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AMPA receptor subunit expression in the endoplasmic reticulum in frontal cortex of elderly patients with schizophrenia.

Hammond JC, Meador-Woodruff JH, Haroutunian V, McCullumsmith RE - PLoS ONE (2012)

Bottom Line: Previous reports have shown potential changes in the trafficking of AMPA receptors based on subunit expression of endosomes, subcellular organelles located near post-synaptic sites.We used Western blot and electron microscopy to show that we isolated an ER enriched fraction.We found no changes in the expression of the AMPA receptor subunits, GluR1-4, in the ER from the dorsolateral prefrontal cortex in schizophrenia.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Alabama Birmingham, Birmingham, Alabama, United States of America. hammondj@uab.edu

ABSTRACT
Several lines of evidence indicate altered trafficking of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors in schizophrenia. Previous reports have shown potential changes in the trafficking of AMPA receptors based on subunit expression of endosomes, subcellular organelles located near post-synaptic sites. We hypothesized that alterations in AMPA receptor trafficking through the endoplasmic reticulum (ER) may also be altered in schizophrenia. Accordingly, we developed a technique to isolate and measure content of the ER from postmortem brain tissue. We used Western blot and electron microscopy to show that we isolated an ER enriched fraction. We found no changes in the expression of the AMPA receptor subunits, GluR1-4, in the ER from the dorsolateral prefrontal cortex in schizophrenia. These data suggest that AMPA receptor trafficking through the ER is largely intact in schizophrenia.

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Related in: MedlinePlus

Neurochemical characterization of subcellular fractions isolated from postmortem human frontal cortex.Fractions enriched for endoplasmic reticulum (P4), cytosol and ‘light’ membranes (S3), mitochondria (P2), and nuclei (P1) were assayed by Western blot for compartment-specific markers: Calnexin (ER), histone3 (Nuc), glucose regulated 75 kDa protein (Grp75, Mito), early endosome antigen 1 (EEA1, endosomes), postsynaptic density 95 (PSD95, PSD), and α1,2 mannosidase (α1,2-Man, Golgi). The PSD segregated with the other ‘heavy’ membranes in the Mit and Nuc fractions. As expected, AMPA receptor subunit 2 (GluR2) was detected in the ER and fractions containing the PSD.
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pone-0039190-g002: Neurochemical characterization of subcellular fractions isolated from postmortem human frontal cortex.Fractions enriched for endoplasmic reticulum (P4), cytosol and ‘light’ membranes (S3), mitochondria (P2), and nuclei (P1) were assayed by Western blot for compartment-specific markers: Calnexin (ER), histone3 (Nuc), glucose regulated 75 kDa protein (Grp75, Mito), early endosome antigen 1 (EEA1, endosomes), postsynaptic density 95 (PSD95, PSD), and α1,2 mannosidase (α1,2-Man, Golgi). The PSD segregated with the other ‘heavy’ membranes in the Mit and Nuc fractions. As expected, AMPA receptor subunit 2 (GluR2) was detected in the ER and fractions containing the PSD.

Mentions: We used differential sucrose gradient centrifugation to obtain an ER enriched fraction from postmortem brain tissue (Figure 1). In addition to an enriched ER fraction, we obtained 3 other fractions. To verify and characterize the identity of each fraction, we used a wide screen of antibodies specific for various subcellular compartments (Figure 2). As expected, bands for each antibody were present in total brain homogenate. In the nuclear fraction (pellet 1), we detected calnexin (an ER marker), histone3 (a nuclear marker), Grp75 (a mitochondrial marker), PSD95 (a post-synaptic density marker), α 1,2-mannosidase (a Golgi marker), and GluR2. In the mitochondrial fraction (pellet 2), we detected calnexin, Grp75, PSD95, α 1,2-mannosidase, and GluR2. In the light membrane/cytosol fraction (supernatant 3), we detected Grp75, and EEA1 (an early endosome marker). The putative ER fraction (pellet 4) contained high levels of calnexin as well as GluR2, which is expected to be in the ER [23]–[25].


