RNA Deregulation in Amyotrophic Lateral Sclerosis: The Noncoding Perspective.

RNA metabolism is central to cellular physiopathology. Almost all the molecular pathways underpinning biological processes are affected by the events governing the RNA life cycle, ranging from transcription to degradation. The deregulation of these processes contributes to the onset and progression of human diseases. In recent decades, considerable efforts have been devoted to the characterization of noncoding RNAs (ncRNAs) and to the study of their role in the homeostasis of the nervous system (NS), where they are highly enriched. Acting as major regulators of gene expression, ncRNAs orchestrate all the steps of the differentiation programs, participate in the mechanisms underlying neural functions, and are crucially implicated in the development of neuronal pathologies, among which are neurodegenerative diseases. This review aims to explore the link between ncRNA dysregulation and amyotrophic lateral sclerosis (ALS), the most frequent motoneuron (MN) disorder in adults. Notably, defective RNA metabolism is known to be largely associated with this pathology, which is often regarded as an RNA disease. We also discuss the potential role that these transcripts may play as diagnostic biomarkers and therapeutic targets.

Mir-34a-5p Mediates Cross-Talk between M2 Muscarinic Receptors and Notch-1/EGFR Pathways in U87MG Glioblastoma Cells: Implication in Cell Proliferation.

Glioblastoma (GBM) is the most aggressive human brain tumor. The high growth potential and decreased susceptibility to apoptosis of the glioma cells is mainly dependent on genetic amplifications or mutations of oncogenic or pro-apoptotic genes, respectively. We have previously shown that the activation of the M2 acetylcholine muscarinic receptors inhibited cell proliferation and induced apoptosis in two GBM cell lines and cancer stem cells. The aim of this study was to delve into the molecular mechanisms underlying the M2-mediated cell proliferation arrest. Exploiting U87MG and U251MG cell lines as model systems, we evaluated the ability of M2 receptors to interfere with Notch-1 and EGFR pathways, whose activation promotes GBM proliferation. We demonstrated that the activation of M2 receptors, by agonist treatment, counteracted Notch and EGFR signaling, through different regulatory cascades depending, at least in part, on p53 status. Only in U87MG cells, which mimic p53-wild type GBMs, did M2 activation trigger a molecular circuitry involving p53, Notch-1, and the tumor suppressor mir-34a-5p. This regulatory module negatively controls Notch-1, which affects cell proliferation mainly through the Notch-1/EGFR axis. Our data highlighted, for the first time, a molecular circuitry that is deregulated in the p53 wild type GBM, based on the cross-talk between M2 receptor and the Notch-1/EGFR pathways, mediated by mir-34a-5p.

FUS stimulates microRNA biogenesis by facilitating co-transcriptional Drosha recruitment.

microRNA abundance has been shown to depend on the amount of the microprocessor components or, in some cases, on specific auxiliary co-factors. In this paper, we show that the FUS/TLS (fused in sarcoma/translocated in liposarcoma) protein, associated with familial forms of Amyotrophic Lateral Sclerosis (ALS), contributes to the biogenesis of a specific subset of microRNAs. Among them, species with roles in neuronal function, differentiation and synaptogenesis were identified. We also show that FUS/TLS is recruited to chromatin at sites of their transcription and binds the corresponding pri-microRNAs. Moreover, FUS/TLS depletion leads to decreased Drosha level at the same chromatin loci. Limited FUS/TLS depletion leads to a reduced microRNA biogenesis and we suggest a possible link between FUS mutations affecting nuclear/cytoplasmic partitioning of the protein and altered neuronal microRNA biogenesis in ALS pathogenesis.

Identification of linc-NeD125, a novel long non coding RNA that hosts miR-125b-1 and negatively controls proliferation of human neuroblastoma cells.

