Transcriptome Analysis of Induced Pluripotent Stem Cells and Neuronal Progenitor Cells, Derived from Discordant Monozygotic Twins with Parkinson’s Disease

Parkinson’s Disease (PD) is a widespread severe neurodegenerative disease that is characterized by pronounced deficiency of the dopaminergic system and disruption of the function of other neuromodulator systems. Although heritable genetic factors contribute significantly to PD pathogenesis, only a small percentage of sporadic cases of PD can be explained using known genetic risk factors. Due to that, it could be inferred that changes in gene expression could be important for explaining a significant percentage of PD cases. One of the ways to investigate such changes, while minimizing the effect of genetic factors on experiment, are the study of PD discordant monozygotic twins. In the course of the analysis of transcriptome data obtained from IPSC and NPCs, 20 and 1906 differentially expressed genes were identified respectively. We have observed an overexpression of TNF in NPC cultures, derived from twin with PD. Through investigation of gene interactions and gene involvement in biological processes, we have arrived to a hypothesis that TNF could play a crucial role in PD-related changes occurring in NPC derived from twins with PD, and identified INHBA, WNT7A and DKK1 as possible downstream effectors of TNF.

A schizophrenia subgroup with elevated inflammation displays reduced microglia, increased peripheral immune cell and altered neurogenesis marker gene expression in the subependymal zone

Inflammation regulates neurogenesis, and the brains of patients with schizophrenia and bipolar disorder have reduced expression of neurogenesis markers in the subependymal zone (SEZ), the birthplace of inhibitory interneurons. Inflammation is associated with cortical interneuron deficits, but the relationship between inflammation and reduced neurogenesis in schizophrenia and bipolar disorder remains unexplored. Therefore, we investigated inflammation in the SEZ by defining those with low and high levels of inflammation using cluster analysis of IL6, IL6R, IL1R1 and SERPINA3 gene expression in 32 controls, 32 schizophrenia and 29 bipolar disorder cases. We then determined whether mRNAs for markers of glia, immune cells and neurogenesis varied with inflammation. A significantly greater proportion of schizophrenia (37%) and bipolar disorder cases (32%) were in high inflammation subgroups compared to controls (10%, p < 0.05). Across the high inflammation subgroups of psychiatric disorders, mRNAs of markers for phagocytic microglia were reduced (P2RY12, P2RY13), while mRNAs of markers for perivascular macrophages (CD163), pro-inflammatory macrophages (CD64), monocytes (CD14), natural killer cells (FCGR3A) and adhesion molecules (ICAM1) were increased. Specific to high inflammation schizophrenia, quiescent stem cell marker mRNA (GFAPD) was reduced, whereas neuronal progenitor (ASCL1) and immature neuron marker mRNAs (DCX) were decreased compared to low inflammation control and schizophrenia subgroups. Thus, a heightened state of inflammation may dampen microglial response and recruit peripheral immune cells in psychiatric disorders. The findings elucidate differential neurogenic responses to inflammation within psychiatric disorders and highlight that inflammation may impair neuronal differentiation in the SEZ in schizophrenia.

Prenatal Octamethylcyclotetrasiloxane Exposure Impaired Proliferation of Neuronal Progenitor, Leading to Motor, Cognition, Social and Behavioral Functions

Cyclic siloxane octamethylcyclotetrasiloxane (D4) has raised concerns as an endocrine-disrupting chemical (EDC). D4 is widely used in detergent products, cosmetics, and personal care products. Recently, robust toxicological data for D4 has been reported, but the adverse effects of D4 on brain development are unknown. Here, pregnant mice on gestational day 9.5 were treated daily with D4 to postnatal day 28, and the offspring mice were studied. The prenatal D4-treated mice exhibited cognitive dysfunction, limited memory, and motor learning defect. Moreover, prenatal D4 exposure reduced the proliferation of neuronal progenitors in the offspring mouse brain. Next, the mechanisms through which D4 regulated the cell cycle were investigated. Aberrant gene expression, such as cyclin-dependent kinases CDK6 and cyclin-dependent kinase inhibitor p27, were found in the prenatal D4-treated mice. Furthermore, the estrogen receptors ERa and ERb were increased in the brain of prenatal D4-treated mice. Overall, these findings suggest that D4 exerts estrogen activity that affects the cell cycle progression of neuronal progenitor cells during neurodevelopment, which may be associated with cognitive deficits in offspring.

Newly-Discovered Neural Features Expand the Pathobiological Knowledge of Blastic Plasmacytoid Dendritic Cell Neoplasm

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive hematologic malignancy originating from plasmacytoid dendritic cells (pDCs). The microRNA expression profile of BPDCN was compared to that of normal pDCs and the impact of miRNA dysregulation on the BPDCN transcriptional program was assessed. MiRNA and gene expression profiling data were integrated to obtain the BPDCN miRNA-regulatory network. The biological process mainly dysregulated by this network was predicted to be neurogenesis, a phenomenon raising growing interest in solid tumors. Neurogenesis was explored in BPDCN by querying different molecular sources (RNA sequencing, Chromatin immunoprecipitation-sequencing, and immunohistochemistry). It was shown that BPDCN cells upregulated neural mitogen genes possibly critical for tumor dissemination, expressed neuronal progenitor markers involved in cell migration, exchanged acetylcholine neurotransmitter, and overexpressed multiple neural receptors that may stimulate tumor proliferation, migration and cross-talk with the nervous system. Most neural genes upregulated in BPDCN are currently investigated as therapeutic targets.

