Vitali Matyash

Heterogeneity in astrocyte morphology and physiology.

Astrocytes as a cell population are not well defined and comprise a heterogeneous population of cells. There are at least 9 different morphological variants which can coexist within one given brain region. Human astrocytes have a considerably more complex morphology as their rodent counterparts. There are also a number of functional differences depending on brain region and developmental stage in the normal (not pathologic) brain. Astrocytes can differ in functional gap junctional coupling, expression of transmitter receptors, membrane currents, and glutamate transporters. We feel that astrocyte heterogeneity has not yet been thoroughly explored and what we report here will just be a beginning of a new field of research.

Gliomas induce and exploit microglial MT1-MMP expression for tumor expansion

Diffuse infiltration of glioma cells into normal brain tissue is considered to be a main reason for the unfavorable outcomes of patients with malignant gliomas. Invasion of glioma cells into the brain parenchyma is facilitated by metalloprotease-mediated degradation of the extracellular matrix. Metalloproteases are released as inactive pro-forms and get activated upon cleavage by membrane bound metalloproteases. Here, we show that membrane type 1 metalloprotease (MT1-MMP) is up-regulated in glioma-associated microglia, but not in the glioma cells. Overexpression of MT1-MMP is even lethal for glioma cells. Glioma-released factors trigger the expression and activity of MT1-MMP via microglial toll-like receptors and the p38 MAPK pathway, as deletion of the toll-like receptor adapter protein MyD88 or p38 inhibition prevented MT1-MMP expression and activity in cultured microglial cells. Microglial MT1-MMP in turn activates glioma-derived pro-MMP-2 and promotes glioma expansion, as shown in an ex vivo model using MT1-MMP-deficient brain tissue and a microglia depletion paradigm. Finally, MyD88 deficiency or microglia depletion largely attenuated glioma expansion in 2 independent in vivo models.

Functional impairment of microglia coincides with beta-amyloid deposition in mice with Alzheimer-like pathology

Microglial cells closely interact with senile plaques in Alzheimer’s disease and acquire the morphological appearance of an activated phenotype. The significance of this microglial phenotype and the impact of microglia for disease progression have remained controversial. To uncover and characterize putative changes in the functionality of microglia during Alzheimer’s disease, we directly assessed microglial behavior in two mouse models of Alzheimer’s disease. Using in vivo two-photon microscopy and acute brain slice preparations, we found that important microglial functions - directed process motility and phagocytic activity - were strongly impaired in mice with Alzheimer’s disease-like pathology compared to age-matched non-transgenic animals. Notably, impairment of microglial function temporally and spatially correlated with Aβ plaque deposition, and phagocytic capacity of microglia could be restored by interventionally decreasing amyloid burden by Aβ vaccination. These data suggest that major microglial functions progressively decline in Alzheimer’s disease with the appearance of Aβ plaques, and that this functional impairment is reversible by lowering Aβ burden, e.g. by means of Aβ vaccination.

Requirement of functional ryanodine receptor type 3 for astrocyte migration

Astrocyte motility plays an important role in the response of the brain to injury and during regeneration. We used two in vitro assays, a wound‐healing model and a chemotaxis assay, to study mechanisms that control astrocyte motility. Ryanodine receptors (RyR), intracellular calcium‐release channels, modulate intracellular Ca2+ levels, and also motility: 1) blocking RyR with antagonizing concentration of ryanodine (200 μM) strongly attenuated motility and 2) motility of astrocytes cultured from homozygous RyR type 3 knockout mice was impaired strongly compared with wild‐type. In contrast, MIP‐1α‐induced chemotaxis was neither impaired in the presence of ryanodine nor in the cells from the knockout animals. Reverse transcription‐polymerase chain reaction (RT‐PCR) analysis combined with Western blotting and immunocytochemistry confirmed the expression of RyR type 3, but not type 1 or 2 in cultured and acutely isolated astrocytes. RyR in astrocytes are linked to Ca2+ signaling because the RyR agonist 4‐chloro‐m‐cresol induced a release of Ca2+ from intracellular stores. These results indicate that astrocytes express only RyR type 3 and that this receptor is important for controlling astrocyte motility.