The human genome contains some thousands of long non coding RNAs (lncRNAs). Many of these transcripts are presently considered crucial regulators of gene expression and functionally implicated in developmental processes in Eukaryotes. Notably, despite a huge number of lncRNAs are expressed in the Central Nervous System (CNS), only a few of them have been characterized in terms of molecular structure, gene expression regulation and function. In the present study, we identify linc-NeD125 as a novel cytoplasmic, neuronal-induced long intergenic non coding RNA (lincRNA). Linc-NeD125 represents the host gene for miR-125b-1, a microRNA with an established role as negative regulator of human neuroblastoma cell proliferation. Here, we demonstrate that these two overlapping non coding RNAs are coordinately induced during in vitro neuronal differentiation, and that their expression is regulated by different mechanisms. While the production of miR-125b-1 relies on transcriptional regulation, linc-NeD125 is controlled at the post-transcriptional level, through modulation of its stability. We also demonstrate that linc-NeD125 functions independently of the hosted microRNA, by reducing cell proliferation and activating the antiapoptotic factor BCL-2.

Mir-23a and mir-125b regulate neural stem/progenitor cell proliferation by targeting Musashi1.

Musashi1 is an RNA binding protein that controls the neural cell fate, being involved in maintaining neural progenitors in their proliferative state. In particular, its downregulation is needed for triggering early neural differentiation programs. In this study, we profiled microRNA expression during the transition from neural progenitors to differentiated astrocytes and underscored 2 upregulated microRNAs, miR-23a and miR-125b, that sinergically act to restrain Musashi1 expression, thus creating a regulatory module controlling neural progenitor proliferation.

The long noncoding RNA nHOTAIRM1 is necessary for differentiation and activity of iPSC-derived spinal motor neurons.

The mammalian nervous system is made up of an extraordinary array of diverse cells that form intricate functional connections. The programs underlying cell lineage specification, identity and function of the neuronal subtypes are managed by regulatory proteins and RNAs, which coordinate the succession of steps in a stereotyped temporal order. In the central nervous system (CNS), motor neurons (MNs) are responsible for controlling essential functions such as movement, breathing, and swallowing by integrating signal transmission from the cortex, brainstem, and spinal cord (SC) towards peripheral muscles. A prime role in guiding the progression of progenitor cells towards the MN fate has been largely attributed to protein factors. More recently, the relevance of a class of regulatory RNAs abundantly expressed in the CNS - the long noncoding RNAs (lncRNAs) - has emerged overwhelmingly. LncRNA-driven gene expression control is key to regulating any step of MN differentiation and function, and its derangement profoundly impacts neuronal pathophysiology. Here, we uncover a novel function for the neuronal isoform of HOTAIRM1 (nHOTAIRM1), a lncRNA specifically expressed in the SC. Using a model system that recapitulates spinal MN (spMN) differentiation, we show that nHOTAIRM1 intervenes in the binary cell fate decision between MNs and interneurons, acting as a pro-MN factor. Furthermore, human iPSC-derived spMNs without nHOTAIRM1 display altered neurite outgrowth, with a significant reduction of both branch and junction numbers. Finally, the expression of genes essential for synaptic connectivity and neurotransmission is also profoundly impaired when nHOTAIRM1 is absent in spMNs. Mechanistically, nHOTAIRM1 establishes both direct and indirect interactions with a number of target genes in the cytoplasm, being a novel post-transcriptional regulator of MN biology. Overall, our results indicate that the lncRNA nHOTAIRM1 is essential for the specification of MN identity and the acquisition of proper morphology and synaptic activity of post-mitotic MNs.

Concerted microRNA control of Hedgehog signalling in cerebellar neuronal progenitor and tumour cells.

MicroRNAs (miRNA) are crucial post-transcriptional regulators of gene expression and control cell differentiation and proliferation. However, little is known about their targeting of specific developmental pathways. Hedgehog (Hh) signalling controls cerebellar granule cell progenitor development and a subversion of this pathway leads to neoplastic transformation into medulloblastoma (MB). Using a miRNA high-throughput profile screening, we identify here a downregulated miRNA signature in human MBs with high Hh signalling. Specifically, we identify miR-125b and miR-326 as suppressors of the pathway activator Smoothened together with miR-324-5p, which also targets the downstream transcription factor Gli1. Downregulation of these miRNAs allows high levels of Hh-dependent gene expression leading to tumour cell proliferation. Interestingly, the downregulation of miR-324-5p is genetically determined by MB-associated deletion of chromosome 17p. We also report that whereas miRNA expression is downregulated in cerebellar neuronal progenitors, it increases alongside differentiation, thereby allowing cell maturation and growth inhibition. These findings identify a novel regulatory circuitry of the Hh signalling and suggest that misregulation of specific miRNAs, leading to its aberrant activation, sustain cancer development.

