Massively parallel characterization of regulatory elements in the developing human cortex.

Nucleotide changes in gene regulatory elements are important determinants of neuronal development and diseases. Using massively parallel reporter assays in primary human cells from mid-gestation cortex and cerebral organoids, we interrogated the cis-regulatory activity of 102,767 open chromatin regions, including thousands of sequences with cell type-specific accessibility and variants associated with brain gene regulation. In primary cells, we identified 46,802 active enhancer sequences and 164 variants that alter enhancer activity. Activity was comparable in organoids and primary cells, suggesting that organoids provide an adequate model for the developing cortex. Using deep learning we decoded the sequence basis and upstream regulators of enhancer activity. This work establishes a comprehensive catalog of functional gene regulatory elements and variants in human neuronal development.

Characterization of enhancer activity in early human neurodevelopment using Massively Parallel Reporter Assay (MPRA) and forebrain organoids.

Regulation of gene expression through enhancers is one of the major processes shaping the structure and function of the human brain during development. High-throughput assays have predicted thousands of enhancers involved in neurodevelopment, and confirming their activity through orthogonal functional assays is crucial. Here, we utilized Massively Parallel Reporter Assays (MPRAs) in stem cells and forebrain organoids to evaluate the activity of ~ 7000 gene-linked enhancers previously identified in human fetal tissues and brain organoids. We used a Gaussian mixture model to evaluate the contribution of background noise in the measured activity signal to confirm the activity of ~ 35% of the tested enhancers, with most showing temporal-specific activity, suggesting their evolving role in neurodevelopment. The temporal specificity was further supported by the correlation of activity with gene expression. Our findings provide a valuable gene regulatory resource to the scientific community.

Characterization of enhancer activity in early human neurodevelopment using Massively parallel reporter assay (MPRA) and forebrain organoids.

Regulation of gene expression through enhancers is one of the major processes shaping the structure and function of the human brain during development. High-throughput assays have predicted thousands of enhancers involved in neurodevelopment, and confirming their activity through orthogonal functional assays is crucial. Here, we utilized Massively Parallel Reporter Assays (MPRAs) in stem cells and forebrain organoids to evaluate the activity of ~7,000 gene-linked enhancers previously identified in human fetal tissues and brain organoids. We used a Gaussian mixture model to evaluate the contribution of background noise in the measured activity signal to confirm the activity of ~35% of the tested enhancers, with most showing temporal-specific activity, suggesting their evolving role in neurodevelopment. The temporal specificity was further supported by the correlation of activity with gene expression. Our findings provide a valuable gene regulatory resource to the scientific community.

Modeling idiopathic autism in forebrain organoids reveals an imbalance of excitatory cortical neuron subtypes during early neurogenesis.

Idiopathic autism spectrum disorder (ASD) is highly heterogeneous, and it remains unclear how convergent biological processes in affected individuals may give rise to symptoms. Here, using cortical organoids and single-cell transcriptomics, we modeled alterations in the forebrain development between boys with idiopathic ASD and their unaffected fathers in 13 families. Transcriptomic changes suggest that ASD pathogenesis in macrocephalic and normocephalic probands involves an opposite disruption of the balance between excitatory neurons of the dorsal cortical plate and other lineages such as early-generated neurons from the putative preplate. The imbalance stemmed from divergent expression of transcription factors driving cell fate during early cortical development. While we did not find genomic variants in probands that explained the observed transcriptomic alterations, a significant overlap between altered transcripts and reported ASD risk genes affected by rare variants suggests a degree of gene convergence between rare forms of ASD and the developmental transcriptome in idiopathic ASD.

Massively parallel characterization of psychiatric disorder-associated and cell-type-specific regulatory elements in the developing human cortex.

Nucleotide changes in gene regulatory elements are important determinants of neuronal development and disease. Using massively parallel reporter assays in primary human cells from mid-gestation cortex and cerebral organoids, we interrogated the cis-regulatory activity of 102,767 sequences, including differentially accessible cell-type specific regions in the developing cortex and single-nucleotide variants associated with psychiatric disorders. In primary cells, we identified 46,802 active enhancer sequences and 164 disorder-associated variants that significantly alter enhancer activity. Activity was comparable in organoids and primary cells, suggesting that organoids provide an adequate model for the developing cortex. Using deep learning, we decoded the sequence basis and upstream regulators of enhancer activity. This work establishes a comprehensive catalog of functional gene regulatory elements and variants in human neuronal development.

FUS Alters circRNA Metabolism in Human Motor Neurons Carrying the ALS-Linked P525L Mutation.

Deregulation of RNA metabolism has emerged as one of the key events leading to the degeneration of motor neurons (MNs) in Amyotrophic Lateral Sclerosis (ALS) disease. Indeed, mutations on RNA-binding proteins (RBPs) or on proteins involved in aspects of RNA metabolism account for the majority of familiar forms of ALS. In particular, the impact of the ALS-linked mutations of the RBP FUS on many aspects of RNA-related processes has been vastly investigated. FUS plays a pivotal role in splicing regulation and its mutations severely alter the exon composition of transcripts coding for proteins involved in neurogenesis, axon guidance, and synaptic activity. In this study, by using in vitro-derived human MNs, we investigate the effect of the P525L FUS mutation on non-canonical splicing events that leads to the formation of circular RNAs (circRNAs). We observed altered levels of circRNAs in FUSP525L MNs and a preferential binding of the mutant protein to introns flanking downregulated circRNAs and containing inverted Alu repeats. For a subset of circRNAs, FUSP525L also impacts their nuclear/cytoplasmic partitioning, confirming its involvement in different processes of RNA metabolism. Finally, we assess the potential of cytoplasmic circRNAs to act as miRNA sponges, with possible implications in ALS pathogenesis.

