Markers of elevated oxidative stress in oligodendrocytes captured from the brainstem and occipital cortex in major depressive disorder and suicide.

Major depressive disorder (MDD) and suicide have been associated with elevated indices of oxidative damage in the brain, as well as white matter pathology including reduced myelination by oligodendrocytes. Oligodendrocytes highly populate white matter and are inherently susceptible to oxidative damage. Pathology of white matter oligodendrocytes has been reported to occur in brain regions that process behaviors that are disrupted in MDD and that may contribute to suicidal behavior. The present study was designed to determine whether oligodendrocyte pathology related to oxidative damage extends to brain areas outside of those that are traditionally considered to contribute to the psychopathology of MDD and suicide. Relative telomere lengths and the gene expression of five antioxidant-related genes, SOD1, SOD2, GPX1, CAT, and AGPS were measured in oligodendrocytes laser captured from two non-limbic brain areas: occipital cortical white matter and the brainstem locus coeruleus. Postmortem brain tissues were obtained from brain donors that died by suicide and had an active MDD at the time of death, and from psychiatrically normal control donors. Relative telomere lengths were significantly reduced in oligodendrocytes of both brain regions in MDD donors as compared to control donors. Three antioxidant-related genes (SOD1, SOD2, GPX1) were significantly reduced and one was significantly elevated (AGPS) in oligodendrocytes from both brain regions in MDD as compared to control donors. These findings suggest that oligodendrocyte pathology in MDD and suicide is widespread in the brain and not restricted to brain areas commonly associated with depression psychopathology.

Rapid and temporary improvement of depression and anxiety observed following niraparib administration: a case report.

BACKGROUND: Cancer patients are disproportionately affected by generalized anxiety and major depression. For many, current treatments for these conditions are ineffective. In this case report, we present a serendipitous case of anxiety and depression improvement following administration of the poly (ADP-ribose) polymerase (PARP) inhibitor niraparib. CASE PRESENTATION: A 61-year old woman with a 20-year history of mild depression developed recurrent ovarian carcinoma and was placed on niraparib for maintenance chemotherapy. With the original onset of ovarian cancer, she experienced an episode of major depression that was resolved with sertraline. After recurrence of ovarian cancer, she experienced a recurrence of major depression and a new onset of generalized anxiety that failed to completely respond to multiple medications. After beginning niraparib therapy the patient noticed a rapid resolution of the symptoms of her anxiety and depression, an effect that was limited to 10-14 days. Due to bone marrow suppression, the patient was taken off and restarted on niraparib several times. Each discontinuation of niraparib resulted in return of her depression and anxiety, while each recontinuation of niraparib resulted in an improvement in her mood and anxiety. CONCLUSIONS: This case demonstrates rapid and temporary improvement of anxiety and depression following niraparib administration. There is ample preclinical data that PARP signaling may play a role in psychiatric illness. A small amount of indirect data from clinical trials also shows that niraparib may have psychiatric benefits. Further research on PARP inhibition and its potential psychoactive effects is sorely needed.

Neuroinflammatory Gene Expression Alterations in Anterior Cingulate Cortical White and Gray Matter of Males With Autism Spectrum Disorder.

