Intramaze and extramaze cue processing in adult APPSWE Tg2576 transgenic mice

The present study examined spatial and nonspatial learning in adult Tg2576 mice. Transgenic mice were impaired in acquisition of a T-maze forced-choice alternation task. However, mutant mice were as sensitive as control mice to the introduction of retention intervals and proactive interference, and this suggested that short-term memory processes were intact in Tg2576 mice. Probe trials revealed that the Tg2576 mice did not use an allocentric strategy to navigate to the goal arm. However, mutant mice acquired an intramaze brightness discrimination, a simple room discrimination, and a contextual biconditional left-right discrimination in a T maze. Results suggest that Tg2576 mice are able to process both intramaze and extramaze stimuli but are impaired in forming an allocentric representation of their environment.     

APPSWE Tg2576小鼠脑屏障结构

目的探讨小鼠的血脑屏障(BBB)及血脑脊液屏障(BCSFB)的基本结构及其在阿尔茨海默病(AD)发生、发展过程中结构、功能及及其超微结构的改变。方法实验用动物采用APPSWE Tg2576鼠,分为APPSWE转基因阳性鼠(模型组)和同窝生野生型小鼠(对照组),每组各20只。饲养16个月后进行全身灌流固定,开颅切取侧脑室室壁及其脉络丛组织。采用免疫荧光及透射电子显微镜技术观察BBB及BCSFB的超微结构,从而观察AD模型脑屏障的改变。结果 AD模型组与对照组相比较,血管密度明显降低; AD小鼠脑屏障正常结构受到损害,主要是脑血管内皮细胞(或脉络丛内皮细胞)之间的连接及其细胞器受损,脉络丛超微结构也出现明显变化,主要表现为细胞间隙增宽,细胞之间的黏附连接等连接结构也有部分碰坏,胞质内出现较多的囊泡状结构等。结论和正常鼠相比,AD鼠脑屏障受到一定的损害,可能致使脑屏障的转运机制出现相应的改变并影响脑内β-淀粉样蛋白(Aβ)的清除,脑屏障中存在的稳态机制,如其分泌物和受体介导的信号传导也可能出现改变,这些因素可能共同参与了AD的形成和进展。     

Intact spatial learning in adult Tg2576 mice

Tg2576 mice, a transgenic model of amyloid pathology associated with Alzheimer's disease (AD), develop measurable levels of soluble amyloid beta1-40 and 1-42 by 6 months of age and amyloid plaque deposition in cortex, hippocampus and amygdala by 10 months of age. To investigate whether non-hippocampal learning strategies would predominate coincident with the age-related increase in Abeta load in the hippocampal region, we measured learning strategies in the T-maze and a redundant cued version of the water maze. Each of these tasks can be solved using either hippocampal or non-hippocampal learning strategies and has proved sensitive to hippocampal disruption in other settings. The results revealed subtle differences in T-maze and water maze performance in Tg2576 mice compared to controls. Surprisingly, however, Tg2576 mice were not impaired relative to non-transgenic littermates on any measures of hippocampal dependent behavior assessed in these tasks. These data suggest that the medial temporal lobe retains considerable function in 15-month-old Tg2576 mice despite significant Abeta pathology.     

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。     

Beta-amyloid-associated expression of intercellular adhesion molecule-1 in brain cortical tissue of transgenic Tg2576 mice

To study the relationship of beta-amyloid-mediated micro- and astrogliosis and inflammation-induced proteins including intercellular adhesion molecule (ICAM-1), brain tissue from transgenic Tg2576 mice expressing the Swedish mutation of the human amyloid precursor protein were examined for ICAM-1 expression. Immunocytochemistry demonstrated a diffuse immunostaining of ICAM-1 in the corona around fibrillary beta-amyloid plaques and an upregulation of ICAM-1 in activated microglial cells located in close proximity to the plaques, an ICAM-1 distribution pattern that partly mimics the situation in the brain of Alzheimer patients. The developmental time course revealed that the rate of cortical ICAM-1 induction was somewhat behind that of the progression of beta-amyloid plaque deposition. The microglial expression of ICAM-1 is a further indicator of the presence of inflammatory reactions in aged transgenic Tg2576 mouse brain, and may be a result of plaque-mediated astrocytic interleukin-1beta upregulation.     

