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Biology subjects

Freitag, K.

Publications and source records attributed to Freitag, K..

8 recordsLinked to original sources

SALL1 and neural microenvironmental contextspecify identity of stem cell derived humanmicroglia-like cells

Microglia, the immune cells of the brain parenchyma, play a pivotal role in neurodevelopment and neuroprotection. Human stem cell-derived microglia-like cells (MGL) offer a valuable, human-relevant system to study microglia in vitro. However, their cell states and intercellular interactions within complex 3-dimensional (3D) tissue contexts in vitro remain underexplored. Here, we analyze the gene expression profiles of MGL generated by two differentiation protocols in conventional 2D cultures. We show that co-culture with neural organoids promotes a more mature, in vivo-like MGL phenotype but this signature is lost during long-term co-culture. By varying media compositions, we modulate 2D MGL states toward more active or more homeostatic identities, which may enhance the similarity to primary microglia identities upon subsequent co-culture. Moreover, forced expression of the microglial lineage regulator SALL1 shifts MGL away from a general myeloid state toward a more defined pre-microglial fate. SALL1 overexpression within a 3D neural organoid environment further amplifies the shift toward a homeostatic developmental microglia population. Collectively, our transcriptomic analysis delineates human MGL states in 2D and 3D contexts, refines existing differentiation protocols toward more authentic MGL, and establishes a framework to investigate the intricate microglia-neural tissue interplay.

developmental biology↗

Synovium-Restricted Armored PD-1-Targeted CAR-T Cells Reprogram Immunity and Resolve Experimental Arthritis

Despite major therapeutic advances, a substantial fraction of patients with autoimmune disease remains refractory to treatment. While B cell-targeted CAR-T therapies have shown considerable efficacy, the central contribution of pathogenic T cells to rheumatoid arthritis (RA) suggests that complementary T cell-directed strategies may enable deeper disease control. Using single-cell multi-omics of human RA and experimental models, PDCD1 was identified as a selective marker of synovial disease-associated T cells. We developed PD-1-directed CAR-T cells that potently eliminate these cells in vitro and in vivo, leading to marked attenuation of synovitis in RA models. To limit off-target activity, we engineered NR4A2-driven CAR-responsive biosensors to restrict CAR activity to inflamed synovium. To couple anti-PD-1 CAR-mediated cytotoxicity with microenvironmental modulation, we further engineered these CAR-T cells to secrete soluble TNF receptor II (sTNFRii), counteracting baseline inflammation and CAR-induced IFN response and promoting a tissue-reparative myeloid state. PD-1-targeted CAR-T therapy thus represents a promising, specific, and safe strategy for autoimmune diseases involving disease-associated T cells.

immunology↗

RNA promotes synapsin coacervation and modulates local translation

Condensates at synapses organize synaptic vesicle (SV) clusters and are essential for efficient neurotransmitter release. While it is established that RNA granules traffic along the axons, the function of RNA at the presynapse remains unclear. Here, we uncover a direct structural role of coding RNAs in organizing presynaptic condensates by focusing on SV clusters, condensates between synapsin-1 and lipid vesicles, a defining feature of nerve terminals. Using in vitro reconstitution systems, we show that RNA drives synapsin-1 coacervation, with bias toward structured RNAs being more effective at promoting phase transitions. The importance of RNA was confirmed in living synapses, where acute disruption of native RNA induces a dispersion of SVs and synapsin. Conversely, ectopically expressed SV-like condensates have the ability to recruit the translational machinery. The microscopy-based in vitro translation assay demonstrates increased translation efficiency within synapsin-1/RNA condensates. Together, our work indicates a novel structural role of RNAs in modulating SV condensates.

cell biology↗

Immunotherapy of cytomegalovirus infection by low-dose adoptive transfer of antiviral CD8 T cells relies on substantial post-transfer expansion of central memory cells but not effector-memory cells