Evidence for putative pathophysiological mechanisms of autism spectrum disorder (ASD), including peripheral inflammation, blood-brain barrier disruption, white matter alterations, and abnormal synaptic overgrowth, indicate a possible involvement of neuroinflammation in the disorder. Neuroinflammation plays a role in the development and maintenance of the dendritic spines involved in glutamatergic and GABAergic neurotransmission, and also influences blood-brain permeability. Cytokines released from microglia can impact the length, location or organization of dendritic spines on excitatory and inhibitory cells as well as recruit and impact glial cell function around the neurons. In this study, gene expression levels of anti- and pro-inflammatory signaling molecules, as well as oligodendrocyte and astrocyte marker proteins, were measured in both gray and white matter tissue in the anterior cingulate cortex from ASD and age-matched typically developing (TD) control brain donors, ranging from ages 4 to 37 years. Expression levels of the pro-inflammatory gene, HLA-DR, were significantly reduced in gray matter and expression levels of the anti-inflammatory gene MRC1 were significantly elevated in white matter from ASD donors as compared to TD donors, but neither retained statistical significance after correction for multiple comparisons. Modest trends toward differences in expression levels were also observed for the pro-inflammatory (CD68, IL1ß) and anti-inflammatory genes (IGF1, IGF1R) comparing ASD donors to TD donors. The direction of gene expression changes comparing ASD to TD donors did not reveal consistent findings implicating an elevated pro- or anti-inflammatory state in ASD. However, altered expression of pro- and anti-inflammatory gene expression indicates some involvement of neuroinflammation in ASD. Autism Res 2020, 13: 870-884. © 2020 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: The anterior cingulate cortex is an integral brain region in modulating social behaviors including nonverbal communication. The study found that inflammatory gene expression levels were altered in this brain region. We hypothesize that the inflammatory changes in this area could impact neuronal function. The finding has future implications in using these molecular markers to identify potential environmental exposures and distinct cell differences in autism.

Altered Expression of Phox2 Transcription Factors in the Locus Coeruleus in Major Depressive Disorder Mimicked by Chronic Stress and Corticosterone Treatment In Vivo and In Vitro.

Phox2a and Phox2b are two homeodomain transcription factors playing a pivotal role in the development of noradrenergic neurons during the embryonic period. However, their expression and function in adulthood remain to be elucidated. Using human postmortem brain tissues, rat stress models and cultured cells, this study aimed to examine the alteration of Phox2a and Phox2b expression. The results show that Phox2a and Phox2b are normally expressed in the human locus coeruleus (LC) in adulthood. Furthermore, the levels of Phox2a protein and mRNA and protein levels of Phox2b were significantly elevated in the LC of brain donors that suffered from the major depressive disorder, as compared to age-matched and psychiatrically normal control donors. Fischer 344 rats subjected to chronic social defeat showed higher mRNA and protein levels of Phox2a and Phox2b in the LC, as compared to non-stressed control rats. In rats chronically administered oral corticosterone, mRNA and protein levels of Phox2b, but not Phox2a, in the LC were significantly increased. In addition, the corticosterone-induced increase in Phox2b protein was reversed by simultaneous treatment with either mifepristone or spironolactone. Exposing SH-SY5Y cells to corticosterone significantly increased expression of Phox2a and Phox2b, which was blocked by corticosteroid receptor antagonists. Taken together, these experiments reveal that Phox2 genes are expressed throughout the lifetime in the LC of humans and Fischer 344 rats. Alterations in their expression may play a role in major depressive disorder and possibly other stress-related disorders through their modulatory effects on the noradrenergic phenotype.

Antidepressant-Like Actions of Inhibitors of Poly(ADP-Ribose) Polymerase in Rodent Models.

Background: Many patients suffering from depressive disorders are refractory to treatment with currently available antidepressant medications, while many more exhibit only a partial response. These factors drive research to discover new pharmacological approaches to treat depression. Numerous studies demonstrate evidence of inflammation and elevated oxidative stress in major depression. Recently, major depression has been shown to be associated with elevated levels of DNA oxidation in brain cells, accompanied by increased gene expression of the nuclear base excision repair enzyme, poly(ADP-ribose) polymerase-1. Given these findings and evidence that drugs that inhibit poly(ADP-ribose) polymerase-1 activity have antiinflammatory and neuroprotective properties, the present study was undertaken to examine the potential antidepressant properties of poly(ADP-ribose) polymerase inhibitors. Methods: Two rodent models, the Porsolt swim test and repeated exposure to psychological stressors, were used to test the poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide, for potential antidepressant activity. Another poly(ADP-ribose) polymerase inhibitor, 5-aminoisoquinolinone, was also tested. Results: Poly(ADP-ribose) polymerase inhibitors produced antidepressant-like effects in the Porsolt swim test, decreasing immobility time, and increasing latency to immobility, similar to the effects of fluoxetine. In addition, 3-aminobenzamide treatment increased sucrose preference and social interaction times relative to vehicle-treated control rats following repeated exposure to combined social defeat and unpredictable stress, mediating effects similar to fluoxetine treatment. Conclusions: The poly(ADP-ribose) polymerase inhibitors 3-aminobenzamide and 5-aminoisoquinolinone exhibit antidepressant-like activity in 2 rodent stress models and uncover poly(ADP-ribose) polymerase as a unique molecular target for the potential development of a novel class of antidepressants.

