[Research Progress of microRNA-7/124/155 in Parkinson's Disease].

Parkinson's disease(PD)is the second most common neurodegenerative disease after Alzheimer's disease,with high morbidity and high disability rate.Since the early symptoms of PD are not typical and often similar to those of normal aging or other diseases.It is easy to missed diagnosis and misdiagnosis,which seriously affects the diagnosis and treatment of this disease and aggravetes the burden on the patients' life.MicroRNAs(miRNA)are a class of endogenous non-coding RNAs that are involved in post-transcriptional regulation by binding to target messenger RNAs(mRNA).They are highly conserved,short,easy to obtain,and can stably exist in peripheral body fluids.They have been used as biomarkers for a variety of diseases.Recent studies have demonstrated that miRNA play an important role in the development of PD.This paper reviews the recent research progress of miR-7/124/155,three mature miRNA in PD,aiming to provide reference for clarifying the pathogenesis and guiding the diagnosis and treatment of PD.

Upregulated DNA Damage-Linked Biomarkers in Parkinson's Disease Model Mice.

SUMMARY STATEMENT: The present study examined expression of DNA damage markers in VMAT2 Lo PD model mice. The results demonstrate there is a significant increase in these DNA damage markers mostly in the brain regions of 18- and 23-month-old model mice, indicating oxidative stress-induced DNA lesion is an important pathologic feature of this mouse model.

Effects of Manipulation of Noradrenergic Activities on the Expression of Dopaminergic Phenotypes in Aged Rat Brains.

This study investigated the effects of the pharmacological manipulation of noradrenergic activities on dopaminergic phenotypes in aged rats. Results showed that the administration of L-threo-3,4-dihydroxyphenylserine (L-DOPS) for 21 days significantly increased the expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the striatum and substantia nigra (SN) of 23-month-old rats. Furthermore, this treatment significantly increased norepinephrine/DA concentrations in the striatum and caused a deficit of sensorimotor gating as measured by prepulse inhibition (PPI). Next, old rats were injected with the α2-adrenoceptor antagonist 2-methoxy idazoxan or ß2-adrenoceptor agonist salmeterol for 21 days. Both drugs produced similar changes of TH and DAT in the striatum and SN. Moreover, treatments with L-DOPS, 2-methoxy idazoxan, or salmeterol significantly increased the protein levels of phosphorylated Akt in rat striatum and SN. However, although a combination of 2-methoxy idazoxan and salmeterol resulted in a deficit of PPI in these rats, the administration of 2-methoxy idazoxan alone showed an opposite behavioral change. The in vitro experiments revealed that treatments with norepinephrine markedly increased mRNAs and proteins of ATF2 and CBP/p300 and reduced mRNA and proteins of HDAC2 and HDAC5 in MN9D cells. A ChIP assay showed that norepinephrine significantly increased CBP/p300 binding or reduced HDAC2 and HDAC5 binding on the TH promoter. The present results indicate that facilitating noradrenergic activity in the brain can improve the functions of dopaminergic neurons in aged animals. While this improvement may have biochemically therapeutic indication for the status involving the degeneration of dopaminergic neurons, it may not definitely include behavioral improvements, as indicated by using 2-methoxy idazoxan only.

Restoration of Noradrenergic Function in Parkinson's Disease Model Mice.

Dysfunction of the central noradrenergic and dopaminergic systems is the primary neurobiological characteristic of Parkinson's disease (PD). Importantly, neuronal loss in the locus coeruleus (LC) that occurs in early stages of PD may accelerate progressive loss of dopaminergic neurons. Therefore, restoring the activity and function of the deficient noradrenergic system may be an important therapeutic strategy for early PD. In the present study, the lentiviral constructions of transcription factors Phox2a/2b, Hand2 and Gata3, either alone or in combination, were microinjected into the LC region of the PD model VMAT2 Lo mice at 12 and 18 month age. Biochemical analysis showed that microinjection of lentiviral expression cassettes into the LC significantly increased mRNA levels of Phox2a, and Phox2b, which were accompanied by parallel increases of mRNA and proteins of dopamine ß-hydroxylase (DBH) and tyrosine hydroxylase (TH) in the LC. Furthermore, there was considerable enhancement of DBH protein levels in the frontal cortex and hippocampus, as well as enhanced TH protein levels in the striatum and substantia nigra. Moreover, these manipulations profoundly increased norepinephrine and dopamine concentrations in the striatum, which was followed by a remarkable improvement of the spatial memory and locomotor behavior. These results reveal that over-expression of these transcription factors in the LC improves noradrenergic and dopaminergic activities and functions in this rodent model of PD. It provides the necessary groundwork for the development of gene therapies of PD, and expands our understanding of the link between the LC-norepinephrine and dopamine systems during the progression of PD.