Intra- and intertask relationships in a behavioral test battery given to Tg2576 transgenic mice and controls

Several transgenic mouse models for Alzheimer's disease (AD) that develop -amyloid deposition have recently been advanced, including the Tg2576 mouse. Thorough behavioral phenotyping of this, or any mouse line/population, requires not only analysis of multiple behavioral measures through a comprehensive battery of tasks, but also a clear understanding of the interrelationships between behavioral measures in such a battery. In our accompanying study (King et al., this volume), Tg2576 transgenic (Tg+) and nontransgenic (Tg-) mice aged 3-19 months were administered an extensive behavioral test battery. The present study involved correlation analysis between those behavioral measures. Numerous correlations were evident for all 169 mice (Tg+ and Tg-) combined, with additional correlations being dependent on genotype or age. For all mice combined, intratask measures in water maze and circular platform were highly correlated; in addition, several measures of activity correlated with each other, as did various measures of balance/agility. A number of correlations between the six cognitive-based tasks of the test battery (e.g. Y-maze, Morris water maze, circular platform, visible platform, passive avoidance, and active avoidance) were also evident, as were correlations between cognitive and sensorimotor measures. In as much as some correlations were found to be exclusive to either Tg+ or Tg- animals alone, separate analysis by genotype is clearly warranted whenever two or more genotypes are involved. Likewise, some correlations were age-dependent, being present either in young adulthood (3 months) or in old age (19 months). These correlation analysis results in mice indicate that: (1) performance in one or several behavioral measures can be predictive of performance in others and (2) both genetic background and age influence the degree and profile of intra-/intertask relationships in an extensive behavioral test battery.     

The "Swedish" mutation of the amyloid precursor protein (APPswe) dissociates components of object-location memory in aged Tg2576 mice

Aged Tg2576 mice show abnormalities in hippocampal morphology and physiology and display behavioral deficits in spatial navigation tasks consonant with a deficit in the functional properties of the hippocampus. However, the nature of the spatial representations disrupted by the "Swedish" mutation of the amyloid precursor protein (APPswe) is unclear. In an effort to characterize the memory deficits in Tg2576 mice, the spontaneous object exploration paradigm was used to interrogate spatial and object memory in mice. With object arrays of comparable size, 16-month-old Tg2576 mice showed a normal object familiarity/novelty effect but impaired memory for the location of objects when 2 objects exchanged locations (topological transformation; Experiment 1). In contrast, Tg2576 mice showed preferential exploration of familiar objects when they were moved to previously unoccupied locations (Experiment 2), irrespective of whether the transformation altered the metric properties of the object array (Experiments 3). These results suggest that Tg2576 mice are able to form representations of the identity of objects and a memory of the spatial organization of objects in an arena. In contrast, conjunctive memory for specific object-location associations is severely impaired in aged Tg2576 mice.     

Perineuronal nets are largely unaffected in Alzheimer model Tg2576 mice

Changes in the molecular organization of the extracellular matrix are key factors in neuropathology. We investigated aggrecan-based perineuronal nets (PNs) in relation to neurodegeneration and activation of glial cells in a transgenic mouse (Tg2576) model of Alzheimer's disease. The formation of amyloid plaques in the cerebral cortex occurred independently of the area-specific distribution of PNs. Matrix components were only affected in the core of plaques in advanced stages of pathology. PNs remained unchanged in the large marginal zone occupied by reactive astrocytic processes. We conclude that the aggrecan-based extracellular matrix of PNs is not enzymatically altered in peripheral plaque territories and is only removed after neuronal death.     

