P2Y13 receptors regulate microglial morphology, surveillance, and resting levels of interleukin 1β release

Microglia sense their environment using an array of membrane receptors. While P2Y12 receptors are known to play a key role in targeting directed motility of microglial processes to sites of damage where ATP/ADP is released, little is known about the role of P2Y13 , which transcriptome data suggest is the second most expressed neurotransmitter receptor in microglia. We show that, in patch-clamp recordings in acute brain slices from mice lacking P2Y13 receptors, the THIK-1 K+ current density evoked by ADP activating P2Y12 receptors was increased by ~50%. This increase suggested that the P2Y12 -dependent chemotaxis response should be potentiated; however, the time needed for P2Y12 -mediated convergence of microglial processes onto an ADP-filled pipette or to a laser ablation was longer in the P2Y13 KO. Anatomical analysis showed that the density of microglia was unchanged, but that they were less ramified with a shorter process length in the P2Y13 KO. Thus, chemotactic processes had to grow further and so arrived later at the target, and brain surveillance was reduced by ~30% in the knock-out. Blocking P2Y12 receptors in brain slices from P2Y13 KO mice did not affect surveillance, demonstrating that tonic activation of these high-affinity receptors is not needed for surveillance. Strikingly, baseline interleukin-1β release was increased fivefold while release evoked by LPS and ATP was not affected in the P2Y13 KO, and microglia in intact P2Y13 KO brains were not detectably activated. Thus, P2Y13 receptors play a role different from that of their close relative P2Y12 in regulating microglial morphology and function

Role of the transcription factor NF-κB in physiology of neurons and disorders of the central nervous system

