Dysregulation of the unfolded protein response (UPR) in the dorsolateral prefrontal cortex in elderly patients with schizophrenia.

Abnormalities in protein localization, function, and posttranslational modifications are targets of schizophrenia (SCZ) research. As a major contributor to the synthesis, folding, trafficking, and modification of proteins, the endoplasmic reticulum (ER) is well-positioned to sense cellular stress. The unfolded protein response (UPR) is an evolutionarily conserved adaptive reaction to environmental and pathological perturbation in ER function. The UPR is a highly orchestrated and complex cellular response, which is mediated through the ER chaperone protein, BiP, three known ER transmembrane stress sensors, protein kinase RNA-like ER kinase (PERK), activating transcription factor-6 (ATF6), inositol requiring enzyme 1α (IRE1α), and their downstream effectors. In this study, we measured protein expression and phosphorylation states of UPR sensor pathway proteins in the dorsolateral prefrontal cortex (DLPFC) of 22 matched pairs of elderly SCZ and comparison subjects. We observed increased protein expression of BiP, decreased PERK, and decreased phosphorylation of IRE1α. We also observed decreased p-JNK2 and increased sXBP1, downstream targets of the IRE1α arm of the UPR. The disconnect between decreased p-IRE1α and increased sXBP1 protein expression led us to measure sXbp1 mRNA. We observed increased expression of the ratio of sXbp1/uXbp1 transcripts, suggesting that splicing of Xbp1 mRNA by IRE1α is increased and drives upregulation of sXBP1 protein expression. These findings suggest an abnormal pattern of UPR activity in SCZ, with specific dysregulation of the IRE1α arm. Dysfunction of this system may lead to abnormal responses to cellular stressors and contribute to protein processing abnormalities previously observed in SCZ.

mTOR kinase activity disrupts a phosphorylation signaling network in schizophrenia brain.

The AKT-mTOR signaling transduction pathway plays an important role in neurodevelopment and synaptic plasticity. mTOR is a serine/threonine kinase that modulates signals from multiple neurotransmitters and phosphorylates specific proteins to regulate protein synthesis and cytoskeletal organization. There is substantial evidence demonstrating abnormalities in AKT expression and activity in different schizophrenia (SZ) models. However, direct evidence for dysregulated mTOR kinase activity and its consequences on downstream effector proteins in SZ pathophysiology is lacking. Recently, we reported reduced phosphorylation of mTOR at an activating site and abnormal mTOR complex formation in the SZ dorsolateral prefrontal cortex (DLPFC). Here, we expand on our hypothesis of disrupted mTOR signaling in the SZ brain and studied the expression and activity of downstream effector proteins of mTOR complexes and the kinase activity profiles of SZ subjects. We found that S6RP phosphorylation, downstream of mTOR complex I, is reduced, whereas PKCα phosphorylation, downstream of mTOR complex II, is increased in SZ DLPFC. In rats chronically treated with haloperidol, we showed that S6RP phosphorylation is increased in the rat frontal cortex, suggesting a potential novel mechanism of action for antipsychotics. We also demonstrated key differences in kinase signaling networks between SZ and comparison subjects for both males and females using kinome peptide arrays. We further investigated the role of mTOR kinase activity by inhibiting it with rapamycin in postmortem tissue and compared the impact of mTOR inhibition in SZ and comparison subjects using kinome arrays. We found that SZ subjects are globally more sensitive to rapamycin treatment and AMP-activated protein kinase (AMPK) contributes to this differential kinase activity. Together, our findings provide new insights into the role of mTOR as a master regulator of kinase activity in SZ and suggest potential targets for therapeutic intervention.

Differential genetic associations and expression of PAPST1/SLC35B2 in bipolar disorder and schizophrenia.

