Screening for Unhealthy Alcohol Use Among Patients With Multiple Chronic Conditions in Primary Care.

Introduction: Unhealthy alcohol use increases the risk for and exacerbation of chronic health conditions. As such, screening, prevention, and management of unhealthy alcohol use is especially critical to improving health outcomes for patients with multiple chronic health conditions. It is unclear to what extent multiple chronic condition status is a barrier to screening for unhealthy alcohol use in the primary care setting. The authors hypothesized that patients with multiple chronic conditions would be at lower odds of being screened for unhealthy alcohol use than patients without multiple chronic conditions. Methods: The authors performed a secondary analysis of electronic health record data for patients from 67 primary care practices in Virginia (2020-2023). Using the Center for Medicare and Medicaid Services' chronic disease framework, they classified patients by multiple chronic condition status: no multiple chronic conditions, physical multiple chronic conditions, mental health multiple chronic conditions, and physical and mental health multiple chronic conditions. They used multiple logistic regressions with an added practice-level random effect to analyze the relationship between multiple chronic condition status and the odds of receiving an alcohol-related assessment, of being screened for unhealthy alcohol use with a U.S. Preventive Services Task Force-recommended instrument, and of screening positive for unhealthy alcohol use within the past 2 years. Results: Within a final cohort of n=11,789, a total of 6,796 patients (58%) had multiple chronic conditions (29% physical multiple chronic conditions, 4% mental health multiple chronic conditions, and 25% physical and mental health multiple chronic conditions). In all, 69% of patients were screened for unhealthy alcohol use, whereas 16% were screened with a U.S. Preventive Services Task Force-recommended instrument, and 7% screened positive for unhealthy alcohol use. Patients with physical and mental health multiple chronic conditions had 0.9 times lower odds of receiving any screening for unhealthy alcohol use than those with no multiple chronic conditions (95% CI=0.8, 1.0; p=0.0240), whereas patients with only physical multiple chronic conditions or only mental health multiple chronic conditions had similar odds. There was no difference in the odds of being screened with a U.S. Preventive Services Task Force-recommended instrument on the basis of multiple chronic condition status. Patients with mental health multiple chronic conditions and physical and mental health multiple chronic conditions had 1.8 and 1.5 times greater odds of screening positive for unhealthy alcohol use, respectively (95% CI=1.3, 2.7; p=0.0014 and 95% CI=1.2, 1.8; p=0.0003). Conclusions: Although patients with chronic mental health conditions were more likely to screen positive for unhealthy alcohol use than patients without multiple chronic conditions, Virginia primary care patients with physical and mental health multiple chronic conditions were less likely to receive an alcohol-related assessment during the past 2 years. Given the overall modest rate of screening with a U.S. Preventive Services Task Force-recommended instrument, further efforts are needed to create the conditions for high-quality alcohol-related preventive service delivery in primary care, particularly for patients with high complexity and/or mental health conditions.

Quality Gap in Long-Stay Antipsychotic Quality Measure Performance Widens Over the Pandemic, Reversing Past Gains.

The Centers for Medicare & Medicaid Services (CMS) grades nursing home performance in antipsychotic prescribing quarterly, publishing findings as a quality measure. While scores have improved since 2011, marked performance variation between facilities persists. To assess quality gap changes between best- and worst-performing deciles, we compared quarterly prescribing changes between these groups pre-pandemic (April 2011 to March 2020) and during the pandemic (April 2020 to March 2022). Antipsychotic quality measure scores, improving pre-pandemic, deteriorated during the pandemic. The pre-pandemic quality gap between the best- and worst-performing deciles narrowed as the worst-performing decile improved faster than the best-performing decile. During the pandemic, the quality gap widened as the worst-performing decile relapsed more than the best-performing decile (p < .0001). The pandemic disrupted quality performance gains and compounded disparities between facilities. A better understanding of the factors allowing high performers to weather pandemic stressors better than poor performers may reveal opportunities to improve nursing home quality and equity for all residents.

Use of population health data to promote equitable recruitment for a primary care practice implementation trial addressing unhealthy alcohol use.

