Control Brain Tissue Research Articles

SETD7-mediated monomethylation is enriched on soluble Tau in Alzheimer\u2019s disease

BackgroundHuman tauopathies including Alzheimer’s disease (AD) are characterized by alterations in the post-translational modification (PTM) pattern of Tau, which parallel the formation of insoluble Tau aggregates, neuronal dysfunction and degeneration. While PTMs on aggregated Tau have been studied in detail, much less is known about the modification patterns of soluble Tau. Furthermore, PTMs other than phosphorylation have only come into focus recently and are still understudied. Soluble Tau species are likely responsible for the spreading of pathology during disease progression and are currently being investigated as targets for immunotherapies. A better understanding of their biochemical properties is thus of high importance.MethodsWe used a mass spectrometry approach to characterize Tau PTMs on a detergent-soluble fraction of human AD and control brain tissue, which led to the discovery of novel lysine methylation events. We developed specific antibodies against Tau methylated at these sites and biochemically characterized methylated Tau species in extracts from human brain, the rTg4510 mouse model and in hiPSC-derived neurons.ResultsOur study demonstrates that methylated Tau levels increase with Tau pathology stage in human AD samples as well as in a mouse model of Tauopathy. Methylated Tau is enriched in soluble brain extracts and is not associated with hyperphosphorylated, high molecular weight Tau species. We also show that in hiPSC-derived neurons and mouse brain, methylated Tau preferentially localizes to the cell soma and nuclear fractions and is absent from neurites. Knock down and inhibitor studies supported by proteomics data led to the identification of SETD7 as a novel lysine methyltransferase for Tau. SETD7 specifically methylates Tau at K132, an event that facilitates subsequent methylation at K130.ConclusionsOur findings indicate that methylated Tau has a specific somatic and nuclear localization, suggesting that the methylation of soluble Tau species may provide a signal for their translocation to different subcellular compartments. Since the mislocalization and depletion of Tau from axons is associated with tauopathies, our findings may shed light onto this disease-associated phenomenon.

Abstract PO-084: Genetic profiling of rat gliomas and cardiac schwannomas from cell phone radiofrequency radiation exposure using a targeted next-generation sequencing gene panel

Abstract The cancer hazard associated with exposure to cell phone radiofrequency radiation (RFR) was examined using lifetime exposure in Sprague-Dawley (SD) rats at the Ramazzini Institute (RI), Italy. There were increased incidences of gliomas in the brain and schwannomas in the heart. In order to understand the translational relevance of these rat tumors for human disease, we examined the top 23 orthologous cancer genes mutated in human gliomas using a custom built a next-generation sequencing gene panel for rats based on Illumina’s TruSeq Custom Amplicon Technology. SD rat tissues (gliomas =15; cardiac schwannomas=9, interim (1 year) sacrificed non-tumor brain tissues from RFR exposed rats =30, control brain and heart control tissues from lifetime exposure=10 each, and age-matched control brain tissues from interim sacrificed rats=10) from the RI-RFR cancer bioassay were examined in this study. The deepSNV R-package with various filtering criteria and the read depth of >1000x were used to identify single-nucleotide variants (SNVs) in the rat gliomas and schwannomas. At 5% allelic frequency, there were an average of 43 SNVs per rat glioma and point mutations were detected in 9 genes (Arid1a, Cic, Tert promoter, Tp53, Atrx, Nf1, Pdgfra, Pi3kr1, and Setd2) in at least 3 or more rat gliomas based on population frequency. Five genes (Nf1, Tert promoter, Setd2, Arid1a, and Pdgfra) harbored SNVs in interim sacrificed brain tissues that were also present in RFR-exposed gliomas from lifetime exposure. Interestingly, no mutations were detected in hotspot regions of Idh1, Idh2, Egfr or Braf. In contrast to most human gliomas which harbor mutations in IDH1 and IDH2 genes, the rat gliomas seem to be Idh1 wild type with mutations in the other glioma-related genes. Primary cardiac tumors are extremely rare in humans. With this targeted NGS panel (at allelic frequency of 2.5%), there were an average of 146 SNVs per rat cardiac schwannoma and unique point mutations were detected in Cic, Egfr, Arid1a, Nf1, Setd2, Cdkn2a, Erbb2, Atrx, Pdgfra, and Notch1 in 3 or more cardiac schwannomas. A subset of the SNVs (Arid1a, Tp53, and Nf1) in rat gliomas was confirmed in human gliomas in the COSMIC database supporting the translational relevance of the rat gliomas for human disease. In addition, several SNVs from rat gliomas and cardiac schwannomas were found in various human cancers including carcinomas, hematopoietic neoplasms, melanomas and neuroendocrine tumors. In conclusion, the rat gliomas appear to share genetic alterations with a subtype of IDH1 wildtype human gliomas and rat primary cardiac schwannomas also harbor mutations in some of the queried cancer genes. Citation Format: Ramesh C. Kovi, Andrea Vornoli, Ashley Brooks, Thai Vu T. Ton, Miaofei Xu, Eva Tibaldi, Federica Gnudi, Jian-Liang Li, Robert C. Sills, John R. Bucher, Fiorella Belpoggi, Arun R. Pandiri. Genetic profiling of rat gliomas and cardiac schwannomas from cell phone radiofrequency radiation exposure using a targeted next-generation sequencing gene panel [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-084.

