Inhomogeneous distribution of Iba‐1 characterizes microglial pathology in Alzheimer's disease

Microglial dystrophy has recently been described as a morphological phenotype of microglia that differs from resting and activated states by spheroid formation and cytorrhexis. In thick sections immunolabeled for HLA‐DR or Iba‐1 dystrophic microglial processes lose their typical, homogeneous staining pattern and appear to be fragmented or clustered. In this study, we performed double immunofluorescence and electron microscopy to determine if this labeling pattern indeed reflects complete separation of microglial processes from the soma. Using Iba‐1/CD68 and Iba‐1/MHC class II, as microglial markers, we observed that isolated Iba‐1 fragments were still connected to each other by segments of the microglial process immune positive for CD68 or MHC class II. Ultrathin serial sections of two Iba‐1 fragments which appeared to be disconnected from each other at the light microscopical level revealed a still existing "bridge" with a diameter of around 0.182 µm. Therefore, microglial dystrophy may reflect alterations of the cytoskeleton ultimately leading to slow cytorrhexis. GLIA 2016;64:1562–1572     

Targeting microglia for the treatment of Alzheimer's Disease

While histological changes in microglia have long been recognized as a pathological feature of Alzheimer's disease (AD), recent genetic association studies have also strongly implicated microglia in the etiology of the disease. Coding and noncoding polymorphisms in several genes expressed in microglia—including APOE, TREM2, CD33, GRN, and IL1RAP—alter AD risk, and therefore could be considered as entry points for therapeutic intervention. Furthermore, microglia may have a substantial effect on current amyloid β (Aβ) and tau immunotherapy approaches, since they are the primary cell type in the brain to mediate Fc receptor‐facilitated antibody effector function. In this review, we discuss the considerations in selecting microglial therapeutic targets from the perspective of drug discovery feasibility, and consider the role of microglia in ongoing immunotherapy clinical strategies. GLIA 2016;64:1710–1732     

MIC-MAC: An automated pipeline for high-throughput characterization and classification of three-dimensional microglia morphologies in mouse and human postmortem brain samples

The phenotypic changes of microglia in brain diseases are particularly diverse and their role in disease progression, beneficial, or detrimental, is still elusive. High-throughput molecular approaches such as single-cell RNA-sequencing can now resolve the high heterogeneity in microglia population for a specific physiological condition, however, the relation between the different microglial signatures and their surrounding brain microenvironment is barely understood. Thus, better tools to characterize the phenotypic variations of microglia in situ are needed, particularly for human brain postmortem samples analysis. To address this challenge, we developed MIC-MAC, a Microglia and Immune Cells Morphologies Analyser and Classifier pipeline that semiautomatically segments, extracts, and classifies all microglia and immune cells labeled in large three-dimensional (3D) confocal image stacks of mouse and human brain samples. Our imaging-based approach enables automatic 3D-morphology characterization and classification of thousands of individual microglia in situ and revealed species- and disease-specific morphological phenotypes in mouse aging, human Alzheimer's disease, and dementia with Lewy Bodie's samples. MIC-MAC is a precision diagnostic tool that allows a rapid, unbiased, and large-scale analysis of microglia morphological states in mouse models and patient brain samples.     

Absence of microglia or presence of peripherally-derived macrophages does not affect tau pathology in young or old hTau mice

Microglia are implicated in the pathophysiology of several neurodegenerative disorders, including Alzheimer's disease. While the role of microglia and peripheral macrophages in regulating amyloid beta pathology has been well characterized, the impact of these distinct cell subsets on tau pathology remains poorly understood. We and others have recently demonstrated that monocytes can engraft the brain and give rise to long-lived parenchymal macrophages, even under nonpathological conditions. We undertook the current study to investigate the regulation of tau pathology by microglia and peripheral macrophages using hTau transgenic mice, which do not exhibit microglial activation/pathology or macrophage engraftment. To assess the direct impact of microglia on tau pathology we developed a protocol for long-term microglial depletion in Cx3cr1^CreERR26^DTA mice and crossed them with hTau mice. We then depleted microglia up to 3 months in both young and old mice, but no net change in forebrain soluble oligomeric tau or total or phosphorylated levels of aggregated tau was recorded. To investigate the consequence of peripherally-derived parenchymal macrophages on tau aggregation we partially repopulated the hTau microglial pool with peripheral macrophages, but this also did not affect levels of tau oligomers or insoluble aggregates. Our study questions the direct involvement of microglia or peripheral macrophages in the development of tau pathology in the hTau model.     