Elevated DNA Oxidation and DNA Repair Enzyme Expression in Brain White Matter in Major Depressive Disorder.

Background: Pathology of white matter in brains of patients with major depressive disorder (MDD) is well-documented, but the cellular and molecular basis of this pathology are poorly understood. Methods: Levels of DNA oxidation and gene expression of DNA damage repair enzymes were measured in Brodmann area 10 (BA10) and/or amygdala (uncinate fasciculus) white matter tissue from brains of MDD (n=10) and psychiatrically normal control donors (n=13). DNA oxidation was also measured in BA10 white matter of schizophrenia donors (n=10) and in prefrontal cortical white matter from control rats (n=8) and rats with repeated stress-induced anhedonia (n=8). Results: DNA oxidation in BA10 white matter was robustly elevated in MDD as compared to control donors, with a smaller elevation occurring in schizophrenia donors. DNA oxidation levels in psychiatrically affected donors that died by suicide did not significantly differ from DNA oxidation levels in psychiatrically affected donors dying by other causes (non-suicide). Gene expression levels of two base excision repair enzymes, PARP1 and OGG1, were robustly elevated in oligodendrocytes laser captured from BA10 and amygdala white matter of MDD donors, with smaller but significant elevations of these gene expressions in astrocytes. In rats, repeated stress-induced anhedonia, as measured by a reduction in sucrose preference, was associated with increased DNA oxidation in white, but not gray, matter. Conclusions: Cellular residents of brain white matter demonstrate markers of oxidative damage in MDD. Medications that interfere with oxidative damage or pathways activated by oxidative damage have potential to improve treatment for MDD.

Erratum to: NTRK2 expression levels are reduced in laser captured pyramidal neurons from the anterior cingulate cortex in males with autism spectrum disorder.

[This corrects the article DOI: 10.1186/s13229-015-0023-2.].

NTRK2 expression levels are reduced in laser captured pyramidal neurons from the anterior cingulate cortex in males with autism spectrum disorder.

BACKGROUND: The anterior cingulate cortex (ACC) is a brain area involved in modulating behavior associated with social interaction, disruption of which is a core feature of autism spectrum disorder (ASD). Functional brain imaging studies demonstrate abnormalities of the ACC in ASD as compared to typically developing control patients. However, little is known regarding the cellular basis of these functional deficits in ASD. Pyramidal neurons in the ACC are excitatory glutamatergic neurons and key cellular mediators of the neural output of the ACC. This study was designed to investigate the potential role of ACC pyramidal neurons in ASD brain pathology. METHODS: Postmortem ACC tissue from carefully matched ASD and typically developing control donors was obtained from two national brain collections. Pyramidal neurons and surrounding astrocytes were separately collected from layer III of the ACC by laser capture microdissection. Isolated RNA was subjected to reverse transcription and endpoint PCR to determine gene expression levels for 16 synaptic genes relevant to glutamatergic neurotransmission. Cells were also collected from the prefrontal cortex (Brodmann area 10) to examine those genes demonstrating differences in expression in the ACC comparing typically developing and ASD donors. RESULTS: The level of NTRK2 expression was robustly and significantly lower in pyramidal neurons from ASD donors as compared to typically developing donors. Levels of expression of GRIN1, GRM8, SLC1A1, and GRIP1 were modestly lower in pyramidal neurons from ASD donors, but statistical significance for these latter genes did not survive correction for multiple comparisons. No significant expression differences of any genes were found in astrocytes laser captured from the same neocortical area. In addition, expression levels of NTRK2 and other synaptic genes were normal in pyramidal neurons laser captured from the prefrontal cortex. CONCLUSIONS: These studies demonstrate a unique pathology of neocortical pyramidal neurons of the ACC in ASD. NTRK2 encodes the tropomyosin receptor kinase B (TrkB), transmission through which neurotrophic factors modify differentiation, plasticity, and synaptic transmission. Reduced pyramidal neuron NTRK2 expression in the ACC could thereby contribute to abnormal neuronal activity and disrupt social behavior mediated by this brain region.

