Double Transgenic Mouse Model Research Articles

Gender differences of peripheral plasma and liver metabolic profiling in APP/PS1 transgenic AD mice

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive cognitive impairment. Currently, there is less knowledge of the involvement of the peripheral biofluid/organ in AD, compared with the central nervous system. In addition, with reported high morbidity in women in particular, it has become very important to explore whether gender difference in the peripheral metabolome is associated with AD. Here, we investigated metabolic responses of both plasma and liver tissues using an APP/PS1 double mutant transgenic mouse model with NMR spectroscopy, as well as analysis from serum biochemistry and histological staining. Fatty acid composition from plasma and liver extracts was analyzed using GC-FID/MS. We found clear gender differences in AD transgenic mice when compared with their wild-type counterparts. Female AD mice displayed more intensive responses, which were highlighted by higher levels of lipids, 3-hydroxybutyrate and nucleotide-related metabolites, together with lower levels of glucose. These observations indicate that AD induces oxidative stress and impairs cellular energy metabolism in peripheral organs. Disturbances in AD male mice were milder with depletion of monounsaturated fatty acids. We also observed a higher activity of delta-6-desaturate and suppressed activity of delta-5-desaturate in female mice, whereas inhibited stearoyl-CoA-desaturase in male mice suggested that AD induced by the double mutant genes results in different fatty acids catabolism depending on gender. Our results provide metabolic clues into the peripheral biofluid/organs involved in AD, and we propose that a gender-specific scheme for AD treatment in men and women may be required.

Kif14 overexpression accelerates murine retinoblastoma development

The mitotic kinesin KIF14 has an essential role in the recruitment of proteins required for the final stages of cytokinesis. Genomic gain and/or overexpression of KIF14 has been documented in retinoblastoma and a number of other cancers, such as breast, lung and ovarian carcinomas, strongly suggesting its role as an oncogene. Despite evidence of oncogenic properties in vitro and in xenografts, Kif14's role in tumor progression has not previously been studied in a transgenic cancer model. Using a novel Kif14 overexpressing, simian virus 40 large T-antigen retinoblastoma (TAg-RB) double transgenic mouse model, we aimed to determine Kif14's role in promoting retinal tumor formation. Tumor initiation and development in double transgenics and control TAg-RB littermates were documented in vivo over a time course by optical coherence tomography, with subsequent ex vivo quantification of tumor burden. Kif14 overexpression led to an accelerated initiation of tumor formation in the TAg-RB model and a significantly decreased tumor doubling time (1.8 vs. 2.9 weeks). Moreover, overall percentage tumor burden was also increased by Kif14 overexpression. These data provide the first evidence that Kif14 can promote tumor formation in susceptible cells in vivo.

Negative role of TAK1 in marginal zone B-cell development incidental to NF-κB noncanonical pathway activation.

The transcription factor nuclear factor-κB (NF-κB) signaling pathway is crucial in B-cell physiology. One key molecule regulating this pathway is the serine/threonine kinase TAK1 (MAP3K7). TAK1 is responsible for positive feedback mechanisms in B-cell receptor signaling that serve as an NF-κB activation threshold. This study aimed to better understand the correlation between TAK1-mediated signaling and B-cell development and humoral immune responses. Here we showed that a B-cell conditional deletion of TAK1 using mb1-cre resulted in a dramatic elimination of the humoral immune response, consistent with the absence of the B-1 B-cell subset. When monitoring the self-reactive B-cell system (the immunoglobulin hen egg lysozyme/soluble hen egg lysozyme double-transgenic mouse model), we found that TAK1-deficient B cells exhibited an enhanced susceptibility to cell death that might explain the disappearance of the B1 subset. In contrast, these mice gained numerous marginal zone (MZ) B cells. We consequently examined the basal and B-cell receptor-induced activity of NF-κB2 that is reported to regulate MZ B-cell development, and demonstrated that the activity of NF-κB2 increased in TAK1-deficient B cells. Thus, our results present a novel in vivo function, the negative role of TAK1 in MZ B-cell development that is likely associated with NF-κB2 activation.

