APP Tg Mice Research Articles

P3-246: Drastic impact on Abeta accumulation in APP Tg mice by genetic background

Background: Though a great deal of effort has been made on the searching for the risk genes of Alzheimer disease (AD), there is little success, except ApoE. Animal model has many advantages (strict control of environmental factors, direct measurement of amyloid pathology, etc) compared to human studies. Methods: APP Tg mice (Tg2576) which are usually maintained by B6/SJL hybrid mice were crossed onto C57BL6/J, SJL/J and DBA2/J inbred strains. The levels of Abeta40 and Abeta42 in Triton and GuHCl fraction from cortex were measured by ELISA. Results: The shortened life span were observed in APP Tg mice with B6 rich genetic background (B6 75%, SJL 25%), which was similar to previous report (Carlson Hum Mol Genet 1997). No significant life span change, however, was observed in the mice with either SJL or DBA rich genetic background. Analysis of 59 APP Tg mice at the age of 12 months showed that lower Abeta accumulation in mice with DBA rich genetic background compared to SJL or B6 rich genetic background. For example, significant difference (p 0.01, Turkey-Kramer) were observed in Abeta40 (-82%) and Abeta42 (-68%) levels in Triton fraction between SJL 84% B6 16% mice and DBA 75% B6 16% SJL 9% mice. Drastic difference (p 0.01, TurkeyKramer) were observed in Abeta40 and Abeta42 levels also from GuHCl fraction between SJL 69% B6 31% and DBA 75% B6 16% SJL 9% mice (-85% and -68%) and between SJL 69% B6 31% and DBA 50% B6 31% SJL 19% mice (-75% and -50%). We focus on SJL and DBA, because the difference of Abeta accumulation was most drastic and both genetic backgrounds have no impact on life span. More animals are being analyzed and amyloid pathology modifier candidate genes will be searched using arrays. Conclusions: Our data demonstrated that genetic background impact on amyloid pathology in APP Tg mice. Mice model can be a useful tools to discover risk genes for human AD.

P1-437: Effects of Icarrin on learning-memory ability and development of β-amyloid in APP transgenic mice

The detailed mechanism and pathological changes of Alzheimer's disease (AD) are not clear as yet. Treated drugs of AD are limited and the effects of them on AD patients are not satisfied. So there is an urgent need for the development of drugs that can ameliorate or delay the onset of symptoms of the disease. Aim of the research is to investigate learning-memory function and pahtological changes related with β-amyloid (Aβ) in brains of APP transgenic (Tg) mice, and to observe the protective effects of Icarrin (ICA), a component of Traditional Chinese Medicine (TCM)_Epimedium, on above-mentioned targets and the expression of α-synuclein which is the precursor protein of non β-amyloid component (NAC) in brains of APP Tg mice. The mice of drug treated group were administered introgastrically by ICA (at doses 18 and 58 mg/kg/d) from 4 to 10 months old. The mice of normal group and negative transgene group were administered of distilled water by the same way. The learning-memory ability as measured by applying Morris water maze (MWM). The expression of APP, β-secretase (BACE), and the expression of α-synuclein in brains were detected by immunohistochemistry and western blot respectively. The content of βamyloid 1–42 (Aβ1–42) were measured by ELISA Assay. The amyloid senile plaques were detected by Congo red staining. The results showed that the escape latency and swimming distance in MWM of APP Tg mice were prolonged. The expression of APP, BACE, the content of Aβ1–42 decreased, the amount of amyloid senile plaques and the expression of α-synuclein increased in brains of APP Tg mice. ICA decreased the latency and swimming distance in MWM test, decreased the expression of APP, BACE in both hippocampus and cortex and the content of Aβ1–42 in hippocampus of model mice, and relieved amyloid plaques burden in brains of model mice. Meanwhile, ICA suppressed the expression of α- synuclein. These results suggest that APP Tg mice could mimic pathological proceedings of AD. Icarrin may have a promising application prospect in treatment of AD.

