RNA-SEQ profiling of dopaminergic neurons from the mid-brain of mice that received intraperitoneal injections of MPTP
Ontology highlight
ABSTRACT: RNA-SEQ of dopaminergic neurons from the mid-brain of mice that received one daily intraperitoneal injection of MPTP-HCl (30 mg/kg free base per day) or saline for five consecutive days. Samples were taken 4 days. Murine midbrain dopaminergic neurons that were treated with MPTP-HCl
Project description:RNA-SEQ profiling of mouse dopaminergic neurons from the mouse mid-brain, with AAV1 injections using a Satb1 shRNA-EGFP construct or a scrambed shRNA-EGFP construct Murine midbrain dopaminergic neurons with Satb1 shRNA treatment or scrambled control
Project description:Engrailed homeoproteins are expressed in adult dopaminergic neurons of the substantia nigra. In Engrailed1 heterozygous mice, these neurons start dying at 6 weeks, are more sensitive to oxidative stress and progressively develop traits similar to those observed following an acute and strong oxidative stress inflected to wild-type neurons. These changes include DNA strand breaks and the modification (intensity and distribution) of several nuclear and nucleolar heterochromatin marks. Engrailed1 and Engrailed2 are biochemically equivalent transducing proteins previously used to antagonize dopaminergic neuron death in Engrailed heterozygous mice and in mouse models of Parkinson disease. Accordingly, we show that, following an acute oxidative stress, a single Engrailed2 injection restores all nuclear and nucleolar heterochromatin marks, decreases the number of DNA strand breaks and protects dopaminergic neurons against apoptosis. RNA-seq data for differentially expressed genes in the SNpc of En1+/- mice, En2 infused mice and 6-OHDA/En2 injection experiments.
Project description:RNA-SEQ of dopaminergic neurons from the mid-brain of mice that received one daily intraperitoneal injection of MPTP-HCl (30 mg/kg free base per day) or saline for five consecutive days. Samples were taken 4 days.
Project description:RNA-SEQ profiling of dopaminergic neurons from the substantia nigra pars compacta and ventral tegmental area regions of the mouse mid-brain Murine midbrain dopaminergic neurons from the SNpc and VTA regions
Project description:Microarray comparison of transgenic mice overexpressing human alpha-synuclein under the PDGF beta promoter to wildtype littermates at 3 months and 9 months of age. RNA was extracted from substantia nigral cells that were isolated by laser capture microdissection.
Project description:SATB1 is a genetic master regulator in dopaminergic neurons. We try to identify the downstream regulated genes and pathways of SATB1 in human dopaminergic and CTX neurons. The RNA-Seq experiment was performed to investigate the role of the genetic master regulator SATB1 in human dopaminergic neurons in comparison to cortical neurons. We generated a human embryonic stem cell knockout clone for SATB1 and differentiated this clone into either dopaminergic or cortical neurons. Immature dopaminergic (day 30 of differentiation), mature dopaminergic (day 50 of differentiation) and mature cortical neurons (day 30 of differentiation) were subsequently subjected to RNA-Seq. We compared wild type and SATB1-KO neurons at the afore mentioned time points, to characterize the regulatory role of SATB1 in the different neuron subtypes.
Project description:Demineralized bone matrix is a widely used allograft where not only the inorganic mineral but also embedded growth factors are removed by hydrochloric acid. The cellular response to the growth factors released during the preparation of demineralized bone matrix, however, has not been studied. Here we investigated the in vitro impact of acid bone lysates (ABL) prepared from porcine cortical bone chips on oral fibroblasts. Proteomic analysis of ABL revealed a large spectrum of bone-derived proteins including TGF-β1. Whole genome microarrays and RT-PCR together with the pharmacologic blocking of TGF-β receptor type I kinase with SB431542 showed that ABL activates the TGF-β target genes interleukin 11, proteoglycan 4, and NADPH oxidase 4. Interleukin 11 expression was confirmed at the protein level by ELISA. Immunofluorescence and Western blot showed the nuclear localization of Smad2/3 and increased phosphorylation of Smad3 with ABL, respectively. This effect was independent of whether ABL was prepared from mandible, calvaria or tibia. ABL prepared with 0.1 N HCL was most effective compared to lysates obtained with 0.01 N and also 1.0 N HCl. These results demonstrate that TGF-β is a major growth factor that is removed upon the preparation of demineralized bone matrix.
