Heparan sulfate accumulation with Abeta deposits in Alzheimer's disease and Tg2576 mice is contributed by glial cells

Abstract Amyloid beta-peptide (Abeta) plaques, one of the major neuropathological lesions in Alzheimer's disease (AD), can be broadly subdivided into two morphological categories: neuritic and diffuse. Heparan sulfate (HS) and HS proteoglycans (HSPGs) are codeposits of multiple amyloidoses, including AD. Although HS has been considered a limiting factor in the initiation of amyloid deposition, the pathological implications of HS in Abeta deposits of AD remain unclear. In this study, immunohistochemistry combined with fluorescence and confocal microscopy was employed to gain deeper insight into the accumulation of HS with Abeta plaques in sporadic and familial AD. Here we demonstrate that HS preferentially accumulated around the Abeta40 dense cores of neuritic plaques, but was largely absent from diffuse Abeta42 plaques, suggesting that Abeta42 deposition may occur independently of HS. A codeposition pattern of HS with Abeta deposits in Tg2576 mice was also examined. We identified the membrane-bound HSPGs, glypican-1 (GPC1) and syndecan-3 (SDC3), in glial cells associated with Abeta deposits, proximal to sites of HS accumulation. In mouse primary glial cultures, we observed increased levels of GPC1 and SDC3 following Abeta stimulation. These results suggest that HS codeposits with Abeta40 in neuritic plaques and is mainly derived from glial cells.     

Impaired long-trace eyeblink conditioning in a Tg2576 mouse model of Alzheimer's disease

Eyeblink conditioning has been used for assessing cognitive performance in cases of human neurodegenerative diseases including Alzheimer's disease (AD). Here, we tested and compared the delay and long-trace interval (TI = 500 ms) eyeblink conditionings in a Tg2576 mouse model of AD, at the age of 3, 6, and 12 months. Tg2576 mice exhibited significant impairment in trace conditioning at 6 months of age. In contrast, delay conditioning was not impaired in Tg2576 mice even at 12 months. These findings indicate that the long-TI eyeblink conditioning is more susceptible to age-related cognitive deterioration than delay conditioning in Tg2576 mice. The long-trace eyeblink conditioning could be a potential tool for detecting early cognitive deficits in AD mouse model.     

Behavioral characterization of the Tg2576 transgenic model of Alzheimer's disease through 19 months

Behavioral characterization of Alzheimer's disease (AD) transgenic models over multiple time points during aging has been largely inadequate, usually being limited to one or two cognitive-based tasks. In this context, the present study utilized a comprehensive 6-week behavioral battery to characterize sensorimotor and cognitive performance of Tg2576 AD transgenic (Tg+) mice and nontransgenic (Tg-) controls aged 3, 9, 14, and 19 months. Compared collectively to Tg- mice over all four time points, Tg+ mice were impaired in Y-maze spontaneous alternation, visible platform recognition, and several sensorimotor tasks; Tg+ mice also showed an overall increase in activity measures. The deficits in visible platform became evident by 9 months of age, while those in sensorimotor tasks became clearly manifest by 14 months. Although the behavioral impairments exhibited by Tg+ mice were usually progressive through 19 months, Tg- animals also showed similar progressive decline in the same behavioral measures; thus, no task revealed a progressive behavioral decline exclusive to Tg+ mice. Moreover, although the 6-week behavioral battery included six cognitively based tasks (i.e., Y-maze, visible platform, Morris water maze, circular platform, passive avoidance, and active avoidance), behavioral analysis through 19 months revealed Tg+ mice to be impaired in only the Y-maze and visible platform tasks. Consequently, Tg2576 mice do not exhibit widespread, profound cognitive impairment, even into old age. This may reflect their predominant C57BL/6 background and an apparent inability of the mutant transgene to profoundly alter performance therein.     