Background: Recruiting underrepresented people and communities in research is essential for generalizable findings. Ensuring representative participants can be particularly challenging for practice-level dissemination and implementation trials. Novel use of real-world data about practices and the communities they serve could promote more equitable and inclusive recruitment. Methods: We used a comprehensive primary care clinician and practice database, the Virginia All-Payers Claims Database, and the HealthLandscape Virginia mapping tool with community-level socio-ecological information to prospectively inform practice recruitment for a study to help primary care better screen and counsel for unhealthy alcohol use. Throughout recruitment, we measured how similar study practices were to primary care on average, mapped where practices' patients lived, and iteratively adapted our recruitment strategies. Results: In response to practice and community data, we adapted our recruitment strategy three times; first leveraging relationships with residency graduates, then a health system and professional organization approach, followed by a community-targeted approach, and a concluding approach using all three approaches. We enrolled 76 practices whose patients live in 97.3% (1844 of 1907) of Virginia's census tracts. Our overall patient sample had similar demographics to the state for race (21.7% vs 20.0% Black), ethnicity (9.5% vs 10.2% Hispanic), insurance status (6.4% vs 8.0% uninsured), and education (26.0% vs 32.5% high school graduate or less). Each practice recruitment approach uniquely included different communities and patients. Discussion: Data about primary care practices and the communities they serve can prospectively inform research recruitment of practices to yield more representative and inclusive patient cohorts for participation.

The Current State of Alcohol Screening and Management in Virginia Primary Care Practices: An Evaluation of Preventive Service Use.

The US Preventive Services Task Force (USPSTF) recommends screening and behavioral counseling for adults over 18 years for unhealthy alcohol use. Recommended screening instruments include the Alcohol Use Disorders Identification Test-Concise and or Single Alcohol Screening Question. Behavioral counseling is feasible in primary care, taking on average 30 minutes. Baseline data for a practice facilitation trial demonstrated clinicians appropriately screened only 10.8% of patients and only identified 9.6% as having risky drinking. Yet, 24% of patients reported risky drinking on a survey, demonstrating the implementation gap of the USPSTF recommendation and opportunity to improve health.

Assessing the effects of antipsychotic medications on schizophrenia functional analysis: a postmortem proteome study.

Antipsychotic drugs (APDs) are effective in treating positive symptoms of schizophrenia (SCZ). However, they have a substantial impact on postmortem studies. As most cohorts lack samples from drug-naive patients, many studies, rather than understanding SCZ pathophysiology, are analyzing the drug effects. We hypothesized that comparing SCZ-altered and APD-influenced signatures derived from the same cohort can provide better insight into SCZ pathophysiology. For this, we performed LCMS-based proteomics on dorsolateral prefrontal cortex (DLPFC) samples from control and SCZ subjects and used statistical approaches to identify SCZ-altered and APD-influenced proteomes, validated experimentally using independent cohorts and published datasets. Functional analysis of both proteomes was contrasted at the biological-pathway, cell-type, subcellular-synaptic, and drug-target levels. In silico validation revealed that the SCZ-altered proteome was conserved across several studies from the DLPFC and other brain areas. At the pathway level, SCZ influenced changes in homeostasis, signal-transduction, cytoskeleton, and dendrites, whereas APD influenced changes in synaptic-signaling, neurotransmitter-regulation, and immune-system processes. At the cell-type level, the SCZ-altered and APD-influenced proteomes were associated with two distinct striatum-projecting layer-5 pyramidal neurons regulating dopaminergic-secretion. At the subcellular synaptic level, compensatory pre- and postsynaptic events were observed. At the drug-target level, dopaminergic processes influenced the SCZ-altered upregulated-proteome, whereas nondopaminergic and a diverse array of non-neuromodulatory mechanisms influenced the downregulated-proteome. Previous findings were not independent of the APD effect and thus require re-evaluation. We identified a hyperdopaminergic cortex and drugs targeting the cognitive SCZ-symptoms and discussed their influence on SCZ pathology in the context of the cortico-striatal pathway.

Corticostriatal dysfunction and social interaction deficits in mice lacking the cystine/glutamate antiporter.