Nasal Extracts from Patients with Alzheimer's Disease Induce Tau Aggregates in a Cellular Model of Tau Propagation.

Background:Emerging evidence indicates that the misfolded tau protein can propagate aggregates between cells in a prion-like manner. This prion activity has been typically studied in brain extracts of patients with Alzheimer’s disease (AD), but not in the olfactory region that can be a potential biomarker in AD.Objective:To investigate the prion seeding activity of tau in nasal mucosa tissues using a cell culture model of tau propagation.Methods:Brain and nasal mucosa homogenates were added to HEK293T cells expressing three repeat or four-repeat domains of tau with the L266V, V337M (3RD*VM) and P301L and V377M mutations (4RD*LM) fused to the enhanced green fluorescence protein (EGFP) respectively. We also measured the level of phosphorylated tau (p-tau), total tau (t-tau), and p-tau/t-tau ratio and performed correlation analysis between tau prion activity and the level of tau.Results:We found that brain and nasal tissue homogenates from patients with AD significantly induced tau aggregation in HEK293T cells either expressing tau 3RD*VM-EGFP or 4RD*LM-EGFP compared with control brain and nasal tissue homogenates. The levels of p-tau and p-tau/t-tau ratio were significantly increased in the brain of patients with AD; however, no significant difference was found in nasal tissue compared with their respective control tissue homogenates.Conclusion:These results suggest that the nasal tissues contain tau seeds, similar to the brain, albeit without changes in the levels of p-tau and t-tau. Therefore, a cellular bioassay using nasal tissues would have great potential as an AD biomarker because of the usefulness of nasal tissue biopsy.

A Profound Basic Characterization of eIFs in Gliomas: Identifying eIF3I and 4H as Potential Novel Target Candidates in Glioma Therapy.

Simple SummaryGliomas are brain tumors with currently limited therapy options. Glioma growth and proliferation is regulated by the mTOR pathway together with eukaryotic initiation factors (eIFs). In this work we show a profound basic characterization of eIFs in human gliomas and demonstrate increased mRNA and protein expressions of several eIFs in gliomas compared to healthy control brain tissue. Moreover, increased eIF3I and eIF4H levels seem to have a negative influence on the survival of patients. Our work suggests eIF3I and eIF4H as potential targets for future glioma therapy.Glioblastoma (GBM) is an utterly devastating cerebral neoplasm and current therapies only marginally improve patients’ overall survival (OS). The PI3K/AKT/mTOR pathway participates in gliomagenesis through regulation of cell growth and proliferation. Since it is an upstream regulator of the rate-limiting translation initiation step of protein synthesis, controlled by eukaryotic initiation factors (eIFs), we aimed for a profound basic characterization of 17 eIFs to identify potential novel therapeutic targets for gliomas. Therefore, we retrospectively analyzed expressions of mTOR-related proteins and eIFs in human astrocytoma samples (WHO grades I–IV) and compared them to non-neoplastic cortical control brain tissue (CCBT) using immunoblot analyses and immunohistochemistry. We examined mRNA expression using qRT-PCR and additionally performed in silico analyses to observe the influence of eIFs on patients’ survival. Protein and mRNA expressions of eIF3B, eIF3I, eIF4A1, eIF4H, eIF5 and eIF6 were significantly increased in high grade gliomas compared to CCBT and partially in low grade gliomas. However, short OS was only associated with high eIF3I gene expression in low grade gliomas, but not in GBM. In GBM, high eIF4H gene expression significantly correlated with shorter patient survival. In conclusion, we identified eIF3I and eIF4H as the most promising targets for future therapy for glioma patients.