Upregulation of tPA/plasminogen proteolytic system in the periphery of amyloid deposits in the Tg2576 mouse model of Alzheimer's disease

Although the tissue plasminogen activator (tPA)/plasminogen/plasmin proteolytic system is thought to modulate the catabolism of amyloid-β (Aβ), in vivo evidence remains insufficient. In the brain of human amyloid precursor protein transgenic Tg2576 mice, we found co-accumulation of tPA and plasminogen at the periphery of compact amyloid deposits, mainly Aβ42-cored plaques, as well as in the walls of blood vessels with cerebral amyloid angiopathy (CAA). This tPA/plasminogen system contained high levels of proteolytic activity. High levels of tPA were also found in reactive astrocytes with increased Aβ42 expression, whereas plasminogen was found only in neurons. When the brain sections of Tg2576 mice were treated with both tPA and plasminogen, levels of thioflavin-S fluorescence, congophilicity and birefringence in the compact amyloid plaques were significantly reduced, and the ultrastructure of Aβ42-fibrils was disrupted. These results suggest that the assembled Aβ42 may promote upregulation of the tPA/plasminogen proteolytic system, which can modulate the deposition of amyloid plaques in vivo.     

Part of membrane-bound Abeta exists in rafts within senile plaques in Tg2576 mouse brain

To clarify whether rafts are the site of abnormal amyloid beta protein (Abeta) deposition, we examined the ultrastructural localization of both flotillin-1 (pre-embedding) and Abeta (post-embedding) in Tg2576 mouse brains. After observing the exact areas of senile plaques by reflection contrast microscopy, we observed these same plaques under an electron microscope. Membrane-bound Abeta was predominantly observed on plasma membranes of small processes in diffuse plaques. Non-fibrillar and fibrillar Abeta was increased in primitive plaques, and the fibrillar form was predominant in mature plaques. The number of flotillin-1-positive rafts per field in mature plaques was prominently less than those outside of the plaques, in diffuse plaques and in primitive plaques. The colocalization of flotillin-1 with Abeta42 appeared approximately 10% of flotillin-1-positive rafts within senile plaques, while there was no colocalization found outside of the plaques. This study ultrastructurally demonstrated that part of membrane-bound Abeta exists in lipid rafts within senile plaques, and suggests that rafts could be one of the sites for initial Abeta deposition.     

Dense-core plaques in Tg2576 and PSAPP mouse models of Alzheimer's disease are centered on vessel walls

Occurrence of amyloid beta (Abeta) dense-core plaques in the brain is one of the chief hallmarks of Alzheimer's disease (AD). It is not yet clear what factors are responsible for the aggregation of Abeta in the formation of these plaques. Using Tg2576 and PSAPP mouse models that exhibit age-related development of amyloid plaques similar to that observed in AD, we showed that approximately 95% of dense plaques in Tg2576 and approximately 85% in PSAPP mice are centered on vessel walls or in the immediate perivascular regions. Stereoscopy and simulation studies focusing on smaller plaques suggested that vascular associations for both Tg2576 and PSAPP mice were dramatically higher than those encountered by chance alone. We further identified ultrastructural microvascular abnormalities occurring in association with dense plaques. Although occurrence of gross cerebral hemorrhage was infrequent, we identified considerable infiltration of the serum proteins immunoglobulin and albumin in association with dense plaques. Together with earlier evidence of vascular clearance of Abeta, our data suggest that perturbed vascular transport and/or perivascular enrichment of Abeta leads to the formation of vasocentric dense plaques in Tg2576 and PSAPP mouse models of AD.     

Impaired rapid eye movement sleep in the Tg2576 APP murine model of Alzheimer's disease with injury to pedunculopontine cholinergic neurons

Impaired rapid eye movement sleep (REMS) is commonly observed in Alzheimer's disease, suggesting injury to mesopontine cholinergic neurons. We sought to determine whether abnormal beta-amyloid peptides impair REMS and injure mesopontine cholinergic neurons in transgenic (hAPP695.SWE) mice (Tg2576) that model brain amyloid pathologies. Tg2576 mice and wild-type littermates were studied at 2, 6, and 12 months by using sleep recordings, contextual fear conditioning, and immunohistochemistry. At 2 months of age, REMS was indistinguishable by genotype but was reduced in Tg2576 mice at 6 and 12 months. Choline acetyltransferase-positive neurons in the pedunculopontine tegmentum of Tg2576 mice at 2 months evidenced activated caspase-3 immunoreactivity, and at 6 and 12 months the numbers of pedunculopontine tegmentum choline acetyltransferase-positive neurons were reduced in the Tg2576 mice. Other cholinergic groups involved in REMS were unperturbed. At 12 months, Tg2576 mice demonstrated increased 3-nitrotyrosine immunoreactivity in cholinergic projection sites but not in cholinergic soma. We have identified a population of selectively compromised cholinergic neurons in young Tg2576 mice that manifest early onset REMS impairment. The differential vulnerability of these cholinergic neurons to Abeta injury provides an invaluable tool with which to understand mechanisms of sleep/wake perturbations in Alzheimer's disease.     

