AMPA receptor subunit localization in schizophrenia anterior cingulate cortex

SCHRES-08679; No of Pages 9 Schizophrenia Research xxx (xxxx) xxx

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AMPA receptor subunit localization in schizophrenia anterior cingulate cortex Jana L. Benesh, Toni M. Mueller ⁎, James H. Meador-Woodruff University of Alabama at Birmingham, Department of Psychiatry and Behavioral Neurobiology, 1720 2nd Ave S., Birmingham, AL 35294, United States of America

a r t i c l e

i n f o

Article history: Received 11 November 2019 Received in revised form 21 January 2020 Accepted 23 January 2020 Available online xxxx Keywords: Postmortem Anterior cingulate cortex Postsynaptic density TARP Subcellular fractionation Stargazin

a b s t r a c t The glutamate hypothesis of schizophrenia suggests that altered glutamatergic transmission occurs in this illness, although precise mechanisms of dysregulation remain elusive. AMPA receptors (AMPARs), a subtype of ionotropic glutamate receptor, are the main facilitators of fast, excitatory neurotransmission in the brain, and changes in AMPAR number or composition at synapses can regulate synaptic strength and plasticity. Prior evidence of abnormal expression of transmembrane AMPAR regulatory proteins (TARPs) in schizophrenia suggests defective trafficking of AMPARs, which we propose could lead to altered AMPAR expression at excitatory synapses. To test this hypothesis, we isolated subcellular fractions enriched for endoplasmic reticulum (ER) and synapses from anterior cingulate cortex (ACC) from schizophrenia (N = 18) and comparison (N = 18) subjects, and measured glutamate receptor subunits (GluA1, GluA2, GluA3, GluA4, NR1, NR2A, NR2B, and NR3A) and TARP member γ2 (stargazin) in homogenates and subcellular fractions by western blot analysis. We found decreased expression of stargazin and an increased ratio of GluA2:stargazin in ACC homogenates, while in the synapse fraction we identified a decrease in GluA1 and reduced ratios of GluA1:stargazin and GluA1:GluA2 in schizophrenia. The amount of stargazin in the ER fraction was not different, but the relative amount of ER/Total stargazin was increased in schizophrenia. Together, these findings suggest that associations between stargazin and AMPA subunits are abnormal, potentially affecting forward trafficking or synaptic stability of GluA1-containing AMPARs. These data provide evidence that altered interactions with trafficking proteins may contribute to glutamate dysregulation in schizophrenia. © 2020 Published by Elsevier B.V.

1. Introduction Many studies in schizophrenia have converged in support of the glutamate hypothesis and have focused on dysregulation of the NMDA subtype of glutamate receptor (NMDAR) (Dean et al., 2015; Javitt, 2007; Nakazawa et al., 2012). The AMPA subtypes of glutamate receptor (AMPAR) are also relevant to the pathophysiology of schizophrenia, given their central role in synaptic events including plasticity, neuronal maturation, memory formation, and synaptogenesis (Hanse et al., 2013; Kumar et al., 2002; Song and Huganir, 2002). Since NMDAR channels are blocked by extracellular Mg2+ that is released following AMPARmediated membrane depolarization, these two populations of glutamate receptor act in concert to regulate the postsynaptic response (Song and Huganir, 2002). More recently, roles of auxiliary proteins have been identified in the dynamic regulation of AMPARs following the identification of stargazin, the prototypical member of the family ⁎ Corresponding author at: University of Alabama at Birmingham, 1719 6th Avenue South, CIRC 576A, Birmingham, AL 35233, United States of America. E-mail address: [email protected] (T.M. Mueller).

of transmembrane AMPAR regulatory proteins (TARPs) (Chen et al., 1999, 2000; Hashimoto et al., 1999; Jackson and Nicoll, 2011; Straub and Tomita, 2012; Tomita et al., 2003). TARPs directly bind to AMPAR complexes during receptor assembly in the endoplasmic reticulum (ER), and traffic the receptor complex through the secretory pathway before forming a physical anchor that docks AMPARs at synapses (Jackson and Nicoll, 2011; Straub and Tomita, 2012). Genetic and pharmacological manipulations of TARPs have shown that these proteins directly control AMPAR number and activity by modulating their intracellular trafficking and biophysical channel properties at synapses (Herring et al., 2013; Sumioka, 2013). We have previously reported altered protein and transcript expression of TARPs in anterior cingulate cortex (ACC) in schizophrenia (Beneyto and Meador-Woodruff, 2006; Drummond et al., 2013), and hypothesized that this may be associated with abnormal numbers of AMPARs reaching postsynaptic membranes due to altered stoichiometry of auxiliary proteins during receptor complex assembly in the ER. To address the possibility that there are altered numbers of synaptic NMDARs or AMPARs in schizophrenia in the face of abnormal auxiliary protein expression, we isolated subcellular fractions from ACC to

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Please cite this article as: J.L. Benesh, T.M. Mueller and J.H. Meador-Woodruff, AMPA receptor subunit localization in schizophrenia anterior cingulate cortex, Schizophrenia Research, https://doi.org/10.1016/j.schres.2020.01.025

