LINE-1 regulates cortical development by acting as long non-coding RNAs.

Long Interspersed Nuclear Elements-1s (L1s) are transposable elements that constitute most of the genome's transcriptional output yet have still largely unknown functions. Here we show that L1s are required for proper mouse brain corticogenesis operating as regulatory long non-coding RNAs. They contribute to the regulation of the balance between neuronal progenitors and differentiation, the migration of post-mitotic neurons and the proportions of different cell types. In cortical cultured neurons, L1 RNAs are mainly associated to chromatin and interact with the Polycomb Repressive Complex 2 (PRC2) protein subunits enhancer of Zeste homolog 2 (Ezh2) and suppressor of zeste 12 (Suz12). L1 RNA silencing influences PRC2's ability to bind a portion of its targets and the deposition of tri-methylated histone H3 (H3K27me3) marks. Our results position L1 RNAs as crucial signalling hubs for genome-wide chromatin remodelling, enabling the fine-tuning of gene expression during brain development and evolution.

Exploratory analysis of L1 retrotransposons expression in autism.

BACKGROUND: Autism spectrum disorder (ASD) is a set of highly heterogeneous neurodevelopmental diseases whose genetic etiology is not completely understood. Several investigations have relied on transcriptome analysis from peripheral tissues to dissect ASD into homogenous molecular phenotypes. Recently, analysis of changes in gene expression from postmortem brain tissues has identified sets of genes that are involved in pathways previously associated with ASD etiology. In addition to protein-coding transcripts, the human transcriptome is composed by a large set of non-coding RNAs and transposable elements (TEs). Advancements in sequencing technologies have proven that TEs can be transcribed in a regulated fashion, and their dysregulation might have a role in brain diseases. METHODS: We exploited published datasets comprising RNA-seq data from (1) postmortem brain of ASD subjects, (2) in vitro cell cultures where ten different ASD-relevant genes were knocked out and (3) blood of discordant siblings. We measured the expression levels of evolutionarily young full-length transposable L1 elements and characterized the genomic location of deregulated L1s assessing their potential impact on the transcription of ASD-relevant genes. We analyzed every sample independently, avoiding to pool together the disease subjects to unmask the heterogeneity of the molecular phenotypes. RESULTS: We detected a strong upregulation of intronic full-length L1s in a subset of postmortem brain samples and in in vitro differentiated neurons from iPSC knocked out for ATRX. L1 upregulation correlated with an high number of deregulated genes and retained introns. In the anterior cingulate cortex of one subject, a small number of significantly upregulated L1s overlapped with ASD-relevant genes that were significantly downregulated, suggesting the possible existence of a negative effect of L1 transcription on host transcripts. LIMITATIONS: Our analyses must be considered exploratory and will need to be validated in bigger cohorts. The main limitation is given by the small sample size and by the lack of replicates for postmortem brain samples. Measuring the transcription of locus-specific TEs is complicated by the repetitive nature of their sequence, which reduces the accuracy in mapping sequencing reads to the correct genomic locus. CONCLUSIONS: L1 upregulation in ASD appears to be limited to a subset of subjects that are also characterized by a general deregulation of the expression of canonical genes and an increase in intron retention. In some samples from the anterior cingulate cortex, L1s upregulation seems to directly impair the expression of some ASD-relevant genes by a still unknown mechanism. L1s upregulation may therefore identify a group of ASD subjects with common molecular features and helps stratifying individuals for novel strategies of therapeutic intervention.

Piwil2 (Mili) sustains neurogenesis and prevents cellular senescence in the postnatal hippocampus.

