Craig A. Stockmeier

Understanding the pathophysiological substrates of schizophrenia is a major challenge for current neuropsychiatric research. As part of a set of multi-omics experiments, we performed an extensive case-control proteomics study on 192 post-mortem tissue sections from prefrontal cortex from 96 individuals, including 47 cases with schizophrenia and 49 healthy controls. Using two independently measured cortical datasets, we identified 387 proteins differentially expressed between schizophrenia cases and controls at a 5% FDR threshold. This significantly regulated set of proteins contains genes located in GWAS-identified schizophrenia loci and proteins identified by pQTL analysis. Gene ontology analysis using GOAT provided evidence for regulation of several major protein categories, emphasizing downregulation of mitochondrial oxidative respiration, ribosomes and the proteasome, upregulation of kinases and (small) GTPases. SynGO analysis supports the notion of synaptic dysfunction in schizophrenia, with major regulators of pre- and postsynaptic function compromised. Our findings highlight the complex molecular dysregulation in schizophrenia, with mitochondrial function downregulated versus signaling and trafficking upregulated, and synapse function disrupted; in combination with prior avenues of research, these finding support a role for energy deficits compromising highly ATP dependent neuronal function as a target for therapeutic interventions.

Contextual triggers are significant factors contributing to relapse in substance use disorders (SUD). Emerging evidence points to a critical role of extracellular matrix (ECM) molecules as mediators of reward memories. Chondroitin sulfate proteoglycans (CSPGs) are a subset of ECM molecules that form perineuronal nets (PNN) around inhibitory neurons. PNNs restrict synaptic connections and help maintain synapses. Rodent models suggest that modulation of PNNs may strengthen contextual reward memories in SUD. However, there is currently a lack of information regarding PNNs in the hippocampus of people with SUD as well as how comorbidity with major depressive disorder (MDD) may affect PNNs. We used postmortem hippocampal tissues from cohorts of human and nonhuman primates with or without chronic alcohol use to test the hypothesis that PNNs are increased in subjects with SUD. We used histochemical labeling and quantitative microscopy to examine PNNs, and qRT-PCR to examine gene expression for ECM molecules, synaptic markers and related markers. We identified increased densities of PNNs and CSPG-labeled glial cells in SUD, coinciding with decreased expression of the ECM protease matrix metalloproteinase 9 (Mmp9), and increased expression for the excitatory synaptic marker vesicle associated membrane protein 2 (Vamp2). Similar increases in PNNs were observed in monkeys with chronic alcohol self-administration. Subjects with MDD displayed changes opposite to SUD, and subjects with SUD and comorbid MDD had minimal changes in any of the outcome measures examined. Our findings demonstrate that PNNs are increased in SUD, possibly contributing to stabilizing contextual reward memories as suggested by preclinical studies. Our results also point to a previously unsuspected role for CSPG expression in glial cells in SUD. Evidence for increased hippocampal PNNs in SUD suggests that targeting PNNs to weaken contextual reward memories is a promising therapeutic approach for SUD, however comorbidity with MDD is a significant consideration.

Schizophrenia is a genetically complex neuropsychiatric disorder with largely unresolved mechanisms of pathology. Identification of genes and pathways associated with schizophrenia is important for understanding the development, progression and treatment of schizophrenia. In this study, pathways associated with schizophrenia were explored at the level of gene expression. The study included post-mortem brain tissue samples from 68 schizophrenia patients and 44 age and sex-matched control subjects. Whole transcriptome poly-A selected paired-end RNA sequencing was performed on tissue from the prefrontal cortex and orbitofrontal cortex. RNA expression differences were detected between case and control individuals, focusing both on single genes and pathways. The results were validated with RT-qPCR. Significant differential expression between patient and controls groups was found for 71 genes. Gene ontology analysis of differentially expressed genes revealed an up-regulation of multiple genes in immune response among the patients (corrected p-value = 0.004). Several genes in the category belong to the complement system, including C1R, C1S, C7, FCN3, SERPING1, C4A and CFI. The increased complement expression is primarily driven by a subgroup of patients with increased expression of immune/inflammatory response genes, pointing to important differences in disease etiology within the patient group. Weighted gene co-expression network analysis highlighted networks associated with both synaptic transmission and activation of the immune response. Our results demonstrate the importance of immune-related pathways in schizophrenia and provide evidence for elevated expression of the complement cascade as an important pathway in schizophrenia pathology.

In recent years, studies have shown higher prevalence of autoantibodies in patients with schizophrenia compared to healthy individuals. This study applies an untargeted and a targeted affinity proteomics approach to explore and characterize the autoantibody repertoire in brain tissues from 73 subjects diagnosed with schizophrenia and 52 control subjects with no psychiatric or neurological disorders. Selected brain tissue lysates were first explored for IgG reactivity on planar microarrays composed of 11,520 protein fragments representing 10,820 unique proteins. Based on these results of ours and other previous studies of autoantibodies related to psychosis, we selected 226 fragments with an average length of 80 amino acids, representing 127 unique proteins. Tissue-based analysis of IgG reactivities using antigen suspension bead arrays was performed in a multiplex and parallel fashion for all 125 subjects. Among the detected autoantigens, higher IgG reactivity in subjects with schizophrenia, as compared to psychiatrically healthy subjects, was found against the glutamate ionotropic receptor NMDA type subunit 2D (anti-GluN2D). In a separate cohort with serum samples from 395 young adults with a wider spectrum of psychiatric disorders, higher levels of serum autoantibodies targeting GluN2D were found when compared to 102 control individuals. By further validating GluN2D and additional potential autoantigens, we will seek insights into how these are associated with severe mental illnesses.

