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. 2014 Apr;154(1-3):1-13.
doi: 10.1016/j.schres.2014.01.028. Epub 2014 Feb 21.

Abnormal partitioning of hexokinase 1 suggests disruption of a glutamate transport protein complex in schizophrenia

Dan Shan  1 , Daniel Mount  1 , Stephen Moore  2 , Vahram Haroutunian  3 , James H Meador-Woodruff  4 , Robert E McCullumsmith  5
Affiliations

Abnormal partitioning of hexokinase 1 suggests disruption of a glutamate transport protein complex in schizophrenia

Dan Shan et al. Schizophr Res. 2014 Apr.

Abstract

Excitatory amino acid transporter 2 (EAAT2) belongs to a family of Na(+) dependent glutamate transporters that maintain a low synaptic concentration of glutamate by removing glutamate from the synaptic cleft into astroglia and neurons. EAAT2 activity depends on Na(+) and K(+) gradients generated by Na(+)/K(+) ATPase and ATP. Hexokinase 1 (HK1), an initial enzyme of glycolysis, binds to mitochondrial outer membrane where it couples cytosolic glycolysis to mitochondrial oxidative phosphorylation, producing ATP utilized by the EAAT2/Na(+)/K(+) ATPase protein complex to facilitate glutamate reuptake. In this study, we hypothesized that the protein complex formed by EAAT2, Na(+)/K(+) ATPase and mitochondrial proteins in human postmortem prefrontal cortex may be disrupted, leading to abnormal glutamate transmission in schizophrenia. We first determined that EAAT2, Na(+)/K(+) ATPase, HK1 and aconitase were found in both EAAT2 and Na(+)/K(+) ATPase interactomes by immunoisolation and mass spectrometry in human postmortem prefrontal cortex. Next, we measured levels of glutamate transport complex proteins in subcellular fractions in the dorsolateral prefrontal cortex and found increases in the EAAT2B isoform of EAAT2 in a fraction containing extrasynaptic membranes and increased aconitase 1 in a mitochondrial fraction. Finally, an increased ratio of HK1 protein in the extrasynaptic membrane/mitochondrial fraction was found in subjects with schizophrenia, suggesting that HK1 protein is abnormally partitioned in this illness. Our findings indicate that the integrity of the glutamate transport protein complex may be disrupted, leading to decreased perisynaptic buffering and reuptake of glutamate, as well as impaired energy metabolism in schizophrenia.

Keywords: Dorsolateral prefrontal cortex (DLPFC); Excitatory amino acid transporter (EAAT); Immunoisolation; Mass spectrometry; Postmortem; Reuptake.

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Conflict of interest statement

Conflict of interest: All authors declare no conflicts of interest.