Cytomegaloviruses (CMVs) are host species-specific in their replication. It is a hallmark of all CMVs that productive primary infection is controlled by concerted innate and adaptive immune responses in the immunocompetent host. As a result, the infection usually passes without overt clinical symptoms and develops into latent infection, referred to as latency. During latency, the virus is maintained in a non-replicative state from which it can reactivate to productive infection under conditions of waning immune surveillance. In contrast, infection of an immunocompromised host causes CMV disease with viral multiple-organ histopathology resulting in organ failure. Primary or reactivated CMV infection of hematopoietic cell transplantation (HCT) recipients in a "window of risk" between therapeutic hematoablative leukemia therapy and immune system reconstitution remains a clinical challenge. Studies in the mouse model of experimental HCT and infection with murine CMV (mCMV), followed by clinical trials in HCT patients with human CMV (hCMV) reactivation, have revealed a protective function of virus-specific CD8 T cells upon adoptive cell transfer (AT). Memory CD8 T cells derived from latently infected hosts are a favored source for immunotherapy by AT. Strikingly low numbers of these cells were found to prevent CMV disease, suggesting either an immediate effector function of few transferred cells or a clonal expansion generating high numbers of effector cells. In the murine model, the memory population consists of resting central memory T cells (TCM), as well as of conventional effector-memory T cells (cTEM) and inflationary effector-memory T cells (iTEM). iTEM, increase in numbers over time in the latently infected host, a phenomenon known as memory inflation (MI). They thus appeared to be a promising source for use in immunotherapy. However, we show here that iTEM contribute little to the control of infection after AT, which rests almost exclusively on a superior proliferation potential of TCM. Author SummaryImmunotherapy of reactivated cytomegalovirus infection in immunocompromised HCT recipients by adoptive transfer (AT) of antiviral CD8 T cells is the last resort to fight virus variants that have acquired resistance to standard antiviral drugs. Provision of cell numbers high enough for clearance of productive infection remains a logistical limitation for AT to become clinical routine. Although use of donor memory CD8 T cells has become the standard in clinical AT, little is known about the relative antiviral efficacies of memory CD8 T-cell activation subsets, such as central memory cells (TCM) and different populations of effector-memory cells (TEM). A reliable quantitative comparison of the antiviral efficacies of memory CD8 T-cell subsets is precluded in clinical investigation, because independent cohorts of AT donors and AT recipients unavoidably differ in many genetical, immunological, and virological variables. Therefore, this is a question for which a preclinical animal model is predestined. We show here in the well-established mouse model of low-dose AT that CMV infection is by far most efficiently controlled by virus-specific TCM, based on a superior potential to proliferate even in extra-lymphoid tissue to prevent virus spread. For clinical AT, our data provide an argument to favor transfer of sorted TCM rather than TEM.

immunology↗

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

Deposition of amyloid beta (A{beta}) along with glia cell-mediated neuroinflammation are prominent pathogenic hallmarks of Alzheimers disease (AD). In recent years, impairment of autophagy has been found to be another important feature, contributing to AD progression and aging. Therefore, we assessed the effect of the autophagy activator Spermidine, a small body-endogenous polyamine often used as dietary supplement and known to promote longevity, on glia cell-mediated neuroinflammation. Spermidine reduced TLR3- and TLR4- mediated inflammatory processes in microglia and astrocytes by decreasing cytotoxicity, inflammasome activity and NF-{kappa}B signaling. In line with these anti-inflammatory effects, oral treatment of the amyloid prone AD-like APPPS1 mice with Spermidine reduced neuroinflammation and neurotoxic soluble A{beta}. Mechanistically, single nuclei sequencing revealed microglia as one of the main targets of Spermidine treatment, with increased expression of genes implicated in cell motility and phagocytosis. Thus, Spermidine provides a promising therapeutic potential to target glia cells in AD progression.

neuroscience↗

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

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.

neuroscience↗

The autophagy activator Spermidine ameliorates Alzheimer's disease pathology and neuroinflammation in mice

Deposition of amyloid beta (A{beta}) and phosphorylated Tau along with microglia- and astrocyte-mediated neuroinflammation are prominent pathogeneic features of Alzheimers Disease (AD). In recent years, impairment of autophagy has also been shown to contribute to AD progression. Here, we provide evidence that oral treatment of amyloid-prone AD-like APPPS1 mice with the autophagy activator Spermidine, a small body-endogenous polyamine often used as dietary supplement, decreased neuroinflammation and reduced neurotoxic soluble A{beta} at both early and late stages of AD. Mechanistically, Spermidine induced autophagy in microglia as well as in astrocytes, which subsequently impacted TLR3- and TLR4-mediated inflammatory processes by decreasing cytotoxicity, inflammasome activity and NF-{kappa}B signalling. Our data highlight that autophagy targets the inflammatory response of glial cells and emphasize the potential of orally administered autophagy-activating drugs such as Spermidine to interfere with AD progression.

neuroscience↗

Systematic Characterization of RhoGEF/RhoGAP Regulatory Proteins Reveals Organization Principles of Rho GTPase Signaling

Rho GTPases control cell morphogenesis and thus fundamental processes in all eukaryotes. They are regulated by 145 RhoGEF and RhoGAP multi-domain proteins in humans. How the Rho signaling system is organized to generate localized responses in cells and prevent their spreading is not understood. Here, we systematically characterized the substrate specificities, localization and interactome of the RhoGEFs/RhoGAPs and revealed their critical role in contextualizing and spatially delimiting Rho signaling. They localize to multiple compartments providing positional information, are extensively interconnected to jointly coordinate their signaling networks and are widely autoinhibited to remain sensitive to local activation. RhoGAPs exhibit lower substrate specificity than RhoGEFs and may contribute to preserving Rho activity gradients. Our approach led us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling a Cdc42/RhoA crosstalk. The spatial organization of Rho signaling thus differs from other small GTPases and expands the repertoire of mechanisms governing localized signaling activity.

cell biology↗