Dataset Information

Astrocyte conditioned media-dependent Phosphatidylinositol 3-kinase interactome in mouse hippocampal neuron cell line Ht22

ABSTRACT: Astrocytes can support neuronal survival through a range of secreted signals that protect against neurotoxicity, oxidative stress, and apoptotic cascades. To identify proteins contributing to protective intracellular neuronal signalling originating from astrocytes, endogenous PI3K was immunoprecipitated from Ht22 cells exposed to primary astrocyte conditioned media (ACM) or cell free media (CFM), followed by iTRAQ-based quantitative proteomic analysis.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain, Hippocampal Neuron

DISEASE(S): Wounds And Injuries

SUBMITTER: Declan Williams

LAB HEAD: Gerold Schmitt-Ulms

PROVIDER: PXD022200 | Pride | 2021-09-09

REPOSITORIES: Pride

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Publications

An endogenous PI3K interactome promoting astrocyte-mediated neuroprotection identifies a novel association with RNA-binding protein ZC3H14.

Alqawlaq Samih S Livne-Bar Izhar I Williams Declan D D'Ercole Joseph J Leung Sara W SW Chan Darren D Tuccitto Alessandra A Datti Alessandro A Wrana Jeffrey L JL Corbett Anita H AH Schmitt-Ulms Gerold G Sivak Jeremy M JM

The Journal of biological chemistry 20201203

Astrocytes can support neuronal survival through a range of secreted signals that protect against neurotoxicity, oxidative stress, and apoptotic cascades. Thus, analyzing the effects of the astrocyte secretome may provide valuable insight into these neuroprotective mechanisms. Previously, we characterized a potent neuroprotective activity mediated by retinal astrocyte conditioned media (ACM) on retinal and cortical neurons in metabolic stress models. However, the molecular mechanism underlying t ...[more]

PMID: 33234594

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Project description:Primary productivity of open ocean environments, such as those inhabited by marine picocyanobacteria Synechococcus sp.WH8102, are often limited by low inorganic phosphate (P). To observe how this organism copes with P starvation, we constructed a full genome microarray and examined differences in gene expression under P-limited and P-replete growth conditions. To determine the temporal nature of the responses, comparisons were made for cells newly entered into P-stress (at a time point corresponding to the induction of extracellular alkaline phosphatase activity) and a later time point (late log phase). In almost all instances the P starvation response was transitory, with 36 genes showing significant upregulation (>log2 fold) while 23 genes were highly downregulated at the early time point; however, these changes in expression were maintained for only five of the upregulated genes. Knockout mutants were constructed for genes SYNW0947 or SYNW0948, comprising a two component regulator hypothesized to play a key role in regulating the response to P-limitation. A high degree of overlap in the sets of genes affected by P-limited conditions and in the knockout mutants supports this hypothesis; however there is some indication that other regulators may play a role in this response in Synechococcus sp. WH8102. Consistent with what has been observed in many other cyanobacteria, the Pho regulon of this strain is comprised largely of genes for alkaline phosphatases, P transport or P metabolism. Interestingly, however, the exact composition and arrangement of the Pho regulon appears highly variable in marine cyanobacteria. In this series four conditions have been analyzed. These are low phosphate stress during early log phase, low phosphate stress during late log phase, SYNW0947 mutation, and SYNW0948 mutation. There are six slides per condition, each with two biological replicates. There are three technical replicates for each biological replicate including one flip-dye comparison. The exception is the low phosphate stress during late log phase experiment which has a total of five slides, two biological replicates with three and two technical replicates, respectively, and one flip-dye comparison for each biological replicate. Each slide contains six replicate spots per gene.

Project description:The genome of the marine Synechococcus sp. WH8102 displays a minimal regulatory network yet physiological and molecular responses of this organism are tuned to episodic limitation for nitrogen and phosphorus. Microarray analyses have demonstrated a key role for the two-component regulatory system, PhoBR, in the regulation of P transport and metabolism in this strain. However, there is some evidence that another regulator, SYNW1019 (designated as ptrA), probably under the control of PhoB, is involved in the wider response to P-depletion. PtrA is one of only two genome encoded DNA binding proteins of the CRP family in Synechococcus sp. WH8102, and a potential transcriptional regulator with homology to NtcA, the global nitrogen regulator in cyanobacteria. To define the precise role of this regulator we constructed a mutant by insertional inactivation and compared the physiology of wild-type Synechcococcus sp. WH8102 with the ptrA mutant under P-replete and P-deplete growth conditions. During P-depletion the ptrA mutant failed to up-regulate phosphatase activity. Microarrays and quantitative RT-PCR indicate that a subset of the Pho regulon is directly controlled by PtrA, including two phosphatase genes (SYNW0196, SYNW2390), a predicted phytase (SYNW0762) and a gene of unknown function (SYNW0165) all of which are highly up regulated during P-limitation. This result was confirmed by electrophoretic mobility shift assays which demonstrated binding of over expressed PtrA to promoter sequences upstream of the induced phosphatases (SYNW0165, SYNW0196 and SYNW2390). This work suggests a two-tiered response to P-depletion in this strain, the first being PhoB-dependent induction of high affinity PO4 transporters, and the second the PtrA-dependent induction of phosphatases for scavenging organic P. The levels of numerous other transcripts are also directly or indirectly controlled by PtrA, including those involved in cell surface modification, metal uptake, photosynthesis, stress responses and other metabolic process, which may indicate a wider role for PtrA in cellular organisation. In an environmental context ptrA is found in a number of picocyanobateria isolated from a range of oceanic provinces, including strains that lack a functional phoBR..These results give broader insight into the regulation of physiological responses that may dictate niche adaptation in genetically diverse lineages of marine Synechococcus, and suggest that signalling networks and coordinated responses to nutrient availability are important, even in oligotrophic ocean environments. In this series gene expression of a ptrA mutant has been analyzed under phosphorus deplete conditions just after the onset of induction of phosphatase activity in wild type. There are six duel-channel slides upon which both ptrA mutant and wild type samples were hybridized. There are three technical replicates for each of two biological replicates including one flip-dye comparison. Each slide contains six replicate spots per gene.

Dataset Information

Astrocyte conditioned media-dependent Phosphatidylinositol 3-kinase interactome in mouse hippocampal neuron cell line Ht22

Publications

An endogenous PI3K interactome promoting astrocyte-mediated neuroprotection identifies a novel association with RNA-binding protein ZC3H14.

Similar Datasets

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