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Although the roles of embryonic yolk sac-derived, resident microglia in neurodevelopment were extensively studied, the possible involvement of bone marrow-derived cells remains elusive. In this work, we used a fate-mapping strategy to selectively label bone marrow-derived cells and their progeny in the brain (FLT3+IBA1+). FLT3+IBA1+ cells were confirmed to be transiently present in the healthy brain during early postnatal development. FLT3+IBA1+ cells have a distinct morphology index at postnatal day(P)0, P7, and P14 compared with neighboring microglia. FLT3+IBA1+ cells also express the microglial markers P2RY12 and TMEM119 and interact with VGLUT1 synapses at P14. Scanning electron microscopy indeed showed that FLT3+ cells contact and engulf pre-synaptic elements. Our findings suggest FLT3+IBA1+ cells might assist microglia in their physiological functions in the developing brain including synaptic pruning which is performed using their purinergic sensors. Our findings stimulate further investigation on the involvement of peripheral macrophages during homeostatic and pathological development.
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Several mental illnesses, characterized by aberrant stress reactivity, often arise after early-life adversity (ELA). However, it is unclear how ELA affects stress-related brain circuit maturation, provoking these enduring vulnerabilities. We find that ELA increases functional excitatory synapses onto stress-sensitive hypothalamic corticotropin-releasing hormone (CRH)-expressing neurons, resulting from disrupted developmental synapse pruning by adjacent microglia. Microglial process dynamics and synaptic element engulfment were attenuated in ELA mice, associated with deficient signaling of the microglial phagocytic receptor MerTK. Accordingly, selective chronic chemogenetic activation of ELA microglia increased microglial process dynamics and reduced excitatory synapse density to control levels. Notably, selective early-life activation of ELA microglia normalized adult acute and chronic stress responses, including stress-induced hormone secretion and behavioral threat responses, as well as chronic adrenal hypertrophy of ELA mice. Thus, microglial actions during development are powerful contributors to mechanisms by which ELA sculpts the connectivity of stress-regulating neurons, promoting vulnerability to stress and stress-related mental illnesses.
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Hormônio Liberador da Corticotropina , Células-Tronco Neurais , Animais , Camundongos , Microglia/fisiologia , Neurônios/fisiologia , Sinapses/fisiologiaRESUMO
Prenatal exposure to maternal high-fat diet (mHFD) acts as a risk factor for various neurodevelopmental alterations in the progeny. Recent studies in mice revealed that mHFD results in both neuroinflammation and hypomyelination in the exposed offspring. Microglia, the brain-resident macrophages, play crucial roles during brain development, notably by modulating oligodendrocyte populations and performing phagocytosis of myelin sheaths. Previously, we reported that mHFD modifies microglial phenotype (i.e., morphology, interactions with their microenvironment, transcripts) in the hippocampus of male and female offspring. In the current study, we further explored whether mHFD may induce myelination changes among the hippocampal-corpus callosum-prefrontal cortex pathway, and result in behavioral outcomes in adolescent offspring of the two sexes. To this end, female mice were fed with control chow or HFD for 4 weeks before mating, during gestation, and until weaning of their litter. Histological and ultrastructural analyses revealed an increased density of myelin associated with a reduced area of cytosolic myelin channels in the corpus callosum of mHFD-exposed male compared to female offspring. Transcripts of myelination-associated genes including Igf1 -a growth factor released by microglia- were also lower, specifically in the hippocampus (without changes in the prefrontal cortex) of adolescent male mouse offspring. These changes in myelin were not related to an altered density, distribution, or maturation of oligodendrocytes, instead we found that microglia within the corpus callosum of mHFD-exposed offspring showed reduced numbers of mature lysosomes and increased synaptic contacts, suggesting microglial implication in the modified myelination. At the behavioral level, both male and female mHFD-exposed adolescent offspring presented loss of social memory and sensorimotor gating deficits. These results together highlight the importance of studying oligodendrocyte-microglia crosstalk and its involvement in the long-term brain alterations that result from prenatal mHFD in offspring across sexes.