Microglia are CNS resident immune cells and a rich source of neuroactive mediators, but their contribution to physiological brain processes such as synaptic plasticity, learning and memory is not fully understood. In this study, we used mice with partial depletion of IκB kinase β, the main activating kinase in the inducible NF-κB pathway, selectively in myeloid lineage cells (mIKKβKO) or excitatory neurons (nIKKβKO) to measure synaptic strength at hippocampal Schaffer collaterals during long term potentiation (LTP) and instrumental conditioning in alert behaving individuals. Resting microglial cells in mIKKβKO mice showed less Iba1-immunoreactivity, and brain IL-1β mRNA levels were selectively reduced compared to controls. Measurement of field excitatory postsynaptic potentials (fEPSPs) evoked by stimulation of the CA3-CA1 synapse in mIKKβKO mice showed higher facilitation in response to paired pulses and enhanced LTP following high frequency stimulation. In contrast, nIKKβKO mice showed normal basic synaptic transmission and LTP induction but impairments in late LTP. To understand the consequences of such impairments in synaptic plasticity for learning and memory, we measured CA1 fEPSPs in behaving mice during instrumental conditioning. IKKβ was not needed in either microglia or neurons for mice to learn lever-pressing (appetitive behavior) to obtain food (consummatory behavior) but was required in both for modification of their hippocampus-dependent appetitive, not consummatory behavior. Our results show that microglia, through IKKβ and therefore NF-κB activity, regulate hippocampal synaptic plasticity and that both microglia and neurons, through IKKβ, are necessary for animals to modify hippocampus-driven behavior during associative learning.Τα μικρογλοιακά κύτταρα αποτελούν τα τοπικά ανοσοποιητικά κύτταρα του Κεντρικού Νευρικού Συστήματος (ΚΝΣ) και μια πλούσια πηγή νευροδραστικών μεσολαβητών, ωστόσο η συνεισφορά τους στις φυσιολογικές διαδικασίες του εγκεφάλου, όπως η συναπτική πλαστικότητα, η μνήμη και η μάθηση δεν έχει κατανοηθεί προς το παρόν. Στην εργασία αυτή, χρησιμοποιήσαμε επίμυες με μερική απάλειψη της ΙκB κινάσης β, την βασικότερη κινάση ενεργοποίησης του επαγόμενου NF-κB μονοπατιού, επιλεκτικά σε κύτταρα μυελοειδικής προέλευσης (mIKKβKO) ή σε διεγερτικούς νευρώνες (nIKKβKO) με σκοπό να μετρήσουμε την αποτελεσματικότητα της συναπτικής διαβίβασης της οδού των παράπλευρων κλάδων Schaffer του ιππόκαμπου κατά τη διάρκεια της μακρόχρονης ενδυνάμωσης (LTP) και της συντελεστικής εξαρτημένης μάθησης. Τα μη ενεργοποιημένα μικρογλοιακά κύτταρα των mIKKβKO επιμύων έδειξαν μειωμένη Iba1-ανοσολογική έκφραση, και χαμηλότερο επίπεδο έκφρασης του mRNA της IL-1β στον εγκέφαλό τους σε σχέση με τους επιμύες ελέγχου. Μετρήσεις των διεγερτικών μετασυναπτικών δυναμικών πεδίου (fEPSPs) που προκλήθηκαν με διέγερση της CA3-CA1 σύναψης στους mIKKβKO επίμυες έδειξαν αυξημένη συναπτική ενδυνάμωση σε απόκριση διπλού διεγερτικού παλμού και ενισχυμένη LTP κατόπιν διέγερσης υψηλής συχνότητας. Σε αντίθεση, οι nIKKβKO επίμυες έδειξαν φυσιολογική συναπτική διαβίβαση και έναρξη της LTP αλλά δυσλειτουργίες στην μεταγενέστερη φάση αυτής (Late LTP). Προκειμένου να κατανοήσουμε τις συνέπειες αυτών των δυσλειτουργιών της συναπτικής πλαστικότητας, στις διαδικασίες της μάθησης και της μνήμης, μετρήσαμε την εξέλιξη του εύρους των πληθυσμιακών δυναμικών (fEPSPs) στην CA1 περιοχή του ιπποκάμπου σε επίμυες κατά τη διάρκεια εκτέλεσης δοκιμής συντελεστικής εξαρτημένης μάθησης. Η IKKβ κινάση δεν ήταν απαραίτητη ούτε στα μικρογλοιακά κύτταρα ούτε στους νευρώνες των επιμύων, προκειμένου να μάθουν να πιέζουν τον μοχλό (ορεκτική συμπεριφορά) για να αποκτήσουν φαγητό (καταναλωτική συμπεριφορά) αλλά και στους δυο τύπους κυττάρων ήταν απαραίτητη για την τροποποίηση της ιπποκαμπο-εξαρτώμενης ορεκτικής, και όχι της καταναλωτικής συμπεριφοράς. Τα αποτελέσματά μας δείχνουν ότι τα μικρογλοιακά κύτταρα, μέσω της IKKβ κινάσης και κατεπέκταση της ενεργοποίησης του NF-κB, συμμετέχουν στη ρύθμιση της συναπτικής πλαστικότητας του ιπποκάμπου, και ότι τόσο τα μικρογλοιακά κύτταρα όσο και οι νευρώνες, μέσω της IKKβ, είναι απαραίτητα για την τροποποίηση των ιπποκαμπο-εξαρτώμενων συμπεριφορών των επιμύων κατά τη διάρκεια της συνειρμικής μάθησης

Mesenchymal Stem Cell Protection of Neurons against Glutamate Excitotoxicity Involves Reduction of NMDA-Triggered Calcium Responses and Surface GluR1, and Is Partly Mediated by TNF

Mesenchymal stem cells (MSC) provide therapeutic effects in experimental CNS disease models and show promise as cell-based therapies for humans, but their modes of action are not well understood. We previously show that MSC protect rodent neurons against glutamate excitotoxicity in vitro, and in vivo in an epilepsy model. Neuroprotection is associated with reduced NMDA glutamate receptor (NMDAR) subunit expression and neuronal glutamate-induced calcium (Ca2+) responses, and increased expression of stem cell-associated genes. Here, to investigate whether MSC-secreted factors modulate neuronal AMPA glutamate receptors (AMPAR) and gene expression, we performed longitudinal studies of enriched mouse cortical neurons treated with MSC conditioned medium (CM). MSC CM did not alter total levels of GluR1 AMPAR subunit in neurons, but its distribution, reducing cell surface levels compared to non-treated neurons. Proportions of NeuN-positive neurons, and of GFAP- and NG2-positive glia, were equal in untreated and MSC CM-treated cultures over time suggesting that neurons, rather than differentially-expanded glia, account for the immature gene profile previously reported in MSC CM-treated cultures. Lastly, MSC CM contained measurable amounts of tumor necrosis factor (TNF) bioactivity and pre-treatment of MSC CM with the TNF inhibitor etanercept reduced its ability to protect neurons. Together these results indicate that MSC-mediated neuroprotection against glutamate excitotoxicity involves reduced NMDAR and GluR1-containing AMPAR function, and TNF-mediated neuroprotection

