Today, Alzheimer's disease (AD) is one of the most devastating neurodegenerative diseases worldwide. Pathologically increased β-amyloid (Aβ) in the brain of AD patients is thought to be one of the main causes for the observed progressive cognitive decline in affected people. The development of new drugs against AD is therefore a main research focus. To be able to test these new drugs, appropriate animal models are needed. The 5xFAD transgenic mouse model mimics the most crucial phenotypic pathologies of amyloidogenic neurodegeneration and is therefore among the best transgenic AD animals available. 5xFAD mice bear 5 mutations, 3 in the APP695 gene as well as 2 mutations in the presenilin 1 gene. The expression of the 5xFAD transgene is driven by the neuron-specific Thy1 promoter. We analyzed the soluble and insoluble fraction of whole brain lysate from 5xFAD mice over age for aggregated Aβ by A4 assay and for Aβ with MSD. Furthermore Aβ aggregates as well as neuroinflammation, as indicated by astrogliosis and activated microglia, were evaluated by immunofluorescent labeling followed by quantification. Our results show an early and progressive increase of Aβ 40 and 42 aggregates as well as neuroinflammation in the cortex and hippocampus of 3 to 9 months old 5xFAD mice. These results suggest that 5xFAD mice are not only a well-suited model for Aβ research but also to analyze AD-related neuroinflammation. Additionally these data give insight into the progression of the most prominent disease hallmarks in 5xFAD mice, providing a good basis for planning possible efficacy studies in those animals.
Alzheimer's Disease (AD) is a severe neurodegenerative disorder and patients suffer from a wide range of symptoms. Early diagnosis of AD is essential to treat the disease as there appears to be no treatment benefit in the fully symptomatic stage. Thus, there is a critical need for a simple and accurate method for detecting AD prior to the onset of these symptoms, when treatment is likely to be most effective. To investigate Alzheimer-related pathophysiology, several transgenic mouse lines have been established in the recent years. Despite their general applicability in basic and applied research, quantitative tools to monitor pathophysiology as well as associated rewiring of metabolic pathways on a systemic level are still lacking. In this study, we carried out different behavioral tests, immunhistology as well as untargeted NMR-based metabolic phenotyping of Tg4-42 and wild-type mice of different ages. In this integrated approach, behavioral tests provide phenotypic read-outs of the disease through anxiety, cognition and spatial learning. Immunhistology provides information on the biochemical alterations of brain areas in form of histological quantification of different biomarkers by specific primary antibodies, e.g. 6E10, GFAP, CD11b, NeuN and/or Synaptophysin to get further information about brain neuropathology, e.g. plaque load, neuroinflammation or synaptic changes. Untargeted NMR spectroscopy is excellently suited to monitor perturbations in a large pool of metabolites in biofluids and tissues and reflects changes downstream of genomic, transcriptomic and proteomic fluctuations. The combination of these techniques in an integrative approach provides a better understanding of (patho-) physiological alterations in health and disease aiding to further understanding of alterations in complex biological networks involved in AD. We will present a NMR-based metabolite biomarker panel of mouse Tg4-42 biofluids and tissues and the integration of these biomarkers with conventional techniques such as histology and behavioral studies. These results provide a deeper insight to understand the (patho-) physiology in this AD mouse model. By performing behavioral studies, immunhistology and NMR-based metabolic phenotyping we present an integrative approach which can be easily extended to other disease models and translated across species since metabolic pathways are conserved through evolution, and are essentially similar in rodents and humans.
Due to the lack of proper biomarkers, in vivo diagnosis of Alzheimer's disease (AD) pathology in patients is still not possible. Major hallmarks of AD comprising extracellular plaque deposits of β-amyloid peptide (Aβ), Tau neurofibrillary tangles and neuroinflammation are concomitantly occurring not only in the brain, but also in other sensory systems such as the retina. Interestingly, a promising clinical study supervised by Dr. Koronyo-Hamaoui was published in 2017 providing the opportunity to detect and track Aβ pathology in the retina of living patients. The here presented research aims to characterize the neuropathological changes occurring both in the brain and retina of different AD animal models and pursues to address suitable biomarkers for early screening tests. Eyes and brain hemispheres from various AD animal models (APP rat, TAU rat, TMHT mouse) aged 6 and 12 months were collected. Quantitative histological analysis of different neuropathological markers was performed in retina, primary and secondary visual cortex (visual system), as well as in the olfactory bulb and piriform cortex (olfactory system). Analyzed AD rodent models display a significant age-dependent shift in neuropathology-related biomarkers when compared to non-transgenic littermates. An overall increase in aggregated Aβ and hyperphosphorylated tau is observed in all sections. Additional analyses revealed pathology-related changes in different neurotransmitters (ChaT, GAD67, TH) and neuroinflammatory markers. Our results strengthen the hypothesis that AD-related pathological biomarkers are present in the retina, unveiling novel analysis parameter for AD mouse models when testing new diagnostic markers.
