, for candidates with slightly reduced chance 11/18 (61%) Engel I and 9/18 (50%) ILAE 1 and for candidates with considerably reduced chance 1/5 (20%) Engel I and 1/5 (20%) ILAE 1.There were no significant differences in discrimination or overall performance between predictions by the clinical team, ESN and m-SFS. Conclusions Preoperative outcome estimates corresponded well with observed outcome indicating adequate patient counseling.

About 95% of patients with Glioblastoma (GBM) show tumor relapse, leaving them with limited therapeutic options as recurrent tumors are most often resistant to the first line chemotherapy standard Temozolomide (TMZ). To identify molecular pathways involved in TMZ resistance, primary GBM Stem-like Cells (GSCs) were isolated, characterized, and selected for TMZ resistance in vitro. Subsequently, RNA sequencing analysis was performed and revealed a total of 49 differentially expressed genes (|log2-fold change| > 0.5 and adjusted p-value < 0.1) in TMZ resistant stem-like cells compared to their matched DMSO control cells. Among up-regulated genes, we identified carbonic anhydrase 2 (CA2) as a candidate gene correlated with glioma malignancy and patient survival. Notably, we describe consistent up-regulation of CA2 not only in TMZ resistant GSCs on mRNA and protein level, but also in patient-matched clinical samples of first manifest and recurrent tumors. Co-treatment with the carbonic anhydrase inhibitor Acetazolamid (ACZ) sensitized cells to TMZ induced cell death. Cumulatively, our findings illustrate the potential of CA2 as a chemosensitizing target in recurrent GBM and provide a rationale for a therapy associated inhibition of CA2 to overcome TMZ induced chemoresistance.

About 95% of patients with Glioblastoma (GBM) show tumor relapse, leaving them with limited therapeutic options as recurrent tumors are most often resistant to the first line chemotherapy standard Temozolomide (TMZ). To identify molecular pathways involved in TMZ resistance, primary GBM Stem-like Cells (GSCs) were isolated, characterized, and selected for TMZ resistance in vitro. Subsequently, RNA sequencing analysis was performed and revealed a total of 49 differentially expressed genes (|log2-fold change| > 0.5 and adjusted p-value < 0.1) in TMZ resistant stem-like cells compared to their matched DMSO control cells. Among up-regulated genes, we identified carbonic anhydrase 2 (CA2) as a candidate gene correlated with glioma malignancy and patient survival. Notably, we describe consistent up-regulation of CA2 not only in TMZ resistant GSCs on mRNA and protein level, but also in patient-matched clinical samples of first manifest and recurrent tumors. Co-treatment with the carbonic anhydrase inhibitor Acetazolamid (ACZ) sensitized cells to TMZ induced cell death. Cumulatively, our findings illustrate the potential of CA2 as a chemosensitizing target in recurrent GBM and provide a rationale for a therapy associated inhibition of CA2 to overcome TMZ induced chemoresistance.

BackgroundCerebral cavernous malformations (CCMs) can cause severe neurological morbidity but our understanding of the mechanisms that drive CCM formation and growth is still incomplete. Recent experimental data suggest that dysfunctional CCM3-deficient endothelial cell clones form cavernous lesions in conjunction with normal endothelial cells.ObjectiveIn this study, we addressed the question whether endothelial cell mosaicism can be found in human cavernous tissue ofCCM1germline mutation carriers.Methods and resultsBringing together single-molecule molecular inversion probes in an ultra-sensitive sequencing approach with immunostaining to visualise the lack of CCM1 protein at single cell resolution, we identified a novel late postzygoticCCM1loss-of-function variant in the cavernous tissue of a de novoCCM1germline mutation carrier. The extended unilateral CCM had been located in the right central sulcus causing progressive proximal paresis of the left arm at the age of 15 years. Immunohistochemical analyses revealed that individual caverns are lined by both heterozygous (CCM1+/−) and compound heterozygous (CCM1−/−) endothelial cells.ConclusionWe here demonstrate endothelial cell mosaicism within single caverns of human CCM tissue. In line with recent in vitro data on CCM1-deficient endothelial cells, our results provide further evidence for clonal evolution in human CCM1 pathogenesis.

Epilepsie ist eine der haufigsten chronischen neurologischen Erkrankungen und stellt fur die Betroffenen und Angehorigen eine erhebliche Belastung dar. Wir berichten uber einen Patienten, bei dem trotz uber 20 Jahren dauernden therapierefraktarem Verlauf und multiplen Voreingriffen eine langjahrige Anfallsfreiheit durch einen epilepsiechirurgischen Eingriff erreicht werden konnte. Der Fallbericht verdeutlicht, dass bei therapierefraktarem Verlauf einer Epilepsie eine zeitnahe prachirurgische Evaluation erfolgen sollte, um den Verlauf der Erkrankung positiv zu beeinflussen und soziale Folgen abzumildern.

