Strategies to gain novel Alzheimer’s disease diagnostics and therapeutics using modulators of ABCA transporters

Adenosine-triphosphate-(ATP)-binding cassette (ABC) transport proteins are ubiquitously present membrane-bound efflux pumps that distribute endo- and xenobiotics across intra- and intercellular barriers. Discovered over 40 years ago, ABC transporters have been identified as key players in various human diseases, such as multidrug-resistant cancer and atherosclerosis, but also neurodegenerative diseases, such as Alzheimer’s disease (AD). Most prominent and well-studied are ABCB1, ABCC1, and ABCG2, not only due to their contribution to the multidrug resistance (MDR) phenotype in cancer, but also due to their contribution to AD. However, our understanding of other ABC transporters is limited, and most of the 49 human ABC transporters have been largely neglected as potential targets for novel small-molecule drugs. This is especially true for the ABCA subfamily, which contains several members known to play a role in AD initiation and progression. This review provides up-to-date information on the proposed functional background and pathological role of ABCA transporters in AD. We also provide an overview of small-molecules shown to interact with ABCA transporters as well as potential in silico, in vitro, and in vivo methodologies to gain novel templates for the development of innovative ABC transporter-targeting diagnostics and therapeutics

Nucleotide-Oligomerization-Domain-2 Affects Commensal Gut Microbiota Composition and Intracerebral Immunopathology in Acute Toxoplasma gondii Induced Murine Ileitis

Background Within one week following peroral high dose infection with Toxoplasma (T.) gondii, susceptible mice develop non-selflimiting acute ileitis due to an underlying Th1-type immunopathology. The role of the innate immune receptor nucleotide-oligomerization-domain-2 (NOD2) in mediating potential extra-intestinal inflammatory sequelae including the brain, however, has not been investigated so far. Methodology/Principal Findings Following peroral infection with 100 cysts of T. gondii strain ME49, NOD2-/- mice displayed more severe ileitis and higher small intestinal parasitic loads as compared to wildtype (WT) mice. However, systemic (i.e. splenic) levels of pro-inflammatory cytokines such as TNF-α and IFN-γ were lower in NOD2-/- mice versus WT controls at day 7 p.i. Given that the immunopathological outcome might be influenced by the intestinal microbiota composition, which is shaped by NOD2, we performed a quantitative survey of main intestinal bacterial groups by 16S rRNA analysis. Interestingly, Bifidobacteria were virtually absent in NOD2-/- but not WT mice, whereas differences in remaining bacterial species were rather subtle. Interestingly, more distinct intestinal inflammation was accompanied by higher bacterial translocation rates to extra- intestinal tissue sites such as liver, spleen, and kidneys in T. gondii infected NOD2-/- mice. Strikingly, intracerebral inflammatory foci could be observed as early as seven days following T. gondii infection irrespective of the genotype of animals, whereas NOD2-/- mice exhibited higher intracerebral parasitic loads, higher F4/80 positive macrophage and microglia numbers as well as higher IFN-γ mRNA expression levels as compared to WT control animals. Conclusion/Significance NOD2 signaling is involved in protection of mice from T. gondii induced acute ileitis. The parasite-induced Th1-type immunopathology at intestinal as well as extra-intestinal sites including the brain is modulated in a NOD2-dependent manner

Development of deep learning models for microglia analyses in brain tissue using DeePathology™ STUDIO

Background Interest in artificial intelligence-driven analysis of medical images has seen a steep increase in recent years. Thus, our paper aims to promote and facilitate the use of this state-of-the-art technology to fellow researchers and clinicians. New method We present custom deep learning models generated in DeePathology™ STUDIO without the need for background knowledge in deep learning and computer science underlined by practical suggestions. Results We describe the general workflow in this commercially available software and present three real-world examples how to detect microglia on IBA1-stained mouse brain sections including their differences, validation results and analysis of a sample slide. Comparison with existing methods Deep-learning assisted analysis of histological images is faster than classical analysis methods, and offers a wide variety of detection possibilities that are not available using methods based on staining intensity. Conclusions Reduced researcher bias, increased speed and extended possibilities make deep-learning assisted analysis of histological images superior to traditional analysis methods for histological images