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BACKGROUND: Maternal nutrition is critical for proper fetal development. While increased nutrient intake is essential during pregnancy, an excessive consumption of certain nutrients, like fat, can lead to long-lasting detrimental consequences on the offspring. Animal work investigating the consequences of maternal high-fat diet (mHFD) revealed in the offspring a maternal immune activation (MIA) phenotype associated with increased inflammatory signals. This inflammation was proposed as one of the mechanisms causing neuronal circuit dysfunction, notably in the hippocampus, by altering the brain-resident macrophages-microglia. However, the understanding of mechanisms linking inflammation and microglial activities to pathological brain development remains limited. We hypothesized that mHFD-induced inflammation could prime microglia by altering their specific gene expression signature, population density, and/or functions. METHODS: We used an integrative approach combining molecular (i.e., multiplex-ELISA, rt-qPCR) and cellular (i.e., histochemistry, electron microscopy) techniques to investigate the effects of mHFD (saturated and unsaturated fats) vs control diet on inflammatory priming, as well as microglial transcriptomic signature, density, distribution, morphology, and ultrastructure in mice. These analyses were performed on the mothers and/or their adolescent offspring at postnatal day 30. RESULTS: Our study revealed that mHFD results in MIA defined by increased circulating levels of interleukin (IL)-6 in the mothers. This phenotype was associated with an exacerbated inflammatory response to peripheral lipopolysaccharide in mHFD-exposed offspring of both sexes. Microglial morphology was also altered, and there were increased microglial interactions with astrocytes in the hippocampus CA1 of mHFD-exposed male offspring, as well as decreased microglia-associated extracellular space pockets in the same region of mHFD-exposed offspring of the two sexes. A decreased mRNA expression of the inflammatory-regulating cytokine Tgfb1 and microglial receptors Tmem119, Trem2, and Cx3cr1 was additionally measured in the hippocampus of mHFD-exposed offspring, especially in males. CONCLUSIONS: Here, we described how dietary habits during pregnancy and nurturing, particularly the consumption of an enriched fat diet, can influence peripheral immune priming in the offspring. We also found that microglia are affected in terms of gene expression signature, morphology, and interactions with the hippocampal parenchyma, in a partially sexually dimorphic manner, which may contribute to the adverse neurodevelopmental outcomes on the offspring.
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Dieta Hiperlipídica , Hipocampo/patologia , Inflamação/patologia , Fenômenos Fisiológicos da Nutrição Materna/fisiologia , Microglia/patologia , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Adolescente , Animais , Receptor 1 de Quimiocina CX3C/metabolismo , Forma Celular/fisiologia , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Inflamação/metabolismo , Interleucina-6/sangue , Lipopolissacarídeos/farmacologia , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Gravidez , Receptores Imunológicos/metabolismo , Fatores Sexuais , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Parkinson's disease (PD) is the most widespread movement disorder with a prevalence of 1 in 1000 individuals above 60 years of age. Until now, understanding the pathological mechanisms of PD to translate them into therapy has remained a high research priority. In this review, we highlight evidence describing the involvement of microglial dysfunction in PD. Thereafter, we provide current knowledge suggesting that the substantia nigra pars compacta and putamen, compared to other brain regions, show a reduced microglial density, as well as altered morphological and functional properties in homeostatic conditions, while presenting dystrophic features associated with aging. Further, we describe that this defective microglial programing emerges as early as the second postnatal week, persists until adulthood and impacts negatively on their transcriptional pattern and provision of local trophic support. We emphasize the role of α-synuclein oligomers as a major dysfunctional signal underlining microglial-mediated phenotypic switch and adaptive response contributing to neurodegeneration. Moreover, we explore available avenues should microglia be considered as target for neuroprotective or restorative strategies including preventing the aggregation of α-synuclein protofibrils formation. However, we provide a note of caution regarding the success of microglial-targeted PD strategies, using minocycline as an example. In conclusion, we discuss putative neuroprotective agents that were unsuccessful in previous trials but could be reconsidered by focusing on the stage of microglial-dependent pathogenic events during PD in suitable cohorts of patients.