Early locus coeruleus degeneration and olfactory dysfunctions in Tg2576 mice

Olfactory deficiency has been reported in the early stages of Alzheimer's disease (AD) in humans but is very poorly understood due to the lack of investigations in animal models of AD. Recent studies point to the noradrenergic system as an important target of the AD pathological process. In addition, noradrenalin has been shown to influence adult neurogenesis which is implicated in cognitive functions. We have therefore investigated the olfactory neurogenesis and cognitive performances in young transgenic Tg2576 mice in relation with the status of the noradrenergic and the cholinergic systems. Tg2576 showed a deficit in neurogenesis in the olfactory bulb evidenced by an increased death of newborn cells and a reduced expression of PSA-NCAM. The locus coeruleus degenerated in Tg2576 between the age of 6.5 and 8 months. These changes were associated with olfactory memory impairments. Our findings indicate that a noradrenergic deficiency could play a role in the early stages of the pathological process in this transgenic model and induce olfactory cognitive impairments through an alteration of olfactory neurogenesis.     

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.     

Antisense inhibition at the beta-secretase-site of beta-amyloid precursor protein reduces cerebral amyloid and acetyl cholinesterase activity in Tg2576

Misprocessing of beta-amyloid precursor protein (APP) leading to the formation of elevated quantities of beta-amyloid peptide (Abeta), derived by a cleavage at the beta-secretase site (N-671/673aa) and by a cleavage at the gamma-secretase site (C-711/713aa) of APP, is considered a key event in the pathogenesis of Alzheimer disease (AD). Point mutations near the beta-secretase site in the human gene for APP, such as in the Swedish mutation-KM670/671NL, lead to a form of dominantly inherited AD. These mutations are known to promote beta-site cleavage and to increase levels of Abeta. Abeta has been shown previously to increase acetyl cholinesterase (AChE) activity in vitro. We wished to test whether translational blocking of APP-mRNA at the mutated beta-site by antisense (AS) oligodeoxynucleotides (ODNs) directed to the mutated site will reduce cerebral amyloid in the Swedish transgenic mouse model (Tg2576). Mice were injected i.c.v. with AS-ODNs directed at the mutated beta-site (AS-beta site) or with AS-ODNs directed at the normal gamma-site (AS-gamma site) of human APP-mRNA, and compared with procedural controls that received i.c.v. injections of sense ODNs at the beta-site (S-beta site), sense ODNs at the gamma-site (S-gamma site) or mismatched ODNs, and with untreated littermates (Lt) and untreated transgenic mice (Tgs). ODNs were injected into the 3rd ventricle once a week for 4 weeks. Brains were processed for enzyme-linked immunosorbent assay analysis of beta- and gamma-cleaved soluble Abeta40 (sAbeta40), beta- and gamma-cleaved soluble Abeta42 (sAbeta42) and alpha-cleaved soluble beta-amyloid precursor protein (sAPPalpha). The physiological relevance of AS ODNs was tested by evaluating the cerebral distribution of AChE before and after the treatment. AChE was found increased about fivefold in Tg cortex as compared with control brain. Results show that compared with untreated and procedural controls, AS-beta increased cerebral levels of sAPPalpha by 43% and reduced sAbeta40/42 by approximately 39%; while simultaneously reducing the cortical density of AChE by approximately fourfold in the treated Tg animals, almost to the level found in the control brain (all values P<0.0001, analysis of variance, unpaired two-tailed Student's t-test), while AS-gamma did not have any effect. These results indicate that AS directed to the mutated beta-site may be an effective approach to treat familial AD.     

