Prodromic Inflammatory-Oxidative Stress in Peritoneal Leukocytes of Triple-Transgenic Mice for Alzheimer's Disease.

Inflammatory-oxidative stress is known to be pivotal in the pathobiology of Alzheimer's disease (AD), but the involvement of this stress at the peripheral level in the disease's onset has been scarcely studied. This study investigated the pro-inflammatory profile and oxidative stress parameters in peritoneal leukocytes from female triple-transgenic mice for AD (3xTgAD) and non-transgenic mice (NTg). Peritoneal leukocytes were obtained at 2, 4, 6, 12, and 15 months of age. The concentrations of TNFα, INFγ, IL-1ß, IL-2, IL-6, IL-17, and IL-10 released in cultures without stimuli and mitogen concanavalin A and lipopolysaccharide presence were measured. The concentrations of reduced glutathione (GSH), oxidized glutathione (GSSG), lipid peroxidation, and Hsp70 were also analyzed in the peritoneal cells. Our results showed that although there was a lower release of pro-inflammatory cytokines by 3xTgAD mice, this response was uncontrolled and overstimulated, especially at a prodromal stage at 2 months of age. In addition, there were lower concentrations of GSH in leukocytes from 3xTgAD and higher amounts of lipid peroxides at 2 and 4 months, as well as, at 6 months, a lower concentration of Hsp70. In conclusion, 3xTgAD mice show a worse pro-inflammatory response and higher oxidative stress than NTg mice during the prodromal stages, potentially supporting the idea that Alzheimer's disease could be a consequence of peripheral alteration in the leukocyte inflammation-oxidation state.

Alterations of Spatial Memory and Gut Microbiota Composition in Alzheimer's Disease Triple-Transgenic Mice at 3, 6, and 9 Months of Age.

Alzheimer's disease (AD) is a progressive neurological disease. Gut microbial dysbiosis is associated with AD. This study involves the comparative assessment of spatial learning, ß-amyloid peptide accumulation, and fecal microbiota alterations in 3×Tg-AD mice from 3 age groups: AD asymptomatic stage (3 m), presymptomatic stage (6 m), and the symptomatic stage of AD (9 m). We demonstrate that spatial memory deficits, brain Aß accumulation, and weight gain in 3×Tg-AD mice gradually appear after 6 months of age. However, the total gut bacterial counts underwent changes from 3 to 6 months of age and were further altered at 9 months of age. Importantly, changes in gut bacteria abundance of Desulfobacterota and Actinobacteriota phyla in 6-month-old mice preceded apparent spatial memory deficits. In summary, Changes in the gut microbial community are one of the mechanisms of early AD pathology.

Alterations of gut microbiota composition and cogni-tive behavior in APP/PS1/tau triple-transgenic mouse model of Alzheimer's disease / 中国药理学与毒理学杂志

OBJECTIVE Alzheimer's disease(AD)is a progressive neurological disease.Given the important role of gut microbiota composition in AD pathology,the observed perturbation in the microbiota composition and diversity may serve as the mechanisms underlying age-dependent APP/PS1/tau triple-transgenic mouse(3×Tg-AD)mice amyloid deposition and memory deficits.Here-in,we intended to investigate the gut microbiota and as-sessed its relationship with the triggering and develop-ment of cognitive impairment of AD.METHODS This study involves the comparative assessment of spatial learning,amyloid β-protein(Aβ)accumulation,and fecal microbiota alterations in 3×Tg-AD mice from three age groups:AD asymptomatic stage(3 m),presymptomatic stage(6 m),and the symptomatic stage of AD(9 m).RE-SULTS We demonstrate that spatial memory deficits,brain Aβ accumulation,and weight gain in 3×Tg-AD mice gradually appear after 6 months of age.However,the total gut bacterial counts underwent changes from 3 to 6 months of age and were further altered at 9 months of age.Importantly,changes in gut bacteria abundance of Desulfobacterota and Actinobacteriota phylain 6-month-old mice preceded apparent spatial memory deficits.CONCLUSION Changes in the gut microbial community are one of the mechanisms of early AD pathology.

Age Related Changes in Muscle Mass and Force Generation in the Triple Transgenic (3xTgAD) Mouse Model of Alzheimer's Disease.

