RESUMO
Microglia, the brain's resident myeloid cells, play central roles in brain defense, homeostasis, and disease. Using a prolonged colony-stimulating factor 1 receptor inhibitor (CSF1Ri) approach, we report an unprecedented level of microglial depletion and establish a model system that achieves an empty microglial niche in the adult brain. We identify a myeloid cell that migrates from the subventricular zone and associated white matter areas. Following CSF1Ri, these amoeboid cells migrate radially and tangentially in a dynamic wave filling the brain in a distinct pattern, to replace the microglial-depleted brain. These repopulating cells are enriched in disease-associated microglia genes and exhibit similar phenotypic and transcriptional profiles to white-matter-associated microglia. Our findings shed light on the overlapping and distinct functional complexity and diversity of myeloid cells of the CNS and provide new insight into repopulating microglia function and dynamics in the mouse brain.
Assuntos
Ventrículos Laterais/fisiologia , Microglia/fisiologia , Substância Branca/fisiologia , Animais , Encéfalo , Modelos Animais de Doenças , Homeostase , Inflamação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/citologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismoRESUMO
BACKGROUND: Microglia, the primary resident myeloid cells of the brain, play critical roles in immune defense by maintaining tissue homeostasis and responding to injury or disease. However, microglial activation and dysfunction has been implicated in a number of central nervous system (CNS) disorders, thus developing tools to manipulate and replace these myeloid cells in the CNS is of therapeutic interest. METHODS: Using whole body irradiation, bone marrow transplant, and colony-stimulating factor 1 receptor inhibition, we achieve long-term and brain-wide (~ 80%) engraftment and colonization of peripheral bone marrow-derived myeloid cells (i.e., monocytes) in the brain parenchyma and evaluated the long-term effects of their colonization in the CNS. RESULTS: Here, we identify a monocyte signature that includes an upregulation in Ccr1, Ms4a6b, Ms4a6c, Ms4a7, Apobec1, Lyz2, Mrc1, Tmem221, Tlr8, Lilrb4a, Msr1, Nnt, and Wdfy1 and a downregulation of Siglech, Slc2a5, and Ccl21a/b. We demonstrate that irradiation and long-term (~ 6 months) engraftment of the CNS by monocytes induces brain region-dependent alterations in transcription profiles, astrocytes, neuronal structures, including synaptic components, and cognition. Although our results show that microglial replacement with peripherally derived myeloid cells is feasible and that irradiation-induced changes can be reversed by the replacement of microglia with monocytes in the hippocampus, we also observe that brain-wide engraftment of peripheral myeloid cells (relying on irradiation) can result in cognitive and synaptic deficits. CONCLUSIONS: These findings provide insight into better understanding the role and complexity of myeloid cells in the brain, including their regulation of other CNS cells and functional outcomes.
Assuntos
Células da Medula Óssea/imunologia , Transplante de Medula Óssea/métodos , Encéfalo/citologia , Encéfalo/imunologia , Células Mieloides/imunologia , Animais , Medula Óssea/imunologia , Medula Óssea/efeitos da radiação , Encéfalo/efeitos da radiação , Sistema Nervoso Central/citologia , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/fisiologia , Monócitos/efeitos da radiação , Células Mieloides/efeitos da radiação , Transcrição Gênica/fisiologia , Transcrição Gênica/efeitos da radiaçãoRESUMO
Batten disease, or juvenile NCL, is a fatal neurodegenerative disorder that occurs due to mutations in the CLN3 gene. Because the function of CLN3 remains unclear, experimental therapies for JNCL have largely concentrated upon the targeting of downstream pathomechanisms. Neuron loss is preceded by localized glial activation, and in this proof-of-concept study we have investigated whether targeting this innate immune response with ibuprofen in combination with the neuroprotective agent lamotrigine improves the previously documented beneficial effects of immunosuppressants alone. Drugs were administered daily to symptomatic Cln3 -/- mice over a 3 month period, starting at 6 months of age, and their impact was assessed using both behavioral and neuropathological outcome measures. During the treatment period, the combination of ibuprofen and lamotrigine significantly improved the performance of Cln3 -/- mice on the vertical pole test, slowing the disease-associated decline, but had less of an impact upon their rotarod performance. There were also moderate and regionally dependent effects upon astrocyte activation that were most pronounced for ibuprofen alone, but there was no overt effect upon microglial activation. Administering such treatments for longer periods will enable testing for any impact upon the neuron loss that occurs later in disease progression. Given the partial efficacy of these treatments, it will be important to test further drugs of this type in order to find more effective combinations.
