APOE genotype-specific differences in the innate immune response

Apolipoprotein-E protein is an endogenous immunomodulatory agent that affects both the innate and the adaptive immune responses. Since individuals with the APOE4 gene demonstrate worsened pathology and poorer outcomes in many neurological disorders, we examined isoform-specific differences in the response of microglia, the primary cellular component of the brain's innate immune response, in detail. Our data demonstrate that microglia derived from APOE4/4 targeted replacement mice demonstrate a pro-inflammatory phenotype that includes altered cell morphology, increased NO production associated with increased NOS2 mRNA levels, and higher pro-inflammatory cytokine production (TNFα, IL-6, IL12p40) compared to microglia derived from APOE3/3 targeted replacement mice. The effect is gene dose-dependent and increases with the number of APOE4 gene alleles. The APOE genotype-specific immune profile observed in the microglial immune response is also observed in the cortex of aged APOE3/3 and APOE4/4 mice treated with lipopolysacchride (LPS) and in peripheral (peritoneal) macrophages. To determine if APOE4's action resulted from an isoform-specific difference in effective levels of the apolipoproteins, we generated mice expressing only a single allele of APOE3. Immune-stimulated macrophages from APOE3/0 mice demonstrated an increased inflammatory response compared to APOE3/3 mice, but less than in APOE4/4 mice. These data suggest that inhibition of inflammation depends upon the dose of apoE3 protein available and that apoE4 protein may alter inflammation partly by dose effects and partly by being qualitatively different than apoE3. Overall, these data emphasize the important role of apolipoprotein E and of the APOE genotype on the immune responses that are evident in most, if not all, neurological disease.     

Sex steroids, APOE genotype and the innate immune system

Microglia are a primary cellular component of the CNS innate immune system. Their response to conserved pathogen motifs is inherent and leads to the release of cytoactive factors that impact surrounding neurons and glia. The microglial response is modified by the local tissue environment and by "global" factors such as gender. Exposure to estrogen and testosterone, in general, down regulate microglia and peripheral macrophage function, promoting an anti-inflammatory phenotype. Other global factors, however, can "override" the gender-based effects demonstrated by estrogen or testosterone. Apolipoprotein E (APOE) genotype and the expression of specific isoforms of apolipoprotein E differentially regulate microglial and peripheral macrophage function. Our studies have shown that the presence of the APOE4 gene, a known risk factor for AD and other neurodegenerative diseases, promotes a pro-inflammatory macrophage phenotype in neonatal microglia. However, in adult mice, the APOE genotype-specific effect depends on gender. Peritoneal macrophages from female adult APOE3 and APOE4 targeted replacement mice do not demonstrate an APOE genotype-specific response, whereas adult male APOE4 targeted replacement mice show enhanced macrophage responsiveness compared to adult male APOE3 mice. At least part of the altered macrophage response in APOE4 male mice may be due to differences in androgen receptor sensitivity to testosterone. These data re-enforce the concept that classical activation in macrophages has multiple levels of regulation, dictated by competing or synergistic factors and genotype.     

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

ABSTRACT: BACKGROUND: Inheritance of the human epsilon4 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 beta (Abeta), 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 Abeta 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 epsilon3 or epsilon4. 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 Abeta. RESULTS: ApoC-I levels varied with APOE genotype in humans and in APOE targeted replacement mice, with epsilon4 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 Abeta. CONCLUSIONS: ApoC-I is immunosuppressive. Our results illuminate a novel potential mechanism for APOE genotype risk for AD; one in which patients with an epsilon4 allele have decreased expression of apoC-I resulting in increased innate immune activity.     

