Lipopolysaccharide-Induced Exosomal miR-146a Is Involved in Altered Expression of Alzheimer's Risk Genes Via Suppression of TLR4 Signaling.

Repeated exposure to toll-like receptor 4 (TLR4) ligands, such as lipopolysaccharide (LPS), reduces responses of monocytes/macrophages to LPS (LPS/endotoxin tolerance). Microglial exposure to Aß deposits, a TLR4 ligand, may cause "Aß/LPS tolerance," leading to decreased Aß clearance. We demonstrated that microglial activation by LPS is diminished in Aß deposit-bearing 12-month-old model mice of Alzheimer's disease (AD), compared with non-AD mice and Aß deposit-free 2-month-old AD mice. Because miR-146a plays a predominant role in inducing TLR tolerance in macrophages and because miR-146a in extracellular vesicles (EVs) shed by inflammatory macrophages increases in circulation, we investigated potential roles of miR-146a and inflammatory EVs in inducing TLR tolerance in microglia and in altering expression of inflammatory AD risk genes. We found that miR-146a upregulation induces TLR tolerance and alters expression of inflammatory AD risk genes in response to LPS treatment in BV2 microglia. LPS brain injection altered expression of the AD risk genes in 12-month-old AD mice but not in non-AD littermates. EVs from inflammatory macrophages polarize BV2 microglia to M1 phenotype and induce TLR tolerance. Microglia exposed to Aß in the brain show reduced cytokine responses to systemic inflammation due to peripheral LPS injection, indicating TLR/Aß tolerance in microglia. Our results suggest that increased miR-146a induces microglial Aß/LPS tolerance and that circulating EVs shed by inflammatory macrophages contribute to microglial Aß/LPS tolerance, leading to reduced Aß clearance. Our study also suggests that altered expression of inflammatory AD risk genes may contribute to AD development via the same molecular mechanism underlying LPS tolerance.

Intracranial IL-17A overexpression decreases cerebral amyloid angiopathy by upregulation of ABCA1 in an animal model of Alzheimer's disease.

Neuroinflammation is a pervasive feature of Alzheimer's disease (AD) and characterized by activated microglia, increased proinflammatory cytokines and/or infiltrating immune cells. T helper 17 (Th17) cells are found in AD brain parenchyma and interleukin-17A (IL-17A) is identified around deposits of aggregated amyloid ß protein (Aß). However, the role of IL-17A in AD pathogenesis remains elusive. We overexpressed IL-17A in an AD mouse model via recombinant adeno-associated virus serotype 5 (rAAV5)-mediated intracranial gene delivery. AD model mice subjected to injection of a vehicle (PBS) or rAAV5 carrying the lacZ gene served as controls. IL-17A did not exacerbate neuroinflammation in IL-17A-overexpressing mice. We found that IL-17A overexpression markedly improved glucose metabolism, decreased soluble Aß levels in the hippocampus and cerebrospinal fluid, drastically reduced cerebral amyloid angiopathy, and modestly but significantly improved anxiety and learning deficits. Moreover, the ATP-binding cassette subfamily A member 1 (ABCA1), which can transport Aß from the brain into the blood circulation, significantly increased in IL-17A-overexpressing mice. In vitro treatment of brain endothelial bEnd.3 cells with IL-17A induced a dose-dependent increase in protein expression of ABCA1 through ERK activation. Our study suggests that IL-17A may decrease Aß levels in the brain by upregulating ABCA1 in blood-brain barrier endothelial cells.

Microglial response to LPS increases in wild-type mice during aging but diminishes in an Alzheimer's mouse model: Implication of TLR4 signaling in disease progression.

