5-Hydroxymethylcytosine (5-hmC) is a newly discovered modified form of cytosine that has been suspected to be an important epigenetic modification in neurodevelopment. While DNA methylation dynamics have already been implicated during neurodevelopment, little is known about hydroxymethylation in this process. Here, we report DNA hydroxymethylation dynamics during cerebellum development in the human brain. Overall, we find a positive correlation between 5-hmC levels and cerebellum development. Genome-wide profiling reveals that 5-hmC is highly enriched on specific gene regions including exons and especially the untranslated regions (UTRs), but it is depleted on introns and intergenic regions. Furthermore, we have identified fetus-specific and adult-specific differentially hydroxymethylated regions (DhMRs), most of which overlap with genes and CpG island shores. Surprisingly, during development, DhMRs are highly enriched in genes encoding mRNAs that can be regulated by fragile X mental retardation protein (FMRP), some of which are disrupted in autism, as well as in many known autism genes. Our results suggest that 5-hmC-mediated epigenetic regulation may broadly impact the development of the human brain, and its dysregulation could contribute to the molecular pathogenesis of neurodevelopmental disorders. Accession number: Sequencing data have been deposited to GEO with accession number GSE40539.

Autosomal recessive cerebellar ataxia (ARCA) comprises a large and heterogeneous group of neurodegenerative disorders. For many affected patients, the genetic cause remains undetermined. Through whole-exome sequencing, we identified compound heterozygous mutations in ubiquitin-like modifier activating enzyme 5 gene (UBA5) in two Chinese siblings presenting with ARCA. Moreover, copy number variations in UBA5 or ubiquitin-fold modifier 1 gene (UFM1) were documented with the phenotypes of global developmental delays and gait disturbances in the ClinVar database. UBA5 encodes UBA5, the ubiquitin-activating enzyme of UFM1. However, a crucial role for UBA5 in human neurological disease remains to be reported. Our molecular study of UBA5-R246X revealed a dramatically decreased half-life and loss of UFM1 activation due to the absence of the catalytic cysteine Cys250. UBA5-K310E maintained its interaction with UFM1, although with less stability, which may affect the ability of this UBA5 mutant to activate UFM1. Drosophila modeling revealed that UBA5 knockdown induced locomotive defects and a shortened lifespan accompanied by aberrant neuromuscular junctions (NMJs). Strikingly, we found that UFM1 and E2 cofactor knockdown induced markedly similar phenotypes. Wild-type UBA5, but not mutant UBA5, significantly restored neural lesions caused by the absence of UBA5. The finding of a UBA5 mutation in cerebellar ataxia suggests that impairment of the UFM1 pathway may contribute to the neurological phenotypes of ARCA.

Background: Intracranial aneurysm (IA) is one of the most lethal forms of cerebrovascular diseases characterized by endothelial dysfunction, vascular smooth muscle cell phenotypic modulation, inflammation and consequently loss of vessel cells and extracellular matrix degradation. Besides environmental factors, genetics seem to be a very important factor in the genesis of this disease. Previous mRNA expression studies revealed a large number of differentially expressed genes between IA and control tissue. However, microRNAs (miRNA), small non-coding RNAs which are post-transcriptional regulators of gene expression, have been barely studied. Studying miRNAs could provide a hypothetical mechanism underlying rupture of IA. Methods: A microarray study was carried out to determine difference in microRNAs and mRNA between patients’ IA tissues and controls. Quantitative RT-PCR assay compared the expression level between two groups (14 IA domes vs. 14 controls) were used for validation. Validated miRNAs were analyzed using Ingenuity Pathway Analysis (IPA) to identify the networks and pathways. Results: 18 miRNAs were confirmed by qPCR to be robustly down-regulated in 14 ruptured IA patients including hsa-mir-133b, hsa-mir-133a, hsa-mir-1, hsa-mir-143-3p, hsa-mir-145-3p, hsa-mir-145-5p, hsa-mir-455-5p, hsa-mir-143-5p, hsa-mir-23b-3p etc., of which 11 miRNAs are clusters: hsa-mir-1/has-mir-133a, hsa-mir-143/hsa-mir-145, hsa-mir-23b/hsa-mir-24-1, and hsa-mir-29b-2/hsa-mir-29c. 12 predicted functions were generated using IPA which showed significant associations with migration of phagocytes, proliferation of mononuclear leukocytes, cell movement of mononuclear leukocytes, cell movement of smooth muscle cells etc. Conclusion: These data support common disease mechanisms that may be under miRNA control and provide exciting directions for further investigations aimed at elucidating the miRNA mechanisms and targets that may yield new therapies for IA.

GGC repeat expansions within NOTCH2NLC have been identified as the genetic cause of neuronal intranuclear inclusion disease (NIID). To understand the molecular pathogenesis of NIID, here, we established both a transgenic mouse model and a human neural progenitor cells (hNPCs) model. Expression of the NOTCH2NLC with expanded GGC repeats produced widespread intranuclear and perinuclear polyglycine (polyG), polyalanine (polyA), and polyarginine (polyR) inclusions, leading to behavioral deficits and severe neurodegeneration, which faithfully mimicked the clinical and pathological features associated with NIID. Furthermore, conserved alternative splicing events were identified between the NIID mouse and hNPC models, among which was the enrichment of the binding motifs of hnRNPM, an RNA binding protein known as alternative splicing regulator. Expanded NOTCH2NLC-polyG and NOTCH2NLC-polyA could interact with and sequester hnRNPM, while overexpression of hnRNPM could ameliorate the cellular toxicity. These results together suggested that dysfunction of hnRNPM could play an important role in the molecular pathogenesis of NIID.

Alzheimer’s disease (AD) is a chronic neurodegenerative disorder characterized by progressive deterioration of cognitive function. Pathogenesis of AD is incompletely understood; evidence suggests a role for epigenetic regulation, in particular the cytosine modifications 5-methylcytosine and 5-hydroxymethylcytosine (5hmC). 5hmC is enriched in the nervous system and displays neurodevelopment and age-related changes. To determine the role of 5hmC in AD, we performed genome-wide analyses of 5hmC in DNA from prefrontal cortex of post-mortem AD patients, and RNA-Seq to correlate changes in 5hmC with transcriptional changes. We identified 325 genes containing differentially hydroxymethylated loci (DhMLs) in both discovery and replication datasets. These are enriched for pathways involved in neuron projection development and neurogenesis. Of these, 140 showed changes in gene expression. Proteins encoded by these genes form direct protein–protein interactions with AD-associated genes, expanding the network of genes implicated in AD. We identified AD-associated single nucleotide polymorphisms (SNPs) located within or near DhMLs, suggesting these SNPs may identify regions of epigenetic gene regulation that play a role in AD pathogenesis. Finally, using an existing AD fly model, we showed some of these genes modulate AD-associated toxicity. Our data implicate neuronal projection development and neurogenesis pathways as potential targets in AD. By incorporating epigenomic and transcriptomic data with genome-wide association studies data, with verification in the Drosophila model, we can expand the known network of genes involved in disease pathogenesis and identify epigenetic modifiers of Alzheimer’s disease.