Elevated GFAP Protein in Anterior Cingulate Cortical White Matter in Males With Autism Spectrum Disorder.

Based on evidence of abnormalities in axon thickness and neuronal disorganization, autism spectrum disorder (ASD) is commonly considered to be a condition resulting from neuronal dysfunction. Yet, recent findings suggest that non-neuronal cell types also contribute to ASD pathology. To investigate the role of glial cells in ASD, a combination of protein and gene expression analyses were used to determine levels of two glial markers, glial fibrillary acidic protein (GFAP) and myelin oligodendrocyte glycoprotein (MOG), in the postmortem brain tissue from control and ASD donors. Levels of GFAP immunoreactivity (ir) were significantly elevated (P = 0.008) in anterior cingulate cortex (Brodmann area 24; BA24) white matter of ASD donors compared to control donors. In contrast, GFAP-ir levels were similar in BA24 gray matter from ASD and control donors. MOG-ir was also similar in both BA24 white and gray matter from ASD and control donors. In anterior prefrontal cortex (BA10), there were no significant differences in GFAP-ir or MOG-ir in either white or gray matter comparing ASD to control donors. Levels of expression of the genes GFAP and MOG also showed no differences between control and ASD donors in BA24 and BA10 white and gray matter. Collectively, these data imply that ASD is associated with an activation of white matter astrocytes in the anterior cingulate cortex as a result of a yet undefined cellular insult. Research is needed to investigate the molecular pathways that underlie this astrocyte reaction and such research may yield important clues regarding the etiology of ASD.

Effects of desipramine treatment on stress-induced up-regulation of norepinephrine transporter expression in rat brains.

RATIONALE: Many studies demonstrate down-regulation of the norepinephrine transporter (NET) by desipramine (DMI) in vitro and in stress-naive rats. Little is known regarding regulation of the NET in stressed animals. OBJECTIVE: The present study was designed to investigate effects of DMI on the expression of NET and protein kinases in the stress rat. METHODS: Adult Fischer 344 rats were subjected to chronic social defeat (CSD) for 4 weeks. DMI (10 mg/kg, intraperitoneal (i.p.)) was administered concurrently with CSD or 1 or 2 weeks after cessation of CSD. Sucrose consumption, NET expression, and protein kinases were measured. RESULTS: CSD significantly increased messenger RNA (mRNA) and protein levels of NET in the locus coeruleus, as well as NET protein levels in the hippocampus, frontal cortex, and amygdala. These effects were nearly abolished when DMI was administered concurrently with CSD. CSD-induced up-regulation of NET expression in the locus coeruleus, hippocampus, and amygdala lasted at least 2 weeks after cessation of CSD, an effect that was significantly attenuated by 1 or 2 weeks of DMI treatment starting from cessation of the CSD. Concurrent administration of DMI with CSD did not markedly interfere with CSD-induced decreases in protein levels of protein kinases A and C in these brain regions, but it did reverse the CSD-induced reduction in phosphorylated cAMP response element-binding (pCREB) protein levels in most brain regions. CONCLUSION: These findings suggest that NET regulation by DMI occurs in both stressed and behaviorally naive rats, and DMI-induced changes in pCREB may be involved.