The age of adult pilocytic astrocytoma cells

Adult pilocytic astrocytomas (PAs) have been regarded as indistinguishable from pediatric PAs in terms of genome-wide expression and methylation patterns. It has been unclear whether adult PAs arise early in life and remain asymptomatic until adulthood, or whether they develop during adulthood. We sought to determine the age and origin of adult human PAs using two types of “marks” in the genomic DNA. First, we analyzed the DNA methylation patterns of adult and pediatric PAs to distinguish between PAs of different anatomic locations (n = 257 PA and control brain tissues). Second, we measured the concentration of nuclear bomb test-derived 14C in genomic DNA (n = 14 cases), which indicates the time point of the formation of human cell populations. Our data suggest that adult and pediatric PAs developing in the infratentorial brain are closely related and potentially develop from precursor cells early in life, whereas supratentorial PAs might show age and location-specific differences.

Molecular changes tracking through multiscale fluorescence microscopy differentiate Meningioma grades and non-tumoral brain tissues

Meningioma is the most common primary intracranial extra-axial tumor. Total surgical removal is the standard therapeutic method to treat this type of brain tumors. However, the risk of recurrence depends on the tumor grade and the extent of the resection including the infiltrated dura mater and, if necessary, the infiltrated bone. Therefore, proper resection of all invasive tumor borders without touching eloquent areas is of primordial in order to decrease the risk of recurrence. Nowadays, none of the intraoperative used tools is able to provide a precise real-time histopathological information on the tumor surrounding areas to help the surgeon to achieve a gross total removal. To respond to this problem, our team is developing a multimodal two-photon fluorescence endomicroscope, compatible with the surgeon tool, to better delimitate tumor boundaries, relying on the endogenous fluorescence of brain tissues. In this context, we are building a tissue database in order to specify each brain tissue, whether healthy or tumoral, with its specific optical signature. In this study, we present a multimodal and multiscale optical measurements on non-tumoral control brain tissue obtained in epilepsy surgery patients and several meningioma grades. We investigated tissue auto-fluorescence to track the molecular changes associated with the tumor grade from deep ultra-violet (DUV) to near infrared (NIR) excitation. Micro-spectroscopy, fluorescence lifetime imaging, two-photon fluorescence imaging and Second Harmonic Generation (SHG) imaging were performed. Several optically derived parameters such as collagen crosslinks fluorescence in DUV, SHG emission in NIR and long lifetime intensity fraction of Nicotinamide Adenine Dinucleotide and Flavins were correlated to discriminate cancerous tissue from control one. While collagen response managed to discriminate meningioma grades from control samples with a 100% sensitivity and 90% specificity through a 3D discriminative algorithm.

Abnormal expression of HOXD11 promotes the malignant behavior of glioma cells and leads to poor prognosis of glioma patients.

BackgroundHomeobox D11 (HOXD11) plays an important role in a variety of cancers, but its precise role in gliomas remains unclear. This study aimed to explore the relationship between HOXD11 and gliomas by combining bioinformatics methods with basic experimental validation.Materials and methodsObtain gene expression information and clinical information of glioma and non-tumor brain tissue samples from multiple public databases such as TCGA (666 glioma samples), CGGA (749 glioma samples), GEPIA(163 glioblastoma samples and 207 normal control samples), GEO (GSE4290 and GSE15824). Nine cases of glioma tissue and five cases of normal control brain tissue were collected from the clinical department of Henan Provincial People’s Hospital for further verification. A series of bioinformatic analysis methods were used to confirm the relationship between HOXD11 expression and overall survival and clinical molecular characteristics of patients with glioma. RT-qPCR was used to verify the change of expression level of HOXD11 in glioma cells and tissues. MTT assay, colony formation assay, wound-healing assay, immunofluorescence staining, flow cytometry and western blotting were used to detect the effect of HOXD11 on the biological behavior of glioma cell line U251.ResultsThe high expression of HOXD11 was significantly related to age, World Health Organization (WHO) grade, chemotherapy status, histological type, and even 1p19q codeletion data and isocitrate dehydrogenase (IDH) mutation. HOXD11, as an independent risk factor, reduces the overall survival of glioma patients and has diagnostic value for the prognosis of glioma. Gene Set Enrichment Analysis (GSEA) showed that HOXD11 was significantly enriched in cell signaling pathway such as cell cycle, DNA replication and so on. Finally, we confirmed that the knockout of HOXD11 can inhibit the proliferation and invasion of U251 glioma cells, and change the biological behavior of tumor cells by preventing the progression of cell cycle.ConclusionsHOXD11 may be used as a candidate biomarker for the clinical application of targeted drug and prognostic assessment treatment of glioma. In addition, This study will help to explore the pathological mechanism of glioma.