Fingolimod as a Treatment in Neurologic Disorders Beyond Multiple Sclerosis

Fingolimod is an approved treatment for relapsing–remitting multiple sclerosis (MS), and its properties in different pathways have raised interest in therapy research for other neurodegenerative diseases. Fingolimod is an agonist of sphingosine-1-phosphate (S1P) receptors. Its main pharmacologic effect is immunomodulation by lymphocyte homing, thereby reducing the numbers of T and B cells in circulation. Because of the ubiquitous expression of S1P receptors, other effects have also been described. Here, we review preclinical experiments evaluating the effects of treatment with fingolimod in neurodegenerative diseases other than MS, such as Alzheimer’s disease or epilepsy. Fingolimod has shown neuroprotective effects in different animal models of neurodegenerative diseases, summarized here, correlating with increased brain-derived neurotrophic factor and improved disease phenotype (cognition and/or motor abilities). As expected, treatment also induced reductions in different neuroinflammatory markers because of not only inhibition of lymphocytes but also direct effects on astrocytes and microglia. Furthermore, fingolimod treatment exhibited additional effects for specific neurodegenerative disorders, such as reduction of amyloid-β production, and antiepileptogenic properties. The neuroprotective effects exerted by fingolimod in these preclinical studies are reviewed and support the translation of fingolimod into clinical trials as treatment in neurodegenerative diseases beyond neuroinflammatory conditions (MS)

Dimethyl fumarate does not mitigate cognitive decline and β-amyloidosis in female APPPS1 mice

Introduction Alzheimer’s disease (AD) is the leading cause of dementia and a major global health issue. Currently, only limited treatment options are available to patients. One possibility to expand the treatment repertoire is repurposing of existing drugs such as dimethyl fumarate (DMF). DMF is approved for treatment of multiple sclerosis and previous animal studies have suggested that DMF may also have a beneficial effect for the treatment of AD. Methods We used an APPPS1 transgenic model of senile β-amyloidosis and treated female mice orally with DMF in two treatment paradigms (pre and post onset). We quantified learning and memory parameters, β-amyloidosis, and neuroinflammation to determine the potential of DMF as AD therapeutics. Results Treatment with DMF had no influence on water maze performance, β-amyloid accumulation, plaque formation, microglia activation, and recruitment of immune cells to the brain. Compared to vehicle-treated animals, oral DMF treatment could not halt or retard disease progression in the mice. Discussion Our results do not favour the use of DMF as treatment for AD. While our results stand in contrast to previous findings in other models, they emphasize the importance of animal model selection and suggest further studies to elucidate the mechanisms leading to conflicting results

Using a qPCR device to screen for modulators of ABC transporter activity: A step-by-step protocol

Introduction Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are transmembrane proteins which actively transport a large variety of substrates across biological membranes. ABC transporter overexpression can be the underlying cause of multidrug resistance in oncology. Moreover, it has been revealed that increased ABCC1 transporter activity can ameliorate behavioural changes and Aβ pathology in a rodent model of Alzheimer's disease and it is currently tested in AD patients. Methods Finding substances that modulate ABC transporter activity (inhibitors and activators) is of high relevance and thus, different methods have been developed to screen for potential modulators. For this purpose, we have developed a cell-based assay to measure the kinetics of ABCC1-mediated efflux of a fluorescent dye using a common qPCR device (Agilent AriaMx). Results We validated the specificity of our method with vanadate and benzbromarone controls. Furthermore, we provide a step-by-step protocol including statistical analysis of the resulting data and suggestions how to modify the protocol specifically to screen for activators of ABCC1. Discussion Our approach is biologically more relevant than cell-free assays. The continuous detection of kinetics allows for a more precise quantification compared with assays with single end-point measurements

A New Tool for the Analysis of the Effect of Intracerebrally Injected Anti-Amyloid-β Compounds

Background: A wide range of techniques has been developed over the past decades to characterize amyloid-β (Aβ) pathology in mice. Until now, no method has been established to quantify spatial changes in Aβ plaque deposition due to targeted delivery of substances using ALZET® pumps. Objective: Development of a methodology to quantify the local distribution of Aβ plaques after intracerebral infusion of compounds. Methods: We have developed a toolbox to quantify Aβ plaques in relation to intracerebral injection channels using Zeiss AxioVision® and Microsoft Excel® software. For the proof of concept, intracerebral stereotactic surgery was performed in 50-day-old APP-transgenic mice injected with PBS. At the age of 100 days, brains were collected for immunhistological analysis. Results: The toolbox can be used to analyze and evaluate Aβ plaques (number, size, and coverage) in specific brain areas based on their location relative to the point of the injection or the injection channel. The tool provides classification of Aβ plaques in pre-defined distance groups using two different approaches. Conclusion: This new analytic toolbox facilitates the analysis of long-term continuous intracerebral experimental compound infusions using ALZET® pumps. This method generates reliable data for Aβ deposition characterization in relation to the distribution of experimental compounds

