Transmission of reduced levels of miR-34/449 from sperm to preimplantation embryos is a key step in the transgenerational epigenetic inheritance of the effects of paternal chronic social instability stress.

The transgenerational effects of exposing male mice to chronic social instability (CSI) stress are associated with decreased sperm levels of multiple members of the miR-34/449 family that persist after their mating through preimplantation embryo (PIE) development. Here we demonstrate the importance of these miRNA changes by showing that restoring miR-34c levels in PIEs derived from CSI stressed males prevents elevated anxiety and defective sociability normally found specifically in their adult female offspring. It also restores, at least partially, levels of sperm miR-34/449 normally reduced in their male offspring who transmit these sex-specific traits to their offspring. Strikingly, these experiments also revealed that inducing miR-34c levels in PIEs enhances the expression of its own gene and that of miR-449 in these cells. The same induction of embryo miR-34/449 gene expression likely occurs after sperm-derived miR-34c is introduced into oocytes upon fertilization. Thus, suppression of this miRNA amplification system when sperm miR-34c levels are reduced in CSI stressed mice can explain how a comparable fold-suppression of miR-34/449 levels can be found in PIEs derived from them, despite sperm containing ~50-fold lower levels of these miRNAs than those already present in PIEs. We previously found that men exposed to early life trauma also display reduced sperm levels of miR-34/449. And here we show that miR-34c can also increase the expression of its own gene, and that of miR-449 in human embryonic stem cells, suggesting that human PIEs derived from men with low sperm miR-34/449 levels may also contain this potentially harmful defect.

Semi-Transparent Luminescent Solar Concentrators Based on Intramolecular Energy Transfer in Polyurethane Matrices.

Luminescent solar concentrators (LSCs) are spectral conversion devices offering interesting opportunities for the integration of photovoltaics into the built environment and portable systems. The Förster-resonance energy transfer (FRET) process can boost the optical response of LSCs by reducing energy losses typically associated to non-radiative processes occurring within the device under operation. In this work, a new class of FRET-based thin-film LSC devices is presented, in which the synthetic versatility of linear polyurethanes (PU) is exploited to control the photophysical properties and the device performance of the resulting LSCs. A series of luminescent linear PUs are synthesized in the presence of two novel bis-hydroxyl-functionalized luminophores of suitable optical properties, used as chain extenders during the step-growth polyaddition reaction for the formation of the linear macromolecular network. By synthetically tuning their composition, the obtained luminescent PUs can achieve a high energy transfer efficiency (≈90%) between the covalently linked luminophores. The corresponding LSC devices exhibit excellent photonic response, with external and internal photon efficiencies as high as ≈4% and ≈37%, respectively. Furthermore, their optimized power conversion efficiency combined with their enhanced average visible-light transmittance highlight their suitability for potential use as transparent solar energy devices.

Preimplantation Embryos Amplify Stress-Induced Changes in Sperm-Derived miRNA Content to Mediate Transgenerational Epigenetic Inheritance.

Chronically stressing male mice can alter the behavior of their offspring across generations. This effect is thought to be mediated by stress-induced changes in the content of specific sperm miRNAs that modify embryo development after their delivery to oocytes at fertilization. A major problem with this hypothesis is that the levels of mouse sperm miRNAs are much lower than those present in preimplantation embryos. This makes it unclear how embryos could be significantly impacted without an amplification system to magnify changes in sperm miRNA content, like those present in lower organisms where transgenerational epigenetic inheritance is well established. Here, we describe such a system for Chronic Social Instability (CSI) stress that can explain how it reduces the levels of the miR-34b,c/449a,b family of miRNAs not only in sperm of exposed males but also in preimplantation embryos ( PIEs ) derived from their mating, as well as in sperm of male offspring. Sperm-derived miR-34c normally positively regulates expression of its own gene and that of miR-449 in PIEs. This feed forward, auto-amplification process is suppressed when CSI stress reduces sperm miR-34c levels. Its suppression is important for the transmission of traits to offspring because restoring miR-34c levels in PIEs from CSI stressed males, which also restores levels of miR-449 in them, suppresses elements of elevated anxiety and defective sociability normally found specifically in their female offspring, as well as reduced sperm miR-34 and miR-449 levels normally found in male offspring, who pass on these traits to their offspring. We previously published that the content of sperm miR-34/449 is also reduced in men raised in highly abusive and/or dysfunctional families. We show here that a similar miRNA auto-amplification system functions in human embryonic stem cells. This raises the possibility that PIEs in offspring of these men also display reduced levels of miR-34/449, enhancing the potential translational significance of these studies.

microRNAs transcriptional profiling of mammary stem cells isolated by PKH26 staining and FACS sorting.

