Heterogeneity of microglia and TNF signaling as determinants for neuronal death or survival

Microglia do not constitute a single, uniform cell population, but rather comprise cells with varied phenotypes, some which are beneficial and others that may require active regulatory control. Thus, gaining a better understanding of the heterogeneity of resident microglia responses will contribute to any interpretation regarding the impact of any such response in the brain. Microglia are the primary source of the pro-inflammatory cytokine, tumor necrosis factor (TNF) that can initiate various effects through the activation of membrane receptors. The TNF p55 receptor contains a death domain and activation normally leads to cellular apoptosis; however, under specific conditions, receptor activation can also lead to the activation of NF-κB and contribute to cell survival. These divergent outcomes have been linked to receptor localization with receptor internalization leading to cell death and membrane localization supporting cell survival. A second TNF receptor, TNF p75 receptor, is normally linked to cell growth and survival, however, it can cooperate with the p55 receptor and contribute to cell death. Thus, while an elevation in TNFα in the brain is often considered an indicator of microglia activation and neuroinflammation, a number of factors come into play to determine the final outcome. Data are reviewed demonstrating that heterogeneity in morphological response of microglia and the expression of TNFα and TNF receptors are critical in identifying and characterizing neurotoxic events as they relate to neuroinflammation, neuronal damage and in stimulating neuroprotection.     

P2X4 receptors control the fate and survival of activated microglia

Microglia, the resident immune cells of the central nervous system, responds to brain disarrangements by becoming activated to contend with brain damage. Here we show that the expression of P2X4 receptors is upregulated in inflammatory foci and in activated microglia in the spinal cord of rats with experimental autoimmune encephalomyelitis (EAE) as well as in the optic nerve of multiple sclerosis patients. To study the role of P2X4 receptors in microgliosis, we activated microglia with LPS in vitro and in vivo. We observed that P2X4 receptor activity in vitro was increased in LPS‐activated microglia as assessed by patch‐clamp recordings. In addition, P2X4 receptor blockade significantly reduced microglial membrane ruffling, TNFα secretion and morphological changes, as well as LPS‐induced microglial cell death. Accordingly, neuroinflammation provoked by LPS injection in vivo induced a rapid microglial loss in the spinal cord that was totally prevented or potentiated by P2X4 receptor blockade or facilitation, respectively. Within the brain, microglia in the hippocampal dentate gyrus showed particular vulnerability to LPS‐induced neuroinflammation. Thus, microglia processes in this region retracted as early as 2 h after injection of LPS and died around 24 h later, two features which were prevented by blocking P2X4 receptors. Together, these data suggest that P2X4 receptors contribute to controlling the fate of activated microglia and its survival.GLIA 2014;62:171–184     

The effect of microglia on embryonic dopaminergic neuronal survival in vitro: diffusible signals from neurons and glia change microglia from neurotoxic to neuroprotective

When embryonic dopaminergic neurons are transplanted into the adult brain, approximately 95% die within a few days. To assess whether microglia activated during transplantation might be responsible for this rapid death, we examined the effect of microglia on rat embryonic dopaminergic neurons in vitro. Conditioned medium from 7-day-old microglia was found to decrease the number of dopamine neurons surviving in primary culture, but activation of the microglia with N-formyl-methionyl-leucyl-phenylalanine (FMLP) or Zymosan A did not increase the toxicity of the conditioned medium. We next tested the effect of coculturing microglia and dopaminergic neurons by placing microglia in semipermeable well inserts over the neuronal cultures. The presence of microglia now increased dopaminergic neuronal survival, microglial activation again having no effect. To increase yet further the possible interactions between microglia and neurons, the mesencephalic cells and microglia were mixed together and placed as a tissue in three-dimensional culture, and here again the presence of microglia increased dopaminergic neuronal survival with no effect of activation. Contact of microglia with the mesencephalic cells therefore converted them from being toxic to dopaminergic neurons to promoting their survival. The change in microglial effect from toxic to protective was caused by soluble molecules secreted by cells in the neuronal cultures, as conditioned medium derived from microglia-neuronal cocultures also had a dopaminergic neuron survival effect, indicating that microglia in cocultures behave differently from microglia removed from neuronal and glial influence. Microglia cocultured with either neurons or astrocytes downregulated inducible nitric oxide synthase (iNOS), indicating a decrease in the production of nitric oxide and possibly other toxic molecules. These findings indicate that in their natural environment, microglia are likely to be beneficial for the survival of embryonic dopaminergic grafts.     

