Dysregulation of the glutamatergic system has been implicated not only in the treatment of major depressive disorder (MDD), but also in the excitotoxic effects of stress and anxiety on the prefrontal cortex, which may precede the onset of a depressive episode. Our previous studies demonstrate marked deficits in prominent postsynaptic proteins involved in glutamate neurotransmission in the prefrontal cortex (PFC), Brodmann’s area 10 (BA 10) from subjects diagnosed with major depressive disorder (MDD). In the same group of subjects we have identified deficits in expression and phosphorylation level of key components of the mammalian target of rapamycin (mTOR) signalling pathway, known to regulate translation initiation. Based on our previous findings, we have postulated that glutamate-dependent dysregulation of mTOR- initiated protein synthesis in the PFC may underlie the pathology of MDD. The aim of this study was to use the NanoString nCounter System to perform analysis of genes coding for glutamate transporters, glutamate metabolizing enzymes, neurotrophic factors and other intracellular signaling markers involved in glutamate signaling that were not previously investigated by our group in the PFC BA10 from subjects with MDD. We have analyzed a total of 200 genes from 16 subjects with MDD and 16 healthy controls. These are part of the same cohort used in our previous studies. Setting our cutoff p-value ≤ 0.01, marked upregulation of genes coding for mitochondrial glutamate carrier (GC1; p=0.0015), neuropilin 1 (NRP-1; p=0.0019), glutamate receptor ionotropic N-methyl-D-aspartate-associated protein 1 (GRINA; p=0.0060), and fibroblast growth factor receptor 1 (FGFR-1; p=0.010) was identified. No significant differences in expression of the remaining 196 genes were observed between MDD subjects and controls. While upregulation of FGFR-1 has been previously shown in MDD; abnormalities in GC-1, GRINA, and NRP-1 have not been reported. Therefore, this postmortem study identifies GC1, GRINA, and NRP-1 as novel factors associated with MDD; however, future studies will be needed to address the significance of these genes in the pathophysiology of depression and antidepressant activity.
prefrontal cortex; major depressive disorder; postmortem; gene expression; digital PCR
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.
mTOR signaling pathway; chronic unpredictable stress; rats; frontal cortex; hippocampus; amygdala; dorsal raphe
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 95 kDa (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, eIF4B). In this study, we examined the expression of mTOR and its core downstream signaling targets: p70S6K, eIF4E, 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.
prefrontal cortex; translation initiation pathway; major depressive disorder; postmortem
Clinical and preclinical evidence suggest a hyperactive glutamatergic system in clinical depression. Recently, the metabotropic glutamate receptor 5 (mGluR5) has been proposed as an attractive target for discovery of novel therapeutic approaches against depression. The goal of this study was to compare mGluR5 binding (PET study) and mGluR5 protein expression (postmortem study) between subjects with major depressive disorder and healthy controls.
Images of mGluR5 receptor binding were acquired using PET and [11C]ABP688 that binds to an allosteric site with high specificity in 11 unmedicated subjects with major depression and 11 matched healthy controls; the amount of mGluR5 protein was investigated using Western blot method in brain samples of 15 depressed subjects and 15 matched controls (postmortem study).
The PET study revealed decreased regional mGluR5 binding in the prefrontal cortex, the cingulate cortex, the insula, the thalamus and the hippocampus of the depressed individuals (uncorrected p<0.001). Severity of depression correlated negatively with mGluR5 binding in the hippocampus (cluster-level corrected p=0.029). The postmortem study showed reduced mGluR5 protein expression in the prefrontal cortex (Brodmann's area 10) in depression (p<0.014), while prefrontal mGluR1 protein expression was unchanged.
The reductions in mGluR5 binding found in the depressed sample are compatible with reduced protein expression in postmortem tissue. Thus, both studies suggest that basal or compensatory changes in excitatory neurotransmission play roles in the pathophysiology of major depression.
Several lines of evidence suggest a dysfunctional glutamate system in major depressive disorder (MDD). Recently, we reported reduced levels of metabotropic glutamate receptor subtype 5 (mGluR5) in postmortem brains in MDD, however the neurobiological mechanisms that induce these abnormalities are unclear. In the present study, we examined the effect of chronic corticosterone (CORT) administration on the expression of mGluR5 protein and mRNA in the rat frontal cortex and hippocampus. Rats were injected with CORT (40 mg/kg, s.c.) or vehicle once daily for 21 days. The expression of mGluR5 protein and mRNA was assessed by Western blotting and quantitative real-time PCR. In addition, mGluR1 protein was measured in the same animals. The results revealed that while there was a significant reduction (− 27 %, p=0.0006) in mGluR5 protein expression in the hippocampus from CORT treated rats, mRNA levels were unchanged. Also unchanged were mGluR5 mRNA and protein levels in the frontal cortex and mGluR1 protein levels in both brain regions. Our findings provide the first evidence that chronic CORT exposure regulates the expression of mGluR5 and are in line with previous postmortem and imaging studies showing reduced mGluR5 in MDD. Our findings suggest that elevated levels of glucocorticoids may contribute to impairments in glutamate neurotransmission in MDD.
hippocampus; frontal cortex; metabotropic glutamate receptor 5; metabotropic glutamate receptor 1; corticosterone; rat
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 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 Brodmann’s area 10 (BA 10) 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.
prefrontal cortex; metabotropic glutamate receptor; major depression; postmortem; fluoxetine; rhesus monkey
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.
