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The neurotrophin Brain-derived neurotrophic factor (BDNF) is encoded by multiple bipartite transcripts. Each BDNF transcript is composed by one out of 11 alternatively spliced exons containing the 5′untranslated region (UTR), and one common exon encompassing the coding sequence (CDS) and the 3′UTR with two variants (short and long). In neurons, BDNF mRNA variants have a distinct subcellular distribution, constituting a “spatial code”, with exon 1, 3, 5, 7 and 8 located in neuronal somata, exon 4 extending into proximal dendrites, and exon 2 and 6 reaching distal dendrites. We previously showed that the CDS encodes constitutive dendritic targeting signals (DTS) and that both the 3′UTR-short and the 3′UTR-long contain activity-dependent DTS. However, the role of individual 5′UTR exons in mRNA sorting remains unclear. Here, we tested the ability of each different BDNF 5′UTRs to affect the subcellular localization of the green fluorescent protein (GFP) reporter mRNA. We found that exon 2 splicing isoforms (2a, 2b, and 2c) induced a constitutive dendritic targeting of the GFP reporter mRNA towards distal dendritic segments. The other isoforms did not affect GFP-mRNA dendritic trafficking. Through a bioinformatic analysis, we identified five unique cis-elements in exon 2a, 2b, and 2c which might contribute to building a DTS. This study provides additional information on the mechanism regulating the cellular sorting of BDNF mRNA variants.

Cardiomyocyte proliferation stops at birth when the heart is no longer exposed to maternal blood and, likewise, to regulatory T cells (Tregs) that are expanded to promote maternal tolerance towards the fetus. Here, we report a role of Tregs in promoting cardiomyocyte proliferation. Treg-conditioned medium promotes cardiomyocyte proliferation, similar to the serum from pregnant animals. Proliferative cardiomyocytes are detected in the heart of pregnant mothers, and Treg depletion during pregnancy decreases both maternal and fetal cardiomyocyte proliferation. Treg depletion after myocardial infarction results in depressed cardiac function, massive inflammation, and scarce collagen deposition. In contrast, Treg injection reduces infarct size, preserves contractility, and increases the number of proliferating cardiomyocytes. The overexpression of six factors secreted by Tregs (Cst7, Tnfsf11, Il33, Fgl2, Matn2, and Igf2) reproduces the therapeutic effect. In conclusion, Tregs promote fetal and maternal cardiomyocyte proliferation in a paracrine manner and improve the outcome of myocardial infarction. Regulatory T cells (Tregs) expand during pregnancy to promote tolerance towards the fetus. Here the authors show that Tregs induce proliferation of fetal and maternal cardiomyocytes during pregnancy and enhance myocardial repair via proliferation-promoting paracrine actions.

Despite the high prevalence of ischemic heart diseases worldwide, no antibody-based treatment currently exists. Starting from the evidence that a specific isoform of the Bone Morphogenetic Protein 1 (BMP1.3) is particularly elevated in both patients and animal models of myocardial infarction, here we assess whether its inhibition by a specific monoclonal antibody reduces cardiac fibrosis. We find that this treatment reduces collagen deposition and cross-linking, paralleled by enhanced cardiomyocyte survival, both in vivo and in primary cultures of cardiac cells. Mechanistically, we show that the anti-BMP1.3 monoclonal antibody inhibits Transforming Growth Factor β pathway, thus reducing myofibroblast activation and inducing cardioprotection through BMP5. Collectively, these data support the therapeutic use of anti-BMP1.3 antibodies to prevent cardiomyocyte apoptosis, reduce collagen deposition and preserve cardiac function after ischemia. Here the authors show that a monoclonal antibody against a soluble isoform of Bone Morphogenetic Protein 1 prevents cardiac cell death, reducing fibrosis and preserving cardiac function after myocardial ischemia.

