ABSTRACT: Transcriptional profiling of the microdissected SVZ from 7-month-old mice Adult neurogenesis is suppressed in the SVZ of 3xTg mice, a model of Alzheimer's disease. To better understand the underlying mechanisms of this suppression, the goals of this experiment were to compare the transcriptional profiles of the SVZ neural stem cell niche in 3xTg-AD mice versus strain controls. We used early middle-aged mice (7-months-old) rather than old mice, in order to identify genetic changes that are not caused secondarily to other degenerative changes occurring in these mice. Two-condition experiment, 3xTg vs WT SVZ. Biological replicates: 4 for each.
Project description:Transcriptional profiling of the microdissected SVZ from 7-month-old mice Adult neurogenesis is suppressed in the SVZ of 3xTg mice, a model of Alzheimer's disease. To better understand the underlying mechanisms of this suppression, the goals of this experiment were to compare the transcriptional profiles of the SVZ neural stem cell niche in 3xTg-AD mice versus strain controls. We used early middle-aged mice (7-months-old) rather than old mice, in order to identify genetic changes that are not caused secondarily to other degenerative changes occurring in these mice.
Project description:Unprecedented bacterial targets are urgently needed for the development of novel antibiotics to overcome the current resistance crisis. Challenges include the limited uptake of compounds as well as prioritizing proteins for their druggability and functional relevance. Especially, the wealth of uncharacterized proteins represents an untapped source for novel targets. However, tools to decipher their function are largely lacking. We here utilize the systematic mining of pyridoxal phosphate dependent enzymes (PLP DEs) in bacteria to focus on a target class, which is known to bind ligands, accesses PLP via active transport from the media and is involved in crucial metabolic processes. For this, we systematically exploited the chemical space of the pyridoxal (PL) scaffold and obtained eight PL probes bearing modifications at various ring positions. These probes were subsequently tested for phosphorylation by cognate kinases and labelling of PLP DEs in clinically relevant Gram-positive (Staphylococcus aureus) as well as Gram-negative (Escherichia coli and Pseudomonas aeruginosa) strains. Overall, the coverage of this diverse set of probes along with refined labelling conditions not only exceeded the performance of a previous probe generation, it also provided a detailed map of binding preferences of certain structural motifs. Although originally conducted in mutant cells devoid of PLP de novo biosynthesis, we here demonstrate efficient PLP DE labelling also in a wild type strain. Overall, the profiling revealed several putative PLP DEs with unknown function, of which we exemplarily characterized five via in-depth enzymatic assays. Finally, we screened a panel of putative PLP binders for antibiotic activity and unravelled the targets of hit molecules via competitive profiling with our probes. Here, an uncharacterized enzyme, essential for bacterial growth, was assigned as PLP dependent cysteine desulfurase and confirmed to be inhibited by the marketed drug phenelzine. Our approach provides a basis for deciphering novel PLP DEs as essential antibiotic targets along with corresponding ways to decipher small molecule inhibitors.
Project description:Sonic hedgehog (Shh) signals via Gli transcription factors to promote maintenance and proliferation of neural stem cells in the adult mouse forebrain. We have analyzed the gene expression pattern in neurogenic Shh-responding astroglia (= neural stem cells ) in the subventicular zone of the lateral ventricle and dentate gyrus of the hippocampus in comparison to the non-neurogenic Shh-responding glia (=Bergman glia) in the cerebellum to identify the genes specifically involved in neurogenic function downstream of Shh signaling. In this dataset, we include the expression data obtained from FACS-sorted Gli1+ GFAP+ cells from microdissected SVZ, hippocampus and cerebellum. GFAP expression is based on hGFAP-GFP reporter line and Gli1 expression is lineage marked using Gli1-CreER;ROSA26-tdTomato mice. 15 Total samples were analyzed. We compared expression levels of SVZ vs. Cerebellum, Hippocampus vs. Cerebelllum to identify genes which had more than 4 fold change in expression levels with p < 0.01. From this narrowed list, we compared between SVZ and Hippocampus to identify the common genes up and down regulated. In addition, we also identified commonly expressed genes in hippocampus and SVZ at high level.
Project description:We have applied a recently developed, highly accurate and sensitive single-cell RNA-seq method (STRT/C1) to perform a molecular census of two regions of the mouse cerebral cortex: the somatosensory cortex and hippocampus CA1. We isolated cells fresh from somatosensory cortex (S1) and hippocampus CA1 area of juvenile (P22 - P32) CD1 mice, 33 males and 34 females. Cells were collected without selection, except that 116 cells were obtained by FACS from 5HT3a-BACEGFP transgenic mice. A total of 76 Fluidigm C1 runs were performed, each attempting 96 cell captures and resulting in 3005 high-quality single-cell cDNAs, containing Unique Molecular Identifiers allowing counting of individual mRNA molecules, even after PCR amplification.
Project description:Transcriptional profiling of subventricular zone (SVZ) progenitors comparing control healthy mice to mice induced to develop an autoimmune demyelination (EAE model). Goal was to unveil genes involved in demyelination-induced reactivity of SVZ progenitors. Two-condition experiment, healthy vs. EAE derived SVZ progenitors. Biological replicates: 2 control replicates, 2 EAE replicates. SVZ progenitors were sorted in two cell populations: neuronal progenitors (PSA-NCAM magnetic sorting) and glial progenitors (NG2 magnetic sorting). Progenitors from healthy mice are reference samples.