Aggregation and misfolding of amyloid beta (Aβ) and tau proteins, suggested to arise from post-translational modification processes, are thought to be the main cause of Alzheimer's disease (AD). Additionally, a plethora of evidence exists that links metabolic dysfunctions such as obesity, type 2 diabetes (T2D), and dyslipidemia to the pathogenesis of AD. We thus investigated the combinatory effect of T2D and human glutaminyl cyclase activity (pyroglutamylation), on the pathology of AD and whether astaxanthin (ASX) treatment ameliorates accompanying pathophysiological manifestations. Male transgenic AD mice, APPxhQC, expressing human APP751 with the Swedish and the London mutation and human glutaminyl cyclase (hQC) enzyme and their non-transgenic (NTG) littermates were used. Both APPxhQC and NTG mice were allocated to 3 groups, control, T2D-control, and T2D-ASX. Mice were fed control or high fat diet ± ASX for 13 weeks starting at an age of 11-12 months. High fat diet fed mice were further treated with streptozocin for T2D induction. Effects of genotype, T2D induction, and ASX treatment were evaluated by analysing glycemic readouts, lipid concentration, Aβ deposition, hippocampus-dependent cognitive function and nutrient sensing using immunosorbent assay, ELISA-based assays, western blotting, immunofluorescence staining, and behavioral testing via Morris water maze (MWM), respectively. APPxhQC mice presented a higher glucose sensitivity compared to NTG mice. T2D-induced brain dysfunction was more severe in NTG compared to the APPxhQC mice. T2D induction impaired memory functions while increasing hepatic LC3B, ABCA1, and p65 levels in NTG mice. T2D induction resulted in a progressive shift of Aβ from the soluble to insoluble form in APPxhQC mice. ASX treatment reversed T2D- induced memory dysfunction in NTG mice and in parallel increased hepatic pAKT while decreasing p65 and increasing cerebral p-S6rp and p65 levels. ASX treatment reduced soluble Aβ38 and Aβ40 and insoluble Aβ40 levels in T2D-induced APPxhQC mice. We demonstrate that T2D induction in APPxhQC mice poses additional risk for AD pathology as seen by increased Aβ deposition. Although ASX treatment reduced Aβ expression in T2D-induced APPxhQC mice and rescued T2D-induced memory impairment in NTG mice, ASX treatment alone may not be effective in cases of T2D comorbidity and AD.
Numerous genetic association studies have identified Neuregulin-1 (NRG1) and its receptor ErbB4 as schizophrenia susceptibility genes.Functionally, NRG-ErbB signaling regulates synaptic plasticity (i.e.LTP induction and maintenance), dendritic spine formation, GAB-Aergic interneuron migration/survival and neural network oscillatory activity, processes believed to be relevant to the pathophysiology underlying schizophrenia.Using acute hippocampal slices from wildtype and knockout mice we found that reversal of LTP at hippocampal Schaeffer collateral-CA1 (SC-CA1) synapses by NRG1 requires ErbB4 activation.Using reverse microdialysis in hippocampi of freely moving rats to measure dopamine and its metabolites HVA and DOPAC by HPLC-EC, we found that NRG1 (1 nM) causes a rapid and dramatic increase of extracellular dopamine levels that reverse LTP by activating D4 receptors (D4Rs).Pharmacological inhibition of D4Rs by selective antagonists, including the antipsychotic clozapine, blocks the effects of NRG1 on LTP reversal; conversely, D4R agonists depotentiate LTP.By means of whole-cell recordings in CA1 and live imaging experiments in dissociated hippocampal neurons using spinning-disk confocal fluorescence microscopy in cells transfected with the pH-sensitive eGFP to measure AMPAR surface expression, we demonstrate that NRG1 and D4R agonists reverse LTP by selectively reducing AMPAR EPSCs at SC-CA1 synapses and promoting the internalization of GluR1-containing receptors, respectively.These results, in combination with prior studies, suggest that dopamine modulates homeostasis at SC-CA1 glutamatergic synapses by regulating the activity of its receptors either positively (D1Rs) or negatively (D4Rs) coupled to adenylate cyclase.In addition to the effects of NRG/ErbB4 signaling on synaptic plasticity, its relevance for neuronal network activity and behaviors associated with psychiatric disorders will be presented.Special emphasis on the importance of ErbB4 signaling in GABAergic interneurons, particularly its function in fast-spiking parvalbuminpositive interneurons (assessed by cre-mediated targeted mutation in these cells), will be discussed.