Transcription Factors Phox2a/2b Upregulate Expression of Noradrenergic and Dopaminergic Phenotypes in Aged Rat Brains.

The present study investigated the effects of forced overexpression of Phox2a/2b, two transcription factors, in the locus coeruleus (LC) of aged rats on noradrenergic and dopaminergic phenotypes in brains. Results showed that a significant increase in Phox2a/2b mRNA levels in the LC region was paralleled by marked enhancement in expression of DBH and TH per se. Furthermore, similar increases in TH protein levels were observed in the substantial nigra and striatum, as well as in the hippocampus and frontal cortex. Overexpression of Phox2 genes also significantly increased BrdU-positive cells in the hippocampal dentate gyrus and NE levels in the striatum. Moreover, this manipulation significantly improved the cognition behavior. The in vitro experiments revealed that norepinephrine treatments may increase the transcription of TH gene through the epigenetic action on the TH promoter. The results indicate that Phox2 genes may play an important role in improving the function of the noradrenergic and dopaminergic neurons in aged animals, and regulation of Phox2 gene expression may have therapeutic utility in aging or disorders involving degeneration of noradrenergic neurons.

Critical Role of Oxidatively Damaged DNA in Selective Noradrenergic Vulnerability.

An important pathology in Parkinson's disease (PD) is the earlier and more severe degeneration of noradrenergic neurons in the locus coeruleus (LC) than dopaminergic neurons in the substantia nigra. However, the basis of such selective vulnerability to insults remains obscure. Using noradrenergic and dopaminergic cell lines, as well as primary neuronal cultures from rat LC and ventral mesencephalon (VM), the present study compared oxidative DNA damage response markers after exposure of these cells to hydrogen peroxide (H2O2). The results showed that H2O2 treatment resulted in more severe cell death in noradrenergic cell lines SK-N-BE(2)-M17 and PC12 than dopaminergic MN9D cells. Furthermore, there were higher levels of oxidative DNA damage response markers in noradrenergic cells and primary neuronal cultures from the LC than dopaminergic cells and primary cultures from the VM. It included increased tail moments and tail lengths in Comet assay, and increased protein levels of phosphor-p53 and γ-H2AX after treatments with H2O2. Consistent with these measurements, exposure of SK-N-BE(2)-M17 cells to H2O2 resulted in higher levels of reactive oxygen species (ROS). Further experiments showed that exposure of SK-N-BE(2)-M17 cells to H2O2 caused an increased level of noradrenergic transporter, reduced protein levels of copper transporter (Ctr1) and 8-oxoGua DNA glycosylase, as well as amplified levels of Cav1.2 and Cav1.3 expression. Taken together, these experiments indicated that noradrenergic neuronal cells seem to be more vulnerable to oxidative damage than dopaminergic neurons, which may be related to the intrinsic characteristics of noradrenergic neuronal cells.

Norepinephrine upregulates the expression of tyrosine hydroxylase and protects dopaminegic neurons against 6-hydrodopamine toxicity.