Reactive retinal microglia,neuronal survival,and the formation of retinal folds and detachments

Reactive microglia and macrophages are prevalent in damaged retinas. Accordingly, we investigate how the activation or ablation of microglia/macrophages influences the survival of neurons in the chick retina in vivo. We applied intraocular injections of interleukin 6 (IL6) to stimulate the reactivity of microglia/macrophages and clodronate‐liposomes to ablate microglia/macrophages. Activation of the microglia/macrophages with IL6 delays the death of retinal neurons from N‐methyl‐D‐aspartate (NMDA) ‐induced excitotoxicity. In addition, activation of microglia/macrophages combined with colchicine‐mediated retinal damage diminished the survival of ganglion cells. Application of IL6 after an excitotoxic insult greatly exacerbates the damage, and causes widespread retinal detachments and folds, accompanied by accumulation of microglia/macrophages in the subretinal space. Damage‐induced retinal folds and detachments were significantly reduced by the ablation of microglia/macrophages. We conclude that microglial reactivity is detrimental to the survival of ganglion cells in colchicine‐damaged retinas and detrimental to the survival of photoreceptors in retinal folds. In addition, we conclude that IL6‐treatment transiently protects amacrine and bipolar cells against an excitotoxic insult. We propose that suppressing reactivity of microglia/macrophages may be an effective means to lessen the damage and vision loss resulting from damage, in particular during retinal detachment injuries. GLIA 2015;63:313–327     

Shifting equilibriums in Alzheimer's disease: the complex roles of microglia in neuroinflammation,neuronal survival and neurogenesis

Alzheimer's disease is the leading cause of dementia.Its increased prevalence in developed countries,due to the sharp rise in ageing populations,presents one of the costliest challenges to modern medicine.In order to find disease-modifying therapies to confront this challenge,a more complete understanding of the pathogenesis of Alzheimer's disease is necessary.Recent studies have revealed increasing evidence for the roles played by microglia,the resident innate immune system cells of the brain.Reflecting the well-established roles of microglia in reacting to pathogens and inflammatory stimuli,there is now a growing literature describing both protective and detrimental effects for individual cytokines and chemokines produced by microglia in Alzheimer's disease.A smaller but increasing number of studies have also addressed the divergent roles played by microglial neurotrophic and neurogenic factors,and how their perturbation may play a key role in the pathogenesis of Alzheimer's disease.Here we review recent findings on the roles played by microglia in neuroinflammation,neuronal survival and neurogenesis in Alzheimer's disease.In each case,landmark studies have provided evidence for the divergent ways in which microglia can either promote neuronal function and survival,or perturb neuronal function,leading to cell death.In many cases,the secreted molecules of microglia can lead to divergent effects depending on the magnitude and context of microglial activation.This suggests that microglial functions must be maintained in a fine equilibrium,in order to support healthy neuronal function,and that the cellular microenvironment in the Alzheimer's disease brain disrupts this fine balance,leading to neurodegeneration.Thus,an understanding of microglial homeostasis,both in health and across the trajectory of the disease state,will improve our understanding of the pathogenic mechanisms underlying Alzheimer's disease,and will hopefully lead to the development of microglial-based therapeutic strategies to restore equilibrium in the Alzheimer's disease brain.     

Respiratory and metabolic acidosis differentially affect the respiratory neuronal network in the ventral medulla of neonatal rats

Two respiratory‐related areas, the para‐facial respiratory group/retrotrapezoid nucleus (pFRG/RTN) and the pre‐Bötzinger complex/ventral respiratory group (preBötC/VRG), are thought to play key roles in respiratory rhythm. Because respiratory output patterns in response to respiratory and metabolic acidosis differ, we hypothesized that the responses of the medullary respiratory neuronal network to respiratory and metabolic acidosis are different. To test these hypotheses, we analysed respiratory‐related activity in the pFRG/RTN and preBötC/VRG of the neonatal rat brainstem–spinal cord in vitro by optical imaging using a voltage‐sensitive dye, and compared the effects of respiratory and metabolic acidosis on these two populations. We found that the spatiotemporal responses of respiratory‐related regional activities to respiratory and metabolic acidosis are fundamentally different, although both acidosis similarly augmented respiratory output by increasing respiratory frequency. PreBötC/VRG activity, which is mainly inspiratory, was augmented by respiratory acidosis. Respiratory‐modulated pixels increased in the preBötC/VRG area in response to respiratory acidosis. Metabolic acidosis shifted the respiratory phase in the pFRG/RTN; the pre‐inspiratory dominant pattern shifted to inspiratory dominant. The responses of the pFRG/RTN activity to respiratory and metabolic acidosis are complex, and involve either augmentation or reduction in the size of respiratory‐related areas. Furthermore, the activation pattern in the pFRG/RTN switched bi‐directionally between pre‐inspiratory/inspiratory and post‐inspiratory. Electrophysiological study supported the results of our optical imaging study. We conclude that respiratory and metabolic acidosis differentially affect activities of the pFRG/RTN and preBötC/VRG, inducing switching and shifts of the respiratory phase. We suggest that they differently influence the coupling states between the pFRG/RTN and preBötC/VRG.     

