Search

In the central nervous system, immunologic surveillance and response are carried out, in large part, by microglia. These resident macrophages derive from myeloid precursors in the embryonic yolk sac, migrating to the brain and eventually populating local tissue prior to blood-brain barrier formation. Preserved for the duration of lifespan, microglia serve the host as more than just a central arm of innate immunity, also contributing significantly to the development and maintenance of neurons and neural networks, as well as neuroregeneration. The critical nature of these varied functions makes the characterization of key roles played by microglia in neurodegenerative disorders, especially Alzheimer’s disease, of paramount importance. While genetic models and rudimentary pharmacologic approaches for microglial manipulation have greatly improved our understanding of central nervous system health and disease, significant advances in the selective and near complete in vitro and in vivo depletion of microglia for neuroscience application continue to push the boundaries of research. Here we discuss the research efficacy and utility of various microglial depletion strategies, including the highly effective CSF1R inhibitor models, noteworthy insights into the relationship between microglia and neurodegeneration, and the potential for therapeutic repurposing of microglial depletion and repopulation.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterized by the deposition of amyloid-beta (Aβ) plaques and widespread neuroinflammation. While the cause of AD remains unknown, multiple factors likely contribute to the disease, including heart disease, diabetes, previous head injury, as well as a number of genetic determinants. Inheritance of the apolipoprotein (APOE) ε4 allele represents the strongest genetic risk factor for development of AD, driving pathogenesis and increasing overall disease severity. APOE has long been recognized as a key regulator of cholesterol homeostasis, although a greater appreciation now exists for its role in various innate immune system processes. Indeed, APOE modulates inflammatory environments in brain in large part by altering gene expression profiles in glia, important mediators of immunity in the CNS. While the association between APOE and AD was first observed nearly three decades ago, the mechanism by which APOE ε4 influences the etiology and pathophysiology of AD is not well characterized. Overwhelming data supports the hypothesis that APOE ε4 dysregulates central amyloid metabolism by an undetermined molecular mechanism, thus laying the foundation for disease. A host of amyloid-degrading enzymes (ADEs) regulate Aβ accumulation in brain, and therefore represent valuable therapeutic targets. Neprilysin (NEP), a metalloendopeptidase expressed by activated microglia and astrocytes, is a broad-spectrum ADE able to degrade a variety of Aβ species. Here we describe in vivo and in vitro experiments designed to investigate the potential for APOE genotype to differentially regulate glial NEP in brain under neuroinflammatory conditions. Our results provide a novel mechanism by which APOE genotype-dependent differential expression of NEP by glia during neuroinflammation may contribute to AD pathogenesis.

As more people live longer, age-related neurodegenerative diseases are an increasingly important societal health issue. Treatments targeting specific pathologies such as amyloid beta in Alzheimer’s disease (AD) have not led to effective treatments, and there is increasing evidence of a disconnect between traditional pathology and cognitive abilities with advancing age, indicative of individual variation in resilience to pathology. Here, we generated a comprehensive neuropathological, molecular, and transcriptomic characterization of hippocampus and two regions cortex in 107 aged donors (median = 90) from the Adult Changes in Thought (ACT) study as a freely-available resource (http://aging.brain-map.org/). We confirm established associations between AD pathology and dementia, albeit with increased, presumably aging-related variability, and identify sets of co-expressed genes correlated with pathological tau and inflammation markers. Finally, we demonstrate a relationship between dementia and RNA quality, and find common gene signatures, highlighting the importance of properly controlling for RNA quality when studying dementia.

Frontotemporal dementia (FTD) is a neurodegenerative disease with devastating changes in behavioral performance and social function. Mutations in the progranulin gene (GRN) are one of the most common causes of inherited FTD due to reduced progranulin expression or activity, including in brain where it is expressed primarily by neurons and microglia. Thus, efforts aimed at enhancing progranulin levels might be a promising therapeutic strategy. Bone marrow-derived cells are able to engraft in the brain and adopt a microglial phenotype under myeloablative irradiation conditioning. This ability makes bone marrow (BM)-derived cells a potential cellular vehicle for transferring therapeutic molecules to the central nervous system. Here, we utilized BM cells from Grn+/+ (wild type or wt) mice labeled with green fluorescence protein for delivery of progranulin to progranulin deficient (Grn−/−) mice. Our results showed that wt bone marrow transplantation (BMT) partially reconstituted progranulin in the periphery and in cerebral cortex of Grn−/− mice. We demonstrated a pro-inflammatory effect in vivo and in ex vivo preparations of cerebral cortex of Grn−/− mice that was partially to fully reversed five months after BMT. Our findings suggest that BMT can be administered as a stem cell-based approach to prevent or to treat neurodegenerative diseases.