As a classic neurotransmitter in the brain, norepinephrine (NE) also is an important modulator to other neuronal systems. Using primary cultures from rat ventral mesencephalon (VM) and dopaminergic cell line MN9D, the present study examined the neuroprotective effects of NE and its effects on the expression of tyrosine hydroxylase (TH). The results showed that NE protected both VM cultures and MN9D cells against 6-hydroxydopamine-caused apoptosis, with possible involvement of adrenal receptors. In addition, treatment with NE upregulated TH protein levels in dose- and time-dependent manner. Further experiments to investigate the potential mechanisms underlying this NE-induced upregulation of TH demonstrated a marked increase in protein levels of the brain-derived neurotrophic factor (BDNF) and the phosphorylated extracellular signal-regulated protein kinase 1 and 2 (pERK1/2) in VM cultures treated with NE. In MN9D cells, a significantly increase of TH and pERK1/2 protein levels were observed after their transfection with BDNF cDNA or exposure to BDNF peptides. Treatment of VM cultures with K252a, an antagonist of the tropomyosin-related kinase B, blocked the upregulatory effects of NE on TH, BDNF and pERK1/2. Administration of MEK1 & MEK2 inhibitors also reversed NE-induced upregulation of TH and pERK1/2. Moreover, ChIP assay showed that treatment with NE or BDNF increased H4 acetylation in the TH promoter. These results suggest that the neuroprotection and modulation of NE on dopaminergic neurons are mediated via BDNF and MAPK/ERK pathways, as well as through epigenetic histone modification, which may have implications for the improvement of therapeutic strategies for Parkinson's disease.

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.

Noradrenergic Modulation on Dopaminergic Neurons.

It is now well accepted that there is a close relationship between noradrenergic and dopaminergic neurons in the brain, especially referring to the modulation of the locus coeruleus-norepinephrine (LC-NE) system on dopamine transmission. The disturbance of this modulation may contribute to neurodegeneration of dopaminergic neurons in Parkinson's disease. In this article, we briefly review evidence related to such modulation. Firstly, we illustrated the noradrenergic innervation and functional implication for the LC-NE system and nigra-striatum dopaminergic system. Furthermore, we depicted neuroprotective effects of the LC-NE on dopaminergic neurons in vivo and in vitro. Moreover, we present data implicating the potential mechanisms underlying the modulation of the LC-NE system on dopaminergic neurons, in particular the effects of NE as a neurotrophic factor and through its ability to stimulate the expression of other neurotrophic factors, such as the brain-derived neurotrophic factor. Finally, we discussed other mechanisms intrinsic to NE's effects. A better understanding of the noradrenergic modulation on dopaminergic neurons may be rewarding by significant advances in etiologic study and promising treatment of Parkinson's disease.

The regulation of corticosteroid receptors in response to chronic social defeat.

Our previous studies demonstrated that chronic social defeat (CSD) up-regulated expression of the serotonin transporter (SERT) and norepinephrine transporter (NET) in the brain, which was mediated by corticosteroid receptors. In the present study we first analyzed the alterations of corticosteroid receptors in different brain regions after the CSD paradigm. The results showed that CSD significantly reduced glucocorticoid receptor (GR) protein levels in the CA1 and dentate gyrus of the hippocampus, as well as in central and basolateral nuclei of the amygdala, which was accompanied by the translocation of GR from cytoplasm to nuclei. CSD also markedly reduced GR mRNA levels and MR immunoreactivity in the CA1, CA3 and dentate gyrus areas of the hippocampus. Conversely, CSD pronouncedly enhanced GR mRNA and protein levels in the dorsal raphe nucleus and locus coeruleus relative to the control. As an extension of our previous studies, in situ hybridization and immunohistochemical staining demonstrated that CSD regimen caused a notable increase of SERT mRNA levels in the dorsal raphe nucleus and increased SERT immunoreactivities in CA1 and CA3 of the hippocampus, as well as those in the basolateral nuclei of the amygdala. Likewise, CSD regimen resulted in an evident enhancement of NET immunoreactivity in the CA1 of the hippocampus and in the basolateral nuclei of the amygdala. Our current findings suggest that GR expressional alterations in response to CSD are complex and brain region-specific, which may correspond to their different functions in these regions.

MicroRNAs 29b and 181a down-regulate the expression of the norepinephrine transporter and glucocorticoid receptors in PC12 cells.