The cellular response in neuroinflammation: The role of leukocytes, microglia and astrocytes in neuronal death and survival

Neuroinflammation is a complex integration of the responses of all cells present within the CNS, including the neurons, macroglia, microglia and the infiltrating leukocytes. The initiating insult, environmental factors, genetic background and age/past experiences all combine to modulate the integrated response of this complex neuroinflammatory circuit. Here, we explore how these factors interact to lead to either neuroprotective versus neurotoxic inflammatory responses. We specifically focus on microglia and astrocytic regulation of autoreactive T cell responses.     

Phagocytosis of apoptotic inflammatory cells by microglia and modulation by different cytokines: mechanism for removal of apoptotic cells in the inflamed nervous system

Apoptosis of autoaggressive T cells in the central nervous system (CNS) is an effective, nonphlogistic mechanism for the termination of autoimmune inflammation in experimental autoimmune encephalomyelitis (EAE). The clearance of apoptotic leukocytes by tissue-specific phagocytes is a critical event in the resolution of the inflammatory attack. To investigate the role of microglia in the removal of apoptotic cells and potential regulatory mechanisms of microglial phagocytosis, an in vitro phagocytosis assay was established, using Lewis rat microglia. Microglia exhibited a high capacity for the uptake of apoptotic autologous thymocytes, as well as apoptotic encephalitogenic myelin basic protein (MBP)-specific T cells, in contrast to nonapoptotic target cells. Pretreatment of microglia with interferon-gamma (IFN-gamma) raised the proportion of microglia capable of phagocytosing apoptotic cells to 75% above the untreated controls. The increased phagocytic activity was selective for apoptotic target cells and was not dependent on phosphatidylserine-mediated recognition mechanisms. In contrast, preincubation of microglia with interleukin-4 (IL-4) inhibited the uptake of apoptotic cells, whereas tumor-necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) did not alter phagocytosis. Phagocytic clearance of apoptotic inflammatory cells by microglia may be an important mechanism for the termination of autoimmune inflammation in the CNS. Augmentation of microglial phagocytosis by the Th-1-type cytokine IFN-gamma suggests a feedback mechanism for the accelerated clearance of the inflammatory infiltrate in the CNS.     

Regulation of prostaglandin E2 synthase expression in activated primary rat microglia: evidence for uncoupled regulation of mPGES-1 and COX-2

Prostaglandin E2 (PGE2) is among the most important mediators involved in neuroinflammatory processes. The final step of its synthesis is regulated by enzymes termed prostaglandin E2 synthases (PGES). Three PGES are known, cytosolic (c)PGES, membrane-associated (m)PGES-1 and mPGES-2. The expression of mPGES-1 is induced by inflammatory stimuli such as lipopolysaccharide (LPS), interleukin (IL)-1beta, and tumor necrosis factor (TNF)-alpha. Although some roles of mPGES-1 have already been suggested, its function in the CNS and the signaling pathways involved in its upregulation are poorly understood. In this study, we examined the regulation of mPGES-1 in primary rat microglia and the signaling pathways involved in its expression. Whereas the expression of cPGES and mPGES-2 was not stimulated by LPS, low doses of LPS (0.1-1 ng/mL) sufficiently stimulated mPGES-1 mRNA expression. A corresponding protein synthesis, however, was obtained only with higher doses (10-100 ng/mL). The LPS-induced increase of mPGES-1 was inhibited by different signaling pathway inhibitors, such as SP600125, LY294002, GF109203X, and SC-514, suggesting the involvement of c-Jun N-terminal kinase (JNK), phosphatidylinositol 3-kinase (PI-3K)/Akt, protein kinase C (PKC) pathways, and the nuclear factor (NF)-kappaB, respectively. In contrast to other reports, LPS-induced mPGES-1 synthesis was not invariably coupled to the synthesis of COX-2, since inhibition of PI-3K with LY294002 decreased mPGES-1 but increased COX-2 levels. This detailed view of the intracellular signaling pathways involved in mPGES-1 expression in activated microglia opens a new avenue in the search for novel potential therapeutic targets to reduce neuroinflammation, and demonstrates that mPGES-1 expression is not strictly coupled to the expression of COX-2.     