Brain Derived Neurotrophic Factor (BDNF) signalling contributes to the formation, maturation and plasticity of Central Nervous System (CNS) synapses. Acute exposure of cultured brain circuits to BDNF leads to up-regulation of glutamatergic neuro-transmission, by the accurate tuning of pre and post synaptic features, leading to structural and functional synaptic changes. Chronic BDNF treatment has been comparatively less investigated, besides it may represent a therapeutic option to obtain rescue of post-injury alterations of synaptic networks. In this study, we used a paradigm of BDNF long-term (4 days) incubation to assess in hippocampal neurons in culture, the ability of such a treatment to alter synapses. By patch clamp recordings we describe the augmented function of excitatory neurotransmission and we further explore by live imaging the presynaptic changes brought about by long-term BDNF. In our study, exogenous long-term BDNF exposure of post-natal neurons did not affect inhibitory neurotransmission. We further compare, by genetic manipulations of cultured neurons and BDNF release, intracellular overexpression of this neurotrophin at the same developmental age. We describe for the first-time differences in synaptic modulation by BDNF with respect to exogenous or intracellular release paradigms. Such a finding holds the potential of influencing the design of future therapeutic strategies.

Pulmonary fibrosis is a devastating disease, in which fibrotic tissue progressively replaces lung alveolar structure, resulting in chronic respiratory failure. Alveolar type II cells act as epithelial stem cells, being able to transdifferentiate into alveolar type I cells, which mediate gas exchange, thus contributing to lung homeostasis and repair after damage. Impaired epithelial transdifferentiation is emerging as a major pathogenetic mechanism driving both onset and progression of fibrosis in the lung. Here, we show that lung endothelial cells secrete angiocrine factors that regulate alveolar cell differentiation. Specifically, we build on our previous data on the anti-fibrotic microRNA-200c and identify the Vascular Endothelial Growth Factor receptor 1, also named Flt1 , as its main functional target in endothelial cells. Endothelial-specific knockout of Flt1 reproduces the anti-fibrotic effect of microRNA-200c against pulmonary fibrosis and results in the secretion of a pool of soluble factors and matrix components able to promote epithelial transdifferentiation in a paracrine manner. Collectively, these data indicate the existence of a complex endothelial-epithelial paracrine crosstalk in vitro and in vivo and position lung endothelial cells as a relevant therapeutic target in the fight against pulmonary fibrosis.

Background New drugs to tackle the next pathway or mutation fueling cancer are constantly proposed, but 97% of them are doomed to fail in clinical trials, largely because they are identified by cellular or in silico screens that cannot predict their in vivo effect. Methods We screened an Adeno-Associated Vector secretome library (> 1000 clones) directly in vivo in a mouse model of cancer and validated the therapeutic effect of the first hit, EMID2, in both orthotopic and genetic models of lung and pancreatic cancer. Results EMID2 overexpression inhibited both tumor growth and metastatic dissemination, consistent with prolonged survival of patients with high levels of EMID2 expression in the most aggressive human cancers. Mechanistically, EMID2 inhibited TGFβ maturation and activation of cancer-associated fibroblasts, resulting in more elastic ECM and reduced levels of YAP in the nuclei of cancer cells. Conclusion This is the first in vivo screening, precisely designed to identify proteins able to interfere with cancer cell invasiveness. EMID2 was selected as the most potent protein, in line with the emerging relevance of the tumor extracellular matrix in controlling cancer cell invasiveness and dissemination, which kills most of cancer patients.

Summary Therapies halting the progression of fibrosis are ineffective and limited. Activated myofibroblasts are emerging as important targets in the progression of fibrotic diseases. Previously, we performed a high-throughput screen on lung fibroblasts and subsequently demonstrated that the inhibition of myofibroblast activation is able to prevent lung fibrosis in bleomycin-treated mice. High-throughput screens are an ideal method of repurposing drugs, yet they contain an intrinsic limitation, which is the size of the library itself. Here, we exploited the data from our “wet” screen and used “dry” machine learning analysis to virtually screen millions of compounds, identifying novel anti-fibrotic hits which target myofibroblast differentiation, many of which were structurally related to dopamine. We synthesized and validated several compounds ex vivo (“wet”) and confirmed that both dopamine and its derivative TS1 are powerful inhibitors of myofibroblast activation. We further used RNAi-mediated knock-down and demonstrated that both molecules act through the dopamine receptor 3 and exert their anti-fibrotic effect by inhibiting the canonical transforming growth factor β pathway. Furthermore, molecular modelling confirmed the capability of TS1 to bind both human and mouse dopamine receptor 3. The anti-fibrotic effect on human cells was confirmed using primary fibroblasts from idiopathic pulmonary fibrosis patients. Finally, TS1 prevented and reversed disease progression in a murine model of lung fibrosis. Both our interdisciplinary approach and our novel compound TS1 are promising tools for understanding and combating lung fibrosis.