Elevated gene expression of glutamate receptors in noradrenergic neurons from the locus coeruleus in major depression.

Glutamate receptors are promising drug targets for the treatment of urgent suicide ideation and chronic major depressive disorder (MDD) that may lead to suicide completion. Antagonists of glutamatergic NMDA receptors reduce depressive symptoms faster than traditional antidepressants, with beneficial effects occurring within hours. Glutamate is the prominent excitatory input to the noradrenergic locus coeruleus (LC). The LC is activated by stress in part through this glutamatergic input. Evidence has accrued demonstrating that the LC may be overactive in MDD, while treatment with traditional antidepressants reduces LC activity. Pathological alterations of both glutamatergic and noradrenergic systems have been observed in depressive disorders, raising the prospect that disrupted glutamate-norepinephrine interactions may be a central component to depression and suicide pathobiology. This study examined the gene expression levels of glutamate receptors in post-mortem noradrenergic LC neurons from subjects with MDD (most died by suicide) and matched psychiatrically normal controls. Gene expression levels of glutamate receptors or receptor subunits were measured in LC neurons collected by laser capture microdissection. MDD subjects exhibited significantly higher expression levels of the NMDA receptor subunit genes, GRIN2B and GRIN2C, and the metabotropic receptor genes, GRM4 and GRM5, in LC neurons. Gene expression levels of these receptors in pyramidal neurons from prefrontal cortex (BA10) did not reveal abnormalities in MDD. These findings implicate disrupted glutamatergic-noradrenergic interactions at the level of the stress-sensitive LC in MDD and suicide, and provide a theoretical mechanism by which glutamate antagonists may exert rapid antidepressant effects.

Shortened telomere length in white matter oligodendrocytes in major depression: potential role of oxidative stress.

Telomere shortening is observed in peripheral mononuclear cells from patients with major depressive disorder (MDD). Whether this finding and its biological causes impact the health of the brain in MDD is unknown. Brain cells have differing vulnerabilities to biological mechanisms known to play a role in accelerating telomere shortening. Here, two glia cell populations (oligodendrocytes and astrocytes) known to have different vulnerabilities to a key mediator of telomere shortening, oxidative stress, were studied. The two cell populations were separately collected by laser capture micro-dissection of two white matter regions shown previously to demonstrate pathology in MDD patients. Cells were collected from brain donors with MDD at the time of death and age-matched psychiatrically normal control donors (N = 12 donor pairs). Relative telomere lengths in white matter oligodendrocytes, but not astrocytes, from both brain regions were significantly shorter for MDD donors as compared to matched control donors. Gene expression levels of telomerase reverse transcriptase were significantly lower in white matter oligodendrocytes from MDD as compared to control donors. Likewise, the gene expression of oxidative defence enzymes superoxide dismutases (SOD1 and SOD2), catalase (CAT) and glutathione peroxidase (GPX1) were significantly lower in oligodendrocytes from MDD as compared to control donors. No such gene expression changes were observed in astrocytes from MDD donors. These findings suggest that attenuated oxidative stress defence and deficient telomerase contribute to telomere shortening in oligodendrocytes in MDD, and suggest an aetiological link between telomere shortening and white matter abnormalities previously described in MDD.

Gene expression deficits in pontine locus coeruleus astrocytes in men with major depressive disorder.

