Astroglial Cell-to-Cell Interaction with Autoreactive Immune Cells in Experimental Autoimmune Encephalomyelitis Involves P2X7 Receptor, 3-Integrin, and Connexin-43

In multiple sclerosis (MS), glial cells astrocytes interact with the autoreactive immune cells that attack the central nervous system (CNS), which causes and sustains neuroinflammation. However, little is known about the direct interaction between these cells when they are in close proximity in the inflamed CNS. By using an experimental autoimmune encephalomyelitis (EAE) model of MS, we previously found that in the proximity of autoreactive CNS-infiltrated immune cells (CNS-IICs), astrocytes respond with a rapid calcium increase that is mediated by the autocrine P2X7 receptor (P2X7R) activation. We now reveal that the mechanisms regulating this direct interaction of astrocytes and CNS-IICs involve the coupling between P2X7R, connexin-43, and β3-integrin. We found that P2X7R and astroglial connexin-43 interact and concentrate in the immediate proximity of the CNS-IICs in EAE. P2X7R also interacts with β3-integrin, and the block of astroglial αvβ3-integrin reduces the P2X7R-dependent calcium response of astrocytes upon encountering CNS-IICs. This interaction was dependent on astroglial mitochondrial activity, which regulated the ATP-driven P2X7R activation and facilitated the termination of the astrocytic calcium response evoked by CNS-IICs. By further defining the interactions between the CNS and the immune system, our findings provide a novel perspective toward expanding integrin-targeting therapeutic approaches for MS treatment by controlling the cell–cell interactions between astrocytes and CNS-IICs.casopis je u kategoriji M2

ASTROCYTE ACTIVITY IN THE CENTRAL NERVOUS SYSTEM AUTOIMMUNITY

Aims: Multiple sclerosis (MS) is an in ammatory autoimmune disorder of the central nervous system (CNS). Complex interactions between inltrating immune cells (IIC) and resident glial cells of the CNS cause myelin loss and neuronal dysfunction in MS. Here we aim to understand how naïve astrocytes functionally respond to the IIC invasion of the CNS. Methods: We measured calcium activity of naïve astrocytes in culture upon application of IIC. An experimental autoimmune encephalomyelitis (EAE) MS rat model was used to isolate IIC from the spinal cord of animals at the symptomatic stage. Naïve astrocytes were isolated from the spinal cord of WT rats. Results: We show that IIC and not the lymph node immune cells evoke vigorous increase in the astrocyte calcium activity. This IIC-induced calcium response depends on an autocrine activation of the purinergic P2X7 receptors on the naïve astrocytes.We also show that IIC induce ATP release from astrocytes by a mechanism that involves gap junctions and/or hemichannels activation and not the vesicular pathway. Our data indicate that ATP release and subsequent increase in the astrocytic calcium activity mainly depends on the cell-cell contact between naïve astrocytes and IIC. Conclusions: These results show that naïve astrocytes functionally respond to the IIC by augmented release of ATP. An increase in ATP release would alter astrocyte-neuron communication and a ect neuronal function in MS.kategorija M3

Dysfunction of oligodendrocyte inwardly rectifying potassium channel in a rat model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by the death of motor neurons in the spinal cord and the brain. Although this disease is characterized by motoneuron degeneration, non-neuronal cells such as oligodendrocytes play an important role in the disease onset and progression. The aim of our study was to examine functional properties of oligodendrocytes in the SOD1(G93A) rat model of ALS with a particular focus on the inwardly rectifying potassium channel Kir4.1 that is abundantly expressed in these glial cells and plays a role in the regulation of extracellular K+. First, we demonstrate that the expression of Kir4.1 is diminished in the spinal cord oligodendrocytes of the SOD1(G93A) rat. Moreover, our data show an elevated number of dysmorphic oligodendrocytes in the ALS spinal cord that is indicative of a degenerative phenotype. In order to assess physiological properties of oligodendrocytes, we prepared cell cultures from the rat spinal cord. Oligodendrocytes isolated from the SOD1(G93A) spinal cord display similar ramification of the processes as the control but express a lower level of Kir4.1. We further demonstrate an impairment of oligodendrocyte functional properties in ALS. Remarkably, whole-cell patch-clamp recordings revealed compromised membrane biophysical properties and diminished inward currents in the SOD1(G93A) oligodendrocytes. In addition, the Ba2+-sensitive Kir currents were decreased in ALS oligodendrocytes. Altogether, our findings provide the evidence of impaired Kir4.1 expression and function in oligodendrocytes of the SOD1(G93A) spinal cord, suggesting oligodendrocyte Kir4.1 channel as a potential contributor to the ALS pathophysiology