A minicircuitry involving REST and CREB controls miR-9-2 expression during human neuronal differentiation.

miRNAs play key roles in the nervous system, where they mark distinct developmental stages. Accordingly, dysregulation of miRNA expression may have profound effects on neuronal physiology and pathology, including cancer. Among the neuronal miRNAs, miR-9 was shown to be upregulated during in vitro neuronal differentiation and downregulated in 50% of primary neuroblastoma tumors, suggesting a potential function as an oncosuppressor gene. In this study we characterized the promoter and the transcriptional regulation of the miR-9-2 gene during neuronal differentiation. We found that, despite its localization inside an exon of a putative host-gene, miR-9-2 is expressed as an independent unit with the promoter located in the upstream intron. By promoter fusion and mutational analyses, together with RNAi and Chromatin immunoprecipitation assays, we demonstrated that the concerted action of the master transcriptional factors RE1-silencing transcription factor (REST) and cAMP-response element binding protein (CREB) on miR-9-2 promoter induces miRNA expression during differentiation. We showed that the repressor REST inhibits the activity of the miR-9-2 promoter in undifferentiated neuroblastoma cells, whereas REST dismissal and phosphorylation of CREB trigger transcription in differentiating cells. Finally, a regulatory feed-back mechanism, in which the reciprocal action of miR-9 and REST may be relevant for the maintenance of the neuronal differentiation program, is shown.

Identification and Functional Characterization of Novel MYC-Regulated Long Noncoding RNAs in Group 3 Medulloblastoma.

The impact of protein-coding genes on cancer onset and progression is a well-established paradigm in molecular oncology. Nevertheless, unveiling the contribution of the noncoding genes-including long noncoding RNAs (lncRNAs)-to tumorigenesis represents a great challenge for personalized medicine, since they (i) constitute the majority of the human genome, (ii) are essential and flexible regulators of gene expression and (iii) present all types of genomic alterations described for protein-coding genes. LncRNAs have been increasingly associated with cancer, their highly tissue- and cancer type-specific expression making them attractive candidates as both biomarkers and therapeutic targets. Medulloblastoma is one of the most common malignant pediatric brain tumors. Group 3 is the most aggressive subgroup, showing the highest rate of metastasis at diagnosis. Transcriptomics and reverse genetics approaches were combined to identify lncRNAs implicated in Group 3 Medulloblastoma biology. Here we present the first collection of lncRNAs dependent on the activity of the MYC oncogene, the major driver gene of Group 3 Medulloblastoma. We assessed the expression profile of selected lncRNAs in Group 3 primary tumors and functionally characterized these species. Overall, our data demonstrate the direct involvement of three lncRNAs in Medulloblastoma cancer cell phenotypes.

The Non-coding Side of Medulloblastoma.

Medulloblastoma (MB) is the most common pediatric brain tumor and a primary cause of cancer-related death in children. Until a few years ago, only clinical and histological features were exploited for MB pathological classification and outcome prognosis. In the past decade, the advancement of high-throughput molecular analyses that integrate genetic, epigenetic, and expression data, together with the availability of increasing wealth of patient samples, revealed the existence of four molecularly distinct MB subgroups. Their further classification into 12 subtypes not only reduced the well-characterized intertumoral heterogeneity, but also provided new opportunities for the design of targets for precision oncology. Moreover, the identification of tumorigenic and self-renewing subpopulations of cancer stem cells in MB has increased our knowledge of its biology. Despite these advancements, the origin of MB is still debated, and its molecular bases are poorly characterized. A major goal in the field is to identify the key genes that drive tumor growth and the mechanisms through which they are able to promote tumorigenesis. So far, only protein-coding genes acting as oncogenic drivers have been characterized in each MB subgroup. The contribution of the non-coding side of the genome, which produces a plethora of transcripts that control fundamental biological processes, as the cell choice between proliferation and differentiation, is still unappreciated. This review wants to fill this major gap by summarizing the recent findings on the impact of non-coding RNAs in MB initiation and progression. Furthermore, their potential role as specific MB biomarkers and novel therapeutic targets is also highlighted.

