Beata Karolewicz

Reduced levels of NR2A and NR2B subunits of NMDA receptor and PSD-95 in the prefrontal cortex in major depression

Recent neuroimaging and postmortem studies have demonstrated abnormalities in glutamatergic transmission in major depression. Glutamate NMDA (N-methyl-d-aspartate) receptors are one of the major mediators of excitatory neurotransmission in the central nervous system. At synaptic sites, NMDA receptors are linked with postsynaptic density protein-95 (PSD-95) that plays a key role in mediating trafficking, clustering, and downstream signaling events, following receptor activation. In this study, we examined the expression of NMDA receptor subunits NR1, NR2A, and NR2B as well as PSD-95 in the anterior prefrontal cortex (PFC) using Western blot method. Cortical samples were obtained from age, gender and postmortem interval matched depressed and psychiatrically healthy controls. The results revealed that there was a reduced expression of the NMDA receptor subunits NR2A (-54%) and NR2B (-48%), and PSD-95 protein level (-40%) in the PFC of depressed subjects relative to controls, with no change in the NR1 subunit. The alterations in NMDA receptor subunits, especially the NR2A and NR2B, as well as PSD-95 suggest an abnormality in the NMDA receptor signaling in the PFC in major depression. Our findings in conjunction with recent clinical, cellular, and neuroimaging studies further implicate the involvement of glutamate neurotransmission in the pathophysiology of depression. This study provides additional evidence that NMDA receptor complex is a target for discovery of novel antidepressants.

The mTOR signaling pathway in the prefrontal cortex is compromised in major depressive disorder

Recent studies demonstrate that rapid antidepressant response to ketamine is mediated by activation of the mammalian target of rapamycin (mTOR) signaling pathway, leading to increased synaptic proteins in the prefrontal cortex (PFC) of rats. Our postmortem studies indicate robust deficits in prominent postsynaptic proteins including N-methyl-d-aspartate (NMDA) receptor subunits (NR2A, NR2B), metabotropic glutamate receptor subtype 5 (mGluR5) and postsynaptic density protein 95kDa (PSD-95) in the PFC in major depressive disorder (MDD). We hypothesize that deficits in the mTOR-dependent translation initiation pathway contribute to the molecular pathology seen in the PFC of MDD subjects, and that a rapid reversal of these abnormalities may underlie antidepressant activity. The majority of known translational regulation occurs at the level of initiation. mTOR regulates translation initiation via its downstream components: p70-kDa ribosomal protein S6 kinase (p70S6K), and eukaryotic initiation factors 4E and 4B (eIF4E and eIF4B). In this study, we examined the expression of mTOR and its core downstream signaling targets: p70S6K, eIF4E, and eIF4B in the PFC of 12 depressed subjects and 12 psychiatrically healthy controls using Western blot. Levels of eIF4E phosphorylated at serine 209 (p-eIF4E-Ser209) and eIF4B phosphorylated at serine 504 (p-eIF4B-Ser504) were also examined. Adjacent cortical tissue samples from both cohorts of subjects were used in our previous postmortem analyses. There was a significant reduction in mTOR, p70S6K, eIF4B and p-eIF4B protein expression in MDD subjects relative to controls. No group differences were observed in eIF4E, p-eIF4E or actin levels. Our findings show deficits in mTOR-dependent translation initiation in MDD particularly via the p70S6K/eIF4B pathway, and indicate a potential association between marked deficits in synaptic proteins and dysregulation of mTOR signaling in MDD.