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measure NMDAR and AMPAR subunits and stargazin (also called TARPγ2) protein expression in fractions enriched for either ER or synapses in schizophrenia and comparison samples, as well as in ACC homogenates. Given our hypothesis that glutamatergic signaling disruptions in schizophrenia arise from altered subcellular distribution of AMPAR and NMDAR subunits due to abnormal interactions with trafficking proteins, we did not expect glutamate receptor subunit expression to be altered in ACC homogenates. Instead, we predicted that total protein levels of each subunit would be normal and expression of one or more subunits would be altered at the synapse. An alternate outcome consistent with this hypothesis is that the stoichiometry of glutamate receptor subunits may be abnormal at the synapse, suggesting differential association of subunit combinations with trafficking molecules. To test our hypothesis, we used western blot analysis to measure protein expression of target molecules in ACC homogenates and subcellular fractions enriched for ER or synapses, representing the proximal and distal ends of the forward trafficking pathway. Based on evidence of altered expression of AMPAR auxiliary proteins in cortical regions in schizophrenia (Beneyto and Meador-Woodruff, 2006; Drummond et al., 2012, 2013), we measured the expression of the prototypical TARP protein, stargazin. Since GluA1, GluA3, and GluA4 form heterotetrameric AMPARs by coassembly with GluA2 (Gan et al., 2015), and GluA1, GluA2, and GluA4 AMPAR subunits interact with stargazin (Chen et al., 2000), we analyzed ratios of protein expression for these AMPAR subunit:subunit/TARP combinations in homogenates and fractions. Prior evidence of altered trafficking of NR2B-containing NMDARs in schizophrenia (Kristiansen et al., 2010a, 2010b) led us to also assess the ratio of NR2A:NR2B in homogenates and fractions. 2. Materials and methods 2.1. Tissue acquisition and preparation Tissue from the full thickness of gray matter from ACC was obtained from the Mount Sinai Medical Center Schizophrenia Brain Collection, and in compliance with the Mount Sinai School of Medicine Institutional Review Board protocol for acquisition of postmortem tissue. Criteria for patient inclusion, and details of tissue preparation have been previously described (Funk et al., 2012). Briefly, patients were diagnosed with schizophrenia using DSM-III-R criteria, and both schizophrenia and comparison brains underwent antemortem clinical assessments. Following brain removal, neuropathological assessment was completed and individuals were excluded from study if there was evidence of neurodegenerative disease, or a history of substance abuse, death by suicide, or coma for N6 h before death. Brain tissue was dissected, snap frozen in liquid nitrogen, and stored at −80 °C prior to homogenization and subcellular fractionation. Schizophrenia (SCZ, N = 18) and comparison groups (COMP, N = 18) were well-matched for sex, age at death, and tissue pH, and the postmortem interval (PMI) was not significantly different between groups (Table 1, Supplementary Table S1, and Supplementary Fig. S1).

Table 1 Subject summary.

N Sex Age Tissue pH PMI (hours) Rx Status

Comparison

Schizophrenia

18 10M/8F 75.6 ± 11.0 6.56 ± 0.23 9.6 ± 6.8

18 10M/8F 75.9 ± 10.0 6.50 ± 0.21 13.2 ± 5.1 13on/5off

Abbreviations: postmortem interval (PMI), antipsychotic treatment within 6 weeks of death (Rx Status), male (M), female (F).

Tissue blocks from each brain were homogenized and subcellular fractions processed in paired groups containing equal numbers of schizophrenia and comparison samples. An aliquot of each fraction was saved during processing and protein concentration measured using a BCA Protein Assay kit (Thermo Scientific, Rockford, IL, USA).

2.2. Subcellular fractionation Subcellular fractionation to obtain enriched fractions of endoplasmic reticulum (ER, ER fraction) and synaptic membranes (SYN fraction) was performed as described previously (Hammond et al., 2012; Mueller et al., 2015). All fractionation steps were performed at 4 °C or on wet ice. Briefly, isotonic extraction buffer (ER0100, Sigma-Aldrich, St. Louis, MO, USA) was added to 50 mg of ACC from each individual and dounce-homogenized, followed by nitrogen cavitation at 450 psi for 8 min to obtain samples of total homogenate. Homogenates were centrifuged at 700 ×g for 10 min to generate pellet (P1) and supernatant (S1). S1 was then centrifuged at 15,000 ×g for 10 min. The resulting supernatant (S2) was stored at −80 °C until ready for further processing and generation of the ER fraction and supernatant (S3). The P2 pellet and P1 were re-suspended in Tris-sucrose buffer (320 mM sucrose, 10 mM Tris (pH 7.4), 1 mM Na3VO4, 5 mM NaF, 1 mM EDTA, 1 mM EGTA) combined, and treated with 8 volumes of Triton X-100 buffer (0.5% (v/v) Triton X-100 in10 mM Tris (pH 7.4), 1 mM Na3VO4, 5 mM NaF, 1 mM EDTA, 1 mM EGTA) for 20 min with gentle rotation before centrifugation at 32,000 ×g for 20 min. The resulting supernatant (S4) was transferred to a new tube and Triton-soluble proteins were precipitated by overnight incubation with acetone at −20 °C. The Tritoninsoluble pellet containing both excitatory and inhibitory synapses (P4, SYN fraction) was rinsed and resuspended in Tris-sucrose buffer and stored at −80 °C until all fractions were generated. Following acetone precipitation of S4, samples were centrifuged at 3000 ×g for 5 min. After decanting and discarding acetone, the resulting pellet (P5, called ExSyn fraction) was air dried, rinsed and resuspended in Trissucrose buffer, and stored at −80 °C. For generation of ER fractions, the S2 supernatant was retrieved from −80 °C storage, thawed on wet ice, then loaded on top of a discontinuous gradient consisting of layered 1.3 M, 1.5 M, and 2.0 M sucrose in 10 mM Tris (pH 7.6) with 0.1 mM EDTA and centrifuged at 126,000 ×g for 70 min. ER membranes segregated to the top interface of the 1.3 M sucrose layer, and were extracted from the gradient following removal of the top layer (S3). MTE/PMSF buffer (270 mM D-mannitol, 10 mM Tris (pH 7.4), 0.1 mM EDTA, 10 mM phenylmethylsulfonyl fluoride) was added to ER membranes, mixed by inversion, and samples centrifuged at 126,000 ×g for 45 min. The resulting pellet (P3, ER fraction) was rinsed with Tris-sucrose buffer before being resuspended in phosphate-buffered saline (PBS) with 0.5% Triton X-100, and stored at −80 °C until all fractions were generated.

