Mikroglia-Phagozytose und purinerge Signalübertragung in der Alzheimer-Krankheit

Microglia, the primary immune cells of the central nervous system, interact through multiple physiological functions with processes in the healthy as well as diseased brain parenchyma. As a cellular link between the immune system and the central nervous system, microglia monitor their environment by using their ramified processes to eventually eliminate harmful exogenous and endogenous waste, pathogens or cell debris through phagocytosis. In the neurodegenerative Alzheimer's disease, microglia change the activation status from a ramified to an amoeboid-activated phenotype and accumulate around amyloid beta plaques. Recent studies show a physiological change in the microglial functions in the Alzheimer mouse model, which manifests itself in the reduction of microglial phagocytosis. Our research presents a link between diminished microglial phagocytosis activity in an Alzheimer mouse model and disruption of purinergic microglial receptor signaling. The measurement of phagocytosis activity by particle uptake after P2Y6 purinergic receptor activation could be stimulated in young 3- to 4-month old healthy control animals as well as in 5xFAD-transgenic Alzheimer's animals, a mouse model of amyloid-beta-plaque deposition. However, this P2Y6R-mediated potentiation of phagocytic activity is reduced in plaque-associated microglia of older 9 to 11-month-old 5xFAD mice. This impairment of phagocytosis activation by the P2Y6-mediated purinergic signaling pathways is thus not congenital and only develops in later stages of amyloidosis of the 5xFAD Alzheimer mouse model. Furthermore, we show that uridine diphosphate, a P2Y6 receptor ligand, induces electrophysiological microglia membrane currents that are altered in response and amplitude in microglia in close regional association with plaques, but not in plaque-free regions of 5xFAD animals. These changes were accompanied by changes in membrane properties and potassium channel activity of plaque-associated microglia in early and late stages of amyloidosis. Thus, our research results show both a change in the phagocytic activity and the electrophysiological properties of the plaque-associated microglia of the 5xFAD Alzheimer mouse in the context of purinergic signaling.Mikroglia, die primären Immunzellen des zentralen Nervensystems, interagieren durch vielfache physiologische Funktionen mit Abläufen im gesunden als auch erkrankten Hirnparenchyma. Als zelluläres Bindestück zwischen dem Immunsystem und dem zentralen Nervensystem überwachen Mikroglia mit Hilfe von ramifizierten Fortsätzen ihre Umgebung, um gegebenenfalls schädliche exogene als auch endogene Abfallstoffe, Pathogene oder Zelltrümmer durch Phagozytose zu beseitigen. In der neurodegenerativen Alzheimer-Erkrankung verändern Mikroglia ihre Morphologie von einem ramifizierten zu einem amoeboiden Phänotyp und akkumulieren in der unmittelbaren Nähe von Amyloid-beta-Plaques. Aktuelle Studien zeigen physiologische Veränderungen der Mikroglia im Alzheimer Mausmodell auf, welche sich unter anderem in der Reduzierung ihrer Phagozytoseaktivität manifestiert. Unsere Forschungsergebnisse präsentieren einen Zusammenhang der verminderten Mikroglia-Phagozytoseaktivität im Alzheimer Mausmodell mit einer Störung der purinergen Mikroglia Rezeptor-Signalgebung. Die Messung der Phagozytoseaktivität von Plaque-assoziierten Mikroglia mittels Partikelaufnahme nach purinerger P2Y6-Rezeptor-Aktivierung konnte in jungen 3 bis 4 Monaten alten gesunden Kontrolltieren als auch in 5xFAD-transgenen Alzheimer Tieren, einem Mausmodell der Amyloid-beta-Plaque Ablagerung, stimuliert werden. Diese P2Y6R-vermittelte Potenzierung der Phagozytoseaktivität ist jedoch in Plaque-assoziierten Mikroglia von älteren 9 bis 11 Monaten alten 5xFAD Mäusen deutlich reduziert. Diese Beeinträchtigung der Phagozytoseaktivierung durch die P2Y6-vermittelten purinergen Signalwege ist also nicht angeboren und entwickelt sich erst in späteren Stadien der Amyloidose des 5xFAD Alzheimer Mausmodells. Des Weiteren führten wir elektrophysiologische Messungen durch. Uridindiphosphat (UDP), ein P2Y6-Rezeptor Ligand, der normalerweise Kalium-selektive Membran¬ströme in Mikroglia induziert, hatte hierbei sehr geringe Auswirkungen auf Mikrogliazellen in enger regionaler Assoziation zu Plaques, nicht aber in plaque-freien Bereichen von 5xFAD-Tieren. Die Membraneigenschaften von Plaque-assoziierten Mikroglia waren ebenfalls verändert, und zwar in frühen und späten Stadien der Amyloidose. Unsere Forschungsergebnisse zeigen also sowohl eine Veränderung der Phagozytoseaktivität als auch der elektrophysiologischen Eigenschaften der Plaque-assoziierten Mikroglia im 5xFAD Alzheimer Mausmodell im Kontext der purinergen Signalgebung auf

Changes in phagocytosis and potassium channel activity in microglia of 5xFAD mice indicate alterations in purinergic signaling in a mouse model of Alzheimer's disease