Effects of the ecto-ATPase apyrase on microglial ramification and surveillance reflect cell depolarization, not ATP depletion

Microglia, the brain’s innate immune cells, have highly motile processes which constantly survey the brain to detect infection, remove dying cells, and prune synapses during brain development. ATP released by tissue damage is known to attract microglial processes, but it is controversial whether an ambient level of ATP is needed to promote constant microglial surveillance in the normal brain. Applying the ATPase apyrase, an enzyme which hydrolyzes ATP and ADP, reduces microglial process ramification and surveillance, suggesting that ambient ATP/ADP maintains microglial surveillance. However, attempting to raise the level of ATP/ADP by blocking the endogenous ecto-ATPase (termed NTPDase1/CD39), which also hydrolyzes ATP/ADP, does not affect the cells’ ramification or surveillance, nor their membrane currents, which respond to even small rises of extracellular [ATP] or [ADP] with the activation of K+ channels. This indicates a lack of detectable ambient ATP/ADP and ecto-ATPase activity, contradicting the results with apyrase. We resolve this contradiction by demonstrating that contamination of commercially available apyrase by a high K+ concentration reduces ramification and surveillance by depolarizing microglia. Exposure to the same K+ concentration (without apyrase added) reduced ramification and surveillance as with apyrase. Dialysis of apyrase to remove K+ retained its ATP-hydrolyzing activity but abolished the microglial depolarization and decrease of ramification produced by the undialyzed enzyme. Thus, applying apyrase affects microglia by an action independent of ATP, and no ambient purinergic signaling is required to maintain microglial ramification and surveillance. These results also have implications for hundreds of prior studies that employed apyrase to hydrolyze ATP/ADP

IKKβ deletion from CNS macrophages increases neuronal excitability and accelerates the onset of EAE, while from peripheral macrophages reduces disease severity

Abstract Background Multiple sclerosis (MS) is a neuroinflammatory demyelinating disease characterized by motor deficits and cognitive decline. Many immune aspects of the disease are understood through studies in the experimental autoimmune encephalomyelitis (EAE) model, including the contribution of the NF-κB transcription factor to neuroinflammation. However, the cell-specific roles of NF-κB to EAE and its cognitive comorbidities still needs further investigation. We have previously shown that the myeloid cell NF-κB plays a role in the healthy brain by exerting homeostatic regulation of neuronal excitability and synaptic plasticity and here we investigated its role in EAE. Methods We used constitutive MφIKKβΚΟ mice, in which depletion of IKKβ, the main activating kinase of NF-κB, was global to CNS and peripheral macrophages, and ΜgΙΚΚβKO mice, in which depletion was inducible and specific to CNS macrophages by 28 days after tamoxifen administration. We subjected these mice to MOG35-55 induced EAE and cuprizone-induced demyelination. We measured pathology by immunohistochemistry, investigated molecular mechanisms by RNA sequencing analysis and studied neuronal functions by in vivo electrophysiology in awake animals. Results Global depletion of IKKβ from myeloid cells in MφIKKβΚΟ mice accelerated the onset and significantly supressed chronic EAE. Knocking out IKKβ only from CNS resident macrophages accelerated the onset and exacerbated chronic EAE, accompanied by earlier demyelination and immune cell infiltration but had no effect in cuprizone-induced demyelination. Peripheral T cell effector functions were not affected by myeloid cell deletion of IKKβ, but CNS resident mechanisms, such as microglial activation and neuronal hyperexcitability were altered from early in EAE. Lastly, depletion of myeloid cell IKKβ resulted in enhanced late long-term potentiation in EAE. Conclusions IKKβ-mediated activation of NF-κΒ in myeloid cells has opposing roles in EAE depending on the cell type and the disease stage. In CNS macrophages it is protective while in peripheral macrophages it is disease-promoting and acts mainly during chronic disease. Although clinically protective, CNS myeloid cell IKKβ deletion dysregulates neuronal excitability and synaptic plasticity in EAE. These effects of IKKβ on brain cognitive abilities deserve special consideration when therapeutic interventions that inhibit NF-κB are used in MS