Evidence for glial-mediated inflammation in aged APP(SW) transgenic mice

Chronic expression of inflammatory cytokines, including interleukin-1β, tumor necrosis factor , and interleukin-6, by glia may underlie the neurodegenerative events that occur within the brains of patients with Alzheimer’s disease (AD). The present study determined whether these markers of inflammation could be observed within the brains of Tg(HuAPP695.K670N/M671L)2576 transgenic mice (Tg2576) that have recently been shown to mimic many features of AD. Interleukin-1β- and tumor necrosis factor -immunopositive microglia were localized with thioflavine-positive (fibrillar) Aβ deposits. Moreover, interleukin-6 immunoreactive astrocytes surrounded fibrillar Aβ deposits. These findings provide evidence that Tg2576 mice exhibit features of the inflammatory pathology seen in AD and suggest that these mice are a useful animal model for studying the role inflammation may play in this disease.     

Abeta-induced inflammatory processes in microglia cells of APP23 transgenic mice

A microglial response is part of the inflammatory processes in Alzheimer's disease (AD). We have used APP23 transgenic mice overexpressing human amyloid precursor protein with the Swedish mutation to characterize this microglia response to amyloid deposits in aged mice. Analyses with MAC-1 and F4/80 antibodies as well as in vivo labeling with bromodeoxyuridine demonstrate that microglia in the plaque vicinity are in an activated state and that proliferation contributes to their accumulation at the plaque periphery. The amyloid-induced microglia activation may be mediated by scavenger receptor A, which is generally elevated, whereas the increased immunostaining of the receptor for advanced glycation end products is more restricted. Although components of the phagocytic machinery such as macrosialin and Fc receptors are increased in activated microglia, efficient clearance of amyloid is missing seemingly because of the lack of amyloid-bound autoantibodies. Similarly, although up-regulation of major histocompatibility complex class II (IA) points toward an intact antigen-presenting function of microglia, lack of T and B lymphocytes does not indicate a cell-mediated immune response in the brains of APP23 mice. The similar characteristics of microglia in the APP23 mice and in AD render the mouse model suitable to study the role of inflammatory processes during AD pathogenesis.     

Intraneuronal APP/A beta trafficking and plaque formation in beta-amyloid precursor protein and presenilin-1 transgenic mice

Neuropil deposition of beta-amyloid peptides A beta40 and A beta42 is believed to be the key event in the neurodegenerative processes of Alzheimer's disease (AD). Since A beta seems to carry a transport signal that is required for axonal sorting of its precursor beta-amyloid precursor protein (APP), we studied the intraneuronal staining profile of A beta peptides in a transgenic mouse model expressing human mutant APP751 (KM670/671NL and V7171) and human mutant presenilin-1 (PS-1 M146L) in neurons. Using surface plasmon resonance we analyzed the A beta antibodies and defined their binding profile to APP, A beta40 and A beta42. Immunohistochemical staining revealed that intraneuronal A beta40 and A beta42 staining preceded plaque deposition, which started at 3 months of age. A beta was observed in the somatodendritic and axonal compartments of many neurons. Interestingly, the striatum, which lacks transgenic APP expression harbored many plaques at 10 months of age. This is most likely due to an APP/A beta transport problem and may be a model region to study APP/A beta trafficking as an early pathological event.     

Interferon-gamma and tumor necrosis factor-alpha regulate amyloid-beta plaque deposition and beta-secretase expression in Swedish mutant APP transgenic mice