AMPA receptor subunit expression in the endoplasmic reticulum in frontal cortex of elderly patients with schizophrenia.

Hammond JC, Meador-Woodruff JH, Haroutunian V, McCullumsmith RE - PLoS ONE (2012)

Neurochemical characterization of subcellular fractions isolated from postmortem human frontal cortex.Fractions enriched for endoplasmic reticulum (P4), cytosol and ‘light’ membranes (S3), mitochondria (P2), and nuclei (P1) were assayed by Western blot for compartment-specific markers: Calnexin (ER), histone3 (Nuc), glucose regulated 75 kDa protein (Grp75, Mito), early endosome antigen 1 (EEA1, endosomes), postsynaptic density 95 (PSD95, PSD), and α1,2 mannosidase (α1,2-Man, Golgi). The PSD segregated with the other ‘heavy’ membranes in the Mit and Nuc fractions. As expected, AMPA receptor subunit 2 (GluR2) was detected in the ER and fractions containing the PSD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375247&req=5

pone-0039190-g002: Neurochemical characterization of subcellular fractions isolated from postmortem human frontal cortex.Fractions enriched for endoplasmic reticulum (P4), cytosol and ‘light’ membranes (S3), mitochondria (P2), and nuclei (P1) were assayed by Western blot for compartment-specific markers: Calnexin (ER), histone3 (Nuc), glucose regulated 75 kDa protein (Grp75, Mito), early endosome antigen 1 (EEA1, endosomes), postsynaptic density 95 (PSD95, PSD), and α1,2 mannosidase (α1,2-Man, Golgi). The PSD segregated with the other ‘heavy’ membranes in the Mit and Nuc fractions. As expected, AMPA receptor subunit 2 (GluR2) was detected in the ER and fractions containing the PSD.
Mentions: We used differential sucrose gradient centrifugation to obtain an ER enriched fraction from postmortem brain tissue (Figure 1). In addition to an enriched ER fraction, we obtained 3 other fractions. To verify and characterize the identity of each fraction, we used a wide screen of antibodies specific for various subcellular compartments (Figure 2). As expected, bands for each antibody were present in total brain homogenate. In the nuclear fraction (pellet 1), we detected calnexin (an ER marker), histone3 (a nuclear marker), Grp75 (a mitochondrial marker), PSD95 (a post-synaptic density marker), α 1,2-mannosidase (a Golgi marker), and GluR2. In the mitochondrial fraction (pellet 2), we detected calnexin, Grp75, PSD95, α 1,2-mannosidase, and GluR2. In the light membrane/cytosol fraction (supernatant 3), we detected Grp75, and EEA1 (an early endosome marker). The putative ER fraction (pellet 4) contained high levels of calnexin as well as GluR2, which is expected to be in the ER [23]–[25].

Bottom Line: Previous reports have shown potential changes in the trafficking of AMPA receptors based on subunit expression of endosomes, subcellular organelles located near post-synaptic sites.We used Western blot and electron microscopy to show that we isolated an ER enriched fraction.We found no changes in the expression of the AMPA receptor subunits, GluR1-4, in the ER from the dorsolateral prefrontal cortex in schizophrenia.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Alabama Birmingham, Birmingham, Alabama, United States of America. hammondj@uab.edu

ABSTRACT
Several lines of evidence indicate altered trafficking of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors in schizophrenia. Previous reports have shown potential changes in the trafficking of AMPA receptors based on subunit expression of endosomes, subcellular organelles located near post-synaptic sites. We hypothesized that alterations in AMPA receptor trafficking through the endoplasmic reticulum (ER) may also be altered in schizophrenia. Accordingly, we developed a technique to isolate and measure content of the ER from postmortem brain tissue. We used Western blot and electron microscopy to show that we isolated an ER enriched fraction. We found no changes in the expression of the AMPA receptor subunits, GluR1-4, in the ER from the dorsolateral prefrontal cortex in schizophrenia. These data suggest that AMPA receptor trafficking through the ER is largely intact in schizophrenia.

Show MeSH
Related in: MedlinePlus