As the immunocompetent cells of the central nervous system, microglia accumulate at amyloid beta plaques in Alzheimer's disease (AD) and acquire a morphological phenotype of activated microglia. Recent functional studies, however, indicate that in mouse models of amyloidosis and AD, these cells are rather dysfunctional indicated by a reduced phagocytic activity. Here, we report that this reduction in phagocytic activity is associated with perturbed purinergic receptor signaling, since phagocytosis could be stimulated by P2Y6 receptor activation in control, but not in 5xFAD transgenic animals, an animal model of amyloid deposition. Impaired phagocytosis is not innate, and develops only at later stages of amyloidosis. Furthermore, we show that membrane currents induced by uridine diphosphate, a ligand activating P2Y6 receptors, are altered in response rate and amplitude in microglia in close vicinity to plaques, but not in plaque-free areas of 5xFAD animals. These changes were accompanied by changes in membrane properties and potassium channel activity of plaque-associated microglia in early and late stages of amyloidosis. As a conclusion, the physiological properties of plaque-associated microglia are altered with a strong impact on purinergic signaling

Athymic mice reveal a requirement for T-cell-microglia interactions in establishing a microenvironment supportive of Nf1 low-grade glioma growth

Pediatric low-grade gliomas (LGGs) frequently do not engraft in immunocompromised mice, limiting their use as an experimental platform. In contrast, murine Neurofibromatosis-1 (Nf1) optic LGG stem cells (o-GSCs) form glioma-like lesions in wild-type, but not athymic, mice following transplantation. Here, we show that the inability of athymic mice to support o-GSC engraftment results from impaired microglia/macrophage function, including reduced expression of Ccr2 and Ccl5, both of which are required for o-GSC engraftment and Nf1 optic glioma growth. Impaired Ccr2 and Ccl5 expression in athymic microglia/macrophages was restored by T-cell exposure, establishing T-cell-microglia/macrophage interactions as critical stromal determinants that support NF1 LGG growth

Cellular heterogeneity contributes to subtype-specific expression of ZEB1 in human glioblastoma.

The transcription factor ZEB1 has gained attention in tumor biology of epithelial cancers because of its function in epithelial-mesenchymal transition, DNA repair, stem cell biology and tumor-induced immunosuppression, but its role in gliomas with respect to invasion and prognostic value is controversial. We characterized ZEB1 expression at single cell level in 266 primary brain tumors and present a comprehensive dataset of high grade gliomas with Ki67, p53, IDH1, and EGFR immunohistochemistry, as well as EGFR FISH. ZEB1 protein expression in glioma stem cell lines was compared to their parental tumors with respect to gene expression subtypes based on RNA-seq transcriptomic profiles. ZEB1 is widely expressed in glial tumors, but in a highly variable fraction of cells. In glioblastoma, ZEB1 labeling index is higher in tumors with EGFR amplification or IDH1 mutation. Co-labeling studies showed that tumor cells and reactive astroglia, but not immune cells contribute to the ZEB1 positive population. In contrast, glioma cell lines constitutively express ZEB1 irrespective of gene expression subtype. In conclusion, our data indicate that immune infiltration likely contributes to differential labelling of ZEB1 and confounds interpretation of bulk ZEB1 expression data

Transcriptional and Translational Differences of Microglia from Male and Female Brains

Summary: Sex differences in brain structure and function are of substantial scientific interest because of sex-related susceptibility to psychiatric and neurological disorders. Neuroinflammation is a common denominator of many of these diseases, and thus microglia, as the brain’s immunocompetent cells, have come into focus in sex-specific studies. Here, we show differences in the structure, function, and transcriptomic and proteomic profiles in microglia freshly isolated from male and female mouse brains. We show that male microglia are more frequent in specific brain areas, have a higher antigen-presenting capacity, and appear to have a higher potential to respond to stimuli such as ATP, reflected in higher baseline outward and inward currents and higher protein expression of purinergic receptors. Altogether, we provide a comprehensive resource to generate and validate hypotheses regarding brain sex differences. : Guneykaya et al. provide transcriptomic, proteomic, and functional data from male and female microglia, providing a resource for further investigation of sex-based differences in microglia. Keywords: microglia, sex differences, transcriptomics, proteomic

Expression of ZEB1 in human glial tumors and normal brain.

<p>(A) Biopsy samples of human glial tumors show abundant nuclear expression of ZEB1. Sections from non-neoplastic normal brain show weak cytoplasmic staining of neurons and nuclear expression in astrocytes. (B) Quantification of ZEB1 in full histological sections of gross total resections of human glial tumors. ZEB1 positive cells were quantified using automated image analysis. Dots represent the mean of multiple regions of interest (ROI) per tumor. Box plots show the distribution of the ZEB1 labelling in tumor with indicated integrated diagnosis according the 2016 WHO classification of CNS tumors [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185376#pone.0185376.ref029" target="_blank">29</a>]. (C) <i>ZEB1</i> mRNA expression in public GBM cDNA microarray datasets. Boxplots show distribution in normal brain vs. GBM. Data were retrieved via the GlioVis portal [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185376#pone.0185376.ref030" target="_blank">30</a>].</p

ZEB1 expression in the tumor microenvironment.

<p>(A) Cell type specific expression of ZEB1 in reactive brain tissue. Human brain biopsy samples from cases of seizure-induced reactive gliosis or subacute infarction were co-stained for ZEB1 and Iba1 for microglia, CD45 for leucocytes, CD68 for macrophages and GFAP for astrocytes, respectively. Scale bars 50 μm. (B) Co-labeling of ZEB1 and CD68 or HLA-DR, respectively in human GBM. (C,D) Correlation of microarray-based gene expression levels of <i>ZEB1</i> and myeloid cell markers <i>CD68</i> (C) and <i>AIF1</i> (D), respectively. Processed log₂-transformed intensities from 157 GBM cases studied by Gravendeel et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185376#pone.0185376.ref034" target="_blank">34</a>] were obtained via the GlioVis portal [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185376#pone.0185376.ref030" target="_blank">30</a>].</p