S2-02-02: The mechanistic involvement of the calpain-calpastatin system in Alzheimer pathology

The mechanism, by which amyloid-β peptide (Aβ) accumulation causes neurodegeneration in Alzheimer disease (AD), remains unresolved. Given that Aβ perturbs calcium homeostasis in neurons, we investigated possible involvement of calpain whose action is irreversible. We first observed close postsynaptic association of calpain activation with Aβ plaque formation in brains from both AD patients and transgenic (Tg) mice overexpressing amyloid precursor protein (APP). The mice also exhibited axonal termini dynamically misdirected to calpain activation-positive Aβ plaques. Consistently, cerebrospinal fluids from Tg mice and AD patients contained greater quantity of calpain-cleaved spectrin than controls. Genetic deficiency of calpastatin (CS), calpain-specific inhibitor protein, augmented Aβ amyloidosis, tau phosphorylation, microgliosis, somato-dendritic dystrophy and mortality in APP-Tg mice. In contrast, brain-specific CS overexpression resulted in opposite phenotypes. These observations indicate that calpain activation may underlie Aβ-triggered pathological cascade and thus shall become a relevant pharmacological target in the treatment of AD.

Novel Aβ Immunogens: Is Shorter Better?

Active and passive Abeta immunotherapy in Alzheimer's disease (AD)-like mouse models lowers cerebral amyloid-beta protein (Abeta) levels, especially if given early in the disease process, and improves cognitive deficits. In 2002, a Phase IIa clinical trial was halted due to meningoencephalitis in approximately 6% of the AD patients. It is hypothesized that the immunogen, full-length Abeta1-42, may have led to an autoimmune response. Currently, we are developing novel Abeta peptide immunogens for active immunization in amyloid precursor protein transgenic mice (APP Tg) to target Abeta B cell epitopes (within Abeta1-15) and avoid Abeta-specific T cell epitopes (Abeta16-42) so as to generate a safe and effective AD vaccine. Intranasal immunization with dendrimeric Abeta1-15 (16 copies of Abeta1-15 on a lysine core) or a tandem repeat of Abeta1-15 joined by 2 lysines and conjugated to an RGD motif with a mutated form of an E. coli-derived adjuvant generated robust Abeta titers in both wildtype and APP Tg mice. The Abeta antibodies recognized a B cell epitope within Abeta1-7, were mostly T-helper 2 associated immunoglobulin isotypes, bound human AD and APP Tg plaques, and detected Abeta oligomers. Splenic T cells reacted to the immunogens but not full-length Abeta. Six months of intranasal immunization (from 6-to-12 months of age) of J20 mice with each immunogen lowered insoluble Abeta42 by 50%, reduced plaque burden and gliosis, and increased Abeta in plasma. Interestingly, Abeta antibody generation was influenced by route of immunization. Transcutaneous immunization with dbeta1-15, but not full-length Abeta, led to high Abeta titers. In summary, our short Abeta immunogens induced robust titers of predominantly Th2 antibodies that were able to clear cerebral Abeta in the absence of Abeta-specific T cell reactivity, indicating the potential for a safer vaccine. We remain optimistic about the potential of such a vaccine for prevention and treatment of AD.

O1-03-01: The role of dietary supplements and herbs in the treatment and prevention of Alzheimer’s disease

induced cognitive deficits and tau pathology. GSK3 inhibition blocked A oligomer-induced JNK and cognitive deficits in oligomer-infused rats. DHA reduced A accumulation in APP Tg mice by a novel mechanism involving APP sorting. APP Tg mice on the high n-6 diet have highly significant loss of the actin-regulatory dendritic spine protein, drebrin. We argue that oligomer-induced drebrin-related defects play a critical role in initiating cognitive deficits. AD clinical trial results found that DHA (as fish oil) had little impact on cognitive decline in established AD patients, but might have been effective at the earliest stages (MMSE 27). Major region-specific drebrin loss has been reported to begin in MCI with a sharp fall-off at very early stages of AD (MMSE 26) -without further progression. Conclusions: DHA can limit A production in vivo by a novel mechanism. Oligomer-induced dendritic spine loss is reflected in drebrin loss and should be a major target. Because it occurs very early, treatments that may mitigate oligomer-induced postsynaptic pathology, including DHA intervention, should begin very early, either in MCI or with primary prevention.