Microglia-mediated clearance of amyloid beta-protein (Aß) via Toll-like receptor 4 (TLR4) signaling may play an important role in the pathogenesis of Alzheimer's disease (AD). However, as the disease progresses, activated microglia appear to become incapable of clearing Aß deposits. Because repeated exposure to a TLR4 ligand leads to a diminished response of monocytes/macrophages to lipopolysaccharide (LPS) and because aggregated Aß is a TLR4 ligand, we hypothesize that chronic exposure of microglia to Aß deposits may induce a state of Toll-like receptor (TLR) signaling dysfunction, leading to decreased Aß clearance and accelerated disease progression. LPS or phosphate-buffered saline (PBS) was injected into the hippocampus of AD-model (TgAPP/PS1) and wild-type (non-Tg) mice before and after the onset of Aß deposition, at age 2 and 12 months, respectively. Brain specimens were collected 7 days post-injection and analyzed for microglial activation and Aß load. While LPS-injected 2-month-old non-Tg mice showed 48-fold and 11-fold greater Iba1 immunoreactivity in the neocortex and hippocampus, respectively, compared with PBS-injected mice, LPS-injected 2-month-old TgAPP/PS1 mice had 61-fold and 13-fold increases in the neocortex and hippocampus, respectively. LPS injection activated microglia more strongly in TgAPP/PS1 mice than in non-Tg mice at 2 months of age. In contrast, at 12 months of age, Iba1 immunoreactivity of microglia was increased 541-fold and 38-fold in the neocortex and hippocampus, respectively, in LPS-injected non-Tg mice and 2.7-fold and 3.3-fold in the neocortex and hippocampus, respectively, in LPS-injected TgAPP/PS1 mice. Surprisingly, LPS injection decreased CD45 immunoreactivity in TgAPP/PS1 mice but increased it in non-Tg mice at 12 months. Although microglia in 12-month-old non-Tg mice showed stronger response to LPS than 2-month-old non-Tg mice, microglia in TgAPP/PS1 mice exhibited diminished immune response to LPS during aging. Our data indicate that microglial TLR4 signaling is altered in an AD mouse model and suggest that altered TLR4 signaling may contribute to Aß accumulation in the brain.

Intracranial delivery of interleukin-17A via adeno-associated virus fails to induce physical and learning disabilities and neuroinflammation in mice but improves glucose metabolism through AKT signaling pathway.

Interleukin-17A (IL-17A) is generally considered as one of the pathogenic factors involved in multiple sclerosis (MS). Indirect evidence for this is that IL-17A-producing T helper 17 (Th17) cells preferentially accumulate in lesions of MS and experimental autoimmune encephalomyelitis (EAE). However, a direct involvement of IL-17A in MS pathogenesis is still an open question. In this study, we overexpressed IL-17A in the brains of mice (IL-17A-in-Brain mice) via recombinant adeno-associated virus serotype 5 (rAAV5)-mediated gene delivery. In spite of high levels of IL-17A expression in the brain and blood, IL-17A-in-Brain mice exhibit no inflammatory responses and no abnormalities in motor coordination and spatial orientation. Unexpectedly, IL-17A-in-Brain mice show decreases in body weight and adipose tissue mass and an improvement in glucose tolerance and insulin sensitivity. IL-17A enhances glucose uptake in PC12 cells by activation of AKT. Our results provide direct evidence for the first time that IL-17A overexpression in the central nervous system does not cause physical and learning disabilities and neuroinflammation and suggest that IL-17A may regulate glucose metabolism through the AKT signaling pathway.

Catalytic immunoglobulin gene delivery in a mouse model of Alzheimer's disease: prophylactic and therapeutic applications.

Accumulation of amyloid beta-peptide (Aß) in the brain is hypothesized to be a causal event leading to dementia in Alzheimer's disease (AD). Aß vaccination removes Aß deposits from the brain. Aß immunotherapy, however, may cause T cell- and/or Fc-receptor-mediated brain inflammation and relocate parenchymal Aß deposits to blood vessels leading to cerebral hemorrhages. Because catalytic antibodies do not form stable immune complexes and Aß fragments produced by catalytic antibodies are less likely to form aggregates, Aß-specific catalytic antibodies may have safer therapeutic profiles than reversibly-binding anti-Aß antibodies. Additionally, catalytic antibodies may remove Aß more efficiently than binding antibodies because a single catalytic antibody can hydrolyze thousands of Aß molecules. We previously isolated Aß-specific catalytic antibody, IgVL5D3, with strong Aß-hydrolyzing activity. Here, we evaluated the prophylactic and therapeutic efficacy of brain-targeted IgVL5D3 gene delivery via recombinant adeno-associated virus serotype 9 (rAAV9) in an AD mouse model. One single injection of rAAV9-IgVL5D3 into the right ventricle of AD model mice yielded widespread, high expression of IgVL5D3 in the unilateral hemisphere. IgVL5D3 expression was readily detectable in the contralateral hemisphere but to a much lesser extent. IgVL5D3 expression was also confirmed in the cerebrospinal fluid. Prophylactic and therapeutic injection of rAAV9-IgVL5D3 reduced Aß load in the ipsilateral hippocampus of AD model mice. No evidence of hemorrhages, increased vascular amyloid deposits, increased proinflammatory cytokines, or infiltrating T-cells in the brains was found in the experimental animals. AAV9-mediated anti-Aß catalytic antibody brain delivery can be prophylactic and therapeutic options for AD.

