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Congenital hydrocephalus (CH), characterized by cerebral ventriculomegaly, is one of the most common reasons for pediatric brain surgery. Recent studies have implicated lin-41 (lineage variant 41)/TRIM71 (tripartite motif 71) as a candidate CH risk gene, however, TRIM71 variants have not been systematically examined in a large patient cohort or conclusively linked with an OMIM syndrome. Through cross-sectional analysis of the largest assembled cohort of patients with cerebral ventriculomegaly, including neurosurgically-treated CH (totaling 2,697 parent-proband trios and 8,091 total exomes), we identified 13 protein-altering de novo variants (DNVs) in TRIM71 in unrelated children exhibiting variable ventriculomegaly, CH, developmental delay, dysmorphic features, and other structural brain defects including corpus callosum dysgenesis and white matter hypoplasia. Eight unrelated patients were found to harbor arginine variants, including two recurrent missense DNVs, at homologous positions in RPXGV motifs of different NHL domains. Seven additional patients with rare, damaging, unphased or transmitted variants of uncertain significance were also identified. NHL-domain variants of TRIM71 exhibited impaired binding to the canonical TRIM71 target CDKN1A; other variants failed to direct the subcellular localization of TRIM71 to processing bodies. Single-cell transcriptomic analysis of human embryos revealed expression of TRIM71 in early first-trimester neural stem cells of the brain. These data show TRIM71 is essential for human brain morphogenesis and that TRIM71 mutations cause a novel neurodevelopmental syndrome featuring ventriculomegaly and CH., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The consumption of processed food is on the rise leading to huge intake of excess dietary salt, which strongly correlates with development of hypertension, often leading to cardiovascular diseases such as stroke and heart attack, as well as activation of the immune system. The effect of salt on macrophages is especially interesting as they are able to sense high sodium levels in tissues leading to transcriptional changes. In the skin, macrophages were shown to influence lymphatic vessel growth which, in turn, enables the transport of excess salt and thereby prevents the development of high blood pressure. Furthermore, salt storage in the skin has been linked to the onset of pro-inflammatory effector functions of macrophages in pathogen defence. However, there is only little known about the mechanisms which are involved in changing macrophage function to salt exposure. Here, we characterize the response of macrophages to excess salt both in vitro and in vivo. Our results validate and strengthen the notion that macrophages exhibit chemotactic migration in response to salt gradients in vitro. Furthermore, we demonstrate a reduction in phagocytosis and efferocytosis following acute salt challenge in vitro. While acute exposure to a high-salt diet in vivo has a less pronounced impact on macrophage core functions such as phagocytosis, our data indicate that prolonged salt challenge may exert a distinct effect on the function of macrophages. These findings suggest a potential role for excessive salt sensing by macrophages in the manifestation of diseases related to high-salt diets and explicitly highlight the need for in vivo work to decipher the physiologically relevant impact of excess salt on tissue and cell function., (© 2024. The Author(s).)

Cochlear hair cell loss is a leading cause of deafness in humans. Neighboring supporting cells have some capacity to regenerate hair cells. However, their regenerative potential sharply declines as supporting cells undergo maturation (postnatal day 5 in mice). We recently reported that reactivation of the RNA-binding protein LIN28B restores the hair cell-regenerative potential of P5 cochlear supporting cells. Here, we identify the LIN28B target Trim71 as a novel and equally potent enhancer of supporting cell plasticity. TRIM71 is a critical regulator of stem cell behavior and cell reprogramming; however, its role in cell regeneration is poorly understood. Employing an organoid-based assay, we show that TRIM71 re-expression increases the mitotic and hair cell-forming potential of P5 cochlear supporting cells by facilitating their de-differentiation into progenitor-like cells. Our mechanistic work indicates that TRIM71's RNA-binding activity is essential for such ability, and our transcriptomic analysis identifies gene modules that are linked to TRIM71 and LIN28B-mediated supporting cell reprogramming. Furthermore, our study uncovers that the TRIM71-LIN28B target Hmga2 is essential for supporting cell self-renewal and hair cell formation., (© 2023 The Authors.)