Boosting with intranasal dendrimeric Aβ1–15 but not Aβ1–15 peptide leads to an effective immune response following a single injection of Aβ1–40/42 in APP-tg mice

BackgroundImmunotherapy for Alzheimer's disease (AD) is emerging as a potential treatment. However, a clinical trial (AN1792) was halted after adverse effects occurred in a small subset of subjects, which may have been caused by a T cell-mediated immunological response. In general, aging limits the humoral immune response, therefore, immunogens and vaccination regimes are required that induce a strong antibody response with less potential for an adverse immune response.MethodIn the current study, we immunized both wildtype and J20 APP-tg mice with a priming injection of Aβ1–40/42, followed by multiple intranasal boosts with the novel immunogen dAβ1–15 (16 copies of Aβ1–15 on a lysine tree), Aβ1–15 peptide or Aβ1–40/42 full length peptide.ResultsJ20 APP-tg mice primed with Aβ1–40/42 subcutaneously and subsequently boosted intranasally with Aβ1–15 peptide did not generate a cellular or humoral immune response. In contrast, J20 APP-tg mice boosted intranasally with dAβ1–15 or full length Aβ1–40/42 produced high levels of anti-Aβ antibodies. Splenocyte proliferation was minimal in mice immunized with dAβ1–15. Wildtype littermates of the J20 APP-tg mice produced higher amounts of anti-Aβ antibodies compared to APP-tg mice but also had low T cell proliferation. The anti-Aβ antibodies were mainly composed of IgG2b and directed to an epitope within the Aβ1–7 region, regardless of the immunogen. Examination of the brain showed a significant reduction in Aβ plaque burden in the J20 APP-tg mice producing antibodies compared to controls. Biochemically, Aβ40 or Aβ42 were also reduced in brain homogenates and elevated in plasma but the changes did not reach significance.ConclusionOur results demonstrate that priming with full length Aβ40/42 followed by boosting with dAβ1–15 but not Aβ1–15 peptide led to a robust humoral immune response with a minimal T cell response in J20 APP-tg mice. In addition, Aβ plaque burden was reduced in mice producing anti-Aβ antibodies. Interestingly, wildtype mice produced higher levels of anti-Aβ antibodies, indicating that immune tolerance may be present in J20 APP-tg mice. Together, these data suggest that dAβ1–15 but not Aβ1–15 peptide may be useful as a boosting immunogen in an AD vaccination regime.

Dendrimeric Aβ1–15 is an effective immunogen in wildtype and APP-tg mice

Immunization of humans and APP-tg mice with full-length β-amyloid (Aβ) results in reduced cerebral Aβ levels. However, due to adverse events in the AN1792 trial, alternative vaccines are required. We investigated dendrimeric Aβ1–15 (dAβ1–15), which is composed of 16 copies of Aβ1–15 peptide on a branched lysine core and thus, includes an Aβ-specific B cell epitope but lacks the reported T cell epitope. Immunization by subcutaneous, transcutaneous, and intranasal routes of B6D2F1 wildtype mice led to anti-Aβ antibody production. Antibody isotypes were mainly IgG1 for subcutaneous or transcutaneous immunization and IgG2b for intranasal immunization, suggestive of a Th2-biased response. All Aβ antibodies preferentially recognized an epitope in Aβ1–7. Intranasal immunization of J20 APP-tg mice resulted in a robust humoral immune response with a corresponding significant reduction in cerebral plaque burden. Splenocyte proliferation against Aβ peptide was minimal indicating the lack of an Aβ-specific cellular immune response. Anti-Aβ antibodies bound monomeric, oligomeric, and fibrillar Aβ. Our data suggest that dAβ1–15 may be an effective and potentially safer immunogen for Alzheimer's disease (AD) vaccination.