Reduced Metabotropic Glutamate Receptor 5 Density in Major Depression Determined by [11C]ABP688 PET and Postmortem Study

OBJECTIVE Clinical and preclinical evidence suggests a hyperactive glutamatergic system in clinical depression. Recently, the metabotropic glutamate receptor 5 (mGluR5) has been proposed as an attractive target for novel therapeutic approaches to depression. The goal of this study was to compare mGluR5 binding (in a positron emission tomography [PET] study) and mGluR5 protein expression (in a postmortem study) between individuals with major depressive disorder and psychiatrically healthy comparison subjects. METHOD Images of mGluR5 receptor binding were acquired using PET with [(11)C]ABP688, which binds to an allosteric site with high specificity, in 11 unmedicated individuals with major depression and 11 matched healthy comparison subjects. The amount of mGluR5 protein was investigated using Western blot in postmortem brain samples of 15 depressed individuals and 15 matched comparison subjects. RESULTS The PET study revealed lower levels of regional mGluR5 binding in the prefrontal cortex, the cingulate cortex, the insula, the thalamus, and the hippocampus in the depression group relative to the comparison group. Severity of depression was negatively correlated with mGluR5 binding in the hippocampus. The postmortem study showed lower levels of mGluR5 protein expression in the prefrontal cortex (Brodmanns area 10) in the depression group relative to the comparison group, while prefrontal mGluR1 protein expression did not differ between groups. CONCLUSIONS The lower levels of mGluR5 binding observed in the depression group are consonant with the lower levels of protein expression in brain tissue in the postmortem depression group. Thus, both studies suggest that basal or compensatory changes in excitatory neurotransmission play roles in the pathophysiology of major depression.

Reduced Level of Glutamic Acid Decarboxylase-67 kDa in the Prefrontal Cortex in Major Depression

Accumulating evidence suggests dysfunction of the gamma-aminobutyric acid (GABA) system in major depressive disorder (MDD). Neuroimaging studies consistently report reductions of cortical GABA in depressed patients. Our post-mortem analyses demonstrate a reduction in the density and size of GABAergic interneurons in the dorsolateral prefrontal cortex (DLPFC) in MDD. The goal of this study was to test whether the level of glutamic acid decarboxylase (GAD), the GABA synthesizing enzyme, will also be reduced in the same cortical region in MDD. Levels of GAD-65 and GAD-67 proteins were investigated by Western blotting in samples from the DLPFC (BA 9) in 13 medication-free subjects with MDD, and 13 psychiatrically healthy controls. The overall amount of GAD-67 was significantly reduced (-34%) in depressed subjects compared to matched controls. Since recent neuroimaging studies have demonstrated that antidepressants modulate GABA levels, additional experiments were performed to examine the levels of GAD in eight depressed subjects treated with antidepressant medications. Levels of GAD-67 were unchanged in these depressed subjects compared to their respective controls (n=8). The overall amounts of GAD-65 were similar in depressed subjects compared to matched controls, regardless of antidepressant medication. Reduced levels of GAD-67, which is localized to somata of GABA neurons, further support our observation of a decreased density of GABAergic neurons in the PFC in depression. It is likely that a decrease in GAD-67 accounts for the reduction in GABA levels revealed by neuroimaging studies. Moreover, our data support previous neuroimaging observations that antidepressant medication normalizes GABA deficits in depression.

Elevated level of metabotropic glutamate receptor 2/3 in the prefrontal cortex in major depression.

Clinical, postmortem and preclinical research strongly implicates dysregulation of glutamatergic neurotransmission in major depressive disorder (MDD). Recently, metabotropic glutamate receptors (mGluRs) have been proposed as attractive targets for the discovery of novel therapeutic approaches against depression. The aim of this study was to examine mGluR2/3 protein levels in the prefrontal cortex (PFC) from depressed subjects. In addition, to test whether antidepressants influence mGluR2/3 expression we also studied levels of mGluR2/3 in fluoxetine-treated monkeys. Postmortem human prefrontal samples containing Brodmanns area 10 (BA10) were obtained from 11 depressed and 11 psychiatrically healthy controls. Male rhesus monkeys were treated chronically with fluoxetine (dose escalated to 3mg/kg, p.o.; n=7) or placebo (n=6) for 39 weeks. The mGluR2/3 immunoreactivity was investigated using Western blot method. There was a robust (+67%) increase in the expression of the mGlu2/3 protein in the PFC of depressed subjects relative to healthy controls. The expression of mGlu2/3 was unchanged in the PFC of monkeys treated with fluoxetine. Our findings provide the first evidence that mGluR2/3 is elevated in the PFC in MDD. This observation is consistent with reports showing that mGluR2/3 antagonists exhibit antidepressant-like activity in animal models and demonstrates that these receptors are promising targets for the discovery of novel antidepressants.

