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Quantification of histone deacetylase isoforms in human frontal cortex, human retina, and mouse brain.

ABSTRACT: Histone deacetylase (HDAC) inhibition has promise as a therapy for Alzheimer's disease (AD) and other neurodegenerative diseases. Currently, therapeutic HDAC inhibitors target many HDAC isoforms, a particularly detrimental approach when HDAC isoforms are known to have different and specialized functions. We have developed a multiple reaction monitoring (MRM) mass spectrometry assay using stable isotope-labeled QconCATs as internal standards to quantify HDAC isoforms. We further determined a quantitative pattern of specific HDACs expressed in various human and mouse neural tissues. In human AD frontal cortex, HDAC1,2 decreased 32%, HDAC5 increased 47%, and HDAC6 increased 31% in comparison to age-matched controls. Human neural retina concentrations of HDAC1, 2, HDAC5, HDAC6, and HDAC7 decreased in age-related macular degeneration (AMD)-affected donors and exhibited a greater decrease in AD-affected donors in comparison to age-matched control neural retinas. Additionally, HDAC concentrations were measured in whole hemisphere of brain of 5XFAD mice, a model of ?-amyloid deposition, to assess similarity to AD in human frontal cortex. HDAC profiles of human frontal cortex and mouse hemisphere had noticeable differences and relatively high concentrations of HDAC3 and HDAC4 in mice, which were undetectable in humans. Our method for quantification of HDAC isoforms is a practical and efficient technique to quantify isoforms in various tissues and diseases. Changes in HDAC concentrations reported herein contribute to the understanding of the pathology of neurodegeneration.

SUBMITTER: Anderson KW

PROVIDER: S-EPMC4427357 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Quantification of histone deacetylase isoforms in human frontal cortex, human retina, and mouse brain.

Anderson Kyle W KW Chen Junjun J Wang Meiyao M Mast Natalia N Pikuleva Irina A IA Turko Illarion V IV

PloS one 20150511 5

Histone deacetylase (HDAC) inhibition has promise as a therapy for Alzheimer's disease (AD) and other neurodegenerative diseases. Currently, therapeutic HDAC inhibitors target many HDAC isoforms, a particularly detrimental approach when HDAC isoforms are known to have different and specialized functions. We have developed a multiple reaction monitoring (MRM) mass spectrometry assay using stable isotope-labeled QconCATs as internal standards to quantify HDAC isoforms. We further determined a quan ...[more]

PMID: 25962138

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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).

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Project description:PURPOSE:The principal excitatory neurotransmitter glutamate plays an important role in many central nervous system disorders. Because glutamate resides predominantly in glutamatergic neurons, its relaxation properties reflect the intracellular environment of glutamatergic neurons. This study developed an improved echo time-independent technique for measuring transverse relaxation time and demonstrated that this radio frequency (RF)-driven longitudinal steady state technique can reliably measure glutamate transverse relaxation in the frontal cortex, where structural and functional abnormalities have been associated with psychiatric symptoms. METHOD:Bloch and Monte Carlo simulations were performed to improve and optimize the RF-driven, longitudinal, steady-state (MARzss) technique to significantly shorten scan time and increase measurement precision. Optimized four-flip angle measurements at 0°,12°, 24°, and 36° with matched repetition time were used in nine human subjects (6F, 3M; 27-49 years old) at 7 Tesla. Longitudinal and transverse relaxation rates for glutamate were measured from a 2 x 2 x 2 cm3 voxel placed in three different brain regions: gray matter-dominated medial prefrontal lobe, white matter-dominated left frontal lobe, and gray matter-dominated occipital lobe. RESULTS:Compared to the original MARzss technique, the scan time per voxel for measuring glutamate transverse relaxation was shortened by more than 50%. In the medial frontal, left frontal, and occipital voxels, the glutamate T2 was found to be 117.5±12.9 ms (mean ± standard deviation, n = 9), 107.3±12.1 (n = 9), and 124.4±16.6 ms (n = 8), respectively. CONCLUSIONS:The improvements described in this study make the MARZSS technique a viable tool for reliably measuring glutamate relaxation from human subjects in a typical clinical setting. It is expected that this improved technique can be applied to characterize the intracellular environment of glutamatergic neurons in a variety of brain disorders.

Project description:BACKGROUND: GABA has important functions in brain plasticity related processes like memory, learning, locomotion and during the development of the nervous system. It is synthesized by the glutamic acid decarboxylase (GAD). There are two isoforms of GAD, GAD1 and GAD2, which are encoded by different genes. During embryonic development the transcription of GAD1 mRNA is regulated by alternative splicing and several alternative transcripts were distinguished in human, mouse and rat. Despite the fact that the structure of GAD1 gene has been extensively studied, knowledge of its exact structural organization, alternative promoter usage and splicing have remained incomplete. RESULTS: In the present study we report the identification and characterization of novel GAD1 splicing isoforms (GenBank: KM102984, KM102985) by analyzing genomic and mRNA sequence data using bioinformatics, cloning and sequencing. Ten mRNA isoforms are generated from GAD1 gene locus by the combined actions of utilizing different promoters and alternative splicing of the coding exons. Using RT-PCR we found that GAD1 isoforms share similar pattern of expression in different mouse tissues and are expressed early during development. Quantitative RT-PCR was used to investigate the expression of GAD1 isoforms and GAD2 in olfactory bulb, cortex, medial and lateral striatum, hippocampus and cerebellum of adult mouse. Olfactory bulb showed the highest expression of GAD1 transcripts. Isoforms 1/2 are the most abundant forms. Their expression is significantly higher in the lateral compared to the medial striatum. Isoforms 3/4, 5/6, 7/8 and 9/10 are barely detectable in all investigated regions except of the high expression in olfactory bulb. When comparing GAD1 expression with GAD2 we found that Isoforms 1/2 are the predominant isoforms. In situ hybridization confirmed the predominant expression of Isoforms 7/8 and 9/10 in the olfactory bulb and revealed their weak expression in hippocampus, cerebellum and some other areas known to express GAD1. CONCLUSIONS: Generation of ten splicing isoforms of GAD1 was described including two so far uncharacterized transcripts. GAD1 splicing isoforms producing the shorter, enzymatically inactive GAD25 protein are expressed at very low level in adult mouse brain except in the olfactory bulb that is associated with neurogenesis and synaptic plasticity even during adulthood.

Dataset Information

Quantification of histone deacetylase isoforms in human frontal cortex, human retina, and mouse brain.

Publications

Quantification of histone deacetylase isoforms in human frontal cortex, human retina, and mouse brain.

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