Muscle-directed anti-Aß single-chain antibody delivery via AAV1 reduces cerebral Aß load in an Alzheimer's disease mouse model.

We previously reported that anti-amyloid-beta (Aß) single-chain antibody (scFv59) brain delivery via recombinant adeno-associated virus (rAAV) was effective in reducing cerebral Aß load in an Alzheimer's disease (AD) mouse model without inducing inflammation. Here, we investigated the prophylactic effects and mechanism of a muscle-directed gene therapy modality in an AD mouse model. We injected rAAV serotype 1 encoding scFv59 into the right thigh muscles of 3-month-old mice. Nine months later, high levels of scFv59 expression were confirmed in the thigh muscles by both immunoblotting and immunohistochemistry. As controls, model mice were similarly injected with rAAV1 encoding antihuman immunodeficiency virus Gag antibody (scFvGag). AAV1-mediated scFv59 gene delivery was effective in decreasing Aß deposits in the brain. Compared with the scFvGag group, levels of Aß in cerebrospinal fluid (CSF) decreased significantly while Aß in serum tended to increase in the scFv59 group. AAV1-mediated scFv59 gene delivery may alter the equilibrium of Aß between the blood and brain, resulting in an increased efflux of Aß from the brain owing to antibody-mediated sequestration/clearance of peripheral Aß. Our results suggest that muscle-directed scFv59 delivery via rAAV1 may be a prophylactic option for AD and that levels of CSF Aß may be used to evaluate the efficacy of anti-Aß immunotherapy.

Combined treatment of Aß immunization with statin in a mouse model of Alzheimer's disease.

We evaluated the therapeutic efficacy of combined treatment of Aß-immunization with simvastatin in an Alzheimer mouse model at age 22 months. DNA prime-adenovirus boost immunization induced modest anti-Aß titers and simvastatin increased the seropositive rate. Aß-KLH was additionally administered to boost the titers. Irrespective of simvastatin, the immunization did not decrease cerebral Aß deposits but increased soluble Aß and tended to exacerbate amyloid angiopathy in the hippocampus. The immunization increased cerebral invasion of leukocytes and simvastatin counteracted the increase. Thus, modest anti-Aß titers can increase soluble Aß and simvastatin may reduce inflammation associated with vaccination in aged Alzheimer mouse models.

The effects of MyD88 deficiency on exploratory activity, anxiety, motor coordination, and spatial learning in C57BL/6 and APPswe/PS1dE9 mice.

Toll-like receptors (TLRs) are a family of pattern-recognition receptors in innate immunity and provide a first line defense against pathogens and tissue injuries. In addition to important roles in infection, inflammation, and immune diseases, recent studies show that TLR signaling is involved in modulation of learning, memory, mood, and neurogenesis. Because MyD88 is essential for the downstream signaling of all TLRs, except TLR3, we investigated the effects of MyD88 deficiency (MyD88-/-) on behavioral functions in mice. Additionally, we recently demonstrated that a mouse model of Alzheimer's disease (AD) deficient for MyD88 had decreases in Aß deposits and soluble Aß in the brain as compared with MyD88 sufficient AD mouse models. Because accumulation of Aß in the brain is postulated to be a causal event leading to cognitive deficits in AD, we investigated the effects of MyD88 deficiency on behavioral functions in the AD mouse model at 10 months of age. MyD88 deficient mice showed more anxiety in the elevated plus-maze. In the motor coordination tests, MyD88 deficient mice remained on a beam and a bar for a longer time, but with slower initial movement on the bar. In the Morris water maze test, MyD88 deficiency appeared to improve spatial learning irrespective of the transgene. Our findings suggest that the MyD88-dependent pathway contributes to behavioral functions in an AD mouse model and its control group.