P2X7R, β3-integrin and Cx-43 mediate interaction between astrocytes and adjacent autoreactive immune cells

Astrocytes form a dense meshwork throughout the central nervous system (CNS) which qualifies them to perform interactive maintenance functions with neighboring cells. In neuroinflammation, this astroglial cell-to-cell interaction varies which can either promote or lessen pathological processes (1,2). In multiple sclerosis (MS), astrocytes engage in an interaction with immune cells which drives neurodegeneration by creating and sustaining an inflammatory CNS environment (3). Previously, we showed that CNS-infiltrated immune cells (CNS-IICs) in the experimental autoimmune encephalomyelitis (EAE) rat, rapidly alter the activity pattern of astrocytes by activating glial P2X7receptor (P2X7R)(4). In the present study, we aimed to identify the properties of astroglial P2X7R in EAE and to identify mechanisms responsible for astrocyte activation in the presence of CNS-IICs (CD4+T cells). In this respect, spinal cords from rats at the peak of EAE and age-matched healthy controls were isolated and protein expression of P2X7R and connexin-43 (Cx-43) were investigated. P2X7R protein expression was decreased in the lumbar spinal cord, while Cx-43 did not change. Next, we found that P2X7R and Cx-43 proteins interact in the lumbar spinal cord since both the monomer and the dimer Cx-43 co-immunoprecipitate with P2X7R. Even though the colocalization of P2X7R and Cx-43 was decreased in EAE compared to the control, the analysis of the distribution of astroglial P2X7R and Cx-43 and their colocalization in the radius of 20 μm from the infiltrated CD4+T cell center showed that astroglial P2X7R and Cx-43 are specifically associated and concentrated in the proximity of CNS-IICs in the EAE spinal cord. Subsequently, to achieve an unambiguous analysis of astrocyte-immune cell interaction, we monitored Ca2+dynamics in Fluo-4 labeled cultured naïve astrocytes following brief bath-application of CNS-IICs isolated and purified from spinal cords of EAE rats. Our data suggest that astroglial αvβ3-integrin acted upstream of P2X7R activation and is likely involved in establishing initial contact of astrocytes with CNS-IICs since astrocytic αvβ3-integrin block reduced the astrocytic Ca2+response to CNS-IIC application. Furthermore, astrocytes challenged with CGP31157 (blocker of mNCLX and HCX) exhibited a prolonged intracellular Ca2+elevation and higher ATP release after brief exposure to CNS-IICs, indicating a regulatory function of mitochondria on this intracellular astrocyte Ca2+response. Collectively these data describing integrin-relevant cellular mechanisms of astroglial P2X7R activation could help to expand integrin-inhibiting therapeutic approaches currently in use for MS treatment toward control ofastrocyte purine-based interaction with immune cells.kategorija M3

αVβ3-Integrin and mitochondria mediate astrocyte response to autoreactive immune cells

The astrocytic network maintains homeostasis in the central nervous system (CNS) through interactions with neighboring cells. In the CNS autoimmune disease, multiple sclerosis (MS), neuroinflammatory conditions modulate these cell-to-cell interactions. Our previous work revealed that the immune cells infiltrated into the CNS (CNS-IICs) of experimental autoimmune encemphalomyelitis (EAE) rat, an animal model of MS, rapidly alter the activity pattern of astrocytes by activating the glial P2X7 receptor (P2X7R). In the present study we further defined the mechanisms responsible for astrocytes’ activation in the presence of CNS-IICs. For this purpose, we used an in vitro experimental setup and monitored Ca2+ dynamics in Fluo-4-labeled cultured naïve astrocytes following brief bath application of CNS-IICs isolated from the spinal cord of the EAE rat. Our data indicate that the astroglial αvβ3-integrin is involved in the initial contact of astrocytes with CNS-IICs, since blocking αvβ3-integrin reduced the expected astrocytic Ca2+ response. Furthermore, blocking of mitochondrial Na+/Ca2+- and H+/Ca2+- exchangers in astrocytes promoted an augmentation of the intracellular Ca2+ increase and a higher ATP release after brief exposure to CNS-IICs, demonstrating that mitochondria regulate the astrocyte-CNS IICs cell-cell interaction. Overall, our study expands the understanding of astrocytes’ interaction with autoreactive immune cells that are present in their local environment in an autoimmune disease. This offers a new conceptual framework for considering direct astrocyte–immune cell interaction to design new strategies for therapy development in the treatment of MS

