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Publikation

Namasivayam, V.; Silbermann, K.; Wiese, M.; Pahnke, J.; Stefan, S. M.; C@PA: Computer-aided pattern analysis to predict multitarget ABC transporter inhibitors J. Med. Chem. 64, 3350-3366, (2021) DOI: 10.1021/acs.jmedchem.0c02199

Based on literature reports of the last two decades, a computer-aided pattern analysis (C@PA) was implemented for the discovery of novel multitarget ABCB1 (P-gp), ABCC1 (MRP1), and ABCG2 (BCRP) inhibitors. C@PA included basic scaffold identification, substructure search and statistical distribution, as well as novel scaffold extraction to screen a large virtual compound library. Over 45,000 putative and novel broad-spectrum ABC transporter inhibitors were identified, from which 23 were purchased for biological evaluation. Our investigations revealed five novel lead molecules as triple ABCB1, ABCC1, and ABCG2 inhibitors. C@PA is the very first successful computational approach for the discovery of promiscuous ABC transporter inhibitors.
Publikation

Dib, S.; Pahnke, J.; Gosselet, F.; Role of ABCA7 in human health and in alzheimer’s disease Int. J. Mol. Sci. 22, 4603, (2021) DOI: 10.3390/ijms22094603

Several studies, including genome wide association studies (GWAS), have strongly suggested a central role for the ATP-binding cassette transporter subfamily A member 7 (ABCA7) in Alzheimer’s disease (AD). This ABC transporter is now considered as an important genetic determinant for late onset Alzheimer disease (LOAD) by regulating several molecular processes such as cholesterol metabolism and amyloid processing and clearance. In this review we shed light on these new functions and their cross-talk, explaining its implication in brain functioning, and therefore in AD onset and development.
Publikation

Bascuñana, P.; Brackhan, M.; Pahnke, J.; Machine learning-supported analyses improve quantitative histological assessments of amyloid-β deposits and activated Microglia J. Alzheimers Dis. 79, 597-605, (2021) DOI: 10.3233/JAD-201120

Background: Detailed pathology analysis and morphological quantification is tedious and prone to errors. Automatic image analysis can help to increase objectivity and reduce time. Here, we present the evaluation of the DeePathology STUDIO™ for automatic analysis of histological whole-slide images using machine learning/artificial intelligence. Objective: To evaluate and validate the use of DeePathology STUDIO for the analysis of histological slides at high resolution. Methods: We compared the DeePathology STUDIO and our current standard method using macros in AxioVision for the analysis of amyloid-β (Aβ) plaques and microglia in APP-transgenic mice at different ages. We analyzed density variables and total time invested with each approach. In addition, we correlated Aβ concentration in brain tissue measured by ELISA with the results of Aβ staining analysis. Results: DeePathology STUDIO showed a significant decrease of the time for establishing new analyses and the total analysis time by up to 90%. On the other hand, both approaches showed similar quantitative results in plaque and activated microglia density in the different experimental groups. DeePathology STUDIO showed higher sensitivity and accuracy for small-sized plaques. In addition, DeePathology STUDIO allowed the classification of plaques in diffuse- and dense-packed, which was not possible with our traditional analysis. Conclusion: DeePathology STUDIO substantially reduced the effort needed for a new analysis showing comparable quantitative results to the traditional approach. In addition, it allowed including different objects (categories) or cell types in a single analysis, which is not possible with conventional methods.
Publikation

Zoufal, V.; Mairinger, S.; Krohn, M.; Wanek, T.; Filip, T.; Sauberer, M.; Stanek, J.; Kuntner, C.; Pahnke, J.; Langer, O.; Measurement of cerebral ABCC1 transport activity in wild-type and APP/PS1-21 mice with positron emission tomography J. Cereb. Blood Flow Metab. 40, 954-965, (2020) DOI: 10.1177/0271678X19854541