MicroRNAs (miRNAs) are an evolutionarily conserved class of small (18-22 nucleotides) noncoding RNAs involved in the regulation of a variety of cellular and developmental processes. MiRNA expression is frequently altered in human cancers compared to normal tissues, potentially contributing to tumorigenesis. Generally, high-throughput profiles of miRNA expression levels are generated using bulk samples, from both normal and cancer tissues. However, cancer tissues are quite heterogeneous and might contain subpopulations critical for tumor development, i.e., cancer stem cells (CSCs) or tumor-initiating cells (TICs) with aberrant stem-like features, such as unlimited self-renewal potential. The isolation of these aberrant subpopulations from solid tumors is a relatively recent achievement, with breast cancer being one of the first solid human cancers in which CSCs have been identified and biologically characterized. Here, we describe a new methodology that can overcome the main challenge in dealing with rare cells such as SCs/CSCs, represented by the paucity of the starting material. Based on previously published protocols, used by both our and other research groups, we used the FACS-sorting approach to isolate mammary normal and cancer stem cells based on the amount of PKH26 fluorescent dye they retained. Depending on the number of SCs/CSCs isolated, we established two different protocols for the reliable and analytically sensitive detection of up to 384 miRNAs using the Taqman Low Density Array (TLDA) platform.

A multifunctional locus controls motor neuron differentiation through short and long noncoding RNAs.

The transition from dividing progenitors to postmitotic motor neurons (MNs) is orchestrated by a series of events, which are mainly studied at the transcriptional level by analyzing the activity of specific programming transcription factors. Here, we identify a post-transcriptional role of a MN-specific transcriptional unit (MN2) harboring a lncRNA (lncMN2-203) and two miRNAs (miR-325-3p and miR-384-5p) in this transition. Through the use of in vitro mESC differentiation and single-cell sequencing of CRISPR/Cas9 mutants, we demonstrate that lncMN2-203 affects MN differentiation by sponging miR-466i-5p and upregulating its targets, including several factors involved in neuronal differentiation and function. In parallel, miR-325-3p and miR-384-5p, co-transcribed with lncMN2-203, act by repressing proliferation-related factors. These findings indicate the functional relevance of the MN2 locus and exemplify additional layers of specificity regulation in MN differentiation.

Prediction and pan-cancer analysis of mammalian transcripts involved in target directed miRNA degradation.

It is currently unknown how many RNA transcripts are able to induce degradation of microRNAs (miRNA) via the mechanism known as target-directed miRNA degradation (TDMD). We developed TDMDfinder, a computational pipeline that identifies 'high confidence' TDMD interactions in the Human and Mouse transcriptomes by combining sequence alignment and feature selection approaches. Our predictions suggested that TDMD is widespread, with potentially every miRNA controlled by endogenous targets. We experimentally tested 37 TDMDfinder predictions, of which 17 showed TDMD effects as measured by RT-qPCR and small RNA sequencing, linking the miR-17, miR-19, miR-30, miR-221, miR-26 and miR-23 families to novel endogenous TDMDs. In some cases, TDMD was found to affect different members of the same miRNA family selectively. Features like complementarity to the miRNA 3' region, bulge size and hybridization energy appeared to be the main factors determining sensitivity. Computational analyses performed using the multiomic TCGA platform substantiated the involvement of many TDMD transcripts in human cancer and highlighted 36 highly significant interactions, suggesting TDMD as a new potential oncogenic mechanism. In conclusion, TDMDfinder provides the first inventory of bona fide human and mouse TDMDs. Available as a free webtool, TDMDfinder allows users to search for any TDMD interaction of interest by customizing its selection criteria.

Microglia-specific overexpression of α-synuclein leads to severe dopaminergic neurodegeneration by phagocytic exhaustion and oxidative toxicity.