Centrosomes play a crucial role during immune cell interactions and initiation of the immune response. In proliferating cells, centrosome numbers are tightly controlled and generally limited to one in G1 and two prior to mitosis. Defects in regulating centrosome numbers have been associated with cell transformation and tumorigenesis. Here, we report the emergence of extra centrosomes in leukocytes during immune activation. Upon antigen encounter, dendritic cells pass through incomplete mitosis and arrest in the subsequent G1 phase leading to tetraploid cells with accumulated centrosomes. In addition, cell stimulation increases expression of polo-like kinase 2, resulting in diploid cells with two centrosomes in G1-arrested cells. During cell migration, centrosomes tightly cluster and act as functional microtubule-organizing centers allowing for increased persistent locomotion along gradients of chemotactic cues. Moreover, dendritic cells with extra centrosomes display enhanced secretion of inflammatory cytokines and optimized T cell responses. Together, these results demonstrate a previously unappreciated role of extra centrosomes for regular cell and tissue homeostasis., (© 2022 Weier et al.)

Navigation of dendritic cells (DCs) from the site of infection to lymphoid organs is guided by concentration gradients of CCR7 ligands. How cells interpret chemokine gradients and how they couple directional sensing to polarization and persistent chemotaxis has remained largely elusive. Previous experimental systems were limited in the ability to control fast de novo formation of the final gradient slope, long-lasting stability of the gradient and to expose cells to dynamic stimulation. Here, we used a combination of microfluidics and quantitative in vitro live cell imaging to elucidate the chemotactic sensing strategy of DCs. The microfluidic approach allows us to generate soluble gradients with high spatio-temporal precision and to analyze actin dynamics, cell polarization, and persistent directional migration in both static and dynamic environments. We demonstrate that directional persistence of DC migration requires steady-state characteristics of the soluble gradient instead of temporally rising CCL19 concentration, implying that spatial sensing mechanisms control chemotaxis of DCs. Kymograph analysis of actin dynamics revealed that the presence of the CCL19 gradient is essential to stabilize leading edge protrusions in DCs and to determine directionality, since both cytoskeletal polarization and persistent chemotaxis are abrogated in the range of seconds when steady-state gradients are perturbed. In contrast to Dictyostelium amoeba, DCs are unable to decode oscillatory stimulation of soluble chemokine traveling waves into a directional response toward the wave source. These findings are consistent with the notion that DCs do not employ adaptive temporal sensing strategies that discriminate temporally increasing and decreasing chemoattractant concentrations in our setting. Taken together, in our experimental system DCs do not depend on increasing absolute chemokine concentration over time to induce persistent migration and do not integrate oscillatory stimulation. The observed capability of DCs to migrate with high directional persistence in stable gradients but not when subjected to periodic temporal cues, identifies spatial sensing as a key requirement for persistent chemotaxis of DCs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Quast, Zölzer, Guu, Alvarez, Küsters, Kiermaier, Kaupp and Kolanus.)