Cross Interaction between M2 Muscarinic Receptor and Notch1/EGFR Pathway in Human Glioblastoma Cancer Stem Cells: Effects on Cell Cycle Progression and Survival.

Glioblastomas (GBM) are the most aggressive form of primary brain tumors in humans. A key feature of malignant gliomas is their cellular heterogeneity. In particular, the presence of an undifferentiated cell population of defined Glioblastoma Stem cells (GSCs) was reported. Increased expression of anti-apoptotic and chemo-resistance genes in GCSs subpopulation favors their high resistance to a broad spectrum of drugs. Our previous studies showed the ability of M2 muscarinic receptors to negatively modulate the cell growth in GBM cell lines and in the GSCs. The aim of this study was to better characterize the inhibitory effects of M2 receptors on cell proliferation and survival in GSCs and investigate the molecular mechanisms underlying the M2-mediated cell proliferation arrest and decreased survival. Moreover, we also evaluated the ability of M2 receptors to interfere with Notch1 and EGFR pathways, whose activation promotes GSCs proliferation. Our data demonstrate that M2 receptors activation impairs cell cycle progression and survival in the primary GSC lines analyzed (GB7 and GB8). Moreover, we also demonstrated the ability of M2 receptor to inhibit Notch1 and EGFR expression, highlighting a molecular interaction between M2 receptor and the Notch-1/EGFR pathways also in GSCs.

HOTAIRM1 regulates neuronal differentiation by modulating NEUROGENIN 2 and the downstream neurogenic cascade.

Neuronal differentiation is a timely and spatially regulated process, relying on precisely orchestrated gene expression control. The sequential activation/repression of genes driving cell fate specification is achieved by complex regulatory networks, where transcription factors and noncoding RNAs work in a coordinated manner. Herein, we identify the long noncoding RNA HOTAIRM1 (HOXA Transcript Antisense RNA, Myeloid-Specific 1) as a new player in neuronal differentiation. We demonstrate that the neuronal-enriched HOTAIRM1 isoform epigenetically controls the expression of the proneural transcription factor NEUROGENIN 2 that is key to neuronal fate commitment and critical for brain development. We also show that HOTAIRM1 activity impacts on NEUROGENIN 2 downstream regulatory cascade, thus contributing to the achievement of proper neuronal differentiation timing. Finally, we identify the RNA-binding proteins HNRNPK and FUS as regulators of HOTAIRM1 biogenesis and metabolism. Our findings uncover a new regulatory layer underlying NEUROGENIN 2 transitory expression in neuronal differentiation and reveal a previously unidentified function for the neuronal-induced long noncoding RNA HOTAIRM1.

MicroRNA profiling in human medulloblastoma.

Medulloblastoma is an aggressive brain malignancy with high incidence in childhood. Current treatment approaches have limited efficacy and severe side effects. Therefore, new risk-adapted therapeutic strategies based on molecular classification are required. MicroRNA expression analysis has emerged as a powerful tool to identify candidate molecules playing an important role in a large number of malignancies. However, no data are yet available on human primary medulloblastomas. A high throughput microRNA expression profiles was performed in human primary medulloblastoma specimens to investigate microRNA involvement in medulloblastoma carcinogenesis. We identified specific microRNA expression patterns which distinguish medulloblastoma differing in histotypes (anaplastic, classic and desmoplastic), in molecular features (ErbB2 or c-Myc overexpressing tumors) and in disease-risk stratification. MicroRNAs expression profile clearly differentiates medulloblastoma from either adult or fetal normal cerebellar tissues. Only a few microRNAs displayed upregulated expression, while most of them were downregulated in tumor samples, suggesting a tumor growth-inhibitory function. This property has been addressed for miR-9 and miR-125a, whose rescued expression promoted medulloblastoma cell growth arrest and apoptosis while targeting the proproliferative truncated TrkC isoform. In conclusion, misregulated microRNA expression profiles characterize human medulloblastomas, and may provide potential targets for novel therapeutic strategies.

Long Noncoding RNAs: Emerging Players in Medulloblastoma.