Learning memory deficit in APP transgenic model of Alzheimer's disease and intervention by Shen-wu capsule and its effective component

目的 观察不同时程拟阿尔茨海默病(Alzheimer's disease,AD)转基因动物模型学习记忆能力改变,以及中药参乌胶囊及其有效成分二苯乙烯苷(TSG)防治AD的药理机制.方法拟AD动物模型为4到16月龄APP695V717I转基因小鼠.4月龄起灌胃给予参乌胶囊及TSG 6个月.另一批未处理的APP转基因小鼠饲养至10月龄开始灌胃给予TSG 6个月.同背景同月龄C57BL/6J小鼠为正常对照组.行为学检测应用Morris 水迷宫实验和物体识别实验.结果早期4月龄APP转基因小鼠即出现学习记忆能力障碍,水迷宫游出时间比对照组延长46.1%,物体识别检测的分辨指数相差2.96倍.增龄至10月龄,正常对照组小鼠学习记忆能力无明显变化,而APP转基因小鼠学习记忆能力急剧下降,水迷宫游出时间与对照组的差异加大为95%,物体识别中分辨指数更相差6.7倍.增龄至晚期16月龄,对照组的老化使其学习记忆能力与10月龄相比也明显下降.虽然APP模型组仍表现明显的学习记忆能力障碍,但差距缩小,水迷宫游出时间与对照组的差异为49.1%,在物体识别中分辨指数只相差2.95倍.参乌胶囊和TSG可明显改善10月龄APP转基因小鼠Morris 水迷宫作业成绩,提高模型鼠物体识别能力.对10月龄已出现病理变化的模型鼠灌服TSG 6个月后,也可明显改善16月龄APP转基因小鼠Morris 水迷宫作业成绩,提高模型鼠物体识别能力.结论 4月龄、10月龄、16月龄APP695V717I转基因小鼠模型较好地模拟早期、中期、晚期AD患者的学习记忆缺陷,可用于AD发病机理和防治AD药物作用机制的研究.参乌胶囊及其有效成分二苯乙烯苷不仅能早期预防学习记忆障碍的发生,还能逆转和治疗已出现的学习记忆障碍,对防治AD等神经退行性疾病具有良好的应用前景。

Minocycline affects microglia activation, Aβ deposition, and behavior in APP‐tg mice

Activated microglia and reactive astrocytes invade and surround cerebral beta amyloid (Abeta) plaques in Alzheimer's disease (AD), but the role of microglia in plaque development is still unclear. In this study, minocycline was administered for 3 months, prior to and early in Abeta plaque formation in amyloid precursor protein transgenic mice (APP-tg). When minocycline was given to younger mice, there was a small but significant increase in Abeta deposition in the hippocampus, concurrent with improved cognitive performance relative to vehicle treated mice. If APP-tg mice received minocycline after Abeta deposition had begun, microglial activation was suppressed but this did not affect Abeta deposition or improve cognitive performance. In vitro studies demonstrated that minocycline suppressed microglial production of IL-1beta, IL-6, TNF, and NGF. Thus, minocycline has different effects on Abeta plaque deposition and microglia activation depending on the age of administration. Our data suggest that this may be due to the effects of minocycline on microglial function. Therefore, anti-inflammatory therapies to suppress microglial activation or function may reduce cytokine production but enhance Abeta plaque formation early in AD.

Mitochondrial failure precedes amyloid beta plaques deposition in APP transgenic mice

Mitochondrial dysfunction has been identified in neurodegenertive disorders including Alzheimer's disease (AD), where accumulation of amyloid beta (A beta) and oxidative stress seem to play central roles in the pathogenesis, by probably directly leading to mitochondrial dysfunction. In order to study the in vivo effect of A beta load during aging, we evaluated the mitochondrial function of brain cells from transgenic (tg) mice bearing mutant amyloid precursor protein (APP, Swedish and London mutation) and non-tg littermate control animals (non-tg) at different ages (1.5, 3, and 6 months). TgAPP mice exhibit onset of A beta plaques at an age of 6 months, but intracellular A (beta) load is already increased at the age of 3 months. Basal mitochondrial transmembrane potential was already decreased in APPtg mice at an age of 1.5 months and decreased further with aging. ATP levels were significantly reduced in tgAPP mice at an age of 3 months compared to age-matched non-tg control mice. Interestingly, cytochrome c oxidase activity was markedly reduced in tgAPP mice at an age of 1.5 and 3 months. The difference was less pronounced at an age of 6 months. In addition, the Bcl-xL/Bax ratio was significantly reduced in brains from tgAPP mice possibly contributing to the mitochondrial dysfunction in tgAPP mice. Taken together, our results indicate that mitochondrial dysfunction is exacerbated by the presence of intracellular A beta as a very early event during pathogenesis.