RESUMO
Microglia are the resident immune cell of the central nervous system (CNS), and serve to protect and maintain the local brain environment. Microglia are critically dependent on signaling through the colony-stimulating factor 1 receptor (CSF1R); administration of CSF1R inhibitors that cross the blood brain barrier (BBB) lead to the elimination of up to 99% of microglia, depending on CNS exposure and treatment duration. Once microglia are depleted, withdrawal of inhibitor stimulates repopulation of the entire CNS with new cells, conceivably enabling a therapeutic strategy for beneficial renewal of the entire microglial tissue. We have explored the kinetics and limits of this repopulation event and show that the rate of microglial repopulation is proportional to the extent of microglial depletion - greater depletion of microglia results in more rapid repopulation. Using a CSF1R inhibitor formulation that eliminates approximately 99% of microglia within 7 days, we subjected mice to multiple rounds of elimination (7 days' treatment) and repopulation (7 days' recovery) and found that the brain only has the capacity for a single complete repopulation event; subsequent elimination and CSF1R inhibitor withdrawal fail to repopulate the brain. However, if the recovery time between, or after, cycles is extended sufficiently then the brain can ultimately repopulate. These kinetic studies define the opportunities and possible limits of the remarkable renewal capacities of microglia.
Assuntos
Encéfalo/citologia , Microglia/fisiologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Aminopiridinas/farmacologia , Animais , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Perfilação da Expressão Gênica , Ontologia Genética , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Pirróis/farmacologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Microglia, the resident immune cell of the brain, can be eliminated via pharmacological inhibition of the colony-stimulating factor 1 receptor (CSF1R). Withdrawal of CSF1R inhibition then stimulates microglial repopulation, effectively replacing the microglial compartment. In the aged brain, microglia take on a "primed" phenotype and studies indicate that this coincides with age-related cognitive decline. Here, we investigated the effects of replacing the aged microglial compartment with new microglia using CSF1R inhibitor-induced microglial repopulation. With 28 days of repopulation, replacement of resident microglia in aged mice (24 months) improved spatial memory and restored physical microglial tissue characteristics (cell densities and morphologies) to those found in young adult animals (4 months). However, inflammation-related gene expression was not broadly altered with repopulation nor the response to immune challenges. Instead, microglial repopulation resulted in a reversal of age-related changes in neuronal gene expression, including expression of genes associated with actin cytoskeleton remodeling and synaptogenesis. Age-related changes in hippocampal neuronal complexity were reversed with both microglial elimination and repopulation, while microglial elimination increased both neurogenesis and dendritic spine densities. These changes were accompanied by a full rescue of age-induced deficits in long-term potentiation with microglial repopulation. Thus, several key aspects of the aged brain can be reversed by acute noninvasive replacement of microglia.
