Many risk genes for the development of Alzheimer’s disease (AD) are exclusively or highly expressed in myeloid cells. Microglia are dependent on colony-stimulating factor 1 receptor (CSF1R) signaling for their survival. We designed and synthesized a highly selective brain-penetrant CSF1R inhibitor (PLX5622) allowing for extended and specific microglial elimination, preceding and during pathology development. We find that in the 5xFAD mouse model of AD, plaques fail to form in the parenchymal space following microglial depletion, except in areas containing surviving microglia. Instead, Aβ deposits in cortical blood vessels reminiscent of cerebral amyloid angiopathy. Altered gene expression in the 5xFAD hippocampus is also reversed by the absence of microglia. Transcriptional analyses of the residual plaque-forming microglia show they exhibit a disease-associated microglia profile. Collectively, we describe the structure, formulation, and efficacy of PLX5622, which allows for sustained microglial depletion and identify roles of microglia in initiating plaque pathogenesis.

BackgroundThe brain’s initial innate response to stroke is primarily mediated by microglia, the resident macrophage of the CNS. However, as early as 4 h after stroke, the blood–brain barrier is compromised and monocyte infiltration occurs. The lack of discriminating markers between these two myeloid populations has led many studies to generate conclusions based on the grouping of these two populations. A growing body of evidence now supports the distinct roles played by microglia and monocytes in many disease models.MethodsUsing a flow cytometry approach, combined with ex-vivo functional assays, we were able to distinguish microglia from monocytes using the relative expression of CD45 and assess the function of each cell type following stroke over the course of 7 days.ResultsWe found that at 72 h after a 90-min middle cerebral artery occlusion (MCAO), microglia populations decrease whereas monocytes significantly increase in the stroke brain compared to sham. After stroke, BRDU incorporation into monocytes in the bone marrow increased. After recruitment to the ischemic brain, these monocytes accounted for nearly all BRDU-positive macrophages. Inflammatory activity peaked at 72 h. Microglia produced relatively higher reactive oxygen species and TNF, whereas monocytes were the predominant IL-1β producer. Although microglia showed enhanced phagocytic activity after stroke, monocytes had significantly higher phagocytic capacity at 72 h. Interestingly, we found a positive correlation between TNF expression levels and phagocytic activity of microglia after stroke.ConclusionsIn summary, the resident microglia population is vulnerable to the effects of severe ischemia, show compromised cell cycle progression, and adopt a largely pro-inflammatory phenotype after stroke. Infiltrating monocytes are primarily involved with early debris clearance of dying cells. These findings suggest that the early wave of infiltrating monocytes may be beneficial to stroke repair and future therapies aimed at mitigating microglia cell death may prove more effective than attempting to elicit targeted anti-inflammatory responses from damaged cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0329-1) contains supplementary material, which is available to authorized users.

The peripheral immune system plays a critical role in aging and in the response to brain injury. Emerging data suggest inflammatory responses are exacerbated in older animals following ischemic stroke; however, our understanding of these age-related changes is poor. In this work, we demonstrate marked differences in the composition of circulating and infiltrating leukocytes recruited to the ischemic brain of old male mice after stroke compared to young male mice. Blood neutrophilia and neutrophil invasion into the brain were increased in aged animals. Relative to infiltrating monocyte populations, brain-invading neutrophils had reduced phagocytic potential, and produced higher levels of reactive oxygen species and extracellular matrix-degrading enzymes (i.e., MMP-9), which were further exacerbated with age. Hemorrhagic transformation was more pronounced in aged versus young mice relative to infarct size. High numbers of myeloperoxidase-positive neutrophils were found in postmortem human brain samples of old (> 71 years) acute ischemic stroke subjects compared to non-ischemic controls. Many of these neutrophils were found in the brain parenchyma. A large proportion of these neutrophils expressed MMP-9 and positively correlated with hemorrhage and hyperemia. MMP-9 expression and hemorrhagic transformation after stroke increased with age. These changes in the myeloid response to stroke with age led us to hypothesize that the bone marrow response to stroke is altered with age, which could be important for the development of effective therapies targeting the immune response. We generated heterochronic bone marrow chimeras as a tool to determine the contribution of peripheral immune senescence to age- and stroke-induced inflammation. Old hosts that received young bone marrow (i.e., Young → Old) had attenuation of age-related reductions in bFGF and VEGF and showed improved locomotor activity and gait dynamics compared to isochronic (Old → Old) controls. Microglia in young heterochronic mice (Old → Young) developed a senescent-like phenotype. After stroke, aged animals reconstituted with young marrow had reduced behavioral deficits compared to isochronic controls, and had significantly fewer brain-infiltrating neutrophils. Increased rates of hemorrhagic transformation were seen in young mice reconstituted with aged bone marrow. This work suggests that age alters the immunological response to stroke, and that this can be reversed by manipulation of the peripheral immune cells in the bone marrow.Electronic supplementary materialThe online version of this article (10.1007/s00401-018-1859-2) contains supplementary material, which is available to authorized users.

Background Microglia, the brain's principal immune cell, are increasingly implicated in Alzheimer's disease (AD), but the molecular interfaces through which these cells contribute to amyloid beta (Aβ)-related neurodegeneration are unclear. We recently identified microglial contributions to the homeostatic and disease-associated modulation of perineuronal nets (PNNs), extracellular matrix structures that enwrap and stabilize neuronal synapses, but whether PNNs are altered in AD remains controversial. Methods Extensive histological analysis was performed on male and female 5xFAD mice at 4, 8, 12, and 18 months of age to assess plaque burden, microgliosis, and PNNs. Findings were validated in postmortem AD tissue. The role of neuroinflammation in PNN loss was investigated via LPS treatment, and the ability to prevent or rescue disease-related reductions in PNNs was assessed by treating 5xFAD and 3xTg-AD model mice with colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 to deplete microglia. Findings Utilizing the 5xFAD mouse model and human cortical tissue, we report that PNNs are extensively lost in AD in proportion to plaque burden. Activated microglia closely associate with and engulf damaged nets in the 5xFAD brain, and inclusions of PNN material are evident in mouse and human microglia, while aggrecan, a critical PNN component, deposits within human dense-core plaques. Disease-associated reductions in parvalbumin (PV)+ interneurons, frequently coated by PNNs, are preceded by PNN coverage and integrity impairments, and similar phenotypes are elicited in wild-type mice following microglial activation with LPS. Chronic pharmacological depletion of microglia prevents 5xFAD PNN loss, with similar results observed following depletion in aged 3xTg-AD mice, and this occurs despite plaque persistence. Interpretation We conclude that phenotypically altered microglia facilitate plaque-dependent PNN loss in the AD brain. Funding The NIH (NIA, NINDS) and the Alzheimer's Association.

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