Age-related alterations in the dynamic behavior of microglia

Microglia, the primary resident immune cells of the central nervous system (CNS), exhibit dynamic behavior involving rapid process motility and cellular migration that is thought to underlie key functions of immune surveillance and tissue repair. Although age-related changes in microglial activation have been implicated in the pathogenesis of neurodegenerative diseases of aging, how dynamic behavior in microglia is influenced by aging is not fully understood. In this study, we employed live imaging of retinal microglia in situ to compare microglial morphology and behavioral dynamics in young and aged animals. We found that aged microglia in the resting state have significantly smaller and less branched dendritic arbors, and also slower process motilities, which probably compromise their ability to survey and interact with their environment continuously. We also found that dynamic microglial responses to injury were age-dependent. While young microglia responded to extracellular ATP, an injury-associated signal, by increasing their motility and becoming more ramified, aged microglia exhibited a contrary response, becoming less dynamic and ramified. In response to laser-induced focal tissue injury, aged microglia demonstrated slower acute responses with lower rates of process motility and cellular migration compared with young microglia. Interestingly, the longer term response of disaggregation from the injury site was retarded in aged microglia, indicating that senescent microglial responses, while slower to initiate, are more sustained. Together, these altered features of microglial behavior at rest and following injury reveal an age-dependent dysregulation of immune response in the CNS that may illuminate microglial contributions to age-related neuroinflammatory degeneration.     

Microglia in depression: current perspectives

Major depressive disorder(MDD) is a prevalent psychiatric disease that involves malfunctions of different cell types in the brain.Accumulating studies started to reveal that microglia, the primary resident immune cells, play an important role in the development and progression of depression. Microglia respond to stress-triggered neuroinflammation, and through the release of proinflammatory cytokines and their metabolic products, microglia may modulate the function of neurons and astrocytes to regulate depression. In this review, we focused on the role of microglia in the etiology of depression. We discussed the dynamic states of microglia; the correlative and causal evidence of microglial abnormalities in depression; possible mechanisms of how microglia sense depression-related stress and modulate depression state; and how antidepressive therapies affect microglia. Understanding the role of microglia in depression may shed light on developing new treatment strategies to fight against this devastating mental illness.     

Microglia - insights into immune system structure, function, and reactivity in the central nervous system

Microglia are essential cellular components of a well-functioning central nervous system (CNS). The development and establishment of the microglial population differs from the other major cell populations in the CNS i.e. neurons and macroglia (astrocytes and oligodendrocytes). This different ontogeny gives microglia unique properties. In recent years detailed studies of the microglial population have been greatly facilitated by the use of bone marrow (BM) chimeric animals. Experimental BM transplants have provided the opportunity to trace and investigate how BM cells migrate into the CNS and settle to become microglia. Furthermore various functional properties of microglia in the normal and pathological CNS are now being revealed because of combinations of BM transplantations and experimental disease models. Here, we describe some of the latest findings in microglial biology and discuss the potential for using microglia in therapeutic interventions.     

Microglia and neuronal cell death

Microglia, the brain's innate immune cell type, are cells of mesodermal origin that populate the central nervous system (CNS) during development. Undifferentiated microglia, also called ameboid microglia, have the ability to proliferate, phagocytose apoptotic cells and migrate long distances toward their final destinations throughout all CNS regions, where they acquire a mature ramified morphological phenotype. Recent studies indicate that ameboid microglial cells not only have a scavenger role during development but can also promote the death of some neuronal populations. In the mature CNS, adult microglia have highly motile processes to scan their territorial domains, and they display a panoply of effects on neurons that range from sustaining their survival and differentiation contributing to their elimination. Hence, the fine tuning of these effects results in protection of the nervous tissue, whereas perturbations in the microglial response, such as the exacerbation of microglial activation or lack of microglial response, generate adverse situations for the organization and function of the CNS. This review discusses some aspects of the relationship between microglial cells and neuronal death/survival both during normal development and during the response to injury in adulthood.     