Conditional inactivation of presenilin 1 prevents amyloid accumulation and temporarily rescues contextual and spatial working memory impairments in amyloid precursor protein transgenic mice.

Accumulation of beta-amyloid (Abeta) peptides in the cerebral cortex is considered a key event in the pathogenesis of Alzheimer's disease (AD). Presenilin 1 (PS1) plays an essential role in the gamma-secretase cleavage of the amyloid precursor protein (APP) and the generation of Abeta peptides. Reduction of Abeta generation via the inhibition of gamma-secretase activity, therefore, has been proposed as a therapeutic approach for AD. In this study, we examined whether genetic inactivation of PS1 in postnatal forebrain-restricted conditional knock-out (PS1 cKO) mice can prevent the accumulation of Abeta peptides and ameliorate cognitive deficits exhibited by an amyloid mouse model that overexpresses human mutant APP. We found that conditional inactivation of PS1 in APP transgenic mice (PS1 cKO;APP Tg) effectively prevented the accumulation of Abeta peptides and formation of amyloid plaques and inflammatory responses, although it also caused an age-related accumulation of C-terminal fragments of APP. Short-term PS1 inactivation in young PS1 cKO;APP Tg mice rescued deficits in contextual fear conditioning and serial spatial reversal learning in a water maze, which were associated with APP Tg mice. Longer-term PS1 inactivation in older PS1 cKO;APP Tg mice, however, failed to rescue the contextual memory and hippocampal synaptic deficits and had a decreasing ameliorative effect on the spatial memory impairment. These results reveal that in vivo reduction of Abeta via the inactivation of PS1 effectively prevents amyloid-associated neuropathological changes and can, but only temporarily, improve cognitive impairments in APP transgenic mice.

Amyloid β-induced Changes in Nitric Oxide Production and Mitochondrial Activity Lead to Apoptosis

Increasing evidence suggests an important role of mitochondrial dysfunction in the pathogenesis of Alzheimer's disease. Thus, we investigated the effects of acute and chronic exposure to increasing concentrations of amyloid beta (Abeta) on mitochondrial function and nitric oxide (NO) production in vitro and in vivo. Our data demonstrate that PC12 cells and human embryonic kidney cells bearing the Swedish double mutation in the amyloid precursor protein gene (APPsw), exhibiting substantial Abeta levels, have increased NO levels and reduced ATP levels. The inhibition of intracellular Abeta production by a functional gamma-secretase inhibitor normalizes NO and ATP levels, indicating a direct involvement of Abeta in these processes. Extracellular treatment of PC12 cells with comparable Abeta concentrations only leads to weak changes, demonstrating the important role of intracellular Abeta. In 3-month-old APP transgenic (tg) mice, which exhibit no plaques but already detectable Abeta levels in the brain, reduced ATP levels can also be observed showing the in vivo relevance of our findings. Moreover, we could demonstrate that APP is present in the mitochondria of APPsw PC12 cells. This presence might be directly involved in the impairment of cytochrome c oxidase activity and depletion of ATP levels in APPsw PC12 cells. In addition, APPsw human embryonic kidney cells, which produce 20-fold increased Abeta levels compared with APPsw PC12 cells, and APP tg mice already show a significantly decreased mitochondrial membrane potential under basal conditions. We suggest a hypothetical sequence of pathogenic steps linking mutant APP expression and amyloid production with enhanced NO production and mitochondrial dysfunction finally leading to cell death.