Interleukin-1 receptor-associated kinase 4 as a potential biomarker: Overexpression predicts poor prognosis in patients with glioma.

The undetectable onset of glioma and the difficulty of surgery lead to a poor prognosis. Appropriate biomarkers for diagnosis and treatment need to be identified. Interleukin-1 receptor-associated kinase 4 (IRAK4) is involved in the initiation and progression of cancer. However, up until now, no report has revealed the relationship between IRAK4 and glioma. The present study aimed to examine the expression of IRAK4 in glioma, and to determine if there was a relationship between IRAK4 expression and clinical outcomes or survival prognosis. Thousands of glioma tissue samples and corresponding clinical information were obtained from various databases. Then a series of bioinformatics methods were used to reveal the role of IRAK4 in glioma. Finally, reverse transcription-quantitative PCR technology was used to verify the bioinformatics results. The study found that the expression of IRAK4 was significantly increased in glioma compared with the control brain tissue samples, and IRAK4, as an independent prognostic factor, shortened the overall survival time of patients with glioma. Gene Set Enrichment Analysis showed that IRAK4 promoted the activation of cell signalling pathways, such as NOD-like and Toll-like receptor signalling pathways. Co-expression analysis showed that the expression of IRAK4 was correlated with CMTM6, MOB1A and other genes. The present study demonstrated the role of IRAK4 as an oncogene in the pathological process of glioma for the first time, and highlights the potential of IRAK4 as a biomarker for prognostic evaluation and treatment of glioma.

Enrichment of Phosphorylated Tau (Thr181) and Functionally Interacting Molecules in Chronic Traumatic Encephalopathy Brain-derived Extracellular Vesicles.

Chronic Traumatic Encephalopathy (CTE) is a tauopathy that affects individuals with a history of mild repetitive brain injury. The initial neuropathologic changes of CTE include perivascular deposition of phosphorylated microtubule-associated protein tau (p-tau). Extracellular vesicles (EVs) are known to carry pathogenic molecules, such as tau in Alzheimer’s disease and CTE suggesting their contribution in pathogenesis. We therefore examined the protein composition of EVs separated from CTE and an age-matched control brain tissues by tandem mass tag -mass spectrometry. The reporter ion intensity was used to quantify the identified molecules. A total of 516 common proteins were identified among three sets of experiments. Weighted protein co-expression network analysis identified 18 unique modules of co-expressed proteins. Two modules were significantly correlated with total tau (t-tau) and p-tau protein in the isolated EVs and enriched in cellular components and biological processes for synaptic vesicle secretion and multivesicular body-plasma membrane fusion. The p-tau (Thr181) level is significantly higher in CTE EVs compared to control EVs and can distinguish the two groups with 73.6% accuracy. A combination of t-tau or p-tau (Thr181) with SNAP-25, PLXNA4 or UBA1, enhanced the accuracy to 96.3, 93.8 and 93.8%, respectively. Bioinformatic protein-protein interaction analysis revealed the functional interaction of SNAP-25 and PLXNA4 with tau, suggesting their interaction in CTE EVs. These data indicate the future application of identified EV proteins for monitoring the CTE risk assessments and understanding the EV-mediated disease progression mechanism.

Imaging epileptic foci in mouse models via a low-density lipoprotein receptor-related protein-1 targeting strategy.