Cannabinoids affect immune responses in ways that may be beneficial for autoimmune diseases. We sought to determine whether chronic Cannabis use differentially modulates a select number of immune parameters in healthy controls and individuals with multiple sclerosis (MS cases). Subjects were enrolled and consented to a single blood draw, matched for age and BMI. We measured monocyte migration isolated from each subject, as well as plasma levels of endocannabinoids and cytokines. Cases met definition of MS by international diagnostic criteria. Monocyte cell migration measured in control subjects and individuals with MS was similarly inhibited by a set ratio of phytocannabinoids. The plasma levels of CCL2 and IL17 were reduced in non-naïve cannabis users irrespective of the cohorts. We detected a significant increase in the endocannabinoid arachidonoylethanolamine (AEA) in serum from individuals with MS compared to control subjects, and no significant difference in levels of other endocannabinoids and signaling lipids irrespective of Cannabis use. Chronic Cannabis use may affect the immune response to similar extent in individuals with MS and control subjects through the ability of phytocannabinoids to reduce both monocyte migration and cytokine levels in serum. From a panel of signaling lipids, only the levels of AEA are increased in individuals with MS, irrespective of Cannabis use or not. Our results suggest that both MS cases and controls respond similarly to chronic Cannabis use with respect to the immune parameters measured in this study.

Apolipoprotein E (apoE) is well known as a regulator of cholesterol homeostasis, and is increasingly recognized to play a prominent role in the modulation of innate immune response, including cell-to-cell communication and migration. Alzheimer's disease (AD) is a slowly progressive neurodegenerative disorder characterized by neuroinflammation that appears to be an important component of the pathophysiology of the disease. Astrocytes are the majority cell type in brain, exerting significant influence over a range of central nervous system activities, including microglial-mediated neuroinflammatory responses. As the resident innate immune effector cells of the brain, microglia respond to soluble chemical signals released from tissue during injury and disease by mobilizing to lesion sites, clearing toxic molecules, and releasing chemical signals of their own. While microglial-mediated neuroinflammation in the AD brain remains an area of intense investigation, the mechanisms underlying reinforcement and regulation of these aberrant microglial responses by astrocytes are largely unstudied. Moreover, although inheritance of APOE ɛ4 represents the greatest genetic risk factor for sporadic AD, the mechanism by which apoE isoforms differentially influence AD pathophysiology is unknown. Here we show that APOE ɛ4 genotype specifically modulates astrocyte secretion of potent microglial chemotactic agents, including CCL3, thus providing evidence that APOE modulation of central nervous system (CNS) innate immune response is mediated through astrocytes. GLIA 2015;63:51–65

Apolipoprotein E4 ( APOE4 ) genotype is the strongest genetic risk factor for late-onset Alzheimer disease and confers a proinflammatory, neurotoxic phenotype to microglia. Here, we tested the hypothesis that bone marrow cell APOE genotype modulates pathological progression in experimental Alzheimer disease. We performed bone marrow transplants (BMT) from green fluorescent protein–expressing human APOE3/3 or APOE4/4 donor mice into lethally irradiated 5-month-old APPswe/PS1ΔE9 mice. Eight months later, APOE4/4 BMT–recipient APPswe/PS1ΔE9 mice had significantly impaired spatial working memory and increased detergent-soluble and plaque Aβ compared with APOE3/3 BMT–recipient APPswe/PS1ΔE9 mice. BMT-derived microglia engraftment was significantly reduced in APOE4/4 recipients, who also had correspondingly less cerebral apoE. Gene expression analysis in cerebral cortex of APOE3/3 BMT recipients showed reduced expression of tumor necrosis factor-α and macrophage migration inhibitory factor (both neurotoxic cytokines) and elevated immunomodulatory IL-10 expression in APOE3/3 recipients compared with those that received APOE4/4 bone marrow. This was not due to detectable APOE -specific differences in expression of microglial major histocompatibility complex class II, C-C chemokine receptor (CCR) type 1, CCR2, CX3C chemokine receptor 1 (CX3CR1), or C5a anaphylatoxin chemotactic receptor (C5aR). Together, these findings suggest that BMT-derived APOE3 -expressing cells are superior to those that express APOE4 in their ability to mitigate the behavioral and neuropathological changes in experimental Alzheimer disease.

