Microglial phagocytosis dysfunction in the dentate gyrus is related to local neuronal activity in a genetic model of epilepsy.

OBJECTIVE: Microglial phagocytosis of apoptotic cells is an essential component of the brain regenerative response during neurodegeneration. Whereas it is very efficient in physiological conditions, it is impaired in mouse and human mesial temporal lobe epilepsy, and now we extend our studies to a model of progressive myoclonus epilepsy type 1 in mice lacking cystatin B (CSTB). METHODS: We used confocal imaging and stereology-based quantification of apoptosis and phagocytosis of the hippocampus of Cstb knockout (KO) mice, an in vitro model of phagocytosis and siRNAs to acutely reduce Cstb expression, and a virtual three-dimensional (3D) model to analyze the physical relationship between apoptosis, phagocytosis, and active hippocampal neurons. RESULTS: Microglial phagocytosis was impaired in the hippocampus of Cstb KO mice at 1 month of age, when seizures arise and hippocampal atrophy begins. This impairment was not related to the lack of Cstb in microglia alone, as shown by in vitro experiments with microglial Cstb depletion. The phagocytosis impairment was also unrelated to seizures, as it was also present in Cstb KO mice at postnatal day 14, before seizures begin. Importantly, phagocytosis impairment was restricted to the granule cell layer and spared the subgranular zone, where there are no active neurons. Furthermore, apoptotic cells (both phagocytosed and not phagocytosed) in Cstb-deficient mice were at close proximity to active cFos+ neurons, and a virtual 3D model demonstrated that the physical relationship between apoptotic cells and cFos+ neurons was specific for Cstb KO mice. SIGNIFICANCE: These results suggest a complex crosstalk between apoptosis, phagocytosis, and neuronal activity, hinting that local neuronal activity could be related to phagocytosis dysfunction in Cstb KO mice. Overall, these data suggest that phagocytosis impairment is an early feature of hippocampal damage in epilepsy and opens novel therapeutic approaches for epileptic patients based on targeting microglial phagocytosis.

Microglia Actively Remodel Adult Hippocampal Neurogenesis through the Phagocytosis Secretome.

During adult hippocampal neurogenesis, most newborn cells undergo apoptosis and are rapidly phagocytosed by resident microglia to prevent the spillover of intracellular contents. Here, we propose that phagocytosis is not merely passive corpse removal but has an active role in maintaining neurogenesis. First, we found that neurogenesis was disrupted in male and female mice chronically deficient for two phagocytosis pathways: the purinergic receptor P2Y12, and the tyrosine kinases of the TAM family Mer tyrosine kinase (MerTK)/Axl. In contrast, neurogenesis was transiently increased in mice in which MerTK expression was conditionally downregulated. Next, we performed a transcriptomic analysis of the changes induced by phagocytosis in microglia in vitro and identified genes involved in metabolism, chromatin remodeling, and neurogenesis-related functions. Finally, we discovered that the secretome of phagocytic microglia limits the production of new neurons both in vivo and in vitro Our data suggest that microglia act as a sensor of local cell death, modulating the balance between proliferation and survival in the neurogenic niche through the phagocytosis secretome, thereby supporting the long-term maintenance of adult hippocampal neurogenesis.SIGNIFICANCE STATEMENT Microglia are the brain professional phagocytes and, in the adult hippocampal neurogenic niche, they remove newborn cells naturally undergoing apoptosis. Here we show that phagocytosis of apoptotic cells triggers a coordinated transcriptional program that alters their secretome, limiting neurogenesis both in vivo and in vitro In addition, chronic phagocytosis disruption in mice deficient for receptors P2Y12 and MerTK/Axl reduces adult hippocampal neurogenesis. In contrast, inducible MerTK downregulation transiently increases neurogenesis, suggesting that microglial phagocytosis provides a negative feedback loop that is necessary for the long-term maintenance of adult hippocampal neurogenesis. Therefore, we speculate that the effects of promoting engulfment/degradation of cell debris may go beyond merely removing corpses to actively promoting regeneration in development, aging, and neurodegenerative diseases.

Quantifying Microglial Phagocytosis of Apoptotic Cells in the Brain in Health and Disease.

Apoptosis is a ubiquitous process occurring in the brain under both physiological and pathological conditions. The central nervous system (CNS) requires an active and efficient clean-up system to prevent the spillover of intracellular contents into the surrounding parenchyma and suppress the initiation of inflammatory and immune responses. Microglia, the resident professional phagocytes of the brain, are the cells in charge of the removal of these dead cells by the process named phagocytosis. Therefore, microglial phagocytosis is a vital mechanism to maintain tissue homeostasis. Traditionally, this process has been assessed using indirect methods, which have resulted in poor or inaccurate estimations of microglial phagocytosis efficiency. In these protocols, we describe a series of parameters to directly quantify microglial phagocytosis efficiency in vivo and in vitro. © 2018 by John Wiley & Sons, Inc.

Clearing the corpses: regulatory mechanisms, novel tools, and therapeutic potential of harnessing microglial phagocytosis in the diseased brain.