Lithium's inhibitory effect on enzymes involved in sulfation process, such as inhibition of 3'(2')-phosphoadenosine 5'-phosphate (PAP) phosphatase, is a possible mechanism of its therapeutic effect for bipolar disorder (BD). 3'-Phosphoadenosine 5'-phosphosulfate (PAPS) is translocated from cytosol to Golgi lumen by PAPS transporter 1 (PAPST1/SLC35B2), where it acts as a sulfa donor. Since SLC35B2 was previously recognized as a molecule that facilitates the release of D-serine, a co-agonist of N-methyl-D-aspartate type glutamate receptor, altered function of SLC35B2 might be associated with the pathophysiology of BD and schizophrenia (SCZ). We performed genetic association analyses of the SLC35B2 gene using Japanese cohorts with 366 BD cases and 370 controls and 2012 SCZ cases and 2170 controls. We then investigated expression of SLC35B2 mRNA in postmortem brains by QPCR using a Caucasian cohort with 33 BD and 34 SCZ cases and 34 controls and by in situ hybridization using a Caucasian cohort with 37 SCZ and 29 controls. We found significant associations between three SNPs (rs575034, rs1875324, and rs3832441) and BD, and significantly reduced SLC35B2 mRNA expression in postmortem dorsolateral prefrontal cortex (DLPFC) of BD. Moreover, we observed normalized SLC35B2 mRNA expression in BD subgroups who were medicated with lithium. While there was a significant association of SLC35B2 with SCZ (SNP rs2233437), its expression was not changed in SCZ. These findings indicate that SLC35B2 might be differentially involved in the pathophysiology of BD and SCZ by influencing the sulfation process and/or glutamate system in the central nervous system.

Intracellular compartment-specific proteasome dysfunction in postmortem cortex in schizophrenia subjects.

Protein homeostasis is an emerging component of schizophrenia (SZ) pathophysiology. Proteomic alterations in SZ are well-documented and changes in transcript expression are frequently not associated with changes in protein expression in SZ brain. The underlying mechanism driving these changes remains unknown, though altered expression of ubiquitin proteasome system (UPS) components have implicated protein degradation. Previous studies have been limited to protein and transcript expression, however, and do not directly test the function of the proteasome. To address this gap in knowledge, we measured enzymatic activity associated with the proteasome (chymotrypsin-, trypsin-, and caspase-like) in the superior temporal gyrus (STG) of 25 SZ and 25 comparison subjects using flourogenic substrates. As localization regulates which cellular processes the proteasome contributes to, we measured proteasome activity and subunit expression in fractions enriched for nucleus, cytosolic, and membrane compartments. SZ subjects had decreased trypsin-like activity in total homogenate. This finding was specific to the nucleus-enriched fraction and was not associated with changes in proteasome subunit expression. Interestingly, both chymotrypsin-like activity and protein expression of 19S RP subunits, which facilitate ubiquitin-dependent degradation, were decreased in the cytosol-enriched fraction of SZ subjects. Intracellular compartment-specific proteasome dysfunction implicates dysregulation of protein expression both through altered ubiquitin-dependent degradation of cytosolic proteins and regulation of protein synthesis due to degradation of transcription factors and transcription machinery in the nucleus. Together, these findings implicate proteasome dysfunction in SZ, which likely has a broad impact on the proteomic landscape and cellular function in the pathophysiology of this illness.

Gene expression of glutamate metabolizing enzymes in the hippocampal formation in human temporal lobe epilepsy.

PURPOSE: Increased interictal concentrations of extracellular hippocampal glutamate have been implicated in the pathophysiology of temporal lobe epilepsy (TLE). Recent studies suggest that perturbations of the glutamate metabolizing enzymes glutamine synthetase (GS) and phosphate activated glutaminase (PAG) may underlie the glutamate excess in TLE. However, the molecular mechanism of the enzyme perturbations remains unclear. A better understanding of the regulatory mechanisms of GS and PAG could facilitate the discovery of novel therapeutics for TLE. METHODS: We used in situ hybridization on histologic sections to assess the distribution and quantity of messenger RNA (mRNA) for GS and PAG in subfields of hippocampal formations from the following: (1) patients with TLE and concomitant hippocampal sclerosis, (2) patients with TLE and no hippocampal sclerosis, and (3) nonepilepsy autopsy subjects. KEY FINDINGS: GS mRNA was increased by ~50% in the CA3 in TLE patients without hippocampal sclerosis versus in TLE patients with sclerosis and in nonepilepsy subjects. PAG mRNA was increased by >100% in the subiculum in both TLE patient categories versus in nonepilepsy subjects. PAG mRNA was also increased in the CA1, CA2, CA3, and dentate hilus in TLE without hippocampal sclerosis versus in TLE with sclerosis. Finally, PAG mRNA was increased in the dentate gyrus in TLE with sclerosis versus in nonepilepsy subjects, and also increased in the hilus in TLE without sclerosis versus in TLE with sclerosis. SIGNIFICANCE: These findings demonstrate complex changes in the expression of mRNAs for GS and PAG in the hippocampal formation in TLE, and raise the possibility that both transcriptional and posttranscriptional mechanisms may underlie the regulation of GS and PAG proteins in the epileptic brain.