A comparative evaluation of treatments with 17β-estradiol and its brain-selective prodrug in a double-transgenic mouse model of Alzheimer's disease

Estrogens are neuroprotective and, thus, potentially useful for the therapy of Alzheimer's disease; however, clinical use of hormone therapy remains controversial due to adverse peripheral effects. The goal of this study was to investigate the benefits of treatment with 10β,17β-dihydroxyestra-1,4-dien-3-one (DHED), a brain-selective prodrug of 17β-estradiol, in comparison with the parent hormone using APPswe/PS1dE9 double transgenic mice to model the pathology of the disease. Ovariectomized and intact females were continuously treated with vehicle, 17β-estradiol, or DHED via subcutaneous osmotic pumps from 6 to 8months of age. We confirmed that this prolonged treatment with DHED did not stimulate uterine tissue, whereas 17β-estradiol treatment increased uterine weight. Amyloid precursor protein decreased in both treatment groups of intact, but not in ovariectomized double transgenic females in which ovariectomy already decreased the expression of this protein significantly. However, reduced brain amyloid-β peptide levels could be observed for both treatments. Consequently, double-transgenic ovariectomized and intact mice had higher cognitive performance compared to untreated control animals in response to both estradiol and DHED administrations. Overall, the tested brain-selective 17β-estradiol prodrug proved to be an effective early-stage intervention in an Alzheimer's disease-relevant mouse model without showing systemic impact and, thus, warrants further evaluation as a potential therapeutic candidate.

396 Induced expression of gasdermin A3 in the skin caused keratinocyte necrosis and anagen delay in the hair cycle

The epidermis forms a critical physical and immune barrier. Dysregulation of keratinocyte to cell death upon external stimuli could compromise the epidermal barrier immunity and cause skin inflammation. Gasdermin A was involved in gastric epithelial apoptosis; its function in the skin was hinted by the skin inflammation and hair loss phenotype in mice with dominant mutations in gasdermin A3 (Gsdma3). The mode of GSDMA3’s action and pathogenesis are in dispute. We and other group have demonstrated that N-terminal portion of Gsdma3 (N-Gsdma3) contains the functional domain, which is auto-regulated by its C-terminal domain. However, overexpression of N-Gsdma3 has been shown to induce autophagy or pyroptosis in some cell culture models. To study the biological function of GSDMA3 in vivo, we generated a doxycycline-inducible double transgenic (DTg) mouse model to express GSDMA3 in the epidermis using K14-rtTA mouse. We found that GSDMA3 overexpression caused epidermal hyperplasia and inflammatory infiltrations, which were associated with increased TNF-α and IL-1α expression. In addition, hair plucking was employed to induce hair cycle reentry at telogen phase. We found a significant delay in anagen initiation in DTg mice but their catagen phase seemed normal. We also observed increased macrophages around and in the bulge region of DTg hair follicles compared with control hair follicles, suggesting that bulge immune privilege was compromised. Regarding the underlying pathogenesis mechanism, we found increased TUNEL-positive staining and spontaneous necrotic-like keratinocytes in DTg mice. In primary keratinocytes isolated from the DTg mice, induced expression of Gsdma3 caused increased mitochondria ROS production, leakage of intracellular components, and spontaneous necrosis. Our data indicated that an innate immune-mediated mechanism could underlie the Gsdma3-mediated skin inflammation phenotype.

Neuroprotective effect of β-asarone against Alzheimer's disease: regulation of synaptic plasticity by increased expression of SYP and GluR1.