Modification of Glial Response in Hibernation: A Patch-Clamp Study on Glial Cells Acutely Isolated from Hibernating Land Snail

Hibernation is a dormant state of some animal species that enables them to survive harsh environmental conditions during the winter seasons. In the hibernating state, preservation of neuronal rhythmic activity at a low level is necessary for maintenance of suspended forms of behavior. As glial cells support rhythmic activity of neurons, preservation of brain function in the hibernating state implies accompanying modification of glial activity. A supportive role of glia in regulating neuronal activity is reflected through the activity of inwardly rectifying K+ channels (Kir). Therefore, we examined electrophysiological response, particularly Kir current response, of glial cells in mixture with neurons acutely isolated from active and hibernating land snail Helix pomatia. Our data show that hibernated glia have significantly lower inward current density, specific membrane conductance, and conductance density compared with active glia. The observed reduction could be attributed to the Kir currents, since the Ba2+-sensitive Kir current density was significantly lower in hibernated glia. Accordingly, a significant positive shift of the current reversal potential indicated a more depolarized state of hibernated glia. Data obtained show that modification of glial current response could be regulated by serotonin (5-HT) through an increase of cGMP as a secondary messenger, since extracellular addition of 5-HT or intracellular administration of cGMP to active glia induced a significant reduction of inward current density and thus mimicked the reduced response of hibernated glia. Lower Kir current density of hibernated glia accompanied the lower electrical activity of hibernated neurons, as revealed by a decrease in neuronal fast inward Na+ current density. Our findings reveal that glial response is reduced in the hibernating state and suggest seasonal modulation of glial activity. Maintenance of low glial activity in hibernation could be important for preservation of brain rhythmic activity and survival of the animal.Ministry of Education, Science and Technological Development, Republic of Serbia {[}173027

Approach for patch-clamping using an upright microscope with z-axis movable stage

We describe an approach for studying the physiology of single live cells using the conceptionally novel upright microscope/patch-clamp configuration. Electrophysiology experiments typically require a microscope with the fixed stage position and the motion control of the microscope objective. Here, we demonstrate that a microscope with a z-axis movable stage and a fixed objective can also be efficiently used in combination with the patch-clamp technique. We define a set of underlying principles governing the operation of this microscope/patch-clamp configuration and demonstrate its performance in practice using cultured astrocytes, microglia, and oligodendrocytes. Experimental results show that our custom configuration provides stable recordings, has a high success rate of the whole-cell patch-clamp trials, can be effectively applied to study cellular physiology of glial cells, and provides comparable performance and usability to the commercially available systems. Our system can be easily replicated or adapted to suit the needs of the research groups and can be cost-effective in reducing the investments in purchasing additional equipment. We provide step-by-step instructions on implementing an upright microscope with z-axis movable stage as a routine workhorse for patch-clamping

Central nervous system-infiltrated immune cells induce calcium increase in astrocytes via astroglial purinergic signaling

Interaction between autoreactive immune cells and astroglia is an important part of the pathologic processes that fuel neurodegeneration in multiple sclerosis. In this inflammatory disease, immune cells enter into the central nervous system (CNS) and they spread through CNS parenchyma, but the impact of these autoreactive immune cells on the activity pattern of astrocytes has not been defined. By exploiting naive astrocytes in culture and CNS-infiltrated immune cells (CNS IICs) isolated from rat with experimental autoimmune encephalomyelitis (EAE), here we demonstrate previously unrecognized properties of immune cell-astrocyte interaction. We show that CNS IICs but not the peripheral immune cell application, evokes a rapid and vigorous intracellular Ca(2+)increase in astrocytes by promoting glial release of ATP. ATP propagated Ca(2+)elevation through glial purinergic P2X7 receptor activation by the hemichannel-dependent nucleotide release mechanism. Astrocyte Ca(2+)increase is specifically triggered by the autoreactive CD4(+)T-cell application and these two cell types exhibit close spatial interaction in EAE. Therefore, Ca(2+)signals may mediate a rapid astroglial response to the autoreactive immune cells in their local environment. This property of immune cell-astrocyte interaction may be important to consider in studies interrogating CNS autoimmune disease