depression; locus coeruleus; cerebellum; glutamate; NMDA receptor
Altered concentrations of dopamine transporter and D2/D3 receptors have been observed in the amygdaloid complex of subjects with major depression. These findings are suggestive of neurochemical abnormalities in the limbic dopamine system in depression. Monoamine oxidase-B (MAO-B) is a key enzyme in the catabolism of biogenic amines, including dopamine, and alterations in this enzyme may underlie dopaminergic abnormalities associated with depression. The specific binding of [3H]lazabemide to MAO-B was measured in the right amygdaloid complex of 15 major depressive subjects and 16 psychiatrically normal controls. Subjects of the two study groups were matched as close as possible for age, sex, and postmortem interval. Examination of the regional distribution of MAO-B revealed lower [3H]lazabemide binding to MAO-B in the lateral and basal nuclei of the amygdala and higher binding in the medial nucleus. A modest elevation in binding to MAO-B observed in all amygdaloid nuclei in major depressive subjects as compared to control subjects failed to reach statistical significance. A significant decrease in binding to MAO-B was observed when cigarette smokers were compared to nonsmoking subjects. The amount of MAO-B binding positively correlated with the age of subjects in all nuclei investigated. A decreased amount of MAO-B in smokers further validates the pharmacological effect of tobacco smoke on this enzyme.
Monoamine oxidase B; Major depression; Amygdala; Postmortem; Smoking; Dopamine; Catecholamine
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 (PFC) 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 dorsolateral PFC (BA9) 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 as compared to matched controls. Since recent neuroimaging studies demonstrate that antidepressants modulate GABA levels, additional experiments were performed to examine the levels of GAD in 8 depressed subjects treated with antidepressant medications. Levels of GAD-67 were unchanged in these depressed subjects as 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.
Post-mortem; GAD; GABA; antidepressants; major depressive disorder; dorsolateral prefrontal cortex
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.
major depressive disorder; glutamate receptors; postmortem; postsynaptic density protein; anterior prefrontal cortex
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 and depressed suicide victims, 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.
Dopamine receptor; gene expression; amygdala; depression; major depression; real-time PCR; human brain; dopamine
It has been postulated that alcoholism is associated with abnormalities in glutamatergic neurotransmission. This study examined the density of glutamate NMDA receptor subunits and its associated proteins in the noradrenergic locus coeruleus (LC) in deceased alcoholic subjects. Our previous research indicated that the NMDA receptor in the human LC is composed of obligatory NR1 and regulatory NR2C subunits. At synapses, NMDA receptors are stabilized through interactions with postsynaptic density protein (PSD-95). PSD-95 provides structural and functional coupling of the NMDA receptor with neuronal nitric oxide synthase (nNOS), an intracellular mediator of NMDA receptor activation. LC tissue was obtained from 10 alcohol-dependent subjects and 8 psychiatrically healthy controls. Concentrations of NR1 and NR2C subunits, as well as PSD-95 and nNOS, were measured using Western blotting. In addition we have examined tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of norepinephrine. The amount of NR1 was lower in the rostral (−30%) and middle (−41%)portions of the LC of alcoholics as compared to control subjects. No differences in the amounts of NR2C, PSD-95, nNOS and TH were detected comparing alcoholic to control subjects. Lower levels of NR1 subunit of the NMDA receptor in the LC implicates altered glutamate-norepinephrine interactions in alcoholism.
glutamate; NMDA receptor; postsynaptic density protein 95; neuronal nitric oxide synthase; tyrosine hydroxylase; locus coeruleus
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 postmortem studies demonstrate that depression is associated with altered concentrations of proteins associated with NMDA receptor signaling in the brain. The present study investigated glutamate signaling 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 postmortem-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 postsynaptic 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 as 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 signaling at the NMDA receptor in the amygdala is disrupted in depression.
NMDA receptor; postsynaptic density protein-95; neuronal nitric oxide synthase; glutamate metabolizing enzymes, amygdala
Major depression and suicide are associated with altered concentrations of specific noradrenergic proteins in the human locus coeruleus (LC). Based on experimental studies that can reproduce these LC abnormalities in laboratory animals, we hypothesized that noradrenergic pathobiology in depression is a result of over-activity of the LC. LC activity is under the control of both excitatory and inhibitory inputs. A major inhibitory input to the LC is GABAergic, arising from the nucleus prepositus hypoglossi. Numerous studies demonstrating low levels of GABA in the CSF and plasma of subjects with major depressive disorder (MDD) raise the possibility that LC over-activity in depression may be secondary to reduced GABAergic input to the LC. Here, GABAergic input to the LC in depression was evaluated by studying the binding of [3H]flunitrazepam to GABAA receptors at three anatomically-defined levels of the human postmortem LC. LC tissues were collected from subjects with MDD, subjects with depressive disorders including MDD that died as a result of suicide, and psychiatrically normal control subjects. A modest rostral-caudal gradient of GABAA receptor binding density was observed amongst all subjects. No significant differences in the amount of binding to GABAA receptors were observed between control subjects (n=21) and MDD subjects (n=9) or depressed suicide victims (n=17). These results demonstrate that GABAA receptor binding in the LC measured with [3H]flunitrazepam is not altered in subjects with depressive illnesses.
postmortem brain; major depressive disorder; suicide, norepinephrine; GABAA receptor; flunitrazepam; locus coeruleus