2.3. Validation of subcellular fractions Protocols that utilize Triton X-100 to isolate synaptic membranes from brain have been previously reported (Billa et al., 2010; GoebelGoody et al., 2009; Hahn et al., 2009; Morón et al., 2007; Mueller et al., 2019), as well as studies demonstrating the effectiveness of nitrogen cavitation in preserving intracellular organelle structure (Hammond et al., 2012; Simpson, 2010). To evaluate the distribution of common marker proteins in samples generated by our fractionation method, samples were prepared and subjected to western blot as described below (Fig. 1A). For additional validation of isolated synapses, pellet P4 (which is resuspended to produce SYN fraction samples) and pellet P5 (which is resuspended to produce an ExSyn fraction) were thin sectioned and post-stained with uranyl acetate and lead citrate for EM imaging (Fig. 1B–H).

Please cite this article as: J.L. Benesh, T.M. Mueller and J.H. Meador-Woodruff, AMPA receptor subunit localization in schizophrenia anterior cingulate cortex, Schizophrenia Research, https://doi.org/10.1016/j.schres.2020.01.025

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Fig. 1. Validation of isolated synaptic membranes. (A) Western blot analysis of the synaptic, Triton-insoluble fraction (SYN) showed enriched expression of a postsynaptic density marker (PSD95) and AMPAR subunit GluA2, but no expression of the presynaptic marker synaptophysin (Syp), and minimal expression of markers for ER membranes (JM4), mitochondria (VDAC), and nuclei (H3). In contrast, the Triton-soluble ExSyn fraction exhibited no detectible PSD95 expression and enrichment of synaptophysin (Syp); however, the presence of other marker proteins in this fraction suggests contamination by other intracellular compartments, likely due to the biochemical methods used in the fractionation protocol. The ER fraction exhibited enrichment of JM4 and Syp, and the GluA2 subunit can be detected in this fraction. Given the role of Syp in the presynaptic forward trafficking pathway, evidence of this marker in the ER fraction is not unexpected. (B-D) Electron microscopy (EM) revealed that no intact synapses were visualized in ExSyn (B, 1650× magnification), but enrichment of synapses was seen in the SYN fraction (C, 2700× magnification, arrowheads indicate synaptic membranes; D, 3200× magnification of top inset). (E–H.) Representative images of isolated synapses in the SYN fraction at 11,000× magnification. No intact structures of other intracellular organelles were identified by EM in the SYN fraction.

2.4. Western blot analysis

2.5. Statistical analyses

To quantitate protein expression in subcellular fractions, samples were prepared with 6× β-mercaptoethanol loading buffer and denatured at 70 °C for 10 min. For each subject, 10 μg of protein from each fraction was loaded per lane onto 4–12% Bis-Tris gels (Invitrogen, Carlsbad, CA, USA) and subjected to SDS-PAGE. Proteins were then transferred to either PVDF or nitrocellulose membranes (Invitrogen, USA) by semi-dry transfer (Bio-Rad, Hercules, CA, USA). Membranes were blocked 1 h in either Li-COR blocking buffer (Li-COR Biosciences, Lincoln, NE, USA), or buffer containing 2% (w/v) bovine serum albumin (BSA) in PBS (pH 7.4) at room temperature depending on the diluent of the first primary antibody probed on the membrane. Commercially available primary antibodies were individually optimized for use in these studies (Supplementary Table S2). Membranes were washed in 0.05% Tween-20 in PBS (pH 7.4) following each primary and secondary antibody incubation, and rinsed with distilled water prior to imaging. Li-COR IR-dye labeled mouse or rabbit secondary antibodies were used to visualize proteins of interest and membranes scanned with the Li-COR Odyssey system (Li-COR Biosciences, USA). Li-COR Odyssey 3.0 analytical software was used to measure the integrated intensity of target protein bands. The integrated intensity value accounts for both the area of the region of membrane measured and any background signal from the membrane. Individual data points were excluded if the integrated intensity was reported as a negative value or if the target protein band was obscured by a technical artifact of the western blot procedure.

For all dependent measures, target protein expression is presented as the integrated intensity of the target band normalized to the integrated intensity of a fraction-specific marker protein: β-tubulin in total homogenates, PSD95 for excitatory synaptic membranes (SYN fraction), and JM4 for ER membranes (ER fraction). Outliers were excluded from protein expression values if the fraction-normalized value was greater than ±2 standard deviations from the mean of all subjects. Ratios of protein expression were calculated from these values. The amount of protein in each fraction relative to total expression (ER/ Total or SYN/Total) is the calculated target protein expression in the ER or SYN fraction normalized to the calculated target protein expression in ACC homogenates. Since ratios could not always be calculated for every individual, in order to maintain sufficient statistical power only extreme outliers with a ratio value greater than ±3 standard deviations from the mean of all subjects were excluded. Each fractionspecific marker was analyzed both as original data or normalized to βtubulin, and no differences were found between schizophrenia and comparison groups for any marker protein in homogenates or the target fraction. All statistical analyses were performed using GraphPad Prism 8.2.1 (La Jolla, CA, USA). Each dependent measure was assessed for normal distribution using the D‘Agostino-Pearson omnibus test, and between group differences were determined by either unpaired two-way Student's t-tests or Mann-Whitney U tests. The Benjamini-Hochberg q-value was used for multiple comparison correction to control the