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.

AMPA receptor subunit localization in schizophrenia anterior cingulate cortex.

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.

Parvalbumin deficiency and GABAergic dysfunction in mice lacking PGC-1alpha.

The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha) is a master regulator of metabolism in peripheral tissues, and it has been proposed that PGC-1alpha plays a similar role in the brain. Recent evidence suggests that PGC-1alpha is concentrated in GABAergic interneurons, so we investigated whether male and female PGC-1alpha -/- mice exhibit abnormalities in interneuron gene expression and/or function. We found a striking reduction in the expression of the Ca(2+)-binding protein parvalbumin (PV), but not other GABAergic markers, throughout the cerebrum in PGC-1alpha +/- and -/- mice. Furthermore, PGC-1alpha overexpression in cell culture was sufficient to robustly induce PV expression. Consistent with a reduction in PV rather than a loss of PV-expressing interneurons, spontaneous synaptic inhibition was not altered in PGC-1alpha -/- mice. However, evoked synaptic responses displayed less paired-pulse depression and dramatic facilitation in response to repetitive stimulation at the gamma frequency. PV transcript expression was also significantly reduced in retina and heart of PGC-1alpha -/- animals, suggesting that PGC-1alpha is required for proper expression of PV in multiple tissues. Together these findings indicate that PGC-1alpha is a novel regulator of interneuron gene expression and function and a potential therapeutic target for neurological disorders associated with interneuron dysfunction.

Expression of the NR2B-NMDA receptor subunit and its Tbr-1/CINAP regulatory proteins in postmortem brain suggest altered receptor processing in schizophrenia.

Several lines of evidence implicate aberrant glutamate neurotransmission in the pathophysiology of schizophrenia. In particular, compromised signaling through the N-methyl-D-aspartate (NMDA) receptor has been linked to positive, negative, and cognitive symptoms of this illness. Studies in postmortem brain have identified altered expression of several structural and signaling molecules of the postsynaptic density (PSD), including the abundantly expressed protein PSD-95, which binds directly to NR2 subunits of the NMDA receptor and regulates its trafficking, membrane expression, and downstream signaling. Several mechanisms for functional regulation of the NR2B-containing NMDA receptor, which have been linked to cognitive dysfunction in schizophrenia, are well known. To analyze whether early events in NR2B processing are affected in schizophrenia, we have isolated a subcellular endoplasmic reticulum (ER)-enriched fraction from postmortem brain and analyzed expression of the NR1 and NR2B NMDA receptor subunits as well as PSD-95 in two areas of prefrontal cortex. We found significantly decreased ER expression of NR2B and PSD-95 in dorsolateral prefrontal cortex in schizophrenia. Analysis in total-cell homogenates from the same subjects of NR2B and PSD-95 expression, as well as of the CINAP and Tbr-1 transcription regulatory proteins, indicate that changes in NR2B processing in schizophrenia involve increased ER exit of NR2B containing NMDA receptors.

Evidence for altered energy metabolism, increased lactate, and decreased pH in schizophrenia brain: A focused review and meta-analysis of human postmortem and magnetic resonance spectroscopy studies.

