Shared patterns of glial transcriptional dysregulation link Huntington's disease and schizophrenia.

Huntington's disease and juvenile-onset schizophrenia have long been regarded as distinct disorders. However, both manifest cell-intrinsic abnormalities in glial differentiation, with resultant astrocytic dysfunction and hypomyelination. To assess whether a common mechanism might underlie the similar glial pathology of these otherwise disparate conditions, we used comparative correlation network approaches to analyse RNA-sequencing data from human glial progenitor cells (hGPCs) produced from disease-derived pluripotent stem cells. We identified gene sets preserved between Huntington's disease and schizophrenia hGPCs yet distinct from normal controls that included 174 highly connected genes in the shared disease-associated network, focusing on genes involved in synaptic signalling. These synaptic genes were largely suppressed in both schizophrenia and Huntington's disease hGPCs, and gene regulatory network analysis identified a core set of upstream regulators of this network, of which OLIG2 and TCF7L2 were prominent. Among their downstream targets, ADGRL3, a modulator of glutamatergic synapses, was notably suppressed in both schizophrenia and Huntington's disease hGPCs. Chromatin immunoprecipitation sequencing confirmed that OLIG2 and TCF7L2 each bound to the regulatory region of ADGRL3, whose expression was then rescued by lentiviral overexpression of these transcription factors. These data suggest that the disease-associated suppression of OLIG2 and TCF7L2-dependent transcription of glutamate signalling regulators may impair glial receptivity to neuronal glutamate. The consequent loss of activity-dependent mobilization of hGPCs may yield deficient oligodendrocyte production, and hence the hypomyelination noted in these disorders, as well as the disrupted astrocytic differentiation and attendant synaptic dysfunction associated with each. Together, these data highlight the importance of convergent glial molecular pathology in both the pathogenesis and phenotypic similarities of two otherwise unrelated disorders, Huntington's disease and schizophrenia.

RIF1 and KAP1 differentially regulate the choice of inactive versus active X chromosomes.

The onset of random X chromosome inactivation in mouse requires the switch from a symmetric to an asymmetric state, where the identities of the future inactive and active X chromosomes are assigned. This process is known as X chromosome choice. Here, we show that RIF1 and KAP1 are two fundamental factors for the definition of this transcriptional asymmetry. We found that at the onset of differentiation of mouse embryonic stem cells (mESCs), biallelic up-regulation of the long non-coding RNA Tsix weakens the symmetric association of RIF1 with the Xist promoter. The Xist allele maintaining the association with RIF1 goes on to up-regulate Xist RNA expression in a RIF1-dependent manner. Conversely, the promoter that loses RIF1 gains binding of KAP1, and KAP1 is required for the increase in Tsix levels preceding the choice. We propose that the mutual exclusion of Tsix and RIF1, and of RIF1 and KAP1, at the Xist promoters establish a self-sustaining loop that transforms an initially stochastic event into a stably inherited asymmetric X-chromosome state.

Dysregulated Glial Differentiation in Schizophrenia May Be Relieved by Suppression of SMAD4- and REST-Dependent Signaling.

Astrocytic differentiation is developmentally impaired in patients with childhood-onset schizophrenia (SCZ). To determine why, we used genetic gain- and loss-of-function studies to establish the contributions of differentially expressed transcriptional regulators to the defective differentiation of glial progenitor cells (GPCs) produced from SCZ patient-derived induced pluripotent cells (iPSCs). Negative regulators of the bone morphogenetic protein (BMP) pathway were upregulated in SCZ GPCs, including BAMBI, FST, and GREM1, whose overexpression retained SCZ GPCs at the progenitor stage. SMAD4 knockdown (KD) suppressed the production of these BMP inhibitors by SCZ GPCs and rescued normal astrocytic differentiation. In addition, the BMP-regulated transcriptional repressor REST was upregulated in SCZ GPCs, and its KD similarly restored normal glial differentiation. REST KD also rescued potassium-transport-associated gene expression and K+ uptake, which were otherwise deficient in SCZ glia. These data suggest that the glial differentiation defect in childhood-onset SCZ, and its attendant disruption in K+ homeostasis, may be rescued by targeting BMP/SMAD4- and REST-dependent transcription.

Nuclear Architecture Organized by Rif1 Underpins the Replication-Timing Program.

DNA replication is temporally and spatially organized in all eukaryotes, yet the molecular control and biological function of the replication-timing program are unclear. Rif1 is required for normal genome-wide regulation of replication timing, but its molecular function is poorly understood. Here we show that in mouse embryonic stem cells, Rif1 coats late-replicating domains and, with Lamin B1, identifies most of the late-replicating genome. Rif1 is an essential determinant of replication timing of non-Lamin B1-bound late domains. We further demonstrate that Rif1 defines and restricts the interactions between replication-timing domains during the G1 phase, thereby revealing a function of Rif1 as organizer of nuclear architecture. Rif1 loss affects both number and replication-timing specificity of the interactions between replication-timing domains. In addition, during the S phase, Rif1 ensures that replication of interacting domains is temporally coordinated. In summary, our study identifies Rif1 as the molecular link between nuclear architecture and replication-timing establishment in mammals.