Emerging evidence suggests that patients with Alzheimer's disease (AD) may show accelerated sarcopenia phenotypes. To investigate whether pathological changes associated with neuronal death and cognitive dysfunction also occur in peripheral motor neurons and muscle as a function of age, we used the triple transgenic mouse model of AD (3xTgAD mice) that carries transgenes for mutant forms of APP, Tau, and presenilin proteins that are associated with AD pathology. We measured changes in motor neurons and skeletal muscle function and metabolism in young (2 to 4 month) female control and 3xTgAD mice and in older (18-20 month) control and 3xTgAD female mice. In older 3xTgAD mice, we observed a number of sarcopenia-related phenotypes, including significantly fragmented and denervated neuromuscular junctions (NMJs) associated with a 17% reduction in sciatic nerve induced vs. direct muscle stimulation induced contractile force production, and a 30% decrease in gastrocnemius muscle mass. On the contrary, none of these outcomes were found in young 3xTgAD mice. We also measured an accumulation of amyloid-ß (Aß) in both skeletal muscle and neuronal tissue in old 3xTgAD mice that may potentially contribute to muscle atrophy and NMJ disruption in the older 3xTgAD mice. Furthermore, the TGF-ß mediated atrophy signaling pathway is activated in old 3xTgAD mice and is a potential contributing factor in the muscle atrophy that occurs in this group. Perhaps surprisingly, mitochondrial oxygen consumption and reactive oxygen species (ROS) production are not elevated in skeletal muscle from old 3xTgAD mice. Together, these results provide new insights into the effect of AD pathological mechanisms on peripheral changes in skeletal muscle.

The effect of exercise on early sensorimotor performance alterations in the 3xTg-AD model of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by a progressive decline in cognitive function; however, recent evidence suggests that non-cognitive sensorimotor and psychomotor symptoms accompany early stages of the disease in humans and AD models. Although exercise is emerging as an important therapeutic to combat AD progression, little is known about the effect of exercise on sensorimotor domain functions. The purpose of this study was to determine if early sensorimotor symptoms accompany deficits in Morris water maze (MWM) performance in the 3xTg-AD model, and investigate if exercise could protect against early behavioral decline. 3xTg-AD and wild-type (WT) control mice were subjected to 12 weeks of moderate intensity wheel running or remained sedentary. At 6 months of age, animals underwent a series of sensorimotor and MWM testing. 3xTg-AD mice displayed deficits in sensorimotor function (beam traversal, spontaneous activity, and adhesive removal) and MWM performance. Interestingly, 3xTg-AD animals exhibited increased freezing and unusual shaking/tremoring behaviors not displayed by WT controls. Exercise improved beam traversal, adhesive removal, and reduced the unusual motor-related behaviors in 3xTg-AD mice. Our study shows that sensorimotor symptoms coincide with deficits in MWM performance, and suggest that exercise may mitigate deficits associated with early disease in 3xTg-AD mice.

Agerelated changes in Alzheimer’s disease-like pathology in male APP/PS1/Tau triple transgenic mice / 中风与神经疾病杂志

@#Objective Immunohistochemical methods were used to observe the age-related changes of Alzheimer’s disease-related pathology in the hippocampus and sensory cortex in male APP/PS1/Tau triple transgenic mice aged 2-15 months.Methods Male 3xTg-AD mice were randomly divided into 7 groups according to the age of 2，4，6，8，10，12 and 15 months，with 3 mice in each group as the experimental group；age-matched male C5B7L/6J mice Randomly divided into 7 groups according to the age of 2，4，6，8，10，12 and 15 months，with 3 mice in each group as a control group. The immunohistochemical method was used to detect the pathological age-related changes in the hippocampus and sensory cortex of each mouse brain tissue with age，closely related to Alzheimer’s disease，including Aβ，phosphorylated tau，transgenic products of human tau P301L mutant，astrogliosis，neuron markers.Results Compared with C5B7L/6J mice，the intensity of immunostaining of Aβ cells（6E10），p-tau（AT8，Ser202/Thr205） and tau（HT7） astrocytes（GFAP） in APP/PS1/Tau triple transgenic mice is significantly enhanced，P<0.0001.The hippocampus and sensory cortex of male APP/PS1/Tau triple transgenic mice aged 2-15 months did not have obvious amyloid plaque deposition. The staining degree of Aβ cells in the CA1 area of the hippocampus gradually increased at the age of 8 months，and the staining degree of the sensory cortex remained unchanged，P<0.05. The staining degree of Tau (HT7) in the CA1 area has weak staining at 2 to 6 months of ageand the staining degree remained stable from 6 months to 15 months；The degree of staining in the sensory cortex increased from 2 to 4 months old，and the degree of staining remained stable at 4 to 15 months，P<0.05.The phosphorylation of AT8 in CA1 area began to increase at 6 months，and the phosphorylation of sensory cortex began to increase at 12 months，P<0.05.Astrocytes（GFAP） in CA1 area and sensory cortex area gradually increase in reactivity from 2 months to 15 months. The number of mature neuron markers（NEUN） in CA1 area gradually decreased from 2 months to 6 months，and was stable from 6 months to 15 months. The number of NEUN in CA-3 area gradually decreased from 2 months to 15 months，P<0.05.Conclusion 3xTg-AD mice show a clear interaction between age and phenotype development，which makes it an important tool for studying the role of aging in disease pathogenesis.