BackgroundIn the setting of drug-resistant epilepsy (DRE), the success of surgery depends on the ability to accurately locate the epileptic foci to be resected or disconnected. However, the epileptic foci in a considerable percentage of the DRE patients cannot be adequately localised. This warrants the need for a reliable imaging strategy to identify the “concealed” epileptic regions.MethodsBrain specimens from DRE patients and kainate-induced epileptic mouse models were immuno-stained to evaluate the integrity of the blood-brain barrier (BBB). The expression of low-density lipoprotein receptor-related protein-1 (LRP1) in the epileptic region of DRE patients and kainate models was studied by immunofluorescence. A micellar-based LRP1-targeted paramagnetic probe (Gd3+-LP) was developed and its ability to define the epileptic foci was investigated by magnetic resonance imaging (MRI).FindingsThe integrity of the BBB in the epileptic region of DRE patients and kainate mouse models were demonstrated. LRP1 expression levels in the epileptic foci of DRE patients and kainate models were 1.70–2.38 and 2.32–3.97 folds higher than in the control brain tissues, respectively. In vivo MRI demonstrated that Gd3+-LP offered 1.68 times higher (P < 0.05) T1-weighted intensity enhancement in the ipsilateral hippocampus of chronic kainite models than the control probe without LRP1 specificity.InterpretationThe expression of LRP1 is up-regulated in vascular endothelium, activated glia in both DRE patients and kainate models. LRP1-targeted imaging strategy may provide an alternative strategy to define the “concealed” epileptic foci by overcoming the intact BBB.FundingThis work was supported by the National Natural Science Foundation, Shanghai Science and Technology Committee, Shanghai Municipal Science and Technology, Shanghai Municipal Health and Family Planning Commission and the National Postdoctoral Program for Innovative Talents.

Unraveling Targetable Systemic and Cell-Type-Specific Molecular Phenotypes of Alzheimer's and Parkinson's Brains With Digital Cytometry.

Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the two most common neurodegenerative disorders worldwide, with age being their major risk factor. The increasing worldwide life expectancy, together with the scarcity of available treatment choices, makes it thus pressing to find the molecular basis of AD and PD so that the causing mechanisms can be targeted. To study these mechanisms, gene expression profiles have been compared between diseased and control brain tissues. However, this approach is limited by mRNA expression profiles derived for brain tissues highly reflecting their degeneration in cellular composition but not necessarily disease-related molecular states. We therefore propose to account for cell type composition when comparing transcriptomes of healthy and diseased brain samples, so that the loss of neurons can be decoupled from pathology-associated molecular effects. This approach allowed us to identify genes and pathways putatively altered systemically and in a cell-type-dependent manner in AD and PD brains. Moreover, using chemical perturbagen data, we computationally identified candidate small molecules for specifically targeting the profiled AD/PD-associated molecular alterations. Our approach therefore not only brings new insights into the disease-specific and common molecular etiologies of AD and PD but also, in these realms, foster the discovery of more specific targets for functional and therapeutic exploration.

LGG-57. SIGNALLING MECHANISMS IN PAEDIATRIC LOW-GRADE GLIOMA

Paediatric low-grade gliomas (pLGGs) constitute the largest group of childhood CNS tumours. They often cause significant disability and morbidity, despite their indolent growth and the good survival rate of patients. The most common genetic alterations in these tumours, KIAA1549:BRAF fusion and BRAFV600E mutation, lead to abnormal activation of MAPK signalling. The central role of this pathway in pLGG development is emphasized by the occasional presence of other MAPK-activating alterations such as RTK mutations. It is not known how these different aberrations can induce the variety of clinical phenotypes seen in pLGG. Here, we compared pilocytic astrocytomas (PAs) containing the KIAA1549:BRAF fusion with glioneuronal tumours (GNTs) containing the BRAFV600E mutation, to identify differentially activated downstream targets of the MAPK pathway. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used as a multi-proteomic approach. Kinase Set Enrichment Analysis (KSEA) using PhosphositePlus and NetworkIN was used to determine relative enrichment of kinase activity in the tumours compared to healthy control brain tissue. Significant similarities and differences were found in the two tumour types. For example, more robust MAPK activation was found in the GNTs than in PAs. However, while PI3K/AKT1/mTOR signalling was active in both PAs and GNTs, there was statistically higher activation in the PAs. In both tumour types, there was significant reduction in casein kinase 2 activity, which likely affects nuclear translocation of ERK and, in turn, alters the range of its phosphorylated substrates. We will present these data together with transcriptomics to further characterise the downstream targets of these genetic alterations.