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.

PsychENCODE and beyond: transcriptomics and epigenomics of brain development and organoids.

Crucial decisions involving cell fate and connectivity that shape the distinctive development of the human brain occur in the embryonic and fetal stages-stages that are difficult to access and investigate in humans. The last decade has seen an impressive increase in resources-from atlases and databases to biological models-that is progressively lifting the curtain on this critical period. In this review, we describe the current state of genomic, transcriptomic, and epigenomic datasets charting the development of normal human brain with a particular focus on recent single-cell technologies. We discuss the emergence of brain organoids generated from pluripotent stem cells as a model to compensate for the limited availability of fetal tissue. Indeed, comparisons of neural lineages, transcriptional dynamics, and noncoding element activity between fetal brain and organoids have helped identify gene regulatory networks functioning at early stages of brain development. Altogether, we argue that large multi-omics investigations have pushed brain development into the "big data" era, and that current and future transversal approaches needed to leverage both fetal brain and organoid resources promise to answer major questions of brain biology and psychiatry.

Exploring the Regulatory Role of Circular RNAs in Neurodegenerative Disorders.

Circular RNAs (circRNAs) are a distinctive class of regulatory non-coding RNAs characterised by the presence of covalently closed ends. They are evolutionary conserved molecules, and although detected in different tissues, circRNAs resulted specifically enriched in the nervous system. Recent studies have shown that circRNAs are dynamically modulated during neuronal development and aging, that circRNAs are enriched at synaptic levels and resulted modulated after synaptic plasticity induction. This has suggested that circRNAs might play an important role in neuronal specification and activity. Despite the exact function of circRNAs is still poorly understood, emerging evidence indicates that circRNAs have important regulatory functions that might extensively contribute to the dynamic modulation of gene expression that supports neuronal pathways. More interestingly, deregulation of circRNAs expression has been linked with various pathological conditions. In this review, we describe current advances in the field of circRNA biogenesis and function in the nervous system both in physiological and in pathological conditions, and we specifically lay out their association with neurodegenerative diseases. Furthermore, we discuss the opportunity to exploit circRNAs for innovative therapeutic approaches and, due to their high stability, to use circRNAs as suitable biomarkers for diagnosis and disease progression.

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.

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.

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.

DOF AFFECTING GERMINATION 2 is a positive regulator of light-mediated seed germination and is repressed by DOF AFFECTING GERMINATION 1.

BACKGROUND: The transcription factor DOF AFFECTING GERMINATION1 (DAG1) is a repressor of the light-mediated seed germination process. DAG1 acts downstream PHYTOCHROME INTERACTING FACTOR3-LIKE 5 (PIL5), the master repressor, and negatively regulates gibberellin biosynthesis by directly repressing the biosynthetic gene AtGA3ox1. The Dof protein DOF AFFECTING GERMINATION (DAG2) shares a high degree of aminoacidic identity with DAG1. While DAG1 inactivation considerably increases the germination capability of seeds, the dag2 mutant has seeds with a germination potential substantially lower than the wild-type, indicating that these factors may play opposite roles in seed germination. RESULTS: We show here that DAG2 expression is positively regulated by environmental factors triggering germination, whereas its expression is repressed by PIL5 and DAG1; by Chromatin Immuno Precipitation (ChIP) analysis we prove that DAG1 directly regulates DAG2. In addition, we show that Red light significantly reduces germination of dag2 mutant seeds. CONCLUSIONS: In agreement with the seed germination phenotype of the dag2 mutant previously published, the present data prove that DAG2 is a positive regulator of the light-mediated seed germination process, and particularly reveal that this protein plays its main role downstream of PIL5 and DAG1 in the phytochrome B (phyB)-mediated pathway.

Independent and interactive effects of DOF affecting germination 1 (DAG1) and the Della proteins GA insensitive (GAI) and Repressor of ga1-3 (RGA) in embryo development and seed germination.

BACKGROUND: The transcription factor DOF AFFECTING GERMINATION1 (DAG1) is a repressor of seed germination acting downstream of the master repressor PHYTOCROME INTERACTING FACTOR3-LIKE 5 (PIL5). Among others, PIL5 induces the expression of the genes encoding the two DELLA proteins GA INSENSITIVE 1 (GAI) and REPRESSOR OF ga1-3 (RGA). RESULTS: Based on the properties of gai-t6 and rga28 mutant seeds, we show here that the absence of RGA severely increases dormancy, while lack of GAI only partially compensates RGA inactivation. In addition, the germination properties of the dag1rga28 double mutant are different from those of the dag1 and rga28 single mutants, suggesting that RGA and DAG1 act in independent branches of the PIL5-controlled germination pathway. Surprisingly, the dag1gai-t6 double mutant proved embryo-lethal, suggesting an unexpected involvement of (a possible complex between) DAG1 and GAI in embryo development. CONCLUSIONS: Rather than overlapping functions as previously suggested, we show that RGA and GAI play distinct roles in seed germination, and that GAI interacts with DAG1 in embryo development.