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  Glial and vascular contributions to neurodegenerative diseases
Human iPSC-derived 3D tissue models to investigate glial and vascular contributions to neurodegenerative and neurovascular diseases
Julien Klimmt1,2, Carolina Cardoso Goncalves1,2,
Judit Gonzalez Gallego1,2, Angelika Dannert1,2,
Sophie Robinson1,2,6, Liliana Pedro1,6,7, Joseph Kroeger1,2, Gernot Kleinberger1,4, Mika Simons1,6,7, Martin Dichgans1,3,6, Christian Haass3,5,6, Dominik Paquet1,3
1Institute for Stroke and Dementia Research (ISD), University Hospital, LMU Munich, Munich, Germany
2Graduate School of Systemic Neurosciences, Munich, Germany
3Munich Cluster for Systems Neurology (SyNergy), Munich, Germany
4ISAR Bioscience GmbH, Planegg, Germany
5Metabolic Biochemistry, Biomedical Center (BMC), Faculty of Medicine, LMU Munich, Germany
6German Center for Neurodegenerative Diseases (DZNE), Germany 7Technical University, Germany
Characterization of amyloid-beta protofibrils in Alzheimer’s disease brain and unique binding properties of lecanemab,
Lars Lannfelt1,2, Linda Söderberg1, Malin Johannesson1, Nicolas Fritz1, Eleni Gkanatsiou1, Adeline Rachalski1, Gunilla Osswald1, Christer Möller1
1 BioArctic AB, Warfvinges väg 35, 112 51 Stockholm, Sweden
2 Department of Public Health/Geriatrics, Uppsala University, Sweden
 Brain research heavily depends on models recapitulating key aspects of human brain physiology and disease pathology. Human iPSCs have great potential to complement existing disease models, as they allow directly studying affected human cell types. In addition, recent developments in CRISPR genome editing revolutionized how impacts of genetic alterations on disease formation can be investigated. Co-culture of disease-relevant iPSC-derived cells with disease-relevant mutations enables studying complex phenotypes involving cellular crosstalk. Combining these technologies we established a new generation of iPSC-based human 3D brain tissue models for neurodegenerative and neurovascular brain diseases. We successfully established a multicellular brain tissue model containing iPSC-derived cortical neurons, astrocytes, microglia, and oligodendrocytes, as well as combinations of cells forming the neurovascular unit (NVU).
Our technology provides highly controllable and reproducible 3-dimensional tissues with typical cell morphologies and functional features, including widespread synapse formation, spontaneous and induced electrical activity, network formation, microglial ramification, tiling and phagocytosis, as well as formation of barrier-containing vessels interacting with astrocytic end feet for the NVU model. Interaction between neurons, glia and vascular cells become evident on morphological and functional levels. The models can be long-term cultured in a postmitotic state without proliferation or cell death, thus providing a more controllable, reproducible, and long-lived alternative to cortical organoids currently used for 3D disease modelling.
To establish human models of AD, FTD-Tau, or neurovascular diseases, we used our efficient CRISPR pipeline to introduce synergistic disease- associated mutations to accelerate naturally occurring disease processes and promote pathology. We will present a first phenotypic characterization of our multicellular tissue models, with an emphasis on glial and vascular interactions.
We expect that these models will support studies elucidating novel, potentially human-specific pathomechanisms and provide a human framework for translation and screening.
Backgrounds: Immunotherapy against amyloid-beta (Aβ) has emerged as a promising treatment option for Alzheimer’s disease (AD). Although many challenges remain, Aβ immunotherapy from late-phase clinical trials have shown promising results. Soluble Aβ aggregates, oligomers and protofibrils, are believed to be the most toxic species of Aβ. Lecanemab is a humanized IgG1 monoclonal antibody, selectively targeting Aβ protofibrils. In a Phase 2b clinical trial in early AD subjects, lecanemab demonstrated potential disease-modifying effects on both clinical endpoints and clearance of Aβ plaques in the brain, with an incidence of the side-effect ARIA-E (amyloid related imaging abnormalities-edema) of approximately 10% at the highest dose. Objectives: We have characterized the main target for lecanemab, i.e. Aβ protofibrils, in AD brain, describing the content of Aβ, as well as levels and size of Aβ protofibrils, in relation to the APOE genotype. The binding of lecanemab to Aβ protofibrils and the mechanism of action in clearance of Aβ was studied.
Methods: Soluble species of Aβ were extracted from post-mortem human AD brain tissue (n=24) and non-demented controls (NDE, n=12), and characterized regarding AD pathologies and APOE status using tissue from the Netherlands brain bank. Aβ protofibril levels were measured by immunoprecipitation (IP) using the protofibril selective antibody mAb158, the murine precursor to lecanemab. Size exclusion chromatography (SEC) and density gradient ultra-centrifugation were used to characterize protofibrils and to evaluate lecanemab’s binding profile. The specificity of lecanemab in binding to synthetic Aβ protofibrils was evaluated by IP in the presence of a 1000-fold excess of Aβ monomers. The lecanemab-mediated uptake of synthetic Aβ protofibrils was also studied in a human monocytic THP-1 cell model. The clearance of Aβ plaques in human AD brain section, mediated by lecanemab, was investigated.
Results: Aβ protofibril levels in AD brain (mean 168 ng/g tissue) were shown to be elevated compared to levels in non-demented control brain (mean 1.5 ng/g tissue). AD subjects, especially APOE E4 carriers, had the highest protofibril levels. Protofibrils were shown to be predominantly composed of Aβ42, with approximately 40 times higher levels of Aβ42 compared to Aβ40. SEC and density gradient ultracentrifugation showed that protofibrils extracted from AD brain were heterogenous in size, between 80 to >500 kDa, and lecanemab was shown to bind similarly to protofibrils of all sizes. Dose-dependent lecanemab-mediated phagocytosis of protofibrils in THP-1 cells (EC50 3 nM) was demonstrated as well as dose-dependent clearance of Aβ plaques in human brain sections. Lecanemab’s binding to protofibrils remained strong and was not affected by a 1000-fold excess of Aβ monomers, further supporting the unique selectivity of the antibody. Conclusion: Soluble aggregated Aβ species, i.e. toxic protofibrils, were significantly elevated in AD brains, especially in ApoE4 carriers when compared to non-demented controls. Lecanemab has a unique binding profile with a high selectivity for protofibrils and has been shown to effectively clear Aβ protofibrils and amyloid plaques.
50 • ISMND 2022

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