Aimβ-asarone, an active component of Acori graminei rhizome, has been reported to have neuroprotective effects in Alzheimer’s disease. As the underlying mechanism is not known, we investigated the neuroprotective effects of β-asarone in an APP/PS1 double transgenic mouse model and in NG108 cells.Materials and methodsAPPswe/PS1dE9 double transgenic male mice were randomly assigned to a model group, β-asarone treatment groups (21.2, 42.4, or 84.8 mg/kg/d), or donepezil treatment group (2 mg/kg/d). Donepezil treatment was a positive control, and background- and age-matched wild-type B6 mice were an external control group. β-asarone (95.6% purity) was dissolved in 0.8% Tween 80 and administered by gavage once daily for 2.5 months. Control and model animals received an equal volume of vehicle. After 2.5 months of treatment, behavior of all animals was evaluated in a Morris water maze. Expression of synaptophysin (SYP) and glutamatergic receptor 1 (G1uR1) in the hippocampus and cortex of the double transgenic mice was assayed by Western blotting. The antagonistic effects of β-asarone against amyloid-β peptide (Aβ) were investigated in vitro in the NG108-15 cell line. After 24 hours of incubation, cells were treated with 10 μm Aβ with or without β-asarone at different concentrations (6.25, 12.5, or 25 μM) for an additional 36 hours. The cytotoxicity of β-asarone was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay of cell viability, and cell morphology was evaluated by bright-field microscopy after 24 hours of treatment. The expression of SYP and GluR1 in cells was detected by Western blot assay in the hippocampus and brain cortex tissues of mice.Resultsβ-asarone at a high dose reduced escape latency and upregulated SYP and GluR1 expression at both medium and high doses. Cell morphology evaluation showed that β-asarone treatment did not result in obvious cell surface spots and cytoplasmic granularity. β-asarone had a dose-dependent effect on cell proliferation.Conclusionβ-asarone antagonized the Aβ neurotoxicity in vivo, improved the learning and memory ability of APP/PS1 mice, and increased the expression of SYP and GluR1 both in vivo and in vitro. Thus, β-asarone may be a potential drug for the treatment of Alzheimer’s disease.

Abstract A06: Novel NF-kB reporter murine model of spontaneous, metastatic breast cancer for spatiotemporal monitoring of local and systemic therapeutic response

Abstract Characterization of therapeutic response in primary and metastatic tumors is limited by endpoint studies and tissue collection. We have successfully developed a unique double-transgenic mouse model, which combines the polyoma virus middle T oncogene (PyVT), a model of human breast cancer, with a nuclear factor-kappaB (NF-kB) reporter model (NGL). PyVT mice spontaneously form primary mammary fat pad tumors, with 80% of mice developing metastatic lesions in the lungs. The reporter is luciferase, driven by NF-kB activation, to provide quantitative bioluminescent measurements of spatially-specific NF-kB activity. The resulting double transgenic (PyNGL) mice were monitored using in vivo imaging systems (IVIS) multiple times, before and after treatment, to quantitatively and spatially characterize NF-kB therapeutic response, both locally and systemically. High intensity focused ultrasound (HIFU) was guided by magnetic resonance (MR) thermometry for tumor-localized hyperthermia which was dosage-controlled by the thermal feedback MR guided HIFU (MRgHIFU) system. A single primary mammary tumor in each PyNGL was treated with sub-ablative hyperthermia of 42⁰ for up to 25 minutes. Pre-treatment IVIS measurements were collected to determine baseline NF-kB activity within each mouse, followed by IVIS imaging every twelve hours after treatment until tissue collection. Treated tumor, contralateral (untreated) tumors, and spleens were collected for immunophenotyping and cytokine analysis using flow cytometry. PyNGL mice exhibited decreased NF-kB activity, spatially correlated with HIFU treatment, as early as 12 hours. In contrast, NF-kB increased in contralateral tumors as early as 24 hours following treatment. Immunophenotyping reveals changes in tumor infiltrating leukocyte populations in HIFU-treated and contralateral tumors, including quantity and phenotype of T cells. The unique murine model developed allowed quantitative, temporal characterization of local and distant tumor response to MFgHIFU. The spontaneous, metastatic nature of PyVT tumor formation in concert with the non-destructive, spatiotemporal imaging capabilities of the NF-kB reporter transgene make the PyNGL model an unparalleled tool for research in metastatic disease. Citation Format: Mary Dockery, Megan Poorman, Stephanie Dudzinski, Whitney Barham, Vandiver Chaplin, Ryan Spears, Jiro Kusunose, Fiona Yull, Charles Caskey, William Grissom, Todd Giorgio. Novel NF-kB reporter murine model of spontaneous, metastatic breast cancer for spatiotemporal monitoring of local and systemic therapeutic response. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A06.

