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

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 MφIKKßΚΟ mice, in which depletion of IKKß, the main activating kinase of NF-κB, was global to CNS and peripheral macrophages, and ΜgΙΚΚßKO 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 IKKß from myeloid cells in MφIKKßΚΟ mice accelerated the onset and significantly supressed chronic EAE. Knocking out IKKß 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 IKKß, but CNS resident mechanisms, such as microglial activation and neuronal hyperexcitability were altered from early in EAE. Lastly, depletion of myeloid cell IKKß resulted in enhanced late long-term potentiation in EAE. CONCLUSIONS: IKKß-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 IKKß deletion dysregulates neuronal excitability and synaptic plasticity in EAE. These effects of IKKß on brain cognitive abilities deserve special consideration when therapeutic interventions that inhibit NF-κB are used in MS.

Novel cell-based analysis reveals region-dependent changes in microglial dynamics in grey matter in a cuprizone model of demyelination.

Microglia are key players in Multiple Sclerosis (MS), expressing many susceptibility genes for this disease. They constantly survey the brain microenvironment, but the precise functional relationships between microglia and pathological processes remain unknown. We performed a detailed assessment of microglial dynamics in three distinct grey matter regions in a cuprizone-induced demyelination model. We found that microglial activation preceded detectable demyelination and showed regional specificities, such as prominent phagocytic activity in cortical layer 5 and early hypertrophic morphology in hippocampal CA1. Demyelination happened earliest in cortical layer 5, although was more complete in CA1. In cortical layer 2/3, microglial activation and demyelination were less pronounced but microglia became hyper-ramified with slower process movement during remyelination, thereby maintaining local brain surveillance. Profiling of microglia using specific morphological and motility parameters revealed region-specific heterogeneity of microglial responses in the grey matter that might serve as sensitive indicators of progression in CNS demyelinating diseases.

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.

P2Y13 receptors regulate microglial morphology, surveillance, and resting levels of interleukin 1ß 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-1ß 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.

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.

Differential contributions of microglial and neuronal IKKß to synaptic plasticity and associative learning in alert behaving mice.

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 ß, the main activating kinase in the inducible NF-κB pathway, selectively in myeloid lineage cells (mIKKßKO) or excitatory neurons (nIKKßKO) to measure synaptic strength at hippocampal Schaffer collaterals during long-term potentiation (LTP) and instrumental conditioning in alert behaving individuals. Resting microglial cells in mIKKßKO mice showed less Iba1-immunoreactivity, and brain IL-1ß mRNA levels were selectively reduced compared with controls. Measurement of field excitatory postsynaptic potentials (fEPSPs) evoked by stimulation of the CA3-CA1 synapse in mIKKßKO mice showed higher facilitation in response to paired pulses and enhanced LTP following high frequency stimulation. In contrast, nIKKßKO 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. IKKß was not necessary 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 IKKß and therefore NF-κB activity, regulate hippocampal synaptic plasticity and that both microglia and neurons, through IKKß, are necessary for animals to modify hippocampus-driven behavior during associative learning.

Analysis of Signaling Mechanisms Regulating Microglial Process Movement.

Microglia, the brain's innate immune cells, are extremely motile cells, continuously surveying the central nervous system (CNS) to serve homeostatic functions and to respond to pathological events. In the healthy brain, microglia exhibit a small cell body with long, branched, and highly motile processes, which constantly extend and retract, effectively "patrolling" the brain parenchyma. Over the last decade, methodological advances in microscopy and the availability of genetically encoded reporter mice have allowed us to probe microglial physiology in situ. Beyond their classical immunological roles, unexpected functions of microglia have been revealed, both in the developing and the adult brain: microglia regulate the generation of newborn neurons, control the formation and elimination of synapses, and modulate neuronal activity. Many of these newly ascribed functions depend directly on microglial process movement. Thus, elucidating the mechanisms underlying microglial motility is of great importance to understand their role in brain physiology and pathophysiology. Two-photon imaging of fluorescently labeled microglia, either in vivo or ex vivo in acute brain slices, has emerged as an indispensable tool for investigating microglial movements and their functional consequences. This chapter aims to provide a detailed description of the experimental data acquisition and analysis needed to address these questions, with a special focus on key dynamic and morphological metrics such as surveillance, directed motility, and ramification.

Amyloid ß oligomers constrict human capillaries in Alzheimer's disease via signaling to pericytes.

Cerebral blood flow is reduced early in the onset of Alzheimer's disease (AD). Because most of the vascular resistance within the brain is in capillaries, this could reflect dysfunction of contractile pericytes on capillary walls. We used live and rapidly fixed biopsied human tissue to establish disease relevance, and rodent experiments to define mechanism. We found that in humans with cognitive decline, amyloid ß (Aß) constricts brain capillaries at pericyte locations. This was caused by Aß generating reactive oxygen species, which evoked the release of endothelin-1 (ET) that activated pericyte ETA receptors. Capillary, but not arteriole, constriction also occurred in vivo in a mouse model of AD. Thus, inhibiting the capillary constriction caused by Aß could potentially reduce energy lack and neurodegeneration in AD.

Microglial Ramification, Surveillance, and Interleukin-1ß Release Are Regulated by the Two-Pore Domain K+ Channel THIK-1.

Microglia exhibit two modes of motility: they constantly extend and retract their processes to survey the brain, but they also send out targeted processes to envelop sites of tissue damage. We now show that these motility modes differ mechanistically. We identify the two-pore domain channel THIK-1 as the main K+ channel expressed in microglia in situ. THIK-1 is tonically active, and its activity is potentiated by P2Y12 receptors. Inhibiting THIK-1 function pharmacologically or by gene knockout depolarizes microglia, which decreases microglial ramification and thus reduces surveillance, whereas blocking P2Y12 receptors does not affect membrane potential, ramification, or surveillance. In contrast, process outgrowth to damaged tissue requires P2Y12 receptor activation but is unaffected by blocking THIK-1. Block of THIK-1 function also inhibits release of the pro-inflammatory cytokine interleukin-1ß from activated microglia, consistent with K+ loss being needed for inflammasome assembly. Thus, microglial immune surveillance and cytokine release require THIK-1 channel activity.