Calpastatin modulates APP processing in the brains of ?-amyloid depositing but not wild-type mice.
ABSTRACT: We report that neuronal overexpression of the endogenous inhibitor of calpains, calpastatin (CAST), in a mouse model of human Alzheimer's disease (AD) ?-amyloidosis, the APP23 mouse, reduces ?-amyloid (A?) pathology and A? levels when comparing aged, double transgenic (tg) APP23/CAST with APP23 mice. Concurrent with A? plaque deposition, aged APP23/CAST mice show a decrease in the steady-state brain levels of the amyloid precursor protein (APP) and APP C-terminal fragments (CTFs) when compared with APP23 mice. This CAST-dependent decrease in APP metabolite levels was not observed in single tg CAST mice expressing endogenous APP or in younger, A? plaque predepositing APP23/CAST mice. We also determined that the CAST-mediated inhibition of calpain activity in the brain is greater in the CAST mice with A? pathology than in non-APP tg mice, as demonstrated by a decrease in calpain-mediated cytoskeleton protein cleavage. Moreover, aged APP23/CAST mice have reduced extracellular signal-regulated kinase 1/2 (ERK1/2) activity and tau phosphorylation when compared with APP23 mice. In summary, in vivo calpain inhibition mediated by CAST transgene expression reduces A? pathology in APP23 mice, with our findings further suggesting that APP metabolism is modified by CAST overexpression as the mice develop A? pathology. Our results indicate that the calpain system in neurons is more responsive to CAST inhibition under conditions of A? pathology, suggesting that in the disease state neurons may be more sensitive to the therapeutic use of calpain inhibitors.
Project description:Transgenic mice that overexpress mutant human amyloid precursor protein (APP) exhibit one hallmark of Alzheimer's disease pathology, namely the extracellular deposition of amyloid plaques. Here, we describe significant deposition of amyloid beta (Abeta) in the cerebral vasculature [cerebral amyloid angiopathy (CAA)] in aging APP23 mice that had striking similarities to that observed in human aging and Alzheimer's disease. Amyloid deposition occurred preferentially in arterioles and capillaries and within individual vessels showed a wide heterogeneity (ranging from a thin ring of amyloid in the vessel wall to large plaque-like extrusions into the neuropil). CAA was associated with local neuron loss, synaptic abnormalities, microglial activation, and microhemorrhage. Although several factors may contribute to CAA in humans, the neuronal origin of transgenic APP, high levels of Abeta in cerebrospinal fluid, and regional localization of CAA in APP23 mice suggest transport and drainage pathways rather than local production or blood uptake of Abeta as a primary mechanism underlying cerebrovascular amyloid formation. APP23 mice on an App-null background developed a similar degree of both plaques and CAA, providing further evidence that a neuronal source of APP/Abeta is sufficient to induce cerebrovascular amyloid and associated neurodegeneration.
Project description:Calpain is activated following myocardial infarction and ablation of calpastatin (CAST), an endogenous inhibitor of calpains, promotes left ventricular remodeling after myocardial infarction (MI). The present study aimed to investigate the effect of transgenic over-expression of CAST on the post-infarction myocardial remodeling process.We established transgenic mice (TG) ubiquitously over-expressing human CAST protein and produced MI in TG mice and C57BL/6J wild-type (WT) littermates.The CAST protein expression was profoundly upregulated in the myocardial tissue of TG mice compared with WT littermates (P < 0.01). Overexpression of CAST significantly reduced the infarct size (P < 0.01) and blunted MI-induced interventricular hypertrophy, global myocardial fibrosis and collagen I and collagen III deposition, hypotension and hemodynamic disturbances at 21 days after MI. Moreover, the MI-induced up-regulation and activation of calpains were obviously attenuated in CAST TG mice. MI-induced down-regulation of CAST was partially reversed in TG mice. Additionally, the MI-caused imbalance of matrix metalloproteinases and their inhibitors was improved in TG mice.Transgenic over-expression of CAST inhibits calpain activation and attenuates post-infarction myocardial remodeling.
