Altered distribution and localization of organellar Na+/H+ exchangers in postmortem schizophrenia dorsolateral prefrontal cortex.

Schizophrenia is a complex and multifactorial disorder associated with altered neurotransmission as well as numerous signaling pathway and protein trafficking disruptions. The pH of intracellular organelles involved in protein trafficking is tightly regulated and impacts their functioning. The SLC9A family of Na+/H+ exchangers (NHEs) plays a fundamental role in cellular and intracellular pH homeostasis. Four organellar NHE isoforms (NHE6-NHE9) are targeted to intracellular organelles involved in protein trafficking. Increased interactions between organellar NHEs and receptor of activated protein C kinase 1 (RACK1) can lead to redistribution of NHEs to the plasma membrane and hyperacidification of target organelles. Given their role in organelle pH regulation, altered expression and/or localization of organellar NHEs could be an underlying cellular mechanism contributing to abnormal intracellular trafficking and disrupted neurotransmitter systems in schizophrenia. We thus characterized organellar NHE expression, co-immunoprecipitation with RACK1, and Triton X-114 (TX-114) phase partitioning in dorsolateral prefrontal cortex of 25 schizophrenia and 25 comparison subjects by Western blot analysis. In schizophrenia after controlling for subject age at time of death, postmortem interval, tissue pH, and sex, there was significantly decreased total expression of NHE8, decreased co-immunoprecipitation of NHE8 (64%) and NHE9 (56%) with RACK1, and increased TX-114 detergent phase partitioning of NHE6 (283%), NHE9 (75%), and RACK1 (367%). Importantly, none of these dependent measures was significantly impacted when comparing those in the schizophrenia group on antipsychotics to those off of antipsychotics for at least 6 weeks at their time of death and none of these same proteins were affected in rats chronically treated with haloperidol. In summary, we characterized organellar NHE expression and distribution in schizophrenia DLPFC and identified abnormalities that could represent a novel mechanism contributing to disruptions in protein trafficking and neurotransmission in schizophrenia.

Protein expression of prenyltransferase subunits in postmortem schizophrenia dorsolateral prefrontal cortex.

The pathophysiology of schizophrenia includes altered neurotransmission, dysregulated intracellular signaling pathway activity, and abnormal dendritic morphology that contribute to deficits of synaptic plasticity in the disorder. These processes all require dynamic protein-protein interactions at cell membranes. Lipid modifications target proteins to membranes by increasing substrate hydrophobicity by the addition of a fatty acid or isoprenyl moiety, and recent evidence suggests that dysregulated posttranslational lipid modifications may play a role in multiple neuropsychiatric disorders, including schizophrenia. Consistent with these emerging findings, we have recently reported decreased protein S-palmitoylation in schizophrenia. Protein prenylation is a lipid modification that occurs upstream of S-palmitoylation on many protein substrates, facilitating membrane localization and activity of key intracellular signaling proteins. Accordingly, we hypothesized that, in addition to palmitoylation, protein prenylation may be abnormal in schizophrenia. To test this, we assayed protein expression of the five prenyltransferase subunits (FNTA, FNTB, PGGT1B, RABGGTA, and RABGGTB) in postmortem dorsolateral prefrontal cortex from patients with schizophrenia and paired comparison subjects (n = 13 pairs). We found decreased levels of FNTA (14%), PGGT1B (13%), and RABGGTB (8%) in schizophrenia. To determine whether upstream or downstream factors may be driving these changes, we also assayed protein expression of the isoprenoid synthases FDPS and GGPS1 and prenylation-dependent processing enzymes RCE and ICMT. We found these upstream and downstream enzymes to have normal protein expression. To rule out effects from chronic antipsychotic treatment, we assayed FNTA, PGGT1B, and RABGGTB in the cortex from rats treated long-term with haloperidol decanoate and found no change in the expression of these proteins. Given the role prenylation plays in localization of key signaling proteins found at the synapse, these data offer a potential mechanism underlying abnormal protein-protein interactions and protein localization in schizophrenia.

Decreased protein S-palmitoylation in dorsolateral prefrontal cortex in schizophrenia.