The astrocytic cystine/glutamate antiporter system xc- represents an important source of extracellular glutamate in the central nervous system, with potential impact on excitatory neurotransmission. Yet, its function and importance in brain physiology remain incompletely understood. Employing slice electrophysiology and mice with a genetic deletion of the specific subunit of system xc-, xCT (xCT-/- mice), we uncovered decreased neurotransmission at corticostriatal synapses. This effect was partly mitigated by replenishing extracellular glutamate levels, indicating a defect linked with decreased extracellular glutamate availability. We observed no changes in the morphology of striatal medium spiny neurons, the density of dendritic spines, or the density or ultrastructure of corticostriatal synapses, indicating that the observed functional defects are not due to morphological or structural abnormalities. By combining electron microscopy with glutamate immunogold labeling, we identified decreased intracellular glutamate density in presynaptic terminals, presynaptic mitochondria, and in dendritic spines of xCT-/- mice. A proteomic and kinomic screen of the striatum of xCT-/- mice revealed decreased expression of presynaptic proteins and abnormal kinase network signaling, that may contribute to the observed changes in postsynaptic responses. Finally, these corticostriatal deregulations resulted in a behavioral phenotype suggestive of autism spectrum disorder in the xCT-/- mice; in tests sensitive to corticostriatal functioning we recorded increased repetitive digging behavior and decreased sociability. To conclude, our findings show that system xc- plays a previously unrecognized role in regulating corticostriatal neurotransmission and influences social preference and repetitive behavior.

Consequences of NMDA receptor deficiency can be rescued in the adult brain.

N-methyl-D-aspartate receptors (NMDARs) are required to shape activity-dependent connections in the developing and adult brain. Impaired NMDAR signalling through genetic or environmental insults causes a constellation of neurodevelopmental disorders that manifest as intellectual disability, epilepsy, autism, or schizophrenia. It is not clear whether the developmental impacts of NMDAR dysfunction can be overcome by interventions in adulthood. This question is paramount for neurodevelopmental disorders arising from mutations that occur in the GRIN genes, which encode NMDAR subunits, and the broader set of mutations that disrupt NMDAR function. We developed a mouse model where a congenital loss-of-function allele of Grin1 can be restored to wild type by gene editing with Cre recombinase. Rescue of NMDARs in adult mice yields surprisingly robust improvements in cognitive functions, including those that are refractory to treatment with current medications. These results suggest that neurodevelopmental disorders arising from NMDAR deficiency can be effectively treated in adults.

Combining Neurobehavioral Analysis and In Vivo Photoaffinity Labeling to Understand Protein Targets of Methamphetamine in Casper Zebrafish.

Photoaffinity labeling (PAL) remains one of the most widely utilized methods of determining protein targets of drugs. Although useful, the scope of this technique has been limited to in vitro applications because of the inability of UV light to penetrate whole organisms. Herein, pigment-free Casper zebrafish were employed to allow in vivo PAL. A methamphetamine-related phenethylamine PAL probe, designated here as 2, demonstrated dose-dependent effects on behavior similar to methamphetamine and permitted concentration-dependent labeling of protein binding partners. Click chemistry was used to analyze binding partners via fluoroimaging. Conjugation to a biotin permitted streptavidin pull-down and proteomic analysis to define direct binding partners of the methamphetamine probe. Bioinformatic analysis revealed the probe was chiefly bound to proteins involved in phagocytosis and mitochondrial function. Future applications of this experimental paradigm combining examination of drug-protein binding interactions alongside neurobehavioral readouts via in vivo PAL will significantly enhance our understanding of drug targets, mechanism(s) of action, and toxicity/lethality.

Adenosine Kinase Expression in the Frontal Cortex in Schizophrenia.

The adenosine hypothesis of schizophrenia posits that reduced availability of the neuromodulator adenosine contributes to dysregulation of dopamine and glutamate transmission and the symptoms associated with schizophrenia. It has been proposed that increased expression of the enzyme adenosine kinase (ADK) may drive hypofunction of the adenosine system. While animal models of ADK overexpression support such a role for altered ADK, the expression of ADK in schizophrenia has yet to be examined. In this study, we assayed ADK gene and protein expression in frontocortical tissue from schizophrenia subjects. In the dorsolateral prefrontal cortex (DLPFC), ADK-long and -short splice variant expression was not significantly altered in schizophrenia compared to controls. There was also no significant difference in ADK splice variant expression in the frontal cortex of rats treated chronically with haloperidol-decanoate, in a study to identify the effect of antipsychotics on ADK gene expression. ADK protein expression was not significantly altered in the DLPFC or anterior cingulate cortex (ACC). There was no significant effect of antipsychotic medication on ADK protein expression in the DLPFC or ACC. Overall, our results suggest that increased ADK expression does not contribute to hypofunction of the adenosine system in schizophrenia and that alternative mechanisms are involved in dysregulation of this system in schizophrenia.