Valproic acid ameliorates olfactory dysfunction in APP/PS1 transgenic mice of Alzheimer's disease: Ameliorations from the olfactory epithelium to the olfactory bulb

Olfactory dysfunction is a common and early symptom of many neurodegenerative diseases, particularly of Alzheimer's disease (AD) and mild cognitive impairment, pointing to the progression to dementia. Recent studies have revealed that valproic acid (VPA) has neuroprotective effects in rodent models of AD. In this study, we investigated the effects of VPA on olfactory dysfunction of APP/PS1 double transgenic mouse models of AD. After continuous treatment with a 100mg/kg daily dose of VPA for 3months, APP/PS1 mice showed improved olfactory performances. In agreement with the behavioral findings, VPA treatment reduced amyloid β (Aβ) burden in the olfactory epithelium (OE) of transgenic mice. And, VPA increased epithelial thickness of the olfactory mucosa through decreased cell apoptosis and increased cell proliferation. In the olfactory bulb (OB), VPA administration also reduced senile plaques and levels of soluble and insoluble Aβ42 peptides. Besides, VPA promoted the increase of mitral cells and decrease of neurofilament immunostaining. In hence, VPA treatment completely improved the olfactory performances and prevented degenerative changes of the OE and OB. Our study raises the possibility of AD diagnosis by OE biopsy. Moreover, VPA may provide a novel therapeutic strategy for the treatment of olfactory dysfunction in AD patients.

A truncated phosphorylated p130Cas substrate domain is sufficient to drive breast cancer growth and metastasis formation in vivo.

Elevated p130Cas (Crk-associated substrate) levels are found in aggressive breast tumors and are associated with poor prognosis and resistance to standard therapeutics in patients. p130Cas signals majorly through its phosphorylated substrate domain (SD) that contains 15 tyrosine motifs (YxxP) which recruit effector molecules. Tyrosine phosphorylation of p130Cas is important for mediating migration, invasion, tumor promotion, and metastasis. We previously developed a Src*/SD fusion molecule approach, where the SD is constitutively phosphorylated. In a polyoma middle T-antigen (PyMT)/Src*/SD double-transgenic mouse model, Src*/SD accelerates PyMT-induced tumor growth and promotes a more aggressive phenotype. To test whether Src*/SD also drives metastasis and which of the YxxP motifs are involved in this process, full-length and truncated SD molecules fused to Src* were expressed in breast cancer cells. The functionality of the Src*/SD fragments was analyzed in vitro, and the active proteins were tested in vivo in an orthotopic mouse model. Breast cancer cells expressing the full-length SD and the functional smaller SD fragment (spanning SD motifs 6-10) were injected into the mammary fat pads of mice. The tumor progression was monitored by bioluminescence imaging and caliper measurements. Compared with control animals, the complete SD promoted primary tumor growth and an earlier onset of metastases. Importantly, both the complete and truncated SD significantly increased the occurrence of metastases to multiple organs. These studies provide strong evidence that the phosphorylated p130Cas SD motifs 6-10 (Y236, Y249, Y267, Y287, and Y306) are important for driving mammary carcinoma progression.

MicroRNA-26b is upregulated in a double transgenic mouse model of Alzheimer's disease and promotes the expression of amyloid-β by targeting insulin-like growth factor 1.