Apoptosis is a widespread phenomenon that occurs in the brain in both physiological and pathological conditions. Dead cells must be quickly removed to avoid the further toxic effects they exert in the parenchyma, a process executed by microglia, the brain professional phagocytes. Although phagocytosis is critical to maintain tissue homeostasis, it has long been either overlooked or indirectly assessed based on microglial morphology, expression of classical activation markers, or engulfment of artificial phagocytic targets in vitro. Nevertheless, these indirect methods present several limitations and, thus, direct observation and quantification of microglial phagocytosis is still necessary to fully grasp its relevance in the diseased brain. To overcome these caveats and obtain a comprehensive, quantitative picture of microglial phagocytosis we have developed a novel set of parameters. These parameters have allowed us to identify the different strategies utilized by microglia to cope with apoptotic challenges induced by excitotoxicity or inflammation. In contrast, we discovered that in mouse and human epilepsy microglia failed to find and engulf apoptotic cells, resulting in accumulation of debris and inflammation. Herein, we advocate that the efficiency of microglial phagocytosis should be routinely tested in neurodegenerative and neurological disorders, in order to determine the extent to which it contributes to apoptosis and inflammation found in these conditions. Finally, our findings point towards enhancing microglial phagocytosis as a novel therapeutic strategy to control tissue damage and inflammation, and accelerate recovery in brain diseases.

Correction: Neuronal Hyperactivity Disturbs ATP Microgradients, Impairs Microglial Motility, and Reduces Phagocytic Receptor Expression Triggering Apoptosis/Microglial Phagocytosis Uncoupling.

[This corrects the article DOI: 10.1371/journal.pbio.1002466.].

Correction: Neuronal Hyperactivity Disturbs ATP Microgradients, Impairs Microglial Motility, and Reduces Phagocytic Receptor Expression Triggering Apoptosis/Microglial Phagocytosis Uncoupling.

[This corrects the article DOI: 10.1371/journal.pbio.1002466.].

Neuronal Hyperactivity Disturbs ATP Microgradients, Impairs Microglial Motility, and Reduces Phagocytic Receptor Expression Triggering Apoptosis/Microglial Phagocytosis Uncoupling.

Phagocytosis is essential to maintain tissue homeostasis in a large number of inflammatory and autoimmune diseases, but its role in the diseased brain is poorly explored. Recent findings suggest that in the adult hippocampal neurogenic niche, where the excess of newborn cells undergo apoptosis in physiological conditions, phagocytosis is efficiently executed by surveillant, ramified microglia. To test whether microglia are efficient phagocytes in the diseased brain as well, we confronted them with a series of apoptotic challenges and discovered a generalized response. When challenged with excitotoxicity in vitro (via the glutamate agonist NMDA) or inflammation in vivo (via systemic administration of bacterial lipopolysaccharides or by omega 3 fatty acid deficient diets), microglia resorted to different strategies to boost their phagocytic efficiency and compensate for the increased number of apoptotic cells, thus maintaining phagocytosis and apoptosis tightly coupled. Unexpectedly, this coupling was chronically lost in a mouse model of mesial temporal lobe epilepsy (MTLE) as well as in hippocampal tissue resected from individuals with MTLE, a major neurological disorder characterized by seizures, excitotoxicity, and inflammation. Importantly, the loss of phagocytosis/apoptosis coupling correlated with the expression of microglial proinflammatory, epileptogenic cytokines, suggesting its contribution to the pathophysiology of epilepsy. The phagocytic blockade resulted from reduced microglial surveillance and apoptotic cell recognition receptor expression and was not directly mediated by signaling through microglial glutamate receptors. Instead, it was related to the disruption of local ATP microgradients caused by the hyperactivity of the hippocampal network, at least in the acute phase of epilepsy. Finally, the uncoupling led to an accumulation of apoptotic newborn cells in the neurogenic niche that was due not to decreased survival but to delayed cell clearance after seizures. These results demonstrate that the efficiency of microglial phagocytosis critically affects the dynamics of apoptosis and urge to routinely assess the microglial phagocytic efficiency in neurodegenerative disorders.

Surveillance, phagocytosis, and inflammation: how never-resting microglia influence adult hippocampal neurogenesis.

Microglia cells are the major orchestrator of the brain inflammatory response. As such, they are traditionally studied in various contexts of trauma, injury, and disease, where they are well-known for regulating a wide range of physiological processes by their release of proinflammatory cytokines, reactive oxygen species, and trophic factors, among other crucial mediators. In the last few years, however, this classical view of microglia was challenged by a series of discoveries showing their active and positive contribution to normal brain functions. In light of these discoveries, surveillant microglia are now emerging as an important effector of cellular plasticity in the healthy brain, alongside astrocytes and other types of inflammatory cells. Here, we will review the roles of microglia in adult hippocampal neurogenesis and their regulation by inflammation during chronic stress, aging, and neurodegenerative diseases, with a particular emphasis on their underlying molecular mechanisms and their functional consequences for learning and memory.