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.

Upregulation of cornichon transcripts in the dorsolateral prefrontal cortex in schizophrenia.

Schizophrenia has been proposed to be associated with abnormal glutamatergic neurotransmission. The AMPA subtype of glutamate receptors (AMPARs) mediates fast excitatory synaptic transmission in the brain, and their trafficking and function is regulated in part by AMPAR auxiliary proteins including the cornichons (CNIH) and transmembrane AMPAR-regulatory proteins. Abnormal regulation of AMPARs through altered expression of these auxiliary proteins could induce changes in glutamatergic neurotransmission and thus the pathophysiology of schizophrenia. In this study, transcript expression of cornichon homologs 1-4 was measured in the dorsolateral prefrontal cortex from schizophrenia (N=25) and comparison (N=25) patient groups by comparative quantitative real-time PCR. Significant upregulation of CNIH-1, CNIH-2, and CNIH-3 mRNA expression was found in schizophrenia, with no change in CNIH-4 expression. To determine the effect of antipsychotic treatment on the expression of these genes, cornichon mRNA expression was assayed in the frontal cortex of rats treated chronically with haloperidol decanoate and no changes in any of the cornichon transcripts were found. Abnormal expression of the CNIH family of genes is consistent with cornichon-mediated AMPAR trafficking abnormalities in schizophrenia, and suggests a new mechanism contributing toward the pathophysiology of this illness.

Glutamatergic gene expression is specifically reduced in thalamocortical projecting relay neurons in schizophrenia.

BACKGROUND: Impairment of glutamate neurons that relay sensory and cognitive information from the medial dorsal thalamus to the dorsolateral prefrontal cortex and other cortical regions may contribute to the pathophysiology of schizophrenia. In this study, we have assessed the cell-specific expression of glutamatergic transcripts in the medial dorsal thalamus. METHODS: We used laser capture microdissection to harvest two populations of medial dorsal thalamic cells, one enriched with glutamatergic relay neurons and the other with gamma-aminobutyric acidergic neurons and astroglia, from postmortem brains of subjects with schizophrenia (n = 14) and a comparison group (n = 20). Quantitative polymerase chain reaction of extracted RNA was used to assay gene expression in the different cell populations. RESULTS: The transcripts encoding the ionotropic glutamate receptor subunits NR2D, GluR3, GluR6, GluR7, and the intracellular proteins GRIP1 and SynGAP1 were significantly decreased in relay neurons but not in the mixed glial and interneuron population in schizophrenia. CONCLUSIONS: Our data suggest that reduced ionotropic glutamatergic expression occurs selectively in neurons, which give rise to the cortical projections of the medial dorsal thalamus in schizophrenia, rather than in thalamic cells that function locally. Our findings indicate that glutamatergic innervation is dysfunctional in the circuitry between the medial dorsal thalamus and cortex.

Increased locomotor activity in mice lacking the low-density lipoprotein receptor.

While the low-density lipoprotein receptor (LDLR) is best known for its role in regulating serum cholesterol, LDLR is expressed in brain, suggesting that it may play a role in CNS function as well. Here, using mice with a null mutation in LDLR (LDLR-/-), we investigated whether the absence of LDLR affects a series of behavioral functions. We also utilized the fact that plasma cholesterol levels can be regulated in LDLR-/- mice by manipulating dietary cholesterol to investigate whether elevated plasma cholesterol might independently affect behavioral performance. LDLR-/- mice showed no major deficits in general sensory or motor function. However, LDLR-/- mice exhibited increased locomotor activity in an open field test without evidence of altered anxiety in either an open field or a light/dark emergence test. By contrast, modulating dietary cholesterol produced only isolated effects. While both C57BL/6J and LDLR-/- mice fed a high cholesterol diet showed increased anxiety in a light/dark task, and LDLR-/- mice fed a high cholesterol diet exhibited longer target latencies in the probe trial of the Morris water maze, no other findings supported a general effect of cholesterol on anxiety or spatial memory. Collectively these studies suggest that while LDLR-/- mice exhibit no major developmental defects, LDLR nevertheless plays a significant role in modulating locomotor behavior in the adult.