Minocycline affects microglia activation, Abeta deposition, and behavior in APP-tg mice

Activated microglia and reactive astrocytes invade and surround cerebral beta amyloid (Abeta) plaques in Alzheimer's disease (AD), but the role of microglia in plaque development is still unclear. In this study, minocycline was administered for 3 months, prior to and early in Abeta plaque formation in amyloid precursor protein transgenic mice (APP-tg). When minocycline was given to younger mice, there was a small but significant increase in Abeta deposition in the hippocampus, concurrent with improved cognitive performance relative to vehicle treated mice. If APP-tg mice received minocycline after Abeta deposition had begun, microglial activation was suppressed but this did not affect Abeta deposition or improve cognitive performance. In vitro studies demonstrated that minocycline suppressed microglial production of IL-1beta, IL-6, TNF, and NGF. Thus, minocycline has different effects on Abeta plaque deposition and microglia activation depending on the age of administration. Our data suggest that this may be due to the effects of minocycline on microglial function. Therefore, anti-inflammatory therapies to suppress microglial activation or function may reduce cytokine production but enhance Abeta plaque formation early in AD.     

Lipid transporter Spns2 promotes microglia pro-inflammatory activation in response to amyloid-beta peptide

Accumulating evidence indicates that neuroinflammation contributes to the pathogenesis and exacerbation of neurodegenerative disorders, such as Alzheimer's disease (AD). Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid that regulates many pathophysiological processes including inflammation. We present evidence here that the spinster homolog 2 (Spns2), a S1P transporter, promotes microglia pro-inflammatory activation in vitro and in vivo. Spns2 knockout (Spns2KO) in primary cultured microglia resulted in significantly reduced levels of pro-inflammatory cytokines induced by lipopolysaccharide (LPS) and amyloid-beta peptide 1–42 oligomers (Aβ42) when compared with littermate controls. Fingolimod (FTY720), a S1P receptor 1 (S1PR1) functional antagonist and FDA approved drug for relapsing–remitting multiple sclerosis, partially blunted Aβ42-induced pro-inflammatory cytokine generation, suggesting that Spns2 promotes microglia pro-inflammatory activation through S1P-signaling. Spns2KO significantly reduced Aβ42-induced nuclear factor kappa B (NFκB) activity. S1P increased, while FTY720 dampened, Aβ42-induced NFκB activity, suggesting that Spns2 activates microglia inflammation through, at least partially, NFκB pathway. Spns2KO mouse brains showed significantly reduced Aβ42-induced microglia activation/accumulation and reduced levels of pro-inflammatory cytokines when compared with age-matched controls. More interestingly, Spns2KO ameliorated Aβ42-induced working memory deficit detected by Y-Maze. In summary, these results suggest that Spns2 promotes pro-inflammatory polarization of microglia and may play a crucial role in AD pathogenesis.     

Apolipoproteins E and J interfere with amyloid‐beta uptake by primary human astrocytes and microglia in vitro

Defective clearance of the amyloid‐β peptide (Aβ) from the brain is considered a strong promoter in Alzheimer's disease (AD) pathogenesis. Astrocytes and microglia are important mediators of Aβ clearance and Aβ aggregation state and the presence of amyloid associated proteins (AAPs), such as Apolipoproteins E and J (ApoE and ApoJ), may influence Aβ clearance by these cells. Here we set out to investigate whether astrocytes and microglia differ in uptake efficiency of Aβ oligomers (Aβ_oligo) and Aβ fibrils (Aβ_fib), and whether the Aβ aggregation state and/or presence of AAPs affect Aβ uptake in these cells in vitro. Adult human primary microglia and astrocytes, isolated from short delay post‐mortem brain tissue, were exposed to either Aβ_oligo or Aβ_fib alone or combined with a panel of certain AAPs whereafter Aβ‐positive cells were quantified using flow cytometry. Upon exposure to Aβ combined with ApoE, ApoJ, α1‐antichymotrypsin (ACT) and a combination of serum amyloid P and complement C1q (SAP‐C1q), a clear reduction in astrocytic but not microglial Aβ_oligo uptake, was observed. In contrast, Aβ_fib uptake was strongly reduced in the presence of AAPs in microglia, but not in astrocytes. These data provide the first evidence of distinct roles of microglia and astrocytes in Aβ clearance. More importantly we show that Aβ clearance by glial cells is negatively affected by AAPs like ApoE and ApoJ. Thus, targeting the association of Aβ with AAPs, such as ApoE and ApoJ, could serve as a therapeutic strategy to increase Aβ clearance by glial cells. GLIA 2014;62:493–503     