Measurement of lactate levels in postmortem brain, iPSCs, and animal models of schizophrenia.

Converging evidence suggests bioenergetic defects contribute to the pathophysiology of schizophrenia and may underlie cognitive dysfunction. The transport and metabolism of lactate energetically couples astrocytes and neurons and supports brain bioenergetics. We examined the concentration of lactate in postmortem brain (dorsolateral prefrontal cortex) in subjects with schizophrenia, in two animal models of schizophrenia, the GluN1 knockdown mouse model and mutant disrupted in schizophrenia 1 (DISC1) mouse model, as well as inducible pluripotent stem cells (iPSCs) from a schizophrenia subject with the DISC1 mutation. We found increased lactate in the dorsolateral prefrontal cortex (p = 0.043, n = 16/group) in schizophrenia, as well as in frontal cortical neurons differentiated from a subject with schizophrenia with the DISC1 mutation (p = 0.032). We also found a decrease in lactate in mice with induced expression of mutant human DISC1 specifically in astrocytes (p = 0.049). These results build upon the body of evidence supporting bioenergetic dysfunction in schizophrenia, and suggests changes in lactate are a key feature of this often devastating severe mental illness.

Connectivity Analyses of Bioenergetic Changes in Schizophrenia: Identification of Novel Treatments.

We utilized a cell-level approach to examine glycolytic pathways in the DLPFC of subjects with schizophrenia (n = 16) and control (n = 16) and found decreased mRNA expression of glycolytic enzymes in pyramidal neurons, but not astrocytes. To replicate these novel bioenergetic findings, we probed independent datasets for bioenergetic targets and found similar abnormalities. Next, we used a novel strategy to build a schizophrenia bioenergetic profile by a tailored application of the Library of Integrated Network-Based Cellular Signatures data portal (iLINCS) and investigated connected cellular pathways, kinases, and transcription factors using Enrichr. Finally, with the goal of identifying drugs capable of "reversing" the bioenergetic schizophrenia signature, we performed a connectivity analysis with iLINCS and identified peroxisome proliferator-activated receptor (PPAR) agonists as promising therapeutic targets. We administered a PPAR agonist to the GluN1 knockdown model of schizophrenia and found it improved long-term memory. Taken together, our findings suggest that tailored bioinformatics approaches, coupled with the LINCS library of transcriptional signatures of chemical and genetic perturbagens, may be employed to identify novel treatment strategies for schizophrenia and related diseases.

Abnormalities of signal transduction networks in chronic schizophrenia.