Though the pathophysiology of schizophrenia remains poorly understood, altered brain energy metabolism is increasingly implicated. Here, we conduct meta-analyses of the available human studies measuring lactate or pH in schizophrenia brain and discuss the accumulating evidence for increased lactate and decreased pH in schizophrenia brain and evidence linking these to negative and cognitive symptom severity. Meta-analysis of six postmortem studies revealed a significant increase in lactate in schizophrenia brain while meta-analysis of 14 magnetic resonance spectroscopy studies did not reveal a significant change in brain pH in schizophrenia. However, only five of these studies were likely sufficiently powered to detect differences in brain pH, and meta-analysis of these five studies found a nonsignificant decrease in pH in schizophrenia brain. Next, we discuss evidence for altered brain energy metabolism in schizophrenia and how this may underlie a buildup of lactate and decreased pH. This alteration, similar to the Warburg effect extensively described in cancer biology, involves diminished tricarboxylic acid cycle and oxidative phosphorylation along with a shift toward increased reliance on glycolysis for energy production. We then explore the role that mitochondrial dysfunction, oxidative stress, and hypoxia-related changes in gene expression likely play in this shift in brain energy metabolism and address the functional consequences of lowered brain pH in schizophrenia including alterations in neurotransmitter regulation, mRNA stability, and overall patterns of gene expression. Finally, we discuss how altered energy metabolism in schizophrenia brain may serve as an effective target in the treatment of this illness.

Post-translational protein modifications in schizophrenia.

Research investigating the pathophysiology of schizophrenia has not yet precisely defined the molecular phenotype of this disorder. Many studies have investigated cellular dysfunction by examining expression levels of molecular targets in postmortem patient brain; however, inconsistencies between transcript and protein measures in schizophrenia are common in the field and represent a challenge to the identification of a unified model of schizophrenia pathogenesis. In humans, >4800 unique proteins are expressed, and the majority of these are modified by glycans and/or lipids. Estimates indicate ~70% of all eukaryotic proteins are modified by at least one type of glycosylation, while nearly 20% of all proteins are known to be lipid-modified. Protein post-translational modification (PTM) by glycosylation and lipidation rely on the spatiotemporal colocalization of enzyme, substrate, and glycan or lipid donor molecule and do not require an upstream "blueprint" or specialized processing machinery for synthesis. Glycan and lipid PTMs can thus facilitate cellular adaptation to environmental signals more rapidly than changes of gene or protein expression, and can significantly impact the localization, function, and interactions of modified substrates, though relatively few studies in schizophrenia have evaluated the PTM status of target proteins. A growing body of literature reports glycosylation and lipidation abnormalities in schizophrenia brain as well as in patient peripheral fluids. In this review, we explain the functional significance of key glycan and lipid PTMs and summarize current findings associated with abnormal glycosylation and lipidation in this illness.

Downregulated AKT-mTOR signaling pathway proteins in dorsolateral prefrontal cortex in Schizophrenia.

Abnormal neurotransmission is central to schizophrenia (SZ). Alterations across multiple neurotransmitter systems in SZ suggest that this illness may be associated with dysregulation of core intracellular processes such as signaling pathways that underlie the regulation and integration of these systems. The AKT-mTOR signaling cascade has been implicated in SZ by gene association, postmortem brain and animal studies. AKT and mTOR are serine/threonine kinases which play important roles in cell growth, proliferation, survival, and differentiation. Both AKT and mTOR require phosphorylation at specific sites for their complete activation. mTOR forms two functionally distinct multiprotein complexes, mTOR Complex 1 (mTORC1) and Complex 2 (mTORC2). mTORC1 mediates ribosome biogenesis, protein translation, and autophagy, whereas mTORC2 contributes to actin dynamics. Altered protein synthesis and actin dynamics can lead to an abnormal neuronal morphology resulting in deficits in learning and memory. Currently, there is a lack of direct evidence to support the hypothesis of disrupted mTOR signaling in SZ, and we have addressed this by characterizing this signaling pathway in SZ brain. We found a reduction in AKT and mTOR protein expression and/or phosphorylation state in dorsolateral prefrontal cortex (DLPFC) from 22 pairs of SZ and matched comparison subjects. We also found reduced protein expression of GßL, a subunit protein common to both mTOR complexes. We further investigated mTOR complex-specific subunit composition and phosphorylation state, and found abnormal mTOR expression in both complexes in SZ DLPFC. These findings provide evidence that proteins associated with the AKT-mTOR signaling cascade are downregulated in SZ DLPFC.