Visualization of microglia in living tissues using Iba1-EGFP transgenic mice

Microglia are thought to play important roles not only in repairing injured tissue but in regulating neuronal activity, and visualizing the cells is very useful as a means of further investigating the function of microglia in vivo. We previously cloned the ionized calcium-binding adaptor molecule 1 (Iba1) gene, which is expressed selectively in microglia/microphages. To generate new transgenic mice to visualize microglia with enhanced green fluorescent protein (EGFP), we here constructed a plasmid carrying EGFP cDNA under control of the Iba1 promoter. This construct was injected into C57B/6 mouse zygotes, and the Iba1-EGFP transgenic line was developed. Fluorescent in-situ hybridization analysis revealed that the Iba1-EGFP transgene was located on chromosome 11D. No obvious defects were observed during development or in adulthood, and the EGFP fluorescence remained invariant over the course of at least four generations. Judging from the immunoreactivity with anti-Iba1 antibody, all EGFP-positive cells in the adult brain were ramified microglia. In the developing transgenic embryos, EGFP signals were detected as early as embryonic Day 10.5. The most prominent EGFP signals were found in forebrain, spinal cord, eye, foreleg, yolk sac, liver, and vessel walls. At postnatal Day 6, clear EGFP signals were observed in the supraventricular corpus callosum, known as "fountain of microglia", where ameboid microglia migrate into the brain parenchyma and mature into ramified microglia. Iba1-EGFP transgenic mice thus permit observation of living microglia under a fluorescence microscope and provide a useful tool for studying the function of microglia in vivo.     

CD11a expression distinguishes infiltrating myeloid cells from plaque-associated microglia in Alzheimer's disease

Alzheimer's disease (AD) is the leading cause of age-related neurodegeneration and is characterized neuropathologically by the accumulation of insoluble beta-amyloid (Aβ) peptides. In AD brains, plaque-associated myeloid (PAM) cells cluster around Aβ plaques but fail to effectively clear Aβ by phagocytosis. PAM cells were originally thought to be brain-resident microglia. However, several studies have also suggested that Aβ-induced inflammation causes peripheral monocytes to enter the otherwise immune-privileged brain. The relationship between AD progression and inflammation in the brain remains ambiguous because microglia and monocyte-derived macrophages are extremely difficult to distinguish from one another in an inflamed brain. Whether PAM cells are microglia, peripheral macrophages, or a mixture of both remains unclear. CD11a is a component of the β2 integrin LFA1. We have determined that CD11a is highly expressed on peripheral immune cells, including macrophages, but is not expressed by mouse microglia. These expression patterns remain consistent in LPS-treated inflamed mice, as well as in two mouse models of AD. Thus, CD11a can be used as a marker to distinguish murine microglia from infiltrating peripheral immune cells. Using CD11a, we show that PAM cells in AD transgenic brains are comprised entirely of microglia. We also demonstrate a novel fluorescence-assisted quantification technique (FAQT), which reveals a significant increase in T lymphocytes, especially in the brains of female AD mice. Our findings support the notion that microglia are the lead myeloid players in AD and that rejuvenating their phagocytic potential may be an important therapeutic strategy.     

Membrane‐type 1 metalloproteinase is upregulated in microglia/brain macrophages in neurodegenerative and neuroinflammatory diseases

We previously reported that glioma cells induce the expression of membrane‐type 1 metalloproteinase (MT1‐MMP or MMP‐14) in tumor‐associated microglia/macrophages and promote tumor growth, whereas MMP‐14 expression in microglia under physiological conditions is very low. Here, we show that the increase in MMP‐14 expression is also found in microglia/macrophages associated with neurodegenerative and neuroinflammatory pathologies in mouse models as well as in human biopsies or post‐mortem tissue. We found that microglial/macrophage MMP‐14 expression was upregulated in Alzheimer's disease tissue, in active lesions of multiple sclerosis, and in tissue from stage II stroke as well as in the corresponding mouse models for the human diseases. In contrast, we observed no upregulation for MMP‐14 in microglia/macrophages in the early phase of stroke or in the corresponding mouse model, in human amyotrophic lateral sclerosis (ALS) tissue or in a mouse model of ALS as well as in human cases of acute brain trauma. These data indicate that MMP‐14 expression is not a general marker for activated microglia/macrophages but is upregulated in defined stages of neuroinflammatory and neurodegenerative diseases and that there is generally a good match between mouse models and human brain pathologies. © 2013 Wiley Periodicals, Inc.     