BACKGROUND: Norepinephrine and glutamate are among several neurotransmitters implicated in the neuropathology of major depressive disorder (MDD). Glia deficits have also been demonstrated in people with MDD, and glia are critical modulators of central glutamatergic transmission. We studied glia in men with MDD in the region of the brain (locus coeruleus; LC) where noradrenergic neuronal cell bodies reside and receive glutamatergic input. METHODS: The expression of 3 glutamate-related genes (SLC1A3, SLC1A2, GLUL) concentrated in glia and a glia gene (GFAP) were measured in postmortem tissues from men with MDD and from paired psychiatrically healthy controls. Initial gene expression analysis of RNA isolated from homogenized tissue (n = 9-10 pairs) containing the LC were followed by detailed analysis of gene expressions in astrocytes and oligodendrocytes (n = 6-7 pairs) laser captured from the LC region. We assessed protein changes in GFAP using immunohistochemistry and immunoblotting (n = 7-14 pairs). RESULTS: Astrocytes, but not oligodendrocytes, demonstrated robust reductions in the expression of SLC1A3 and SLC1A2, whereas GLUL expression was unchanged. GFAP expression was lower in astrocytes, and we confirmed reduced GFAP protein in the LC using immunostaining methods. LIMITATIONS: Reduced expression of protein products of SLC1A3 and SLC1A2 could not be confirmed because of insufficient amounts of LC tissue for these assays. Whether gene expression abnormalities were associated with only MDD and not with suicide could not be confirmed because most of the decedents who had MDD died by suicide. CONCLUSION: Major depressive disorder is associated with unhealthy astrocytes in the noradrenergic LC, characterized here by a reduction in astrocyte glutamate transporter expression. These findings suggest that increased glutamatergic activity in the LC occurs in men with MDD.

Low gene expression of bone morphogenetic protein 7 in brainstem astrocytes in major depression.

The noradrenergic locus coeruleus (LC) is the principal source of brain norepinephrine, a neurotransmitter thought to play a major role in the pathology of major depressive disorder (MDD) and in the therapeutic action of many antidepressant drugs. The goal of this study was to identify potential mediators of brain noradrenergic dysfunction in MDD. Bone morphogenetic protein 7 (BMP7), a member of the transforming growth factor-ß superfamily, is a critical mediator of noradrenergic neuron differentiation during development and has neurotrophic and neuroprotective effects on mature catecholaminergic neurons. Real-time PCR of reversed transcribed RNA isolated from homogenates of LC tissue from 12 matched pairs of MDD subjects and psychiatrically normal control subjects revealed low levels of BMP7 gene expression in MDD. No differences in gene expression levels of other members of the BMP family were observed in the LC, and BMP7 gene expression was normal in the prefrontal cortex and amygdala in MDD subjects. Laser capture microdissection of noradrenergic neurons, astrocytes, and oligodendrocytes from the LC revealed that BMP7 gene expression was highest in LC astrocytes relative to the other cell types, and that the MDD-associated reduction in BMP7 gene expression was limited to astrocytes. Rats exposed to chronic social defeat exhibited a similar reduction in BMP7 gene expression in the LC. BMP7 has unique developmental and trophic actions on catecholamine neurons and these findings suggest that reduced astrocyte support for pontine LC neurons may contribute to pathology of brain noradrenergic neurons in MDD.

Eszopiclone facilitation of the antidepressant efficacy of fluoxetine using a social defeat stress model.

This study analyzed the interaction of the sleep aid eszopiclone (ESZ) and antidepressant fluoxetine (FLX) on social defeat stress (SDS) in the mouse. Beta adrenoreceptors, brain-derived neurotrophic factor (BDNF) and cAMP response element binding protein (CREB) expression in the hippocampus and frontal cortex were also analyzed. Subjects were adult male 'intruder' C57/B6 mice that were exposed to a retired 'resident' male breeder ICR mouse in this animal's home cage for a 5 min period for each of 10 consecutive days, and the resident established physical dominance. The following day, all animals were assigned to one of four drug treatment groups, and treatment was given for up to 18 days: vehicle, ESZ only (3mg/kg), FLX (10mg/kg) only, or ESZ+FLX. A social interaction test was given on days 1, 5, 10, and 15 of drug treatment to assess SDS. Results showed that the ESZ+FLX group spent less time in avoidance zones during the interaction test at days 1 and 5, and more time in the interaction zone at day 5 compared to defeated mice given vehicle. All drug treatment groups spent more time in the interaction zone compared to defeated mice given vehicle on day 1 as well as day 10. SDS completely dissipated by the fourth interaction test according to both behavioral measures. Neurochemically, SDS did not produce changes in any marker analyzed. This study shows the combination of ESZ and FLX alleviated SDS, but a neurochemical correlate remains elusive.