Adult neural progenitor cells (aNPCs) ensure lifelong neurogenesis in the mammalian hippocampus. Proper regulation of aNPC fate has thus important implications for brain plasticity and healthy aging. Piwi proteins and the small noncoding RNAs interacting with them (piRNAs) have been proposed to control memory and anxiety, but the mechanism remains elusive. Here, we show that Piwil2 (Mili) is essential for proper neurogenesis in the postnatal mouse hippocampus. RNA sequencing of aNPCs and their differentiated progeny reveal that Mili and piRNAs are dynamically expressed in neurogenesis. Depletion of Mili and piRNAs in the adult hippocampus impairs aNPC differentiation toward a neural fate, induces senescence, and generates reactive glia. Transcripts modulated upon Mili depletion bear sequences complementary or homologous to piRNAs and include repetitive elements and mRNAs encoding essential proteins for proper neurogenesis. Our results provide evidence of a critical role for Mili in maintaining fitness and proper fate of aNPCs, underpinning a possible involvement of the piRNA pathway in brain plasticity and successful aging.

Towards SINEUP-based therapeutics: Design of an in vitro synthesized SINEUP RNA.

SINEUPs are a novel class of natural and synthetic non-coding antisense RNA molecules able to increase the translation of a target mRNA. They present a modular organization comprising an unstructured antisense target-specific domain, which sets the specificity of each individual SINEUP, and a structured effector domain, which is responsible for the translation enhancement. In order to design a fully functional in vitro transcribed SINEUP for therapeutics applications, SINEUP RNAs were synthesized in vitro with a variety of chemical modifications and screened for their activity on endogenous target mRNA upon transfection. Three combinations of modified ribonucleotides-2'O methyl-ATP (Am), N6 methyl-ATP (m6A), and pseudo-UTP (ψ)-conferred SINEUP activity to naked RNA. The best combination tested in this study was fully modified with m6A and ψ. Aside from functionality, this combination conferred improved stability upon transfection and higher thermal stability. Common structural determinants of activity were identified by circular dichroisms, defining a core functional structure that is achieved with different combinations of modifications.

Analysis of LINE1 Retrotransposons in Huntington's Disease.

Transposable elements (TEs) are mobile genetic elements that made up about half the human genome. Among them, the autonomous non-LTR retrotransposon long interspersed nuclear element-1 (L1) is the only currently active TE in mammals and covers about 17% of the mammalian genome. L1s exert their function as structural elements in the genome, as transcribed RNAs to influence chromatin structure and as retrotransposed elements to shape genomic variation in somatic cells. L1s activity has been shown altered in several diseases of the nervous system. Huntington disease (HD) is a dominantly inherited neurodegenerative disorder caused by an expansion of a CAG repeat in the HTT gene which leads to a gradual loss of neurons most prominently in the striatum and, to a lesser extent, in cortical brain regions. The length of the expanded CAG tract is related to age at disease onset, with longer repeats leading to earlier onset. Here we carried out bioinformatic analysis of public RNA-seq data of a panel of HD mouse models showing that a decrease of L1 RNA expression recapitulates two hallmarks of the disease: it correlates to CAG repeat length and it occurs in the striatum, the site of neurodegeneration. Results were then experimentally validated in Htt Q111 knock-in mice. The expression of L1-encoded proteins was independent from L1 RNA levels and differentially regulated in time and tissues. The pattern of expression L1 RNAs in human HD post-mortem brains showed similarity to mouse models of the disease. This work suggests the need for further study of L1s in HD and adds support to the current hypothesis that dysregulation of TEs may be involved in neurodegenerative diseases.

The miRNA Content of Exosomes Released from the Glioma Microenvironment Can Affect Malignant Progression.

Low-grade gliomas (LGG) are infiltrative primary brain tumors that in 70% of the cases undergo anaplastic transformation, deeply affecting prognosis. However, the timing of progression is heterogeneous. Recently, the tumor microenvironment (TME) has gained much attention either as prognostic factor or therapeutic target. Through the release of extracellular vesicles, the TME contributes to tumor progression by transferring bioactive molecules such as microRNA. The aim of the study was to take advantage of glioma-associated stem cells (GASC), an in vitro model of the glioma microenvironment endowed with a prognostic significance, and their released exosomes, to investigate the possible role of exosome miRNAs in favoring the anaplastic transformation of LGG. Therefore, by deep sequencing, we analyzed and compared the miRNA profile of GASC and exosomes obtained from LGG patients characterized by different prognosis. Results showed that exosomes presented a different signature, when compared to their cellular counterpart and that, although sharing several miRNAs, exosomes of patients with a bad prognosis, selectively expressed some miRNAs possibly responsible for the more aggressive phenotype. These findings get insights into the value of TME and exosomes as potential biomarkers for precision medicine approaches aimed at improving LGG prognostic stratification and therapeutic strategies.