CXCR7, CXCR4, and Their Ligand Expression Profile in Traumatic Brain Injury

Traumatic brain injury (TBI) is a health problem worldwide, and therapeutic strategies to enhance brain tissue repair to lessen neurologic sequels are imperative. We aimed to analyze the impact of the inflammatory process in TBI through CXCR4 and CXCR7 chemokine receptors and their ligands' CXCL11 and CXCL12 expression profile in search for potential new druggable targets. Twelve pericontusional tissues from severe TBI patients submitted to surgical treatment, and 20 control brain tissues from normal autopsy were analyzed for expression profile by real-time quantitative-polymerase chain reaction. CXCR7 and CXCR4 protein expressions were analyzed by immunohistochemistry. The findings were correlated with the clinical evolution. Increased gene expression of both receptors and their ligands was observed in TBI compared with controls, presenting high sensitivity and specificity to differentiate TBI from normal control (area under the curve ranging from 0.85 to 0.98, P < 0.001). In particular, CXCR7 expression highly correlated with CXCR4 and both ligands' expressions in TBI. Higher immunoreactions for CXCR7 and CXCR4 were identified in neurons and endothelial cells of TBI samples compared with controls. The patients presenting upregulated chemokine expression levels showed a trend toward favorable clinical evolution at up to 6 months of follow-up. The neuroprotective trend of CXCR4, CXCR7, CXCL11, and CXCL12 in TBI observed in this initial analysis warrants further studies with more patients, analyzing the involved signaling pathways for the development of new therapeutic strategies for TBI.

Characterization of MK6240, a tau PET tracer, in autopsy brain tissue from Alzheimer\u2019s disease cases

PurposeMK6240 is a second-generation tau PET tracer designed to detect the neurofibrillary tangles in the brains of patients with Alzheimer’s disease (AD). The aim of the study was to characterize 3H-MK6240 in AD and control brain tissue and to compare its binding properties with those of first-generation tau PET tracers.MethodsSaturation binding assays with 3H-MK6240 were carried out in the temporal and parietal cortices of AD brains to determine the maximum number of binding sites (Bmax) and the dissociation constants (Kd) at these sites. Competitive binding assays were carried out between 3H-MK6240 and unlabelled MK6240, AV-1451 (aka T807, flortaucipir) and THK5117, and between 3H-THK5351 and unlabelled MK6240. Regional binding studies with 3H-MK6240 were carried out in homogenates from six AD and seven control brains and, using autoradiography, on large frozen sections from two AD brains and one control brain.ResultsThe saturation binding assays gave Bmax and Kd values of 59.2 fmol/mg and 0.32 nM in the temporal cortex and 154.7 fmol/mg and 0.15 nM in the parietal cortex. The competitive binding assays revealed two binding sites with affinities in the picomolar and nanomolar range shared by 3H-MK6240 and all the tested unlabelled compounds. There were no binding sites in common between 3H-THK5351 and unlabelled MK6240. Regional binding of 3H-MK6240 was significantly higher in AD brain tissue than in controls. Binding in brain tissue from AD patients with early-onset AD was significantly higher than in brain tissue from patients with late-onset AD. Binding of 3H-MK6240 was not observed in off-target regions. Autoradiography showed high regional cortical binding in the two AD brains and very low binding in the control brain.Conclusions3H-MK6240 has a high binding affinity for tau deposits in AD brain tissue but also has different binding characteristics from those of the first-generation tau tracers. This confirms the complexity of tau tracer binding on tau deposits with different binding affinities for different binding sites.

Glutamine uptake and utilization of human mesenchymal glioblastoma in orthotopic mouse model

BackgroundGlioblastoma (GBM) are highly heterogeneous on the cellular and molecular basis. It has been proposed that glutamine metabolism of primary cells established from human tumors discriminates aggressive mesenchymal GBM subtype to other subtypes.MethodsTo study glutamine metabolism in vivo, we used a human orthotopic mouse model for GBM. Tumors evolving from the implanted primary GBM cells expressing different molecular signatures were analyzed using mass spectrometry for their metabolite pools and enrichment in carbon 13 (13C) after 13C-glutamine infusion.ResultsOur results showed that mesenchymal GBM tumors displayed increased glutamine uptake and utilization compared to both control brain tissue and other GBM subtypes. Furthermore, both glutamine synthetase and transglutaminase-2 were expressed accordingly to GBM metabolic phenotypes.ConclusionThus, our results outline the specific enhanced glutamine flux in vivo of the aggressive mesenchymal GBM subtype.