Reserpine-induced reduction in norepinephrine transporter function requires catecholamine storage vesicles.

Treatment of rats with reserpine, an inhibitor of the vesicular monoamine transporter (VMAT), depletes norepinephrine (NE) and regulates NE transporter (NET) expression. The present study examined the molecular mechanisms involved in regulation of the NET by reserpine using cultured cells. Exposure of rat PC12 cells to reserpine for a period as short as 5min decreased [(3)H]NE uptake capacity, an effect characterized by a robust decrease in the V(max) of the transport of [(3)H]NE. As expected, reserpine did not displace the binding of [(3)H]nisoxetine from the NET in membrane homogenates. The potency of reserpine for reducing [(3)H]NE uptake was dramatically lower in SK-N-SH cells that have reduced storage capacity for catecholamines. Reserpine had no effect on [(3)H]NE uptake in HEK-293 cells transfected with the rat NET (293-hNET), cells that lack catecholamine storage vesicles. NET regulation by reserpine was independent of trafficking of the NET from the cell surface. Pre-exposure of cells to inhibitors of several intracellular signaling cascades known to regulate the NET, including Ca(2+)/Ca(2+)-calmodulin dependent kinase and protein kinases A, C and G, did not affect the ability of reserpine to reduce [(3)H]NE uptake. Treatment of PC12 cells with the catecholamine depleting agent, alpha-methyl-p-tyrosine, increased [(3)H]NE uptake and eliminated the inhibitory effects of reserpine on [(3)H]NE uptake. Reserpine non-competitively inhibits NET activity through a Ca(2+)-independent process that requires catecholamine storage vesicles, revealing a novel pharmacological method to modify NET function. Further characterization of the molecular nature of reserpine's action could lead to the development of alternative therapeutic strategies for treating disorders known to be benefitted by treatment with traditional competitive NET inhibitors.

Gene expression analyses of neurons, astrocytes, and oligodendrocytes isolated by laser capture microdissection from human brain: detrimental effects of laboratory humidity.

Laser capture microdissection (LCM) is a versatile computer-assisted dissection method that permits collection of tissue samples with a remarkable level of anatomical resolution. LCM's application to the study of human brain pathology is growing, although it is still relatively underutilized, compared with other areas of research. The present study examined factors that affect the utility of LCM, as performed with an Arcturus Veritas, in the study of gene expression in the human brain using frozen tissue sections. LCM performance was ascertained by determining cell capture efficiency and the quality of RNA extracted from human brain tissue under varying conditions. Among these, the relative humidity of the laboratory where tissue sections are stained, handled, and submitted to LCM had a profound effect on the performance of the instrument and on the quality of RNA extracted from tissue sections. Low relative humidity in the laboratory, i.e., 6-23%, was conducive to little or no degradation of RNA extracted from tissue following staining and fixation and to high capture efficiency by the LCM instrument. LCM settings were optimized as described herein to permit the selective capture of astrocytes, oligodendrocytes, and noradrenergic neurons from tissue sections containing the human locus coeruleus, as determined by the gene expression of cell-specific markers. With due regard for specific limitations, LCM can be used to evaluate the molecular pathology of individual cell types in post-mortem human brain.

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.

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.

Glutamate signaling proteins and tyrosine hydroxylase in the locus coeruleus of alcoholics.

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 eight 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.