Schizophrenia is a serious neuropsychiatric disorder characterized by disruptions of brain cell metabolism, microstructure, and neurotransmission. All of these processes require coordination of multiple kinase-mediated signaling events. We hypothesize that imbalances in kinase activity propagate through an interconnected network of intracellular signaling with potential to simultaneously contribute to many or all of the observed deficits in schizophrenia. We established a workflow distinguishing schizophrenia-altered kinases in anterior cingulate cortex using a previously published kinome array data set. We compared schizophrenia-altered kinases to haloperidol-altered kinases, and identified systems, functions, and regulators predicted using pathway analyses. We used kinase inhibitors with the kinome array to test hypotheses about imbalance in signaling and conducted preliminary studies of kinase proteins, phosphoproteins, and activity for kinases of interest. We investigated schizophrenia-associated single nucleotide polymorphisms in one of these kinases, AKT, for genotype-dependent changes in AKT protein or activity. Kinome analyses identified new kinases as well as some previously implicated in schizophrenia. These results were not explained by chronic antipsychotic treatment. Kinases identified in our analyses aligned with cytoskeletal arrangement and molecular trafficking. Of the kinases we investigated further, AKT and (unexpectedly) JNK, showed the most dysregulation in the anterior cingulate cortex of schizophrenia subjects. Changes in kinase activity did not correspond to protein or phosphoprotein levels. We also show that AKT single nucleotide polymorphism rs1130214, previously associated with schizophrenia, influenced enzyme activity but not protein or phosphoprotein levels. Our data indicate subtle changes in kinase activity and regulation across an interlinked kinase network, suggesting signaling imbalances underlie the core symptoms of schizophrenia. DISEASE MECHANISMS: A SIGNALING IMBALANCE: A study by US scientists indicates that changes in the activity of key signaling proteins may underlie core symptoms of schizophrenia. Protein kinases mediate the activation of intracellular signaling events and analyses of the kinome, the complete set of protein kinases encoded in the genome, previously revealed significant changes in phosphorylation patterns in postmortem brain tissue from patients with schizophrenia. Based on these findings, Jennifer McGuire at the University of Cincinnati and colleagues investigated the upstream regulation of these proteins. They identified both established and novel proteins associated with schizophrenia in the anterior cingulate cortex, with JNK and AKT activity being the most disrupted in schizophrenia patients. Their findings highlight how subtle changes in the activity of a small number of signaling proteins can propagate and have major consequences for mental health.

Postsynaptic Density-95 Isoform Abnormalities in Schizophrenia.

Background: Postsynaptic density-95 (PSD-95) protein expression is dysregulated in schizophrenia in a variety of brain regions. We have designed experiments to examine PSD-95 mRNA splice variant expression in the dorsolateral prefrontal cortex from subjects with schizophrenia. Methods: We performed quantitative PCR and western blot analysis to measure PSD-95 expression in schizophrenia vs control subjects, rodent haloperidol treatment studies, rodent postmortem interval studies, and GluN1 knockdown (KD) mice vs controls. Results: We found decreased mRNA expression of beta (t = 4.506, df = 383, P < .0001) and truncated (t = 3.378, df = 383, P = .0008) isoforms of PSD-95, whereas alpha was unchanged. Additionally, we found decreased PSD-95 protein expression in schizophrenia (t = 2.746, df = 71, P = .0076). We found no correlation between PSD-95 protein and alpha, beta, or truncated mRNA isoforms in schizophrenia. PSD-95 beta transcript was increased (t = 3.346, df = 14, P < .05) in the GluN1 KD mouse model of schizophrenia. There was an increase in PSD-95 alpha mRNA expression (t = 2.905, df = 16, P < .05) in rats following long-term haloperidol administration. Conclusions: Our findings describe a unique pathophysiology of specific PSD-95 isoform dysregulation in schizophrenia, chronic neuroleptic treatment, and a genetic lesion mouse model of drastically reduced N-methyl-d-aspartate receptor (NMDAR) complex expression. These data indicate that regulation of PSD-95 is multifaceted, may be isoform specific, and biologically relevant for synaptic signaling function. Specifically, NMDAR-mediated synaptic remodeling, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor trafficking and interaction may be impaired in schizophrenia by decreased PSD-95 beta and truncated expression (respectively). Further, increased PSD-95 beta transcript in the GluN1 KD mouse model poses a potential compensatory rescue of NMDAR-mediated function via increased postsynaptic throughput of the severely reduced GluN1 signal. Together, these data propose that disruption of excitatory signaling complexes through genetic (GluN1 KD), pharmacologic (antipsychotics), or disease (schizophrenia) mechanisms specifically dysregulates PSD-95 expression.

Systematic evaluation of data-independent acquisition for sensitive and reproducible proteomics-a prototype design for a single injection assay.

