Die Auswirkung der Spermidin-induzierten Autophagie auf die Pathogenese der Alzheimer Erkrankung im Mausmodell

Alzheimer´s disease (AD), the most common cause of dementia, is not only characterized by extracellular amyloid β (Aβ) plaque formation and neurofibrillary tangles deposition, but also by microglia- and astrocyte-mediated neuroinflammation. In the last decade, impairment of autophagy has been found to be yet another important feature of AD linking neuroinflammation and Aβ pathology. The nutritional supplement spermidine is a known autophagy activator which has been shown to induce longevity in various model organisms. Here, we investigated spermidine´s therapeutic potential in the AD-like mouse model APPPS1. Oral treatment with spermidine resulted in a reduction of neurotoxic soluble Aβ at both early and late stages of pathology in APPPS1 mice. Subsequent single nuclei sequencing of hemispheres of spermidinetreated APPPS1 mice and H2O controls revealed that spermidine affected diseaseassociated microglia by increasing migratory and phagocytic genes. Spermidine also altered the transcriptome of oligodendrocytes. Similar to microglia, spermidine modified genes associated with anti-inflammation, proliferation, transcription and the cytoskeleton and additionally reduced heat shock response genes in oligodendrocytes. In vitro treatment of activated microglia, astrocytes and oligodendrocytes with spermidine reduced the release of cytokines dose-dependently, thus confirming the anti-inflammatory properties of spermidine. To complement the transcriptomics approach, a proteomics analysis of isolated microglia from spermidine-treated APPPS1 mice was performed. Spermidine reversed AD-associated changes in APPPS1 mice by promoting anti-inflammatory, cytoskeletal, autophagic and endocytic effects. An in-depth assessment of the antiinflammatory effects of spermidine in microglia in vitro elucidated that spermidine targeted NF-κB-mediated cytokine transcription upon LPS/ATP, poly I:C and Aβ stimulation as well as the assembly of the NLRP3 inflammasome, thus reducing the release of IL-1β and IL-18. The anti-inflammatory effects of spermidine were further substantiated by a spermidinemediated CNS-wide reduction in cytokine levels in the hemispheres of APPPS1 mice at late stages of the pathology. As a translational approach, activated microglia derived from human induced pluripotent stem cells were treated with spermidine validating the antiinflammatory effects of spermidine. Taken together, spermidine reduced glial-mediated neuroinflammation and induced cell migration, autophagy and Aβ degradation in microglia of APPPS1 mice resulting in reduced levels of soluble Aβ. Thus, spermidine supplementation presents an intriguing approach that should be further validated in AD patients.Die Alzheimer-Krankheit (AD), eine der häufigsten Ursache für Demenz, ist nicht nur durch die Bildung von extrazellulären Amyloid β (Aβ)-Plaques und der Ablagerung von neurofibrillären Tangles gekennzeichnet, sondern auch durch eine von Mikroglia und Astrozyten vermittelte Neuroinflammation. Im letzten Jahrzehnt wurde die Beeinträchtigung der Autophagie als ein weiteres Merkmal für die Alzheimer-Krankheit identifiziert, welches die Neuroinflammation und Aβ-Pathologie miteinander verbindet. Das Nahrungsergänzungsmittel Spermidin ist ein bekannter Autophagie-Aktivator, welcher in verschiedenen Modellorganismen Langlebigkeit induziert. Hier haben wir den therapeutischen Effekt von Spermidin im AD-ähnlichen Mausmodell APPPS1 untersucht. Die orale Behandlung mit Spermidin führte zu einer Reduktion des neurotoxischen löslichen Aβ in der frühen und späten Pathologie der APPPS1 Mäuse. Die Einzel-Nukleus RNASequenzierung von Hemisphären der mit Spermidin behandelten APPPS1 Mäuse und H2OKontrollen ergab, dass Spermidin krankheitsassoziierte Mikroglia durch Erhöhung von Migrations- und Phagozytosegenen beeinflusst. Spermidin veränderte auch das Transkriptom von Oligodendrozyten. Ähnlich wie in Mikroglia modifizierte Spermidin auch in Oligodendrozyten Gene, die mit Entzündungshemmung, Proliferation, Transkription und dem Zytoskelett in Verbindung stehen und reduzierte zusätzlich Hitzeschockreaktionsgene. In-vitro-Behandlung von aktivierten Mikroglia, Astrozyten und Oligodendrozyten mit Spermidin verringerte die Freisetzung von Zytokinen dosisabhängig und bestätigte die entzündungshemmenden Wirkung von Spermidin. Neben der Transkriptomanalyse wurde eine Proteomanalyse von isolierten Mikroglia aus Spermidin behandelten APPPS1 Mäusen durchgeführt. Spermidin kehrte die AD-assoziierten Veränderungen in APPPS1 Mäusen um, indem es entzündungshemmende, zytoskelettale, autophagische und endozytische Effekte förderte. Eine tiefgreifende Analyse der entzündungshemmenden Wirkung von Spermidin in Mikroglia in vitro ergab, dass Spermidin gezielt die NF-κB-vermittelte Zytokintranskription nach LPS/ATP, poly I:C und Aβ Stimulation sowie das NLRP3-Inflammasom hemmte und so die Freisetzung von IL-1β und IL-18 reduzierte. Die entzündungshemmende Wirkung von Spermidin wurden durch eine ZNS-weite Reduktion der Zytokinspiegel in den Hemisphären der mit Spermidin behandelten APPPS1 Mäuse weiter untermauert. Als translationaler Ansatz wurden aktivierte Mikroglia aus menschlichen induzierten pluripotenten Stammzellen mit Spermidin behandelt, welches ebenfalls eine entzündungshemmende Wirkung von Spermidin zeigte. Zusammenfassend reduzierte Spermidin die gliavermittelte Neuroinflammation und induzierte Zellmigration, Autophagie und den Aβ-Abbau in Mikroglia von APPPS1 Mäusen, was zu einer Reduktion der löslichen Aβ-Konzentration führte. Somit stellt die Spermidin- Supplementierung einen faszinieren