Fish oil enhances anti-amyloidogenic properties of green tea EGCG in Tg2576 mice

Extracellular plaques of β-amyloid (Aβ) peptides are implicated in Alzheimer's Disease (AD) pathogenesis. Aβ formation is precluded by α-secretase, which cleaves within the Aβ domain of APP generating soluble APP-α (sAPP-α). Thus, α-secretase upregulation may be a target AD therapy. We previously showed green tea derived EGCG increased sAPP-α in AD mouse models. However, the comparable effective dose of EGCG in humans may exceed clinical convenience and/or safety. Epidemiological studies suggested fish oil consumption is associated with reduced dementia risk. Here we investigated whether oral co-treatment with fish oil (8 mg/kg/day) and EGCG (62.5 mg/kg/day or 12.5 mg/kg/day) would reduce AD-like pathology in Tg2576 mice. In vitro co-treatment of N2a cells with fish oil and EGCG enhanced sAPP-α production compared to either compound alone (P < 0.001). Fish oil enhanced bioavailability of EGCG versus EGCG treatment alone (P < 0.001). Fish oil and EGCG had a synergetic effect on inhibition of cerebral Aβ deposits (P < 0.001) suggesting moderate supplementation with EGCG and fish oil having significant therapeutic potential for the 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.     

Lovastatin enhances Abeta production and senile plaque deposition in female Tg2576 mice

A recent clinical study showed that statins, which are inhibitors of cholesterol biosynthesis pathway, reduced the prevalence of Alzheimer's disease (AD). Animal studies that have employed high cholesterol diet indicate significant relationship between cholesterol level and senile plaque deposition. Here, we investigated the effects of lovastatin on beta-amyloid production and senile plaque deposition in an animal model of AD (Tg2576 mice). As expected, lovastatin treatment reduced plasma cholesterol level in both male and female mice. However, lovastatin enhanced the amounts of beta-amyloid and other beta-secretase derived peptides in females, but not in males. Likewise, lovastatin increased the number of plaques in the hippocampus and cortex of females, but not in males. Lovastatin did not change the amounts of full-length or alpha-secretase processed amyloid precursor protein (APP), or presenilin 1 (PS1) in either sex. Thus, lovastatin lowers cholesterol level in both genders, but enhances beta-amyloid production and senile plaque deposition only in brains of female Tg2576 mice. Our results suggest that low plasma cholesterol levels might be a risk factor for AD in females.     

Transient enriched housing before amyloidosis onset sustains cognitive improvement in Tg2576 mice

Levels of educational and occupational attainment, as components of cognitive reserve, may modify the relationship between the pathological hallmarks and cognition in Alzheimer's disease (AD). We examined whether exposure of a Tg2576 transgenic mouse model of AD to environmental enrichment (EE) at a specific period during the amyloidogenic process favored the establishment of a cognitive reserve. We found that exposure to EE during early adulthood of Tg2576 mice—before amyloidogenesis has started—reduced the severity of AD-related cognitive deficits more efficiently than exposure later in life, when the pathology is already present. Interestingly, early-life exposure to EE, while slightly reducing forebrain surface covered by amyloid plaques, did not significantly impact aberrant inhibitory remodeling in the hippocampus of Tg2576 mice. Thus, transient early-life exposure to EE exerts long-lasting protection against cognitive impairment during AD pathology. In addition, these data define the existence of a specific life time frame during which stimulatory activity most efficiently builds a cognitive reserve, limiting AD progression and favoring successful aging.     

Mannan-Abeta28 conjugate prevents Abeta-plaque deposition, but increases microhemorrhages in the brains of vaccinated Tg2576 (APPsw) mice

Background

New pre-clinical trials in AD mouse models may help to develop novel immunogen-adjuvant configurations with the potential to avoid the adverse responses that occurred during the clinical trials with AN-1792 vaccine formulation. Recently, we have pursued an alternative immunization strategy that replaces QS21 the Th1 type adjuvant used in the AN-1792 clinical trial with a molecular adjuvant, mannan that can promote a Th2-polarized immune response through interactions with mannose-binding and CD35/CD21 receptors of the innate immune system. Previously we established that immunization of wild-type mice with mannan-Aβ₂₈ conjugate promoted Th2-mediated humoral and cellular immune responses. In the current study, we tested the efficacy of this vaccine configuration in amyloid precursor protein (APP) transgenic mice (Tg2576).