Elevated levels of NR2A and PSD-95 in the lateral amygdala in depression.

Compelling evidence suggests that major depression is associated with dysfunction of the brain glutamatergic transmission, and that the glutamatergic N-methyl-d-aspartate (NMDA) receptor plays a role in antidepressant activity. Recent post-mortem studies demonstrate that depression is associated with altered concentrations of proteins associated with NMDA receptor signalling in the brain. The present study investigated glutamate signalling proteins in the amygdala from depressed subjects, given strong evidence for amygdala pathology in depression. Lateral amygdala samples were obtained from 13-14 pairs of age- sex-, and post-mortem-interval-matched depressed and psychiatrically healthy control subjects. Concentrations of NR1 and NR2A subunits of the NMDA receptor, as well as NMDA receptor-associated proteins such as post-synaptic density protein-95 (PSD-95) and neuronal nitric oxide synthase (nNOS) were measured by Western immunoblotting. Additionally, levels of enzymes involved in glutamate metabolism, including glutamine synthetase and glutamic acid decarboxylase (GAD-67), were measured in the same amygdala samples. NR2A protein levels were markedly and significantly elevated (+115%, p=0.03) in depressed subjects compared to controls. Interestingly, PSD-95 levels were also highly elevated (+128%, p=0.01) in the same depressed subjects relative to controls. Amounts of NR1, nNOS, glutamine synthetase, and GAD-67 were unchanged. Increased levels of NR2A and PSD-95 suggest that glutamate signalling at the NMDA receptor in the amygdala is disrupted in depression.

Reduced phosphorylation of the mTOR signaling pathway components in the amygdala of rats exposed to chronic stress

The activity of the mammalian target of rapamycin (mTOR), an ubiquitously expressed serine/threonine kinase, is central to the regulation of translation initiation and, consequently protein synthesis required for long-term potentiation and new synaptic connections. Recent studies show that activation of the mTOR signaling pathway is required for the rapid antidepressant actions of glutamate N-methyl-d-aspartate (NMDA) receptor antagonists such as ketamine. Our prior work documented the first evidence of robust deficits in the mTOR signaling pathway in the prefrontal cortex (PFC) from subjects diagnosed with major depressive disorder (MDD). The goal of this study was to determine whether alterations in mTOR signaling can be observed in rats exposed to the chronic unpredictable stress (CUS) model of depression. In the present study, we examined the effect of CUS on the expression of phosphorylated mTOR and its downstream signaling components in the frontal cortex, hippocampus, amygdala, and dorsal raphe. We also examined the effect of CUS on the expression of kinases that phosphorylate mTOR such as extracellular signal-regulated kinase (ERK1/2) and protein kinase B/Akt (Akt1). In addition, we examined the effect of stress on the phosphorylation of GluR1 an, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit. We found that eight-weeks of CUS exposure significantly decreased the phosphorylation levels of mTOR and its downstream signaling components in the amygdala. Reduced level of phospho-mTOR in the amygdala was accompanied by decreased phosphorylation of ERK-1/2, Akt-1, and GluR1. No significant changes were seen in the frontal cortex, hippocampus, or dorsal raphe. Our study demonstrates that long-term stress exposure results in brain region-specific abnormalities in signaling pathways previously linked to novel mechanisms for rapid antidepressant effects. These observations are in line with evidence showing that mTOR and its upstream and downstream signaling partners could be important targets for the development of novel antidepressants.

Group housing of mice increases immobility and antidepressant sensitivity in the forced swim and tail suspension tests.