Data-independent acquisition (DIA)-based proteomics has become increasingly complicated in recent years because of the vast number of workflows described, coupled with a lack of studies indicating a rational framework for selecting effective settings to use. To address this issue and provide a resource for the proteomics community, we compared 12 DIA methods that assay tryptic peptides using various mass-isolation windows. Our findings indicate that the most sensitive single injection LC-DIA method uses 6 m/z isolation windows to analyze the densely populated tryptic peptide range from 450 to 730 m/z, which allowed quantification of 4465 Escherichia coli peptides. In contrast, using the sequential windowed acquisition of all theoretical fragment-ions (SWATH) approach with 26 m/z isolation windows across the entire 400-1200 m/z range, allowed quantification of only 3309 peptides. This reduced sensitivity with 26 m/z windows is caused by an increase in co-eluting compounds with similar precursor values detected in the same tandem MS spectra, which lowers the signal-to-noise of peptide fragment-ion chromatograms and reduces the amount of low abundance peptides that can be quantified from 410 to 920 m/z. Above 920 m/z, more peptides were quantified with 26 m/z windows because of substantial peptide (13) C isotope distributions that parse peptide ions into separate isolation windows. Because reproducible quantification has been a long-standing aim of quantitative proteomics, and is a so-called trait of DIA, we sought to determine whether precursor-level chromatograms used in some methods rather than their fragment-level counterparts have similar precision. Our data show that extracted fragment-ion chromatograms are the reason DIA provides superior reproducibility. Copyright © 2015 John Wiley & Sons, Ltd.

Decreased chloride channel expression in the dorsolateral prefrontal cortex in schizophrenia.

Alterations in GABAergic neurotransmission are implicated in several psychiatric illnesses, including schizophrenia. The Na-K-Cl and K-Cl cotransporters regulate intracellular chloride levels. Abnormalities in cotransporter expression levels could shift the chloride electrochemical gradient and impair GABAergic transmission. In this study, we performed Western blot analysis to investigate whether the Na-K-Cl and K-Cl cotransporter protein is abnormally expressed in the dorsal lateral prefrontal cortex and the anterior cingulate cortex in patients with schizophrenia versus a control group. We found decreased K-Cl cotransporter protein expression in the dorsal lateral prefrontal cortex, but not the anterior cingulate cortex, in subjects with schizophrenia, supporting the hypothesis of region level abnormal GABAergic function in the pathophysiology of schizophrenia. Subjects with schizophrenia off antipsychotic medication at the time of death had decreased K-Cl cotransporter protein expression compared to both normal controls and subjects with schizophrenia on antipsychotics. Our results provide evidence for KCC2 protein abnormalities in schizophrenia and suggest that antipsychotic medications might reverse deficits of this protein in the illness.

Increased G protein-coupled receptor kinase (GRK) expression in the anterior cingulate cortex in schizophrenia.

BACKGROUND: Current pharmacological treatments for schizophrenia target G protein-coupled receptors (GPCRs), including dopamine receptors. Ligand-bound GPCRs are regulated by a family of G protein-coupled receptor kinases (GRKs), members of which uncouple the receptor from heterotrimeric G proteins, desensitize the receptor, and induce receptor internalization via the arrestin family of scaffolding and signaling molecules. GRKs initiate the activation of downstream signaling pathways, can regulate receptors and signaling molecules independent of GPCR phosphorylation, and modulate epigenetic regulators like histone deacetylases (HDACs). We hypothesize that the expression of GRK proteins is altered in schizophrenia, consistent with previous findings of alterations upstream and downstream from this family of molecules that facilitate intracellular signaling processes. METHODS: In this study, we measured protein expression via Western blot analysis for GRKs 2, 3, 5, and 6 in the anterior cingulate cortex of patients with schizophrenia (n=36) and a comparison group (n=33). To control for antipsychotic treatment, we measured these same targets in haloperidol-treated vs. untreated rats (n=10 for both). RESULTS: We found increased levels of GRK5 in schizophrenia. No changes were detected in GRK protein expression in rats treated with haloperidol decanoate for 9 months. CONCLUSION: These data suggest that increased GRK5 expression may contribute to the pathophysiology of schizophrenia via abnormal regulation of the cytoskeleton, endocytosis, signaling, GPCRs, and histone modification.

Recent advances in targeting the ionotropic glutamate receptors in treating schizophrenia.

The treatment of schizophrenia has been focused on modulation of dopamine receptors for over 50 years. Recent developments have implicated other neurotransmitter systems in the pathophysiology of this illness. The discovery and characterization of glutamate receptors and their roles in the brain has lead to novel approaches for the treatment of schizophrenia. In this article, we review drugs that modulate ionotropic gluamate receptors and discuss their efficacy for the treatment of this often debilitating severe mental illness.