Impact of IVIg on the interaction between activated T cells and microglia

When human microglia are co-cultured with activated human T lymphocytes, several cytokines become up-regulated in significant quantities. This condition can also occur at sites of inflammation in autoimmune inflammatory diseases of the central nervous system (CNS), including multiple sclerosis (MS), where T cells infiltrate the brain tissue and come in proximity to microglia. Therefore, T cell-microglia interaction is a potential avenue of drug therapy to decrease neuroinflammation. An immunomodulator used in autoimmune disorders is intravenous immunoglobulins (IVIg). The mechanisms of IVIg activity in diseases such as MS remain unclear. Here, we report that the application of IVIg to activated T cells leads to their decreased ability to engage microglia. As a result of IVIg treatment of T cells, there were reduced levels of tumor necrosis factor-alpha a and interleukin-10 in T cell-microglia co-culture. Our results add to the understanding of how IVIg may affect inflammation of the CNS.     

Evidence for the presence of N-acetylaspartylglutamate in cultured oligodendrocytes and LPS activated microglia

The levels of N-acetylaspartylglutamate (NAAG) were determined by HPLC in untreated or lipopolysaccharide (LPS) activated pure astrocyte, oligodendrocyte, and microglial cultures derived from developing rat brain. Oligodendrocyte cultures expressed 1.52±0.12 nmol/μg protein of NAAG, whereas astrocyte cultures (0.04±0.08 nmol/μg protein) and untreated microglial cultures (0.05±0.09 nmol/μg protein) contained only trace amounts of the dipeptide. After stimulation of microglial cultures for 24 h with LPS, NAAG levels increased significantly to 0.37±0.12 SD nmol/μg protein. NAAG levels in astrocyte and oligodendrocyte cultures remained unchanged after LPS treatment. The findings indicate that NAAG is localized to specific glial cell types. Further our results suggest that NAAG biosynthesis is induced in microglia, activated by specific stimuli.     

Time dependence of aequorin-indicated calcium levels in stimulated and unstimulated platelets: evidence for multiple aequorin environments in platelets

Aequorin-induced calcium signals were examined in human unstimulated platelets and platelets stimulated with various agonists as a function of time. The total aequorin response in unstimulated platelets, obtained by Triton x-100 lysis in the presence of 1 mM Ca, decreased in a distinctly non-linear manner over 20-60 min. This decrease was slightly, but significantly, greater in platelets maintained in the continuous presence of 1 mM Ca than in platelets maintained without external Ca, and could not be accounted for completely by leakage of aequorin from the cells. Basal Ca levels in unstimulated platelets also decreased in a non-linear manner, with a similar sensitivity to the continuous presence or absence of external Ca. These observed changes in aequorin response thus appear to be at least partially due to an intracellular discharge of aequorin, and are therefore consistent with the view that aequorin in platelets is heterogeneously distributed among localized environments differing in Ca concentration. The aequorin signals observed initially in platelets stimulated by ADP or epinephrine were lost completely over a period of 30-60 min in almost all cases studied, while initial rates of aggregation were either unchanged (epinephrine) or only partially decreased (ADP) over this same time period. In contrast, thrombin- and A23187-induced aequorin signals were virtually unchanged over periods up to 90 min. Minimal changes with time also occurred in the aequorin signals induced by phorbol ester or by collagen in the presence of indomethacin. These differences in time dependence suggest that the signals generated by ADP and epinephrine may derive from different sources of aequorin than those associated with the signals induced by other agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effective connectivity during processing of facial affect: evidence for multiple parallel pathways

The perception of facial affect engages a distributed cortical network. We used functional magnetic resonance imaging and dynamic causal modeling to characterize effective connectivity during explicit (conscious) categorization of affective stimuli in the human brain. Specifically, we examined the modulation of connectivity from posterior regions of the face-processing network to the lateral ventral prefrontal cortex (VPFC) during affective categorization and we tested for a potential role of the amygdala (AMG) in mediating this modulation. We found that explicit processing of facial affect led to prominent modulation (increase) in the effective connectivity from the inferior occipital gyrus (IOG) to the VPFC, while there was less evidence for modulation of the afferent connections from fusiform gyrus and AMG to VPFC. More specifically, the forward connection from IOG to the VPFC exhibited a selective increase under anger (as opposed to fear or sadness). Furthermore, Bayesian model comparison suggested that the modulation of afferent connections to the VPFC was mediated directly by facial affect, as opposed to an indirect modulation mediated by the AMG. Our results thus suggest that affective information is conveyed to the VPFC along multiple parallel pathways and that AMG activity is not sufficient to account for the gating of information transfer to the VPFC during explicit emotional processing.