The human apolipoprotein E (APOE) gene plays an important role in lipid metabolism. It has three common genetic variants, alleles ɛ2/ɛ3/ɛ4, which translate into three protein isoforms of apoE2, E3 and E4. These isoforms can differentially influence total serum cholesterol levels; therefore, APOE has been linked with cardiovascular disease. Additionally, its ɛ4 allele is strongly associated with the risk of Alzheimer's disease (AD), whereas the ɛ2 allele appears to have a modest protective effect for AD. Despite decades of research having illuminated multiple functional differences among the three apoE isoforms, the precise mechanisms through which different APOE alleles modify diseases risk remain incompletely understood. In this study, we examined the genomic structure of APOE in search for properties that may contribute novel biological consequences to the risk of disease. We identify one such element in the ɛ2/ɛ3/ɛ4 allele-carrying 3′-exon of APOE. We show that this exon is imbedded in a well-defined CpG island (CGI) that is highly methylated in the human postmortem brain. We demonstrate that this APOE CGI exhibits transcriptional enhancer/silencer activity. We provide evidence that this APOE CGI differentially modulates expression of genes at the APOE locus in a cell type-, DNA methylation- and ɛ2/ɛ3/ɛ4 allele-specific manner. These findings implicate a novel functional role for a 3′-exon CGI and support a modified mechanism of action for APOE in disease risk, involving not only the protein isoforms but also an epigenetically regulated transcriptional program at the APOE locus driven by the APOE CGI.

A major therapeutic target for Parkinson's disease (PD) is providing increased glial-derived neurotrophic factor (GDNF) to dopaminergic neurons. We tested the hypothesis that innate immune activation increases astrocyte GDNF production and that this is regulated by specific eicosanoid receptors. Innate immune-activated primary murine astrocytes were assayed for GDNF expression and secretion. Controls were agent vehicle exposure and wild-type mice. Rank order for up to 10-fold selectively increased GDNF expression was activators of TLR3 > TLR2 or TLR4 > TLR9. TLR3 activator-stimulated GDNF expression was selectively JNK-dependent, followed cyclooxygenase (COX)-2, was coincident with membranous PGE2 synthase, and was not significantly altered by a nonspecific COX- or a COX-2-selective inhibitor. Specific eicosanoid receptors had opposing effects on TLR3 activator-induced GDNF expression: ∼60% enhancement by blocking or ablating of PGE2 receptor subtype 1 (EP1), ∼30% enhancement by activating PGF2α receptor or thromboxane receptor, or ∼15% enhancement by activating EP4. These results demonstrate functionally antagonistic eicosanoid receptor subtype regulation of innate immunity-induced astrocyte GDNF expression and suggest that selective inhibition of EP1 signaling might be a means to augment astrocyte GDNF secretion in the context of innate immune activation in diseased regions of brain in PD.—Li, X., Cudaback, E., Breyer, R. M., Montine, K. S., Keene, C. D., Montine, T. J. Eicosanoid receptor subtype-mediated opposing regulation of Toll-like receptor-stimulated expression of astrocyte glial-derived neurotrophic factor.

The largest genetic risk for late-onset Alzheimer's disease (AD) resides at the apolipoprotein E gene (APOE) locus, which has three common alleles (ɛ2, ɛ3, ɛ4) that encode three isoforms (apoE2, apoE3, apoE4). The very strong association of the APOE ɛ4 allele with AD risk and its role in the accumulation of amyloid β in brains of people and animal models solidify the biological relevance of apoE isoforms but do not provide mechanistic insight. The innate immune response is consistently observed in AD and is a likely contributor to neuronal injury and response to injury. Here we review emerging data showing that apoE isoform regulation of multiple facets of the innate immune response in the brain may alter AD not only through amyloid β-dependent mechanisms, but also through other, amyloid β-independent mechanisms.