Plaque‐dependent morphological and electrophysiological heterogeneity of microglia in an Alzheimer's disease mouse model

Microglia, the central nervous system resident innate immune cells, cluster around Aβ plaques in Alzheimer's disease (AD). The activation phenotype of these plaque‐associated microglial cells, and their differences to microglia distant to Aβ plaques, are incompletely understood. We used novel three‐dimensional cell analysis software to comprehensively analyze the morphological properties of microglia in the TgCRND8 mouse model of AD in spatial relation to Aβ plaques. We found strong morphological changes exclusively in plaque‐associated microglia, whereas plaque‐distant microglia showed only minor changes. In addition, patch‐clamp recordings of microglia in acute cerebral slices of TgCRND8 mice revealed increased K⁺ currents in plaque‐associated but not plaque‐distant microglia. Within the subgroup of plaque‐associated microglia, two different current profiles were detected. One subset of cells displayed only increased inward currents, while a second subset showed both increased inward and outward currents, implicating that the plaque microenvironment differentially impacts microglial ion channel expression. Using pharmacological channel blockers, multiplex single‐cell PCR analysis and RNA fluorescence in situ hybridization, we identified Kir and Kv channel types contributing to the in‐ and outward K⁺ conductance in plaque‐associated microglia. In summary, we have identified a previously unrecognized level of morphological and electrophysiological heterogeneity of microglia in relation to amyloid plaques, suggesting that microglia may display multiple activation states in AD.     

Developmental profile of microglia distribution in nuclei of the superior olivary complex

In the brain, microglia are involved in immune responses and synaptic maturation. During early development, these cells invade the brain, proliferate, and morphologically mature to achieve coverage of the surrounding tissue with their fine processes. Their developmental proliferation overlaps with the postnatal development of neuronal circuits. Within the superior olivary complex (SOC), an auditory brainstem structure, microglia, and their early postnatal development have been documented. A quantification over the full developmental profile of the arrangement and morphological changes in single microglia cells is missing. Here, we used immunofluorescence labeling to quantify their distribution, morphological changes, and coverage during early and late postnatal development in the SOC of Mongolian gerbils. Microglia distributed rather homogenously within each nucleus with a bias to the nucleus borders at postnatal day (P) 5 and more centrally in the nucleus in mature stages. We found a nucleus-specific transient increase in microglia cell number and density reaching its peak at P17 with a subsequent decline to P55 values. Length and branching of microglia protrusions increased especially after P12. The stronger ramification together with the increase in cell density allows coverage of the surrounding tissue from P5 to mature stages, despite the large developmental increase in nucleus size. The transient increase in density during synaptic refinement in SOC nuclei suggests that microglia are important during the pruning period, compensating for developmental increase in tissue volume, and that in mature stages their main function appears tissue surveillance.     

Estrogen,brain structure,and cognition in postmenopausal women

Declining estrogen levels before, during, and after menopause can affect memory and risk for Alzheimer's disease. Undesirable side effects of hormone variations emphasize a role for hormone therapy (HT) where possible benefits include a delay in the onset of dementia—yet findings are inconsistent. Effects of HT may be mediated by estrogen receptors found throughout the brain. Effects may also depend on lifestyle factors, timing of use, and genetic risk. We studied the impact of self‐reported HT use on brain volume in 562 elderly women (71–94 years) with mixed cognitive status while adjusting for aforementioned factors. Covariate‐adjusted voxelwise linear regression analyses using a model with 16 predictors showed HT use as positively associated with regional brain volumes, regardless of cognitive status. Examinations of other factors related to menopause, oophorectomy and hysterectomy status independently yielded positive effects on brain volume when added to our model. One interaction term, HTxBMI, out of several examined, revealed significant negative association with overall brain volume, suggesting a greater reduction in brain volume than BMI alone. Our main findings relating HT to regional brain volume were as hypothesized, but some exploratory analyses were not in line with existing hypotheses. Studies suggest lower levels of estrogen resulting from oophorectomy and hysterectomy affect brain volume negatively, and the addition of HT modifies the relation between BMI and brain volume positively. Effects of HT may depend on the age range assessed, motivating studies with a wider age range as well as a randomized design.     

Reactive microglia in the developing brain

Summary Reactive microglia in the developing brain after stab wound was studied by morphological, cytochemical, and autoradiographic methods. Morphologically, early reactive cells are of the M cell type (Matthews 1974). They show an activated nucleus, cytoplasm rich in ribosomes with wide Golgi complex and variable numbers of lipid inclusions. Big clear vacuoles are found in many of these cells. Microtubules not associated with centrioles and filaments may or may not be present. Junctional complexes of the zonula or puncta adherentia types are occasionally found. Strong NADPH dehydrogenase, weak NADH dehydrogenase, strong ATPase, and strong acid phosphatase, in addition to nonspecific esterase activites were demonstrated in many reactive cells. Intravenous infusion of labelled bone marrow cells from a donor showed labelled macrophages and labelled perivascular cells at the site of injury. Intracerebral injection of a small dose of tritiated thymidine at the time of injury resulted in the appearance of labelled macrophages in the following days. These data suggest that many of the reactive cells have an exogenous, more probably monocytic, origin; but a certain amount of endogenous cells also act as macrophages in brain injuries.     