TLR4 mutation reduces microglial activation, increases Aß deposits and exacerbates cognitive deficits in a mouse model of Alzheimer's disease.

BACKGROUND: Amyloid plaques, a pathological hallmark of Alzheimer's disease (AD), are accompanied by activated microglia. The role of activated microglia in the pathogenesis of AD remains controversial: either clearing Aß deposits by phagocytosis or releasing proinflammatory cytokines and cytotoxic substances. Microglia can be activated via toll-like receptors (TLRs), a class of pattern-recognition receptors in the innate immune system. We previously demonstrated that an AD mouse model homozygous for a loss-of-function mutation of TLR4 had increases in Aß deposits and buffer-soluble Aß in the brain as compared with a TLR4 wild-type AD mouse model at 14-16 months of age. However, it is unknown if TLR4 signaling is involved in initiation of Aß deposition as well as activation and recruitment of microglia at the early stage of AD. Here, we investigated the role of TLR4 signaling and microglial activation in early stages using 5-month-old AD mouse models when Aß deposits start. METHODS: Microglial activation and amyloid deposition in the brain were determined by immunohistochemistry in the AD models. Levels of cerebral soluble Aß were determined by ELISA. mRNA levels of cytokines and chemokines in the brain and Aß-stimulated monocytes were quantified by real-time PCR. Cognitive functions were assessed by the Morris water maze. RESULTS: While no difference was found in cerebral Aß load between AD mouse models at 5 months with and without TLR4 mutation, microglial activation in a TLR4 mutant AD model (TLR4M Tg) was less than that in a TLR4 wild-type AD model (TLR4W Tg). At 9 months, TLR4M Tg mice had increased Aß deposition and soluble Aß42 in the brain, which were associated with decrements in cognitive functions and expression levels of IL-1ß, CCL3, and CCL4 in the hippocampus compared to TLR4W Tg mice. TLR4 mutation diminished Aß-induced IL-1ß, CCL3, and CCL4 expression in monocytes. CONCLUSION: This is the first demonstration of TLR4-dependent activation of microglia at the early stage of ß-amyloidosis. Our results indicate that TLR4 is not involved in the initiation of Aß deposition and that, as Aß deposits start, microglia are activated via TLR4 signaling to reduce Aß deposits and preserve cognitive functions from Aß-mediated neurotoxicity.

MyD88 deficiency ameliorates ß-amyloidosis in an animal model of Alzheimer's disease.

The accumulation of ß-amyloid protein (Aß) in the brain is thought to be a primary etiologic event in Alzheimer's disease (AD). Fibrillar Aß plaques, a hallmark of AD abnormality, are closely associated with activated microglia. Activated microglia have contradictory roles in the pathogenesis of AD, being either neuroprotective (by clearing harmful Aß and repairing damaged tissues) or neurotoxic (by producing proinflammatory cytokines and reactive oxygen species). Aß aggregates can activate microglia by interacting with multiple toll-like receptors (TLRs), the pattern-recognition receptors of the innate immune system. Because the adapter protein MyD88 is essential for the downstream signaling of all TLRs, except TLR3, we investigated the effects of MyD88 deficiency (MyD88(-/-)) on Aß accumulation and microglial activation in an AD mouse model. MyD88 deficiency decreased Aß load and microglial activation in the brain. The decrease in Aß load in an MyD88(-/-) AD mouse model was associated with increased and decreased protein expression of apolipoprotein E (apoE) and CX3CR1, respectively, compared with that in an MyD88 wild-type AD mouse model. These results suggest that MyD88 deficiency may reduce Aß load by enhancing the phagocytic capability of microglia through fractalkine (the ligand of CX3CR1) signaling and by promoting apoE-mediated clearance of Aß from the brain. These findings also suggest that chronic inflammatory responses induced by Aß accumulation via the MyD88-dependent signaling pathway exacerbate ß-amyloidosis in AD.

Anti-Amyloid-ß Single-Chain Antibody Brain Delivery Via AAV Reduces Amyloid Load But May Increase Cerebral Hemorrhages in an Alzheimer's Disease Mouse Model.