Re-evaluating the role of nucleosomal bivalency in early development

Nucleosomes, composed of DNA and histone proteins, represent the fundamental repeating unit of the eukaryotic genome1; posttranslational modifications of these histone proteins influence the activity of the associated genomic regions to regulate cell identity2–4. Traditionally, trimethylation of histone 3-lysine 4 (H3K4me3) is associated with transcriptional initiation5–10, whereas trimethylation of H3K27 (H3K27me3) is considered transcriptionally repressive11–15. The apparent juxtaposition of these opposing marks, termed “bivalent domains”16–18, was proposed to specifically demarcate of small set transcriptionally-poised lineage-commitment genes that resolve to one constituent modification through differentiation, thereby determining transcriptional status19–22. Since then, many thousands of studies have canonized the bivalency model as a chromatin hallmark of development in many cell types. However, these conclusions are largely based on chromatin immunoprecipitations (ChIP) with significant methodological problems hampering their interpretation. Absent direct quantitative measurements, it has been difficult to evaluate the strength of the bivalency model. Here, we present reICeChIP, a calibrated sequential ChIP method to quantitatively measure H3K4me3/H3K27me3 bivalency genome-wide, addressing the limitations of prior measurements. With reICeChIP, we profile bivalency through the differentiation paradigm that first established this model16,18: from naïve mouse embryonic stem cells (mESCs) into neuronal progenitor cells (NPCs). Our results cast doubt on every aspect of the bivalency model; in this context, we find that bivalency is widespread, does not resolve with differentiation, and is neither sensitive nor specific for identifying poised developmental genes or gene expression status more broadly. Our findings caution against interpreting bivalent domains as specific markers of developmentally poised genes.

Locus specific epigenetic modalities of random allelic expression imbalance

Most autosomal genes are thought to be expressed from both alleles, with some notable exceptions, including imprinted genes and genes showing random monoallelic expression (RME). The extent and nature of RME has been the subject of debate. Here we investigate the expression of several candidate RME genes in F1 hybrid mouse cells before and after differentiation, to define how they become persistently, monoallelically expressed. Clonal monoallelic expression is not present in embryonic stem cells, but we observe high frequencies of monoallelism in neuronal progenitor cells by assessing expression status in more than 200 clones. We uncover unforeseen modes of allelic expression that appear to be gene-specific and epigenetically regulated. This non-canonical allelic regulation has important implications for development and disease, including autosomal dominant disorders and opens up therapeutic perspectives.

Distinct regulation of hippocampal neuroplasticity and ciliary genes by corticosteroid receptors

Glucocorticoid hormones (GCs) — acting through hippocampal mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) — are critical to physiological regulation and behavioural adaptation. We conducted genome-wide MR and GR ChIP-seq and Ribo-Zero RNA-seq studies on rat hippocampus to elucidate MR- and GR-regulated genes under circadian variation or acute stress. In a subset of genes, these physiological conditions resulted in enhanced MR and/or GR binding to DNA sequences and associated transcriptional changes. Binding of MR at a substantial number of sites however remained unchanged. MR and GR binding occur at overlapping as well as distinct loci. Moreover, although the GC response element (GRE) was the predominant motif, the transcription factor recognition site composition within MR and GR binding peaks show marked differences. Pathway analysis uncovered that MR and GR regulate a substantial number of genes involved in synaptic/neuro-plasticity, cell morphology and development, behavior, and neuropsychiatric disorders. We find that MR, not GR, is the predominant receptor binding to >50 ciliary genes; and that MR function is linked to neuronal differentiation and ciliogenesis in human fetal neuronal progenitor cells. These results show that hippocampal MRs and GRs constitutively and dynamically regulate genomic activities underpinning neuronal plasticity and behavioral adaptation to changing environments.

Regulation of neuronal progenitor delamination by dynein-driven post-Golgi apical transport

Radial glial (RG) cells are the neural stem cells of the developing neocortex. Apical RG (aRG) cells can delaminate to generate basal RG (bRG) cells, a cell type associated with human brain expansion. Here, we report that this delamination is regulated by the post-Golgi secretory pathway. Using in situ subcellular live imaging, we show that post-Golgi transport of RAB6+ vesicles occurs toward the minus ends of microtubules and depends on dynein. We demonstrate that the apical determinant Crumbs3 (CRB3) is also transported by dynein. Double knockout of RAB6A/A' and RAB6B impairs apical localization of CRB3, and induces a retraction of aRG cell apical process, leading to delamination and ectopic division. These defects are phenocopied by knock-out of the dynein activator LIS1. Overall, our results identify a RAB6-dynein-LIS1 complex for Golgi to apical surface transport in aRG cells, and highlights the role of this pathway in the maintenance of neuroepithelial integrity.

Identification of PAX6 and NFAT4 as the transcriptional regulators of lncRNA Mrhl in neuronal progenitors

LncRNA Mrhlhas been shown to be involved in regulating meiotic commitment of mouse spermatogonial progenitors and coordinating differentiation events in mouse embryonic stem cells. Here we have characterized the interplay of Mrhlwith lineage-specific transcription factors during mouse neuronal lineage development. Our results demonstrate that Mrhl is predominantly expressed in the neuronal progenitor populations in mouse embryonic brains and in retinoic acid derived radial-glia like neuronal progenitor cells. Mrhl levels are significantly down regulated in postnatal brains and in maturing neurons. In neuronal progenitors, a master transcription factor, PAX6, acts to regulate the expression of Mrhl through direct physical binding at a major site in the distal promoter, located at 2.9kb usptream of the TSS of Mrhl. Furthermore, NFAT4 occupies the Mrhl proximal promoter at two sites, at 437bp and 143bp upstream of the TSS. ChIP studies reveal that PAX6 and NFAT4 interact with each other, suggesting co-regulation of lncRNA Mrhl expression in neuronal progenitors. Our studies herewith are crucial towards understanding how lncRNAs are regulated by major lineage-specific TFstowardsdefining specific development and differentiation events.