Transthyretin accelerates vascular Abeta deposition in a mouse model of Alzheimer's disease

Transthyretin (TTR) binds amyloid-β (Aβ) and prevents Aβ fibril formation in vitro . It was reported that the lack of neurodegeneration in a transgenic mouse model of Alzheimer's disease (AD) (Tg2576 mouse) was associated with increased TTR level in the hippocampus, and that chronic infusion of anti-TTR antibody into the hippocampus of Tg2576 mice led to increased local Aβ deposits, tau hyperphosphorylation and apoptosis. TTR is, therefore, speculated to prevent Aβ pathology in AD. However, a role for TTR in Aβ deposition is not yet known. To investigate the relationship between TTR and Aβ deposition, we generated a mouse line carrying a null mutation at the endogenous TTR locus and the human mutant amyloid precursor protein cDNA responsible for familial AD (Tg2576 /TTR ^−/− mouse) by crossing Tg2576 mice with TTR-deficient mice. We asked whether Aβ deposition was accelerated in Tg2576/ TTR ^−/− mice relative to the heterozygous mutant Tg2576 (Tg2576/ TTR ^+/−) mice. Contrary to our expectations, the degree of total and vascular Aβ burdens in the aged Tg2576/ TTR ^−/− mice was significantly reduced relative to the age-matched Tg2576/ TTR ^+/− mice. Our experiments present, for the first time, compelling evidence that TTR does not suppress but rather accelerates vascular Aβ deposition in the mouse model of AD.     

Intraneuronal Abeta accumulation and origin of plaques in Alzheimer's disease

Plaques are a defining neuropathological hallmark of Alzheimer's disease (AD) and the major constituent of plaques, the beta-amyloid peptide (Abeta), is considered to play an important role in the pathophysiology of AD. But the biological origin of Abeta plaques and the mechanism whereby Abeta is involved in pathogenesis have been unknown. Abeta plaques were thought to form from the gradual accumulation and aggregation of secreted Abeta in the extracellular space. More recently, the accumulation of Abeta has been demonstrated to occur within neurons with AD pathogenesis. Moreover, intraneuronal Abeta accumulation has been reported to be critical in the synaptic dysfunction, cognitive dysfunction and the formation of plaques in AD. Here we provide a historical overview on the origin of plaques and a discussion on potential biological and therapeutic implications of intraneuronal Abeta accumulation for AD.     

Tg2576小鼠海马发育过程中小胶质细胞和血管的变化

目的 探讨Tg2576转基因小鼠发育过程中海马CA1区小胶质细胞增殖和血管变化的规律。方法 取不同发育时间(P0、P7、P30、P180、P360) Tg2576转基因模型鼠与同时间点野生鼠,通过应用免疫组织化学、TUNEL、墨汁灌注、RT-PCR和透射电镜等方法研究海马发育过程中小胶质细胞和血管的变化。结果 随着小鼠的生长发育,P180后转基因组海马CA1区小胶质细胞密度和血管体密度高于对照组小鼠,RT-PCR结果显示,P360时转基因组海马CA1区小胶质细胞更多处于激活状态。 结论 小胶质细胞与血管改变的共同作用加重了阿尔茨海默病。     

A transgenic rat model of Alzheimer's disease with extracellular Abeta deposition

Many transgenic mouse models of Alzheimer's disease (AD) that deposit amyloid (Abeta) have been produced, but development of an Abeta-depositing rat model has not been successful. Here, we describe a rat model with extracellular fibrillar Abeta deposition. Two lines of Sprague Dawley rats with transgenes expressing human amyloid precursor protein (APP) with the familial AD (FAD) mutations K670N/M671L and K670N/M671L/V717I were crossed. Abeta production in the double homozygous rats was sufficient for deposition by 17-18 months of age. The age of onset of Abeta deposition was reduced by crossing in a third rat line carrying a human presenilin-1 (PS-1) transgene with the FAD M146V mutation. The triple homozygous line had an onset of Abeta deposition by 7 months of age. Deposits appeared similar to those observed in the mouse models and displayed surrounding glial and phosphorylated tau reactivity. Abeta levels measured by ELISA were comparable to those reported in mouse models, suggesting that substantially greater amounts of soluble Abeta are not required in the rat to generate Abeta deposition.     