Alzheimer's disease (AD) is the most common form of dementia among the aging population. It is pathologically characterized by synaptic impairment, accumulation of neurofibrillary tangles and amyloid‑β (Aβ) deposition. MicroRNA‑26b (miR‑26b) has been observed to be upregulated in the human temporal cortex in AD, however, the function of miR‑26b has not been verified. Reverse transcription‑quantitative polymerase chain reaction was conducted to investigate the expression levels of miR‑26b in a double transgenic mouse model of AD. Following transfection of miR‑26b or an miR‑26b inhibitor, western blot analysis, enzyme‑linked immunosorbent assay and luciferase assays were performed. The present study demonstrated that the expression levels of miR‑26b were upregulated in a double transgenic mouse model of AD. It was also demonstrated that upregulation of miR‑26b in N2a/APP cells downregulated the insulin‑like growth factor 1 (IGF‑1) protein expression level and promoted Aβ production, whereas inhibition of miR‑26b in N2a/APP cells upregulated the IGF‑1 protein level and suppressed Aβ production. Furthermore, miR‑26b target sites in IGF‑1 were confirmed using a luciferase assay in HEK293 cells. The present study may be useful in the development of effective therapeutic strategies against AD.

Pharmacological removal of serum amyloid P component from intracerebral plaques and cerebrovascular Aβ amyloid deposits in vivo.

Human amyloid deposits always contain the normal plasma protein serum amyloid P component (SAP), owing to its avid but reversible binding to all amyloid fibrils, including the amyloid β (Aβ) fibrils in the cerebral parenchyma plaques and cerebrovascular amyloid deposits of Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA). SAP promotes amyloid fibril formation in vitro, contributes to persistence of amyloid in vivo and is also itself directly toxic to cerebral neurons. We therefore developed (R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC), a drug that removes SAP from the blood, and thereby also from the cerebrospinal fluid (CSF), in patients with AD. Here we report that, after introduction of transgenic human SAP expression in the TASTPM double transgenic mouse model of AD, all the amyloid deposits contained human SAP. Depletion of circulating human SAP by CPHPC administration in these mice removed all detectable human SAP from both the intracerebral and cerebrovascular amyloid. The demonstration that removal of SAP from the blood and CSF also removes it from these amyloid deposits crucially validates the strategy of the forthcoming ‘Depletion of serum amyloid P component in Alzheimer's disease (DESPIAD)’ clinical trial of CPHPC. The results also strongly support clinical testing of CPHPC in patients with CAA.

Molecular links between early energy metabolism alterations and Alzheimer's disease.

Recent studies suggest that the neurobiology of Alzheimer's disease (AD) pathology could not be explained solely by an increase in beta-amyloid levels. In fact, success with potential therapeutic drugs that inhibit the generation of beta amyloid has been low. Therefore, due to therapeutic failure in recent years, the scientists are looking for alternative hypotheses to explain the causes of the disease and the cognitive loss. Accordingly, alternative hypothesis propose a link between AD and peripheral metabolic alteration. Then, we review in depth changes related to insulin signalling and energy metabolism in the context of the APPSwe/PS1dE9 (APP/PS1) mice model of AD. We show an integrated view of the changes that occur in the early stages of the amyloidogenic process in the APP/PS1 double transgenic mice model. These early changes affect several key metabolic processes related to glucose uptake and insulin signalling, cellular energy homeostasis, mitochondrial biogenesis and increased Tau phosphorylation by kinase molecules like mTOR and Cdk5.

Effects of triptolide on hippocampal microglial cells and astrocytes in the APP/PS1 double transgenic mouse model of Alzheimer's disease.