Central Nervous System tumors are the leading cause of cancer-related death in children, and medulloblastoma has the highest incidence rate. The current therapies achieve a 5-year survival rate of 50-80%, but often inflict severe secondary effects demanding the urgent development of novel, effective, and less toxic therapeutic strategies. Historically identified on a histopathological basis, medulloblastoma was later classified into four major subgroups-namely WNT, SHH, Group 3, and Group 4-each characterized by distinct transcriptional profiles, copy-number aberrations, somatic mutations, and clinical outcomes. Additional complexity was recently provided by integrating gene- and non-gene-based data, which indicates that each subclass can be further subdivided into specific subtypes. These deeper classifications, while getting over the typical tumor heterogeneity, indicate that different forms of medulloblastoma hold different molecular drivers that can be successfully exploited for a greater diagnostic accuracy and for the development of novel, targeted treatments. Long noncoding RNAs are transcripts that lack coding potential and play relevant roles as regulators of gene expression in mammalian differentiation and developmental processes. Their cell type- and tissue-specificity, higher than mRNAs, make them more informative about cell- type identity than protein-coding genes. Remarkably, about 40% of long noncoding RNAs are expressed in the brain and their aberrant expression has been linked to neuro-oncological disorders. However, while their involvement in gliomas and neuroblastomas has been extensively studied, their role in medulloblastoma is still poorly explored. Here, we present an overview of current knowledge regarding the function played by long noncoding RNAs in medulloblastoma biology.

A Regulatory Circuitry Between Gria2, miR-409, and miR-495 Is Affected by ALS FUS Mutation in ESC-Derived Motor Neurons.

Mutations in fused in sarcoma (FUS) cause amyotrophic lateral sclerosis (ALS). FUS is a multifunctional protein involved in the biogenesis and activity of several types of RNAs, and its role in the pathogenesis of ALS may involve both direct effects of disease-associated mutations through gain- and loss-of-function mechanisms and indirect effects due to the cross talk between different classes of FUS-dependent RNAs. To explore how FUS mutations impinge on motor neuron-specific RNA-based circuitries, we performed transcriptome profiling of small and long RNAs of motor neurons (MNs) derived from mouse embryonic stem cells carrying a FUS-P517L knock-in mutation, which is equivalent to human FUS-P525L, associated with a severe and juvenile-onset form of ALS. Combining ontological, predictive and molecular analyses, we found an inverse correlation between several classes of deregulated miRNAs and their corresponding mRNA targets in both homozygous and heterozygous P517L MNs. We validated a circuitry in which the upregulation of miR-409-3p and miR-495-3p, belonging to a brain-specific miRNA subcluster implicated in several neurodevelopmental disorders, produced the downregulation of Gria2, a subunit of the glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor with a significant role in excitatory neurotransmission. Moreover, we found that FUS was involved in mediating such miRNA repression. Gria2 alteration has been proposed to be implicated in MN degeneration, through disturbance of Ca2+ homeostasis, which triggers a cascade of damaging "excitotoxic" events. The molecular cross talk identified highlights a role for FUS in excitotoxicity and in miRNA-dependent regulation of Gria2. This circuitry also proved to be deregulated in heterozygosity, which matches the human condition perfectly.

Characterization of the lncRNA transcriptome in mESC-derived motor neurons: Implications for FUS-ALS.

Long non-coding RNAs (lncRNAs) are currently recognized as crucial players in nervous system development, function and pathology. In Amyotrophic Lateral Sclerosis (ALS), identification of causative mutations in FUS and TDP-43 or hexanucleotide repeat expansion in C9ORF72 point to the essential role of aberrant RNA metabolism in neurodegeneration. In this study, by taking advantage of an in vitro differentiation system generating mouse motor neurons (MNs) from embryonic stem cells, we identified and characterized the long non-coding transcriptome of MNs. Moreover, by using mutant mouse MNs carrying the equivalent of one of the most severe ALS-associated FUS alleles (P517L), we identified lncRNAs affected by this mutation. Comparative analysis with human MNs derived in vitro from induced pluripotent stem cells indicated that candidate lncRNAs are conserved between mouse and human. Our work provides a global view of the long non-coding transcriptome of MN, as a prerequisite toward the comprehension of the still poorly characterized non-coding side of MN physiopathology.

Drosophila CG3303 is an essential endoribonuclease linked to TDP-43-mediated neurodegeneration.

