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Proteomic analysis of isolated murine neurons

ABSTRACT: Muscle isoform of fructose 1,6-bisphosphatase (FBP2) is a regulatory enzyme of glycogen synthesis from non-carbohydrates and a multifunctional protein indispensable for induction and maintenance of the long-term potentiation (the LTP), a molecular/cellular mechanism of memory formation in the hippocampus. During the LTP formation FBP2 affects phosphorylation status of the CAMKIV and CAMKII, protein kinases involved directly and indirectly in regulation of gene transcription in neurons. The results of the study demonstrate the effect of partial FBP2 silencing (using the shRNA technique; three days of the silencing) in 17-day-old culture of mice hippocampal neurons on their proteome. Hippocampal neurons were isolated from newborn BALB/c mice and cultured at 37°C in a humidified atmosphere with 5% CO2 as described previously [Mozrzymas et al.] with a minor modification - the glucose concentration in the culture medium was 2.5 mM. Mozrzymas J., Szczesny T., Rakus, D. The effect of glycogen phosphorolysis on basal glutaminergic transmission. Biochem. Biophys. Res. Commun. 2011, 404, 652–655.

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ORGANISM(S): Rattus Norvegicus (rat)

SUBMITTER: Jacek Wisniewski

LAB HEAD: Jacek R. Wisniewski

PROVIDER: PXD038676 | Pride | 2026-01-24

REPOSITORIES: Pride

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			Action	DRS
	20211125_EXPL4_JRW_SA_L_YY18_20211130114753.raw	Raw
	20211125_EXPL4_JRW_SA_L_YY19.raw	Raw
	20211125_EXPL4_JRW_SA_L_YY20.raw	Raw
	20211125_EXPL4_JRW_SA_L_YY24.raw	Raw
	20211125_EXPL4_JRW_SA_L_YY25.raw	Raw

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Project description:Expression of VP16-CREB, a constitutively active form of CREB, in hippocampal neurons of the CA1 region lowers the threshold for eliciting the late, persistent phase of long-term potentiation (L-LTP) in the Schaffer collateral pathway. This VP16-CREB-mediated L-LTP differs from the conventional late phase of LTP in not being dependent on new transcription. This finding suggests that in the transgenic mice the mRNA transcript(s) encoding the protein(s) necessary for this form of L-LTP might already be present in CA1 neurons in the basal condition. We used high-density oligonucleotide arrays to identify the mRNAs differentially expressed in the hippocampus of transgenic and wild-type mice. Experiment Overall Design: To identify the specific gene whose induction in hippocampal neurons correlated with the facilitated L-LTP phenotype, we harvested hippocampal mRNA at time points assessed in our physiological study and carried out a gene expression analysis using oligonucleotide microarrays. To obtain a sufficient quantity of poly(A)-RNA and reduce the effect of the biological variability of the sample, hippocampi from 6 to 10 mice matched for genotype and time of induction were pooled together in each sample. The final data set included genechips for animals on dox (gene Off) and for animals that were one, two, and five weeks after removal of dox (gene On). We also included samples from wild-type mice maintained under identical conditions and from transgenic mice that expressed VP16-CREB for three weeks before turning the transgene off again for two weeks with dox (Rev, gene Off). Since the isolation of mRNA from the whole hippocampus favors genes that are more broadly over-expressed in the hippocampus of transgenic mice and, therefore, may lead to an underestimation of the total complement of genes showing an altered expression in specific hippocampal subregions, we extended our comparison between transgenic and wild-type expression profiles using microdissected CA1 regions. We obtained two samples corresponding to VP16-CREB mice three weeks after induction and one sample corresponding to wild-type mice kept in the same conditions. Experiment Overall Design: These twelve mRNA samples were analyzed using Affymetrix genechips MG-U74v2 setA.

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Proteomic analysis of isolated murine neurons

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