Neural precursor cells induce cell death of high-grade astrocytomas through stimulation of TRPV1

Primary astrocytomas of grade 3 or 4 according to the classification system of the World Health Organization (high-grade astrocytomas or HGAs) are preponderant among adults and are almost invariably fatal despite the use of multimodal therapy. Here we show that the juvenile brain has an endogenous defense mechanism against HGAs. Neural precursor cells (NPCs) migrate to HGAs, reduce glioma expansion and prolong survival time by releasing endovanilloids that activate the vanilloid receptor (transient receptor potential vanilloid subfamily member-1 or TRPV1) on HGA cells. TRPV1 is highly expressed in tumor and weakly expressed in tumor-free brain. TRPV1 stimulation triggers tumor cell death through the branch of the endoplasmic reticulum stress pathway that is controlled by activating transcription factor-3 (ATF3). The antitumorigenic response of NPCs is lost with aging. NPC-mediated tumor suppression can be mimicked in the adult brain by systemic administration of the synthetic vanilloid arvanil, suggesting that TRPV1 agonists have potential as new HGA therapeutics.

Glutamate-triggered calcium signalling in mouse Bergmann glial cells in situ: Role of inositol-1,4,5-trisphosphate-mediated intracellular calcium release

The mechanisms of glutamate-induced changes in intracellular free calcium concentration in Bergmann glial cells in mouse cerebellar slices were investigated by Fura-2-based microfluorimetry. Extracellular applications of glutamate, quisqualate and kainate triggered an increase in cytoplasmic calcium concentration, whereas N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate were ineffective. The calcium elevation triggered by kainate was completely blocked by removal of calcium ions from the external solutions or by slice incubation with 6-cyano-7-nitroquinoxaline-2,3-dione. Conversely, both glutamate- and quisqualate-induced intracellular calcium transients were only slightly attenuated by slice incubation with either 6-cyano-7-nitroquinoxaline-2,3-dione or calcium-free solution, suggesting the intracellular origin for calcium ions. The glutamate-triggered cytosolic calcium increases were inhibited by slice incubation with thapsigargin, the inhibitor of intracellular calcium pumps, or by intracellular perfusion of Bergmann glial cells with heparin, the antagonist of inositol-1,4,5-trisphosphate-gated calcium release channels. Therefore the calcium release from inositol-1,4,5-trisphosphate-sensitive intracellular stores plays the major role in glutamate-induced calcium signalling. We concluded that Bergmann glial cells express calcium permeable ionotropic glutamate receptors, which might be important for generation of fast calcium signals. However, slow glutamate-evoked calcium signals are mostly determined by inositol-1,4,5-trisphosphate-dependent intracellular signalling chain.

Nitric Oxide Signals Parallel Fiber Activity to Bergmann Glial Cells in the Mouse Cerebellar Slice

Stimulation of parallel fibers in the cerebellar cortex triggers a transient calcium increase in Bergmann glial cells, a special form of astrocytes. Using patch-clamping and imaging techniques we have found that this form of neuron-glia interaction is mediated by nitric oxide (NO) since the response is blocked by the NO-synthase inhibitor N omega-nitro-l-arginine and mimicked by NO donors. None of the neurotransmitter receptors of Bergmann glia identified so far participates in or interferes with this signaling cascade. The NO-triggered increases in [Ca(2+)](i), as studied in Bergmann glial cells in the slice or in cultured astrocytes, are due to Ca(2+) influx and not to release from cytoplasmic stores. Thus, NO released from parallel fibers serves as a signaling substance to the neighboring glial elements.