Recent findings in human samples and animal models support the involvement of inflammation in the development of Parkinson's disease. Nevertheless, it is currently unknown whether microglial activation constitutes a primary event in neurodegeneration. We generated a new mouse model by lentiviral-mediated selective α-synuclein (αSYN) accumulation in microglial cells. Surprisingly, these mice developed progressive degeneration of dopaminergic (DA) neurons without endogenous αSYN aggregation. Transcriptomics and functional assessment revealed that αSYN-accumulating microglial cells developed a strong reactive state with phagocytic exhaustion and excessive production of oxidative and proinflammatory molecules. This inflammatory state created a molecular feed-forward vicious cycle between microglia and IFNγ-secreting immune cells infiltrating the brain parenchyma. Pharmacological inhibition of oxidative and nitrosative molecule production was sufficient to attenuate neurodegeneration. These results suggest that αSYN accumulation in microglia induces selective DA neuronal degeneration by promoting phagocytic exhaustion, an excessively toxic environment and the selective recruitment of peripheral immune cells.

miR-146 connects stem cell identity with metabolism and pharmacological resistance in breast cancer.

Although ectopic overexpression of miRNAs can influence mammary normal and cancer stem cells (SCs/CSCs), their physiological relevance remains uncertain. Here, we show that miR-146 is relevant for SC/CSC activity. MiR-146a/b expression is high in SCs/CSCs from human/mouse primary mammary tissues, correlates with the basal-like breast cancer subtype, which typically has a high CSC content, and specifically distinguishes cells with SC/CSC identity. Loss of miR-146 reduces SC/CSC self-renewal in vitro and compromises patient-derived xenograft tumor growth in vivo, decreasing the number of tumor-initiating cells, thus supporting its pro-oncogenic function. Transcriptional analysis in mammary SC-like cells revealed that miR-146 has pleiotropic effects, reducing adaptive response mechanisms and activating the exit from quiescent state, through a complex network of finely regulated miRNA targets related to quiescence, transcription, and one-carbon pool metabolism. Consistent with these findings, SCs/CSCs display innate resistance to anti-folate chemotherapies either in vitro or in vivo that can be reversed by miR-146 depletion, unmasking a "hidden vulnerability" exploitable for the development of anti-CSC therapies.

Frataxin gene editing rescues Friedreich's ataxia pathology in dorsal root ganglia organoid-derived sensory neurons.

Friedreich's ataxia (FRDA) is an autosomal-recessive neurodegenerative and cardiac disorder which occurs when transcription of the FXN gene is silenced due to an excessive expansion of GAA repeats into its first intron. Herein, we generate dorsal root ganglia organoids (DRG organoids) by in vitro differentiation of human iPSCs. Bulk and single-cell RNA sequencing show that DRG organoids present a transcriptional signature similar to native DRGs and display the main peripheral sensory neuronal and glial cell subtypes. Furthermore, when co-cultured with human intrafusal muscle fibers, DRG organoid sensory neurons contact their peripheral targets and reconstitute the muscle spindle proprioceptive receptors. FRDA DRG organoids model some molecular and cellular deficits of the disease that are rescued when the entire FXN intron 1 is removed, and not with the excision of the expanded GAA tract. These results strongly suggest that removal of the repressed chromatin flanking the GAA tract might contribute to rescue FXN total expression and fully revert the pathological hallmarks of FRDA DRG neurons.

Correction: Dual role for miR-34a in the control of early progenitor proliferation and commitment in the mammary gland and in breast cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

LncRNA EPR controls epithelial proliferation by coordinating Cdkn1a transcription and mRNA decay response to TGF-ß.

Long noncoding RNAs (lncRNAs) are emerging as regulators of fundamental biological processes. Here we report on the characterization of an intergenic lncRNA expressed in epithelial tissues which we termed EPR (Epithelial cell Program Regulator). EPR is rapidly downregulated by TGF-ß and its sustained expression largely reshapes the transcriptome, favors the acquisition of epithelial traits, and reduces cell proliferation in cultured mammary gland cells as well as in an animal model of orthotopic transplantation. EPR generates a small peptide that localizes at epithelial cell junctions but the RNA molecule per se accounts for the vast majority of EPR-induced gene expression changes. Mechanistically, EPR interacts with chromatin and regulates Cdkn1a gene expression by affecting both its transcription and mRNA decay through its association with SMAD3 and the mRNA decay-promoting factor KHSRP, respectively. We propose that EPR enables epithelial cells to control proliferation by modulating waves of gene expression in response to TGF-ß.

MiR-135a-5p Is Critical for Exercise-Induced Adult Neurogenesis.