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Microglia/patologia , Doença de Parkinson/patologia , Parte Compacta da Substância Negra/patologia , Putamen/patologia , Animais , Humanos , FenótipoRESUMO
BACKGROUND: Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder that affects cognitive and motor abilities by primarily targeting the striatum and cerebral cortex. HD is caused by a mutation elongating the CAG repeats within the Huntingtin gene, resulting in HTT protein misfolding. Although the genetic cause of HD has been established, the specific susceptibility of neurons within various brain structures has remained elusive. Microglia, which are the brain's resident macrophages, have emerged as important players in neurodegeneration. Nevertheless, few studies have examined their implication in HD. METHODS: To provide novel insights, we investigated the maturation and dysfunction of striatal microglia using the R6/2 mouse model of HD. This transgenic model, which presents with 120+/-5 CAG repeats, displays progressive motor deficits beginning at 6 weeks of age, with full incapacitation by 13 weeks. We studied microglial morphology, phagocytic capacity, and synaptic contacts in the striatum of R6/2 versus wild-type (WT) littermates at 3, 10, and 13 weeks of age, using a combination of light and transmission electron microscopy. We also reconstructed dendrites and determined synaptic density within the striatum of R6/2 and WT littermates, at nanoscale resolution using focused ion beam scanning electron microscopy. RESULTS: At 3 weeks of age, prior to any known motor deficits, microglia in R6/2 animals displayed a more mature morphological phenotype than WT animals. Microglia from R6/2 mice across all ages also demonstrated increased phagocytosis, as revealed by light microscopy and transmission electron microscopy. Furthermore, microglial processes from 10-week-old R6/2 mice made fewer contacts with synaptic structures than microglial processes in 3-week-old R6/2 mice and age-matched WT littermates. Synaptic density was not affected by genotype at 3 weeks of age but increased with maturation in WT mice. The location of synapses was lastly modified in R6/2 mice compared with WT controls, from targeting dendritic spines to dendritic trunks at both 3 and 10 weeks of age. CONCLUSIONS: These findings suggest that microglia may play an intimate role in synaptic alteration and loss during HD pathogenesis.
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Microglia/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Animais , Forma Celular/fisiologia , Modelos Animais de Doenças , Feminino , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Masculino , Camundongos , Camundongos Transgênicos , Microglia/patologia , Neurônios/patologia , Sinapses/patologiaRESUMO
Synapse loss is the strongest correlate for cognitive decline in Alzheimer's disease. The mechanisms underlying synapse loss have been extensively investigated using mouse models expressing genes with human familial Alzheimer's disease mutations. In this review, we summarize how multiphoton in vivo imaging has improved our understanding of synapse loss mechanisms associated with excessive amyloid in the living animal brain. We also discuss evidence obtained from these imaging studies for the role of cell-intrinsic calcium dyshomeostasis and cell-extrinsic activities of microglia, which are the immune cells of the brain, in mediating synapse loss.
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Microglia, the brain's resident macrophages, are incredibly plastic and dynamic cells. In this chapter, we aim to describe and classify the many morphological changes they can display in normal development, aging, and disease. Although microglia in healthy adult brain tissue are often ramified with small somas, they can undergo massive and rapid morphological shifts in response to stimuli, becoming amoeboid or hypertrophic. Older animals occasionally contain dystrophic, senescent, and gitter cell-like microglia, and brain injury can be accompanied by an increase in rod cells. By a careful study of microglial morphology, coupled with ultrastructural insights gleaned using electron microscopy, insights can be provided into the functions performed by these various morphological phenotypes.
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Lesões Encefálicas , Encéfalo , Microglia , Animais , Encéfalo/imunologia , Encéfalo/ultraestrutura , Lesões Encefálicas/imunologia , Lesões Encefálicas/patologia , Humanos , Microglia/imunologia , Microglia/ultraestruturaRESUMO
Studies using rewilded laboratory mice have begun to provide important clues into the complex relationship between environment, immunity, and behavior. In a recent paper, Cope and colleagues (Hippocampus, 2019) showed that exposing laboratory mice to outdoor living, either with or without peripheral worm infection, increased adult neurogenesis and had major effects on microglia, but only outdoor living coupled with worm infection increased anxiety.