MicroRNAs are short non-coding RNAs that provide global regulation of gene expression at the post-transcriptional level. Such regulation has been found to play a role in stress-induced epigenetic responses in the brain. The norepinephrine transporter (NET) and glucocorticoid receptors are closely related to the homeostatic integration and regulation after stress. Our previous studies demonstrated that NET mRNA and protein levels in rats are regulated by chronic stress and by administration of corticosterone, which is mediated through glucocorticoid receptors. Whether miRNAs are intermediaries in the regulation of these proteins remains to be elucidated. This study was undertaken to determine possible regulatory effects of miRNAs on the expression of NET and glucocorticoid receptors in the noradrenergic neuronal cell line. Using computational target prediction, we identified several candidate miRNAs potentially targeting NET and glucocorticoid receptors. Western blot results showed that over-expression of miR-181a and miR-29b significantly repressed protein levels of NET, which is accompanied by a reduced [3 H] norepinephrine uptake, and glucocorticoid receptors in PC12 cells. Luciferase reporter assays verified that both miR-181a and miR-29b bind the 3'UTR of mRNA of NET and glucocorticoid receptors. Furthermore, exposure of PC12 cells to corticosterone markedly reduced the endogenous levels of miR-29b, which was not reversed by the application of glucocorticoid receptor antagonist mifepristone. These observations indicate that miR-181a and miR-29b can function as the negative regulators of NET and glucocorticoid receptor translation in vitro. This regulatory effect may be related to stress-induced up-regulation of the noradrenergic phenotype, a phenomenon observed in stress models and depressive patients. This study demonstrated that miR-29b and miR-181a, two short non-coding RNAs that provide global regulation of gene expression, markedly repressed protein levels of norepinephrine (NE) transporter and glucocorticoid receptor (GR), as well as NE uptake by binding the 3'UTR of their mRNAs in PC12 cells. Also, exposure of cells to corticosterone significantly reduced miR-29b levels through a GR-independent way.

DNA Damage in Major Psychiatric Diseases.

Human cells are exposed to exogenous insults and continuous production of different metabolites. These insults and unwanted metabolic products might interfere with the stability of genomic DNA. Recently, many studies have demonstrated that different psychiatric disorders show substantially high levels of oxidative DNA damage in the brain accompanied with morphological and functional alterations. It reveals that damaged genomic DNA may contribute to the pathophysiology of these mental illnesses. In this article, we review the roles of oxidative damage and reduced antioxidant ability in some vastly studied psychiatric disorders and emphasize the inclusion of treatment options involving DNA repair. In addition, while most currently used antidepressants are based on the manipulation of the neurotransmitter regulation in managing different mental abnormalities, they are able to prevent or reverse neurotoxin-induced DNA damage. Therefore, it may be plausible to target on genomic DNA alterations for psychiatric therapies, which is of pivotal importance for future antipsychiatric drug development.

Corticotropin releasing factor up-regulates the expression and function of norepinephrine transporter in SK-N-BE (2) M17 cells.

Corticotropin releasing factor (CRF) has been implicated to act as a neurotransmitter or modulator in central nervous activation during stress. In this study, we examined the regulatory effect of CRF on the expression and function of the norepinephrine transporter (NET) in vitro. SK-N-BE (2) M17 cells were exposed to different concentrations of CRF for different periods. Results showed that exposure of cells to CRF significantly increased mRNA and protein levels of NET in a concentration- and time-dependent manner. The CRF-induced increase in NET expression was mimicked by agonists of either CRF receptor 1 or 2. Furthermore, similar CRF treatments induced a parallel increase in the uptake of [(3) H] norepinephrine. Both increased expression and function of NET caused by CRF were abolished by simultaneous administration of CRF receptor antagonists, indicating a mediation by CRF receptors. However, there was no additive effect for the combination of both receptor antagonists. Chromatin immunoprecipitation assays confirm an increased acetylation of histone H3 on the NET promoter following treatment with CRF. Taken together, this study demonstrates that CRF up-regulates the expression and function of NET in vitro. This regulation is mediated through CRF receptors and an epigenetic mechanism related to histone acetylation may be involved. This CRF-induced regulation on NET expression and function may play a role in development of stress-related depression and anxiety. This study demonstrated that corticotropin release factor (CRF) up-regulated the expression and function of norepinephrine transporter (NET) in a concentration- and time-dependent manner, through activation of CRF receptors and possible histone acetylation in NET promoter. The results indicate that their interaction may play an important role in stress-related physiological and pathological status.