Please cite this article as: J.L. Benesh, T.M. Mueller and J.H. Meador-Woodruff, AMPA receptor subunit localization in schizophrenia anterior cingulate cortex, Schizophrenia Research, https://doi.org/10.1016/j.schres.2020.01.025

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false discovery rate (FDR) within each hypothesis family. For all statistical tests α = 0.05 and for multiple comparisons q* = 0.05. For significantly different dependent variables that survived multiple testing correction, post hoc analyses were performed to assess potential covariates. Linear regression was performed to determine associations between protein measures and subject age, tissue pH, or PMI. Dependent measures found to covary with any continuous variables were subsequently assessed by ANCOVA. To assess potential effects of sex, unpaired two-way Student's t-tests or Mann-Whitney U tests compared dependent variables in males versus females. Dependent variables found different between males and females were subsequently assessed by two-way ANOVA and multiple comparisons were performed using the Benjamini-Hochberg FDR method if the two-way ANOVA revealed a significant effect of sex or diagnosis. One-way ANOVA or non-parametric Kruskal-Wallis tests were used to detect differences between COMP individuals (N = 18), schizophrenia patients “on” (SCZON) antipsychotics (N = 13), and schizophrenia patients “off” (SCZOFF) antipsychotics (N = 5); and no effects of antipsychotic treatment were detected. Patients are characterized as “off” antipsychotics if no antipsychotic treatment occurred within 6 weeks of death; additional comment on this limitation is included in the discussion. 3. Results 3.1. Enrichment of synapses from postmortem brain To determine if AMPARs are abnormally localized in synapses in schizophrenia, we adapted a method that capitalizes on synaptic membrane insolubility in Triton X-100 (Goebel-Goody et al., 2009), to enrich synapses from brain. Consistent with previous reports (Carlin et al., 1980; Cho et al., 1992; Ferrario et al., 2011; Goebel-Goody et al., 2009; Goebel et al., 2005), the Triston-insoluble SYN fraction showed specific expression of the postsynaptic density marker PSD95, and the Tritonsoluble ExSyn fraction showed expression of synaptophysin (SYP), a protein associated with presynaptic vesicles and extrasynaptic membranes

(Fig. 1A). The ExSyn fraction also showed expression of other marker proteins not associated with extrasynaptic cellular membranes. EM analyses found no detectible synapses in the ExSyn fraction (Fig. 1B), and enrichment of both excitatory and inhibitory synapses in the SYN fraction (Fig. 1C-H). No evidence of other intact organelles was found by EM in either fraction. 3.2. AMPAR and NMDAR subunit expression in ACC homogenates Total protein expression levels of AMPAR subunits (GluA1–4) and NMDAR subunits (NR1, NR2A, NR2B, and NR3A) were not different in ACC homogenates between schizophrenia and comparison groups (Fig. 2, Table 2). 3.3. Stargazin expression in ACC homogenates Stargazin protein expression was reduced ~45% in schizophrenia relative to comparison individuals in ACC homogenates (Fig. 3A, Table 2). Post hoc analysis revealed a significant difference in stargazin protein expression between males and females [t (31) = 2.4, p = 0.02], with stargazin levels ~63% higher in females (Fig. 3B). Analysis of stargazin expression by two-way ANOVA found no interaction of sex and diagnosis [FINT (1, 29) = 0.09]; however, independent effects of diagnosis [FDx (1, 29) = 12.3, p = 0.002] and sex [FSEX (1, 29) = 9.6, p = 0.004] were identified (Fig. 3B). Comparisons using the Benjamini-Hochberg FDR method confirmed that stargazin expression is reduced ~55% in males and ~41% in females with schizophrenia [p(male) = 0.02, p(female) = 0.018; q = 0.02]. Stargazin protein expression in females was ~60% higher than males in the comparison group and more than double the stargazin expression level of males in the schizophrenia group [p(COMP) = 0.03, p(SCZ) = 0.0498; q = 0.0498]. Stargazin is known to influence trafficking of AMPARs via an interaction with the GluA2 subunit. In ACC homogenates, the ratio of GluA2: stargazin was increased [t (29) = 3.7, p = 0.0008] in schizophrenia (Fig. 3E, Table 2). The ratios of other AMPAR subunits (GluA1, GluA3,

Fig. 2. AMPAR and NMDAR subunits are expressed normally in schizophrenia ACC. Protein expression of (A) AMPAR subunits (GluA1–4) and (B) NMDAR subunits (NR1, NR2A, NR2B, and NR3A) was measured in schizophrenia (SCZ) and comparison (COMP) ACC homogenates by Western blot analysis. Data are expressed as the integrated intensity of the target protein normalized to the integrated intensity of intralane β-tubulin. No significant differences were detected. Error bars indicate the mean and S.E.M. for each group.

Please cite this article as: J.L. Benesh, T.M. Mueller and J.H. Meador-Woodruff, AMPA receptor subunit localization in schizophrenia anterior cingulate cortex, Schizophrenia Research, https://doi.org/10.1016/j.schres.2020.01.025

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Table 2 Protein expression and ratios of protein expression in total ACC homogenates. Comparison

Schizophrenia

Mean ± S.D.

Mean ± S.D.