Reactive astrocytes and microglia in Alzheimer's disease surround amyloid plaques and secrete proinflammatory cytokines that affect neuronal function. Relationship between cytokine signaling and amyloid-beta peptide (Abeta) accumulation is poorly understood. Thus, we generated a novel Swedish beta-amyloid precursor protein mutant (APP) transgenic mouse in which the interferon (IFN)-gamma receptor type I was knocked out (APP/GRKO). IFN-gamma signaling loss in the APP/GRKO mice reduced gliosis and amyloid plaques at 14 months of age. Aggregated Abeta induced IFN-gamma production from co-culture of astrocytes and microglia, and IFN-gamma elicited tumor necrosis factor (TNF)-alpha secretion in wild type (WT) but not GRKO microglia co-cultured with astrocytes. Both IFN-gamma and TNF-alpha enhanced Abeta production from APP-expressing astrocytes and cortical neurons. TNF-alpha directly stimulated beta-site APP-cleaving enzyme (BACE1) expression and enhanced beta-processing of APP in astrocytes. The numbers of reactive astrocytes expressing BACE1 were increased in APP compared with APP/GRKO mice in both cortex and hippocampus. IFN-gamma and TNF-alpha activation of WT microglia suppressed Abeta degradation, whereas GRKO microglia had no changes. These results support the idea that glial IFN-gamma and TNF-alpha enhance Abeta deposition through BACE1 expression and suppression of Abeta clearance. Taken together, these observations suggest that proinflammatory cytokines are directly linked to Alzheimer's disease pathogenesis.     

Silver impregnation of microglial cells and astrocytes in paraffin sections

A method based on mordanting with sulphuric acid-potassium ferrocyanide solution and on physical development is described.     

Nitric oxide in the cerebral cortex of amyloid-precursor protein (SW) Tg2576 transgenic mice

Changes in the amyloid-peptide (Abeta), neuronal and inducible nitric oxide (NO)synthase (nNOS, iNOS), nitrotyrosine, glial fibrillary acidic protein, and lectin from Lycopersicon esculentum (tomato) were investigated in the cerebral cortex of transgenic mice (Tg2576) to amyloid precursor protein (APP), by immunohistochemistry (bright light, confocal, and electron microscopy). The expression of nitrergic proteins and synthesis of nitric oxide were analyzed by immunoblotting and NOS activity assays, respectively. The cerebral cortex of these transgenic mice showed an age-dependent progressive increase in intraneuronal aggregates of Abeta-peptide and extracellular formation of senile plaques surrounded by numerous microglial and reactive astrocytes. Basically, no changes to nNOS reactivity or expression were found in the cortical mantle of either wild or transgenic mice. This reactivity in wild mice corresponded to numerous large type I and small type II neurons. The transgenic mice showed swollen, twisted, and hypertrophic preterminal and terminal processes of type I neurons, and an increase of the type II neurons. The calcium-dependent NOS enzymatic activity was higher in wild than in the transgenic mice. The iNOS reactivity, expression and calcium-independent enzymatic activity increased in transgenic mice with respect to wild mice, and were related to cortical neurons and microglial cells. The progressive elevation of NO production resulted in a specific pattern of protein nitration in reactive astrocytes. The ultrastructural study carried out in the cortical mantle showed that the neurons contained intracellular aggregates of Abeta-peptide associated with the endoplasmic reticulum, mitochondria, and Golgi apparatus. The endothelial vascular cells also contained Abeta-peptide deposits. This transgenic model might contribute to understand the role of the nitrergic system in the biological changes related to neuropathological progression of Alzheimer's disease.     

The microglial phagocytic role with specific plaque types in the Alzheimer disease brain

Alzheimer disease (AD) involves glial inflammation associated with amyloid plaques. The role of the microglial cells in the AD brain is controversial, as it remains unclear if the microglia form the amyloid fibrils of plaques or react to them in a macrophage-phagocytic role. Also, it is not known why microglia are preferentially associated with some amyloid plaque types. This review will provide substantial evidence to support the phagocytic role of microglia in the brain as well as explain why microglia are generally associated with specific plaque types that may be explained through their unique mechanisms of formation. In summary, the data presented suggests that plaque associated microglial activation is typically subsequent to specific amyloid plaque formations in the AD brain.     