Nonhealing wounds place a significant burden on both quality of life of affected patients and health systems. Skin substitutes are applied to promote the closure of nonhealing wounds, although their efficacy is limited by inadequate vascularization. The stromal vascular fraction (SVF) from the adipose tissue is a promising therapy to overcome this limitation. Despite a few successful clinical trials, its incorporation in the clinical routine has been hampered by their inconsistent results. All these studies concluded by warranting pre-clinical work aimed at both characterizing the cell types composing the SVF and shedding light on their mechanism of action. Here, we established a model of nonhealing wound, in which we applied the SVF in combination with a clinical-grade skin substitute. We purified the SVF cells from transgenic animals to trace their fate after transplantation and observed that it gave rise to a mature vascular network composed of arteries, capillaries, veins, as well as lymphatics, structurally and functionally connected with the host circulation. Then we moved to a human-in-mouse model and confirmed that SVF-derived endothelial cells formed hybrid human-mouse vessels, that were stabilized by perivascular cells. Mechanistically, SVF-derived endothelial cells engrafted and expanded, directly contributing to the formation of new vessels, while a population of fibro-adipogenic progenitors stimulated the expansion of the host vasculature in a paracrine manner. These data have important clinical implications, as they provide a steppingstone toward the reproducible and effective adoption of the SVF as a standard care for nonhealing wounds.

Sorting of mRNAs in neuronal dendrites relies upon inducible transport mechanisms whose molecular bases are poorly understood. We investigated here the mechanism of inducible dendritic targeting of rat brain-derived neurotrophic factor (BDNF) mRNAs as a paradigmatic example. BDNF encodes multiple mRNAs with either short or long 3' UTR, both hypothesized to harbor inducible dendritic targeting signals. However, the mechanisms of sorting of the two 3' UTR isoforms are controversial. We found that dendritic localization of BDNF mRNAs with short 3' UTR was induced by depolarization and NT3 in vitro or by seizures in vivo and required CPEB-1, -2 and ELAV-2, -4. Dendritic targeting of long 3' UTR was induced by activity or BDNF and required CPEB-1 and the relief of soma-retention signals mediated by ELAV-1, -3, -4, and FXR proteins. Thus, long and short 3' UTRs, by using different sets of RNA-binding proteins provide a mechanism of selective targeting in response to different stimuli which may underlay distinct roles of BDNF variants in neuronal development and plasticity.

Dysregulated sensing of self–nucleic acid is a leading cause of autoimmunity in multifactorial and monogenic diseases. Mutations in Wiskott-Aldrich syndrome protein (WASp), a key regulator of cytoskeletal dynamics in immune cells, cause autoimmune manifestations and increased production of type I IFNs by innate cells. Here we show that immune complexes of self-DNA and autoantibodies (DNA-ICs) contribute to elevated IFN levels via activation of the cGAS/STING pathway of cytosolic sensing. Mechanistically, lack of endosomal F-actin nucleation by WASp caused a delay in endolysosomal maturation and prolonged the transit time of ingested DNA-ICs. Stalling in maturation-defective organelles facilitated leakage of DNA-ICs into the cytosol, promoting activation of the TBK1/STING pathway. Genetic deletion of STING and STING and cGAS chemical inhibitors abolished IFN production and rescued systemic activation of IFN-stimulated genes in vivo. These data unveil the contribution of cytosolic self–nucleic acid sensing in WAS and underscore the importance of WASp-mediated endosomal actin remodeling in preventing innate activation.