CD200 maintains the region-specific phenotype of microglia in the midbrain and its role in Parkinson's disease

Microglia are a specialized population of tissue macrophages in the mammalian brain. Microglial phenotype is tightly regulated by local environmental factors, although little is known about these factors and their region-preferred roles in regulating local neuroinflammatory responses. We hypothesized that microglia in different brain regions respond differently to neuroinflammatory stimulation and that CD200, an anti-inflammatory protein mainly originated from neurons, acts as a local cue inhibiting microglia activation in the midbrain. We utilized a CD200-deficient mouse line to analyze the phenotypic role of CD200 in the regulation of normal neuron–microglia homeostasis in the midbrain and in the dopaminergic degeneration in an α-synuclein overexpression model of PD. We found that systemic administration of an endotoxin lipopolysaccharide induced a region-preferred change in CD200 expression in the midbrain. Similarly, CD200^−/− mice showed a regional preference in an enhancement of microglia activation and baseline inflammatory levels in the midbrain and dopamine neuron loss in the substantia nigra (SN). In a mouse model of Parkinson's disease (PD) induced by rAAV-hSYN injection into the SN, CD200^−/− mice showed more dopamine neuron loss in the SN than wild type mice. Activation of CD200 receptors with a CD200 fusion protein alleviated the neuroinflammation and neuronal death in the SN of PD mice. These findings demonstrate that CD200 is essential for the midbrain homeostasis and acts as a critical local regulator in controlling microglial properties related to the PD pathogenesis.     

Chlamydia pneumoniae infection promotes the transmigration of monocytes through human brain endothelial cells

We have investigated the effects of Chlamydia pneumoniae on human brain endothelial cells (HBMECs) and human monocytes as a mechanism for breaching the blood-brain barrier (BBB) in Alzheimer's disease (AD). HBMECs and peripheral blood monocytes may be key components in controlling the entry of C. pneumoniae into the human brain. Our results indicate that C. pneumoniae infects blood vessels and monocytes in AD brain tissues compared with normal brain tissue. C. pneumoniae infection stimulates transendothelial entry of monocytes through HBMECs. This entry is facilitated by the up-regulation of VCAM-1 and ICAM-1 on HBMECs and a corresponding increase of LFA-1, VLA-4, and MAC-1 on monocytes. C. pneumoniae infection in HBMECs and THP-1 monocytes up-regulates monocyte transmigration threefold in an in vitro brain endothelial monolayer. In this way, C. pneumoniae infection in these cell types may contribute to increased monocyte migration and promote inflammation within the CNS resulting from infection at the level of the vasculature. Thus, infection at the level of the vasculature may be a key initiating factor in the pathogenesis of neurodegenerative diseases such as sporadic AD.     

P2Y12 receptor is expressed on human microglia under physiological conditions throughout development and is sensitive to neuroinflammatory diseases

Microglia are resident immune cells in the central nervous system (CNS), which are essential for immune defence and critically contribute to neuronal functions during homeostasis. Until now, little is known about microglia biology in humans in part due to the lack of microglia‐specific markers. We therefore investigated the expression of the purinergic receptor P2Y₁₂ in human brain tissue. Compared to classical markers used to identify microglia such as Iba1, CD68 or MHCII, we found that P2Y₁₂ is expressed on parenchymal microglia but is absent from perivascular or meningeal macrophages. We further demonstrate that P2Y₁₂ expression is stable throughout human brain development, including fetal phases, and quantification of P2 microglia revealed that the density of human microglia is constant throughout lifetime. In contrast, CD68 expression increases during aging in cerebellar but not in cortical microglia, indicating regional heterogeneity. CNS pathologies such as Alzheimer's disease or multiple sclerosis—but not schizophrenia—result in decreased P2Y₁₂ immunoreactivity in plaque‐ or lesion‐associated myeloid cells, whereas Iba1 expression remains detectable. Our results suggest that P2Y₁₂ is a useful marker for the identification of human microglia throughout the lifespan. Moreover, P2Y₁₂ expression might help to discriminate activated microglia and infiltrating myeloid cells from quiescent microglia in the human CNS. GLIA 2017;65:375–387