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.

Identification of novel targets for PGC-1alpha and histone deacetylase inhibitors in neuroblastoma cells.

Recent evidence suggests that the transcriptional coactivator peroxisome proliferator activated receptor gamma coactivator 1alpha (PGC-1alpha) is involved in the pathology of Huntington's Disease (HD). While animals lacking PGC-1alpha express lower levels of genes involved in antioxidant defense and oxidative phosphorylation in the brain, little is known about other targets for PGC-1alpha in neuronal cells and whether there are ways to pharmacologically target PGC-1alpha in neurons. Here, PGC-1alpha overexpression in SH-SY5Y neuroblastoma cells upregulated expression of genes involved in mitochondrial function, glucose transport, fatty acid metabolism, and synaptic function. Overexpression also decreased vulnerability to hydrogen peroxide-induced cell death and caspase 3 activation. Treatment of cells with the histone deacetylase inhibitors (HDACi's) trichostatin A and valproic acid upregulated PGC-1alpha and glucose transporter 4 (GLUT4). These results suggest that PGC-1alpha regulates multiple pathways in neurons and that HDACi's may be good candidates to target PGC-1alpha and GLUT4 in HD and other neurological disorders.

Expression of transcripts for myelin related genes in postmortem brain from cocaine abusers.

Chronic abuse of cocaine is known to cause neuroadaptive changes in the nucleus accumbens (NAc) and ventral tegmental area (VTA). In addition, altered expression of the myelin-related genes MBP, MOBP, PLP1 as well as of MAL2 in NAc was recently reported by gene array analysis in brains from cocaine abusers. In the present study we used in situ hybridization to quantify transcript expression of these four genes, as well as for the myelin-related transcripts encoding quaking, EDG2, claudin-11, transferrin, CNP, and MAG in caudate, putamen, internal capsule, and NAc in postmortem brain from cocaine abusers and matched comparison subjects. Most transcripts were not different between these groups in these striatal regions, and contrary to previous reports, we did not detect any changes in the NAc. However, expression of the transcript encoding PLP1 was significantly decreased in ventral and dorsal regions of the caudate, putamen, and in the internal capsule. Additionally, expression of claudin-11 and transferrin was decreased in the caudate and internal capsule, respectively. PLP1 is expressed at very high levels in oligodendrocytes and is essential in maintaining stability of myelin sheets. Based on these findings, altered expression of PLP1 in most areas of the striatum suggests that widespread changes to the myelin structure could be associated with the adaptive changes following chronic cocaine abuse.

Vesicular glutamate transporter mRNA expression in the medial temporal lobe in major depressive disorder, bipolar disorder, and schizophrenia.

BACKGROUND: Altered glutamate transmission has been found in the medial temporal lobe in severe psychiatric illnesses, including major depressive disorder (MDD) and bipolar disorder (BD). The vesicular glutamate transporters (VGLUTs) have a pivotal role in presynaptic release of glutamate into the synaptic cleft. We investigated this presynaptic marker in major psychiatric illness by measuring transcript expression of the VGLUTs in the medial temporal lobe. METHODS: The study sample comprised four groups of 13 subjects with MDD, BD, or schizophrenia (SCZ), and a comparison group from the Stanley Foundation Neuropathology Consortium. In situ hybridization was performed to quantify messenger RNA (mRNA) expression of VGLUT 1, 2, and 3 in medial temporal lobe structures. We also examined the same areas of rats treated with antidepressants, a mood stabilizer, and antipsychotics to assess the effects of these medications on VGLUT mRNA expression. RESULTS: We found decreased VGLUT1 mRNA expression in both MDD and BD in the entorhinal cortex (ERC), decreased VGLUT2 mRNA expression in MDD in the middle temporal gyrus, and increased VGLUT2 mRNA expression in SCZ in the inferior temporal gyrus (ITG). We also found a negative correlation between age and VGLUT1 mRNA expression in BD in the ERC and ITG. We did not find any changes in VGLUT mRNA expression in the hippocampus in any diagnostic group. We found decreased VGLUT1 mRNA expression in rats treated with haloperidol in the temporal cortex. CONCLUSIONS: These data indicate region-specific alterations of presynaptic glutamate innervation in the medial temporal lobe in the mood disorders.