Apolipoprotein E receptors and amyloid expression are modulated in an apolipoprotein E-dependent fashion in response to hippocampal deafferentation in rodent

The entorhinal cortex lesion paradigm is a widely accepted and efficient method to provoke reactive synaptogenesis and terminal remodeling in the adult CNS. This approach has been used successfully to contrast the profile of reactivity from various proteins associated with Alzheimer's disease pathophysiology in wild-type and apolipoprotein E (apoE)-deficient (APOE ko) mice. Results indicate that the production of the beta-amyloid 1-40 peptide (A beta 40) is increased in response to neuronal injury, with a timing that is different between wild-type and APOE ko animals. Moreover, we report that baseline levels of the A beta 40 peptide are significantly higher in the APOE ko mice. The expression of the apolipoprotein E receptor type 2 (apoER2) is also modulated by the deafferentation process in the hippocampus, but only in APOE ko mice. These results provide novel insights as to the molecular mechanisms responsible for the poor plastic response reported in apoE4-expressing and apoE deficient mice in response to hippocampal injury.     

The APOE4 genotype alters the response of microglia and macrophages to 17beta-estradiol

The apolipoprotein E4 (APOE4) gene is a well-known risk factor for Alzheimer's disease (AD) and other neurological disorders. Post-menopausal women with AD who express at least one APOE4 gene have more severe neuropathology and worsened cognitive scores than their non-expressing counterparts. Since 17β-estradiol down-regulates inflammation as part of its neuroprotective role, we examined the effect of 17β-estradiol on the response of microglia to immune activation as a function of APOE genotype. Our data show that the anti-inflammatory activity of 17β-estradiol is significantly reduced in APOE4 targeted replacement mice compared to APOE3 mice. A significant interaction between APOE genotype and the response to 17β-estradiol was observed for NO and cytokine production by immune activated microglia. The genotype specific effect was not restricted to brain macrophages since peritoneal macrophages from APOE4 ovariectomized mice also demonstrated a significant difference in 17β-estradiol responsiveness. ERβ protein levels in APOE4 microglia were higher than APOE3 microglia, suggesting a difference in post-translational protein regulation in the presence of the APOE4 gene. Overall, our data indicate that the APOE genotype may be a critical component in assessing the effectiveness of 17β-estradiol's action and may impact the neuroprotective role of 17β-estradiol and of hormone replacement therapy on brain function when the APOE4 gene is expressed.     

Microglial Activation in Alzheimer Disease: Association with APOE Genotype

Microglial cells are considered to play an important role in the pathogenesis of Alzheimer disease. Apart from producing the Alzheimer amyloid precursor (APP) as an acute phase protein, microglial cells seem to be involved in the deposition of its amyloidogenic cleavage product, the amyloid-β peptide (Aβ). Aβ is bound by apolipoprotein E (APOE) in an isoform-specific manner, and it has been demonstrated that inheritance of the AD susceptibility allele, APOE ε4, is associated with increased deposition of Aβ in the cerebral cortex. However, the relationship between APOE ε4 gene dose and microglial activation is unknown. Using microglial expression of major histocompatibility complex class II molecules as a marker, we have performed a quantitative genotype-phenotype analysis on microglial activation in frontal and temporal cortices of 20 APOE genotyped AD brains. The number of activated microglia and the tissue area occupied by these cells increased significantly with APOE ε4 gene dose. When a model of multiple linear regression was used to compare the relative influence of APOE genotype, sex, disease duration, age at death, diffuse and neuritic plaques as well as neurofibrillary tangles on microglial activation, only APOE genotype was found to have a significant effect. Thus, the APOE gene product represents an important determinant of microglial activity in AD. Since microglial activation by APP has been shown to be modulated by apoE in vitro, a direct role of microglia in AD pathogenesis is conceivable.     

