Jinghong Kou

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

BackgroundAmyloid plaques, a pathological hallmark of Alzheimers disease (AD), are accompanied by activated microglia. The role of activated microglia in the pathogenesis of AD remains controversial: either clearing Aβ 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 Aβ deposits and buffer-soluble Aβ 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 Aβ 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 Aβ deposits start.MethodsMicroglial activation and amyloid deposition in the brain were determined by immunohistochemistry in the AD models. Levels of cerebral soluble Aβ were determined by ELISA. mRNA levels of cytokines and chemokines in the brain and Aβ-stimulated monocytes were quantified by real-time PCR. Cognitive functions were assessed by the Morris water maze.ResultsWhile no difference was found in cerebral Aβ 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 Aβ deposition and soluble Aβ42 in the brain, which were associated with decrements in cognitive functions and expression levels of IL-1β, CCL3, and CCL4 in the hippocampus compared to TLR4W Tg mice. TLR4 mutation diminished Aβ-induced IL-1β, CCL3, and CCL4 expression in monocytes.ConclusionThis is the first demonstration of TLR4-dependent activation of microglia at the early stage of β-amyloidosis. Our results indicate that TLR4 is not involved in the initiation of Aβ deposition and that, as Aβ deposits start, microglia are activated via TLR4 signaling to reduce Aβ deposits and preserve cognitive functions from Aβ-mediated neurotoxicity.

MyD88 Deficiency Ameliorates β-Amyloidosis in an Animal Model of Alzheimer's Disease

The accumulation of β-amyloid protein (Aβ) in the brain is thought to be a primary etiologic event in Alzheimers disease (AD). Fibrillar Aβ 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 Aβ and repairing damaged tissues) or neurotoxic (by producing proinflammatory cytokines and reactive oxygen species). Aβ 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 Aβ accumulation and microglial activation in an AD mouse model. MyD88 deficiency decreased Aβ load and microglial activation in the brain. The decrease in Aβ 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 Aβ load by enhancing the phagocytic capability of microglia through fractalkine (the ligand of CX3CR1) signaling and by promoting apoE-mediated clearance of Aβ from the brain. These findings also suggest that chronic inflammatory responses induced by Aβ accumulation via the MyD88-dependent signaling pathway exacerbate β-amyloidosis in AD.

Catalytic Immunoglobulin Gene Delivery in a Mouse Model of Alzheimer's Disease: Prophylactic and Therapeutic Applications

Accumulation of amyloid beta-peptide (Aβ) in the brain is hypothesized to be a causal event leading to dementia in Alzheimer’s disease (AD). Aβ vaccination removes Aβ deposits from the brain. Aβ immunotherapy, however, may cause T cell- and/or Fc-receptor-mediated brain inflammation and relocate parenchymal Aβ deposits to blood vessels leading to cerebral hemorrhages. Because catalytic antibodies do not form stable immune complexes and Aβ fragments produced by catalytic antibodies are less likely to form aggregates, Aβ-specific catalytic antibodies may have safer therapeutic profiles than reversibly-binding anti-Aβ antibodies. Additionally, catalytic antibodies may remove Aβ more efficiently than binding antibodies because a single catalytic antibody can hydrolyze thousands of Aβ molecules. We previously isolated Aβ-specific catalytic antibody, IgVL5D3, with strong Aβ-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 Aβ 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-Aβ catalytic antibody brain delivery can be prophylactic and therapeutic options for AD.

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 Alzheimers disease (AD) deficient for MyD88 had decreases in Aβ deposits and soluble Aβ in the brain as compared with MyD88 sufficient AD mouse models. Because accumulation of Aβ 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.

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 (Aβ) via Toll-like receptor 4 (TLR4) signaling may play an important role in the pathogenesis of Alzheimers disease (AD). However, as the disease progresses, activated microglia appear to become incapable of clearing Aβ deposits. Because repeated exposure to a TLR4 ligand leads to a diminished response of monocytes/macrophages to lipopolysaccharide (LPS) and because aggregated Aβ is a TLR4 ligand, we hypothesize that chronic exposure of microglia to Aβ deposits may induce a state of Toll-like receptor (TLR) signaling dysfunction, leading to decreased Aβ 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 Aβ deposition, at age 2 and 12 months, respectively. Brain specimens were collected 7 days post-injection and analyzed for microglial activation and Aβ 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 Aβ accumulation in the brain.

Anti-amyloid-β 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-β protein (Aβ) in the brain is thought to be a causal event in Alzheimers disease (AD). Immunotherapy targeting Aβ holds great promise for reducing Aβ in the brain. Here, we evaluated the efficacy and safety of anti-Aβ single-chain antibody (scFv59) delivery via recombinant adeno-associated virus (rAAV) on reducing Aβ deposits in an AD mouse model (TgAβPPswe/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 TgAβPPswe/PS1dE9 mice. rAAV5 was similarly injected into the ventricle of 10-month-old TgAβPPswe/PS1dE9 mice and 5 months later its efficacy and safety were evaluated. Immunoreactive Aβ deposits reduced in the hippocampus. Aβ42 levels in cerebrospinal fluid (CSF) tended to increase and the Aβ40 : 42 ratio decreased in CSF, suggesting that Aβ42 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 Aβ, caution for cerebrovascular effects should be exercised when rAAV-mediated anti-Aβ immunotherapy is applied.

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

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

Muscle-Directed Anti-Aβ Single-Chain Antibody Delivery via AAV1 Reduces Cerebral Aβ Load in an Alzheimer’s Disease Mouse Model

We previously reported that anti-amyloid-beta (Aβ) single-chain antibody (scFv59) brain delivery via recombinant adeno-associated virus (rAAV) was effective in reducing cerebral Aβ 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 Aβ deposits in the brain. Compared with the scFvGag group, levels of Aβ in cerebrospinal fluid (CSF) decreased significantly while Aβ in serum tended to increase in the scFv59 group. AAV1-mediated scFv59 gene delivery may alter the equilibrium of Aβ between the blood and brain, resulting in an increased efflux of Aβ from the brain owing to antibody-mediated sequestration/clearance of peripheral Aβ. Our results suggest that muscle-directed scFv59 delivery via rAAV1 may be a prophylactic option for AD and that levels of CSF Aβ may be used to evaluate the efficacy of anti-Aβ immunotherapy.

Simvastatin enhances immune responses to Aβ 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 Alzheimers 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 Aβ1-6 fused to the receptor-binding domain (Ia) of Pseudomonas exotoxin A, AdPEDI-(Aβ1-6)(11), is effective in inducing an immune response against amyloid-β protein (Aβ) and reducing brain Aβ load in Alzheimers 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-(Aβ1-6)(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-(Aβ1-6)(11). Immunoglobulin isotyping revealed that the vaccination predominantly induced Th2 immune responses. Simvastatin treatment prevented Aβ-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-(Aβ1-6)(11). Simvastatin may be effective in preventing behavioral changes associated with vaccination.

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 Alzheimers 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 β protein (Aβ). 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 Aβ 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 Aβ 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 Aβ levels in the brain by upregulating ABCA1 in blood-brain barrier endothelial cells.