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Hipocampo , Infecções por Nematoides , Animais , Ansiedade , Camundongos , Microglia , NeurogêneseRESUMO
BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disease, characterized by the deposition of extracellular fibrillar amyloid ß (fΑß) and the intracellular accumulation of neurofibrillary tangles. As AD progresses, Aß drives a robust and prolonged inflammatory response via its recognition by microglia, the brain's immune cells. Microglial reactivity to fAß plaques may impair their normal surveillance duties, facilitating synaptic loss and neuronal death, as well as cognitive decline in AD. METHODS: In the current study, we performed correlative light, transmission, and scanning electron microscopy to provide insights into microglial structural and functional heterogeneity. We analyzed microglial cell bodies and processes in areas containing fAß plaques and neuronal dystrophy, dystrophy only, or appearing healthy, among the hippocampus CA1 of 14-month-old APPSwe-PS1Δe9 mice versus wild-type littermates. RESULTS: Our quantitative analysis revealed that microglial cell bodies in the AD model mice were larger and displayed ultrastructural signs of cellular stress, especially nearby plaques. Microglial cell bodies and processes were overall less phagocytic in AD model mice. However, they contained increased fibrillar materials and non-empty inclusions proximal to plaques. Microglial cell bodies and processes in AD model mice also displayed reduced association with extracellular space pockets that contained debris. In addition, microglial processes in healthy subregions of AD model mice encircled synaptic elements more often compared with plaque-associated processes. These observations in mice were qualitatively replicated in post-mortem hippocampal samples from two patients with AD (Braak stage 5). CONCLUSION: Together, our findings identify at the ultrastructural level distinct microglial transformations common to mouse and human in association with amyloid pathology.
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Doença de Alzheimer/patologia , Microglia/patologia , Microglia/ultraestrutura , Idoso , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides , Animais , Hipocampo/patologia , Hipocampo/ultraestrutura , Humanos , CamundongosRESUMO
Sickness behavior is a set of behavioral changes induced by infections and mediated by pro-inflammatory cytokines. It is characterized by fatigue, decreased appetite and weight loss, changes in sleep patterns, cognitive functions, and lost interest in social activity. It can expedite recovery by conserving energy to mount an immune response involving innate immunity. To provide insights into microglial implication in sickness behavior with special focus on cognitive and social impairment, we investigated changes in their ultrastructure and interactions with synapses using a toxemia mouse model. Adult mice were injected with 1 mg/kg lipopolysaccharide (LPS) or saline, and assayed for signs of sickness behavior. LPS treated mice displayed reduced activity in open-field tests 24 h post-injection, while social avoidance and weight gain/loss were not significantly different between treatment groups. Microglia were investigated using electron microscopy to describe changes in their structure and function at nanoscale resolution. Microglial cell bodies and processes were investigated in the hippocampus CA1, a region responsible for learning and memory that is often impacted after peripheral LPS administration. Microglia in LPS treated animals displayed larger cell bodies as well as less complex processes at the time point examined. Strikingly, microglial processes in LPS injected animals were also more likely to contact excitatory synapses and contained more phagocytic material compared with saline injected controls. We have identified at the ultrastructural level significant changes in microglia-synapse interactions shortly after LPS administration, which draws attention to studying the roles of microglia in synaptic rewiring after inflammatory stimuli.
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For patients with incurable neurodegenerative disorders such as Huntington's (HD) and Parkinson's disease, cell transplantation has been explored as a potential treatment option. Here, we present the first clinicopathological study of a patient with HD in receipt of cell-suspension striatal allografts who took part in the NEST-UK multicenter clinical transplantation trial. Using various immunohistochemical techniques, we found a discrepancy in the survival of grafted projection neurons with respect to grafted interneurons as well as major ongoing inflammatory and immune responses to the grafted tissue with evidence of mutant huntingtin aggregates within the transplant area. Our results indicate that grafts can survive more than a decade post-transplantation, but show compromised survival with inflammation and mutant protein being observed within the transplant site. Ann Neurol 2018;84:950-956.
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Aloenxertos/patologia , Doença de Huntington/cirurgia , Acetilcolinesterase/metabolismo , Adulto , Antígenos CD/metabolismo , Encéfalo/patologia , Transplante de Tecido Encefálico/métodos , Calbindina 2/metabolismo , Humanos , Proteína Huntingtina/genética , Doença de Huntington/genética , Interneurônios/metabolismo , Interneurônios/patologia , Masculino , Microglia/metabolismo , Microglia/patologia , Proteínas do Tecido Nervoso/metabolismo , Parvalbuminas/metabolismoRESUMO
The first electron microscope was constructed in 1931. Several decades later, techniques were developed to allow the first ultrastructural analysis of microglia by transmission electron microscopy (EM). In the 50 years that followed, important roles of microglia have been identified, specifically due to the ultrastructural resolution currently available only with EM. In particular, the addition of electron-dense staining using immunohistochemical EM methods has allowed the identification of microglial cell bodies, as well as processes, which are difficult to recognize in EM, and to uncover their complex interactions with neurons and synapses. The ability to recognize neuronal, astrocytic, and oligodendrocytic compartments in the neuropil without any staining is another invaluable advantage of EM over light microscopy for studying intimate cell-cell contacts. The technique has been essential in defining microglial interactions with neurons and synapses, thus providing, among other discoveries, important insights into their roles in synaptic stripping and pruning via phagocytosis of extraneous synapses. Recent technological advances in EM including serial block-face imaging and focused-ion beam scanning EM have also facilitated automated acquisition of large tissue volumes required to reconstruct neuronal circuits in 3D at nanometer-resolution. These cutting-edge techniques which are now becoming increasingly available will further revolutionize the study of microglia across stages of the lifespan, brain regions, and contexts of health and disease. In this mini-review, we will focus on defining the distinctive ultrastructural features of microglia and the unique insights into their function that were provided by EM.