An apolipoprotein E-based therapeutic improves outcome and reduces Alzheimer's disease pathology following closed head injury: evidence of pharmacogenomic interaction

Apolipoprotein E (apoE) modifies glial activation and the CNS inflammatory response in an isoform-specific manner. Peptides derived from the receptor-binding region of apoE have been demonstrated to maintain the functional activity of the intact protein, and to improve histological and functional deficits after closed head injury. In the current study, APOE2, APOE3, and APOE4 targeted replacement (TR) mice expressing the human apoE protein isoforms (apoE2, apoE3 and apoE4) were used in a clinically relevant model of closed head injury to assess the interaction between the humanized apoE background and the therapeutic apoE mimetic peptide, apoE(133-149). Treatment with the apoE-mimetic peptide reduced microglial activation and early inflammatory events in all of the targeted replacement animals and was associated with histological and functional improvement in the APOE2TR and APOE3TR animals. Similarly, brain beta amyloid protein (Abeta)(1-42) levels were increased as a function of head injury in all of the targeted replacement mice, while treatment with apoE peptide suppressed Abeta(1-42) levels in the APOE2TR and APOE3TR animals. These results suggest a pharmacogenomic interaction between the therapeutic effects of the apoE mimetic peptide and the human apoE protein isoforms. Furthermore, they suggest that administration of apoE-mimetic peptides may serve as a novel therapeutic strategy for the treatment of acute and chronic neurological disease.     

Apolipoprotein E-specific innate immune response in astrocytes from targeted replacement mice

Background  

Inheritance of the three different alleles of the human apolipoprotein (apo) E gene (APOE) are associated with varying risk or clinical outcome from a variety of neurologic diseases. ApoE isoform-specific modulation of several pathogenic processes, in addition to amyloid β metabolism in Alzheimer's disease, have been proposed: one of these is innate immune response by glia. Previously we have shown that primary microglia cultures from targeted replacement (TR) APOE mice have apoE isoform-dependent innate immune activation and paracrine damage to neurons that is greatest with TR by the ε4 allele (TR APOE4) and that derives from p38 mitogen-activated protein kinase (p38MAPK) activity.     

The Involvement of Collagen Triple Helix Repeat Containing 1 in Muscular Dystrophies

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.Humans uniquely have three different apolipoprotein E (APOE) alleles (ɛ2, ɛ3, and ɛ4). APOE4 is the single greatest genetic risk factor for late-onset Alzheimer disease (AD), and there is a gene dosage effect.¹ However, genetic association does not inform function/pathogenesis. Multiple mechanisms have been postulated that predominantly focus on production, metabolism, or clearance of amyloid-β (Aβ) and that are variably supported by multiple observations, including: i) APOE genotype is strongly related to Aβ levels in brain and cerebrospinal fluid of AD patients^2,3; ii) modulation of apolipoprotein E (apoE) protein levels in brain results in alterations of Aβ burden^4,5; iii) Aβ degradation is at least partially apoE dependent^6,7; and iv) Aβ clearance is differentially modulated by apoE isoforms, with APOE4 mice exhibiting reduced central and peripheral Aβ clearance compared with APOE3 mice.^8–10 Aβ degradation and clearance is at least partially dependent on microglia, the innate immune effector cells of the brain. Microglia have migratory and phagocytic capacity, are increased in the vicinity of Aβ plaques, and phagocytose Aβ.^11–13 APOE genotype modulates central nervous system innate immune function in culture,¹⁴ including astrocyte and microglia elaboration of cytokines and chemokines,^15,16 microglia production of reactive oxygen species,¹⁷ microglia-mediated paracrine neurotoxicity,¹⁸ microglia migration,¹⁹ and other functions.²⁰ However, the specific contribution of microglial APOE genotype to AD pathophysiology in vivo is largely unknown.To address this critical question and to test a potential therapeutic application, we used the fact that bone marrow transplantation (BMT) results in the gradual replacement of endogenous (host) microglia (to the near exclusion of other cell types) with microglia derived from donor marrow, in both wild-type mice and transgenic mouse models of AD.^21–24 We used targeted-replacement (TR) APOE mice homozygous for either the APOE3 or APOE4 gene inserted into the mouse APOE regulatory elements^25,26 that coexpressed green fluorescent protein (GFP). We transplanted whole bone marrow (BM) isolated from TR APOE3/3;GFP or TR APOE4/4;GFP mice into lethally irradiated APPswe/PS1ΔE9 mice to determine the specific role of microglial APOE genotype in the pathological progression of AD.     