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.     

Regulation of microglia by ionotropic glutamatergic and GABAergic neurotransmission

Recent studies have indicated that constitutive functions of microglia in the healthy adult central nervous system (CNS) involve immune surveillance, synapse maintenance and trophic support. These functions have been related to the ramified structure of 'resting' microglia and the prominent motility in their processes that provide extensive coverage of the entire extracellular milleu. In this review, we examine how external signals, and in particular, ionotropic neurotransmission, regulate features of microglial morphology and process motility. Current findings indicate that microglial physiology in the healthy CNS is constitutively and reciprocally regulated by endogenous ionotropic glutamatergic and GABAergic neurotransmission. These influences do not act directly on microglial cells but indirectly via the activity-dependent release of ATP, likely through a mechanism involving pannexin channels. Microglia in the 'resting' state are not only dynamically active, but also constantly engaged in ongoing communication with neuronal and macroglial components of the CNS in a functionally relevant way.     

Involvement of heparanase in migration of microglial cells

Heparanase, a matrix-degrading enzyme that cleaves heparan sulfate side chains from heparan sulfate proteoglycans (HSPGs), has been shown to facilitate cell invasion, migration, and extravasation of metastatic tumor cells or immune cells. In this study, the expression and functions of heparanase were investigated using rat primary cultured microglia, the resident macrophages in the brain. The microglia were found to express heparanase mRNA and protein. Microglia treated with lipopolysaccharide (LPS) were activated, expressed induced nitric oxide synthase and elevated the expression of heparanase. Heparanase has two molecular weights: a 65 kDa latent form and an active 50 kDa. Both forms were expressed by LPS-treated activated microglia; however, untreated microglia primarily expressed the latent form. Cell lysates from microglia actually degraded Matrigel containing HSPG. Heparanase was colocalized with the actin cytoskeleton in microglial leading edges or ruffled membranes. Microglia transmigrated through a Matrigel-coated pored membrane. This process was inhibited by SF-4, a specific heparanase inhibitor, in a concentration-dependent manner. Degraded HSPG was generated when microglia transmigrated through the coated membrane, and this was also inhibited by SF-4. The results suggest the involvement of heparanase in the migration or invasion of microglia or brain macrophages across basement membrane around brain vasculature.     

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1^GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.     

Microglia arise from extra-embryonic yolk sac primitive progenitors

Microglia are the resident macrophage population of the central nervous system (CNS). Adequate microglia function is crucial for the homeostasis of the CNS in health and disease, as they represent the first line of defence against pathogens, contributing to immune responses, but are also involved in tissue repair and remodeling. It is therefore crucial to better understand microglia origin and homeostasis. Much controversy remains regarding the nature of microglial progenitors, as the exact contribution and persistence of embryonic and post-natal hematopoietic progenitors to the adult microglial pool in the steady state remained unclear. In this study, we show that post-natal hematopoietic progenitors do not significantly contribute to microglia homeostasis in the adult brain in mice. In vivo lineage tracing studies established that adult microglia derives from primitive hematopoietic progenitors that arise before embryonic day 8. These results identify microglia as an ontogenically distinct population in the mononuclear phagocyte system and have implications for the use of embryonically-derived microglial progenitors for the treatment of various brain disorders.     