Accumulation of amyloid-ß protein (Aß) in the brain is thought to be a causal event in Alzheimer's disease (AD). Immunotherapy targeting Aß holds great promise for reducing Aß in the brain. Here, we evaluated the efficacy and safety of anti-Aß single-chain antibody (scFv59) delivery via recombinant adeno-associated virus (rAAV) on reducing Aß deposits in an AD mouse model (TgAßPPswe/PS1dE9). First, delivery of scFv59 to the brain was optimized by injecting rAAV serotypes 1, 2, and 5 into the right lateral ventricle. Symmetrical high expression of scFv59 was found throughout the hippocampus and partly in the neocortex in both hemispheres via rAAV1 or rAAV5, while scFv59 expression via rAAV2 was mostly limited to one hemisphere. rAAV1, however, induced apoptosis and microglial activation but rAAV5 did not. Therefore, rAAV5 was selected for therapeutic scFv59 delivery in TgAßPPswe/PS1dE9 mice. rAAV5 was similarly injected into the ventricle of 10-month-old TgAßPPswe/PS1dE9 mice and 5 months later its efficacy and safety were evaluated. Immunoreactive Aß deposits reduced in the hippocampus. Aß42 levels in cerebrospinal fluid (CSF) tended to increase and the Aß40 : 42 ratio decreased in CSF, suggesting that Aß42 was relocated from the parenchyma to CSF. Hemorrhages associated with a focal increase in blood vessel amyloid were found in the brain. While immunotherapy has great potential for clearing cerebral Aß, caution for cerebrovascular effects should be exercised when rAAV-mediated anti-Aß immunotherapy is applied.

Simvastatin enhances immune responses to Aß vaccination and attenuates vaccination-induced behavioral alterations.

Statins are widely used to lower cholesterol levels by inhibiting cholesterol biosynthesis. Some evidence has indicated that statins might have therapeutic and preventive benefits for Alzheimer's disease (AD). We and others also have shown the beneficial effect of statin treatment in reversing learning and memory deficits in animal models of AD. However, data from clinical trials are inconclusive. We previously documented that the adenovirus vector encoding 11 tandem repeats of Aß1-6 fused to the receptor-binding domain (Ia) of Pseudomonas exotoxin A, AdPEDI-(Aß1-6)(11), is effective in inducing an immune response against amyloid-ß protein (Aß) and reducing brain Aß load in Alzheimer's mouse models. In the present study, we examined whether the administration of simvastatin can modulate immune and behavioral responses of C57BL/6 mice to vaccination. Simvastatin was given to the animals as a diet admixture for four weeks, followed by nasal vaccination with AdPEDI-(Aß1-6)(11) once per week for four weeks. The cholesterol-lowering action of simvastatin was monitored by measuring the cholesterol levels in plasma. Simvastatin significantly increased the number of the mice responding to vaccination compared with the mice receiving only AdPEDI-(Aß1-6)(11). Immunoglobulin isotyping revealed that the vaccination predominantly induced Th2 immune responses. Simvastatin treatment prevented Aß-induced production of IFN-γ in splenocytes. The adenovirus vaccination altered mouse behavior in T- and elevated plus-maze tests and simvastatin counteracted such behavioral changes. Our results indicate that simvastatin clearly enhances the immune responses of C57BL/6 mice to the nasal vaccination with AdPEDI-(Aß1-6)(11). Simvastatin may be effective in preventing behavioral changes associated with vaccination.

Isoform-specific enhancement of adenylyl cyclase activity by n-alkanols.

BACKGROUND: Numerous studies suggest that cAMP signaling pathways play important roles in the development of and predisposition to alcoholism. Our previous study showed that cAMP generation by various isoforms of adenylyl cyclase (AC) exhibits a broad spectrum of responses to ethanol in the human embryonic kidney (HEK) 293 cell system overexpressing individual AC isoforms. These findings suggest that the target of ethanol's action in the cAMP-generating system is AC. However, it is unknown if the action of ethanol is direct or indirect. METHODS: The effect of a series of n-alkanols (ethanol to decanol) on dopamine (DA)-stimulated activity of AC isoforms type 6, 7, and 9 (AC6, AC7, and AC9) were examined in transfected HEK293 cells by cAMP accumulation assay. RESULTS: n-Alkanols increased DA-stimulated cAMP production in an AC isoform-specific manner, and displayed the alcohol cutoff phenomenon (defined as the carbon chain length beyond which there is no further increase in the potency of an ascending series of n-alkanols). The n-alkanol cutoffs for AC6, AC7, and AC9 are butanol (C4), pentanol (C5), and equal to or greater than decanol (C10), respectively. CONCLUSION: The results clearly indicate that, in the HEK293 expression system, the alcohol cutoff effect for n-alkanol potentiation of DA-stimulated AC activity is AC isoform specific. These results strongly suggest that alcohols interact directly with AC molecules.