Mechanisms of hypoxic tolerance in presymptomatic APP23 transgenic mice

In the B6-Tg (ThylAPP)23Sdz (APP23tg) transgenic mouse model of Alzheimer's disease hypoxic tolerance is impaired prior to amyloid deposition. We therefore investigated mechanisms known to mediate resistance to hypoxic episodes in presymptomatic APP23tg and appropriate control strains. The mRNA expression levels in the hippocampus of adenosine receptor subtypes A1 and A3, estrogen receptors alpha and beta, progesterone receptor, and neuronal and endothelial nitric oxide synthase were investigated with semi-quantitative RT-PCR. Mice were pretreated in vivo with a low dose of 3-nitropropionate, an inhibitor of succinic dehydrogenase, known to mediate hypoxic tolerance within 1h. We found increased expression levels in presymptomatic, untreated APP23tg animals of adenosine A3 receptor mRNA and estrogen receptor alpha mRNA. In addition, we observed an increase in nNOS expression levels upon mild cellular hypoxia induced by 3-NP in transgenic but not in wild-type animals. We conclude that overexpression of human APP results in differential expression of receptors conferring hypoxic tolerance prior to amyloid deposition. Up-regulation of nNOS expression levels upon hypoxic challenge in APP23tg transgenic animals may therefore reflect a selective vulnerability in these animals even before amyloid deposition.     

HuD在N2aAPP细胞和APP/PS1转基因小鼠中的表达

目的研究RNA结合蛋白HuD在AD细胞和动物模型中的表达水平,探讨HuD与AD的关系;明确PI3K/AKT通路对神经细胞HuD表达的调控作用。方法免疫荧光检测HuD在C57BL/6小鼠脑组织中的表达水平;构建过表达APP的N2a APP细胞模型,Western blot检测HuD的表达情况;购买APP/PS1转基因小鼠,Western blot检测HuD在脑组织中的表达情况;应用LY294002处理3种神经细胞(N2a、SH-SY5Y和HT22),阻断PI3K/AKT通路,观察该通路对HuD的调控作用。结果 HuD在整个C57BL/6小鼠脑组织中均有表达,且在海马体的颗粒细胞尤为明显;在N2a APP细胞,HuD的表达水平明显低于对照组;APP/PS1转基因小鼠脑组织中的HuD水平也低于野生对照鼠;LY294002处理下调了HuD在神经细胞中的表达水平。结论 HuD在N2a APP细胞和APP/PS1转基因小鼠中均呈低表达;HuD在神经细胞中受PI3K/AKT通路的调控,为研究HuD与AD的关系及寻找新的AD诊断和治疗靶点提供了理论基础。     

Progressive age-related impairment of cognitive behavior in APP23 transgenic mice

Transgenic APP23 mice expressing human APP(751) with the K670N/M671L mutation, were compared at ages 3, 18 or 25 months to non-transgenic littermates in passive avoidance and in a small and large Morris maze. The task in the smaller pool habituated their flight response to the platform. Impairments in passive avoidance and small pool performance in APP23 mice were clearly age-related. In the larger Morris maze APP23 mice at all ages were impaired in latency and distance swum before finding the platform. Identical performance of 18-month APP23 and controls in a visible platform condition indicates that the Morris maze performance deficit was not due to sensory, motor or motivational alterations. At age 3 months both groups initially unexpectedly avoided the visible platform, suggesting that in young mice neophobia may contribute significantly to performance in cognitive tests. In conclusion, APP23 mice exhibit both early behavioral impairment in the large Morris maze as well as impairments in passive avoidance and small pool performance that are marked only in old age.     

Expression and significance of DSCAM in the cerebral cortex of APP transgenic mice

Patients with schizophrenia have been shown to display decreased sensitivity to pain, which can severely compound the impact of injuries and illnesses. Alterations in the sensory and affective systems of pain processing have been proposed as mechanisms, but the unique contribution of each of these systems has not been elucidated. The aim of this study was to investigate these two components of pain using the NMDA receptor antagonist, phencyclidine (PCP), an established animal model of schizophrenia. Animals underwent L5 spinal nerve ligation surgery in order to provoke a condition of ongoing pain responding, followed by treatment with 2.58 mg/kg of PCP, or saline, and 20 mg/kg of the atypical antipsychotic clozapine, or vehicle, in a block design. Responses to mechanical stimuli were assessed to determine changes in sensory processing, and affective pain processing was examined with the place escape avoidance paradigm. The results showed animals receiving PCP exhibited decreased sensitivity to mechanical stimulation and unaltered behavior in the avoidance paradigm. These findings corroborate and strengthen the human literature investigating schizophrenia and alterations in pain perception. More importantly, the differential findings between the tests of sensory and affective pain processing provide a novel means of understanding schizophrenia-related pain insensitivity.     