Ionotropic glutamate receptor mRNA expression in the human thalamus: absence of change in schizophrenia.

Abnormalities in glutamate neurotransmission are thought to be among the major contributing factors to the pathophysiology of schizophrenia. Although schizophrenia has been regarded mostly as a disorder of higher cortical function, the cortex and thalamus work as a functional unit. Existing data regarding alterations of glutamate receptor subunit expression in the thalamus in schizophrenia remain equivocal. This postmortem study examined mRNA expression of ionotropic glutamate receptor (iGluR) subunits and PSD95 in 5 precisely defined and dissected thalamic subdivisions (medial and lateral sectors of the mediodorsal nucleus; and the ventral lateral posterior, ventral posterior, and centromedian nuclei) of persons with schizophrenia and matched controls using quantitative PCR with normalization to multiple endogenous controls. Among 15 genes examined (NR1 and NR2A-D subunits of the NMDA receptor; GluR1-4 subunits of the AMPA receptor; GluR5-7 and KA1-2 subunits of the kainate receptor; PSD95), all but two (GluR4 and KA1) were expressed at quantifiable levels. Differences in iGluR gene expression were seen between different thalamic nuclei but not between diagnostic groups. The relative abundance of transcripts was: NR1>>NR2A>NR2B>NR2D>NR2C for NMDA, GluR2>GluR1>GluR3 for AMPA, and KA2>GluR5>GluR7>GluR6 for kainate receptors. The expression of PSD95 correlated with the expression of NR1, NR2A, NR2B, NR2D and GluR6 in all nuclei. These results provide detailed and quantitative information on iGluR subunit expression in multiple nuclei of the human thalamus but suggest that alterations in their expression are not a prominent feature of schizophrenia.

Up-regulation of NMDA receptor subunit and post-synaptic density protein expression in the thalamus of elderly patients with schizophrenia.

Numerous studies have described structural and functional abnormalities of the thalamus in schizophrenia, but surprisingly few studies have examined neurochemical abnormalities that accompany these pathological changes. We previously identified abnormalities of multiple molecules associated with glutamatergic neurotransmission, including changes in NMDA receptor subunit transcripts and binding sites and NMDA receptor-associated post-synaptic density (PSD) protein transcripts in the thalamus of elderly patients with schizophrenia. In the present study, we performed western blot analysis to determine whether protein levels of NMDA receptor subunits (NR1, NR2A, NR2B) and associated PSD proteins (NF-L, PSD95, SAP102) are altered in schizophrenia. Thalamic tissue from each subject was grossly dissected into two regions: a dorsomedial region containing limbic-associated dorsomedial, anterior and central medial thalamic nuclei; and a ventral thalamus region that primarily consisted of the ventral lateral nucleus. We observed increased protein expression of the NR2B NMDA receptor subunit and its associated intracellular protein, PSD95, in the dorsomedial thalamus of patients with schizophrenia, but the other molecules were unchanged, and we found no changes in the ventral thalamus. These data provide additional evidence of thalamic neurochemical abnormalities, particularly in thalamic nuclei which project to limbic regions of the brain. Further, these findings provide additional evidence of NMDA receptor alterations in schizophrenia, which may play an important role in the neurobiology of the illness.

Expression of excitatory amino acid transporter interacting protein transcripts in the thalamus in schizophrenia.