Protein GluA1 GluA2 GluA3 GluA4 NR1 NR2A NR2B NR3A Stargazin

1.379 1.053 0.251 0.619 0.192 0.299 0.078 0.045 0.186

1.034 1.094 0.253 0.630 0.194 0.223 0.057 0.044 0.104

Ratio GluA1:GluA2 GluA3:GluA2 GluA4:GluA2 GluA2:Stargazin GluA1:Stargazin GluA4:Stargazin NR2A:NR2B

1.365 ± 0.64 0.257 ± 0.155 0.679 ± 0.282 6.677 ± 4.730 10.380 ± 9.388 4.653 ± 4.573 5.937 ± 4.172

± ± ± ± ± ± ± ± ±

0.826 0.466 0.091 0.201 0.044 0.146 0.049 0.033 0.089

± ± ± ± ± ± ± ± ±

0.581 0.355 0.074 0.163 0.029 0.095 0.031 0.034 0.076

0.956 ± 0.505 0.242 ± 0.112 0.706 ± 0.352 14.660 ± 6.883 15.860 ± 14.790 10.470 ± 9.013 4.344 ± 3.213

Test statistic

p-Value

q-Value

t (32) = t (33) = t (28) = t (27) = t (28) = t (32) = t (31) = U = 56 U = 57

0.004

0.006

0.0008

0.007

0.041

0.014

1.42 0.29 0.07 0.15 0.15 1.83 1.43

t (30) = 2.02 U = 95 t (25) = 0.22 t (29) = 3.74 U = 106 U = 44 U = 81

Protein values are the β-tubulin normalized integrated intensity of each protein of interest. Ratio values are the ratios of β-tubulin normalized integrated intensities of proteins with functional relevance to one another. Two-tailed unpaired Student's t-tests were used to assess normally distributed data; non-normally distributed data were assessed by Mann-Whitney U tests. P-values and q-values which met the threshold for significance (α =0.05, q* = 0.05) are listed in bold.

and GluA4) to GluA2 in ACC homogenates and the ratio of GluA1: stargazin were not different between diagnostic groups (Table 2). 3.4. Stargazin expression in subcellular fractions enriched for ER membranes or synapses The protein level of stargazin was not different between schizophrenia and comparison individuals in either the ER or SYN fractions; however, the amount of stargazin protein expressed in the ER fraction relative to total stargazin levels (ER/Total) was increased [U = 42, p =

0.005] in schizophrenia (Fig. 3D, Table 3). The relative amount of stargazin expressed in the SYN fraction (SYN/Total) was also increased in schizophrenia [t (29) = 2.13, p = 0.04, q = 0.006], but this observation failed to survive multiple testing correction (Fig. 3F, Table 4). 3.5. Protein expression of AMPAR and NMDAR subunits in subcellular fractions enriched for ER or synapses Protein expression levels and proportional protein expression levels in the ER fraction for GluA1–4, NR1, NR2A, and NR2B were not different

Fig. 3. Stargazin protein expression levels in ACC homogenates and subcellular fractions. Protein expression was measured in schizophrenia (SCZ) and comparison (COMP) subjects by western blot analysis in total ACC homogenates and subcellular fractions. Total protein expression is expressed as the integrated intensity of stargazin normalized to the integrated intensity of intralane β-tubulin in total homogenates. Ratio of total protein expression is the ratio of β-tubulin normalized protein expression levels in total homogenates. ER fraction protein expression is the integrated intensity of stargazin normalized to intralane JM4 in ER fractions. ER/total protein expression is the ER fraction protein expression normalized to total protein expression. Error bars represent the group mean and S.E.M. on all graphs. (A) Stargazin is reduced in schizophrenia ACC homogenates. (B) Stargazin expression is higher in females (open shapes; ○, □) relative to males (filled shapes; ●, ■) independent of diagnosis, and is reduced in both males and females with schizophrenia (squares; □, ■) relative to comparison individuals (circles; ○, ●). (C) The ratio of GluA2:stargazin is increased in schizophrenia ACC homogenates. (D) In the ER fraction, stargazin protein levels are not different from comparison (left), but the amount of stargazin in the ER fraction relative to total stargazin levels (right) is increased in schizophrenia. (E) In the SYN fraction, stargazin expression is not different between diagnostic groups. *p b 0.05, **p b 0.01, ***p b 0.001.

Please cite this article as: J.L. Benesh, T.M. Mueller and J.H. Meador-Woodruff, AMPA receptor subunit localization in schizophrenia anterior cingulate cortex, Schizophrenia Research, https://doi.org/10.1016/j.schres.2020.01.025

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Table 3 Protein expression and ratios of protein expression in the ER fraction. Comparison

Schizophrenia

Mean ± S.D.

Mean ± S.D.

Protein GluA1 GluA2 GluA3 GluA4 NR1 NR2A NR2B Stargazin

0.063 0.077 0.364 0.108 0.032 0.015 0.006 0.011

± ± ± ± ± ± ± ±

0.047 0.034 0.301 0.061 0.016 0.004 0.005 0.007

0.053 0.065 0.403 0.071 0.043 0.017 0.010 0.012

± ± ± ± ± ± ± ±

0.032 0.019 0.171 0.018 0.023 0.005 0.008 0.006

U = 101 t (31) = 1.36 U = 78 t (28) = 0.58 t (28) = 1.47 t (30) = 0.88 U = 99 U = 108

ER/total GluA1 GluA2 GluA3 GluA4 NR1 NR2A NR2B Stargazin

0.054 0.085 1.609 0.175 0.171 0.066 0.133 0.071

± ± ± ± ± ± ± ±

0.030 0.048 1.094 0.093 0.083 0.045 0.133 0.067

0.058 0.062 1.580 0.214 0.231 0.083 0.164 0.152

± ± ± ± ± ± ± ±

0.038 0.028 0.749 0.135 0.111 0.044 0.110 0.103

t (27) = 0.03 t (29) = 1.65 U = 87 t (26) = 0.88 U = 59 U = 86 U = 65 U = 42

Ratios GluA1:GluA2 GluA3:GluA2 GluA4:GluA2 GluA2:Stargazin GluA1:Stargazin GluA4:Stargazin NR2A:NR2B