Combined Effect of Fatty Diet and Cognitive Decline on Brain Metabolism, Food Intake, Body Weight, and Counteraction by Intranasal Insulin Therapy in 3×Tg Mice.

Obesity and cognitive decline can occur in association. Brain dysmetabolism and insulin resistance might be common underlying traits. We aimed to examine the effect of high-fat diet (HFD) on cognitive decline, and of cognitive impairment on food intake and body-weight, and explore efficacy of chronic intranasal insulin (INI) therapy. We used control (C) and triple transgenic mice (3×Tg, a model of Alzheimer's pathology) to measure cerebral mass, glucose metabolism, and the metabolic response to acute INI administration (cerebral insulin sensitivity). Y-Maze, positron emission-computed tomography, and histology were employed in 8 and 14-month-old mice, receiving normal diet (ND) or HFD. Chronic INI therapy was tested in an additional 3×Tg-HFD group. The 3×Tg groups overate, and had lower body-weight, but similar BMI, than diet-matched controls. Cognitive decline was progressive from HFD to 3×Tg-ND to 3×Tg-HFD. At 8 months, brain fasting glucose uptake (GU) was increased by C-HFD, and this effect was blunted in 3×Tg-HFD mice, also showing brain insulin resistance. Brain mass was reduced in 3×Tg mice at 14 months. Dentate gyrus dimensions paralleled cognitive findings. Chronic INI preserved cognition, dentate gyrus and metabolism, reducing food intake, and body weight in 3×Tg-HFD mice. Peripherally, leptin was suppressed and PAI-1 elevated in 3×Tg mice, correlating inversely with cerebral GU. In conclusion, 3×Tg background and HFD exert additive (genes*lifestyle) detriment to the brain, and cognitive dysfunction is accompanied by increased food intake in 3×Tg mice. PAI-1 levels and leptin deficiency were identified as potential peripheral contributors. Chronic INI improved peripheral and central outcomes.

Redox Proteomic Profiling of Specifically Carbonylated Proteins in the Serum of Triple Transgenic Alzheimer's Disease Mice.

Oxidative stress is a key event in the onset and progression of neurodegenerative diseases, including Alzheimer's disease (AD). To investigate the role of oxidative stress in AD and to search for potential biomarkers in peripheral blood, serums were collected in this study from the 3-, 6-, and 12-month-old triple transgenic AD mice (3×Tg-AD mice) and the age- and sex-matched non-transgenic (non-Tg) littermates. The serum oxidized proteins were quantified by slot-blot analysis and enzyme-linked immunosorbent assay (ELISA) to investigate the total levels of serum protein carbonyl groups. Western blotting, in conjunction with two-dimensional gel electrophoresis (2D-Oxyblot), was employed to identify and quantify the specifically-carbonylated proteins in the serum of 3×Tg-AD mice. The results showed that the levels of serum protein carbonyls were increased in the three month old 3×Tg-AD mice compared with the non-Tg control mice, whereas no significant differences were observed in the six and 12 months old AD mice, suggesting that oxidative stress is an early event in AD progression. With the application of 2D-Oxyblot analysis, (immunoglobin) Ig gamma-2B chain C region (IGH-3), Ig lambda-2 chain C region (IGLC2), Ig kappa chain C region (IGKC), and Ig kappa chain V-V region HP R16.7 were identified as significantly oxidized proteins compared with the control. Among them IGH-3 and IGKC were validated via immunoprecipitation and Western blot analysis. Identification of oxidized proteins in the serums of 3×Tg-AD mice can not only reveal potential roles of those proteins in the pathogenesis of AD but also provide potential biomarkers of AD at the early stage.

Liver X Receptor Agonist Modifies the DNA Methylation Profile of Synapse and Neurogenesis-Related Genes in the Triple Transgenic Mouse Model of Alzheimer's Disease.