The excitatory amino acid transporters (EAATs) are a family of plasma membrane proteins that maintain synaptic glutamate concentration by removing glutamate from the synaptic cleft. EAATs are expressed by glia (EAAT1 and EAAT2) and neurons (EAAT3 and EAAT4) throughout the brain. Glutamate reuptake is regulated, in part, by EAAT-interacting proteins that modulate subcellular localization and glutamate transport activity of the EAATs. Several lines of investigation support the hypothesis of glutamatergic abnormalities in schizophrenia. Previous work in our laboratory demonstrated increased expression of EAAT1 and EAAT2 transcripts in the thalamus, suggesting that alterations in synaptic glutamate levels may contribute to the pathophysiology of schizophrenia. Since EAAT-interacting proteins regulate EAAT function, directly impacting glutamatergic neurotransmission, we hypothesized that expression of EAAT-interacting proteins may also be altered in schizophrenia. Using in situ hybridization in subjects with schizophrenia and a comparison group, we detected increased expression of JWA and KIAA0302, molecules that regulate EAAT3 and EAAT4, respectively, in the thalamus in schizophrenia. In contrast, we did not find changes in the expression of transcripts for the EAAT2 and EAAT4 regulatory proteins GPS-1 and ARHGEF11. To address prior antipsychotic treatment in our schizophrenic subjects, we treated rats with haloperidol and clozapine for 4 weeks, and found changes in transcript expression of the EAAT-interacting proteins in clozapine-, but not haloperidol-, treated rats. These findings suggest that proteins associated with the regulation of glutamate reuptake may be abnormal in this illness, supporting the hypothesis of altered thalamic glutamatergic neurotransmission in schizophrenia.

Increased expression of glutaminase and glutamine synthetase mRNA in the thalamus in schizophrenia.

Numerous molecules enable the handling of glutamate that is destined for neurotransmitter release, including transporters, receptors and glutamatergic enzymes. Previous work in our lab has shown altered levels of transcript expression of excitatory amino acid transporters and a vesicular glutamate transporter in the thalamus in schizophrenia. These changes suggest that molecules that facilitate the release and reuptake of glutamate may be abnormal in schizophrenia. In this study we determined the levels of expression of phosphate activated glutaminase (PAG), which converts glutamine to glutamate, and glutamine synthetase (GS), which converts glutamate to glutamine, with the hypothesis that thalamic PAG and GS transcript expression is altered in schizophrenia. We investigated expression of PAG and GS mRNA using in situ hybridization in six different thalamic nuclei (anterior, dorsomedial, centromedial, ventral anterior, ventral and reticular) from 13 persons with schizophrenia and 8 comparison subjects and found that transcripts for PAG and GS were significantly increased in schizophrenia. Increased PAG and GS transcripts suggest enhanced glutamatergic neurotransmission in the thalamus and its efferent targets in schizophrenia.

Altered transcript expression of NMDA receptor-associated postsynaptic proteins in the thalamus of subjects with schizophrenia.

OBJECTIVE: NMDA receptor dysfunction has been implicated in the pathophysiology of schizophrenia. The NMDA receptor is a multimeric ligand-gated ion channel, and the obligate NR(1) subunit is expressed as one of eight isoforms due to the alternative splicing of exons 5, 21, and 22. Alternative splicing of NR(1) subunits modulates receptor function by influencing the association of NR(1) with other NMDA receptor subunits and myriad intracellular molecules, such as the postsynaptic density family of proteins that target NMDA receptors to the synaptic membrane and couple it to numerous signal transduction enzymes. Recently, the authors reported that the NMDA receptor subunits NR(1) and NR(2C) are abnormally expressed in the thalamus in schizophrenia. They hypothesized that this reduction is associated with specific NR(1) isoforms and that NMDA receptor-related postsynaptic density proteins are abnormally expressed. METHOD: Using in situ hybridization, the authors examined expression of the transcripts encoding NR(1) isoforms containing exons 5, 21, or 22, and the NMDA receptor-related postsynaptic density proteins NF-L, PSD93, PSD95, and SAP102. RESULTS: Reduced NR(1) subunit transcript expression was restricted to exon 22-containing isoforms. Increased expression of the NMDA receptor-associated postsynaptic density proteins NF-L, PSD95, and SAP102 was also detected in the thalamus of subjects with schizophrenia. CONCLUSIONS: These data support the hypothesis of glutamatergic abnormalities in schizophrenia and suggest that glutamatergic dysfunction may occur not only at the level of receptor expression but also within intracellular pathways associated with glutamate receptor-associated signal transduction.