Recent reports suggest abnormalities of neurotransmitter receptor trafficking, targeting, dendritic localization, recycling, and degradation in the brain in schizophrenia. We hypothesized that a potential explanation for these findings may be abnormal posttranslational modifications that influence intracellular targeting and trafficking of proteins between subcellular compartments. Dysregulation of protein palmitoylation is a strong candidate for such a process. S-palmitoylation is a reversible thioesterification of palmitoyl-groups to cysteine residues that can regulate trafficking and targeting of intracellular proteins. Using a biotin switch assay to study S-palmitoylation of proteins in human postmortem brain, we identified a pattern of palmitoylated proteins that cluster into 17 bands of discrete molecular masses, including numerous proteins associated with receptor signal transduction. Using mass spectrometry, we identified 219 palmitoylated proteins in human frontal cortex, and individually validated palmitoylation status of a subset of these proteins. Next, we assayed protein palmitoylation in dorsolateral prefrontal cortex from 16 schizophrenia patients and paired comparison subjects. S-palmitoylation was significantly reduced for proteins in most of the 17 schizophrenia bands. In rats chronically treated with haloperidol, the same pattern of palmitoylation was observed but the extent of palmitoylation was unchanged, suggesting that the diminution in protein palmitoylation in schizophrenia is not due to chronic antipsychotic treatment. These results indicate there are changes in the extent of S-palmitoylation of many proteins in the frontal cortex in schizophrenia. Given the roles of this posttranslational modification, these data suggest a potential mechanism reconciling previous observations of abnormal intracellular targeting and trafficking of neurotransmitter receptors in this illness.

Evolutionarily conserved pattern of AMPA receptor subunit glycosylation in Mammalian frontal cortex.

Protein glycosylation may contribute to the evolution of mammalian brain complexity by adapting excitatory neurotransmission in response to environmental and social cues. Balanced excitatory synaptic transmission is primarily mediated by glutamatergic neurotransmission. Previous studies have found that subunits of the AMPA subtype of glutamate receptor are N-glycosylated, which may play a critical role in AMPA receptor trafficking and function at the cell membrane. Studies have predominantly used rodent models to address altered glycosylation in human pathological conditions. Given the rate of mammalian brain evolution and the predicted rate of change in the brain-specific glycoproteome, we asked if there are species-specific changes in glycoprotein expression, focusing on the AMPA receptor. N-glycosylation of AMPA receptor subunits was investigated in rat (Rattus norvegicus), tree shrew (Tupaia glis belangeri), macaque (Macaca nemestrina), and human frontal cortex tissue using a combination of enzymatic deglycosylation and Western blot analysis, as well as lectin binding assays. We found that two AMPA receptor subunits, GluA2 and GluA4, are sensitive to deglycosylation with Endo H and PNGase F. When we enriched for glycosylated proteins using lectin binding assays, we found that all four AMPA receptor subunits are glycosylated, and were predominantly recognized by lectins that bind to glucose or mannose, N-acetylglucosamine (GlcNAc), or 1-6αfucose. We found differences in glycosylation between different subunits, as well as modest differences in glycosylation of homologous subunits between different species.

Alterations of the myristoylated, alanine-rich C kinase substrate (MARCKS) in prefrontal cortex in schizophrenia.

Abnormal synaptic plasticity has been implicated in the cognitive deficits seen in schizophrenia, where alterations have been found in neurotransmission, signaling and dendritic dynamics. Rapid rearrangement of the actin cytoskeleton is critical for plasticity and abnormalities of molecular regulators of this process are candidates for understanding mechanisms underlying these changes in schizophrenia. The myristoylated, alanine-rich C-kinase substrate (MARCKS) is crucial for many roles associated with synaptic plasticity, including facilitation of neurotransmission, dendritic branching and in turn cognitive function. Accordingly, we hypothesized that this protein is abnormally expressed or regulated in schizophrenia. We measured protein expression of MARCKS by Western blot analysis in postmortem samples of dorsolateral prefrontal cortex (DLPFC) from elderly schizophrenia patients (N=16) and a comparison group (N=20). We also assayed phosphorylated-MARCKS (pMARCKS), given the role of phosphorylation in reversing membrane association by MARCKS. We found decreased expression of both MARCKS and pMARCKS in schizophrenia. Altered myristoylation may be a mechanism that explains this down-regulation of MARCKS, so we also assayed expression of the two isoforms of the key myristoylation enzyme, NMT, and an enzymatic inhibitor of this enzyme, NMT-inhibitor protein (NIP71) by Western blotting in these same subjects. Expression did not change between groups for these proteins, suggesting a mechanism other than myristoylation is responsible for decreased MARCKS expression in schizophrenia. These data suggest a potential mechanism underlying aspects of altered synaptic plasticity observed in schizophrenia.