Tubulin cofactor B regulates microtubule densities during microglia transition to the reactive states

Microglia are highly dynamic cells of the CNS that continuously survey the welfare of the neural parenchyma and play key roles modulating neurogenesis and neuronal cell death. In response to injury or pathogen invasion parenchymal microglia transforms into a more active cell that proliferates, migrates and behaves as a macrophage. The acquisition of these extra skills implicates enormous modifications of the microtubule and actin cytoskeletons. Here we show that tubulin cofactor B (TBCB), which has been found to contribute to various aspects of microtubule dynamics in vivo, is also implicated in microglial cytoskeletal changes. We find that TBCB is upregulated in post-lesion reactive parenchymal microglia/macrophages, in interferon treated BV-2 microglial cells, and in neonate amoeboid microglia where the microtubule densities are remarkably low. Our data demonstrate that upon TBCB downregulation both, after microglia differentiation to the ramified phenotype in vivo and in vitro, or after TBCB gene silencing, microtubule densities are restored in these cells. Taken together these observations support the view that TBCB functions as a microtubule density regulator in microglia during activation, and provide an insight into the understanding of the complex mechanisms controlling microtubule reorganization during microglial transition between the amoeboid, ramified, and reactive phenotypes.     

Expression and Contributions of TRPM7 and KCa2.3/SK3 Channels to the Increased Migration and Invasion of Microglia in Anti-Inflammatory Activation States

Microglia rapidly respond to CNS injury and disease and can assume a spectrum of activation states. While changes in gene expression and production of inflammatory mediators have been extensively described after classical (LPS-induced) and alternative (IL4-induced) microglial activation, less is known about acquired de-activation in response to IL10. It is important to understand how microglial activation states affect their migration and invasion; crucial functions after injury and in the developing CNS. We reported that LPS-treated rat microglia migrate very poorly, while IL4-treated cells migrate and invade much better. Having discovered that the lamellum of migrating microglia contains a large ring of podosomes – microscopic structures that are thought to mediate adhesion, migration and invasion – we hypothesized that IL4 and IL10 would differentially affect podosome expression, gene induction, migration and invasion. Further, based on the enrichment of the KCa2.3/SK3 Ca²⁺-activated potassium channel in microglial podosomes, we predicted that it regulates migration and invasion. We found both similarities and differences in gene induction by IL4 and IL10 and, while both cytokines increased migration and invasion, only IL10 affected podosome expression. KCa2.3 currents were recorded in microglia under all three activation conditions and KCNN3 (KCa2.3) expression was similar. Surprisingly then, of three KCa2.3 inhibitors (apamin, tamapin, NS8593), only NS8593 abrogated the increased migration and invasion of IL4 and IL10-treated microglia (and invasion of unstimulated microglia). This discrepancy was explained by the observed block of TRPM7 currents in microglia by NS8593, which occurred under all three activation conditions. A similar inhibition of both migration and invasion was seen with a TRPM7 inhibitor (AA-861) that does not block KCa2.3 channels. Thus, we conclude that TRPM7 (not KCa2.3) contributes to the enhanced ability of microglia to migrate and invade when in anti-inflammatory states. This will be an important consideration in developing TRPM7 inhibitors for treating CNS injury.     

Human NK cells kill resting but not activated microglia via NKG2D- and NKp46-mediated recognition

Microglia are resident macrophage-like APCs of the CNS. To avoid escalation of inflammatory processes and bystander damage within the CNS, microglia-driven inflammatory responses need to be tightly regulated and both spatially and temporally restricted. Following traumatic, infectious, and autoimmune-mediated brain injury, NK cells have been found in the CNS, but the functional significance of NK cell recruitment and their mechanisms of action during brain inflammation are not well understood. In this study, we investigated whether and by which mechanisms human NK cells might edit resting and activated human microglial cells via killing in vitro. IL-2-activated NK cells efficiently killed both resting allogeneic and autologous microglia in a cell-contact-dependent manner. Activated NK cells rapidly formed synapses with human microglial cells in which perforin had been polarized to the cellular interface. Ab-mediated NKG2D and NKp46 blockade completely prevented the killing of human microglia by activated NK cells. Up-regulation of MHC class I surface expression by TLR4 stimulation protected microglia from NK cell-mediated killing, whereas MHC class I blockade enhanced cytotoxic NK cell activity. These data suggest that brain-infiltrating NK cells might restrict innate and adaptive immune responses within the human CNS via elimination of resting microglia.     