Cationic liposomes are versatile lipid nanocarriers to improve the pharmacological properties of drug payloads. Recent advantages include the application of their intrinsic immunostimulatory effects to enhance immune activation. Herein, we report for the first time the structural effect of cationic lipids in promoting T cell activation and differentiation in vitro. Two types of cationic liposomes R3C14 and R5C14 were prepared from single type of lipids Arg-C3-Clu2C14 or Arg-C5-Clu2C14, which bear arginine head group and ditetradecyl tails but vary in the carbon number of the spacer in between. Murine CD8 or CD4 T cells were pretreated with 50 μM of each type of liposomes for 2 h, followed by stimulation with anti-CD3/CD28 antibodies for 24 h. In comparison to liposome-untreated T cells, R5C14-pretreatment induced a robust T cell activation (IL-2, CD25 + ) and differentiation into effector cells (CD44 high , CD62L low ), whereas R3C14 did not show comparable effect. Furthermore, a weak activation of nuclear factor of activated T cells (NFAT) was detected in Jurkat-Lucia NFAT cells (InvivoGen), suggesting a potential signaling pathway for the liposomal effect. Although R5C14 liposomes did not activate T cells without subsequent CD3/CD28 stimulation, this study implied a recessive effect of some cationic adjuvant in priming T cells to enhance their responsiveness to antigens., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Hydrocephalus, characterized by cerebral ventricular dilatation, is routinely attributed to primary defects in cerebrospinal fluid (CSF) homeostasis. This fosters CSF shunting as the leading reason for brain surgery in children despite considerable disease heterogeneity. In this study, by integrating human brain transcriptomics with whole-exome sequencing of 483 patients with congenital hydrocephalus (CH), we found convergence of CH risk genes in embryonic neuroepithelial stem cells. Of all CH risk genes, TRIM71/lin-41 harbors the most de novo mutations and is most specifically expressed in neuroepithelial cells. Mice harboring neuroepithelial cell-specific Trim71 deletion or CH-specific Trim71 mutation exhibit prenatal hydrocephalus. CH mutations disrupt TRIM71 binding to its RNA targets, causing premature neuroepithelial cell differentiation and reduced neurogenesis. Cortical hypoplasia leads to a hypercompliant cortex and secondary ventricular enlargement without primary defects in CSF circulation. These data highlight the importance of precisely regulated neuroepithelial cell fate for normal brain-CSF biomechanics and support a clinically relevant neuroprogenitor-based paradigm of CH., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Cell survival, tissue integrity and organismal health depend on the ability to maintain functional protein networks even under conditions that threaten protein integrity. Protection against such stress conditions involves the adaptation of folding and degradation machineries, which help to preserve the protein network by facilitating the refolding or disposal of damaged proteins. In multicellular organisms, cells are permanently exposed to stress resulting from mechanical forces. Yet, for long time mechanical stress was not recognized as a primary stressor that perturbs protein structure and threatens proteome integrity. The identification and characterization of protein folding and degradation systems, which handle force-unfolded proteins, marks a turning point in this regard. It has become apparent that mechanical stress protection operates during cell differentiation, adhesion and migration and is essential for maintaining tissues such as skeletal muscle, heart and kidney as well as the immune system. Here, we provide an overview of recent advances in our understanding of mechanical stress protection., (© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Interferon (IFN)-induced guanosine triphosphate hydrolysing enzymes (GTPases) have been identified as cornerstones of IFN-mediated cell-autonomous defence. Upon IFN stimulation, these GTPases are highly expressed in various host cells, where they orchestrate anti-microbial activities against a diverse range of pathogens such as bacteria, protozoan and viruses. IFN-induced GTPases have been shown to interact with various host pathways and proteins mediating pathogen control via inflammasome activation, destabilising pathogen compartments and membranes, orchestrating destruction via autophagy and the production of reactive oxygen species as well as inhibiting pathogen mobility. In this mini-review, we provide an update on how the IFN-induced GTPases target pathogens and mediate host defence, emphasising findings on protection against bacterial pathogens., (© 2021 The Author(s).)

Three-dimensional (3D) culture bridges and minimizes the gap between in vitro and in vivo states of cells and various 3D culture systems have been developed according to different approaches. However, most of these approaches are either complicated to operate, or costive to scale up. Therefore, a simple method for stem cell spheroid formation and preservation was proposed using poly(D,L-lactic acid) porous thin film (porous nanosheet), which were fabricated by a roll-to-roll gravure coating method combining a solvent etching process. The obtained porous nanosheet was less than 200 nm in thickness and had an average pore area of 6.6 μm 2 with a porosity of 0.887. It offered a semi-adhesive surface for stem cells to form spheroids and maintained the average spheroid diameter below 100 μm for 5 days. In comparison to the spheroids formed in suspension culture, the porous nanosheets improved cell viability and cell division rate, suggesting the better feasibility to be applied as 3D culture scaffolds., Competing Interests: ST was an inventor of patent (PCT/JP2013/056823) of porous nanosheets and collaborating with Nanotheta Co., Ltd., which is holding the patent. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tsai, Li, Torres-Fernández, Weise, Kolanus and Takeoka.)