Characterization of 7- and 19-month-old Tg2576 mice using multimodal in vivo imaging: limitations as a translatable model of Alzheimer's disease

With 90% of neuroscience clinical trials failing to see efficacy, there is a clear need for the development of disease biomarkers that can improve the ability to predict human Alzheimer's disease (AD) trial outcomes from animal studies. Several lines of evidence, including genetic susceptibility and disease studies, suggest the utility of fluorodeoxyglucose positron emission tomography (FDG-PET) as a potential biomarker with congruency between humans and animal models. For example, early in AD, patients present with decreased glucose metabolism in the entorhinal cortex and several regions of the brain associated with disease pathology and cognitive decline. While several of the commonly used AD mouse models fail to show all the hallmarks of the disease or the limbic to cortical trajectory, there has not been a systematic evaluation of imaging-derived biomarkers across animal models of AD, contrary to what has been achieved in recent years in the Alzheimer's Disease Neuroimaging Initiative (ADNI) (Miller, 2009). If animal AD models were found to mimic endpoints that correlate with the disease onset, progression, and relapse, then the identification of such markers in animal models could afford the field a translational tool to help bridge the preclinical-clinical gap. Using a combination of FDG-PET and functional magnetic resonance imaging (fMRI), we examined the Tg2576 mouse for global and regional measures of brain glucose metabolism at 7 and 19 months of age. In experiment 1 we observed that at younger ages, when some plaque burden and cognitive deficits have been reported, Tg2576 mice showed hypermetabolism as assessed with FDG-PET. This hypermetabolism decreased with age to levels similar to wild type (WT) counterparts such that the 19-month-old transgenic (Tg) mice did not differ from age matched WTs. In experiment 2, using cerebral blood volume (CBV) fMRI, we demonstrated that the hypermetabolism observed in Tg mice at 7 months could not be explained by changes in hemodynamic parameters as no differences were observed when compared with WTs. Taken together, these data identify brain hypermetabolism in Tg2576 mice which cannot be accounted for by changes in vascular compliance. Instead, the hypermetabolism may reflect a neuronal compensatory mechanism. Our data are discussed in the context of disease biomarker identification and target validation, suggesting little or no utility for translational based studies using Tg2576 mice.     

Molecular polymorphism of Abeta in Alzheimer's disease

Alzheimer's disease is defined pathologically by the presence of senile plaques, which consist primarily of extracellular aggregates of fibrillar Abeta peptide, and neurofibrillary tangles, which are abnormal, intracellular bundles of fibrillar tau protein. The advent of amyloid binding agents as diagnostic imaging probes for Alzheimer's disease (AD) has made it imperative to understand at a molecular and disease level what these ligands are reporting. In addition to improving the accuracy of diagnosis, we argue that these selective ligands can serve as probes for molecular polymorphisms that may govern the pathogenicity of abnormal protein aggregates.     

C4b-binding protein in Alzheimer's disease: binding to Abeta1-42 and to dead cells

In the Alzheimer's disease (AD) brain, binding of Clq within the Cl complex, the initiating molecule of the classical complement pathway, to apoptotic cells, DNA and amyloid-beta (Abeta), the major constituent of senile plaques, can initiate complement activation. However, the extent of activation is determined by the balance between activation and inhibition. Fluid-phase complement inhibitor C4b-binding protein (C4BP) was immunohistochemically detected in Abeta plaques and on apoptotic cells in AD brain. In vitro, C4BP bound apoptotic and necrotic but not viable brain cells (astrocytes, neurons and oligodendrocytes) and limited complement activation on dead brain cells. C4BP also bound Abeta1-42 peptide directly, via the C4BP alpha-chain, and limited the extent of complement activation by Abeta. C4BP levels in cerebrospinal fluid (CSF) of dementia patients and controls were low compared to levels in plasma and correlated with CSF levels of other inflammation-related factors. In conclusion, C4BP binds to dead brain cells and Abeta peptide in vitro, is present in CSF and possibly protects against excessive complement activation in AD brains.     