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Microglia/ultraestrutura , Microscopia Eletrônica/história , Microscopia Eletrônica/métodos , Animais , História do Século XX , História do Século XXI , Humanos , Imageamento Tridimensional/métodos , FenótipoRESUMO
Microglia, the immune cells of the central nervous system, continuously survey the brain to detect alterations and maintain tissue homeostasis. The motility of microglial processes is indicative of their surveying capacity in normal and pathological conditions. The gold standard technique to study motility involves the use of two-photon microscopy to obtain time-lapse images from brain slices or the cortex of living animals. This technique generates four dimensionally-coded images which are analyzed manually using time-consuming, non-standardized protocols. Microglial process motility analysis is frequently performed using Z-stack projections with the consequent loss of three-dimensional (3D) information. To overcome these limitations, we developed ProMoIJ, a pack of ImageJ macros that perform automatic motility analysis of cellular processes in 3D. The main core of ProMoIJ is formed by two macros that assist the selection of processes, automatically reconstruct their 3D skeleton, and analyze their motility (process and tip velocity). Our results show that ProMoIJ presents several key advantages compared with conventional manual analysis: (1) reduces the time required for analysis, (2) is less sensitive to experimenter bias, and (3) is more robust to varying numbers of processes analyzed. In addition, we used ProMoIJ to demonstrate that commonly performed 2D analysis underestimates microglial process motility, to reveal that only cells adjacent to a laser injured area extend their processes toward the lesion site, and to demonstrate that systemic inflammation reduces microglial process motility. ProMoIJ is a novel, open-source, freely-available tool which standardizes and accelerates the time-consuming labor of 3D analysis of microglial process motility.
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Imageamento Tridimensional/métodos , Microglia/citologia , Reconhecimento Automatizado de Padrão/métodos , Animais , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Lasers , Camundongos Transgênicos , Córtex Motor/citologia , Software , Córtex Somatossensorial/citologia , Córtex Somatossensorial/lesões , Córtex Somatossensorial/patologiaRESUMO
Microglia are the only immune cells that permanently reside in the central nervous system (CNS) alongside neurons and other types of glial cells. The past decade has witnessed a revolution in our understanding of their roles during normal physiological conditions. Cutting-edge techniques revealed that these resident immune cells are critical for proper brain development, actively maintain health in the mature brain, and rapidly adapt their function to physiological or pathophysiological needs. In this review, we highlight recent studies on microglial origin (from the embryonic yolk sac) and the factors regulating their differentiation and homeostasis upon brain invasion. Elegant experiments tracking microglia in the CNS allowed studies of their unique roles compared with other types of resident macrophages. Here we review the emerging roles of microglia in brain development, plasticity and cognition, and discuss the implications of the depletion or dysfunction of microglia for our understanding of disease pathogenesis. Immune activation, inflammation and various other conditions resulting in undesirable microglial activity at different stages of life could severely impair learning, memory and other essential cognitive functions. The diversity of microglial phenotypes across the lifespan, between compartments of the CNS, and sexes, as well as their crosstalk with the body and external environment, is also emphasised. Understanding what defines particular microglial phenotypes is of major importance for future development of innovative therapies controlling their effector functions, with consequences for cognition across chronic stress, ageing, neuropsychiatric and neurological diseases.
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Encéfalo/fisiologia , Cognição/fisiologia , Microglia/fisiologia , Envelhecimento/fisiologia , Animais , Encéfalo/crescimento & desenvolvimento , Homeostase , HumanosRESUMO
[This corrects the article DOI: 10.1371/journal.pbio.1002466.].