Methods

Mannan was purified, activated and chemically conjugated to Aβ₂₈ peptide. Humoral immune responses induced by the immunization of mice with mannan-Aβ₂₈ conjugate were analyzed using a standard ELISA. Aβ₄₂ and Aβ₄₀ amyloid burden, cerebral amyloid angiopathy (CAA), astrocytosis, and microgliosis in the brain of immunized and control mice were detected using immunohistochemistry. Additionally, cored plaques and cerebral vascular microhemorrhages in the brains of vaccinated mice were detected by standard histochemistry.

Results

Immunizations with low doses of mannan-Aβ₂₈ induced potent and long-lasting anti-Aβ humoral responses in Tg2576 mice. Even 11 months after the last injection, the immunized mice were still producing low levels of anti-Aβ antibodies, predominantly of the IgG1 isotype, indicative of a Th2 immune response. Vaccination with mannan-Aβ₂₈ prevented Aβ plaque deposition, but unexpectedly increased the level of microhemorrhages in the brains of aged immunized mice compared to two groups of control animals of the same age either injected with molecular adjuvant fused with an irrelevant antigen, BSA (mannan-BSA) or non-immunized mice. Of note, mice immunized with mannan-Aβ₂₈ showed a trend toward elevated levels of CAA in the neocortex and in the leptomeninges compared to that in mice of both control groups.

Conclusion

Mannan conjugated to Aβ₂₈ provided sufficient adjuvant activity to induce potent anti-Aβ antibodies in APP transgenic mice, which have been shown to be hyporesponsive to immunization with Aβ self-antigen. However, in old Tg2576 mice there were increased levels of cerebral microhemorrhages in mannan-Aβ₂₈ immunized mice. This effect was likely unrelated to the anti-mannan antibodies induced by the immunoconjugate, because control mice immunized with mannan-BSA also induced antibodies specific to mannan, but did not have increased levels of cerebral microhemorrhages compared with non-immunized mice. Whether these anti-mannan antibodies increased the permeability of the blood brain barrier thus allowing elevated levels of anti-Aβ antibodies entry into cerebral perivascular or brain parenchymal spaces and contributed to the increased incidence of microhemorrhages remains to be investigated in the future studies.     

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.     

Chronic dietary alpha-lipoic acid reduces deficits in hippocampal memory of aged Tg2576 mice

Oxidative stress may play a key role in Alzheimer's disease (AD) neuropathology. Here, the effects of the antioxidant, alpha-lipoic acid (ALA) were tested on the Tg2576 mouse, a transgenic model of cerebral amyloidosis associated with AD. Ten-month old Tg2576 and wild type mice were fed an ALA-containing diet (0.1%) or control diet for 6 months and then assessed for the influence of diet on memory and neuropathology. ALA-treated Tg2576 mice exhibited significantly improved learning, and memory retention in the Morris water maze task compared to untreated Tg2576 mice. Twenty-four hours after contextual fear conditioning, untreated Tg2576 mice exhibited significantly impaired context-dependent freezing. ALA-treated Tg2576 mice exhibited significantly more context freezing than the untreated Tg2576 mice. Assessment of brain soluble and insoluble beta-amyloid levels revealed no differences between ALA-treated and untreated Tg2576 mice. Brain levels of nitrotyrosine, a marker of nitrative stress, were elevated in Tg2576 mice, while F2 isoprostanes and neuroprostanes, oxidative stress markers, were not elevated in the Tg2576 mice relative to wild type. These data indicate that chronic dietary ALA can reduce hippocampal-dependent memory deficits of Tg2576 mice without affecting beta-amyloid levels or plaque deposition.     

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.