Human APOE protein localized in brains of transgenic mice

Transgenic mice carrying the three common human apolipoprotein E (APOE) alleles have been developed. In this study, brains of the transgenic mice have been analyzed by in situ histohybridization, immunohistochemistry, and immunoblots to determine sites of gene expression, to identify specific brain cells associated with human apoE protein, and to determine the relative concentrations of the human apoE. Results indicate that (1) human APOE mRNA and apoE protein occur in the gray and white matter of transgenic mouse brains; (2) in the hippocampus of transgenic brains, human apoE protein reacts immunologically within the same cells as the glial fibrillary acidic protein (GFAP), a specific marker for astrocytes; and (3) concentrations of the apoE isoforms determined in three heterozygous transgenic brains range from 22 to 250 pmol/g wet weight of brain.     

Human apoE isoforms differentially regulate brain amyloid-β peptide clearance

The apolipoprotein E (APOE) ε4 allele is the strongest genetic risk factor for late-onset, sporadic Alzheimer's disease (AD). The APOE ε4 allele markedly increases AD risk and decreases age of onset, likely through its strong effect on the accumulation of amyloid-β (Aβ) peptide. In contrast, the APOE ε2 allele appears to decrease AD risk. Most rare, early-onset forms of familial AD are caused by autosomal dominant mutations that often lead to overproduction of Aβ(42) peptide. However, the mechanism by which APOE alleles differentially modulate Aβ accumulation in sporadic, late-onset AD is less clear. In a cohort of cognitively normal individuals, we report that reliable molecular and neuroimaging biomarkers of cerebral Aβ deposition vary in an apoE isoform-dependent manner. We hypothesized that human apoE isoforms differentially affect Aβ clearance or synthesis in vivo, resulting in an apoE isoform-dependent pattern of Aβ accumulation later in life. Performing in vivo microdialysis in a mouse model of Aβ-amyloidosis expressing human apoE isoforms (PDAPP/TRE), we find that the concentration and clearance of soluble Aβ in the brain interstitial fluid depends on the isoform of apoE expressed. This pattern parallels the extent of Aβ deposition observed in aged PDAPP/TRE mice. ApoE isoform-dependent differences in soluble Aβ metabolism are observed not only in aged but also in young PDAPP/TRE mice well before the onset of Aβ deposition in amyloid plaques in the brain. Additionally, amyloidogenic processing of amyloid precursor protein and Aβ synthesis, as assessed by in vivo stable isotopic labeling kinetics, do not vary according to apoE isoform in young PDAPP/TRE mice. Our results suggest that APOE alleles contribute to AD risk by differentially regulating clearance of Aβ from the brain, suggesting that Aβ clearance pathways may be useful therapeutic targets for AD prevention.     

Comparison of astrocytic and myocytic metabolic dysregulation in apolipoprotein E deficient and human apolipoprotein E transgenic mice

The accumulation of tubular aggregates in type II skeletal muscle fibres and fibrillo-granular inclusions in hippocampal protoplasmic astrocytes are characteristic lesions of apolipoprotein E deficient mice. Moreover these inclusions reacted immunocytochemically with an antibody specific to fragment 17-24 of the published sequence of Alzheimer's amyloid peptide. In an effort to evaluate the role of apolipoprotein E in the formation of these abnormal structures, we examined the tibialis anterior muscle and the hippocampus of several groups of animals including: (i) apolipoprotein E "knockout" mice which had been whole body irradiated with 1200 rads and bone marrow replenished with apolipoprotein E sufficient marrow; and (ii) three transgenic murine strains that had been genetically engineered to express either human apolipoprotein E2, E3 or E4 protein on an apoE deficient background. The results of this study showed that the presence of murine apolipoprotein E (even in subnormal levels in the serum) in irradiated bone marrow replenished mice and in all three (E2, E3 or E4) human apoE transgenic strains was sufficient to prevent the aggregation of sarcoplasmic tubules in the tibialis anterior type II muscle fibres. Similarly apolipoprotein E "knockout" bone marrow replenished mice and all three transgenic strains expressing the different human apolipoprotein E alleles reduced the number of the astrocytic inclusions in the hippocampus to levels not significantly different to those observed in control C57Bl6J animals.The data obtained in this study indicate that neurological and neuromuscular abnormalities found in apoE deficient mice are reversed when apoE protein is replaced in the circulation, either by bone marrow transplantation of normal apoE sufficient marrow, or by gene therapy with the apoE gene, albeit of human origin and irrespective of the allele used.     