Mitochondrial Exchanger NCLX Plays a Major Role in the Intracellular Ca2+ Signaling, Gliotransmission, and Proliferation of Astrocytes

Mitochondria not only provide cells with energy, but are central to Ca2+ signaling. Powered by the mitochondrial membrane potential, Ca2+ enters the mitochondria and is released into the cytosol through a mitochondrial Na+/Ca2+ exchanger. We established that NCLX, a newly discovered mitochondrial Na+/Ca2+ exchanger, is expressed in astrocytes isolated from mice of either sex. Immunoblot analysis of organellar fractions showed that the location of NCLX is confined to mitochondria. Using pericam-based mitochondrial Ca2+ imaging and NCLX inhibition either by siRNA or by the pharmacological blocker CGP37157, we demonstrated that NCLX is responsible for mitochondrial Ca2+ extrusion. Suppression of NCLX function altered cytosolic Ca2+ dynamics in astrocytes and this was mediated by a strong effect of NCLX activity on Ca2+ influx via store-operated entry. Furthermore, Ca2+ influx through the store-operated Ca2+ entry triggered strong, whereas ER Ca2+ release triggered only modest mitochondrial Ca2+ transients, indicating that the functional cross talk between the plasma membrane and mitochondrial domains is particularly strong in astrocytes. Finally, silencing of NCLX expression significantly reduced Ca2+-dependent processes in astrocytes (i.e., exocytotic glutamate release, in vitro wound closure, and proliferation), whereas Ca2+ wave propagation was not affected. Therefore, NCLX, by meditating astrocytic mitochondrial Na+/Ca2+ exchange, links between mitochondria and plasma membrane Ca2+ signaling, thereby modulating cytoplasmic Ca2+ transients required to control a diverse array of astrocyte functions.

GDNF mediates glioblastoma-induced microglia attraction but not astrogliosis

High-grade gliomas are the most common primary brain tumors. Their malignancy is promoted by the complex crosstalk between different cell types in the central nervous system. Microglia/brain macrophages infiltrate high-grade gliomas and contribute to their progression. To identify factors that mediate the attraction of microglia/macrophages to malignant brain tumors, we established a glioma cell encapsulation model that was applied in vivo. Mouse GL261 glioma cell line and human high-grade glioma cells were seeded into hollow fibers (HF) that allow the passage of soluble molecules but not cells. The glioma cell containing HF were implanted into one brain hemisphere and simultaneously HF with non-transformed fibroblasts (controls) were introduced into the contralateral hemisphere. Implanted mouse and human glioma- but not fibroblast-containing HF attracted microglia and up-regulated immunoreactivity for GFAP, which is a marker of astrogliosis. In this study, we identified GDNF as an important factor for microglial attraction: (1) GL261 and human glioma cells secret GDNF, (2) reduced GDNF production by siRNA in GL261 in mouse glioma cells diminished attraction of microglia, (3) over-expression of GDNF in fibroblasts promoted microglia attraction in our HF assay. In vitro migration assays also showed that GDNF is a strong chemoattractant for microglia. While GDNF release from human or mouse glioma had a profound effect on microglial attraction, the glioma-induced astrogliosis was not affected. Finally, we could show that injection of GL261 mouse glioma cells with GDNF knockdown by shRNA into mouse brains resulted in reduced tumor expansion and improved survival as compared to injection of control cells.

Activation of serotonin receptors promotes microglial injury-induced motility but attenuates phagocytic activity.

Microglia, the brain immune cell, express several neurotransmitter receptors which modulate microglial functions. In this project we studied the impact of serotonin receptor activation on distinct microglial properties as serotonin deficiency not only has been linked to a number of psychiatric disease like depression and anxiety but may also permeate from the periphery through blood-brain barrier openings seen in neurodegenerative disease. First, we tested the impact of serotonin on the microglial response to an insult caused by a laser lesion in the cortex of acute slices from Cx3Cr1-GFP-/+ mice. In the presence of serotonin the microglial processes moved more rapidly towards the laser lesion which is considered to be a chemotactic response to ATP. Similarly, the chemotactic response of cultured microglia to ATP was also enhanced by serotonin. Quantification of phagocytic activity by determining the uptake of microspheres showed that the amoeboid microglia in slices from early postnatal animals or microglia in culture respond to serotonin application with a decreased phagocytic activity whereas we could not detect any significant change in ramified microglia in situ. The presence of microglial serotonin receptors was confirmed by patch-clamp experiments in culture and amoeboid microglia and by qPCR analysis of RNA isolated from primary cultured and acutely isolated adult microglia. These data suggest that microglia express functional serotonin receptors linked to distinct microglial properties.