Abnormal activity of the MAPK- and cAMP-associated signaling pathways in frontal cortical areas in postmortem brain in schizophrenia.

Recent evidence suggests that schizophrenia may result from alterations of integration of signaling mediated by multiple neurotransmitter systems. Abnormalities of associated intracellular signaling pathways may contribute to the pathophysiology of schizophrenia. Proteins and phospho-proteins comprising mitogen activated protein kinase (MAPK) and 3'-5'-cyclic adenosine monophosphate (cAMP)-associated signaling pathways may be abnormally expressed in the anterior cingulate (ACC) and dorsolateral prefrontal cortex (DLPFC) in schizophrenia. Using western blot analysis we examined proteins of the MAPK- and cAMP-associated pathways in these two brain regions. Postmortem samples were used from a well-characterized collection of elderly patients with schizophrenia (ACC=36, DLPFC=35) and a comparison (ACC=33, DLPFC=31) group. Near-infrared intensity of IR-dye labeled secondary antisera bound to targeted proteins of the MAPK- and cAMP-associated signaling pathways was measured using LiCor Odyssey imaging system. We found decreased expression of Rap2, JNK1, JNK2, PSD-95, and decreased phosphorylation of JNK1/2 at T183/Y185 and PSD-95 at S295 in the ACC in schizophrenia. In the DLPFC, we found increased expression of Rack1, Fyn, Cdk5, and increased phosphorylation of PSD-95 at S295 and NR2B at Y1336. MAPK- and cAMP-associated molecules constitute ubiquitous intracellular signaling pathways that integrate extracellular stimuli, modify receptor expression and function, and regulate cell survival and neuroplasticity. These data suggest abnormal activity of the MAPK- and cAMP-associated pathways in frontal cortical areas in schizophrenia. These alterations may underlie the hypothesized hypoglutamatergic function in this illness. Together with previous findings, these data suggest that abnormalities of intracellular signaling pathways may contribute to the pathophysiology of schizophrenia.

Evidence for abnormal forward trafficking of AMPA receptors in frontal cortex of elderly patients with schizophrenia.

Several lines of evidence point to alterations of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor trafficking in schizophrenia. Multiple proteins, including synapse-associated protein 97 (SAP97), glutamate receptor-interacting protein 1 (GRIP1), and N-ethylmaleimide sensitive factor (NSF), facilitate the forward trafficking of AMPA receptors toward the synapse. Once localized to the synapse, AMPA receptors are trafficked in a complex endosomal system. We hypothesized that alterations in the expression of these proteins and alterations in the subcellular localization of AMPA receptors in endosomes may contribute to the pathophysiology of schizophrenia. Accordingly, we measured protein expression of SAP97, GRIP1, and NSF in the dorsolateral prefrontal cortex and found an increase in the expression of SAP97 and GRIP1 in schizophrenia. To determine the subcellular localization of AMPA receptor subunits, we developed a technique to isolate early endosomes from post-mortem tissue. We found increased GluR1 receptor subunit protein in early endosomes in subjects with schizophrenia. Together, these data suggest that there is an alteration of forward trafficking of AMPA receptors as well as changes in the subcellular localization of an AMPA receptor subunit in schizophrenia.

Decreased expression of NMDA receptor-associated proteins in frontal cortex of elderly patients with schizophrenia.

Converging evidence suggests too few activation-ready N-methyl-D-aspartic acid (NMDA) receptor complexes in the postsynaptic density in schizophrenia. Postsynaptic density protein 95 (PSD95), Synaptic GTPase-activating protein (SynGAP), and Multiple PDZ domain protein (MUPP1) are integral components of the NMDA receptor signaling complex, and help facilitate signaling, trafficking, and stabilization. We hypothesized that deficits involving these molecules may contribute to the pathophysiology of schizophrenia. To test our hypothesis, we measured protein expression of PSD95, SynGAP, and MUPP1 in the anterior cingulate cortex and dorsolateral prefrontal cortex. We found decreased PSD95 expression in the anterior cingulate cortex. Antipsychotic medication analyses showed decreased SynGAP expression in the anterior cingulate cortex in patients off medication when analyzed against our comparison group. These data suggest that NMDA receptor complex formation, localization, and downstream signaling may be abnormal in schizophrenia.