Progressive loss of synaptic integrity in human apolipoprotein E4 targeted replacement mice and attenuation by apolipoprotein E2

Inheritance of the APOE4 allele is a well established genetic risk factor linked to the development of late onset Alzheimer's disease. As the major lipid transport protein in the central nervous system, apolipoprotein (apo) E plays an important role in the assembly and maintenance of synaptic connections. Our previous work showed that 7 month old human apoE4 targeted replacement (TR) mice displayed significant synaptic deficits in the principal neurons of the lateral amygdala, a region that is critical for memory formation and also one of the primary regions affected in Alzheimer's disease, compared to apoE3 TR mice. In the current study, we determined how age and varying APOE genotype affect synaptic integrity of amygdala neurons by comparing electrophysiological and morphometric properties in C57BL6, apoE knockout, and human apoE3, E4 and E2/4 TR mice at 1 month and 7 months. The apoE4 TR mice exhibited the lowest level of excitatory synaptic activity and dendritic arbor compared to other cohorts at both ages, and became progressively worse by 7 months. In contrast, the apoE3 TR mice exhibited the highest synaptic activity and dendritic arbor of all cohorts at both ages. C57BL6 mice displayed virtually identical synaptic activity to apoE3 TR mice at 1 month; however this activity decreased by 7 months. ApoE knockout mice exhibited a similar synaptic activity profile with apoE4 TR mice at 7 months. Consistent with previous reports that APOE2 confers protection, the apoE4-dependent deficits in excitatory activity were significantly attenuated in apoE2/4 TR mice at both ages. These findings demonstrate that expression of human apoE4 contributes to functional deficits in the amygdala very early in development and may be responsible for altering neuronal circuitry that eventually leads to cognitive and affective disorders later in life.     

Reduced levels of human apoE4 protein in an animal model of cognitive impairment

The APOE4 allele is the most common genetic determinant for Alzheimer's disease (AD) in the developed world. APOE genotype specific differences in brain apolipoprotein E protein levels have been observed in numerous studies since the discovery of APOE4's link to AD. Since the human apoE4 targeted replacement mice display characteristics of cognitive impairment we sought to determine if reduced levels of apoE might provide one explanation for this impairment. We developed a novel mass spectrometry method to measure apoE protein levels in plasma. Additionally, we developed an ELISA that replicates the mass spectrometry data and enables the rapid quantitation of apoE in plasma, brain and cerebrospinal fluid. We detected a significant decrease in plasma, brain and cerebrospinal fluid apoE levels in the apoE4 mice compared to apoE2 and E3 mice. We also measured a small (∼19%) decrease in brain apoE levels from aged, non-demented APOE4 carriers. Our findings suggest that a fraction of APOE4-linked AD may be due to insufficient levels of functional apoE required to maintain neuronal health.     