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[This corrects the article DOI: 10.1371/journal.pbio.1002466.].
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A detailed protocol is provided here to identify amyloid Aß plaques in brain sections from Alzheimer's disease mouse models before pre-embedding immunostaining (specifically for ionized calcium-binding adapter molecule 1 (IBA1), a calcium binding protein expressed by microglia) and tissue processing for electron microscopy (EM). Methoxy-X04 is a fluorescent dye that crosses the blood-brain barrier and selectively binds to ß-pleated sheets found in dense core Aß plaques. Injection of the animals with methoxy-X04 prior to sacrifice and brain fixation allows pre-screening and selection of the plaque-containing brain sections for further processing with time-consuming manipulations. This is particularly helpful when studying early AD pathology within specific brain regions or layers that may contain very few plaques, present in only a small fraction of the sections. Post-mortem processing of tissue sections with Congo Red, Thioflavin S, and Thioflavin T (or even with methoxy-X04) can label ß-pleated sheets, but requires extensive clearing with ethanol to remove excess dye and these procedures are incompatible with ultrastructural preservation. It would also be inefficient to perform labeling for Aß (and other cellular markers such as IBA1) on all brain sections from the regions of interest, only to yield a small fraction containing Aß plaques at the right location. Importantly, Aß plaques are still visible after tissue processing for EM, allowing for a precise identification of the areas (generally down to a few square millimeters) to examine with the electron microscope.
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Microglia , Placa Amiloide , Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides , Animais , Barreira Hematoencefálica , Encéfalo , Camundongos , Microscopia EletrônicaRESUMO
BACKGROUND: Organelle remodeling processes are evolutionarily conserved and involved in cell functions during development, aging, and cell death. Some endogenous and exogenous molecules can modulate these processes. Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid, has mainly been considered as a modulator of plasma membrane fluidity in brain development and aging, while DHA's role in organelle remodeling in specific neural cell types at the ultrastructural level remains largely unexplored. DHA is notably incorporated into dynamic organelles named lipid bodies (LBs). We hypothesized that DHA could attenuate the inflammatory response in lipopolysaccharide (LPS)-activated microglia by remodeling LBs and altering their functional interplay with mitochondria and other associated organelles. RESULTS: We used electron microscopy to analyze at high spatial resolution organelle changes in N9 microglial cells exposed to the proinflammogen LPS, with or without DHA supplementation. Our results revealed that DHA reverses several effects of LPS in organelles. In particular, a large number of very small and grouped LBs was exclusively found in microglial cells exposed to DHA. In contrast, LBs in LPS-stimulated cells in the absence of DHA were sparse and large. LBs formed in the presence of DHA were generally electron-dense, suggesting DHA incorporation into these organelles. The accumulation of LBs in microglial cells from mouse and human was confirmed in situ. In addition, DHA induced numerous contacts between LBs and mitochondria and reversed the frequent disruption of mitochondrial integrity observed upon LPS stimulation. Dilation of the endoplasmic reticulum lumen was also infrequent following DHA treatment, suggesting that DHA reduces oxidative stress and protein misfolding. Lipidomic analysis in N9 microglial cells treated with DHA revealed an increase in phosphatidylserine, indicating the role of this phospholipid in normalization and maintenance of physiological membrane functions. This finding was supported by a marked reduction of microglial filopodia and endosome number and significant reduction of LPS-induced phagocytosis. CONCLUSIONS: DHA attenuates the inflammatory response in LPS-stimulated microglial cells by remodeling LBs and altering their interplay with mitochondria and other associated organelles. Our findings point towards a mechanism by which omega-3 DHA participates in organelle reorganization and contributes to the maintenance of neural cell homeostasis.
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Ácidos Docosa-Hexaenoicos/farmacologia , Gotículas Lipídicas/efeitos dos fármacos , Microglia/efeitos dos fármacos , Animais , Linhagem Celular Transformada , Citocinas/metabolismo , Citocinas/ultraestrutura , Relação Dose-Resposta a Droga , Ácidos Graxos/metabolismo , Gotículas Lipídicas/ultraestrutura , Lipopolissacarídeos/farmacologia , Camundongos , Microglia/ultraestrutura , Microscopia Eletrônica de Transmissão , Organelas/efeitos dos fármacos , Organelas/ultraestrutura , Fagocitose/efeitos dos fármacos , Fatores de Tempo , Triglicerídeos/metabolismoRESUMO
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.