Mounting evidence points to critical roles for DNA modifications, including 5-methylcytosine (5mC) and its oxidized forms, in the development, plasticity and disorders of the mammalian nervous system. The novel DNA base 5- hydroxymethylcytosine (5hmC) is known to be capable of initiating passive or active DNA demethylation, but whether and how extensively 5hmC functions in shaping the post-mitotic neuronal DNA methylome is unclear. Here we report the genome-wide distribution of 5hmC in dentate granule neurons from adult mouse hippocampus in vivo. 5hmC in the neuronal genome is highly enriched in gene bodies, especially in exons, and correlates with gene expression. Direct genome-wide comparison of 5hmC distribution between embryonic stem cells and neurons reveals extensive differences, reflecting the functional disparity between these two cell types. Importantly, integrative analysis of 5hmC, overall DNA methylation and gene expression profiles of dentate granule neurons in vivo reveals the genome-wide antagonism between these two states of cytosine modifications, supporting a role for 5hmC in shaping the neuronal DNA methylome by promoting active DNA demethylation.

DNA methylation has been studied comprehensively and linked to both normal neurodevelopment and neurological diseases. The recent identification of several new DNA modifications, including 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine, has given us a new perspective on the previously observed plasticity in 5mC-dependent regulatory processes. Here, we review the latest research into these cytosine modifications, focusing mainly on their roles in neurodevelopment and diseases.

MicroRNA (miRNA)/RNA interference (RNAi) is recognized as one of the most important mechanisms regulating gene expression at the posttranscriptional level in eukaryotic cells. The main components within the miRNA/RNAi pathway are now known and well characterized, but studies on the molecular mechanisms that regulate the activity of the miRNA/RNAi pathway are just beginning to emerge. High-throughput reporter assays have been developed to monitor the activity of the miRNA/RNAi pathway and applied in a proof-of-concept pilot screening, which has led to the identification of some inhibitors and activators that either generally or specifically regulate the activity of the miRNA/RNAi pathway. In addition, combined with multidisciplinary approaches like proteomics, biochemistry, and genetics, some protein co-factors were found to play important roles in the regulation of the miRNA/RNAi pathway. Herein we highlight the high-throughput reporter assays developed in recent years and the resulting discovery of miRNA/RNAi enhancers and inhibitors.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) share phenotypic and pathologic overlap. Recently, an expansion of GGGGCC repeats in the first intron of C9orf72 was found to be a common cause of both illnesses; however, the molecular pathogenesis of this expanded repeat is unknown. Here we developed both Drosophila and mammalian models of this expanded hexanucleotide repeat and showed that expression of the expanded GGGGCC repeat RNA (rGGGGCC) is sufficient to cause neurodegeneration. We further identified Pur α as the RNA-binding protein of rGGGGCC repeats and discovered that Pur α and rGGGGCC repeats interact in vitro and in vivo in a sequence-specific fashion that is conserved between mammals and Drosophila. Furthermore, overexpression of Pur α in mouse neuronal cells and Drosophila mitigates rGGGGCC repeat-mediated neurodegeneration, and Pur α forms inclusions in the fly eye expressing expanded rGGGGCC repeats, as well as in cerebellum of human carriers of expanded GGGGCC repeats. These data suggest that expanded rGGGGCC repeats could sequester specific RNA-binding protein from their normal functions, ultimately leading to cell death. Taken together, these findings suggest that the expanded rGGGGCC repeats could cause neurodegeneration, and that Pur α may play a role in the pathogenesis of amyotrophic lateral sclerosis and frontotemporal dementia.

The somatic epigenome can be reprogrammed to a pluripotent state by a combination of transcription factors. Altering cell fate involves transcription factors cooperation, epigenetic reconfiguration, such as DNA methylation and histone modification, posttranscriptional regulation by microRNAs, and so on. Nevertheless, such reprogramming is inefficient. Evidence suggests that during the early stage of reprogramming, the process is stochastic, but by the late stage, it is deterministic. In addition to conventional reprogramming methods, dozens of small molecules have been identified that can functionally replace reprogramming factors and significantly improve induced pluripotent stem cell (iPSC) reprogramming. Indeed, iPS cells have been created recently using chemical compounds only. iPSCs are thought to display subtle genetic and epigenetic variability; this variability is not random, but occurs at hotspots across the genome. Here we discuss the progress and current perspectives in the field. Research into the reprogramming process today will pave the way for great advances in regenerative medicine in the future.

Epigenetic regulation, such as DNA methylation and histone modification, is implicated in the aberrant changes in gene expression that occur during the progression of neurodegeneration. Many epigenetics-based drugs have been developed recently for the treatment of some neurodegenerative disorders, including Alzheimer’s, Parkinson’s, and Huntington’s diseases. Here we review recent studies that highlight the role of epigenetic modifications in neurodegeneration, among them DNA methylation and demethylation and histone acetylation and deacetylation; we also explore the possibility of using epigenetics-based therapeutics to treat neurodegenerative disorders.

Neurogenesis, the process of generating functional neurons from neural stem cells, is tightly controlled by many intrinsic and extrinsic mechanisms. Uncovering these regulatory mechanisms is crucial for understanding the functions and plasticity of the human brain. Recent studies in both invertebrates and vertebrates point to the importance of small regulatory RNAs in regulating lineage-specific gene expression and determining neuronal identity during neurogenesis. These new observations suggest that small regulatory RNAs could function at many levels to regulate self-renewal of neural stem cells and neuronal fate specification, implicating small regulatory RNAs in the complexity of neurogenesis.