Effects of Antidepressants on DSP4/CPT-Induced DNA Damage Response in Neuroblastoma SH-SY5Y Cells.

DNA damage is a form of cell stress and injury. Increased systemic DNA damage is related to the pathogenic development of neurodegenerative diseases. Depression occurs in a relatively high percentage of patients suffering from degenerative diseases, for whom antidepressants are often used to relieve depressive symptoms. However, few studies have attempted to elucidate why different groups of antidepressants have similar effects on relieving symptoms of depression. Previously, we demonstrated that neurotoxins N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4)- and camptothecin (CPT) induced the DNA damage response in SH-SY5Y cells, and DSP4 caused cell cycle arrest which was predominately in the S-phase. The present study shows that CPT treatment also resulted in similar cell cycle arrest. Some classic antidepressants could reduce the DNA damage response induced by DSP4 or CPT in SH-SY5Y cells. Cell viability examination demonstrated that both DSP4 and CPT caused cell death, which was prevented by spontaneous administration of some tested antidepressants. Flow cytometric analysis demonstrated that a majority of the tested antidepressants protect cells from being arrested in S-phase. These results suggest that blocking the DNA damage response may be an important pharmacologic characteristic of antidepressants. Exploring the underlying mechanisms may allow for advances in the effort to improve therapeutic strategies for depression appearing in degenerative and psychiatric diseases.

Neurotoxin-induced DNA damage is persistent in SH-SY5Y cells and LC neurons.

Degeneration of the noradrenergic neurons has been reported in the brain of patients suffering from neurodegenerative diseases. However, their pathological characteristics during the neurodegenerative course and underlying mechanisms remain to be elucidated. In the present study, we used the neurotoxin camptothecin (CPT) to induce the DNA damage response in neuroblastoma SH-SY5Y cells, normal fibroblast cells, and primarily cultured locus coeruleus (LC) and raphe neurons to examine cellular responses and repair capabilities after neurotoxin exposure. To our knowledge, the present study is the first to show that noradrenergic SH-SY5Y cells are more sensitive to CPT-induced DNA damage and deficient in DNA repair, as compared to fibroblast cells. Furthermore, similar to SH-SY5Y cells, primarily cultured LC neurons are more sensitive to CPT-induced DNA damage and show a deficiency in repairing this damage. Moreover, while N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) exposure also results in DNA damage in cultured LC neurons, neither CPT nor DSP4 induce DNA damage in neuronal cultures from the raphe nuclei. Taken together, noradrenergic SH-SY5Y cells and LC neurons are sensitive to CPT-induced DNA damage and exhibit a repair deficiency, providing a mechanistic explanation for the pathological characteristics of LC degeneration when facing endogenous and environmental DNA-damaging insults in vivo.

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.

Corticosterone administration up-regulated expression of norepinephrine transporter and dopamine ß-hydroxylase in rat locus coeruleus and its terminal regions.