0.748 ± 0.475 4.719 ± 2.830 1.473 ± 0.942 8.483 ± 3.022 7.442 ± 5.945 12.760 ± 8.757 3.311 ± 1.802

0.839 ± 0.491 7.436 ± 5.224 1.868 ± 1.520 6.290 ± 2.963 6.054 ± 5.645 10.980 ± 8.265 3.416 ± 3.589

Test statistic

t (25) = 0.49 t (27) = 1.76 U = 100 t (30) = 2.07 U = 93 U = 89 U = 86

p-Value

q-Value

0.005

0.006

0.047

0.007

Protein values are the JM4 normalized integrated intensity of each protein of interest. ER/Total values are the JM4 normalized integrated intensity in the ER fraction normalized to the βtubulin normalized integrated intensity in total homogenates for each target protein. Ratio values are the ratios JM4 normalized integrated intensities of proteins with functional relevance to one another in the ER fraction. Two-tailed unpaired Student's t-tests were used to assess normally distributed data; non-normally distributed data were assessed by Mann-Whitney U tests. P-values and q-values which met the threshold for significance (α =0.05, q* = 0.05) are listed in bold.

Table 4 Protein expression and ratios of protein expression in the SYN fraction. Comparison

Schizophrenia

Test statistic

p-Value

q-Value

0.001

0.006

0.023

0.011

0.042

0.006

0.011

0.014

0.004

0.007

Mean ± S.D.

Mean ± S.D.

Protein GluA1 GluA2 GluA3 GluA4 NR1 NR2A NR2B NR3A Stargazin

0.398 0.200 1.090 0.035 0.644 0.154 0.036 0.075 0.045

± ± ± ± ± ± ± ± ±

0.222 0.127 0.641 0.022 0.317 0.051 0.018 0.045 0.027

0.199 0.205 1.132 0.042 0.503 0.115 0.055 0.077 0.041

± ± ± ± ± ± ± ± ±

0.139 0.241 0.514 0.030 0.296 0.045 0.062 0.049 0.033

U = 52 U = 118 U = 125 U = 125 U = 105 t (32) = 2.38 U = 144 U = 141 U = 130

SYN/total GluA1 GluA2 GluA3 GluA4 NR1 NR2A NR2B NR3A Stargazin

0.347 0.215 1.215 0.980 13.89 0.709 0.689 1.842 0.287

± ± ± ± ± ± ± ± ±

0.547 0.124 1.605 0.647 6.833 0.434 0.630 1.150 0.192

0.259 0.185 1.632 1.324 11.19 0.614 0.834 2.403 0.499

± ± ± ± ± ± ± ± ±

0.193 0.173 1.546 0.957 7.118 0.324 0.622 1.532 0.344

U = 87 U = 109 U = 45 t (19) = 0.97 t (22) = 0.95 t (30) = 0.70 U = 105 U = 48 t (29) = 2.13

Ratios GluA1:GluA2 GluA3:GluA2 GluA4:GluA2 GluA2:Stargazin GluA1:Stargazin GluA4:Stargazin NR2A:NR2B

2.247 6.120 0.209 4.625 10.73 0.989 5.296

± ± ± ± ± ± ±

0.904 3.795 0.155 1.989 5.772 0.677 2.781

1.498 9.157 0.305 4.575 5.659 1.062 3.971

± ± ± ± ± ± ±

0.68 5.334 0.212 2.570 2.839 0.732 2.579

t (32) = 2.70 U = 85 U = 96 U = 119 t (31) = 3.10 U = 123 t (31) = 1.41

Protein values are the PSD95 normalized integrated intensity of each protein of interest. SYN/Total values are the PSD95 normalized integrated intensity in the SYN fraction normalized to β-tubulin normalized integrated intensity in total homogenates for each protein. Ratio values are the ratios of PSD95 normalized integrated intensities of proteins with functional relevance to one another in the SYN fraction. Two-tailed unpaired Student's t-tests were used to assess normally distributed data; non-normally distributed data were assessed by Mann-Whitney U tests. P-values and q-values which met the threshold for significance (α =0.05, q* = 0.05) are listed in bold.

Please cite this article as: J.L. Benesh, T.M. Mueller and J.H. Meador-Woodruff, AMPA receptor subunit localization in schizophrenia anterior cingulate cortex, Schizophrenia Research, https://doi.org/10.1016/j.schres.2020.01.025

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Fig. 4. GluA1 protein expression in the SYN fraction. GluA1 protein expression was measured in schizophrenia (SCZ) and comparison (COMP) subjects by western blot analysis in total ACC homogenates and subcellular fractions. Protein expression data are expressed as the integrated intensity of GluA1 normalized to the intralane integrated intensity of PSD95 in the SYN fraction. Ratios of protein expression in the SYN fraction are calculated using the PSD95 normalized protein expression values. Protein expression of GluA1 and the ratios of GluA1: GluA2 and GluA1:stargazin are reduced in the SYN fraction in schizophrenia. *p b 0.05, **p b 0.01.