Impact of polymorphic transposable elements on transcription in lymphoblastoid cell lines from public data.

BACKGROUND: Transposable elements (TEs) are DNA sequences able to mobilize themselves and to increase their copy-number in the host genome. In the past, they have been considered mainly selfish DNA without evident functions. Nevertheless, currently they are believed to have been extensively involved in the evolution of primate genomes, especially from a regulatory perspective. Due to their recent activity they are also one of the primary sources of structural variants (SVs) in the human genome. By taking advantage of sequencing technologies and bioinformatics tools, recent surveys uncovered specific TE structural variants (TEVs) that gave rise to polymorphisms in human populations. When combined with RNA-seq data this information provides the opportunity to study the potential impact of TEs on gene expression in human. RESULTS: In this work, we assessed the effects of the presence of specific TEs in cis on the expression of flanking genes by producing associations between polymorphic TEs and flanking gene expression levels in human lymphoblastoid cell lines. By using public data from the 1000 Genome Project and the Geuvadis consortium, we exploited an expression quantitative trait loci (eQTL) approach integrated with additional bioinformatics data mining analyses. We uncovered human loci enriched for common, less common and rare TEVs and identified 323 significant TEV-cis-eQTL associations. SINE-R/VNTR/Alus (SVAs) resulted the TE class with the strongest effects on gene expression. We also unveiled differential functional enrichments on genes associated to TEVs, genes associated to TEV-cis-eQTLs and genes associated to the genomic regions mostly enriched in TEV-cis-eQTLs highlighting, at multiple levels, the impact of TEVs on the host genome. Finally, we also identified polymorphic TEs putatively embedded in transcriptional units, proposing a novel mechanism in which TEVs may mediate individual-specific traits. CONCLUSION: We contributed to unveiling the effect of polymorphic TEs on transcription in lymphoblastoid cell lines.

An NF-κB signature predicts low-grade glioma prognosis: a precision medicine approach based on patient-derived stem cells.

Background: While recent genome-wide association studies have suggested novel low-grade glioma (LGG) stratification models based on a molecular classification, we explored the potential clinical utility of patient-derived cells. Specifically, we assayed glioma-associated stem cells (GASC) that are patient-derived and representative of the glioma microenvironment. Methods: By next-generation sequencing, we analyzed the transcriptional profile of GASC derived from patients who underwent anaplastic transformation either within 48 months (GASC-BAD) or ≥7 years (GASC-GOOD) after surgery. Gene set enrichment and pathway enrichment analyses were applied. The prognostic role of a nuclear factor-kappaB (NF-κB) signature derived from GASC-BAD was tested in 530 newly diagnosed diffuse LGG patients comprised within The Cancer Genome Atlas (TCGA) database. The prognostic value of the GASC upstream regulator p65 NF-κB was assessed, by univariate and multivariate Cox analyses, in a single center case study, including 146 grade II LGGs. Results: The key elements differentiating the transcriptome of GASC isolated from LGG with different prognoses were mostly related to hallmarks of cancer (eg, inflammatory/immune process, NF-κB activation). Consistently, the NF-κB signature extrapolated from the GASC study was prognostic in the dataset of TCGA. Finally, the nuclear expression of the NF-kB-p65 protein, assessed using an inexpensive immunohistochemical method, was an independent predictor of both overall survival and malignant progression-free survival in 146 grade II LGGs. Conclusion: This study demonstrates for the first time the independent prognostic role of NF-kB activation in LGG and outlines the role of patient-based stem cell models as a tool for precision medicine approaches.