Physical exercise stimulates adult hippocampal neurogenesis and is considered a relevant strategy for preventing age-related cognitive decline in humans. The underlying mechanisms remains controversial. Here, we show that exercise increases proliferation of neural precursor cells (NPCs) of the mouse dentate gyrus (DG) via downregulation of microRNA 135a-5p (miR-135a). MiR-135a inhibition stimulates NPC proliferation leading to increased neurogenesis, but not astrogliogenesis, in DG of resting mice, and intriguingly it re-activates NPC proliferation in aged mice. We identify 17 proteins (11 putative targets) modulated by miR-135 in NPCs. Of note, inositol 1,4,5-trisphosphate (IP3) receptor 1 and inositol polyphosphate-4-phosphatase type I are among the modulated proteins, suggesting that IP3 signaling may act downstream miR-135. miR-135 is the first noncoding RNA essential modulator of the brain's response to physical exercise. Prospectively, the miR-135-IP3 axis might represent a novel target of therapeutic intervention to prevent pathological brain aging.

Dual role for miR-34a in the control of early progenitor proliferation and commitment in the mammary gland and in breast cancer.

The role of the tumour-suppressor miR-34 family in breast physiology and in mammary stem cells (MaSCs) is largely unknown. Here, we revealed that miR-34 family, and miR-34a in particular, is implicated in mammary epithelium homoeostasis. Expression of miR-34a occurs upon luminal commitment and differentiation and serves to inhibit the expansion of the pool of MaSCs and early progenitor cells, likely in a p53-independent fashion. Mutant mice (miR34-KO) and loss-of-function approaches revealed two separate functions of miR-34a, controlling both proliferation and fate commitment in mammary progenitors by modulating several pathways involved in epithelial cell plasticity and luminal-to-basal conversion. In particular, miR-34a acts as endogenous inhibitor of the Wnt/beta-catenin signalling pathway, targeting up to nine upstream regulators at the same time, thus modulating the expansion of the MaSCs/early progenitor pool. These multiple roles of miR-34a are maintained in a model of human breast cancer, in which chronic expression of miR-34a in triple-negative mesenchymal-like cells (enriched in cancer stem cells-CSCs) could promote a luminal-like differentiation programme, restrict the CSC pool, and inhibit tumour propagation. Hence, activation of miR-34a-dependent programmes could provide a therapeutic opportunity for the subset of breast cancers, which are rich in CSCs and respond poorly to conventional therapies.

Endogenous transcripts control miRNA levels and activity in mammalian cells by target-directed miRNA degradation.

Little is known about miRNA decay. A target-directed miRNA degradation mechanism (TDMD) has been suggested, but further investigation on endogenous targets is necessary. Here, we identify hundreds of targets eligible for TDMD and show that an endogenous RNA (Serpine1) controls the degradation of two miRNAs (miR-30b-5p and miR-30c-5p) in mouse fibroblasts. In our study, TDMD occurs when the target is expressed at relatively low levels, similar in range to those of its miRNAs (100-200 copies per cell), and becomes more effective at high target:miRNA ratios (>10:1). We employ CRISPR/Cas9 to delete the miR-30 responsive element within Serpine1 3'UTR and interfere with TDMD. TDMD suppression increases miR-30b/c levels and boosts their activity towards other targets, modulating gene expression and cellular phenotypes (i.e., cell cycle re-entry and apoptosis). In conclusion, a sophisticated regulatory layer of miRNA and gene expression mediated by specific endogenous targets exists in mammalian cells.

Uncovering the Stability of Mature miRNAs by 4-Thio-Uridine Metabolic Labeling.

MicroRNAs (miRNAs) are an evolutionary conserved class of short, single-stranded noncoding RNAs (<18-22 nt in length) that act in posttranscriptional regulation of gene expression in higher eukaryotes. The abundance of a miRNA is a key feature in control of its activity and, therefore, a number of mechanisms finely regulate miRNA levels, acting at both transcriptional and posttranscriptional level. Recent evidences, including our research, highlighted the role of miRNA decay as a mechanism controlling the miRNA pool. We describe in this chapter an optimized methodology to determine miRNA degradation rates in mammalian cells. Our approach is based on metabolic pulse labeling with 4-thiouridine (4sU), a uridine analog that is incorporated in nascent RNA and allows thiol-specific biotinylation and selective pull-down of labeled RNA. In particular, given the long average half-life and the complex biogenetic process of miRNAs, we developed a "pulse-chase" protocol where 4sU is removed from the medium after a long labeling period (2-3 h pulse), and labeled RNA is purified at different time points to measure the decay of labeled molecules. By combining the 4sU-based "pulse-chase" approach with high-throughput small RNA sequencing (sRNAseq), it is possible to quantify at genome-wide level miRNA degradation rates.

Synergic Functions of miRNAs Determine Neuronal Fate of Adult Neural Stem Cells.