between diagnostic groups (Table 3). In the SYN fraction, GluA1 protein levels were reduced ~50% in the SYN fraction [U = 52, p = 0.001] in schizophrenia (Fig. 4A, Table 4). Protein expression in the SYN fraction and SYN/Total protein expression of GluA2–4, NR1, NR2A, and NR2B were not different between diagnostic groups (Table 4). No ratios of protein expression were found different in the ER fraction in schizophrenia (Table 3). However, the ratio of GluA1:GluA2 was ~66% lower in the SYN fraction in schizophrenia [t (32) = 2.7, p = 0.01] (Fig. 4B, Table 4). This ratio was found to covary with PMI [F (1, 32) = 8.1, p = 0.008]. Subsequent analysis by ANCOVA found that the difference due to diagnosis remains significant when controlling for PMI [F (1, 31) = 5.3, p = 0.03]. No other ratios of protein expression were different in the SYN fraction in schizophrenia (Table 4). 4. Discussion In this study, we identified reduced expression of stargazin, and an increased ratio of GluA2:stargazin in total ACC homogenates in schizophrenia. Along with reduced total levels of stargazin, ER/Total stargazin was increased in schizophrenia. Although we did not identify any differences in the total levels of AMPAR or NMDAR subunits in total homogenates, GluA1 was reduced in a subcellular fraction enriched for synapses. Also in the SYN fraction, the ratios of GluA1:stargazin and GluA1:GluA2 were reduced in schizophrenia. These findings support a role for GluA1 in the synaptic pathophysiology of schizophrenia, and expand on previous studies of abnormalities of cortical AMPAR protein expression in this illness (Breese et al., 1995; Corti et al., 2011; Hammond et al., 2010, 2012; Tucholski et al., 2013), and are consistent with studies of transgenic GluA1 mice that exhibit phenotypic features similar to some aspects of schizophrenia (Inta et al., 2010; Wiedholz et al., 2008; Zamanillo, 1999). The current findings also support the hypothesis that abnormal compartment-specific localization and trafficking of AMPARs, rather than discrete alterations in the total number of AMPARs expressed, may underlie AMPA-associated defects in schizophrenia. Tetrameric AMPARs are first assembled and modified in the ER before associating with TARPs such as stargazin. AMPARs associated with TARP proteins are then trafficked as a complex to the synapse (Bredt and Nicoll, 2003; Jackson and Nicoll, 2011). Our current findings indicate abnormal AMPAR subunit-TARP associations in schizophrenia and suggest that synaptic AMPAR dysregulation in this illness occurs downstream of altered complex assembly or processing in the ER. Consistent with this, we have reported abnormal N-linked glycosylation of GluA2 in schizophrenia which we speculated was consistent with accelerated ER exit of this subunit (Tucholski et al., 2013). While we found in the current study that GluA2 protein levels in ER and SYN fractions were unchanged in schizophrenia, the reduced ratio of GluA1:GluA2 in the SYN

fraction is consistent with this model, reflecting a shift to more GluA2containing receptor complexes at synapses in schizophrenia. We did not observe changes in the ER in schizophrenia of either GluA2 expression or the ratio of GluA1:GluA2, however, an increased ratio of GluA2: stargazin in total homogenates and increased ER/Total stargazin levels suggest that despite lower total levels of stargazin, once stargazin molecules are expressed they actively participate in AMPAR complex trafficking and may preferentially assemble with GluA2 containing AMPARs in this illness. GluA2 primarily associates with GluA3 in ER, while synaptic GluA2 is present in complexes containing GluA1 and to a lesser extent GluA3 (Greger et al., 2002; Lu et al., 2009; Wenthold et al., 1996). Unlike GluA1, insertion of GluA2 at the synapse is not activity-dependent, but rather is driven by constitutive trafficking pathways thought to underlie homeostatic regulation (Greger et al., 2002; Hayashi et al., 2000; Passafaro et al., 2001; Shi et al., 2001). Recent studies have highlighted the importance of AMPAR auxiliary proteins in AMPAR regulation and trafficking (Bedoukian et al., 2006; Jackson and Nicoll, 2011; Vandenberghe et al., 2005; Ziff, 2007), and their abnormal expression in schizophrenia (Drummond et al., 2012, 2013). In this study, we did not find abnormal subcellular expression levels of stargazin in schizophrenia; however, the ratio of GluA2: stargazin is increased in homogenates and the ratio of GluA1:stargazin is reduced in the SYN fraction, suggesting perturbations of stargazin expression may contribute to reduced synaptic expression of GluA1. In light of evidence that suggests auxiliary protein-mediated trafficking of AMPARs occurs independently of other modulatory effects on receptor targeting and biophysical properties (Bedoukian et al., 2008), the effects of reduced total stargazin levels may contribute to preferential TARP-AMPAR subunit interactions and in turn alterations at the synapse. We have previously reported data that was consistent with accelerated ER exit and forward trafficking of NMDARs in schizophrenia. NMDAR trafficking proteins including CASK and Veli-3 were decreased in ACC in schizophrenia (Kristiansen et al., 2010a, 2010b). We also found that the GluN1-C2′ splice variant was increased in ACC in schizophrenia, while the GluN1-C2 variant was not changed (Kristiansen et al., 2006). This is intriguing given that the C2′ variant accelerates forward trafficking of NMDARs from the ER in an activity-dependent manner (Mu et al., 2003). We have also reported increased GluA1 expression in early endosomes (Hammond et al., 2010), but no differences of AMPAR subunit expression in late endosomes or an ER fraction (Hammond et al., 2011, 2012). Whether the GluA1 changes we observed in the SYN fraction in schizophrenia may reflect dysfunction of mechanisms mediated by TARP-AMPAR interactions, these findings are consistent with abnormal forward trafficking and synaptic expression of glutamate receptors in schizophrenia. Reduced levels of

Please cite this article as: J.L. Benesh, T.M. Mueller and J.H. Meador-Woodruff, AMPA receptor subunit localization in schizophrenia anterior cingulate cortex, Schizophrenia Research, https://doi.org/10.1016/j.schres.2020.01.025