The principal pathology of Alzheimer's disease includes neuronal extracellular deposition of amyloid-beta peptides and formation of senile plaques, which in turn induce neuroinflammation in the brain. Triptolide, a natural extract from the vine-like herb Tripterygium wilfordii Hook F, has potent anti-inflammatory and immunosuppressive efficacy. Therefore, we determined if triptolide can inhibit activation and proliferation of microglial cells and astrocytes in the APP/PS1 double transgenic mouse model of Alzheimer's disease. We used 1 or 5 μg/kg/d triptolide to treat APP/PS1 double transgenic mice (aged 4-4.5 months) for 45 days. Unbiased stereology analysis found that triptolide dose-dependently reduced the total number of microglial cells, and transformed microglial cells into the resting state. Further, triptolide (5 μg/kg/d) also reduced the total number of hippocampal astrocytes. Our in vivo test results indicate that triptolide suppresses activation and proliferation of microglial cells and astrocytes in the hippocampus of APP/PS1 double transgenic mice with Alzheimer's disease.

Generation of a double transgenic humanized neonatal Fc receptor (FcRn)/albumin mouse to study the pharmacokinetics of albumin-linked drugs

Human serum albumin (HSA) is a natural carrier protein possessing multiple ligand binding sites with a plasma half-life ~19days, facilitated by interaction with the human neonatal Fc receptor (FcRn), that promotes it as a highly attractive drug delivery technology. A lack of adequate rodent models, however, is a major challenge in the preclinical development of albumin-linked therapeutics. This work describes the first double transgenic mouse model bearing both human FcRn and HSA genes (hFcRn+/+, hAlb+/+) under the control of an endogenous promoter. Human FcRn was shown by immunohistochemical and qPCR analysis to be ubiquitously expressed in the major organs. Physiological levels of HSA were detected in the blood that exhibited similar FcRn binding kinetics to recombinant or human serum-derived HSA. The circulatory half-life (t1/2) was shown to be dependent on FcRn binding affinity that increased from low affinity (t1/2 29h), to wild type (t1/2 50h), to high affinity (t1/2 80h) variants, that validates the application of the model for optimizing the pharmacokinetics of drug carriers who's circulatory half-life is dependent in some manner upon interaction with endogenous FcRn. This study presents a novel mouse model that better mimics the human physiological conditions and, thus, has potential wide applications in the development of albumin-linked drugs or conventional drugs whose action is influenced by reversible binding to endogenous HSA.

Model-Based Tumor Growth Dynamics and Therapy Response in a Mouse Model of De Novo Carcinogenesis.

Tumorigenesis is a complex, multistep process that depends on numerous alterations within the cell and contribution from the surrounding stroma. The ability to model macroscopic tumor evolution with high fidelity may contribute to better predictive tools for designing tumor therapy in the clinic. However, attempts to model tumor growth have mainly been developed and validated using data from xenograft mouse models, which fail to capture important aspects of tumorigenesis including tumor-initiating events and interactions with the immune system. In the present study, we investigate tumor growth and therapy dynamics in a mouse model of de novo carcinogenesis that closely recapitulates tumor initiation, progression and maintenance in vivo. We show that the rate of tumor growth and the effects of therapy are highly variable and mouse specific using a Gompertz model to describe tumor growth and a two-compartment pharmacokinetic/ pharmacodynamic model to describe the effects of therapy in mice treated with 5-FU. We show that inter-mouse growth variability is considerably larger than intra-mouse variability and that there is a correlation between tumor growth and drug kill rates. Our results show that in vivo tumor growth and regression in a double transgenic mouse model are highly variable both within and between subjects and that mathematical models can be used to capture the overall characteristics of this variability. In order for these models to become useful tools in the design of optimal therapy strategies and ultimately in clinical practice, a subject-specific modelling strategy is necessary, rather than approaches that are based on the average behavior of a given subject population which could provide erroneous results.

Ganglioside-Dependent Neural Stem Cell Proliferation in Alzheimer's Disease Model Mice.