Diverse but unique astrocytic phenotypes during embryonic stem cell differentiation, culturing and development

Astrocytes are resident glia cells of the central nervous system (CNS) that play complex and heterogeneous roles in brain development, homeostasis and disease. Since their vast involvement in health and disease is becoming increasingly recognized, suitable and reliable tools for studying these cells in vivo and in vitro are of utmost importance. One of the key challenges hereby is to adequately mimic their context-dependent in vivo phenotypes and functions in vitro. To better understand the spectrum of astrocytic variations in defined settings we performed a side-by-side-comparison of embryonic stem cell (ESC)-derived astrocytes as well as primary neonatal and adult astrocytes, revealing major differences on a functional and transcriptomic level, specifically on proliferation, migration, calcium signaling and cilium activity. Our results highlight the need to carefully consider the choice of astrocyte origin and phenotype with respect to age, isolation and culture protocols based on the respective biological question

Diverse but unique astrocytic phenotypes during embryonic stem cell differentiation, culturing and development

Functional and transcriptomic comparison of primary neonatal, adult and ESC-derived astrocytes reveals major variations, highlighting the importance for an informed choice of astrocyte origin for research questions

Development of Beta-Amyloid-Specific CAR-Tregs for the Treatment of Alzheimer’s Disease

Background: Alzheimer’s disease (AD) is a neurodegenerative disease that remains uncured. Its pathogenesis is characterized by the formation of β-amyloid (Aβ) plaques. The use of antigen-specific regulatory T cells (Tregs) through adoptive transfer has shown promise for the treatment of many inflammatory diseases, although the effectiveness of polyspecific Tregs is limited. Obtaining a sufficient number of antigen-specific Tregs from patients remains challenging. Aims and Methods: To address this problem, we used an antibody-like single-chain variable fragment from a phage library and subsequently generated a chimeric antigen receptor (CAR) targeting β-amyloid. Results: The β-amyloid-specific CARs obtained were stimulated by both recombinant and membrane-bound Aβ isolated from the murine brain. The generated CAR-Tregs showed a normal Treg phenotype, were antigen-specific activatable, and had suppressive capacity. Conclusion: This study highlights the potential of CAR technology to generate antigen-specific Tregs and presents novel approaches for developing functional CARs

Systems analysis of RhoGEF and RhoGAP regulatory proteins reveals spatially organized RAC1 signalling from integrin adhesions.

Rho GTPases are central regulators of the cytoskeleton and, in humans, are controlled by 145 multidomain guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). How Rho signalling patterns are established in dynamic cell spaces to control cellular morphogenesis is unclear. Through a family-wide characterization of substrate specificities, interactomes and localization, we reveal at the systems level how RhoGEFs and RhoGAPs contextualize and spatiotemporally control Rho signalling. These proteins are widely autoinhibited to allow local regulation, form complexes to jointly coordinate their networks and provide positional information for signalling. RhoGAPs are more promiscuous than RhoGEFs to confine Rho activity gradients. Our resource enabled us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling CDC42-RHOA crosstalk. Moreover, we show that integrin adhesions spatially segregate GEFs and GAPs to shape RAC1 activity zones in response to mechanical cues. This mechanism controls the protrusion and contraction dynamics fundamental to cell motility. Our systems analysis of Rho regulators is key to revealing emergent organization principles of Rho signalling