Project description:The polymorphic ?-amyloid lesions present in individuals with Alzheimer's disease are collectively known as cerebral ?-amyloidosis. Amyloid precursor protein (APP) transgenic mouse models similarly develop ?-amyloid depositions that differ in morphology, binding of amyloid conformation-sensitive dyes, and A?40/A?42 peptide ratio. To determine the nature of such ?-amyloid morphotypes, ?-amyloid-containing brain extracts from either aged APP23 brains or aged APPPS1 brains were intracerebrally injected into the hippocampus of young APP23 or APPPS1 transgenic mice. APPPS1 brain extract injected into young APP23 mice induced ?-amyloid deposition with the morphological, conformational, and A?40/A?42 ratio characteristics of ?-amyloid deposits in aged APPPS1 mice, whereas APP23 brain extract injected into young APP23 mice induced ?-amyloid deposits with the characteristics of ?-amyloid deposits in aged APP23 mice. Injecting the two extracts into the APPPS1 host revealed a similar difference between the induced ?-amyloid deposits, although less prominent, and the induced deposits were similar to the ?-amyloid deposits found in aged APPPS1 hosts. These results indicate that the molecular composition and conformation of aggregated A? in APP transgenic mice can be maintained by seeded conversion.
Project description:BACKGROUND: The deposition of the amyloid ?-peptide (A?) in the brain is one of the hallmarks of Alzheimer's disease (AD). It is not yet clear whether A? always leads to similar changes or whether it induces different features of neurodegeneration in relation to its intra- and/or extracellular localization or to its intracellular trafficking routes. To address this question, we have analyzed two transgenic mouse models: APP48 and APP23 mice. The APP48 mouse expresses A?1-42 with a signal sequence in neurons. These animals produce intracellular A? independent of amyloid precursor protein (APP) but do not develop extracellular A? plaques. The APP23 mouse overexpresses human APP with the Swedish mutation (KM670/671NL) in neurons and produces APP-derived extracellular A? plaques and intracellular A? aggregates. RESULTS: Tracing of commissural neurons in layer III of the frontocentral cortex with the DiI tracer revealed no morphological signs of dendritic degeneration in APP48 mice compared to littermate controls. In contrast, the dendritic tree of highly ramified commissural frontocentral neurons was altered in 15-month-old APP23 mice. The density of asymmetric synapses in the frontocentral cortex was reduced in 3- and 15-month-old APP23 but not in 3- and 18-month-old APP48 mice. Frontocentral neurons of 18-month-old APP48 mice showed an increased proportion of altered mitochondria in the soma compared to wild type and APP23 mice. A? was often seen in the membrane of neuronal mitochondria in APP48 mice at the ultrastructural level. CONCLUSIONS: These results indicate that APP-independent intracellular A? accumulation in APP48 mice is not associated with dendritic and neuritic degeneration but with mitochondrial alterations whereas APP-derived extra- and intracellular A? pathology in APP23 mice is linked to dendrite degeneration and synapse loss independent of obvious mitochondrial alterations. Thus, A? aggregates in APP23 and APP48 mice induce neurodegeneration presumably by different mechanisms and APP-related production of A? may, thereby, play a role for the degeneration of neurites and synapses.