Microglial phagocytosis attenuated by short-term exposure to exogenous ATP through P2X7 receptor action

Microglia, the CNS resident macrophages responsible for the clearance of degenerating cellular fragments, are essential to tissue remodeling and repair after CNS injury. ATP can be released in large amounts after CNS injury and may mediate microglial activity through the ionotropic P2X and the metabotropic P2Y receptors. This study indicates that exposure to a high concentration of ATP for 30 min rapidly induces changes of the microglial cytoskeleton, and significantly attenuates microglial phagocytosis. A pharmacological approach showed that ATP-induced inhibition of microglial phagocytotic activity was due to P2X₇R activation, rather than that of P2YR. Activation of P2X₇R by its agonist, 2'-3'- O -(4-benzoyl)benzoyl-ATP (BzATP), produced a Ca²⁺-independent reduction in microglial phagocytotic activity. In addition, the knockdown of P2X₇R expression by lentiviral-mediated shRNA interference or the blockade of P2X₇R activation by the specific antagonists, oxidized ATP (oxATP) and brilliant blue G, has efficiently restored the phagocytotic activity of ATP and BzATP-treated microglia. Our results reveal that P2X₇R activation may induce the formation of a Ca²⁺-independent signaling complex, which results in the reduction of microglial phagocytosis. This suggests that exposure to ATP for a short-term period may cause insufficient clearance of tissue debris by microglia through P2X₇R activation after CNS injury, and that blockade of this receptor may preserve the phagocytosis of microglia and facilitate CNS tissue repair.     

Dynamics of neuron-glia interplay upon exposure to unconjugated bilirubin

Microglia are the main players of the brain immune response. They act as active sensors that rapidly respond to injurious insults by shifting into different activated states. Elevated levels of unconjugated bilirubin (UCB) induce cell death, immunostimulation and oxidative stress in both neurons and astrocytes. We recently reported that microglial phagocytic phenotype precedes the release of pro-inflammatory cytokines upon UCB exposure. We investigated whether and how microglia microenvironment influences the response to UCB. Our findings revealed that conditioned media derived from UCB-treated astrocytes reduce microglial inflammatory reaction and cell death, suggesting an attempt to curtail microglial over activation. Conditioned medium from UCB-challenged neurons, although down-regulating tumor necrosis factor-α and interleukin-1β promoted the release of interleukin-6 and nitric oxide, the activation of matrix metalloproteinase-9, and cell death, as compared with UCB-direct effects on microglia. Moreover, soluble factors released by UCB-treated neurons intensified the phagocytic properties manifested by microglia under direct exposure to UCB. Results from neuron-microglia mixed cultures incubated with UCB evidenced that sensitized microglia were able to prevent neurite outgrowth impairment and cell death. In conclusion, our data indicate that stressed neurons signal microglial clearance functions, but also overstimulate its inflammatory potential ultimately leading to microglia demise.     

FimH adhesin of Escherichia coli K1 type 1 fimbriae activates BV-2 microglia

The generation of intense inflammation in the subarachnoid space in response to meningitis-causing bacteria contributes to brain dysfunction and neuronal injury in bacterial meningitis. Microglia, the major immune effector cells in the central nervous system (CNS), become activated by bacterial components to produce proinflammatory immune mediators. In this study, we showed that FimH adhesin, a tip component of type 1 fimbriae of meningitis-causing Escherichia coli K1, activated the murine microglial cell line, BV-2, which resulted in the production of nitric oxide and the release of tumor necrosis factor-alpha. Mitogen-activated protein kinases, ERK and p-38, and nuclear factor-kappaB were involved in FimH adhesin-mediated microglial activation. These findings suggest that FimH adhesin contributes to the CNS inflammatory response by virtue of activating microglia in E. coli meningitis.     