The aggregation and formation of amyloid plaques by amyloid β-peptides (Aβs) is believed to be one of the pathological hallmarks of Alzheimer’s disease (AD). Intriguingly, Aβs have also been shown to possess proliferative effects on neural stem cells (NSCs). Many essential cellular processes in NSCs, such as fate determination and proliferation, are heavily influenced by cell surface glycoconjugates, including gangliosides. It has recently been shown that Aβ1-42 alters several key glycosyltransferases and glycosidases. To further define the effects of Aβs and to clarify the potential mechanisms of action of those peptides on NSCs, NSCs were cultured from embryonic brains of the double-transgenic mouse model of AD [B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J] coexpressing mutants of amyloid precursor protein (APPswe) and presenilin1 (PSEN1dE9). We found that Aβs not only promoted cell proliferation but also altered expression of several key glycogenes for glycoconjugate metabolism, such as sialyltransferases II and III (ST-II & -III) in AD NSCs. In addition, we found upregulation of epidermal growth factor receptor and Notch1 intracellular domain. Moreover, the increased expression of ST-II and -III coincided with the elevated levels of c-series gangliosides (A2B5+ antigens) in AD NSCs. Further, we revealed that epidermal growth factor signaling and gangliosides are necessary components on Aβ-stimulated NSC proliferation. Our present study has thus provided a novel mechanism for the upregulation of c-series ganglioside expression and increases in several NSC markers to account for the proliferative effect of Aβs on NSCs in AD mouse brain. These observations support the potential beneficial effects of Aβs and gangliosides in promoting neurogenesis in AD brain.

Protective Effects of Dietary Supplementation with a Combination of Nutrients in a Transgenic Mouse Model of Alzheimer's Disease.

ObjectiveThis study investigated the effects of intervention with a combination of nutrients in the amyloid precursor protein-presenilin (APP-PSN) C57BL/6J double transgenic mouse model of Alzheimer’s disease (AD).MethodsA total of 72 2-month-old APP-PSN mice were randomly assigned to three groups. The model group (MG) was fed regular, unsupplemented chow, while the low- and high-dose treatment groups (LG and HG, respectively) were given a combination of nutrients that included phosphatidylserine, blueberry extracts, docosahexaenoic acid, and eicosapentaenoic acid as part of their diet. An additional 24 wild-type littermates that were fed unsupplemented chow served as the negative control group (NG). After 3 and 7 months of treatment, the cognitive performance was assessed with the Morris water maze and the shuttle box escape/avoidance task, and the biochemical parameters and oxidative stress were evaluated in both the blood and brain.ResultsAn improvement in antioxidant capacity was observed in the treatment groups relative to the MG at 3 months, while superior behavioral test results were observed in the mice of the HG and NG groups. In the MG, pycnosis was detected in neuronal nuclei, and a loss of neurons was observed in the cerebral cortex and the hippocampus. At 7 months, the β-amyloid1–42 peptide accumulation was significantly elevated in the MG but was markedly lower in the mice fed the nutrient combination. The antioxidant capacity and behavioral test scores were also higher in these mice.ConclusionsEarly intervention with a combination of nutrients should be considered as a strategy for preventing cognitive decline and other symptoms associated with AD.

Magnetic resonance T2 relaxation time at 7 Tesla associated with amyloid β pathology and age in a double-transgenic mouse model of Alzheimer’s disease

The aim of this study was to better understand the effect of amyloid-β plaques on magnetic resonance T2 relaxation time. We investigated these changes associated with age in an APP/PS1 mouse model of AD at 7 Tesla, combined with histology. Ten double-transgenic AD and ten wild type (WT) female mice (aged 12–20 months) were used in a cross-sectional study. Mean T2 values and standard deviations were calculated for each Regions of interest (ROIs) on T2 map. Immunohistochemistry for amyloid plaques and fluorescence staining with thioflavine S were performed of brain sections after imaging. The results showed that mean T2 values of the hippocampus, cortex, corpus callosum, and thalamus of older mice were significantly lower than of the younger. Compared to WT mice, the T2 values of the hippocampus, corpus callosum, and thalamus in younger AD mice were significantly greater, while the T2 values of the hippocampus and cortex in older AD mice were significantly less. Aβ-40 immunohistochemistry and thioflavine S stainging were positive in the matched region both for younger and older AD mice, while neither Aβ-40 nor thioflavine S were observed in WT mice. These findings suggest that regional T2 values of AD mice may decrease with age, and changes in T2 values in AD mice may be influenced by many factors besides amyloid-β plaque accumulation. Furthermore, they support that the standard deviation of the mean T2 value should be considered as well as the mean.