Single X-ray irradiation modulates proteoglycan expression in brain tissue: investigation using mouse model.

Radiotherapy is an integral part of glioblastoma treatment affecting both cancer cells and tumour microenvironment, where proteoglycans (PGs) are key extracellular components. However, the molecular effects of radiotherapy on PGs expression and functional activity in brain tissue are poorly understood. Here, we aimed to study the short-term effects of X-ray irradiation on PGs expression in normal brain tissue in mouse model in vivo. Two-month-old male CBL/6Bl mice (n = 54) were used in this study, animals' brains were irradiated using either research synchrotron VEPP-4 or clinical linear accelerator ElektaAxesse. Control (n = 18) and irradiated (n = 36) brain tissues were analysed at 24h, 48h and 72h after irradiation. Morphology of the cortex and hippocampus was accessed by H&E staining, and expression of PGs (syndecan-1, glypican-1, HSPG2/perlecan, versican, brevican, neurocan, NG2/CSPG4, CD44, decorin, biglycan) was determined by RT-PCR. Single irradiation of mouse brain with a 7Gy dose did not affect tissue morphology and mRNA levels of most highly-expressed PGs decorin and neurocan, although resulted in significant downregulation of brevican (3-10-fold) and NG2/CSPG4 (8-9-fold) expression both in cerebral cortex and subcortex. Research synchrotron and clinical linear accelerators demonstrated minor variability in their effects. Single X-ray irradiation with a 7Gy dose does not significantly affect the mouse brain tissue morphology but selectively decreases expression levels of some PGs. The downregulation of brevican and NG2/CSPG4 but not decorin and neurocan reflects alteration of extracellular matrix in irradiated brain tissue, which might contribute to the formation of a permissive microenvironment for glioblastoma relapse development.

Brain Proteomic Profiling in Intractable Epilepsy Caused by TSC1 Truncating Mutations: A Small Sample Study.

Tuberous sclerosis complex (TSC) is a genetic disease characterized by seizures, mental deficiency, and abnormalities of the skin, brain, kidney, heart, and lungs. TSC is inherited in an autosomal dominant manner and is caused by variations in either the TSC1 or TSC2 gene. TSC-related epilepsy (TRE) is the most prevalent and challenging clinical feature of TSC, and more than half of the patients have refractory epilepsy. In clinical practice, we found several patients of intractable epilepsy caused by TSC1 truncating mutations. To study the changes of protein expression in the brain, three cases of diseased brain tissue with TSC1 truncating mutation resected in intractable epilepsy operations and three cases of control brain tissue resected in craniocerebral trauma operations were collected to perform protein spectrum detection, and then the data-independent acquisition (DIA) workflow was used to analyze differentially expressed proteins. As a result, there were 55 up- and 55 down-regulated proteins found in the damaged brain tissue with TSC1 mutation compared to the control. Further bioinformatics analysis revealed that the differentially expressed proteins were mainly concentrated in the synaptic membrane between the patients with TSC and the control. Additionally, TSC1 truncating mutations may affect the pathway of amino acid metabolism. Our study provides a new idea to explore the brain damage mechanism caused by TSC1 mutations.

M1 muscarinic acetylcholine receptor dysfunction in moderate Alzheimer's disease pathology.