Adult neurogenesis requires the precise control of neuronal versus astrocyte lineage determination in neural stem cells. While microRNAs (miRNAs) are critically involved in this step during development, their actions in adult hippocampal neural stem cells (aNSCs) has been unclear. As entry point to address that question we chose DICER, an endoribonuclease essential for miRNA biogenesis and other RNAi-related processes. By specific ablation of Dicer in aNSCs in vivo and in vitro, we demonstrate that miRNAs are required for the generation of new neurons, but not astrocytes, in the adult murine hippocampus. Moreover, we identify 11 miRNAs, of which 9 have not been previously characterized in neurogenesis, that determine neurogenic lineage fate choice of aNSCs at the expense of astrogliogenesis. Finally, we propose that the 11 miRNAs sustain adult hippocampal neurogenesis through synergistic modulation of 26 putative targets from different pathways.

MicroRNA-independent functions of DGCR8 are essential for neocortical development and TBR1 expression.

Recent evidence indicates that the miRNA biogenesis factors DROSHA, DGCR8, and DICER exert non-overlapping functions, and have also roles in miRNA-independent regulatory mechanisms. However, it is currently unknown whether miRNA-independent functions of DGCR8 play any role in the maintenance of neuronal progenitors and during corticogenesis. Here, by phenotypic comparison of cortices from conditional Dgcr8 and Dicer knockout mice, we show that Dgcr8 deletion, in contrast to Dicer depletion, leads to premature differentiation of neural progenitor cells and overproduction of TBR1-positive neurons. Remarkably, depletion of miRNAs upon DCGR8 loss is reduced compared to DICER loss, indicating that these phenotypic differences are mediated by miRNA-independent functions of DGCR8. We show that Dgcr8 mutations induce an earlier and stronger phenotype in the developing nervous system compared to Dicer mutants and that miRNA-independent functions of DGCR8 are critical for corticogenesis. Finally, our data also suggest that the Microprocessor complex, with DROSHA and DGCR8 as core components, directly regulates the Tbr1 transcript, containing evolutionarily conserved hairpins that resemble miRNA precursors, independently of miRNAs.

Optimization and Standardization of Circulating MicroRNA Detection for Clinical Application: The miR-Test Case.

BACKGROUND: The identification of circulating microRNAs (miRNAs) in the blood has been recently exploited for the development of minimally invasive tests for the early detection of cancer. Nevertheless, the clinical transferability of such tests is uncertain due to still-insufficient standardization and optimization of methods to detect circulating miRNAs in the clinical setting. METHODS: We performed a series of tests to optimize the quantification of serum miRNAs that compose the miR-Test, a signature for lung cancer early detection, and systematically analyzed variables that could affect the performance of the test. We took advantage of a large-scale (>1000 samples) validation study of the miR-Test that we recently published, to evaluate, in clinical samples, the effects of analytical and preanalytical variables on the quantification of circulating miRNAs and the clinical output of the signature (risk score). RESULTS: We developed a streamlined and standardized pipeline for the processing of clinical serum samples that allows the isolation and analysis of circulating miRNAs by quantitative reverse-transcription PCR, with a throughput compatible with screening trials. The major source of analytical variation came from RNA isolation from serum, which could be corrected by use of external (spike-in) or endogenous miRNAs as a reference for normalization. We also introduced standard operating procedures and QC steps to check for unspecific fluctuations that arise from the lack of standardized criteria in the collection or handling of the samples (preanalytical factors). CONCLUSIONS: We propose our methodology as a reference for the development of clinical-grade blood tests on the basis of miRNA detection.

Degradation dynamics of microRNAs revealed by a novel pulse-chase approach.

The regulation of miRNAs is critical to the definition of cell identity and behavior in normal physiology and disease. To date, the dynamics of miRNA degradation and the mechanisms involved in remain largely obscure, in particular, in higher organisms. Here, we developed a pulse-chase approach based on metabolic RNA labeling to calculate miRNA decay rates at genome-wide scale in mammalian cells. Our analysis revealed heterogeneous miRNA half-lives, with many species behaving as stable molecules (T1/2> 24 h), while others, including passenger miRNAs and a number (25/129) of guide miRNAs, are quickly turned over (T1/2= 4-14 h). Decay rates were coupled with other features, including genomic organization, transcription rates, structural heterogeneity (isomiRs), and target abundance, measured through quantitative experimental approaches. This comprehensive analysis highlighted functional mechanisms that mediate miRNA degradation, as well as the importance of decay dynamics in the regulation of the miRNA pool under both steady-state conditions and during cell transitions.