Figures

Fig. 1
Fig. 1
Immunoisolation of EAAT2 and Na+/K+ ATPase from human postmortem prefrontal cortex. A) Western blot (WB) of EAAT2 immunoisolated with rabbit anti-EAAT2 antibody. The black arrows indicate a monomer (62KD), dimer (120KD) and trimer (200KD) of EAAT2. B) Western blot of Na+/K+ ATPase α1 immunoisolated with rabbit pan anti–Na+/K+ ATPase antibody (black arrow). Prefrontal cortical lysates (600 μg protein) prepared with 1% SDS were used for the immunoisolation. 10 μg of lysate (Lys) was loaded as a positive control, Rabbit IgG (IgG) was used as a negative control in the immunoisolation. Abbreviations: Immunoisolation (II), Western blot (WB), excitatory amino acid transporter (EAAT), tissue lysate (Lys), Na+/K+ ATPase α1 (NaKAα1).
Fig. 2
Fig. 2
Identification of glutamate transport protein complex in human postmortem prefrontal cortex. Identified proteins were grouped based on software of Database for Annotation, Visualization and Integrated Discovery (DAVID). The left box shows grouped proteins identified from immunoaffinity purified EAAT2 (EAAT2 interactome). The right box shows grouped proteins identified from immunoaffinity purified Na+/K+ ATPase (Na+/K+ ATPase interactome). The lower box shows proteins common to the EAAT2 and Na+/K+ ATPase interactomes.
Fig. 3
Fig. 3
Western blot analysis of glutamate transport protein complex in DLPFC total homogenate in paired schizophrenia and comparison subjects. Data are expressed as mean ± standard error. Expression of EAAT2 (A), EAAT2B (B), EAAT2 exon 9 skipping (C), Na+/K+ ATPase α1 (NaKA α1, D), Na+/K+ ATPase β (NaKA β, E), Hexokinase1 (HK1, F), aconitase 1 (G) and aconitase 2 (H) proteins in total homogenate was not significantly changed in subjects with schizophrenia. Schizophrenia (SCZ); control (CTL).
Fig. 4
Fig. 4
Assessment of extrasynaptic membrane and mitochondrial fractions isolated from human postmortem prefrontal cortex by Western blot and electron microscopy. A) Western blot shows enriched expression of the mitochondrial marker, voltage-dependent ion channel (VDAC), in the mitochondrial fraction (MT). VDAC was not detected in the extrasynaptic membrane (ES) fraction. The black arrow indicates the VDAC band on the Western blot. B) Electron microscope image (left, 4400×) showing that the extrasynaptic membrane fraction contains structures resembling light membranes not contaminated with other subcellular organelles. Electron microscope image (right, 4400×) showing an enrichment of intact mitochondria in the mitochondrial fraction (red arrows and inset).
Fig. 5
Fig. 5
Western blot analysis of EAAT2 and EAAT2 splice variants in subcellular fractions. Data are reported as mean ± standard error of the values generated for each subject expressed as relative expression in the fraction normalized to total homogenate within each assay. EAAT2B (B), a splice variant of EAAT2, was significantly increased in ES fraction in subjects with schizophrenia. No significant changes were observed for EAAT2 (A) or EAAT2 exon 9 skipping (C). Representative Western blot images shown on the left for one subject pair. *P < 0.05.
Fig. 6
Fig. 6
Western blot analysis of Na+/K+ ATPase subunits, HK1 and aconitase subunits in extrasynaptic membrane/cytosol (ES) and mitochondrial (MT) fractions. Data are reported as mean ± standard error of the values generated for each subject expressed as relative expression in the fraction normalized to total homogenate within each assay. Expression of aconitase 1 protein (D) was significantly increased in MT fraction in subjects with schizophrenia. No significant changes were found in the expression of Na+/K+ ATPase α1 (A), β (B), HK1(C) or aconitase 2 (E) proteins in subjects with schizophrenia. Representative Western blot images shown on the left for one subject pair. Arrows indicate the bands of interest. *P < 0.05.
Fig. 7
Fig. 7
Partitioning of HK1, aconitase 1 and 2 in extrasynaptic membrane/cytosol (ES) and mitochondrial (MT) fractions. Data expressed as the mean ± standard error of the ratios of the ES to MT fractions for each subject. A significantly increased ratio of HK1 in the ES/MT fractions was found in the DLPFC subjects with schizophrenia. The ES/MT ratio for aconitase 1 and 2 was not significantly changed in subjects with schizophrenia. *P < 0.05.
Fig. 8
Fig. 8
We characterized a protein complex containing EAAT2, Na+/K+ATPase, hexokinase 1 (HK1), aconitase 1 (AC1), and aconitase 2 (AC2) in human prefrontal cortex. In subjects with schizophrenia, expression of the EAAT2 splice variant EAAT2B (E2B) was significantly increased in a fraction containing extrasynaptic membranes and cytosol (ES), while and expression of aconitase 1 was significantly increased in the mitochondrial fraction (MT). An increased ratio of hexokinase 1 protein expression in ES/MT fraction was also found in subjects with schizophrenia.
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