Adipose tissue derived stem cells: in vitro and in vivo analysis of a standard and three commercially available cell-assisted lipotransfer techniques.

INTRODUCTION: Autologous fat grafting is commonly used to correct soft-tissue contour deformities. However, results are impaired by a variable and unpredictable resorption rate. Autologous adipose-derived stromal cells in combination with lipoinjection (cell-assisted lipotransfer) seem to favor a long-term persistence of fat grafts, thus fostering the development of devices to be used in the operating room at the point of care, to isolate the stromal vascular fraction (SVF) and produce SVF-enhanced fat grafts with safe and standardized protocols. Focusing on patients undergoing breast reconstruction by lipostructure, we analyzed a standard technique, a modification of the Coleman's procedure, and three different commercially available devices (Lipokit, Cytori, Fastem), in terms of 1) ability to enrich fat grafts in stem cells and 2) clinical outcome at 6 and 12 months. METHODS: To evaluate the ability to enrich stem cells, we compared, for each patient (n=20), the standard lipoaspirate with the respective stem cell-enriched one, analyzing yield, immunophenotype and colony-forming capacity of the SVF cells as well as immunophenotype, clonogenicity and multipotency of the obtained adipose stem cells (ASCs). Regarding the clinical outcome, we compared, by ultrasonography imaging, changes at 6 and 12 months in the subcutaneous thickness of patients treated with stem-cell enriched (n=14) and standard lipoaspirates (n=16). RESULTS: Both methods relying on the enzymatic isolation of primitive cells led to significant increase in the frequency, in the fat grafts, of SVF cells as well as of clonogenic and multipotent ASCs, while the enrichment was less prominent for the device based on the mechanical isolation of the SVF. From a clinical point of view, patients treated with SVF-enhanced fat grafts demonstrated, at six months, a significant superior gain of thickness of both the central and superior-medial quadrants with respect to patients treated with standard lipotransfer. In the median-median quadrant the effect was still persistent at 12 months, confirming an advantage of lipotransfer technique in enriching improving long-term fat grafts. CONCLUSIONS: This comparative study, based on reproducible biological and clinical parameters and endpoints, showed an advantage of lipotransfer technique in enriching fat grafts in stem cells and in favoring, clinically, long-term fat grafts.

Glioma-associated stem cells: a novel class of tumor-supporting cells able to predict prognosis of human low-grade gliomas.

BACKGROUND: Translational medicine aims at transferring advances in basic science research into new approaches for diagnosis and treatment of diseases. Low-grade gliomas (LGG) have a heterogeneous clinical behavior that can be only partially predicted employing current state-of-the-art markers, hindering the decision-making process. To deepen our comprehension on tumor heterogeneity, we dissected the mechanism of interaction between tumor cells and relevant components of the neoplastic environment, isolating, from LGG and high-grade gliomas (HGG), proliferating stem cell lines from both the glioma stroma and, where possible, the neoplasm. METHODS AND FINDINGS: We isolated glioma-associated stem cells (GASC) from LGG (n=40) and HGG (n=73). GASC showed stem cell features, anchorage-independent growth, and supported the malignant properties of both A172 cells and human glioma-stem cells, mainly through the release of exosomes. Finally, starting from GASC obtained from HGG (n=13) and LGG (n=12) we defined a score, based on the expression of 9 GASC surface markers, whose prognostic value was assayed on 40 subsequent LGG-patients. At the multivariate Cox analysis, the GASC-based score was the only independent predictor of overall survival and malignant progression free-survival. CONCLUSIONS: The microenvironment of both LGG and HGG hosts non-tumorigenic multipotent stem cells that can increase in vitro the biological aggressiveness of glioma-initiating cells through the release of exosomes. The clinical importance of this finding is supported by the strong prognostic value associated with the characteristics of GASC. This patient-based approach can provide a groundbreaking method to predict prognosis and to exploit novel strategies that target the tumor stroma.