A Role for apoE in Regulating the Levels of α-1-Antichymotrypsin in the Aging Mouse Brain and in Alzheimer’s Disease

This study was designed to explore the possible functional relationships between apolipoprotein E (apoE) and the protease inhibitor alpha-1-antichymotrypsin in the aging mouse brain and in Alzheimer's disease. For this purpose, levels of EB22/5 (the mouse homologue to human alpha-1-antichymotrypsin) mRNA expression was studied in apoE-deficient mice. These mice showed an age-dependent increase of EB22/5 mRNA expression in the brain. Furthermore, overexpression of allele 3 of human APOE gene in transgenic mice (in an apoE-deficient background) resulted in normalization of levels of EB22/5 mRNA expression compatible with levels found in control mice. In contrast, overexpression of human APOE4 allele or down-regulation of the apoE receptor low density lipoprotein receptor-related protein by deletion of the receptor-associated protein was associated with elevated levels of EB22/5 similar to apoE-deficient mice. Consistent with the findings in murine models, human alpha-1-antichymotrypsin protein was increased in brain homogenates from patients with Alzheimer's disease, and levels of this serpin were the highest in patients with the APOE4 allele. In summary, the present study showed evidence supporting a role for apoE in regulating alpha-1-antichymotrypsin expression. This is relevant to Alzheimer's disease because these two molecules appear to be closely associated with the pathogenesis of this disorder.     

Apolipoprotein E isoform-specific effects on cytokine and nitric oxide production from mouse Schwann cells after inflammatory stimulation

Previously, we reported that apolipoprotein E (apoE) deficiency increased the susceptibility to experimental autoimmune neuritis (EAN), an inflammatory autoimmune disorder of the peripheral nervous system (PNS) and an animal model for human Guillain-Barré syndrome (GBS) by affecting the antigen-presenting function of Schwann cells (SCs) via influence upon IL-6 production. To further elucidate the role of apoE in inflammation of the PNS, here we studied the effect of different isoforms of apoE on SCs in response to inflammatory stimulation. SCs from apoE2, E3 and E4 transgenic (Tg) and wild type (WT) mice were cultured, and their responses to stimulation by lipopolysaccharide (LPS) plus interferon (IFN)-γ were compared. Upon stimulation, the morphology of cultured SCs changed. Pronounced production of interleukin (IL)-6 and IL-10 within SCs, and of IL-6 and nitric oxide (NO) in the supernatants were found in an isoform-dependent manner (apoE3>apoE2≈apoE4). Further results indicated that both nuclear factor (NF) κB and Akt signaling pathways were involved in the process by the same isoform-dependent pattern. However, the expression of co-stimulatory molecules as showing the antigen-presenting capacity of SCs was not significantly different among these groups. In conclusion, SCs respond to inflammatory insults accompanied by increased productions of IL-6, IL-10 and NO in an apoE-isoform-dependent manner. SCs from apoE2 and apoE4 Tg mice seem to bear some dysfunction in producing cytokines (IL-6 and IL-10) and NO as compared with their apoE3 counterparts, probably resulting from their insufficiency to suppress the activation of NFκB and Akt pathways. Our findings may help to understand the role of different isoforms of apoE in inflammatory disorders of the PNS.     

Apolipoprotein E-genotype dependent hippocampal and cortical responses to traumatic brain injury

The different alleles of the apolipoprotein E gene (APOE-gene, ApoE-protein) have been reported to influence recovery after traumatic brain injury (TBI) in both human patients and animal models, with the e4 allele typically conferring poorer prognosis for recovery. How the E4 allele, and consequently the ApoE4 isoform, affects recovery is unknown, but proposed mechanisms include neurogenesis, inflammatory response and amyloid processing or metabolism. Using the controlled cortical impact (CCI) model of brain injury and microarray technology we have characterized the genomic response to injury in the brains of APOE2, APOE3 and APOE4 transgenic mice and identified quantitatively and qualitatively significantly different profiles of gene expression in both the hippocampus and the cortex of the APOE3 mice compared to APOE4. The observed gene regulation predicts functional consequences including effects on inflammatory processes, cell growth and proliferation, and cellular signaling, and may suggest that the poor recovery post-TBI in APOE4 animals and human patients is less likely to result from a specific activation of neurodegenerative mechanisms than a loss of reparative capability.     

