RNA-seq investigating the influence of astrocytes on developing cortical neurons
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ABSTRACT: Astrocytes are implicated in neuronal development, particularly excitatory synaptogenesis, but their genome-wide impact is unclear. Using cell-type specific RNA-seq we show that cortical astrocytes induce widespread transcriptomic changes in developing cortical neurons. Rat cortical neurons were maintained in the presence or absence of mouse astrocytes, RNA-seq performed, and mixed-species RNA-seq reads sorted according to species. Cultures were also treated with TTX to abolish neuronal firing activity, to investigate the effects of the presence or absence activity-dependent signalling.
Project description:Astrocytes are implicated in neuronal development, particularly excitatory synaptogenesis, but their genome-wide impact is unclear. Using cell-type specific ChIP-seq we show that cortical astrocytes induce widespread changes in developing cortical neurons in histone marks associated with active open chromatin and repressed/condensed chromatin. Rat cortical neurons were maintained in the presence or absence of mouse astrocytes, ChIP-seq performed, and mixed-species ChIP-seq reads sorted according to species.
Project description:We measure changes in the proteome after 48 hours of activity deprivation with tetrodotoxin (TTX) and the NMDA receptor antagonist (2R)-amino-5-phosphonovaleric acid (APV). We used wild type and FMRP mutant primary cortical neurons treated with vehicle or treated with TTX-APV.
Project description:Rett syndrome (RTT; OMIM#312750) is a rare devastating neurodevelopmental disorder that represents the most common genetic cause of severe intellectual disability in girls. Mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene have been reported in over 95% cases of classical forms of RTT. Although initial studies supported a role for MeCP2 exclusively in neurons, recent data indicate a function also in astrocytes, which emerged as critical players involved in RTT pathogenesis through non-cell autonomous effects. Indeed, Mecp2 knock-out (KO) astrocytes cannot properly support neuronal maturation of wilt-type (WT) neurons and our data demonstrated a detrimental effect also on synaptogenesis and synaptic maintainence. Nevertheless, the molecular mechanisms by which RTT astrocytes can impact on neuronal health remains unknown. In comparison to previous studies exploring the transcriptomic and proteomic profiles of KO astrocytes per se, we used an indirect strategy to unveil the molecular mechanisms responsible for their negative action on neurons. We thus analysed the molecular pathways deregulated in WT neurons cultivated under the influence of KO (n=8) versus WT (n=7) astrocytes, in a transwell-based co-culture system, that allows the exchange of paracrine signals preventing cell-to-cell contact. Astrocytes were seeded on transwell inserts and transferred on neurons at Div0; the co-cultures were maintained until Div14. WT cortical neurons cultivated alone were also included (n=5).
Project description:General anesthesia is a common clinical procedure yet can result in long term CNS-adverse effects, particularly in the elderly, or dementia patients. Suppression of cortical activity is a key feature of the anesthetic-induced unconscious state, with activity being a well-described regulator of pathways important for brain health. However the extent to which the effects of anesthesia go beyond simple suppression of neuronal activity is incompletely understood, as are its effects on non-neuronal cell types such as astrocytes, which are important integrators of both neuronal activity and inflammatory signalling. In order to address these questions, we performed a combination of RNA-seq and TRAP-seq (Translating Ribosome Affinity Purification, where astrocyte-specific translating mRNAs are sequenced) on mouse cortical tissue and primary cortical neurons.
Project description:Induced pluripotent stem cell (iPSC)-derived cortical neurons present a powerful new model of neurological disease. Previous work has established that differentiation protocols produce cortical neurons but little has been done to characterise these at cellular resolution. In particular, it is unclear to what extent in vitro two-dimensional, relatively disordered culture conditions recapitulate the development of in vivo cortical layer identity. Single cell multiplex RT-qPCR was used to interrogate the expression of genes previously implicated in cortical layer or phenotypic identity in individual cells. Unexpectedly, 22.7% of neurons analysed frequently co-expressed canonical fetal deep and upper cortical layer markers, and this co-expression was also present at the level of translated protein. By comparing our results to available single cell RNA-seq data from human fetal and adult brain, we observed that this co-expression of layer markers was also seen in primary tissue. These results suggest that establishing neuronal layer identity in iPSC-derived or primary cortical neurons using canonical marker genes transcripts is unlikely to be informative. Single cell RNA-seq of 16 iPSC-derived cortical neurons. This dataset was used for normalization purposes for GSE67835.
Project description:To investigate the functional and molecular effects of neuronal activity suppresion at the network level, we co-cultured human-derived iPSCs neurons with rat astrocytes and treated them with TTX.
Project description:This experiment comprises RNA-seq data used to study evolutionary differences between humans and mice in neuronal activity-dependent transcriptional responses. Activity-dependent transcriptional responses in developing human stem cell-derived cortical neurons were compared with those induced in developing primary- or stem cell-derived mouse cortical neurons 4 hours after KCl-induced membrane depolarisation. Activity-dependent transcriptional responses were also measured in aneuploid mouse neurons carrying human chromosome 21, allowing study of the regulation of Hsa21 genes, plus their mouse orthologs, side-by-side in the same cellular environment of a mouse primary neuron.
Project description:Activation of neurons is one of the fundamental events for the functioning of nervous system. Neuronal activation relays information to next neurons. On the other hand, the activated neurons themselves are also influenced by neuronal activation. Depending on the type and condition of neuronal activation, these activated neurons change their gene expressions, thereby being able to process information more or less efficiently. We applied the microarray technology to identify hither-to-uncharacterized as activity-dependent genes. Especially, we screened the transcription factors, because early changes in the transcription factors should result in alterations of gene expression profiles and subsequent neuronal properties. Experiment Overall Design: Rat primary cortical neurons with or without KCl treatment were selected for RNA extraction and hybridization on Affymetrix microarrays. To identify the genes whose expression was induced by depolarization, we first compared gene expression profiles in control vs. 4 hr after KCl (25 mM) treated cortical neurons using Affymetrix Genechips specified for neurobiology. All four hybridizations were analyzed for correlation accuracy between the replicates of the same treatments .Control replicates (control 1, 2) KCl-treated replicates (KCl 1, 2)
Project description:tRNA-derived stress-induced RNAs (tiRNAs) are a new class of small non-coding RNA that have emerged as important regulators of cellular stress responses. tiRNAs are derived from specific tRNA cleavage by the stress-induced ribonuclease angiogenin (ANG). Loss-of-function mutations in the ANG gene are linked to amyotrophic lateral sclerosis (ALS), and elevated levels of specific tiRNAs were recently identified in ALS patient serum samples. However, the biological role of tiRNA production in neuronal stress responses and neurodegeneration remains largely unknown. This study aimed to evaluate the genome-wide regulation of neuronal stress responses by an ALS-associated tiRNA; 5’tiRNAGly-GCC. To this end, we performed whole-transcript RNA-seq on mouse primary cortical neurons transfected with either a synthetic mimic of endogenous 5’tiRNAGly-GCC or a scrambled tiRNA control. This dataset contains the RNA-seq data used to identify the effects of 5’tiRNAGly-GCC on global gene expression in primary neurons.