RNA interference (RNAi) is a well-conserved mechanism that uses small noncoding RNAs to silence gene expression posttranscriptionally. Gene regulation by RNAi is now recognized as one of the major regulatory pathways in eukaryotic cells. Although the main components of the RNAi/miRNA pathway have been identified, the molecular mechanisms regulating the activity of the RNAi/miRNA pathway have only begun to emerge within the last couple of years. Recently, high-throughput reporter assays to monitor the activity of the RNAi/miRNA pathway have been developed and used for proof-of-concept pilot screens. Both inhibitors and activators of the RNAi/miRNA pathway have been found. Although still in its infancy, a chemical biology approach using high-throughput chemical screens should open up a new avenue for dissecting the RNAi/miRNA pathway, as well as developing novel RNAi- or miRNA-based therapeutic interventions.

An expanding assortment of small, noncoding RNAs identified in the nervous system suggests a strong connection between their combinatorial regulatory potential and the complexity of the nervous system. Misregulation of these small regulatory RNAs could contribute to the abnormalities in brain development that are associated with neurodevelopmental disorders. Here we give an overview of the diversity and unexpected abundance of small RNAs, as well as specific examples that illustrate their functional significance in neurodevelopmental disorders. We also discuss an intriguing, albeit elusive area of study: the potential impact of newly discovered classes of small RNAs in the nervous system.

Fragile X syndrome (FXS), the most common form of inherited mental retardation, is caused by the loss of functional fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein that can regulate the translation of specific mRNAs. It is known to regulate synaptic development through the regulation of local protein synthesis in synapses. MicroRNAs (miRNAs) are a class of small noncoding RNAs involved in almost every biological process. They exhibit spatiotemporal expression during brain development, and some miRNAs play important roles in neural development. A growing body of evidence now implicates the miRNA pathway in the molecular pathogenesis of FXS. Here we review the current state of knowledge about the microRNA pathway in neural development and the emergence of possible roles for miRNAs in FXS.

5-Hydroxymethylcytosine (5hmC), a novel modified cytosine, is oxidized from 5-methylcytosine (5mC) by the ten-eleven translocation (Tet) protein family. The specific distribution of 5hmC in mammalian brain and its roles in gene regulation suggest that 5hmC is important in brain development. 5hmC may also contribute to the mechanisms underlying neurological diseases. Here, we summarize the current knowledge of 5hmC, with an emphasis on its roles in neurodevelopmental and neurodegenerative disorders.

Noncoding RNAs play important and diverse regulatory roles throughout the genome and make major contributions to disease pathogenesis. The FMR1 gene is involved in three different syndromes: fragile X syndrome (FXS), primary ovarian insufficiency (POI), and fragile X-associated tremor/ataxia syndrome (FXTAS) in older patients. Noncoding RNAs have been implicated in the molecular pathogenesis of both FXS and FXTAS. Here we will review our current knowledge on the role(s) of noncoding RNAs in FXS and FXTAS, particularly the role of the microRNA pathway in FXS and the role of noncoding riboCGG (rCGG) repeat in FXTAS.

RNA-mediated mechanisms of disease pathogenesis in neurological disorders have been recognized in the context of certain repeat expansion disorders. This RNA-initiated neurodegeneration may play a more pervasive role in disease pathology beyond the classic dynamic mutation disorders. Here, we review the mechanisms of RNA toxicity and aberrant RNA processing that have been implicated in ageing-related neurological disorders. We focus on diseases with aberrant sequestration of RNA-binding proteins, bi-directional transcription, aberrant translation of repeat expansion RNA transcripts (repeat-associated non-ATG (RAN) translation), and the formation of pathological RNA:DNA secondary structure (R-loop). It is likely that repeat expansion disorders arise from common mechanisms caused by the repeat expansion mutations. However, the context of the repeat expansion determines the specific molecular consequences, leading to clinically distinct disorders.

•A combination of modified reprogramming factors (OySyNyK) is developed•A highly efficient and rapid reprogramming system is established•TET1/2 proteins are involved in rapid iPSC induction by OySyNyK•OySyNyK factors coordinate with TET proteins to promote rapid reprogrammingSomatic cell reprogramming toward induced pluripotent stem cells (iPSCs) holds great promise in future regenerative medicine. However, the reprogramming process mediated by the traditional defined factors (OSMK) is slow and extremely inefficient. Here, we develop a combination of modified reprogramming factors (OySyNyK) in which the transactivation domain of the Yes-associated protein is fused to defined factors and establish a highly efficient and rapid reprogramming system. We show that the efficiency of OySyNyK-induced iPSCs is up to 100-fold higher than the OSNK and the reprogramming by OySyNyK is very rapid and is initiated in 24 hr. We find that OySyNyK factors significantly increase Tet1 expression at the early stage and interact with Tet1/2 to promote reprogramming. Our studies not only establish a rapid and highly efficient iPSC reprogramming system but also uncover a mechanism by which engineered factors coordinate with TETs to regulate 5hmC-mediated epigenetic control.Download high-res image (231KB)Download full-size image