525. Therapy for Alzheimer's Disease by Neprilysin Gene Transfer

Accumulation of the amyloid beta peptide (A|[beta]|) is believed to be a key component in the pathogenesis of Alzheimer's disease (AD). Deregulation of the anabolism and / or catabolism of A|[beta]| likely contribute to progression of this disease. Endopeptidases expressed in the brain are now thought to control A|[beta]| concentrations. Neprilysin is a metalloendopeptidase that has been shown to be an important mediator of A|[beta]| degradation. This discovery has opened the way for potentially new therapeutic strategies designed to augment protease activity. To investigate this approach we constructed a lentiviral gene transfer vector that efficiently produces biologically active neprilysin (Lenti-Nep), and intracranially injected this vector into mouse models of amyloidosis (APPtg). Previously we had shown that one-month after Lenti-Nep administration amyloid deposits were significantly reduced in mice with advanced amyloidosis (43% reduction) in addition to improvements in dendritic density (16% increase in MAP2 immunoreactivity). We now show that long-term experiments (five to six months) in younger APPtg mice resulted in further reduced amyloid pathology (54% to 65% reductions in deposited amyloid) and improvements in MAP2, synaptophysin, and NeuN immunoreactivity (|[sim]|20%). Memory test performance in the Morris Water Maze was also improved by Lenti-Nep administration. These data further support the potential use of neprilysin gene transfer for the treatment on AD.

Species-specific immune response to immunization with human versus rodent Aβ peptide

Amyloid beta (Aβ) immunization of amyloid precursor protein (APP)-transgenic (tg) mice with human Aβ induces humoral immunity, however, the immune response to endogenous rodent Aβ is unknown. Fourteen-month J20 APP-tg mice and non-tg littermates were immunized subcutaneously followed by chronic intranasal boosting with human or rodent Aβ peptide and adjuvant LT(R192G). Rodent Aβ-immunized APP-tg mice had anti-rodent Aβ antibody levels of 257.8 μg/ml and those immunized with human Aβ had anti-human Aβ antibodies of 120.8 μg/ml. Non-tg littermates had anti-rodent and anti-human Aβ antibody concentrations of 98.8 and 231.1 μg/ml, respectively. Inter-species cross-reactivity was minimal. Anti-human Aβ antibodies were predominately IgG1 and IgG2b, while anti-rodent Aβ antibodies were equally IgG1, IgG2a, and IgG2b. Anti-human Aβ antibodies recognized an epitope within human Aβ1–9. Anti-rodent Aβ antibodies did not stain Alzheimer’s disease (AD) plaques but bound some plaques in APP-tg mice. Splenocytes proliferated modestly to their respective antigen and secreted low levels of IL-2 and IFN-γ. Therefore, immunizing APP-tg and non-tg mice with rodent Aβ resulted in a species-specific humoral response with modest T cell reactivity.

Amyloid-beta immunization in Alzheimer's disease transgenic mouse models and wildtype mice.

Alzheimer's disease is the most prevalent form of dementia worldwide. Therapies are desperately needed to prevent and cure the disease. Mouse models of amyloid-beta deposition [APP and PSAPP transgenic (tg) mice] have been useful in determining the role of amyloid-beta (A beta) in both the pathogenesis and cognitive changes in AD. In addition, they have allowed scientists to investigate potential AD therapies in living animals. Active and passive A beta immunizations have been employed successfully in APP and PSAPP tg mice to lower cerebral A beta levels and improve cognition. Optimization of immunization protocols and characterization of immune responses in wildtype mice have been reported. Based on the promising results of A beta immunization studies in mice, a clinical trial was initiated for A beta vaccination in humans with AD. Although no adverse effects were reported in the Phase I safety trials, about 5% of AD patients in the phase II clinical trial developed meningoencephalitis, ending the trial prematurely in March 2002. Studies in AD mouse models and wildtype mice may help elucidate the mechanism for these unwanted side effects and will be useful for testing newer, safer vaccines for future use in human clinical trials.