Eiron Cudaback

Identification of a novel endocannabinoid-hydrolyzing enzyme expressed by microglial cells

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.

The Expression Level of CB1 and CB2 Receptors Determines Their Efficacy at Inducing Apoptosis in Astrocytomas

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.

Apolipoprotein E isoforms and regulation of the innate immune response in brain of patients with Alzheimer's disease

The largest genetic risk for late-onset Alzheimers 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.

Apolipoprotein E isoform-dependent microglia migration

Complement component C5a and ATP are potent effectors of microglial movement and are increased in diverse neurodegenerative diseases and at sites of injury. Apolipoprotein E (apoE) influences microglial function, and different human apoE iso‐forms confer variable risk for development of neurodegenerative disorders, especially Alzheimers disease. The purpose of this investigation was to test the hypothesis that mouse apoE and human apoE isoforms influence microglial migration. Using primary wild‐type and apoE‐deficient microglia, we show that C5a‐ and ATP‐stimulated chemotaxis are largely apoE‐dependent processes with different molecular bases. Although the C5a‐dependent chemotaxis of wild‐type microglia was completely blocked by receptor‐associated protein (RAP), suggesting apoE receptor involvement, ATP‐stimu‐lated migration was unaffected by RAP but was associated with differential ERK phosphorylation. Studies using primary microglia derived from targeted replacement mice “humanized” for the coding exons (protein isoform) of human ε2 (apoE2), ε3 (apoE3), or ε4 (apoE4) allele of APOE revealed that primary mouse microglia expressing apoE4 or apoE2 exhibited significantly reduced C5a‐ and ATP‐stimu‐lated migration compared with microglia expressing human apoE3. This study, for the first time, demonstrates apoE dependence and apoE isoform‐specific modulation of microglial migration in response to distinct chemotactic stimuli commonly associated with neurodegenerative disease.—Cudaback, E., Li, X., Montine, K. S., Montine, T. J., Keene, C. D. Apolipoprotein E isoform‐dependent microglia migration. FASEB J. 25, 2082‐2091 (2011). www.fasebj.org

Suppressed microglial E prostanoid receptor 1 signaling selectively reduces tumor necrosis factor alpha and interleukin 6 secretion from toll-like receptor 3 activation

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.

APOE genotype-dependent modulation of astrocyte chemokine CCL3 production

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. Alzheimers 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 C-I is an APOE genotype-dependent suppressor of glial activation

BackgroundInheritance 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.MethodsApoC-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.ResultsApoC-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.ConclusionsApoC-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.

Cerebrospinal Fluid Particles in Alzheimer Disease and Parkinson Disease

Abstract 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) &bgr;42-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 &egr;4/&egr;4 participants had CSF apoE-positive particles that were more frequently larger but at an 88% lower level versus those in APOE &egr;3/&egr;3 or APOE &egr;3/&egr;4 patients; this finding was reproduced in conditioned medium from mouse primary glial cell cultures with targeted replacement of apoE. Cerebrospinal fluid apoE-positive and &bgr;-amyloid (A&bgr;42)-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.

Eicosanoid receptor subtype-mediated opposing regulation of TLR-stimulated expression of astrocyte glial-derived neurotrophic factor

A major therapeutic target for Parkinsons 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. FASEB J. 26, 3075–3083 (2012). www.fasebj.org

Binding of NIR-conPK and NIR-6T to Astrocytomas and Microglial Cells: Evidence for a Protein Related to TSPO

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, [11C]-PK 11195 and [11C]-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 [3H]-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 [3H]-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-γ (but not TNFα and TGFβ) 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.