Expression of four housekeeping proteins in elderly patients with schizophrenia.

We compared protein expression by Western blot analysis in four areas of postmortem brain from patients with schizophrenia and control subjects for several proteins that are often used as controls for Western blot studies: beta-tubulin, actin, glyceraldehyde-3-phosphate dehydrogenase, and valosin-containing protein. We did not detect any differences in expression between subjects with schizophrenia and a comparison group. These results suggest that all four proteins are suitable loading controls for postmortem studies of schizophrenia.

Fractionation of Subcellular Compartments from Human Brain Tissue.

Subcellular fractionation methods permit the isolation, purification, and/or enrichment of specific cellular compartments from complex tissue samples. Enrichment of multiple subcellular compartments from the same tissue sample permits comparisons of the spatial distribution of target proteins between specific intracellular compartments and, in some cases, can provide information about spatiotemporal processing of key cellular components. Here we describe a method to generate subcellular fractions enriched for heavy membranes and nuclei, rough and smooth endoplasmic reticulum membranes, light membranes and cytosol, synapses, and other intermediate cellular membranes from postmortem human brain tissue. These subcellular fractions can be used in a variety of downstream applications to assess the localization, relative abundance, and stoichiometry of glutamate receptor subunits along the forward trafficking pathway.

Abnormal ER quality control of neural GPI-anchored proteins via dysfunction in ER export processing in the frontal cortex of elderly subjects with schizophrenia.

Abnormalities of posttranslational protein modifications (PTMs) have recently been implicated in the pathophysiology of schizophrenia. Glycosylphosphatidylinositols (GPIs) are a class of complex glycolipids, which anchor surface proteins and glycoproteins to the cell membrane. GPI attachment to proteins represents one of the most common PTMs and GPI-associated proteins (GPI-APs) facilitate many cell surface processes, including synapse development and maintenance. Mutations in the GPI processing pathway are associated with intellectual disability, emphasizing the potential role of GPI-APs in cognition and schizophrenia-associated cognitive dysfunction. As initial endoplasmic reticulum (ER)-associated protein processing is essential for GPI-AP function, we measured protein expression of molecules involved in attachment (GPAA1), modification (PGAP1), and ER export (Tmp21) of GPI-APs, in homogenates and in an ER enriched fraction derived from dorsolateral prefrontal cortex (DLPFC) of 15 matched pairs of schizophrenia and comparison subjects. In total homogenate we found a significant decrease in transmembrane protein 21 (Tmp21) and in the ER-enriched fraction we found reduced expression of post-GPI attachment protein (PGAP1). PGAP1 modifies GPI-anchors through inositol deacylation, allowing it to be recognized by Tmp21. Tmp21 is a component of the p24 complex that recognizes GPI-anchored proteins, senses the status of the GPI-anchor, and regulates incorporation into COPII vesicles for export to the Golgi apparatus. Together, these proteins are the molecular mechanisms underlying GPI-AP quality control and ER export. To investigate the potential consequences of a deficit in export and/or quality control, we measured cell membrane-associated expression of known GPI-APs that have been previously implicated in schizophrenia, including GPC1, NCAM, MDGA2, and EPHA1, using Triton X-114 phase separation. Additionally, we tested the sensitivity of those candidate proteins to phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme that cleaves GPI from GPI-APs. While we did not observe a difference in the amount of these GPI-APs in Triton X-114 phase separated membrane fractions, we found decreased NCAM and GPC1 within the PI-PLC sensitive fraction. These findings suggest dysregulation of ER-associated GPI-AP protein processing, with impacts on post-translational modifications of proteins previously implicated in schizophrenia such as NCAM and GPC1. These findings provide evidence for a deficit in ER protein processing pathways in this illness.