Project description:Alzheimer's disease (AD) is thought to be caused by accumulation of amyloid-? protein (A?), which is a cleavage product of amyloid precursor protein (APP). Transgenic mice overexpressing APP have been used to recapitulate amyloid-? pathology. Among them, APP23 and APPswe/PS1deltaE9 (deltaE9) mice are extensively studied. APP23 mice express APP with Swedish mutation and develop amyloid plaques late in their life, while cognitive deficits are observed in young age. In contrast, deltaE9 mice with mutant APP and mutant presenilin-1 develop amyloid plaques early but show typical cognitive deficits in old age. To unveil the reasons for different progressions of cognitive decline in these commonly used mouse models, we analyzed the number and turnover of dendritic spines as important structural correlates for learning and memory. Chronic in vivo two-photon imaging in apical tufts of layer V pyramidal neurons revealed a decreased spine density in 4-5-month-old APP23 mice. In age-matched deltaE9 mice, in contrast, spine loss was only observed on cortical dendrites that were in close proximity to amyloid plaques. In both cases, the reduced spine density was caused by decreased spine formation. Interestingly, the patterns of alterations in spine morphology differed between these two transgenic mouse models. Moreover, in APP23 mice, APP was found to accumulate intracellularly and its content was inversely correlated with the absolute spine density and the relative number of mushroom spines. Collectively, our results suggest that different pathological mechanisms, namely an intracellular accumulation of APP or extracellular amyloid plaques, may lead to spine abnormalities in young adult APP23 and deltaE9 mice, respectively. These distinct features, which may represent very different mechanisms of synaptic failure in AD, have to be taken into consideration when translating results from animal studies to the human disease.
Project description:Much evidence indicates that women have a higher risk of developing Alzheimer's disease (AD) than do men. The reason for this gender difference is unclear. We hypothesize that estrogen deficiency in the brains of women with AD may be a key risk factor. In rapidly acquired postmortem brains from women with AD, we found greatly reduced estrogen levels compared with those from age- and gender-matched normal control subjects; AD and control subjects had comparably low levels of serum estrogen. We examined the onset and severity of AD pathology associated with estrogen depletion by using a gene-based approach, by crossing the estrogen-synthesizing enzyme aromatase gene knockout mice with APP23 transgenic mice, a mouse model of AD, to produce estrogen-deficient APP23 mice. Compared with APP23 transgenic control mice, estrogen-deficient APP23 mice exhibited greatly reduced brain estrogen and early-onset and increased beta amyloid peptide (Abeta) deposition. These mice also exhibited increased Abeta production, and microglia cultures prepared from the brains of these mice were impaired in Abeta clearance/degradation. In contrast, ovariectomized APP23 mice exhibited plaque pathology similar to that observed in the APP23 transgenic control mice. Our results indicate that estrogen depletion in the brain may be a significant risk factor for developing AD neuropathology.
Project description:Transgenic (Tg) mouse models of Alzheimer's disease have served as valuable tools for investigating pathogenic mechanisms related to A? accumulation. However, assessing disease status in these animals has required time-consuming behavioral assessments or postmortem neuropathological analysis. Here, we report a method for tracking the progression of A? accumulation in vivo using bioluminescence imaging (BLI) on two lines of Tg mice, which express luciferase (luc) under control of the Gfap promoter as well as mutant human amyloid precursor protein. Bigenic mice exhibited an age-dependent increase in BLI signals that correlated with the deposition of A? in the brain. Bioluminescence signals began to increase in 7-mo-old Tg(CRND8:Gfap-luc) mice and 14-mo-old Tg(APP23:Gfap-luc) mice. When Tg(APP23:Gfap-luc) mice were inoculated with brain homogenates from aged Tg(APP23) mice, BLI detected the accelerated disease onset and induced A? deposition at 11 mo of age. Because of its rapid, noninvasive, and quantitative format, BLI permits the objective repeated analysis of individual mice at multiple time points, which is likely to facilitate the testing of A?-directed therapeutics.