Previous data suggest a possible link between multidrug resistance-associated protein 1 (ABCC1) and brain clearance of beta-amyloid (Aβ). We used PET with 6-bromo-7-[11C]methylpurine ([11C]BMP) to measure cerebral ABCC1 transport activity in a beta-amyloidosis mouse model (APP/PS1-21) and in wild-type mice aged 50 and 170 days, without and with pretreatment with the ABCC1 inhibitor MK571. One hundred seventy days-old-animals additionally underwent [11C]PiB PET scans to measure Aβ load. While baseline [11C]BMP PET scans detected no differences in the elimination slope of radioactivity washout from the brain (kelim) between APP/PS1-21 and wild-type mice of both age groups, PET scans after MK571 pretreatment revealed significantly higher kelim values in APP/PS1-21 mice than in wild-type mice aged 170 days, suggesting increased ABCC1 activity. The observed increase in kelim occurred across all investigated brain regions and was independent of the presence of Aβ plaques measured with [11C]PiB. Western blot analysis revealed a trend towards increased whole brain ABCC1 levels in 170 days-old-APP/PS1-21 mice versus wild-type mice and a significant positive correlation between ABCC1 levels and kelim. Our data point to an upregulation of ABCC1 in APP/PS1-21 mice, which may be related to an induction of ABCC1 in astrocytes as a protective mechanism against oxidative stress.
Publikation

Zoufal, V.; Mairinger, S.; Brackhan, M.; Krohn, M.; Filip, T.; Sauberer, M.; Stanek, J.; Wanek, T.; Tournier, N.; Bauer, M.; Pahnke, J.; Langer, O.; Imaging P-Glycoprotein Induction at the Blood–Brain Barrier of a β-Amyloidosis Mouse Model with 11C-Metoclopramide PET J. Nucl. Med. 61, 1050-1057, (2020) DOI: 10.2967/jnumed.119.237198

P-glycoprotein (ABC subfamily B member 1, ABCB1) plays an important role at the blood-brain barrier (BBB) in promoting clearance of neurotoxic β-amyloid (Aβ) peptides from the brain into the blood. ABCB1 expression and activity were found to be decreased in the brains of Alzheimer disease patients. Treatment with drugs that induce cerebral ABCB1 activity may be a promising approach to delay the build-up of Aβ deposits in the brain by enhancing clearance of Aβ peptides from the brain. The aim of this study was to investigate whether PET with the weak ABCB1 substrate radiotracer 11C-metoclopramide can measure ABCB1 induction at the BBB in a β-amyloidosis mouse model (APP/PS1-21 mice) and in wild-type mice. Methods: Groups of wild-type and APP/PS1-21 mice aged 50 or 170 d underwent 11C-metoclopramide baseline PET scans or scans after intraperitoneal treatment with the rodent pregnane X receptor activator 5-pregnen-3β-ol-20-one-16α-carbonitrile (PCN, 25 mg/kg) or its vehicle over 7 d. At the end of the PET scans, brains were harvested for immunohistochemical analysis of ABCB1 and Aβ levels. In separate groups of mice, radiolabeled metabolites of 11C-metoclopramide were determined in plasma and brain at 15 min after radiotracer injection. As an outcome parameter of cerebral ABCB1 activity, the elimination slope of radioactivity washout from the brain (k E,brain) was calculated. Results: PCN treatment resulted in an increased clearance of radioactivity from the brain as reflected by significant increases in k E,brain (from +26% to +54% relative to baseline). Immunohistochemical analysis confirmed ABCB1 induction in the brains of PCN-treated APP/PS1-21 mice with a concomitant decrease in Aβ levels. There was a significant positive correlation between k E,brain and ABCB1 levels in the brain. In wild-type mice, a significant age-related decrease in k E,brain was found. Metabolite analysis showed that most radioactivity in the brain comprised unmetabolized 11C-metoclopramide in all animal groups. Conclusion: 11C-metoclopramide can measure ABCB1 induction in the mouse brain without the need to consider an arterial input function and may find potential application in Alzheimer disease patients to noninvasively evaluate strategies to enhance the clearance properties of the BBB.
Publikation

Zoufal, V.; Wanek, T.; Krohn, M.; Mairinger, S.; Filip, T.; Sauberer, M.; Stanek, J.; Pekar, T.; Bauer, M.; Pahnke, J.; Langer, O.; Age dependency of cerebral P-glycoprotein function in wild-type and APPPS1 mice measured with PET J. Cereb. Blood Flow Metab. 40, 150-162, (2020) DOI: 10.1177/0271678X18806640