Downregulated microRNA-222 is correlated with increased p27Kip¹ expression in a double transgenic mouse model of Alzheimer's disease.

MicroRNAs (miRNAs) are a group of endogenous non-coding small RNAs that regulate protein expression by binding to the 3' untranslated region (UTR) of target genes. miRNAs are abundantly expressed in the central nervous system and participate in neuronal differentiation and synaptic plasticity. However, the possible roles and associated target genes of miRNAs in Alzheimer's disease (AD) are largely unknown. In the current study, miR‑222 was observed to be downregulated in APPswe/PSΔE9 mice (a model for AD) compared with age‑matched controls. Furthermore, the downregulation of miR‑222 was correlated with increased p27Kip1 protein levels. Bioinformatic analysis showed that there was one highly‑conserved putative binding site for miR‑222 in the 3'‑UTR of p27Kip1. Luciferase reporter assays confirmed that p27Kip1 was a direct target of miR‑222. Consistently, there was an inverse correlation between p27Kip1 and miR‑222 expression levels in SH‑SY5Y cells. In conclusion, these results suggest that the abnormal expression of miR‑222 may contribute to dysregulation of the cell‑cycle in AD, at least in part by affecting the expression of p27Kip1.

Abstract P071: Mineralocorticoid Receptor Overexpression in Adipose Tissue Leads to Metabolic Syndrome and Induction of Prostaglandin D2 Synthase PTGDS and lipocalin-2 NGAL

Objectives: Metabolic Syndrome (MetS) is a cluster of metabolic risk factors associated with a higher risk for cardiovascular events. Adipose tissue (AT) plays a central role in the obesity-related metabolic abnormalities and it’s known that Mineralocorticoid Receptor (MR) activation affects adipocyte differentiation and function. The purposes of this study were to evaluate the molecular and metabolic consequences of adipocyte-targeted increase in MR expression in mice. Results: In our study we showed that MR expression is increased in visceral adipose tissue (VAT) in a preclinical mouse model of MetS. Thus, we generated a double transgenic mouse model with a conditional and inducible overexpression of MR in AT (Adipo-MORE), demonstrating that increased expression of MR in AT contributes to MetS development with multiple metabolic abnormalities, including visceral obesity (VAT mass, control-MR 5.0±0.6 adipo-MORE 10.4±1.8, p<0.05), insulin resistance (HOMA index, control-MR 8.9±0.8 adipo-MORE 14.5±2.2, p<0.05) as well as dyslipidemia (total cholesterol, mg/dL control-MR 86.0±6.0 adipo-MR 109±6, p<0.05). We also identified prostaglandin D2 synthase (PTGDS) as a novel mediator of adipogenic effects of MR activation in adipocytes. Since adipokines play pivotal roles in the regulation of insulin sensitivity in obesity, we also focused on the role of lipocalin-2 NGAL, recently identified in our laboratory as a target of aldosterone action in cardiovascular system, as new adipocyte MR target. It’s known that NGALKO mice fed a high fat diet are protected from obesity-induced cardiovascular dysfunctions. In our model we observed an increase of NGAL mRNA levels (3 fold increase, p<0.05) in VAT. Moreover, NGAL was induced (2-8 fold increase, p<0.05) in primary cultures of adipocytes differentiated ex vivo from adipo-MORE mice in presence of aldosterone stimulation compared to controls. Conclusions: We demonstrated that PTGDS is a novel direct MR target in rodent adipocytes mediating adipogenic effects of MR activation. We also found that NGAL is induced in presence of MR overexpression in adipocytes in vivo and ex vivo. In consideration of our results, other analyses are necessary to better understand the role of NGAL as adipocyte MR target.