Project description:Parkinson’s disease (PD) is one of the most common neurodegenerative disease caused by diminution of neurons in the substantia nigra (SN) which projects dopaminergic (DA) axons to the striatum and other target areas. Recently, accumulating data demonstrated the prospects of cell replacement therapy by using neural stem cell (NSCs) transplantation. However the mechanisms underlying the potential efficacy are not fully understood. To gain new insights into the mechanisms of 6-OHDA induced lesion and potential efficacy of hNSCs transplantation, Intrastriatal 6-Hydroxydopamine (6-OHDA) injected parkinsonian mice were unilaterally engrafted with undifferentiated human NSCs to striatum (ST). High-throughput quantitative proteomic approach was utilized to characterize the proteome profiles of PD related brain regions in these mice including SN, ST, olfactory bulb (OB) and subventricular zone (SVZ). The abundance of more than 5000 proteins with high confidence in each region was determined in this study which represents the most extensive proteomic study of PD mouse models to date. In addition to the disruption of the DA system, the quantitative analysis demonstrated the profound disturbance of SVZ proteome after 6-OHDA insult, in which the abundance of more than 20% proteins was significantly changed. After hNSCs engraftment, the proteome of SVZ was restored and the astrocytes in ST was greatly activated in companion with the increase in neurotrophic factors. Furthermore, bioinformatics analysis demonstrated that changes in proteome was not caused by the proliferation of hNSCs or their progeny, but rather by the reaction of endogenous cells. Overall, this study reveals that hNSCs benefits parkinsonian animals by eliciting endogenous responses in multiple brain regions, and discovers the unexpected role of SVZ cells in PD progress and treatment, providing new therapeutic targets.
Project description:The 3xTg-AD mouse is a widely used model in the study of Alzheimer’s Disease (AD). It has been extensively characterized both from the anatomical and behavioral point of view but poorly studied at the transcriptomic level. For the first time, this study characterizes the whole blood transcriptome of the 3xTg-AD mouse at three and six months of age and evaluates how gene expression is modulated by transcranial direct current stimulation (tDCS). RNA-seq analysis revealed 183 differentially expressed genes (DEGs) that were a direct signature of the genetic background of the mouse. The expression profile of age-related genes in the 3 months-old 3xTg-AD mice was more similar to that of 6 months rather than 3 months-control mice, suggesting a premature aging of the 3xTg-AD mice. Moreover, in the 6 months-old 3xTg-AD mice, we observed a high number of DEGs that could represent good peripheral biomarkers of AD progression. Finally, tDCS was associated with gene expression changes in the 3xTg-AD but not in the control mice. In conclusion, this study provides a better molecular characterization of the 3xTg-AD mouse and suggests that blood gene expression can be used to identify new biomarkers of AD progression and treatments effect.
Project description:In this study, using a microarray approach, we investigated the age-dependent changes in the gene expression profile of hippocampi obtained from young and old 3xTg-AD and WT control mice, in order to identify the molecular mechanisms involved in the development of AD and assess the role of aging in the development of the disease. A global gene expression profile in the hippocampi obtained from 3xTgAD (expressing mutant human APP, PS1, and tau) and WT mice at 3 and 12 months of age was studied by employing a Mouse OneArray Whole Genome DNA microarray. Data were analyzed with the Ingenuity Pathways Analysis (IPA) in order to achieve a classification of the results on the basis of their biological functions and disclose functional networks and/or pathways. In this study we performed gene expression profiles for a total of 9 experiments. Hippocampi were collected from 2 3xTG and 2 WT mice at 3 months of age and from 2 3xTG and 2 WT mice at 12 months of age. Three MicroArray experiments were performed for each condition (WT 12 moa versus WT 3 moa; 3xTG 3 moa versus WT 3 moa; 3xTG 12 moa versus WT 12moa). On the total of 9 experiments 6 were biological replicates and 3 were technical replicates.
Project description:Neural stem cells (NSCs) generate new neurons throughout life in two distinct areas of the mammalian brain: the subventricular zone (SVZ) lining the lateral ventricles and the hippocampal dentate gyrus (DG). How gene expression signatures differ among NSCs and immature neurons within and between these adult neurogenic regions is unknown. We isolated NSCs and their progeny using transgenic mice expressing GFP under the control of the Sox2 promoter (labeling NSCs) and transgenic mice expressing DsRed under the control of the doublecortin (Dcx) promoter (labeling immature neurons). Comparison of the transcriptomes of SOX2+ cells derived from both neurogenic areas revealed that NSCs are highly similar but that functionally significant differences in gene expression exist: IGF2, which is expressed only in SOX2+ cells in the DG but not in the SVZ, is required for proliferation of DG-derived but not SVZ-derived NSCs. Gene expression profiles strongly diverged in immature neurons, and we provide evidence that ephrinB3, which was up-regulated only in the DG but not in the SVZ during neuronal differentiation, regulates the survival of newborn granule cells. Thus, the data provided here show that stem cell populations in the adult DG and SVZ are similar but have unique properties that manifest themselves later during neural differentiation, resulting in distinct neuronal populations Hippocampi and SVZ from 6 week old DCX-DsRed and Sox2-GFP Reporter mice were dissected and cell sorted using FACS. cDNA were generated and analysed using Agilent Platform.