Human cerebrospinal fluid (CSF) contains diverse lipid particles, including lipoproteins that are distinct from their plasma counterparts and contain apolipoprotein (apo) E isoforms, apoJ, and apoAI, and extracellular vesicles, which can be detected by annexin V binding. The aim of this study was to develop a method to quantify CSF particles and evaluate their relationship to aging and neurodegenerative diseases. We used a flow cytometric assay to detect annexin V-, apoE-, apoAI-, apoJ-, and amyloid (A) β42-positive particles in CSF from 131 research volunteers who were neurologically normal or had mild cognitive impairment (MCI), Alzheimer disease (AD) dementia, or Parkinson disease. APOE e4/e4 participants had CSF apoE-positive particles that were more frequently larger but at an 88% lower level versus those in APOE e3/e3 or APOE e3/e4 patients; this finding was reproduced in conditioned medium from mouse primary glial cell cultures with targeted replacement of apoE. Cerebrospinal fluid apoE-positive and β-amyloid (Aβ42)-positive particle concentrations were persistently reduced one-third to one-half in middle and older age subjects; apoAI-positive particle concentration progressively increased approximately 2-fold with age. Both apoAI-positive and annexin V-positive CSF particle levels were reduced one-third to one-half in CSF of MCI and/or AD dementia patients versus age-matched controls. Our approach provides new methods to investigate CNS lipid biology in relation to neurodegeneration and perhaps develop new biomarkers for diagnosis or treatment monitoring.

Focal cerebral ischemia and traumatic brain injury induce an escalating amount of cell death because of harmful mediators diffusing from the original lesion site. Evidence suggests that healthy cells surrounding these lesions attempt to protect themselves by producing endocannabinoids (eCBs) and activating cannabinoid receptors, the molecular target for marijuana-derived compounds. Indeed, activation of cannabinoid receptors reduces the production and diffusion of harmful mediators. Here, we provide evidence that an exception to this pattern is found in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. We show that cell damage induced by EAE does not lead to increase in eCBs, even though cannabinoid receptors are functional because synthetic cannabinoid agonists are known to confine EAE-induced lesions. This lack of eCB increase is likely due to IFN-γ, which is released by primed T cells invading the CNS. We show that IFN-γ disrupts the functionality of purinergic P2X 7 receptors, a key step controlling eCB production by microglia, the main source of eCBs in brain. Accordingly, induction of EAE in P2X 7 −/− mice results in even lower eCB levels and more pronounced cell damage than in wild-type mice. Our data suggest that the high level of CNS IFN-γ associated with EAE disrupts eCB-mediated neuroprotection while maintaining functional cannabinoid receptors, thus providing additional support for the use of cannabinoid-based medicine to treat multiple sclerosis.

Endophilin-B1, also known as Bax-interacting factor 1 (Bif-1, and encoded by SH3GLB1), is a multifunctional protein involved in apoptosis, autophagy and mitochondrial function. We recently described a unique neuroprotective role for neuron-specific alternatively spliced isoforms of endophilin-B1. To examine whether endophilin-B1-mediated neuroprotection could be a novel therapeutic target for Alzheimer's disease we used a double mutant amyloid precursor protein and presenilin 1 (APPswe/PSEN1dE9) mouse model of Alzheimer's disease and observed that expression of neuron-specific endophilin-B1 isoforms declined with disease progression. To determine if this reduction in endophilin-B1 has a functional role in Alzheimer's disease pathogenesis, we crossed endophilin-B1(-/-) mice with APPswe/PSEN1dE9 mice. Deletion of endophilin-B1 accelerated disease onset and progression in 6-month-old APPswe/PSEN1dE9/endophilin-B1(-/-) mice, which showed more plaques, astrogliosis, synaptic degeneration, cognitive impairment and mortality than APPswe/PSEN1dE9 mice. In mouse primary cortical neuron cultures, overexpression of neuron-specific endophilin-B1 isoforms protected against amyloid-β-induced apoptosis and mitochondrial dysfunction. Additionally, protein and mRNA levels of neuron-specific endophilin-B1 isoforms were also selectively decreased in the cerebral cortex and in the synaptic compartment of patients with Alzheimer's disease. Flow sorting of synaptosomes from patients with Alzheimer's disease demonstrated a negative correlation between amyloid-β and endophilin-B1 levels. The importance of endophilin-B1 in neuronal function was further underscored by the development of synaptic degeneration and cognitive and motor impairment in endophilin-B1(-/-) mice by 12 months. Our findings suggest that endophilin-B1 is a key mediator of a feed-forward mechanism of Alzheimer's disease pathogenesis where amyloid-β reduces neuron-specific endophilin-B1, which in turn enhances amyloid-β accumulation and neuronal vulnerability to stress.