Expression and significance of DSCAM in the cerebral cortex of APP transgenic mice

Down syndrome cell adhesion molecule (DSCAM) plays important roles in the regulation of synaptogenesis, neurite outgrowth, axon guidance and synapse formation. Overexpression of DSCAM in Down syndrome (DS) may be involved in the pathogenesis of mental retardation through an inhibitory action on synaptogenesis/neurite outgrowth, and in the precocious dementia associated with an amyloid precursor protein (APP) dosage effect with enhanced plaque formation. In this report we examined the expression of DSCAM in the cerebral cortex of APP transgenic mice versus age-matched wild-type mice. We found that the level of DSCAM expression increased with increasing age in both groups of mice, up to a maximum at 3 months old. The level of DSCAM expression in APP transgenic mice was significantly higher than in the age-matched wild types. We propose that overexpression of DSCAM in the cerebral cortex might play an important role in the learning and memory defects of APP transgenic mice.     

Spatial and temporal control of age-related APP processing in genomic-based beta-secretase transgenic mice

Genetic mutations associated with Alzheimer's disease (AD) in the Amyloid Precursor Protein (APP) gene specifically alter the production of the APP processing product, amyloid-beta (Abeta) peptide, generated by beta- and gamma-secretases. The accumulation and deposition of Abeta is hypothesized to cause AD pathogenesis, leading to the debilitating neurological deficits observed in AD patients. However, it is unclear how processing of APP to generate Abeta corresponds with the age-dependent pattern of brain-regional neurodegeneration common in AD. We have previously shown that overexpression of BACE1, the primary beta-secretase gene, in mice expressing an AD mutant form of APP leads to significantly elevated regional Abeta levels, which coincide with the regional pattern of Abeta deposition. In the current study, we have used our genomic-based beta-secretase transgenic mice to determine how BACE1 regulates the spatial and temporal pattern of Abeta production throughout post-natal development. Specifically, we observed unique differences in the brain-regional expression pattern between neonatal and adult BACE1 transgenic mice. These alterations in the BACE1 expression profile directly corresponds with age-related differences in regional Abeta production and deposition. These studies indicate that modulation of BACE1 expression leads to dramatic alterations in APP processing and AD-like neuropathology. Furthermore, our studies provide further evidence that BACE1 plays a major role in the regulation of the APP processing pathway, influencing the age-dependent onset of AD pathogenesis.     

Focal glial activation coincides with increased BACE1 activation and precedes amyloid plaque deposition in APP[V717I] transgenic mice

Background  

Inflammation is suspected to contribute to the progression and severity of neurodegeneration in Alzheimer's disease (AD). Transgenic mice overexpressing the london mutant of amyloid precursor protein, APP [V717I], robustly recapitulate the amyloid pathology of AD.     

Rapid detection of Abeta deposits in APP transgenic mice by Hoechst 33342

The accumulation of beta-amyloid (Abeta) is the earliest event seen in the neocortex and hippocampus of Alzheimer's disease (AD) patients. Transgenic mouse models of Abeta deposition are excellent tools for validating pharmacological therapies for reducing Abeta burden. Sensitive and rapid probes should be needed for detecting Abeta plaques ex vivo and in vivo in the transgenic mouse models. However, a thioflavin derivative, Pittsburgh Compound-B (PIB), which is a successful PET tracer for detecting Abeta plaques in AD brains, does not visualize Abeta plaques in APP and PS1/APP transgenic mice. Here, we report that Hoechst 33342, a cell-permeable fluorescent probe for staining DNA and nuclei, also detects Abeta plaques in APP Tg mouse. These findings could allow us to rapidly detect Abeta plaques in AD mouse models, and to develop improved compounds for detecting Abeta plaques in vivo in mouse models.     

