Wip1 regulates adult neurogenesis and Wnt signaling during aging
ABSTRACT: The number of newly-formed neurons declines rapidly during aging. Here we describe an important mechanism that contributes to this decline via Wip1-dependent regulation of neuronal differentiation. We found that Wip1 is expressed in neural stem/progenitor cells (NPCs) of the mouse subventricular zone and its upregulation at physiological levels maintained higher NPC numbers and neuronal differentiation in old mice. This resulted in markedly improved neuron formation and rescued a functional defect in fine odor discrimination in old mice. We identified Dkk3 as a key downstream target of Wip1 and found that its expression in SVZ is restricted to NPCs. Functionally, Dkk3 inhibited neuroblast formation by suppressing Wnt signaling, while deletion of Dkk3 or pharmacological reactivation of the Wnt pathway improved neuron formation and olfactory function in aged mice. We propose that Wip1 controls a Dkk3-dependent inhibition of neuronal differentiation during aging and thus regulating Wip1 levels could prevent certain aspects of functional decline of the aging brain. We found if neurospheres were derived from 18 months old mice, Wip1 transgenic neurospheres were more neurogenic than wt ones. This microarray was a pilot experiment to search the mechanism how Wip1 Transgene promoted neurogenesis, and found Dkk3 as a potential mediator. WT vs Wip1Tg neurospheres were cultured from mouse brain, and gene expression was compared using Illumina mouseWG-6 array
Project description:Analysis of dopaminergic neuronal gene expression changes by Nurr1 and/or Foxa2 overexpression. Result provides that Foxa2 potentiates Nurr1-induced DA neuronal phenotype gene expression. To identify the syergism of Nurr1 and Foxa2 for developing DA neural precursors, neural precusor cells (NPCs) isolated from embryonic brain were treated control, Nurr1, Foxa2 and Nurr1-Foxa2 retrovirus. After treatment of retroviruses, NPCs were cultrued in N2 media withdrawn mitogen (bFGF, EGF) for differetiation of DA neuron. Total RNA was obtained from NPCs in differentiation day 2.
Project description:Hematopoietic stem cell (HSC) aging underlies many age-related hematopoietic disorders. Accumulation of DNA damage is a hallmark of HSC aging. Wild-type p53-induced phosphatase 1 (Wip1) is a homeostatic regulator of DNA damage response. We used microarrays to detail the global programme of gene expression in Wip1 KO HSC Wild-type p53-induced phosphatase 1 (Wip1) knockout HSC and Wild type HSC were selected for RNA extraction and hybridization on Affymetrix microarrays.
Project description:Hematopoietic stem cell (HSC) aging underlies many age-related hematopoietic disorders. Accumulation of DNA damage is a hallmark of HSC aging. Wild-type p53-induced phosphatase 1 (Wip1) is a homeostatic regulator of DNA damage response. We used microarrays to detail the global programme of gene expression in Wip1 KO HSC Overall design: Wild-type p53-induced phosphatase 1 (Wip1) knockout HSC and Wild type HSC were selected for RNA extraction and hybridization on Affymetrix microarrays.
Project description:To isolate neuronal progenitor cells (NPCs), forebrains of E13.5 Miz1+/+ or Miz1-delta-POZ embryos were cut in small pieces, digested with trypsin and filtered through sterile gauze. Cells were cultivated in 2:1 DMEM/F12 supplemented with 1xB27 (Life technologies), 20 ng/ul EGF (Biomol), 20 ng/?l basic FGF (Biomol), 1 ug/ml fungizone (Gibco) and Penicillin/Streptomycin (PAA). NPCs were passaged every seven days. RNA expression of different genotypes was compared in sec. and quart. neurospheres.
Project description:Neural precursor cells (NPCs) in the mammalian neocortex generate various neuronal and glial cell types in a developmental stage-dependent manner. Most neocortical NPCs lose their neurogenic potential after birth. We have previously shown that high mobility group A (HMGA) proteins confer the neurogenic potential on early-stage NPCs during the midgestation period, although the underlying mechanisms are not fully understood. Here we performed microarray analysis and compared expression profiles between control and HMGA2-overexpressed NPCs. Mouse neocortical neuroepithelial cells were isolated at embryonic day 11.5 and cultured as neurospheres for 9 days in vitro in the presence of fibroblast growth factor (FGF) 2 and epidermal growth factor (EGF). These cells were infected with control or HMGA2-expressing retrovirus, cultured in the presence of FGF and EGF for 3 days. Half of them were collected immediately for microarray analysis, and the other half were cultured in the absence of FGF for 12 h and then collected for microarray analysis.