Mouse Rif1 is a key regulator of the replication-timing programme in mammalian cells.

The eukaryotic genome is replicated according to a specific spatio-temporal programme. However, little is known about both its molecular control and biological significance. Here, we identify mouse Rif1 as a key player in the regulation of DNA replication timing. We show that Rif1 deficiency in primary cells results in an unprecedented global alteration of the temporal order of replication. This effect takes place already in the first S-phase after Rif1 deletion and is neither accompanied by alterations in the transcriptional landscape nor by major changes in the biochemical identity of constitutive heterochromatin. In addition, Rif1 deficiency leads to both defective G1/S transition and chromatin re-organization after DNA replication. Together, these data offer a novel insight into the global regulation and biological significance of the replication-timing programme in mammalian cells.

Parkinson disease-associated DJ-1 is required for the expression of the glial cell line-derived neurotrophic factor receptor RET in human neuroblastoma cells.

Mutations in PARK7/DJ-1 are associated with autosomal recessive, early onset Parkinson disease (PD). DJ-1 is an atypical peroxiredoxin-like peroxidase that may act as a redox-dependent chaperone and a regulator of transcription. Here we show that DJ-1 plays an essential role in the expression of rearranged during transfection (RET), a receptor for the glial cell line-derived neurotrophic factor, a neuroprotective molecule for dopaminergic neurons, the main target of degeneration in PD. The inducible loss of DJ-1 triggers the establishment of hypoxia and the production of reactive oxygen species that stabilize the hypoxia-inducible factor-1alpha (HIF-1a). HIF-1a expression is required for RET down-regulation. This study establishes for the first time a molecular link between the lack of functional DJ-1 and the glial cell line-derived neurotrophic factor signaling pathway that may explain the adult-onset loss of dopaminergic neurons. Furthermore, it suggests that hypoxia may play an important role in PD.

A proteomic approach to the bilirubin-induced toxicity in neuronal cells reveals a protective function of DJ-1 protein.

Unconjugated bilirubin (UCB) is a powerful antioxidant and a modulator of cell growth through the interaction with several signal transduction pathways. Although newborns develop a physiological jaundice, in case of severe hyperbilirubinemia UCB may become neurotoxic causing severe long-term neuronal damages, also known as bilirubin encephalopathy. To investigate the mechanisms of UCB-induced neuronal toxicity, we used the human neuroblastoma cell line SH-SY5Y as an in vitro model system. We verified that UCB caused cell death, in part due to oxidative stress, which leads to DNA damage and cell growth reduction. The mechanisms of cytotoxicity and cell adaptation to UCB were studied through a proteomic approach that identified differentially expressed proteins involved in cell proliferation, intracellular trafficking, protein degradation and oxidative stress response. In particular, the results indicated that cells exposed to UCB undertake an adaptive response that involves DJ-1, a multifunctional neuroprotective protein, crucial for cellular oxidative stress homeostasis. This study sheds light on the mechanisms of bilirubin-induced neurotoxicity and might help to design a strategy to prevent or ameliorate the neuronal damages leading to bilirubin encephalopathy.

A transcriptome analysis identifies molecular effectors of unconjugated bilirubin in human neuroblastoma SH-SY5Y cells.

BACKGROUND: The deposition of unconjugated bilirubin (UCB) in selected regions of the brain results in irreversible neuronal damage, or Bilirubin Encephalopathy (BE). Although UCB impairs a large number of cellular functions in other tissues, the basic mechanisms of neurotoxicity have not yet been fully clarified. While cells can accumulate UCB by passive diffusion, cell protection may involve multiple mechanisms including the extrusion of the pigment as well as pro-survival homeostatic responses that are still unknown. RESULTS: Transcriptome changes induced by UCB exposure in SH-SY5Y neuroblastoma cell line were examined by high density oligonucleotide microarrays. Two-hundred and thirty genes were induced after 24 hours. A Gene Ontology (GO) analysis showed that at least 50 genes were directly involved in the endoplasmic reticulum (ER) stress response. Validation of selected ER stress genes is shown by quantitative RT-PCR. Analysis of XBP1 splicing and DDIT3/CHOP subcellular localization is presented. CONCLUSION: These results show for the first time that UCB exposure induces ER stress response as major intracellular homeostasis in surviving neuroblastoma cells in vitro.