Genomic and proteomic microglial profiling: pathways for neuroprotective inflammatory responses following nerve fragment clearance and activation

Microglia, a primary immune effector cell of the central nervous system (CNS) affects homeostatic, neuroprotective, regenerative and degenerative outcomes in health and disease. Despite these broad neuroimmune activities linked to specific environmental cues, a precise cellular genetic profile for microglia in the context of disease and repair has not been elucidated. To this end we used nucleic acid microarrays, proteomics, immunochemical and histochemical tests to profile microglia in neuroprotective immune responses. Optic and sciatic nerve (ON and SN) fragments were used to stimulate microglia in order to reflect immune consequences of nervous system injury. Lipopolysaccharide and latex beads-induced microglial activation served as positive controls. Cytosolic and secreted proteins were profiled by surface enhanced laser desorption ionization-time of flight (SELDI-TOF) ProteinChip, 1D and 2D difference gel electrophoresis. Proteins were identified by peptide sequencing with tandem mass spectrometry, ELISA and western blot tests. Temporal expression of pro-inflammatory cytokines, antioxidants, neurotrophins, and lysosomal enzyme expression provided, for the first time, a unique profile of secreted microglia proteins with neuroregulatory functions. Most importantly, this molecular and biochemical signature supports a broad range of microglial functions for debris clearance and promotion of neural repair after injury.     

Lysophosphatidic acid receptor activation affects the C13NJ microglia cell line proteome leading to alterations in glycolysis,motility, and cytoskeletal architecture

Microglia, the immunocompetent cells of the CNS, are rapidly activated in response to injury and microglia migration towards and homing at damaged tissue plays a key role in CNS regeneration. Lysophosphatidic acid (LPA) is involved in signaling events evoking microglia responses through cognate G protein‐coupled receptors. Here we show that human immortalized C13NJ microglia express LPA receptor subtypes LPA₁, LPA₂, and LPA₃ on mRNA and protein level. LPA activation of C13NJ cells induced Rho and extracellular signal‐regulated kinase activation and enhanced cellular ATP production. In addition, LPA induced process retraction, cell spreading, led to pronounced changes of the actin cytoskeleton and reduced cell motility, which could be reversed by inhibition of Rho activity. To get an indication about LPA‐induced global alterations in protein expression patterns a 2‐D DIGE/LC‐ESI‐MS proteomic approach was applied. On the proteome level the most prominent changes in response to LPA were observed for glycolytic enzymes and proteins regulating cell motility and/or cytoskeletal dynamics. The present findings suggest that naturally occurring LPA is a potent regulator of microglia biology. This might be of particular relevance in the pathophysiological context of neurodegenerative disorders where LPA concentrations can be significantly elevated in the CNS.     

Hierarchical interactions among cytokines establish the functional state of microglia

Microglia rapidly respond to CNS injury yet the mechanisms leading to their activation and inactivation remain poorly defined. In particular, few studies have established how interactions among inflammatory mediators affect the innate immune response of microglia. To begin to understand the hierarchy of cytokine signalling we examined the effects of several cytokines on purified newborn and adult rat microglia in vitro, and we have examined the microglial response to injury in mice deficient in the IL‐1 type 1 receptor (IL‐1R1). Using several indices of activation, we find that IL‐1β, TNF‐α, and IL‐6 are potent microglial activators. By contrast, TGF‐β1 did not activate the cells and when TGFβ1 was administered prior to IL‐1β, it blocked the effects of IL‐1β. However, TGFβ1 was ineffective in antagonizing IL‐6. In null mice lacking the IL‐1R1, microglia inefficiently responded to injury, and IL‐6 induction was severely curtailed. These data establish a model of hierarchical signalling, whereby constitutive expression of TGF‐β1 in the CNS maintains microglia in a resting state. IL‐1, while an important microglial activator, is modifiable, whereas, the downstream cytokine, IL‐6, is a strong stimulus that is unaffected by other modifiers of the innate immune response. Acknowledgements: Supported by NMSS award #RG 3837.