Dataset Information

Relationship between somatostatin and death receptor expression in the orbital frontal cortex in schizophrenia: a postmortem brain mRNA study.

ABSTRACT:

Background

Recently, we provided evidence showing reductions in GAD67 and Dlx mRNAs in the orbital frontal cortex (OFC) in schizophrenia. It is unknown whether these reductions relate mainly to somatostatin (SST) or parvalbumin (PV) mRNA expression changes, and/or whether these reductions are related to decreased SST mRNA+ interneuron density.

Aims

To determine whether inhibitory interneuron deficits in the OFC from people with schizophrenia are greatest for SST or PV mRNAs, and whether any such deficits relate to mRNAs encoding cell death signalling molecules.

Methods

Inhibitory interneuron mRNAs (SST; PV: in situ hybridization, quantitative PCR (qPCR)) and death signaling mRNAs [FAS receptor (FASR); TNFSF13: qPCR] were measured in control and schizophrenia subjects (38/38). SST mRNA+ interneuron-like cells were quantified in layer II in the gyrus rectus. Gray matter SST and PV mRNAs were correlated with interstitial white matter neuron (IWMN) density (GAD65/67; NeuN) and death signaling mRNAs.

Results

SST mRNA was reduced in OFC layers I-VI in schizophrenia (both in situ and qPCR), with greatest deficit in layer II (67%). Layer II SST mRNA+ neuron density was reduced in schizophrenia (~29%). PV mRNA was reduced in layers III (18%) and IV (31%) with no significant diagnostic difference in PV mRNA measured by qPCR. FASR mRNA was increased in schizophrenia (34%). SST, but not PV, expression correlated negatively with FASR and TNFSF13 expressions and with IWMN density.

Conclusions

Our study demonstrates that SST interneurons are predominantly linked to the inhibitory interneuron pathology in the OFC in schizophrenia and that increased death receptor signaling mRNAs relate to prominent laminar deficits in SST mRNA in the OFC in schizophrenia. We suggest that SST interneurons may be more vulnerable to increased death receptor signaling than PV interneurons.

SUBMITTER: Joshi D

PROVIDER: S-EPMC4849439 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Publications

Relationship between somatostatin and death receptor expression in the orbital frontal cortex in schizophrenia: a postmortem brain mRNA study.

Joshi Dipesh D Catts Vibeke S VS Olaya Juan C JC Shannon Weickert Cynthia C

NPJ schizophrenia 20150304

<h4>Background</h4>Recently, we provided evidence showing reductions in GAD67 and Dlx mRNAs in the orbital frontal cortex (OFC) in schizophrenia. It is unknown whether these reductions relate mainly to somatostatin (SST) or parvalbumin (PV) mRNA expression changes, and/or whether these reductions are related to decreased SST mRNA+ interneuron density.<h4>Aims</h4>To determine whether inhibitory interneuron deficits in the OFC from people with schizophrenia are greatest for SST or PV mRNAs, and w ...[more]

PMID: 27336026

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Project description:Prefrontal cortex tissue slices from a cognitively and neurpathologically normal human subject were obtained from the University of Pennsylvania (UPenn) Brain Bank. 300-350 mg grey matter was homogenized in 1.5 ml solution A (0.32 M sucrose, 1mM MgCl2 and 0.1mM CaCl2) with a Teflon pestle. ~100 ?l of the homogenate was saved, solubilized with 1% SDS and clarified by centrifugation. To prepare the [13C6]brain ISTD, 400 mg labeled MouseExpress brain tissue (Cambridge Isotopes, MA) was homogenized in 4 ml buffer A as described above, centrifuged at 1,000 x g for 10 min to clarify, agitated on a rocker at 4�C for 30 min, and centrifuged at 10,000 x g for 20 min. The pellet, a crude membrane fraction, was dissolved in 2 ml 20 mM Tris pH 7.4 with 1% SDS. Total protein in all preparations was quantified with the micro BCA assay (Pierce) and all solutions were supplemented with protease and phosphate inhibitor cocktails (Sigma) as well as 1 mM sodium fluoride and sodium orthovanadate (Sigma). Human cortex homogenate was mixed with the [13C6]brain ISTD (1?g/?l) at a ratio of 2:1(?g/?g) and processed for LC-MS/MS: 40 ?l preparations were heated with lithium dodecyl sulfate (LDS) (Invitrogen) buffer at 95�C for 20 min, separated on a 1.5 mm 4-12% Bis-Tris Gel (Invitrogen), cut into five fractions, chopped into ?2 mm cubes, washed in 200 ?l 50% acetonitrile (ACN) containing 25 mM NH4HCO3, reduced in 300 ?l 10 mM DTT, alkylated in 300 ?l 55 mM iodoacetamide (Sigma), and digested with 80-120 ?l trypsin (.025 ?g/?l) (Promega) overnight at 37�C, so that the amount of trypsin was 1:5 of the total protein to be digested by mass. Peptides were recovered from gel cubes into 200 ?l 50/50 H2O/ACN with 3% formic acid by vortex-mixing and sonication for 20 min each, twice. Samples were then evaporated to a 100 ?l volume, brought to 1ml in H20 with 0.1% formic acid, desalted with Oasis� HLB cartridges (Waters), evaporated almost to completion and suspended in ~ 45?l H2O with 0.1% formic acid, and filtered with 0.22 �m Ultra free-MC filter cartridges (Millipore). LC-data dependant -MS/MS analyses were conducted on a Q Exactive (ThermoFisher Scientific) quadrupole orbi-trap hybrid instrument with an Easy nLC-II (ThermoFisher Scientific) nano-pump/autosampler. 3?l peptide sample (~ 1 ug) was loaded and resolved on a 20 cm x 75 �m proteopepII C18 packed tip column over a 90 min gradient at a flow rate of 350 nL/min, 0-35% mobile phase B (ACN, 0.1 formic acid). A data-dependent top 10 method was used to acquire a 70,000 resolution full scan to trigger ten 17,500 resolution HCD scans. Ions were isolated for MS/MS analysis with a 2.0 Da window on the quadrupole. Average cycle times were 1.2 sec. Each sample was analyzed in triplicate. Raw files from triplicate injections were searched together within Proteome Discoverer 1.3 (ThermoFisher Scientific) using SEQUEST and the human refseq. database (release 47, ftp://ftp.ncbi.nih.gov/refseq/H_sapiens/mRNA_Prot/). Search parameters allowed trypsin to cleave after Lysine and Arginine and have 2 missed cleavages. Precursor ion mass tolerance was set to 15 ppm and fragment ion mass tolerance was 20 mmu. The dynamic modification of methionine (oxidation = 15.995 Da) and lysine (13C6 = 6.020 Da), in addition to the static modification of cysteine (carbamidomethylation = 57.021 Da) was accepted on up to 4 residues per peptide. Within the Proteome Discoverer Software, SILAC pairs were identified using the �2 plex� workflow node, and all peptides were rescored using the Percolator algorithm node. Finally, peptides were filtered at 1% false discovery rate (FDR).

Dataset Information

Relationship between somatostatin and death receptor expression in the orbital frontal cortex in schizophrenia: a postmortem brain mRNA study.

Background

Aims

Methods

Results

Conclusions

Publications

Relationship between somatostatin and death receptor expression in the orbital frontal cortex in schizophrenia: a postmortem brain mRNA study.

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