Mutations affecting the germline can result in infertility or the generation of germ cell tumors (GCT), highlighting the need to identify and characterize the genes controlling germ cell development. The RNA-binding protein and E3 ubiquitin ligase TRIM71 is essential for embryogenesis, and its expression has been reported in GCT and adult mouse testes. To investigate the role of TRIM71 in mammalian germ cell embryonic development, we generated a germline-specific conditional Trim71 knockout mouse (cKO) using the early primordial germ cell (PGC) marker Nanos3 as a Cre-recombinase driver. cKO mice are infertile, with male mice displaying a Sertoli cell-only (SCO) phenotype which in humans is defined as a specific subtype of non-obstructive azoospermia characterized by the absence of germ cells in the seminiferous tubules. Infertility in male Trim71 cKO mice originates during embryogenesis, as the SCO phenotype was already apparent in neonatal mice. The in vitro differentiation of mouse embryonic stem cells (ESCs) into PGC-like cells (PGCLCs) revealed reduced numbers of PGCLCs in Trim71 -deficient cells. Furthermore, TCam-2 cells, a human GCT-derived seminoma cell line which was used as an in vitro model for PGCs, showed proliferation defects upon TRIM71 knockdown. Additionally, in vitro growth competition assays, as well as proliferation assays with wild type and CRISPR/Cas9-generated TRIM71 mutant NCCIT cells showed that TRIM71 also promotes proliferation in this malignant GCT-derived non-seminoma cell line. Importantly, the PGC-specific markers BLIMP1 and NANOS3 were consistently downregulated in Trim71 KO PGCLCs, TRIM71 knockdown TCam-2 cells and TRIM71 mutant NCCIT cells. These data collectively support a role for TRIM71 in PGC development. Last, via exome sequencing analysis, we identified several TRIM71 variants in a cohort of infertile men, including a loss-of-function variant in a patient with an SCO phenotype. Altogether, our work reveals for the first time an association of TRIM71 deficiency with human male infertility, and uncovers further developmental roles for TRIM71 in the germline during mouse embryogenesis., Competing Interests: LT-F held a stipend which was donated by Bayer AG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Torres-Fernández, Emich, Port, Mitschka, Wöste, Schneider, Fietz, Oud, Di Persio, Neuhaus, Kliesch, Hölzel, Schorle, Friedrich, Tüttelmann and Kolanus.)