Background Inheritance of the human ϵ4 allele of the apolipoprotein (apo) E gene (APOE) significantly increases the risk of developing Alzheimer’s disease (AD), in addition to adversely influencing clinical outcomes of other neurologic diseases. While apoE isoforms differentially interact with amyloid β (Aβ), a pleiotropic neurotoxin key to AD etiology, more recent work has focused on immune regulation in AD pathogenesis and on the mechanisms of innate immunomodulatory effects associated with inheritance of different APOE alleles. APOE genotype modulates expression of proximal genes including APOC1, which encodes a small apolipoprotein that is associated with Aβ plaques. Here we tested the hypothesis that APOE-genotype dependent innate immunomodulation may be mediated in part by apoC-I. Methods ApoC-I concentration in cerebrospinal fluid from control subjects of differing APOE genotypes was quantified by ELISA. Real-time PCR and ELISA were used to analyze apoC-I mRNA and protein expression, respectively, in liver, serum, cerebral cortex, and cultured primary astrocytes derived from mice with targeted replacement of murine APOE for human APOE ϵ3 or ϵ4. ApoC-I direct modulation of innate immune activity was investigated in cultured murine primary microglia and astrocytes, as well as human differentiated macrophages, using specific toll-like receptor agonists LPS and PIC as well as Aβ. Results ApoC-I levels varied with APOE genotype in humans and in APOE targeted replacement mice, with ϵ4 carriers showing significantly less apoC-I in both species. ApoC-I potently reduced pro-inflammatory cytokine secretion from primary murine microglia and astrocytes, and human macrophages, stimulated with LPS, PIC, or Aβ. Conclusions ApoC-I is immunosuppressive. Our results illuminate a novel potential mechanism for APOE genotype risk for AD; one in which patients with an ϵ4 allele have decreased expression of apoC-I resulting in increased innate immune activity.

Activation of innate immunity via toll-like receptors (TLRs) is associated with neurodegenerative diseases, and some effectors, like tumor necrosis factor alpha (TNFα) and interleukin 6 (IL-6), directly contribute to neurodegeneration. We tested the hypothesis that prostaglandin (PG) E2 receptor subtype 1 (EP1) was necessary for the induction of microglial cytokines following the activation of innate immunity. Primary murine microglia had cytokine secretion by activators of TLR3 > TLR9 > TLR4 > TLR2. TLR3 activation induced early expression of cyclooxygenase 2 (COX2) and delayed expression of membranous PGE synthase and secretion of PGE2. Nonselective and COX2-selective inhibitors blocked TLR3 induction of TNFα and IL-6. Moreover, of the nine of twenty cytokines and chemokines induced by TLR3 activation, only TNFα and IL-6 were significantly dependent on EP1 signaling as determined using microglia from mice homozygous deficient for EP1 gene or wild-type microglia coincubated with an EP1 antagonist. These results were confirmed by blocking intracellular Ca2+ release with 2-aminoethoxy-diphenyl borate or Xestospongin C, inhibitors of IP3 receptors. Our results show that suppression of microglial EP1 signaling achieves much of the desired effect of COX inhibitors by selectively blocking TLR3-induced microglial secretion of two major effectors of paracrine neuron damage. In combination with the ability of EP1 suppression to ameliorate excitotoxicity, these data point to blockade of EP1 as an attractive candidate therapeutic for neurodegenerative diseases. © 2011 Wiley-Liss, Inc.

