Beclin1-driven autophagy modulates the inflammatory response of microglia via NLRP3.

Alzheimer's disease is characterized not only by extracellular amyloid plaques and neurofibrillary tangles, but also by microglia-mediated neuroinflammation. Recently, autophagy has been linked to the regulation of the inflammatory response. Thus, we investigated how an impairment of autophagy mediated by BECN1/Beclin1 reduction, as described in Alzheimer's disease patients, would influence cytokine production of microglia. Acutely stimulated microglia from Becn1+/- mice exhibited increased expression of IL-1beta and IL-18 compared to wild-type microglia. Becn1+/-APPPS1 mice also contained enhanced IL-1beta levels. The investigation of the IL-1beta/IL-18 processing pathway showed an elevated number of cells with inflammasomes and increased levels of NLRP3 and cleaved CASP1/Caspase1 in Becn1+/- microglia. Super-resolation microscopy revealed a very close association of NLRP3 aggregates and LC3-positive vesicles. Interestingly, CALCOCO2 colocalized with NLRP3 and its downregulation increased IL-1beta release. These data support the notion that selective autophagy can impact microglia activation by modulating IL-1beta and IL-18 production via NLRP3 degradation and thus present a mechanism how impaired autophagy could contribute to neuroinflammation in Alzheimer's disease.

Spermidine reduces neuroinflammation and soluble amyloid beta in an Alzheimer's disease mouse model.

BACKGROUND: Deposition of amyloid beta (Aß) and hyperphosphorylated tau along with glial cell-mediated neuroinflammation are prominent pathogenic hallmarks of Alzheimer's disease (AD). In recent years, impairment of autophagy has been identified as another important feature contributing to AD progression. Therefore, the potential of the autophagy activator spermidine, a small body-endogenous polyamine often used as dietary supplement, was assessed on Aß pathology and glial cell-mediated neuroinflammation. RESULTS: Oral treatment of the amyloid prone AD-like APPPS1 mice with spermidine reduced neurotoxic soluble Aß and decreased AD-associated neuroinflammation. Mechanistically, single nuclei sequencing revealed AD-associated microglia to be the main target of spermidine. This microglia population was characterized by increased AXL levels and expression of genes implicated in cell migration and phagocytosis. A subsequent proteome analysis of isolated microglia confirmed the anti-inflammatory and cytoskeletal effects of spermidine in APPPS1 mice. In primary microglia and astrocytes, spermidine-induced autophagy subsequently affected TLR3- and TLR4-mediated inflammatory processes, phagocytosis of Aß and motility. Interestingly, spermidine regulated the neuroinflammatory response of microglia beyond transcriptional control by interfering with the assembly of the inflammasome. CONCLUSIONS: Our data highlight that the autophagy activator spermidine holds the potential to enhance Aß degradation and to counteract glia-mediated neuroinflammation in AD pathology.

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

Astrocytes are resident glial 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 murine 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.

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 ß-amyloid (Aß) 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 ß-amyloid. RESULTS: The ß-amyloid-specific CARs obtained were stimulated by both recombinant and membrane-bound Aß 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.

NanoString technology distinguishes anti-TIF-1γ+ from anti-Mi-2+ dermatomyositis patients.

Dermatomyositis (DM) is a systemic idiopathic inflammatory disease affecting skeletal muscle and skin, clinically characterized by symmetrical proximal muscle weakness and typical skin lesions. Recently, myositis-specific autoantibodies (MSA) became of utmost importance because they strongly correlate with distinct clinical manifestations and prognosis. Antibodies against transcription intermediary factor 1γ (TIF-1γ) are frequently associated with increased risk of malignancy, a specific cutaneous phenotype and limited response to therapy in adult DM patients. Anti-Mi-2 autoantibodies, in contrast, are typically associated with classic DM rashes, prominent skeletal muscle weakness, better therapeutic response and prognosis, and less frequently with cancer. Nevertheless, the sensitivity of autoantibody testing is only moderate, and alternative reliable methods for DM patient stratification and prediction of cancer risk are needed. To further investigate these clinically distinct DM subgroups, we herein analyzed 30 DM patients (n = 15 Mi-2+ and n = 15 TIF-1 γ+ ) and n = 8 non-disease controls (NDC). We demonstrate that the NanoString technology can be used as a very sensitive method to clearly differentiate these two clinically distinct DM subgroups. Using the nCounter PanCancer Immune Profiling Panel™, we identified a set of significantly dysregulated genes in anti-TIF-1γ+ patient muscle biopsies including VEGFA, DDX58, IFNB1, CCL5, IL12RB2, and CD84. Investigation of type I IFN-regulated transcripts revealed a striking type I interferon signature in anti-Mi-2+ patient biopsies. Our results help to stratify both subgroups and predict, which DM patients require an intensified diagnostic procedure and might have a poorer outcome. Potentially, this could also have implications for the therapeutic approach.

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.