Project description:Despite the advances in our understanding of aging-associated behavioral decline, we know relatively little about how aging affect neural circuits that underlie specific behaviors. Specifically, we know little about how aging affect expression of genes in specific neural circuits. We have now addressed this problem by exploring a cholinergic neuron R15, an identified neuron of marine snail Aplysia. R15 is characterized by bursting action potentials and is implicated in reproduction, osmoregulation and locomotion. We examined changes in gene expression in R15 neurons during aging by microarray analyses of RNAs prepared from two different age groups, mature and old animals. Specifically we find that 1083 ESTs are differentially regulated in mature and old R15 neurons. Bioinformatics analyses of these genes have identified specific biological pathways and molecular processes that are up or down regulated in mature and old neurons. Comparison with human signaling networks using pathway analyses have identified three major networks that are altered in old R15 neurons. Furthermore, by single neuron qRTPCR we examined expression levels of candidate regulators involved in transcription (CREB1) and translation (S6 kinase) and find that aging is associated with a decrease in expression of these regulators. We next studied expression of CREB1 and S6 kinase in two different motor neurons (L7 and L11) and another cholinergic neuron R2 and find that these neurons have characteristic changes in gene expression during aging
Project description:The neocortex contains an unparalleled diversity of neuronal subtypes responsible for complex behavior. Each cortical neuron has distinct traits, which are developmentally acquired under the control of batteries of neuron subtype-specific and pan-neuronal genes. The cis-regulatory logic that orchestrates the coordinated regulation of each unique combination of genes is not known for any class of neurons of the neocortex. We report that Fezf2, a transcription factor able to induce defining features of corticospinal motor neurons (CSMN), associates with the proximal promoters and regulates expression of series of CSMN signature genes. Fezf2 targets are functionally relevant as demonstrated by the finding that Fezf2 governs expression of the axon guidance receptor EphB1 to execute the ipsilateral extension of the corticospinal tract. Our data indicate that co-regulated expression of neuron subtype-specific gene batteries by a common transcription factor is one component of the regulatory logic responsible for the establishment of CSMN identity. Overall design: Fezf2 binding is characterized in neurospheres using ChIP-seq targeted against 3Flag-tagged Fezf2. Two types of control experiments were performed: mock IP and GFP-3Flag ChIP-seq. All experiments were performed in biological duplicate.
Project description:WIP1 phosphatase is emerging as an important regulator of tumorigenesis, but no unifying mechanistic network has been proposed. Here we found that WIP1 plays a key role in the transcriptional regulation of heterochromatin-associated DNA sequences in germ-line and cancer cells. WIP1 was required for epigenetic remodeling of repetitive DNA elements within the heterochromatin, including L1 LINE retrotransposons. Mechanistically, WIP1regulated an ATM-dependent increase in BRCA1 occupancy on L1 LINEs, resulting in closed chromatin without ubiquitination of histone H2A. This mechanism appeared to be dependent on the ability of BRCA1 to bind the heterochromatin protein HP1, the recruitment of DNA methyltransferases, and subsequent DNA methylation. Attenuation of ATM, in turn, reversed heterochromatin methylation in both germ-line and cancer cells. DNA methylation plays a central role in the generation of mutations in human tumors and we found that WIP1 levels strongly correlated with C-to-T substitutions and a total mutation load in primary breast cancers. We propose that WIP1 plays an important role in the regulation of DNA methylation and global heterochromatin silencing, and thus is critical in maintaining genome integrity during development and in cancer. Total RNA was extracted from control spermatids, Wip1-/- and Wip1-/- Atm+/- spermatids. The final cRNA samples were hybridized in triplicates to Illumina Mouse WG-6 v2.0 Expression arrays.
Project description:Exposure to lead (Pb) during childhood can result in learning disabilities and behavioral problems. Although described in animal models, whether Pb exposure also alters neuronal differentiation in the developing brains of exposed children is unknown. Here, we investigated the effects of physiologically relevant concentrations of Pb (from 0.4 to 1.9 µM or 0 to 40µg/dl) on the capacity of human embryonic stem cells (hESCs) to progress to a neuronal fate. We found that neither acute nor chronic exposure to Pb prevented hESCs from generating neural precursor cells (NPCs). NPCs derived from hESCs chronically exposed to 1.9 µM or 40µg/dl Pb throughout the neural differentiation process generated 2.5 times more TUJ1-positive neurons than those derived from control hESCs. Pb exposure of hESCs during the stage of neural rosette formation resulted in a significant decrease in the expression levels of the neural marker genes PAX6 and MSI1. Furthermore, the resulting NPCs differentiated into neurons with shorter neurites and less branching than control neurons, as assessed by Sholl analysis. DNA methylation studies of control, acutely treated hESCs and NPCs derived from chronically exposed hESCs using the Illumina HumanMethylation450 BeadChip® demonstrated that Pb exposure induced changes in the methylation status of genes involved in neurogenetic signaling pathways. In summary, our study shows that exposure to Pb subtly alters the neuronal differentiation of exposed hESCs and that these changes could be partly mediated by modifications in the DNA methylation status of genes crucial to brain development. We analyzed the methylation profile of undifferentiated (n=2 independent experiments) and differentiating (n=2 independent experiments) human embryonic stem cells (hESCs) acutely exposed to losed to lead (Pb) and neural precursor cells derived from hESCs chronically exposed to Pb throughout the neural differentiation process (n=3 independent experiments).
Project description:Decline in tissue NAD levels during aging has been linked to aging-associated diseases, such as age-related metabolic disease, physical decline, and Alzheimers disease. However, the mechanism for aging-associated NAD decline remains unclear. Here we report that pro-inflammatory M1 macrophages, but not naive or M2 macrophages, highly express the NAD consuming enzyme CD38 and have enhanced CD38-dependent NADase activity. Furthermore, we show that aging is associated with enhanced inflammation due to increased senescent cells, and the accumulation of CD38 positive M1 macrophages in visceral white adipose tissue. We also find that inflammatory cytokines found in the supernatant from senescent cells (Senescence associated secretory proteins, SASP) induces macrophages to proliferate and express CD38. As senescent cells progressively accumulate in adipose tissue during aging, these results highlight a new causal link between visceral tissue senescence and tissue NAD decline during aging and may present a novel therapeutic opportunity to maintain NAD levels during aging.