Project description:In a positron-emission tomography (PET) study with the ?-amyloid (A?) tracer [(18)F]-florbetaben, we previously showed that A? deposition in transgenic mice expressing Swedish mutant APP (APP-Swe) mice can be tracked in vivo. ?-Secretase modulators (GSMs) are promising therapeutic agents by reducing generation of the aggregation prone A?42 species without blocking general ?-secretase activity. We now aimed to investigate the effects of a novel GSM [8-(4-Fluoro-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]-[1-(3-methyl-[1,2,4]thiadiazol-5-yl)-piperidin-4-yl]-amine (RO5506284) displaying high potency in vitro and in vivo on amyloid plaque burden and used longitudinal A?-microPET to trace individual animals. Female transgenic (TG) APP-Swe mice aged 12 months (m) were assigned to vehicle (TG-VEH, n=12) and treatment groups (TG-GSM, n=12), which received daily RO5506284 (30?mg?kg(-1)) treatment for 6 months. A total of 131 A?-PET recordings were acquired at baseline (12 months), follow-up 1 (16 months) and follow-up 2 (18 months, termination scan), whereupon histological and biochemical analyses of A? were performed. We analyzed the PET data as VOI-based cortical standard-uptake-value ratios (SUVR), using cerebellum as reference region. Individual plaque load assessed by PET remained nearly constant in the TG-GSM group during 6 months of RO5506284 treatment, whereas it increased progressively in the TG-VEH group. Baseline SUVR in TG-GSM mice correlated with ?%-SUVR, indicating individual response prediction. Insoluble A?42 was reduced by 56% in the TG-GSM versus the TG-VEH group relative to the individual baseline plaque load estimates. Furthermore, plaque size histograms showed differing distribution between groups of TG mice, with fewer small plaques in TG-GSM animals. Taken together, in the first A?-PET study monitoring prolonged treatment with a potent GSM in an AD mouse model, we found clear attenuation of de novo amyloidogenesis. Moreover, longitudinal PET allows non-invasive assessment of individual plaque-load kinetics, thereby accommodating inter-animal variations.
Project description:BACKGROUND: The progressive development of Alzheimer's disease (AD) pathology follows a spatiotemporal pattern in the human brain. In a transgenic (Tg) mouse model of AD expressing amyloid precursor protein (APP) with the arctic (E693G) mutation, pathology spreads along anatomically connected structures. Amyloid-? (A?) pathology first appears in the subiculum and is later detected in interconnected brain regions, including the retrosplenial cortex. We investigated whether the spatiotemporal pattern of A? pathology in the Tg APP arctic mice to interconnected brain structures can be interrupted by destroying neurons using a neurotoxin and thereby disconnecting the neural circuitry. RESULTS: We performed partial unilateral ibotenic acid lesions of the subiculum (first structure affected by A? pathology) in young Tg APParc mice, prior to the onset of pathology. We assessed A?/C99 pathology in mice aged up to 6 months after injecting ibotenate into the subiculum. Compared to the brains of intact Tg APP arctic mice, we observed significantly decreased A?/C99 pathology in the ipsilateral dorsal subiculum, CA1 region of the hippocampus and the retrosplenial cortex; regions connecting to and from the dorsal subiculum. By contrast, A?/C99 pathology was unchanged in the contralateral hippocampus in the mice with lesions. CONCLUSION: These results, obtained in an animal model of AD, support the notion that A?/C99 pathology is transmitted between interconnected neurons in AD.
Project description:Pathological aggregation of amyloid-? (A?) is a main hallmark of Alzheimer's disease (AD). Recent genetic association studies have linked innate immune system actions to AD development, and current evidence suggests profound gender differences in AD pathogenesis. Here, we characterise gender-specific pathologies in the APP23 AD-like mouse model and find that female mice show stronger amyloidosis and astrogliosis compared with male mice. We tested the gender-specific effect of lack of IL12p40, the shared subunit of interleukin (IL)-12 and IL-23, that we previously reported to ameliorate pathology in APPPS1 mice. IL12p40 deficiency gender specifically reduces A? plaque burden in male APP23 mice, while in female mice, a significant reduction in soluble A?1-40 without changes in A? plaque burden is seen. Similarly, plasma and brain cytokine levels are altered differently in female versus male APP23 mice lacking IL12p40, while glial properties are unchanged. These data corroborate the therapeutic potential of targeting IL-12/IL-23 signalling in AD, but also highlight the importance of gender considerations when studying the role of the immune system and AD.