Xianwu Li

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.

Suppressed Accumulation of Cerebral Amyloid β Peptides in Aged Transgenic Alzheimer’s Disease Mice by Transplantation with Wild-Type or Prostaglandin E2 Receptor Subtype 2-Null Bone Marrow

A complex therapeutic challenge for Alzheimers disease (AD) is minimizing deleterious aspects of microglial activation while maximizing beneficial actions, including phagocytosis/clearance of amyloid beta (Abeta) peptides. One potential target is selective suppression of microglial prostaglandin E(2) receptor subtype 2 (EP2) function, which influences microglial phagocytosis and elaboration of neurotoxic cytokines. To test this hypothesis, we transplanted bone marrow cells derived from wild-type mice or mice homozygous deficient for EP2 (EP2(-/-)) into lethally irradiated 5-month-old wild-type or APPswe-PS1DeltaE9 double transgenic AD mouse model recipients. We found that cerebral engraftment by bone marrow transplant (BMT)-derived wild-type or EP2(-/-) microglia was more efficient in APPswe-PS1DeltaE9 than in wild-type mice, and APPswe-PS1DeltaE9 mice that received EP2(-/-) BMT had increased cortical microglia compared with APPswe-PS1DeltaE9 mice that received wild-type BMT. We found that myeloablative irradiation followed by bone marrow transplant-derived microglia engraftment, rather than cranial irradiation or BMT alone, was responsible for the approximate one-third reduction in both Abeta plaques and potentially more neurotoxic soluble Abeta species. An additional 25% reduction in cerebral cortical Abeta burden was achieved in mice that received EP2(-/-) BMT compared with mice that received wild-type BMT. Our results provide a foundation for an adult stem cell-based therapy to suppress soluble Abeta peptide and plaque accumulation in the cerebrum of patients with AD.

Different mechanisms of apolipoprotein E isoform–dependent modulation of prostaglandin E2 production and triggering receptor expressed on myeloid cells 2 (TREM2) expression after innate immune activation of microglia

Several lines of evidence support immune response in brain as a mechanism of injury in Alzheimer disease (AD). Moreover, immune activation is heightened in apolipoprotein E (APOE) ɛ4 carriers; inhibitors of prostaglandin (PG) synthesis show a partially protective effect on AD risk from APOE ɛ4; and genetic variants in triggering receptor expressed on myeloid cells 2 (TREM2) are a rare but potent risk for AD. We tested the hypothesis that APOE ɛ4 inheritance modulates both the PGE2 pathway and TREM2 expression using primary murine microglia from targeted replacement (TR) APOE3/3 and APOE4/4 mice. Microglial cyclooxygenase‐2, microsomal PGE synthase, and PGE2 expression were increased 2‐ to 25‐fold in both genotypes by TLR activators; however, this induction was significantly (P < 0.01) greater in TR APOE4/4 microglia with TLR3 and TLR4 activators. Microglial TREM2 expression was reduced approximately 85% by all TLR activators; this reduction was approximately one‐third greater in microglia from TR APOE4/4 mice. Importantly, both receptor‐associated protein and a nuclear factor κ‐Hght‐chain‐enhancer inhibitor blocked TR APOE4/4‐dependent effects on the PGE2 pathway but not on TREM2 expression. These data demonstrate complementary, but mechanistically distinct, regulation of pro‐ and anti‐inflammatory mediators in TR APOE4/4 murine microglia that yields a more proinflammatory state than with TR APOE3/3.—Li, X., Montine, K. S., Keene, C. D., Montine, T. J. Different mechanisms of apolipoprotein E isoform‐dependent modulation of prostaglandin E2 production and triggering receptor expressed on myeloid cells 2 (TREM2) expression after innate immune activation of microglia. FASEB J. 29, 1754‐1762 (2015). www.fasebj.org

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.

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.

Ablation of the microglial protein DOCK2 reduces amyloid burden in a mouse model of Alzheimer's disease

Alzheimers disease (AD) neuropathology is characterized by innate immune activation primarily through prostaglandin E2 (PGE2) signaling. Dedicator of cytokinesis 2 (DOCK2) is a guanyl nucleotide exchange factor expressed exclusively in microglia in the brain and is regulated by PGE2 receptor EP2. DOCK2 modulates microglia cytokine secretion, phagocytosis, and paracrine neurotoxicity. EP2 ablation in experimental AD results in reduced oxidative damage and amyloid beta (Aβ) burden. This discovery led us to hypothesize that genetic ablation of DOCK2 would replicate the anti-Aβ effects of loss of EP2 in experimental AD. To test this hypothesis, we crossed mice that lacked DOCK2 (DOCK2-/-), were hemizygous for DOCK2 (DOCK2+/-), or that expressed two DOCK2 genes (DOCK2+/+) with APPswe-PS1Δe9 mice (a model of AD). While we found no DOCK2-dependent differences in cortex or in hippocampal microglia density or morphology in APPswe-PS1Δe9 mice, cerebral cortical and hippocampal Aβ plaque area and size were significantly reduced in 10-month-old APPswe-PS1Δe9/DOCK2-/- mice compared with APPswe-PS1Δe9/DOCK2+/+ controls. DOCK2 hemizygous APPswe-PS1Δe9 mice had intermediate Aβ plaque levels. Interestingly, soluble Aβ42 was not significantly different among the three genotypes, suggesting the effects were mediated specifically in fibrillar Aβ. In combination with earlier cell culture results, our in vivo results presented here suggest DOCK2 contributes to Aβ plaque burden via regulation of microglial innate immune function and may represent a novel therapeutic target for AD.

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