N-linked glycosylation of cortical N-methyl-D-aspartate and kainate receptor subunits in schizophrenia.

Dysfunctional glutamate neurotransmission has been implicated in the pathophysiology of schizophrenia. Abnormal expressions in schizophrenia of ionotropic glutamate receptors (iGluRs) and the proteins that regulate their trafficking have been found to be region and subunit specific in brain, suggesting that abnormal trafficking of iGluRs may contribute toward altered glutamatergic neurotransmission. The post-translational modification N-glycosylation of iGluR subunits can be used as a proxy for their intracellular localization. Receptor complexes assemble in the lumen of the endoplasmic reticulum, where N-glycosylation begins with the addition of N-linked oligomannose glycans, and is subsequently trimmed and replaced by more elaborate glycans while trafficking through the Golgi apparatus. Previously, we found abnormalities in N-glycosylation of the GluR2 AMPA receptor subunit in schizophrenia. Here, we investigated N-glycosylation of N-methyl-D-aspartate and kainate (KA) receptor subunits in the dorsolateral prefrontal cortex from patients with schizophrenia and a comparison group. We used enzymatic deglycosylation with two glycosidases: endoglycosidase H (Endo H), which removes immature high mannose-containing sugars, and peptide-N-glycosidase F (PNGase F), which removes all N-linked sugars. The NR1, NR2A, NR2B, GluR6, and KA2 subunits were all sensitive to treatment with Endo H and PNGase F. The GluR6 KA receptor subunit was significantly more sensitive to Endo H-mediated deglycosylation in schizophrenia, suggesting a larger molecular mass of N-linked high mannose and/or hybrid sugars on GluR6. This finding, taken with our previous work, suggests that a cellular mechanism underlying abnormal glutamate neurotransmission in schizophrenia may involve abnormal trafficking of both AMPA and KA receptors.

Abnormal N-linked glycosylation of cortical AMPA receptor subunits in schizophrenia.

Numerous studies have demonstrated brain region- and subunit-specific abnormalities in the expression of subunits of the AMPA subtype of glutamate receptors in schizophrenia. In addition, abnormalities in the expression of proteins that regulate the forward trafficking of AMPA receptors through the cell have been reported. These findings suggest abnormal trafficking of AMPA receptors as a mechanism underlying dysregulated glutamate neurotransmission in schizophrenia. AMPA receptor subunits (GluR1-4) assemble to form AMPA receptor complexes in the lumen of the endoplasmic reticulum (ER). These subunits undergo the posttranslational modification of N-linked glycosylation in the ER and the Golgi apparatus before the assembled receptors are transported to the plasma membrane. In this study, we measured expression of AMPA receptors and the extent of their N-glycosylation using Western blot analysis in the dorsolateral prefrontal cortex in subjects with schizophrenia (N = 35) and a comparison group (N = 31). N-glycosylation was assessed using molecular mass shift assays following digestion with endoglycosidase H (Endo H), which removes immature high mannose-containing sugars, and with peptide-N-glycosidase F (PNGase F), which removes all N-linked sugars. Of the four AMPA receptor subunits, only GluR4 was significantly increased in schizophrenia. GluR2 and GluR4 were both sensitive to Endo H and PNGase F treatment. Endo H-mediated deglycosylation of GluR2 resulted in a significantly smaller pool of GluR2 protein to shift in schizophrenia, reflecting less N-linked high mannose and/or hybrid sugars on the GluR2 protein in this illness. This was confirmed by immunoisolation of GluR2 and probing with Concanavalin A, a mannose specific lectin; in subjects with schizophrenia GluR2 was significantly less reactive to Concanavalin A. Altered N-linked glycosylation of the GluR2 subunit in schizophrenia suggests abnormal trafficking of AMPA receptors from the ER to the synaptic membrane in schizophrenia.