The stem cell-specific RNA-binding protein TRIM71/LIN-41 was the first identified target of the pro-differentiation and tumor suppressor miRNA let-7. TRIM71 has essential functions in embryonic development and a proposed oncogenic role in several cancer types, such as hepatocellular carcinoma. Here, we show that TRIM71 regulates let-7 expression and activity via two independent mechanisms. On the one hand, TRIM71 enhances pre-let-7 degradation through its direct interaction with LIN28 and TUT4, thereby inhibiting let-7 maturation and indirectly promoting the stabilization of let-7 targets. On the other hand, TRIM71 represses the activity of mature let-7 via its RNA-dependent interaction with the RNA-Induced Silencing Complex (RISC) effector protein AGO2. We found that TRIM71 directly binds and stabilizes let-7 targets, suggesting that let-7 activity inhibition occurs on active RISCs. MiRNA enrichment analysis of several transcriptomic datasets from mouse embryonic stem cells and human hepatocellular carcinoma cells suggests that these let-7 regulatory mechanisms shape transcriptomic changes during developmental and oncogenic processes. Altogether, our work reveals a novel role for TRIM71 as a miRNA repressor and sheds light on a dual mechanism of let-7 regulation., (Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Inflammation and infection can trigger local tissue Na+ accumulation. This Na+-rich environment boosts proinflammatory activation of monocyte/macrophage-like cells (MΦs) and their antimicrobial activity. Enhanced Na+-driven MΦ function requires the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5), which augments nitric oxide (NO) production and contributes to increased autophagy. However, the mechanism of Na+ sensing in MΦs remained unclear. High extracellular Na+ levels (high salt [HS]) trigger a substantial Na+ influx and Ca2+ loss. Here, we show that the Na+/Ca2+ exchanger 1 (NCX1, also known as solute carrier family 8 member A1 [SLC8A1]) plays a critical role in HS-triggered Na+ influx, concomitant Ca2+ efflux, and subsequent augmented NFAT5 accumulation. Moreover, interfering with NCX1 activity impairs HS-boosted inflammatory signaling, infection-triggered autolysosome formation, and subsequent antibacterial activity. Taken together, this demonstrates that NCX1 is able to sense Na+ and is required for amplifying inflammatory and antimicrobial MΦ responses upon HS exposure. Manipulating NCX1 offers a new strategy to regulate MΦ function., Competing Interests: The authors have declared that no competing interests exist.

Nonsense-mediated decay (NMD) plays a fundamental role in the degradation of premature termination codon (PTC)-containing transcripts, but also regulates the expression of functional transcripts lacking PTCs, although such 'non-canonical' functions remain ill-defined and require the identification of factors targeting specific mRNAs to the NMD machinery. Our work identifies the stem cell-specific mRNA repressor protein TRIM71 as one of these factors. TRIM71 plays an essential role in embryonic development and is linked to carcinogenesis. For instance, TRIM71 has been correlated with advanced stages and poor prognosis in hepatocellular carcinoma. Our data shows that TRIM71 represses the mRNA of the cell cycle inhibitor and tumor suppressor CDKN1A/p21 and promotes the proliferation of HepG2 tumor cells. CDKN1A specific recognition involves the direct interaction of TRIM71 NHL domain with a structural RNA stem-loop motif within the CDKN1A 3'UTR. Importantly, CDKN1A repression occurs independently of miRNA-mediated silencing. Instead, the NMD factors SMG1, UPF1 and SMG7 assist TRIM71-mediated degradation of CDKN1A mRNA, among other targets. Our data sheds light on TRIM71-mediated target recognition and repression mechanisms and uncovers a role for this stem cell-specific factor and oncogene in non-canonical NMD, revealing the existence of a novel mRNA surveillance mechanism which we have termed the TRIM71/NMD axis., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Insulin plays a central role in regulating metabolic homeostasis and guanine-nucleotide exchange factors of the cytohesin family have been suggested to be involved in insulin signal transduction. The Drosophila homolog of cytohesin-3, steppke, has been shown to be essential for insulin signaling during larval development. However, genetic evidence for the functional importance of cytohesin-3 in mammals is missing. We therefore analyzed the consequences of genetic cytohesin-3-deficiency on insulin signaling and function in young and aged mice, using normal chow or high-fat diet (HFD). Insulin-receptor dependent signaling events are significantly reduced in liver and adipose tissue of young cytohesin-3-deficient mice after insulin-injection, although blood glucose levels and other metabolic parameters remain normal in these animals. Interestingly, however, cytohesin-3-deficient mice showed a reduced age- and HFD-induced weight gain with a significant reduction of body fat compared to wild-type littermates. Furthermore, cytohesin-3-deficient mice on HFD displayed no alterations in energy expenditure, but had an increased lipid excretion instead, as well as a reduced expression of genes essential for bile acid synthesis. Our findings show for the first time that an intact cyth3 locus is required for full insulin signaling in mammals and might constitute a novel therapeutic target for weight reduction.