Background Cannabinoids represent unique compounds for treating tumors, including astrocytomas. Whether CB1 and CB2 receptors mediate this therapeutic effect is unclear. Principal Findings We generated astrocytoma subclones that express set levels of CB1 and CB2, and found that cannabinoids induce apoptosis only in cells expressing low levels of receptors that couple to ERK1/2. In contrast, cannabinoids do not induce apoptosis in cells expressing high levels of receptors because these now also couple to the prosurvival signal AKT. Remarkably, cannabinoids applied at high concentration induce apoptosis in all subclones independently of CB1, CB2 and AKT, but still through a mechanism involving ERK1/2. Significance The high expression level of CB1 and CB2 receptors commonly found in malignant astrocytomas precludes the use of cannabinoids as therapeutics, unless AKT is concomitantly inhibited, or cannabinoids are applied at concentrations that bypass CB1 and CB2 receptors, yet still activate ERK1/2.

The endocannabinoids (eCBs) anandamide and 2-arachidonoyl glycerol (2-AG) are inactivated by a two-step mechanism. First, they are carried into cells, and then anandamide is hydrolyzed by fatty acid amide hydrolase (FAAH) and 2-AG by monoacylglycerol lipase (MGL). Here we provide evidence for a previously undescribed MGL activity expressed by microglial cells. We found that the mouse microglial cell line BV-2 does not express MGL mRNA and yet efficiently hydrolyzes 2-AG. URB597 (3′-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate) reduces this hydrolysis by 50%, suggesting the involvement of FAAH. The remaining activity is blocked by classic MGL inhibitors [[1,1-biphenyl]-3-yl-carbamic acid, cyclohexyl ester (URB602) and MAFP (methylarachidonyl fluorophosphate)] and is unaffected by inhibitors of COXs (cyclooxygenases), LOXs (lipooxygenases), and DGLs (diacylglycerol lipases), indicating the involvement of a novel MGL activity. Accordingly, URB602 leads to selective accumulation of 2-AG in intact BV-2 cells. Although MGL expressed in neurons is equally distributed between the cytosolic, mitochondrial, and nuclear fractions, the novel MGL activity expressed by BV-2 cells is enriched in mitochondrial and nuclear fractions. A screen for novel inhibitors of eCB hydrolysis identified several compounds that differentially block MGL, FAAH, and the novel MGL activity. Finally, we provide evidence for expression of the novel MGL by mouse primary microglia in culture. Our results suggest the presence of a novel, pharmacologically distinct, MGL activity that controls 2-AG levels in microglia.

PK 11195 and DAA1106 bind with high-affinity to the translocator protein (TSPO, formerly known as the peripheral benzodiazepine receptor). TSPO expression in glial cells increases in response to cytokines and pathological stimuli. Accordingly, [(11)C]-PK 11195 and [(11)C]-DAA1106 are recognized molecular imaging (MI) agents capable of monitoring changes in TSPO expression occurring in vivo and in response to various neuropathologies.Here we tested the pharmacological characteristics and TSPO-monitoring potential of two novel MI agents: NIR-conPK and NIR-6T. NIR-conPK is an analogue of PK 11195 conjugated to the near-infrared (NIR) emitting fluorophore: IRDye 800CW. NIR-6T is a DAA1106 analogue also conjugated to IRDye 800CW.We found that NIR-6T competed for [(3)H]-PK 11195 binding in astrocytoma cell homogenates with nanomolar affinity, but did not exhibit specific binding in intact astrocytoma cells in culture, indicating that NIR-6T is unlikely to constitute a useful MI agent for monitoring TSPO expression in intact cells. Conversely, we found that NIR-conPK did not compete for [(3)H]-PK 11195 binding in astrocytoma cell homogenate, but exhibited specific binding in intact astrocytoma cells in culture with nanomolar affinity, suggesting that NIR-conPK binds to a protein distinct, but related to, TSPO. Accordingly, treating intact astrocytoma cells and microglia in culture with cytokines led to significant changes in the amount of NIR-conPK specific binding without corresponding change in TSPO expression. Remarkably, the cytokine-induced changes in the protein targeted by NIR-conPK in intact microglia were selective, since IFN-gamma (but not TNFalpha and TGFbeta) increased the amount of NIR-conPK specific binding in these cells.Together these results suggest that NIR-conPK binds to a protein that is related to TSPO, and expressed by astrocytomas and microglia. Our results also suggest that the expression of this protein is increased by specific cytokines, and thus allows for the monitoring of a particular subtype of microglia activation.