Aggregation of amyloid beta and loss of cholinergic innervation in the brain are predominant components of Alzheimer’s disease pathology and likely underlie cognitive impairment. Acetylcholinesterase inhibitors are one of the few treatment options for Alzheimer’s disease, where levels of available acetylcholine are enhanced to counteract the cholinergic loss. However, these inhibitors show limited clinical efficacy. One potential explanation for this is a concomitant dysregulation of cholinergic receptors themselves as a consequence of the amyloid beta pathology. We tested this hypothesis by examining levels of M1 muscarinic acetylcholine receptors in the temporal cortex from seven Alzheimer’s disease and seven non-disease age-matched control brain tissue samples (control: 85 ± 2.63 years old, moderate Alzheimer’s disease: 84 ± 2.32 years old, P-value = 0.721; eight female and six male patients). The samples were categorized into two groups: ‘control’ (Consortium to Establish a Registry for Alzheimer’s Disease diagnosis of ‘No Alzheimer’s disease’, and Braak staging pathology of I–II) and ‘moderate Alzheimer’s disease’ (Consortium to Establish a Registry for Alzheimer’s Disease diagnosis of ‘possible/probable Alzheimer’s disease’, and Braak staging pathology of IV). We find that in comparison to age-matched controls, there is a loss of M1 muscarinic acetylcholine receptors in moderate Alzheimer’s disease tissue (control: 2.17 ± 0.27 arbitrary units, n = 7, Mod-AD: 0.83 ± 0.16 arbitrary units, n = 7, two-tailed t-test, t = 4.248, P = 0.00113). Using a functional rat cortical brain slice model, we find that postsynaptic muscarinic acetylcholine receptor function is dysregulated by aberrant amyloid beta-mediated activation of metabotropic glutamate receptor 5. Crucially, blocking metabotropic glutamate receptor 5 restores muscarinic acetylcholine receptor function and object recognition memory in 5XFAD transgenic mice. This indicates that the amyloid beta-mediated activation of metabotropic glutamate receptor 5 negatively regulates muscarinic acetylcholine receptor and illustrates the importance of muscarinic acetylcholine receptors as a potential disease-modifying target in the moderate pathological stages of Alzheimer’s disease.

Aluminum and Amyloid-β in Familial Alzheimer's Disease.

Genetic predispositions associated with metabolism of the amyloid-β protein precursor underlie familial Alzheimer's disease; a form of dementia characterized by early disease onset and elevated levels of cortical amyloid-β. Human exposure to aluminum is linked to the etiology of Alzheimer's disease and recent research measured a high content of aluminum in brain tissue in familial Alzheimer's disease. To elaborate upon this finding, we have obtained brain tissues from a Colombian cohort of donors with familial Alzheimer's disease. We have used established methods to measure the aluminum content of these tissues and we have compared the data with a recently measured dataset for control brain tissues. We report significantly higher levels of aluminum in brain tissues in donors with familial Alzheimer's disease than in control tissues from donors without neurological impairment or neurodegeneration. We have used aluminum-specific fluorescence microscopy along with complementary imaging for amyloid-β to demonstrate a very high degree of co-localization of these two risk factors in brain tissue in familial Alzheimer's disease. Aluminum and amyloid-β were co-located in senile plaques as well as vasculature, the latter resembling cerebral amyloid angiopathy. Aluminum was also found separately from amyloid-β in intracellular compartments including glia and neuronal axons. The research has identified an arguably unique association between high brain aluminum content and amyloid-β and allows postulation that genetic predispositions defining familial Alzheimer's disease underlie this relationship.

Soluble protein tyrosine phosphatase receptor type Z (PTPRZ) in cerebrospinal fluid is a potential diagnostic marker for glioma.

BackgroundHigh-grade glioma is the most pervasive and lethal of all brain malignancies. Despite advances in imaging technologies, discriminating between gliomas and other brain diseases such as multiple sclerosis (MS) often requires brain biopsy. Several reports show that protein tyrosine phosphatase receptor Z (PTPRZ) is highly expressed in glioblastoma, and we found that a soluble cleaved form of PTPRZ (sPTPRZ) was present in the cerebrospinal fluid (CSF). The aim of this study was to determine whether the sPTPRZ level in CSF has utility as a diagnostic marker for glioma.MethodsMicroarray datasets from normal brain tissue and brain tumors were obtained from the Gene Expression Omnibus. PTPRZ protein expression in clinical specimens was evaluated by immunohistochemistry. Semiquantitative western blotting was used to measure sPTPRZ levels in CSF samples from patients with glioma, schwannoma, MS, or nontumor disorders.ResultsExpression of PTPRZ mRNA and protein was markedly increased in glioblastoma, astrocytoma, oligodendroglioma, and schwannoma tissues compared with control brain tissue. sPTPRZ was present at significantly elevated levels in the CSF of patients with glioma (grades 1–4), but not in patients with schwannoma or MS, compared with the control samples. Receiver operating characteristic curve analysis showed that sPTPRZ in CSF could discriminate between glioma and MS patients (area under the curve 0.9676; P < .0001).ConclusionssPTPRZ in CSF is a promising diagnostic biomarker for glioma and could reduce the need for a surgical biopsy.