Decreased expression of striatal signaling genes in a mouse model of Huntington's disease

To understand gene expression changes mediated by a polyglutamine repeat expansion in the human huntingtin protein, we used oligonucleotide DNA arrays to profile approximately 6000 striatal mRNAs in the R6/2 mouse, a transgenic Huntington's disease (HD) model. We found diminished levels of mRNAs encoding components of the neurotransmitter, calcium and retinoid signaling pathways at both early and late symptomatic time points (6 and 12 weeks of age). We observed similar changes in gene expression in another HD mouse model (N171-82Q). These results demonstrate that mutant huntingtin directly or indirectly reduces the expression of a distinct set of genes involved in signaling pathways known to be critical to striatal neuron function.     

Gene expression correlates of neurofibrillary tangles in Alzheimer's disease

Neurofibrillary tangles (NFT) constitute one of the cardinal histopathological features of Alzheimer's disease (AD). To explore in vivo molecular processes involved in the development of NFTs, we compared gene expression profiles of NFT-bearing entorhinal cortex neurons from 19 AD patients, adjacent non-NFT-bearing entorhinal cortex neurons from the same patients, and non-NFT-bearing entorhinal cortex neurons from 14 non-demented, histopathologically normal controls (ND). Of the differentially expressed genes, 225 showed progressively increased expression (AD NFT neurons > AD non-NFT neurons > ND non-NFT neurons) or progressively decreased expression (AD NFT neurons < AD non-NFT neurons < ND non-NFT neurons), raising the possibility that they may be related to the early stages of NFT formation. Immunohistochemical studies confirmed that many of the implicated proteins are dysregulated and preferentially localized to NFTs, including apolipoprotein J, interleukin-1 receptor-associated kinase 1, tissue inhibitor of metalloproteinase 3, and casein kinase 2, beta. Functional validation studies are underway to determine which candidate genes may be causally related to NFT neuropathology, thus providing therapeutic targets for the treatment of AD.     

Intramuscular delivery of a single chain antibody gene reduces brain Abeta burden in a mouse model of Alzheimer's disease

Anti-beta-amyloid (Abeta) immunotherapy has been well documented to effectively elicit amyloid plaque clearance and slow cognitive decline in experimental and clinical studies. However, anti-Abeta immunotherapy was associated with detrimental effects of brain inflammation and microhemorrhage, presumably induced by T-cell-mediated and/or Fc-mediated inflammatory responses. In the present study, a single chain antibody (scFv) against Abeta could effectively inhibit the aggregation of Abeta and promote the disaggregation of preformed Abeta fibrils. The recombined adeno-associated virus vectors carrying the scFv gene were produced to delivery the scFv gene. Hippocampus delivery of the scFv gene was effective in reducing the amyloid plaque in the hippocampus of an Alzheimer's disease (AD) mouse model. Further studies demonstrated that intramuscular delivery of the scFv gene was as effective as intracranial delivery in reducing the total Abeta level in the brain with a concomitant elevated Abeta level in serum. No enhanced microglial activation, discernable T lymphocyte infiltration, and increased microhemorrhage were found after intracranial and intramuscular delivery of the scFv gene. Our results suggest that intramuscular delivery of the scFv gene would be a novel peripheral noninflammatory immunological modality targeting Abeta clearance and be promising in future drug development for the prevention and treatment of AD.     

Cell proliferation and total granule cell number in dentate gyrus of transgenic Tg2576 mouse

This report provides in vivo evidence of adult neurogenesis and the total granule cell count in the dentate gyrus of the Tg2576 mouse model of Alzheimer's disease. Mice were deeply anaesthetized and perfused with 4 percent buffered paraformaldehyde. Brains were removed and post-fixed in the same fixative overnight. Following equilibration in 30 percent sucrose, 30 micrometer sections were cut in sagittal plane in freezing microtome for immunohistochemistry and 20 micrometer from plastic embedded brains. Thioflavin-S confirmed the presence of amyloid plaques in the Tg2576 mice. Cell proliferation in the subventricular zone and dentate gyrus of hippocampus were observed with Ki-67 and doublecortin markers. Using optical fractionator, total granule number was estimated to be 445,280 per hemisphere in the 18-month-old Tg2576 mouse. Cell proliferation tends to end in the dentate gyrus but continues in the SVZ and the total granule cell number was less compared to normal laboratory and wild rodents.