Neurotoxicity in murine striatal dopaminergic pathways following long-term application of low doses of permethrin and MPTP.

The long-term effects of permethrin (PM) and its interaction with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on striatal dopaminergic pathways were investigated in C57BL/6 mice. In a 3-month exposure, technical PM (1.5mg/kg) was administered once per week, with MPTP (20mg/kg) given once on either the 7th week or 11th week. In a 6-month exposure, PM (0.8mg/kg or 1.5mg/kg) was administered once per week for 26 weeks, with MPTP (20mg/kg) given once, on week 24. Alterations in the expression of tyrosine hydroxylase (TH), dopamine transporter (DAT), and alpha-synuclein proteins were analyzed 1 day after the last PM treatment using western blot assay. PM had no significant effect on striatal dopaminergic pathways by itself, whereas MPTP significantly reduced the expression of TH and DAT proteins. In both exposure paradigms, weekly 1.5mg/kg PM treatments antagonized the toxic effect of MPTP on TH and DAT expression (p<0.05). There was no significant alteration of alpha-synuclein expression following any exposure to PM and/or MPTP. [(3)H]Tetrabenazine (TBZ) binding assay for expression of striatal vesicular monoamine transporter (VMAT) showed no effect of PM, but the reduction in this protein caused by MPTP was attenuated by PM, consistent with effects on other dopaminergic biomarkers. The overall findings demonstrate that long-term, low-dose exposure to PM alone did not cause signs of neurotoxicity to striatal dopaminergic neural terminals, or enhance the effects of MPTP. We conclude that under typical use conditions, PM poses little Parkinsonian hazard to humans, including when impregnated into clothing for control of biting flies.

Potentiating effect of the ATP-sensitive potassium channel blocker glibenclamide on complex I inhibitor neurotoxicity in vitro and in vivo.

Previous studies have demonstrated a deficiency in mitochondrial function in Parkinson's disease. We measured the ability of mitochondrial inhibitors of complexes I (rotenone, MPP(+), and HPP(+)), II (amdro), IV (Na cyanide), and an uncoupler (dinoseb) to release preloaded dopamine from murine striatal synaptosomes. These compounds were potent dopamine releasers, and the effect was calcium-dependent. The striatum also contains a significant density of K(ATP)(+) channels, which play a protective role during ATP decline. Blockage of these channels with glibenclamide only potentiated the dopamine release by complex I inhibitors, and a selective potentiating effect of glibenclamide on the toxicity of MPTP was also observed, in vivo, using C57BL/6 mice. Western blots of striatal dopamine transporter (DAT) and tyrosine hydroxylase (TH) proteins demonstrated that 30 mg/kg of glibenclamide alone did not affect the expression of DAT and TH after two weeks of daily treatments, but it significantly enhanced the reduction of DAT and TH by a single dose of 20 mg/kg of MPTP. Amdro or dinoseb alone, or in conjunction with glibenclamide did not alter the expression of DAT and TH. The possible mechanisms underlying dopamine release and the selectivity of glibenclamide were further evaluated, in vitro. (86)Rb efflux assay showed that glibenclamide inhibited rotenone-induced K(+) efflux, but not dinoseb-induced K(+) efflux. Analysis of ATP titers in treated synaptosomes did not support a correlation between mitochondrial inhibition and K(ATP)(+) channel activation. However, assay of reactive oxygen species (ROS) showed that greater amounts of ROS generated by complex I inhibitors was a contributory factor to K(ATP)(+) channel activation and glibenclamide potentiation. Overall, these findings suggest that co-exposure to mitochondrial complex I inhibitors and glibenclamide or a genetic defect in K(ATP)(+) channel function, may increase neurotoxicity in the striatal dopaminergic system.