骨髓基质细胞移植改善APP/PS1转基因小鼠的认知功能

目的探究骨髓基质细胞(Bone marrow stromal cells,BMSCs)移植治疗对APP/PS转基因的阿尔兹海默病(Alzheimer's disease,AD)模型小鼠认知功能障碍的改善作用。方法分离培养C57BL/6小鼠的BMSCs,诱导其分化为神经细胞,通过相差显微镜技术和免疫细胞化学荧光染色技术鉴定其表型与分化。通过显微注射技术,将绿色荧光蛋白(Enhanced green fluorescent protein,eGFP)转基因小鼠(生后3个月)来源的BMSCs注入APP/PS转基因小鼠(生后12个月)侧脑室内后,以荧光显微镜技术检测移植细胞的存活和整合情况,HE染色检测AD模型小鼠海马区萎缩的恢复程度并以Morris水迷宫试验与旷场试验检测移植治疗后小鼠认知功能障碍的改善情况。结果分离自C57BL/6小鼠的BMSCs在增殖培养基中贴壁生长,形态呈纺锤形,免疫表型CD271+/CD45-++,经神经分化诱导培养可形成β-tubulin-III神经元和GFAP星形胶质细胞。GFP转基因小鼠来源的BMSCs移植入APP/PS1转基因小鼠脑内可存活并与宿主海马区神经组织有效整合。移植治疗3周后宿主小鼠海马区面积显著增加(<0.001),水迷宫实验逃生潜伏期明显缩短(<0.01),旷场试验评分显著提高(<0.05)。结论骨髓基质细胞移植治疗可有效改善APP/PS1转基因的AD模型小鼠的认知功能障碍。     

Type-specific evolution of amyloid plaque and angiopathy in APPsw mice

To clarify how Aβ deposits start in the brain, we examined the early to late stages of senile plaques and amyloid angiopathy in APPsw mice. All types of human senile plaques were observed in the mouse brains. The premature forms of cored plaques appeared first in the cerebral cortex of mice at 7–8 months old. Then, amyloid angiopathy emerged, followed by diffuse plaques consisting of Aβ1–42. Modifications of the N-terminus of Aβ were late phase phenomena. The premature forms of cored plaques were composed of central Aβ1–40 amyloid cores, surrounding amorphous Aβ1–42 deposits, and accumulation of Aβ1–42 in some peripheral cells. These cells were incorporated in amyloid cores, and these plaques developed to large cored plaques composed of Aβ1–40 and Aβ1–42. The size and number of cored plaques were increased with age. These findings indicate different evolution paths for cored plaques and diffuse plaques, and suggest the presence of a pathway that initiates with the intracellular accumulation of Aβ1–42 and leads to the development of classic plaques in human brain tissues.     

Associative and motor learning in 12-month-old transgenic APP+PS1 mice

Doubly transgenic 12-month-old amyloid precursor protein and presenilins 1 (APP+PS1) mice (n=14) and littermate control mice (n=17) were tested on eyeblink classical conditioning-a task impaired in humans with Alzheimer's disease (AD). Mice were also tested on a motor learning task (rotorod) and on sensory tasks (prepulse inhibition [PPI] and acoustic startle). Transgenic mice had impaired motor performance on rotorod. Overall, APP+PS1 mice performed similarly to controls on both 500ms delay and 500ms trace eyeblink conditioning as well as on prepulse inhibition (PPI) and acoustic startle. However, within the transgenic group, cortical amyloid burden correlated significantly with decreased trace eyeblink conditioning. Moreover, cortical amyloid burden and hippocampal microglia activation correlated significantly with decreased PPI. These data suggest that only those transgenic mice with the most severe amyloid pathology exhibited deficits in hippocampus-dependent tasks. Transgenic mouse models of amyloid deposition differ from Alzheimer patients not only by the absence of major neuronal loss, but also by the general absence of severe impairments in eyeblink conditioning, except for mice with the greatest amyloid pathology.     

Expression of complement system components during aging and amyloid deposition in APP transgenic mice

Background  

A causal role of the complement system in Alzheimer's disease pathogenesis has been postulated based on the identification of different activated components up to the membrane attack complex at amyloid plaques in brain. However, histological studies of amyloid plaque bearing APP transgenic mice provided only evidence for an activation of the early parts of the complement cascade. To better understand the contribution of normal aging and amyloid deposition to the increase in complement activation we performed a detailed characterization of the expression of the major mouse complement components.