Endoribonucleases participate in almost every step of eukaryotic RNA metabolism, acting either as degradative or biosynthetic enzymes. We previously identified the founding member of the Eukaryotic EndoU ribonuclease family, whose components display unique biochemical features and are flexibly involved in important biological processes, such as ribosome biogenesis, tumorigenesis and viral replication. Here we report the discovery of the CG3303 gene product, which we named DendoU, as a novel family member in Drosophila. Functional characterisation revealed that DendoU is essential for Drosophila viability and nervous system activity. Pan-neuronal silencing of dendoU resulted in fly immature phenotypes, highly reduced lifespan and dramatic motor performance defects. Neuron-subtype selective silencing showed that DendoU is particularly important in cholinergic circuits. At the molecular level, we unveiled that DendoU is a positive regulator of the neurodegeneration-associated protein dTDP-43, whose downregulation recapitulates the ensemble of dendoU-dependent phenotypes. This interdisciplinary work, which comprehends in silico, in vitro and in vivo studies, unveils a relevant role for DendoU in Drosophila nervous system physio-pathology and highlights that DendoU-mediated neurotoxicity is, at least in part, contributed by dTDP-43 loss-of-function.

FUS affects circular RNA expression in murine embryonic stem cell-derived motor neurons.

The RNA-binding protein FUS participates in several RNA biosynthetic processes and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Here we report that FUS controls back-splicing reactions leading to circular RNA (circRNA) production. We identified circRNAs expressed in in vitro-derived mouse motor neurons (MNs) and determined that the production of a considerable number of these circRNAs is regulated by FUS. Using RNAi and overexpression of wild-type and ALS-associated FUS mutants, we directly correlate the modulation of circRNA biogenesis with alteration of FUS nuclear levels and with putative toxic gain of function activities. We also demonstrate that FUS regulates circRNA biogenesis by binding the introns flanking the back-splicing junctions and that this control can be reproduced with artificial constructs. Most circRNAs are conserved in humans and specific ones are deregulated in human-induced pluripotent stem cell-derived MNs carrying the FUSP525L mutation associated with ALS.

The long noncoding RNA linc-NeD125 controls the expression of medulloblastoma driver genes by microRNA sponge activity.

Long noncoding RNAs (lncRNAs) are major regulators of physiological and disease-related gene expression, particularly in the central nervous system. Dysregulated lncRNA expression has been documented in several human cancers, and their tissue-specificity makes them attractive candidates as diagnostic/prognostic biomarkers and/or therapeutic agents. Here we show that linc-NeD125, which we previously characterized as a neuronal-induced lncRNA, is significantly overexpressed in Group 4 medulloblastomas (G4 MBs), the largest and least well characterized molecular MB subgroup. Mechanistically, linc-NeD125 is able to recruit the miRNA-induced silencing complex (miRISC) and to directly bind the microRNAs miR-19a-3p, miR-19b-3p and miR-106a-5p. Functionally, linc-NeD125 acts as a competing endogenous RNA (ceRNA) that, sequestering the three miRNAs, leads to de-repression of their targets CDK6, MYCN, SNCAIP, and KDM6A, which are major driver genes of G4 MB. Accordingly, linc-NeD125 downregulation reduces G4 cell proliferation. Moreover, we also provide evidence that linc-NeD125 ectopic expression in the aggressive Group 3 MB cells attenuates their proliferation, migration and invasion.This study unveils the first lncRNA-based ceRNA network in central nervous system tumours and provides a novel molecular circuit underlying the enigmatic Group 4 medulloblastoma.

Large-scale purification and crystallization of the endoribonuclease XendoU: troubleshooting with His-tagged proteins.

XendoU is the first endoribonuclease described in higher eukaryotes as being involved in the endonucleolytic processing of intron-encoded small nucleolar RNAs. It is conserved among eukaryotes and its viral homologue is essential in SARS replication and transcription. The large-scale purification and crystallization of recombinant XendoU are reported. The tendency of the recombinant enzyme to aggregate could be reversed upon the addition of chelating agents (EDTA, imidazole): aggregation is a potential drawback when purifying and crystallizing His-tagged proteins, which are widely used, especially in high-throughput structural studies. Purified monodisperse XendoU crystallized in two different space groups: trigonal P3(1)21, diffracting to low resolution, and monoclinic C2, diffracting to higher resolution.