Saccharomyces cerevisiae harbors a family of GPI-anchored cell wall proteins for interaction with its environment. The flocculin Flo11, a major representative of these fungal adhesins, confers formation of different types of multicellular structures such as biofilms, flors, or filaments. To understand these environment-dependent growth phenotypes on a molecular level, we solved the crystal structure of the N-terminal Flo11A domain at 0.89-Å resolution. Besides a hydrophobic apical region, the Flo11A domain consists of a β sandwich of the fibronectin type III domain (FN3). We further show that homophilic Flo11-Flo11 interactions and heterophilic Flo11-plastic interactions solely depend on the Flo11A domain and are strongly pH dependent. These functions of Flo11A involve an apical region with its surface-exposed aromatic band, which is accompanied by acidic stretches. Together with electron microscopic reconstructions of yeast cell-cell contact sites, our data suggest that Flo11 acts as a spacer-like, pH-sensitive adhesin that resembles a membrane-tethered hydrophobin.

Familial cerebral cavernous malformations (CCMs) predispose to seizures and hemorrhagic stroke. Molecular genetic analyses of CCM1, CCM2, and CCM3 result in a mutation detection rate of up to 98%. However, only whole genome sequencing (WGS) in combination with the Manta algorithm for analyses of structural variants revealed a heterozygous 24 kB inversion including exon 1 of CCM2 in a 12-year-old boy with familial CCMs. Its breakpoints were fine-mapped, and quantitative analysis on RNA confirmed reduced CCM2 expression. Our data expand the spectrum of CCM mutations and indicate that the existence of a fourth CCM disease gene is rather unlikely.

Metastatic breast cancer affects long-term survival and is a major cause of cancer death for women worldwide. The Metalloprotease-Disintegrin ADAM8 promotes breast cancer development and brain metastasis in a mouse breast cancer model. Here, abundant ADAM8 expression was detected in primary human breast tumors and associated brain metastases. To investigate the function of ADAM8 in metastasis, MB-231 breast cancer cells with ADAM8 knockdown (MB-231_shA8) and scramble control cells (MB-231_shCtrl) were analyzed for their capability to develop metastases. In vitro, formation of metastatic complexes in hanging drops is dependent on ADAM8 and blocked by ADAM8 inhibition. MB-231_shA8 in contrast to MB-231_shCtrl cells were impaired in transmigration through an endothelial and a reconstituted blood-brain barrier. Out of 23 MMP and 22 ADAM genes, only the MMP-9 gene was affected by ADAM8 knockdown in MB-231_shA8 cells. Following re-expression of wild-type ADAM8 in contrast to ADAM8 lacking the cytoplasmic domain in MB-231_shA8 cells caused increased levels of activated pERK1/2 and pCREB (S133) that were associated with elevated MMP-9 transcription. Application of ADAM8 and MMP-9 antibodies reduced transmigration of MB-231 cells suggesting that ADAM8 affects transmigration of breast cancer cells by MMP-9 regulation. ADAM8-dependent transmigration was confirmed in Hs578t cells overexpressing ADAM8. Moreover, transmigration of MB-231 and Hs578t cells was significantly reduced for cells treated with an antibody directed against P-selectin glycoprotein ligand (PSGL-1), a substrate of ADAM8. From these data we conclude that ADAM8 promotes early metastatic processes such as transendothelial migration by upregulation of MMP-9 and shedding of PSGL-1 from breast cancer cells.

The valosin-containing protein (VCP) is involved in a plethora of cellular processes including membrane dynamics, DNA damage response, and protein quality control.1 Its essential role in humans is highlighted by diverse clinical phenotypes linked to VCP mutations: (1) inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD); (2) amyotrophic lateral sclerosis; (3) Charcot-Marie-Tooth disease type 2; and (4) hereditary spastic paraplegia (reviewed by Evangelista et al.2). Moreover, mutant VCP has been implicated in the pathogenesis of Parkinson disease (PD).3 Acknowledgment: The authors thank the patient and his family for consent to publish this study, Andreas Odparlik (University Clinic Halle/Saale) for SPECT images, and Frank Bittner for picture editing.