Emerging evidence suggests that there is a reduction in overall cortical excitatory to inhibitory balance in major depressive disorder (MDD), which afflicts ∼14%-20% of individuals. Reduced pyramidal cell arborization occurs with stress and MDD, and may diminish excitatory neurotransmission. Enhanced deposition of perineuronal net (PNN) components also occurs with stress. Since parvalbumin-expressing interneurons are the predominant cell population that is enveloped by PNNs, which enhance their ability to release GABA, excess PNN deposition likely increases pyramidal cell inhibition. In the present study, we investigate the potential for matrix metalloprotease-9 (MMP-9), an endopeptidase secreted in response to neuronal activity, to contribute to the antidepressant efficacy of the serotonin/norepinephrine reuptake inhibitor venlafaxine in male mice. Chronic venlafaxine increases MMP-9 levels in murine cortex, and increases both pyramidal cell arborization and PSD-95 expression in the cortex of WT but not MMP-9-null mice. We have previously shown that venlafaxine reduces PNN deposition and increases the power ofex vivoγ oscillations in conventionally housed mice. γ power is increased with pyramidal cell disinhibition and with remission from MDD. Herein we observe that PNN expression is increased in a corticosterone-induced stress model of disease and reduced by venlafaxine. Compared with mice that receive concurrent venlafaxine, corticosterone-treated mice also display reducedex vivoγ power and impaired working memory. Autopsy-derived PFC samples show elevated MMP-9 levels in antidepressant-treated MDD patients compared with controls. These preclinical and postmortem findings highlight a link between extracellular matrix regulation and MDD.SIGNIFICANCE STATEMENTReduced excitatory neurotransmission occurs with major depressive disorder, and may be normalized by antidepressant treatment. Underlying molecular mechanisms are, however, not well understood. Herein we investigate a potential role for an extracellular protease, released from neurons and known to play a role in learning and memory, in antidepressant-associated increases in excitatory transmission. Our data suggest that this protease, matrix metalloprotease-9, increases branching of excitatory neurons and concomitantly attenuates the perineuronal net to potentially reduce inhibitory input to these neurons. Matrix metalloprotease-9 may thus enhance overall excitatory/inhibitory balance and neuronal population dynamics, which are important to mood and memory.

Suicide is a major public health concern; nevertheless, its neurobiology remains unknown. An area of interest in suicide research is the dorsolateral prefrontal cortex (DLPFC). We aimed to identify altered proteins and potential biological pathways in the DLPFC of individuals who died by suicide employing mass spectrometry-based untargeted proteomics. Postmortem DLPFC from age-matched male suicide mood disorder cases (n = 5) and non-suicide mood disorder cases (n = 5) were compared. The proteins that differed between groups at false discovery rate (FDR) adjusted p-values (Benjamini–Hochberg–Yekutieli) |0.4| were considered statistically significant and were subjected to pathway analysis by Qiagen Ingenuity software. Thirty-three of the 5162 detected proteins showed significantly altered expression levels in the suicide cases and two of them after adjustment for body mass index. The top differentially expressed protein was potassium voltage-gated channel subfamily Q member 3 (KCNQ3) (Log₂FC = −0.481, p = 2.10 × 10⁻⁰⁹, FDR = 5.93 × 10⁻⁰⁶), which also showed a trend to downregulation in Western blot (p = 0.045, Bonferroni adjusted p = 0.090). The most notably enriched pathway was the GABA receptor signaling pathway (p < 0.001). Here, we report a reduction trend of KCNQ3 levels in the DLPFC of male suicide victims with mood disorders. Further studies with a larger sample size and equal sex representation are needed.

Emerging evidence suggests that there is a reduction in overall cortical excitatory to inhibitory balance in major depressive disorder (MDD), which afflicts approximately 14-20% of individuals. Reduced pyramidal cell arborization occurs with stress and MDD, and may diminish excitatory neurotransmission. Enhanced deposition of perineuronal net (PNN) components also occurs with stress. Since parvalbumin-expressing interneurons are the predominant cell population that is enveloped by PNNs, which enhance their ability to release GABA, excess PNN deposition likely increases pyramidal cell inhibition. In the present study we investigate the potential for matrix metalloprotease-9 (MMP-9), an endopeptidase secreted in response to neuronal activity, to contribute to the antidepressant efficacy of venlafaxine, a serotonin/norepinephrine reuptake inhibitor. Chronic venlafaxine increases MMP-9 levels in murine cortex, and increases both pyramidal cell arborization and PSD-95 expression in the cortex of wild-type but not MMP-9 null mice. We have previously shown that venlafaxine reduces PNN deposition and increases the power of ex vivo gamma oscillations in conventionally-housed mice. Gamma power is increased with pyramidal cell disinhibition and with remission from MDD. Herein we observe that PNN expression is increased in a corticosterone-induced stress model of disease and reduced by venlafaxine. As compared to mice that receive concurrent venlafaxine, corticosterone treated mice also display reduced ex vivo gamma power and impaired working memory. Autopsy-derived prefrontal cortex samples show elevated MMP-9 levels in anti-depressant treated MDD patients as compared to controls. These preclinical and postmortem findings highlight a link between extracellular matrix regulation and MDD.