The liver X receptor agonist, GW3965, improves cognition in Alzheimer's disease (AD) mouse models. Here, we determined if short-term GW3965 treatment induces changes in the DNA methylation state of the hippocampus, which are associated with cognitive improvement. Twenty-four-month-old triple-transgenic AD (3xTg-AD) mice were treated with GW3965 (50 mg/kg/day for 6 days). DNA methylation state was examined by modified bisulfite conversion and hybridization on Illumina Infinium Methylation BeadChip 450 k arrays. The Morris water maze was used for behavioral analysis. Our results show in addition to improvement in cognition methylation changes in 39 of 13,715 interrogated probes in treated 3xTg-AD mice compared with untreated 3xTg-AD mice. These changes in methylation probes include 29 gene loci. Importantly, changes in methylation status were mainly from synapse-related genes (SYP, SYN1, and DLG3) and neurogenesis-associated genes (HMGB3 and RBBP7). Thus, our results indicate that liver X receptors (LXR) agonist treatment induces rapid changes in DNA methylation, particularly in loci associated with genes involved in neurogenesis and synaptic function. Our results suggest a new potential mechanism to explain the beneficial effect of GW3965.

Redox proteomics identification of specifically carbonylated proteins in the hippocampi of triple transgenic Alzheimer's disease mice at its earliest pathological stage.

Alzheimer's disease (AD) is the most common cause of dementia in the elderly population. Attempts to develop therapies for the treatment of the late stage AD have been unsuccessful. Increasing evidences indicate that oxidative stress is an early event of neurodegeneration, however the pathogenic mechanism of AD remains unclarified. In the present study, slot-blot analysis was used to determine the levels of protein carbonyls in the hippocampi of 3-month-old triple transgenic AD mice (3 × Tg-AD). The increased levels of protein carbonyls were observed in the hippocampi of 3 × Tg-AD mice as compared to the non-transgenic controls (non-Tg). Using a redox-proteomic approach, twelve proteins were found to be significantly altered in the levels of protein carbonyls in the hippocampus. These proteins are crucial in energy metabolism, protein folding, cell structure, signal transduction and excitotoxicity. Immunoprecipitation and Western blot were used to validate two proteins identified by the redox proteomics. In addition, increased expression level of carbonyl reductase 1 (CBR1) was observed in the hippocampi of 3 × Tg-AD mice. These results demonstrate that significant protein carbonylation occurs early in the 3-month-old 3 × Tg-AD mice, which support the viewpoint that oxidative stress is an early event in AD progression. BIOLOGICAL SIGNIFICANCE: In this study, we have observed increased levels of protein carbonyls in the hippocampi of 3 × Tg-AD mice before the appearance of Aß plaques and neurofibrillary tangles (NFTs). By redox proteomics, twelve specifically carbonylated proteins were identified. Among them, alpha-enolase (ENO1) and glutamine synthetase (GS) were identified as the common targets of oxidation in the brains of 3 × Tg-AD mice, mild cognitive impairment (MCI) sufferers and AD patients. For the first time, the oxidation of t-complex protein 1 subunit epsilon (CCT5) and protein disulfide-isomerase A3 (PDIA3) were reported to be associated with AD. These results indicated that the combination of monoclonal anti-DNP antibody with digital imaging system could enhance the specificity and accuracy of redox proteomics analysis. Those data support the viewpoint that oxidative stress occurs at the early pathological stage of AD. In addition, this paper provides new information for understanding the pathological process of AD and for developing more appropriate therapies to intervene AD progression.

Flow cytometric analysis of the graft-versus-Leukemia-effect after hematopoietic stem cell transplantation in mice.

Acute Graft-versus-Host-Disease (aGvHD) is one of the major complications following allogeneic hematopoietic stem cell transplantation (HSCT). Although rather helpful, the use of conventional immunosuppressive drugs leads to general immunosuppression and is toxic. The effects of CD4(+) T-cells, in respect to the development of aGvHD, can be altered by administration of antihuman CD4 monoclonal antibodies, here MAX.16H5 IgG1 . This approach must be tested for possible interference with the Graft-versus-Leukemia-Effect (GvL). Thus, in vitro experiments were conducted, exposing P815 leukemic cells to bone marrow and splenocytes from cd4(-/-) -C57Bl/6 mice transgenic for human CD4 and HLA-DR3 (triple transgenic mice, [TTG]) as well as previously irradiated splenocytes from Balb/c(wt) mice. Using flow cytometry, the vitality of the various malignant and graft cells was analyzed over the course of 4 days. The survival rate of P815 cells did not change significantly when exposed to MAX.16H5 IgG1 , neither did the viability of the graft cells. This provides evidence that MAX.16H5 IgG1 does not impair the GvL effect in vitro. Additionally, P815-Balb/c(wt) leukemic mice were transplanted with P815(GFP) cells, bone marrow, and splenocytes from TTG mice with and without MAX.16H5 IgG1 . Without transplantation, P815(GFP) leukemic cells could be detected by flow cytometry in the liver, the bone marrow, and the spleen of recipients. The antibodies prevented aGvHD while leaving the GvL effect intact. These findings indicate no negative effect of MAX.16H5 IgG1 on the GvL effect in vitro and in vivo after HSCT in a murine model.

