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.

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.

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.

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.

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.

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.

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.

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.

Conventional and nonconventional roles of the nucleolus.

As the most prominent of subnuclear structures, the nucleolus has a well-established role in ribosomal subunit assembly. Additional nucleolar functions, not related to ribosome biogenesis, have been discovered within the last decade. Built around multiple copies of the genes for preribosomal RNA (rDNA), nucleolar structure is largely dependent on the process of ribosome assembly. The nucleolus is disassembled during mitosis at which time preribosomal RNA transcription and processing are suppressed; it is reassembled at the end of mitosis in part from components preserved from the previous cell cycle. Expression of preribosomal RNA (pre-rRNA) is regulated by the silencing of individual rDNA genes via alterations in chromatin structure or by controlling RNA polymerase I initiation complex formation. Preribosomal RNA processing and posttranscriptional modifications are guided by a multitude of small nucleolar RNAs. Nearly completed ribosomal subunits are exported to the cytoplasm by an established nuclear export system with the aid of specialized adapter molecules. Some preribosomal and nucleolar components are transiently localized in Cajal bodies, presumably for modification or assembly. The nonconventional functions of nucleolus include roles in viral infections, nuclear export, sequestration of regulatory molecules, modification of small RNAs, RNP assembly, and control of aging, although some of these functions are not well established. Additional progress in defining the mechanisms of each step in ribosome biogenesis as well as clarification of the precise role of the nucleolus in nonconventional activities is expected in the next decade.

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.

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.

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.].

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.

Protein NPM3 interacts with the multifunctional nucleolar protein B23/nucleophosmin and inhibits ribosome biogenesis.

Protein B23/nucleophosmin is a multifunctional protein that plays roles in ribosome biogenesis, control of centrosome duplication, and regulation of p53 expression. A yeast two-hybrid screen was performed in a search for interaction partners of B23. The complementary DNA for a highly acidic protein, nucleoplasmin 3 (NPM3), was found in multiple positive clones. Protein NPM3 and its interaction with B23 were further characterized. Endogenous B23 was able to be co-immunoprecipitated with NPM3, and this complex was resistant to ribonuclease treatment and high concentrations of salt. The N-terminal 35-90 amino acids of B23 were found to be required for their interaction. Separate co-immunoprecipitation studies of B23 and NPM3 suggested the existence of two different complexes, one containing B23 and 28 S ribosomal RNA (rRNA) and another composed of B23, NPM3, and other proteins, but no RNA. NPM3 was localized in the nucleolus, and its nucleolar localization depended on active rRNA transcription. In the cells overexpressing NPM3, there were decreased rates of pre-rRNA synthesis and processing. Overexpression of a mutant of NPM3 that did not interact with B23 did not alter pre-rRNA synthesis and processing, suggesting that the interaction of NPM3 with B23 plays a role in the ribosome biogenesis.

Role of protein kinase CK2 phosphorylation in the molecular chaperone activity of nucleolar protein b23.

Protein B23 is a multifunctional nucleolar protein whose molecular chaperone activity is proposed to play role in ribosome assembly. Previous studies (Szebeni, A., and Olson, M. O. J. (1999) Protein Sci. 8, 905-912) showed that protein B23 has several characteristics typical of molecular chaperones, including anti-aggregation activity, promoting the renaturation of denatured proteins, and preferential binding to denatured substrates. However, until now there has been no proposed mechanism for release of a bound substrate. Protein B23 can be phosphorylated by protein kinase CK2 (CK2) in a segment required for chaperone activity. The presence of bound substrate enhanced the rate of CK2 phosphorylation of protein B23 by 2-3-fold, and this enhancement was dependent on a nonpolar region in its N-terminal end. Formation of a complex between B23 and chaperone test substrates (rhodanese or citrate synthase) was inhibited by CK2 phosphorylation. Furthermore, CK2 phosphorylation of a previously formed B23-substrate complex promoted its dissociation. The dissociation of complexes between B23 and the human immunodeficiency virus-Rev protein required both CK2 phosphorylation and competition with a Rev nuclear localization signal peptide, suggesting that Rev binds B23 at two separate sites. These studies suggest that unlike many molecular chaperones, which directly hydrolyze ATP, substrate release by protein B23 is dependent on its phosphorylation by CK2.