The aim of these two case reports is to demonstrate that a predefined, structured, multimodal clinical bed-side testing during seizures in a long-term video-EEG monitoring setting facilitates diagnosis of complex neuropsychological syndromes. To the best of our knowledge, we present the first case of conduction aphasia as the sole ictal semiology, and a patient with focal seizures producing an angular gyrus syndrome in the speech dominant hemisphere. The relevance of diagnosing ictal aphasic and angular gyrus syndromes and localizing the symptomatogenic zone is discussed. Current pathophysiological concepts are presented regarding conduction aphasia and Gerstmann's syndrome.

Poster: "ECR 2012 / C-0059 / Cancer and Inflammation: Differentiation by USPIO-Enhanced MR Imaging" by: "P. Seyfer, K. J. Klose, J. Heverhagen; Marburg/DE"

SummaryFour molecules of the tumor suppressor p53 assemble to cooperatively bind proapoptotic target genes. The structural basis for cooperativity consists of interactions between adjacent DNA binding domains. Mutations at the interaction interface that compromise cooperativity were identified in cancer patients, suggesting a requirement of cooperativity for tumor suppression. We report on an analysis of cooperativity mutant p53E177R mice. Apoptotic functions of p53 triggered by DNA damage and oncogenes were abolished in these mice, whereas functions in cell-cycle control, senescence, metabolism, and antioxidant defense were retained and were sufficient to suppress development of spontaneous T cell lymphoma. Cooperativity mutant mice are nevertheless highly cancer prone and susceptible to different oncogene-induced tumors. Our data underscore the relevance of DNA binding cooperativity for p53-dependent apoptosis and tumor suppression and highlight cooperativity mutations as a class of p53 mutations that result in a selective loss of apoptotic functions due to an altered quaternary structure of the p53 tetramer.

IL-17–producing CD8 + T (Tc17) cells are detectible in multiple sclerosis (MS) lesions; however, their contribution to the disease is unknown. To identify functions of Tc17 cells, we induced EAE, a murine model of MS, in mice lacking IFN regulatory factor 4 (IRF4). IRF4-deficient mice failed to generate Tc17 and Th17 cells and were resistant to EAE. After adoptive transfer of WT CD8 + T cells and subsequent immunization for EAE induction in these mice, the CD8 + T cells developed a Tc17 phenotype in the periphery but could not infiltrate the CNS. Similarly, transfer of small numbers of WT CD4 + T cells alone did not evoke EAE, but when transferred together with CD8 + T cells, IL-17–producing CD4 + (Th17) T cells accumulated in the CNS and mice developed severe disease. Th17 accumulation and development of EAE required IL-17A production by CD8 + T cells, suggesting that Tc17 cells are required to promote CD4 + T cell–mediated induction of EAE. Accordingly, patients with early-stage MS harbored a greater number of Tc17 cells in the cerebrospinal fluid than in peripheral blood. Our results reveal that Tc17 cells contribute to the initiation of CNS autoimmunity in mice and humans by supporting Th17 cell pathogenicity.

Malignant tumors with neuroendocrine differentiation of the nasal cavity include neuroendocrine carcinoma, small cell undifferentiated carcinoma, olfactory neuroblastoma (esthesioneuroblastoma), and sinonasal undifferentiated carcinoma (SNUC). The rarity of these tumors and overlapping clinical, histological and immunohistochemical findings make the correct diagnosis difficult. However accurate diagnosis is critical for therapy and the outcome differs significantly between these entities. Here we describe the case of a 62-year-old woman with an intranasal tumor extending into the adjacent anatomical compartments and with infiltration of the meninges. The suspected preoperative diagnosis was olfactory neuroblastoma. Histological and immunohistochemical analysis showed a lobulated tumor with neuroendocrine differentiation and we ultimately diagnosed the process as a SNUC. In this report, we discuss the differential diagnosis at hand and present histological findings found in the specimen indicating that the process originated from local glands present in the nasal mucosa.

Adaptor proteins allow temporal and spatial coordination of signalling. In this study, we show SUMOylation of the adaptor protein TANK and its interacting kinase TANK-binding kinase 1 (TBK1). Modification of TANK by the small ubiquitin-related modifier (SUMO) at the evolutionarily conserved Lys 282 is triggered by the kinase activities of IκB kinase ɛ (IKKɛ) and TBK1. Stimulation of TLR7 leads to inducible SUMOylation of TANK, which in turn weakens the interaction with IKKɛ and thus relieves the negative function of TANK on signal propagation. Reconstitution experiments show that an absence of TANK SUMOylation impairs inducible expression of distinct TLR7-dependent target genes, providing a molecular mechanism that allows the control of TANK function.