Detection and Prediction of Mild Cognitive Impairment in Alzheimer's Disease Mice

Background: The recent failure of clinical trials to treat Alzheimer’s disease (AD) indicates that the current approach of modifying disease is either wrong or is too late to be efficient. Mild cognitive impairment (MCI) denotes the phase between the preclinical phase and clinical overt dementia. AD mouse models that overexpress human amyloid-β (Aβ) are used to study disease pathogenesis and to conduct drug development/testing. However, there is no direct correlation between the Aβ deposition, the age of onset, and the severity of cognitive dysfunction. Objective: To detect and predict MCI when Aβ plaques start to appear in the hippocampus of an AD mouse. Methods: We trained wild-type and AD mice in a Morris water maze (WM) task with different inter-trial intervals (ITI) at 3 months of age and assessed their WM performance. Additionally, we used a classification algorithm to predict the genotype (APPtg versus wild-type) of an individual mouse from their respective WM data. Results: MCI can be empirically detected using a short-ITI protocol. We show that the ITI modulates the spatial learning of AD mice without affecting the formation of spatial memory. Finally, a simple classification algorithm such as logistic regression on WM data can give an accurate prediction of the cognitive dysfunction of a specific mouse. Conclusion: MCI can be detected as well as predicted simultaneously with the onset of Aβ deposition in the hippocampus in AD mouse model. The mild cognitive impairment prediction can be used for assessing the efficacy of a treatment

Time- and Sex-Dependent Effects of Fingolimod Treatment in a Mouse Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common cause of dementia. Fingolimod has previously shown beneficial effects in different animal models of AD. However, it has shown contradictory effects when it has been applied at early disease stages. Our objective was to evaluate fingolimod in two different treatment paradigms. To address this aim, we treated male and female APP-transgenic mice for 50 days, starting either before plaque deposition at 50 days of age (early) or at 125 days of age (late). To evaluate the effects, we investigated the neuroinflammatory and glial markers, the Aβ load, and the concentration of the brain-derived neurotrophic factor (BDNF). We found a reduced Aβ load only in male animals in the late treatment paradigm. These animals also showed reduced microglia activation and reduced IL-1β. No other treatment group showed any difference in comparison to the controls. On the other hand, we detected a linear correlation between BDNF and the brain Aβ concentrations. The fingolimod treatment has shown beneficial effects in AD models, but the outcome depends on the neuroinflammatory state at the start of the treatment. Thus, according to our data, a fingolimod treatment would be effective after the onset of the first AD symptoms, mainly affecting the neuroinflammatory reaction to the ongoing Aβ deposition

Time- and Sex-Dependent Effects of Fingolimod Treatment in a Mouse Model of Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common cause of dementia. Fingolimod has previously shown beneficial effects in different animal models of AD. However, it has shown contradictory effects when it has been applied at early disease stages. Our objective was to evaluate fingolimod in two different treatment paradigms. To address this aim, we treated male and female APP-transgenic mice for 50 days, starting either before plaque deposition at 50 days of age (early) or at 125 days of age (late). To evaluate the effects, we investigated the neuroinflammatory and glial markers, the Aβ load, and the concentration of the brain-derived neurotrophic factor (BDNF). We found a reduced Aβ load only in male animals in the late treatment paradigm. These animals also showed reduced microglia activation and reduced IL-1β. No other treatment group showed any difference in comparison to the controls. On the other hand, we detected a linear correlation between BDNF and the brain Aβ concentrations. The fingolimod treatment has shown beneficial effects in AD models, but the outcome depends on the neuroinflammatory state at the start of the treatment. Thus, according to our data, a fingolimod treatment would be effective after the onset of the first AD symptoms, mainly affecting the neuroinflammatory reaction to the ongoing Aβ deposition