Melanocortins protect against progression of Alzheimer's disease in triple-transgenic mice by targeting multiple pathophysiological pathways.

Besides specific triggering causes, Alzheimer's disease (AD) involves pathophysiological pathways that are common to acute and chronic neurodegenerative disorders. Melanocortins induce neuroprotection in experimental acute neurodegenerative conditions, and low melanocortin levels have been found in occasional studies performed in AD-type dementia patients. Here we investigated the possible neuroprotective role of melanocortins in a chronic neurodegenerative disorder, AD, by using 12-week-old (at the start of the study) triple-transgenic (3xTg-AD) mice harboring human transgenes APPSwe, PS1M146V, and tauP301L. Treatment of 3xTg-AD mice, once daily until the end of the study (30 weeks of age), with the melanocortin analog [Nle(4),D-Phe(7)]-α-melanocyte-stimulating hormone (NDP-α-MSH) reduced cerebral cortex/hippocampus phosphorylation/level of all AD-related biomarkers investigated (mediators of amyloid/tau cascade, oxidative/nitrosative stress, inflammation, apoptosis), decreased neuronal loss, induced over-expression of the synaptic activity-dependent gene Zif268, and improved cognitive functions, relative to saline-treated 3xTg-AD mice. Pharmacological blockade of melanocortin MC4 receptors prevented all neuroprotective effects of NDP-α-MSH. Our study identifies, for the first time, a class of drugs, MC4 receptor-stimulating melanocortins, that are able to counteract the progression of experimental AD by targeting pathophysiological mechanisms up- and down-stream of ß-amyloid and tau. These data could have important clinical implications.

Neural stem cell transplantation improves spatial learning and memory via neuronal regeneration in amyloid-ß precursor protein/presenilin 1/tau triple transgenic mice.

Neural stem cell (NSC) transplantation has recently become a main research target for Alzheimer's disease (AD) treatment. In the present study, we transplanted NSCs from C57BL/6 mice into the hippocampus in the 12-month-old triple transgenic model of AD (3 × Tg) and determined whether NSC transplantation can alleviate impairments in spatial learning and memory via neuronal regeneration in AD mice. Two months after transplantation, Morris water maze tests suggested that spatial learning and memory in the 3 × Tg mice receiving NSCs was significantly improved compared to 3 × Tg mice not receiving NSCs. Furthermore, quantification of Nissl staining revealed that the number of neurons in the hippocampus of 3 × Tg mice receiving NSCs was significantly greater than that in 3 × Tg mice not receiving NSCs, indicating that new neurons were generated. These results may demonstrate that NSC transplantation can improve spatial learning and memory via neuronal regeneration in amyloid-ß precursor protein/presenilin 1/tau 3 × Tg mice.

[Premature immunosenescence in triple-transgenic mice for Alzheimer's disease]. / Inmunosenescencia prematura en ratones triple-transgénicos para la enfermedad del Alzheimer.

INTRODUCTION: A deterioration of the neuroimmunoendocrine network has been observed in Alzheimer's disease (AD). However, the peripheral immune response has hardly been investigated in this pathology. Since some immune function parameters have been established as good markers of the rate of ageing, and can predict longevity, the aim of the present work was to study some of these functions in splenic leucocytes in transgenic mice for AD of different ages. MATERIAL AND METHODS: Young female (4 ± 1 months), adult (9 ± 1 months), and mature (12 ± 1 months) triple-transgenic mice for AD (3 xTgAD) and non-transgenic (NTg) control mice of the same ages were used. The chemotaxis, the anti-tumour activity of « natural killer ¼ (NK) cells and the lymphoproliferative response in the presence of the mitogens concanavalin A and lipopolysaccharide, functions that decrease with age, were determined in splenic leucocytes. In addition, the differences in lifespan between 3 xTgAD and NTg were studied in parallel using other animals, until their death through natural causes. RESULTS: In 3 xTgAD, with respect to NTg, chemotaxis decreased at all ages studied, whereas in lymphoproliferative response this reduction was shown at 4 months and 9 months. NK activity was diminished only in young 3 xTgAD with respect to NTg. The 3 xTgAD showed a shorter lifespan than the NTg control group. CONCLUSIONS: The 3 xTgAD mice show a premature immunosenescence, which could explain their early mortality. The determination of these immune functions at peripheral level could serve as a marker of the progression of the Alzheimer's disease.