Assuntos
Envelhecimento/metabolismo , Cognição/fisiologia , Microglia/metabolismo , Neurônios/metabolismo , Animais , Contagem de Células , Forma Celular/efeitos dos fármacos , Cognição/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/genética , Inflamação/patologia , Lipopolissacarídeos/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/metabolismoRESUMO
Microglia mediate chronic neuroinflammation following central nervous system (CNS) disease or injury, and in doing so, damage the local brain environment by impairing recovery and contributing to disease processes. Microglia are critically dependent on signaling through the colony-stimulating factor 1 receptor (CSF1R) and can be eliminated via administration of CSF1R inhibitors. Resolving chronic neuroinflammation represents a universal goal for CNS disorders, but long-term microglial elimination may not be amenable to clinical use. Notably, withdrawal of CSF1R inhibitors stimulates new microglia to fully repopulate the CNS, affording an opportunity to renew this cellular compartment. To that end, we have explored the effects of acute microglial elimination, followed by microglial repopulation, in a mouse model of extensive neuronal loss. Neuronal loss leads to a prolonged neuroinflammatory response, characterized by the presence of swollen microglia expressing CD68 and CD45, as well as elevated levels of cytokines, chemokines, complement, and other inflammatory signals. These collective responses are largely resolved by microglial repopulation. Furthermore, microglial repopulation promotes functional recovery in mice, with elevated plus maze performance matching that of uninjured mice, despite the loss of 80% of hippocampal neurons. Analyses of synaptic surrogates revealed increases in PSD95 and synaptophysin puncta with microglial repopulation, suggesting that these cells sculpt and regulate the synaptic landscape. Thus, our results show that short-term microglial elimination followed by repopulation may represent a clinically feasible and novel approach to resolve neuroinflammatory events and promote brain recovery.
Assuntos
Encéfalo/fisiopatologia , Proliferação de Células/fisiologia , Encefalite/fisiopatologia , Microglia/fisiologia , Recuperação de Função Fisiológica/fisiologia , Animais , Astrócitos/patologia , Astrócitos/fisiologia , Encéfalo/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Morte Celular , Modelos Animais de Doenças , Encefalite/patologia , Encefalite/psicologia , Feminino , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Microglia/patologia , Neuroimunomodulação/fisiologia , Neurônios/patologia , Neurônios/fisiologia , Sinapses/patologia , Sinapses/fisiologiaRESUMO
Cranial irradiation for the treatment of brain cancer elicits progressive and severe cognitive dysfunction that is associated with significant neuropathology. Radiation injury in the CNS has been linked to persistent microglial activation, and we find upregulation of pro-inflammatory genes even 6 weeks after irradiation. We hypothesize that depletion of microglia in the irradiated brain would have a neuroprotective effect. Adult mice received acute head only irradiation (9 Gy) and were administered a dietary inhibitor (PLX5622) of colony stimulating factor-1 receptor (CSF1R) to deplete microglia post-irradiation. Cohorts of mice maintained on a normal and PLX5662 diet were analyzed for cognitive changes using a battery of behavioral tasks 4-6 weeks later. PLX5622 treatment caused a rapid and near complete elimination of microglia in the brain within 3 days of treatment. Irradiation of animals given a normal diet caused characteristic behavioral deficits designed to test medial pre-frontal cortex (mPFC) and hippocampal learning and memory and caused increased microglial activation. Animals receiving the PLX5622 diet exhibited no radiation-induced cognitive deficits, and exhibited near complete loss of IBA-1 and CD68 positive microglia in the mPFC and hippocampus. Our data demonstrate that elimination of microglia through CSF1R inhibition can ameliorate radiation-induced cognitive deficits in mice.