The forced swim test and tail suspension test are often used in laboratory practice to identify compounds that possess antidepressant-like activity. This experiment was conducted to determine whether housing conditions per se influence the response of mice in these antidepressant screening procedures. Male NIH Swiss mice were housed individually or in groups (five per cage) for 8 weeks prior to testing. After 8 weeks, the animals were exposed to the forced swim and tail-suspension tests. Group housed mice displayed high levels of immobility in the forced swim and tail suspension tests. Desipramine injection 60 min prior testing, in doses 7.5 and 15 mg/kg, produced significant reductions in the immobility time in forced swimming and tail suspension tests. Individually housed mice, when exposed to these tests, displayed lower levels of immobility with a magnitude comparable to the effect of desipramine in group housed mice. Desipramine given to individually housed mice did not reduce the duration of immobility either in the forced swim test or in the tail suspension test. These results indicate that both tests are sensitive to housing conditions. This observation suggests that long lasting group housing may be critical to the behavioral response in these preclinical screening procedures in mice.

Elevated Levels of the NR2C Subunit of the NMDA Receptor in the Locus Coeruleus in Depression

Low levels of the intracellular mediator of glutamate receptor activation, neuronal nitric oxide synthase (nNOS) were previously observed in locus coeruleus (LC) from subjects diagnosed with major depression. This finding implicates abnormalities in glutamate signaling in depression. Receptors responding to glutamate in the LC include ionotropic N-methyl-D-aspartate receptors (NMDARs). The functional NMDAR is a hetero-oligomeric structure composed of NR1 and NR2 (A–D) subunits. Tissue containing the LC and a nonlimbic LC projection area (cerebellum) was obtained from 13 and 9 matched pairs, respectively, of depressed subjects and control subjects lacking major psychiatric diagnoses. NMDAR subunit composition in the LC was evaluated in a psychiatrically normal subject. NR1 and NR2C subunit immunoreactivities in LC homogenates showed prominent bands at 120 and 135 kDa, respectively. In contrast to NR1 and NR2C, very weak immunoreactivity of NR2A and NR2B subunits was observed in the LC. Possible changes in concentrations of NR1 and NR2C that might occur in depression were assessed in the LC and cerebellum. The overall amount of NR1 immunoreactivity was normal in the LC and cerebellum in depressed subjects. Amounts of NR2C protein were significantly higher (+61%, p=0.003) in the LC and modestly, but not significantly, elevated in the cerebellum (+35%) of depressives as compared to matched controls. Higher levels of NR2C subunit implicate altered glutamatergic input to the LC in depressive disorders.

Dopamine receptor gene expression in human amygdaloid nuclei: elevated D4 receptor mRNA in major depression.

Previous findings from this laboratory demonstrating changes in dopamine (DA) transporter and D2 receptors in the amygdaloid complex of subjects with major depression indicate that disruption of dopamine neurotransmission to the amygdala may contribute to behavioral symptoms associated with depression. Quantitative real-time RT-PCR was used to investigate the regional distribution of gene expression of DA receptors in the human amygdala. In addition, relative levels of mRNA of DA receptors in the basal amygdaloid nucleus were measured postmortem in subjects with major depression and normal control subjects. All five subtypes of DA receptor mRNA were detected in all amygdaloid subnuclei, although D1, D2, and D4 receptor mRNAs were more abundant than D3 and D5 mRNAs by an order of magnitude. The highest level of D1 mRNA was found in the central nucleus, whereas D2 mRNA was the most abundant in the basal nucleus. Levels of D4 mRNA were highest in the basal and central nuclei. In the basal nucleus, amounts of D4, but not D1 or D2, mRNAs were significantly higher in subjects with major depression as compared to control subjects. These findings demonstrate that the D1, D2 and D4 receptors are the major subtypes of DA receptors in the human amygdala. Elevated DA receptor gene expression in depressive subjects further implicates altered dopaminergic transmission in the amygdala in depression.