Alzheimer's disease can be modelled by different transgenic mouse strains. To gain deeper insight into disease model mechanisms, the previously described Tg4-42 mouse was analysed for transgene integration. On RNA/DNA level the transgene integration resulted in more than 20 copy numbers and further caused a deletion of exon 2 of the retinoic acid receptor beta. These findings were also confirmed on protein level with highly decreased retinoic acid receptor beta protein levels in homozygous Tg4-42 mice and may have an impact on the previously described phenotype of homozygous Tg4-42 mice to be solely dependent on amyloid-ß 4-42 expression. Since hemizygous mice show no changes in RARB protein levels it can be concluded that the previously described phenotype of these mice should not be affected by the retinoic acid receptor beta gene knockout. In order to fully understand the results of transgenesis, it is extremely advisable to determine the genome integration site and the basic structure of the inserted transgenes. This can be carried out for instance by next-generation sequencing techniques. Our results thus suggest that a detailed characterization of new disease models using the latest genomics technologies prior to functional studies could be a valuable tool to avoid an unexpected genetic influence on the animals' phenotype that is not only based on the inserted transgene. This would also significantly improve the selection of mouse models that are best suited for therapeutic development and basic research.
Environmental experience and drugs are two parameters that affect the maturation of neurotransmitter systems. The influence of impoverished rearing (IR) versus enriched rearing (ER) was compared in conjunction with postnatal methamphetamine (MA) treatment. The densities of immunostained 5-HT fibres were quantified in septal and temporal regions of the hippocampal dentate gyrus (DG) in young adult gerbils. In the IR group, 5-HT fibre densities were significantly increased in the molecular, granular and polymorphic layers of the DG in the temporal plane. After postnatal MA treatment, the 5-HT fibre density in the ER group reached a level equivalent to that of the IR group in nearly all respects. Under IR conditions, the pharmacological intervention significantly increased the maturation of fibre densities in septal layers only in the right hemisphere with no significant alterations in the left hemisphere and in temporal regions of either hemisphere. According to our previous studies on hippocampal neurogenesis, adaptations of 5-HT fibre densities partly proved to be positively correlated to cell proliferation rates for each of the specific conditions. Thus, the induced MA sensitivity, caused by pharmacological intervention at day 14, was manifested as direct interaction of 5-HT fibre maturation and cell proliferation in dependence of environmental factors. Both IR and MA together give us a better understanding of raphe-hippocampal plasticity and offer new perspectives for pharmacological studies on the 5-HT participation in mental disorders.
Abstract Background Senile plaques frequently contain pyroglutamate amyloid β (Aβ‐pE(3)), a N‐terminally truncated Aβ species that is more closely linked to Alzheimer’s Disease (AD) compared to other Aβ species. Tau protein is highly phosphorylated at several residues in AD, specifically tau protein phosphorylated (ptau) at Ser202/Thr205 is known to be increased in AD. First studies suggest that the two pathologies of plaques and tau phosphorylation might be linked to each other. Method To evaluate if Aβ‐pE(3) and ptau Ser202/Thr205 levels correlate in human and transgenic AD mouse models we analyzed human cortical and hippocampal brain tissue of different Braak stages as well as murine brain tissue of two transgenic mouse models for levels of Aβ‐pE(3) and ptau Ser202/Thr205 and correlated the data. Result Our results show that Aβ‐pE(3) formation increases already at early Braak stages while ptau Ser202/Thr205 mostly increases at later stages. Further analyses revealed strong correlations between the two pathologies in the temporal, frontal, cingulate and occipital cortex. However, correlation in the hippocampus was weaker. Evaluation of murine transgenic brain tissue demonstrated a slow but steady increase of Aβ‐pE(3) from 6 to 12 months of age in the cortex and hippocampus of APPSL mice and a very early and strong Aβ‐pE(3) increase in 5xFAD mice. ptau Ser202/Thr205 levels increased at the age of 9 months in APPSL mice and already at 6 months in 5xFAD mice. Conclusion Our results show that Aβ‐pE(3) and ptau Ser202/Thr205 levels strongly correlate in human as well as murine brain tissues, suggesting that tau phosphorylation might be amplified by Aβ‐pE(3).