Assuntos
Comportamento Animal/efeitos da radiação , Cognição/efeitos da radiação , Irradiação Craniana , Hipocampo , Microglia/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/fisiopatologia , Neoplasias Encefálicas/radioterapia , Hipocampo/metabolismo , Hipocampo/patologia , Hipocampo/fisiopatologia , Masculino , Camundongos , Microglia/patologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismoRESUMO
In addition to amyloid-ß plaque and tau neurofibrillary tangle deposition, neuroinflammation is considered a key feature of Alzheimer's disease pathology. Inflammation in Alzheimer's disease is characterized by the presence of reactive astrocytes and activated microglia surrounding amyloid plaques, implicating their role in disease pathogenesis. Microglia in the healthy adult mouse depend on colony-stimulating factor 1 receptor (CSF1R) signalling for survival, and pharmacological inhibition of this receptor results in rapid elimination of nearly all of the microglia in the central nervous system. In this study, we set out to determine if chronically activated microglia in the Alzheimer's disease brain are also dependent on CSF1R signalling, and if so, how these cells contribute to disease pathogenesis. Ten-month-old 5xfAD mice were treated with a selective CSF1R inhibitor for 1 month, resulting in the elimination of â¼80% of microglia. Chronic microglial elimination does not alter amyloid-ß levels or plaque load; however, it does rescue dendritic spine loss and prevent neuronal loss in 5xfAD mice, as well as reduce overall neuroinflammation. Importantly, behavioural testing revealed improvements in contextual memory. Collectively, these results demonstrate that microglia contribute to neuronal loss, as well as memory impairments in 5xfAD mice, but do not mediate or protect from amyloid pathology.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/prevenção & controle , Peptídeos beta-Amiloides/metabolismo , Microglia/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Doença de Alzheimer/patologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Feminino , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Placa Amiloide/prevenção & controleRESUMO
BACKGROUND: Microglia are dependent upon colony-stimulating factor 1 receptor (CSF1R) signaling for their survival in the adult brain, with administration of the dual CSF1R/c-kit inhibitor PLX3397 leading to the near-complete elimination of all microglia brainwide. Here, we determined the dose-dependent effects of a specific CSF1R inhibitor (PLX5622) on microglia in both wild-type and the 3xTg-AD mouse model of Alzheimer's disease. METHODS: Wild-type mice were treated with PLX5622 for up to 21 days, and the effects on microglial numbers were assessed. 3xTg-AD mice were treated with PLX5622 for 6 or 12 weeks and effects on microglial numbers and pathology subsequently assessed. RESULTS: High doses of CSF1R inhibitor eliminate most microglia from the brain, but a 75% lower-dose results in sustained elimination of ~30 of microglia in both wild-type and 3xTg-AD mice. No behavioral or cognitive deficits were found in mice either depleted of microglia or treated with lower CSF1R inhibitor concentrations. Aged 3xTg-AD mice treated for 6 or 12 weeks with lower levels of PLX5622 resulted in improved learning and memory. Aß levels and plaque loads were not altered, but microglia in treated mice no longer associated with plaques, revealing a role for the CSF1R in the microglial reaction to plaques, as well as in mediating cognitive deficits. CONCLUSIONS: We find that inhibition of CSF1R alone is sufficient to eliminate microglia and that sustained microglial elimination is concentration-dependent. Inhibition of the CSF1R at lower levels in 3xTg-AD mice prevents microglial association with plaques and improves cognition.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/psicologia , Transtornos Cognitivos/tratamento farmacológico , Microglia/patologia , Placa Amiloide/patologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Doença de Alzheimer/patologia , Animais , Ansiedade/psicologia , Encéfalo/efeitos dos fármacos , Contagem de Células , Linhagem Celular , Quimiotaxia/efeitos dos fármacos , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/psicologia , Relação Dose-Resposta a Droga , Humanos , Aprendizagem/efeitos dos fármacos , Memória/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacosRESUMO
The colony-stimulating factor 1 receptor (CSF1R) is a key regulator of myeloid lineage cells. Genetic loss of the CSF1R blocks the normal population of resident microglia in the brain that originates from the yolk sac during early development. However, the role of CSF1R signaling in microglial homeostasis in the adult brain is largely unknown. To this end, we tested the effects of selective CSF1R inhibitors on microglia in adult mice. Surprisingly, extensive treatment results in elimination of â¼99% of all microglia brain-wide, showing that microglia in the adult brain are physiologically dependent upon CSF1R signaling. Mice depleted of microglia show no behavioral or cognitive abnormalities, revealing that microglia are not necessary for these tasks. Finally, we discovered that the microglia-depleted brain completely repopulates with new microglia within 1 week of inhibitor cessation. Microglial repopulation throughout the CNS occurs through proliferation of nestin-positive cells that then differentiate into microglia.