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.
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.
Endothelial lipase (EL) is a potent modulator of the structural and functional properties of HDL. Impact of EL on cholesterol efflux capacity (CEC) of serum and isolated HDL is not well understood and apparently contradictory data were published. Here, we systematically examined the impact of EL on composition and CEC of serum and isolated HDL, in vitro and in vivo, using EL-overexpressing cells and EL-overexpressing mice. CEC was examined in a validated assay using 3H-cholesterol labelled J774 macrophages. In vitro EL-modification of serum resulted in complex alterations, including enrichment of serum with lipid-free/-poor apoA-I, decreased size of human (but not mouse) HDL and altered HDL lipid composition. EL-modification of serum increased CEC, in line with increased lipid-free/-poor apoA-I formation. In contrast, CEC of isolated HDL was decreased likely through altered lipid composition. In contrast to in vitro results, EL-overexpression in mice markedly decreased HDL-cholesterol and apolipoprotein A-I serum levels associated with a decreased CEC of serum. HDL lipid composition was altered, but HDL particle size and CEC were not affected. Our study highlights the multiple and complex effects of EL on HDL composition and function and may help to clarify the seemingly contradictory data found in published articles.
Abstract Background Alzheimer’s disease (AD) and other tauopathies are neurodegenerative disorders characterized by cellular accumulation of aggregated tau protein. Tau pathology within these disorders is accompanied by chronic neuroinflammation, such as activation of the classical complement pathway by complement initiation factor C1q. Additionally, about half of the AD cases present with inclusions composed of aggregated alpha-synuclein called Lewy bodies. Lewy bodies in disorders such as Parkinson’s disease and Lewy body dementia also frequently occur together with tau pathology. Immunotherapy is currently the most promising treatment strategy for tauopathies. However, the presence of multiple pathological processes within tauopathies makes it desirable to simultaneously target more than one disease pathway. Methods Herein, we have developed three bispecific antibodies based on published antibody binding region sequences. One bispecific antibody binds to tau plus alpha-synuclein and two bispecific antibodies bind to tau plus C1q. Results The affinity of the bispecific antibodies to their targets compared to their monospecific counterparts ranged from nearly identical to one order of magnitude lower. All bispecific antibodies retained binding to aggregated protein in patient-derived brain sections. The bispecific antibodies also retained their ability to inhibit aggregation of recombinant tau, regardless of whether the tau binding sites were in IgG or scFv format. Mono- and bispecific antibodies inhibited cellular seeding induced by AD-derived pathological tau with similar efficacy. Finally, both Tau-C1q bispecific antibodies completely inhibited the classical complement pathway. Conclusion Bispecific antibodies that bind to multiple pathological targets may therefore present a promising approach to treat tauopathies and other neurodegenerative disorders.
The microtubule associated protein tau plays a crucial role in the pathology of several neurodegenerative diseases, especially Alzheimer's. Thus, the development of new compounds capable of inhibiting or preventing tau aggregation, hyperphosphorylation or uptake is moving in the focus of Alzheimer disease treatment. Consequently, dependable and in optimal case complementary in vitro and in vivo models that mirror tau pathology in human diseases are needed. We established and optimized several in vitro methods to monitor tau pathology: (1) A cell-free Thioflavin-T aggregation assay, (2) stably transfected tau overexpressing SHSY5Y cell lines to monitor tau phosphorylation and aggregation, as well as (3) tau uptake assays using mouse primary neurons in combination with different tau seeds. Several positive controls like methylene blue or tau targeting antibodies will be tested with those assays to further validate them as drug screening tools. Our results will show the potency of several known tau interacting compounds within our models. In vitro methods to screen for the activity of compounds are of high relevance for the early stages of drug development. The here presented assays were developed as complementary tools to our transgenic and induced in vivo tauopathy models TMHT and hTau as well as seed or virus injection, respectively.
Defective degradation and clearance of amyloid-β as well as inflammation per se are crucial players in the pathology of Alzheimer's disease (AD). A defective transport across the blood-brain barrier is causative for amyloid-β (Aβ) accumulation in the brain, provoking amyloid plaque formation. Using primary porcine brain capillary endothelial cells and murine organotypic hippocampal slice cultures as in vitro models of AD, we investigated the effects of the antioxidant astaxanthin (ASX) on Aβ clearance and neuroinflammation. We report that ASX enhanced the clearance of misfolded proteins in primary porcine brain capillary endothelial cells by inducing autophagy and altered the Aβ processing pathway. We observed a reduction in the expression levels of intracellular and secreted amyloid precursor protein/Aβ accompanied by an increase in ABC transporters ABCA1, ABCG1 as well as low density lipoprotein receptor-related protein 1 mRNA levels. Furthermore, ASX treatment increased autophagic flux as evidenced by increased lipidation of LC3B-II as well as reduced protein expression of phosphorylated S6 ribosomal protein and mTOR. In LPS-stimulated brain slices, ASX exerted anti-inflammatory effects by reducing the secretion of inflammatory cytokines while shifting microglia polarization from M1 to M2 phenotype. Our data suggest ASX as potential therapeutic compound ameliorating AD-related blood brain barrier impairment and inflammation.
Abstract Background Neurofilament‐light chain (NF‐L) is known as biomarker of many neurodegenerative diseases and their progression. By analyzing amyotrophic lateral sclerosis (ALS) patients CSF or plasma progression of NF‐L levels can forecast conversion from the presymptomatic to symptomatic stage and the time of survival. So far, detailed analyses of NF‐L expression in neurodegenerative disease patient’s samples were performed, while NF‐L levels of preclinical mouse models of ALS, Alzheimer’s and Parkinson’s disease as well as lysosomal storage diseases are still unknown. Method We therefore evaluated NF‐L levels in the plasma of the ALS models SOD1‐G93A low expressor and TAR6/6 mice, the Alzheimer’s disease model 5xFAD, the Parkinson’s disease model Line 61 and the Gaucher disease model 4L/PS‐NA and the CSF of selected models using a commercially available ELISA kit from UmanDiagnostics. Result Our results show that NF‐L levels are highly increased in the plasma of ALS, Alzheimer’s and Gaucher disease mouse models, while in the analyzed PD model NF‐L plasma levels barely changed. Most of the analyzed models show a progressive increase of NF‐L levels over age. Conclusion NF‐L measurements in the plasma of the neurodegenerative disease mouse models of ALS and Alzheimer’s disease are thus a good tool to evaluate disease progression. Compared to analyses in human tissues, our results suggest that murine NF‐L levels and their progression have a high translation value. Furthermore, our data indicate that NF‐L might also be a good biomarker for diseases that are not classical neurodegenerative diseases but disorders with a neuronal component like some lysosomal storage diseases.