P-glycoprotein (P-gp, ABCB1) is an efflux transporter at the blood–brain barrier (BBB), which mediates clearance of beta-amyloid (Aβ) from brain into blood. We used (R)-[11C]verapamil PET in combination with partial P-gp inhibition with tariquidar to measure cerebral P-gp function in a beta-amyloidosis mouse model (APPtg) and in control mice at three different ages (50, 200 and 380 days). Following tariquidar pre-treatment (4 mg/kg), whole brain-to-plasma radioactivity concentration ratios (Kp,brain) were significantly higher in APPtg than in wild-type mice aged 50 days, pointing to decreased cerebral P-gp function. Moreover, we found an age-dependent decrease in cerebral P-gp function in both wild-type and APPtg mice of up to −50%. Alterations in P-gp function were more pronounced in Aβ-rich brain regions (hippocampus, cortex) than in a control region with negligible Aβ load (cerebellum). PET results were confirmed by immunohistochemical staining of P-gp in brain microvessels. Our results confirm previous findings of reduced P-gp function in Alzheimer’s disease mouse models and show that our PET protocol possesses adequate sensitivity to measure these functional changes in vivo. Our PET protocol may find use in clinical studies to test the efficacy of drugs to induce P-gp function at the human BBB to enhance Aβ clearance.
Publikation

Stefan, K.; Wen Leck, L. Y.; Namasivayam, V.; Bascuñana, P.; Huang, M. L.-H.; Riss, P. J.; Pahnke, J.; Jansson, P. J.; Stefan, S. M.; Vesicular ATP-binding cassette transporters in human disease: relevant aspects of their organization for future drug development Future Drug Discovery 4, FDD51, (2020) DOI: 10.4155/fdd-2020-0025

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Publikation

Rai, S. P.; Krohn, M.; Pahnke, J.; Early Cognitive Training Rescues Remote Spatial Memory but Reduces Cognitive Flexibility in Alzheimer’s Disease Mice J. Alzheimers Dis. 75, 1301-1317, (2020) DOI: 10.3233/JAD-200161

Background:Spatial memory dysfunction has been demonstrated in mouse models of Alzheimer’s disease (AD) which is consistent with the clinical finding that the early signature of AD includes difficulties in the formation and/or storage of a memory. A stored memory—a long term memory—can be modulated via process called as memory retrieval that can either lead toward memory reconsolidation or even memory extinction.Objective:We aim to shed light on the fate of the spatial memory during memory reactivation and memory extinction using a water maze task.Methods:In Set-up I, we trained 3-month-old mice (wild-type mice and mice with cerebral β-amyloidosis) and assessed the fate of remote memory after four months of retention interval (RI). In Set-up II, we performed an early-extensive training at 2 months of age, retrained the same mice at 3 months of age, introduced four months of RI, and finally assessed remote spatial memory at 7 months of age.Results:We find in β-amyloidosis mice that memory reactivation problems were detectable at 7 months of age and were alleviated by cognitive overtraining. Similarly, forgetting of remote spatial memory was also minimized by cognitive overtraining. Finally, we show that the cognitive training facilitates the recovery of the reactivated spatial memory while reducing the ability to form new spatial memory in AD mice.Conclusion:This result may explain the rationality behind the cognitive reserve observed in AD patients and elderly with severe β-amyloidosis not corresponding to the actual low dementia symptoms.
Publikation

Rai, S. P.; Bascuñana, P.; Brackhan, M.; Krohn, M.; Möhle, L.; Paarmann, K.; Pahnke, J.; Detection and Prediction of Mild Cognitive Impairment in Alzheimer’s Disease Mice J. Alzheimers Dis. 77, 1209-1221, (2020) DOI: 10.3233/jad-200675

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.
Publikation

Möhle, L.; Schwarzová, B.; Krohn, M.; Stefan, S. M.; Pahnke, J.; Using a qPCR device to screen for modulators of ABC transporter activity: A step-by-step protocol J. Pharmacol. Toxicol. Methods 104, 106882, (2020) DOI: 10.1016/j.vascn.2020.106882

IntroductionAdenosine 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.MethodsFinding 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).ResultsWe 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.DiscussionOur 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.
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