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J.L. Benesh et al. / Schizophrenia Research xxx (xxxx) xxx

GluA1 at the synapse concurrent with increased GluA1 in early endosomes that we previously reported suggest that mechanisms regulating the activity-dependent subcellular distribution and synaptic targeting of GluA1 are impaired in this illness. Corroborating our finding that AMPAR subunits are normally expressed in total homogenates and ER fractions, few changes in AMPAR transcript levels have been previously reported in schizophrenia (reviewed in Rubio et al., 2012) and lack of expression differences in total homogenates or the proximal end of the forward trafficking pathway suggest that decreased synaptic GluA1 is not due to decreased gene expression or impaired subunit translation, but rather mislocalization of GluA1 after AMPAR exit from the ER. As the ratio of GluA2:stargazin is increased in total homogenates and the ratios of GluA1:stargazin and GluA1:GluA2 are decreased in the SYN fraction, an alternative hypothesis is that preferential association of stargazin with GluA2 and reduced levels of stargazin in schizophrenia may limit the amount of stargazin available to interact with GluA1. Failure to associate with stargazin could lead to instability of GluA1-containing receptors which lack GluA2 subunits in the synapse and enhanced endocytosis of this subset of GluA1-containing AMPARs into early endosomes. Future studies examining whether abnormalities in GluA1 localization might be due to other mechanisms of distal dysregulation would be intriguing given the importance of rapid GluA1 availability at proximal sites of membrane insertion for cellular response to synaptic stimuli. Our current findings are consistent with altered glutamatergic synaptic activity in schizophrenia. The glutamate hypothesis of schizophrenia posits decreased NMDAR-mediated neurotransmission in this illness. Since activation of AMPARs is required for removal of the magnesium block and subsequent activation of NMDARs (Song and Huganir, 2002), NMDAR hypofunction could be secondary to dysregulated AMPAR subunit localization in schizophrenia. On the other hand, animal studies that have examined reduced NMDAR function have shown that this can cause paradoxical strengthening of synapses, as well as increased numbers of functional synapses containing AMPARs (Hanse et al., 2013; Herring et al., 2013; Myers et al., 1999). AMPAR activation in the face of NMDAR inactivity can promote internalization of AMPARs, and specifically target them to endosomes for lysosomal degradation (Ehlers, 2000). However, since AMPAR subunits have been found normally expressed in late endosomes (Hammond et al., 2011) which target proteins to the lysosome for degradation, it is more likely that GluA1-containing receptors which are not expressed at the synapse remain available for synaptic reinsertion via endosomal recycling. Our finding of decreased GluA1 in the SYN fraction in schizophrenia could reflect increased synaptic strength and subsequent endocytosis of GluA1, leaving fewer GluA1-containing receptors in this synaptic compartment, but more GluA1 localized to endosomes. A proteomic study on isolated synapses from ACC found alterations in schizophrenia of proteins involved in the regulation of endocytosis, NMDARs, calcium signaling, and long-term potentiation (Föcking et al., 2014) consistent with this proposed model. There are limitations to this work that are common to all studies in postmortem brain in neuropsychiatric illness. A challenge in studies of schizophrenia is that nearly all patients have been treated with chronic antipsychotic medications. To begin to address this confound, we performed post hoc analyses for schizophrenia patients on or off antipsychotic treatment at the time of death, and found no differences in any dependent measures for which we found changes in the entire schizophrenia group. Although this analysis is relatively underpowered (NSCZ-ON = 13; NSCZ-OFF = 5), it suggests that changes we observed may not be due to antipsychotic treatment, but rather associated with the illness. Previous studies have reported no changes in GluA1 or TARP protein expression in animals treated chronically with haloperidol (Drummond et al., 2012, 2013; Eastwood et al., 1996; O'Connor et al., 2007). In summary, these data suggest abnormal synaptic targeting of GluA1 in schizophrenia, and support a model of AMPAR dysfunction in

this illness that may be due, in part, to abnormal association of this subunit with TARP proteins such as stargazin. Since our findings were specific to the GluA1 subtype of AMPAR, this may be an indication that cellular machinery associated with the regulation of this subunit during activity-driven events is altered in this illness. Whether decreased synaptic GluA1 is due to accelerated ER exit and abnormal AMPAR complex assembly, synaptic instability and enhanced endocytosis of GluA1 containing AMPARs, or is a secondary consequence to altered synaptic activity, a key finding is that regardless of the underlying mechanism, there are fewer GluA1 subunits in the synapse in schizophrenia. Contributors JLB and JHM-W designed the study. JLB executed experimental protocols, collected and analyzed data, created figures and tables, and wrote the first draft of the manuscript. TMM performed statistical analyses and created figures and tables. JLB and TMM performed literature searches and collaborated on data interpretation and manuscript draft revisions. All authors contributed to and have approved the final manuscript. Funding Research reported in this publication was funded by the National Institute of Mental Health of the National Institutes of Health under award number R01MH53327. Additional support was provided by the Department of Psychiatry and Behavioral Neurobiology at the University of Alabama at Birmingham. Declaration of competing interest The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors have no conflicts of interest to disclose. Acknowledgements The authors gratefully acknowledge Dr. Rosalinda Roberts and the Alabama Brain Collection for postmortem cortical samples used for protocol development and antibody optimization.

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Please cite this article as: J.L. Benesh, T.M. Mueller and J.H. Meador-Woodruff, AMPA receptor subunit localization in schizophrenia anterior cingulate cortex, Schizophrenia Research, https://doi.org/10.1016/j.schres.2020.01.025