Microglial MHC class II is dispensable for experimental autoimmune encephalomyelitis and cuprizone‐induced demyelination

Microglia are resident immune cells in the CNS, strategically positioned to clear dead cells and debris, and orchestrate CNS inflammation and immune defense. In steady state, these macrophages lack MHC class II (MHCII) expression, but microglia activation can be associated with MHCII induction. Whether microglial MHCII serves antigen presentation for critical local T‐cell restimulation in CNS auto‐immune disorders or modulates microglial signaling output remains under debate. To probe for such scenarios, we generated mice harboring an MHCII deficiency in microglia, but not peripheral myeloid cells. Using the CX₃CR1^CreER‐based approach we report that microglial antigen presentation is obsolete for the establishment of EAE, with disease onset, progression, and severity unaltered in mutant mice. Antigen presentation‐independent roles of microglial MHCII were explored using a demyelination model induced by the copper chelator cuprizone. Absence of microglial I‐A^b did not affect the extent of these chemically induced white matter alterations, nor did it affect microglial proliferation or gene expression associated with locally restricted de‐ and remyelination.     

Apolipoprotein E isoform-dependent dendritic recovery of hippocampal neurons following activation of innate immunity

Background  

Innate immune activation, including a role for cluster of differentiation 14/toll-like receptor 4 co-receptors (CD14/TLR-4) co-receptors, has been implicated in paracrine damage to neurons in several neurodegenerative diseases that also display stratification of risk or clinical outcome with the common alleles of the apolipoprotein E gene (APOE): APOE2, APOE3, and APOE4. Previously, we have shown that specific stimulation of CD14/TLR-4 with lipopolysaccharide (LPS) leads to greatest innate immune response by primary microglial cultures from targeted replacement (TR) APOE4 mice and greatest p38MAPK-dependent paracrine damage to neurons in mixed primary cultures and hippocampal slice cultures derived from TR APOE4 mice. In contrast, TR APOE2 astrocytes had the highest NF-kappaB activity and no neurotoxicity. Here we tested the hypothesis that direct activation of CD14/TLR-4 in vivo would yield different amounts of paracrine damage to hippocampal sector CA1 pyramidal neurons in TR APOE mice.     

Gx‐50 reduces β‐amyloid‐induced TNF‐α, IL‐1β, NO,and PGE2 expression and inhibits NF‐κB signaling in a mouse model of Alzheimer's disease

Chronic inflammation, which is regulated by overactivated microglia in the brain, accelerates the occurrence and development of Alzheimer's disease (AD). Gx‐50 has been investigated as a novel drug for the treatment of AD in our previous studies. Here, we investigated whether gx‐50 possesses anti‐inflammatory effects in primary rat microglia and a mouse model of AD, amyloid precursor protein (APP) Tg mice. The expression of TNF‐α, IL‐1β, NO, prostaglandin E2, and the expression of iNOS and COX2 were inhibited by gx‐50 in amyloid β (Aβ) treated rat microglia; additionally, microglial activation and the expression of IL‐1β, iNOS, and COX2 were also significantly suppressed by gx‐50 in APP⁺ transgenic mice. Furthermore, gx‐50 inhibited the activation of NF‐κB and MAPK cascades in vitro and in vivo in APP‐Tg mice. Moreover, the expression of TLR4 and its downstream signaling proteins MyD88 and tumor necrosis factor receptor associated factor 6 (TRAF6) was reduced by gx‐50 in vitro and in vivo. Interestingly, silencing of TLR4 reduced Aβ‐induced upregulation of IL‐1β and TRAF6 to levels similar to gx‐50 inhibition; moreover, overexpression of TLR4 increased the expression of MyD88 and TRAF6, which was significantly reduced by gx‐50. These findings provide strong evidence that gx‐50 has anti‐inflammatory effects against Aβ‐triggered microglial overactivation via a mechanism that involves the TLR4‐mediated NF‐κBB/MAPK signaling cascade.