Project description:We have conducted quantitative proteomic analyses of the axons of cultured rat cortical neurons. Axons are isolated by using glass chips that enable the axons and their cell bodies of neurons to grow in separated regions on the chip surface. Proteins extracted from the isolated axons, as well as those extracted from whole cortical neurons are subjected to two-dimensional liquid chromatography (2D-LC)-mass spectrometry (MS)-MS analyses. The abundances of proteins in the axon are found to be strongly correlated with their average abundances in whole neurons. Based upon these data, a quantitative description of the protein distribution among various subcellular structures in the axon has been generated. The proteins extracted from the axons and whole neurons are also subjected to stable isotope dimethyl labeling reaction and then to 2D-LC-MS/MS analysis. Proteins enriched in the axon compartment of rat cortical neurons are thus identified.
Project description:To develop more potent cell transplantation therapy for neurodegenerative disorder such as Parkinson’s disease (PD), the condition of the host brain environment should be considered to improve the outcome of grafted neurons. However, we never know which condition of host brain environment is suitable and supportive for the donor cells. In addition, what endogenous factor(s) do contribute to improve the engraftment of donor cells in host brain? Therefore, the identification of such effective factor(s) strongly contribute to improve the overcome of cell transplantation therapy. Here, we constructed the experimental approach to identify the effective soluble factor(s) for cell-grafting by comparison between various parkinsonian mouse brain condition and transplantation outcome using induced pluripotent stem cell (iPSC)-derived dopaminergic (DA) neuron progenitors. According to our experimental approach, we have identified secreted peptide, neurexophilin 3 (NXPH3) that enhance the survival of grafted-iPSC-derived DA neurons. Grafted-iPSC-derived DA neurons were increased by local supplement of NXPH3 protein. In addition, the expression level of NXPH3 in putamen of PD patients was significantly decreased than that of normal controls by using postmortem samples. These findings would be expected to contribute the new experimental strategy to indentify the endogenous effective factors for cell-grafting as in vivo application of stem cell technology. Mice were divided into three groups as follows: (i) 1 week after acute administration (four times by every 2 h) of free base MPTP HCl (20 mg/kg, 80 mg/kg in total, intraperitoneally). (ii) 8 weeks after chronic administration (once a day for 20 consecutive days) of free base MPTP HCl (4 mg/kg per day, 80 mg/kg in total). (iii) 1 week after injection (four times by every 2 h) of saline (control).
Project description:The proper subcellular localization of RNAs and local translational regulation is crucial in highly compartmentalized cells, such as neurons. RNA localization is mediated by specific cis-regulatory elements usually found in mRNA 3'UTRs. Therefore, processes that generate alternative 3'UTRs – alternative splicing and polyadenylation – have the potential to diversify mRNA localization patterns in neurons. Here, we performed mapping of alternative 3'UTRs in neurites and soma isolated from mESC-derived neurons. Our analysis identified 593 genes with differentially localized 3'UTR isoforms. In particular, we have shown that two isoforms of Cdc42 gene with distinct functions in neuronal polarity are differentially localized between neurites and soma of mESC-derived and mouse primary cortical neurons, at both mRNA and protein level. Using reporter assays and 3'UTR swapping experiments, we have identified the role of alternative 3’UTRs and mRNA transport in differential localization of alternative CDC42 protein isoforms. Moreover, we used SILAC to identify isoform-specific Cdc42 3'UTR-bound proteome with potential role in Cdc42 localization and translation. Our analysis points to usage of alternative 3'UTR isoforms as a novel mechanism to provide for differential localization of functionally diverse alternative protein isoforms.