Stress has been reported to activate the locus coeruleus (LC)-noradrenergic system. In this study, corticosterone (CORT) was orally administrated to rats for 21 days to mimic stress status. In situ hybridization measurements showed that CORT ingestion significantly increased mRNA levels of norepinephrine transporter (NET) and dopamine ß-hydroxylase (DBH) in the LC region. Immunofluorescence staining and western blotting revealed that CORT treatment also increased protein levels of NET and DBH in the LC, as well as NET protein levels in the hippocampus, the frontal cortex and the amygdala. However, CORT-induced increase in DBH protein levels only appeared in the hippocampus and the amygdala. Elevated NET and DBH expression in most of these areas (except for NET protein levels in the LC) was abolished by simultaneous treatment with combination of corticosteroid receptor antagonist mifepristone and spironolactone (s.c. for 21 days). Also, treatment with mifepristone alone prevented CORT-induced increases of NET expression and DBH protein levels in the LC. In addition, behavioral tasks showed that CORT ingestion facilitated escape in avoidance trials using an elevated T-maze, but interestingly, there was no significant effect on the escape trial. Corticosteroid receptor antagonists failed to counteract this response in CORT-treated rats. In the open-field task, CORT treatment resulted in less activity in a defined central zone compared to controls and corticosteroid receptor antagonist treatment alleviated this increase. In conclusion, this study demonstrates that chronic exposure to CORT results in a phenotype that mimics stress-induced alteration of noradrenergic phenotypes, but the effects on behavior are task dependent. As the sucrose consumption test strongly suggests CORT ingestion-induced depression-like behavior, further elucidation of underlying mechanisms may improve our understanding of the correlation between stress and the development of depression.

Effects of DSP4 on the noradrenergic phenotypes and its potential molecular mechanisms in SH-SY5Y cells.

Dopamine ß-hydroxylase (DBH) and norepinephrine (NE) transporter (NET) are the noradrenergic phenotypes for their functional importance to noradrenergic neurons. It is known that in vivo N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) treatment induces degeneration of noradrenergic terminals by interacting with NET and depleting intracellular NE. However, DSP4's precise mechanism of action remains unclear. In this study various biochemical approaches were employed to test the hypothesis that DSP4 down-regulates the expression of DBH and NET, and to determine molecular mechanisms that may be involved. The results showed that treatment of SH-SY5Y neuroblastoma cells with DSP4 significantly decreased mRNA and protein levels of DBH and NET. DSP4-induced reduction of DBH mRNA and proteins, as well as NET proteins showed a time- and concentration-dependent manner. Flow cytometric analysis demonstrated that DSP4-treated cells were arrested predominantly in the S-phase, which was reversible. The arrest was confirmed by several DNA damage response markers (phosphorylation of H2AX and p53), suggesting that DSP4 causes replication stress which triggers cell cycle arrest via the S-phase checkpoints. Moreover, the comet assay verified that DSP4 induced single-strand DNA breaks. In summary, the present study demonstrated that DSP4 down-regulates the noradrenergic phenotypes, which may be mediated by its actions on DNA replication, leading to replication stress and cell cycle arrest. These action mechanisms of DSP4 may account for its degenerative consequence after systematic administration for animal models.

Effects of chronic social defeat on expression of dopamine ß-hydroxylase in rat brains.

It is documented that stress activates the locus coeruleus-norepinephrine system. However, there are far few reports regarding effects of stress on the expression of dopamine ß-hydroxylase, a hallmark enzyme of the noradrenergic neuron. In the present study, adult Fischer 344 rats were subjected to chronic social defeat for 4 weeks. Dopamine ß-hydroxylase expressional levels in the locus coeruleus and its terminal regions were measured by in situ hybridization and western blotting. The results showed that immediately following chronic social defeat there are significantly increased mRNA and protein levels of dopamine ß-hydroxylase in the locus coeruleus, and dopamine ß-hydroxylase protein levels in the hippocampus, frontal cortex and amygdala, compared with those in the control. This chronic social defeat-induced upregulation of dopamine ß-hydroxylase was completely abolished by adrenalectomy, and/or by treatment with corticosteroid receptor antagonists, mifepristone and spironolactone, either alone or in combination. Furthermore, treatment with desipramine, an antidepressant with specific inhibitory effects on norepinephrine transport, prevented an increased dopamine ß-hydroxylase expression by chronic social defeat in the locus coeruleus and its main terminal regions such as the hippocampus, frontal cortex and amygdala. However, treatment with fluoxetine, an antidepressant with specific inhibition for serotonin transport, only selectively blocked increased dopamine ß-hydroxylase protein levels in the hippocampus caused by CSD. The present findings indicate that chronic social defeat activates the locus coeruleus-norepinephrine system by upregulating the expression of dopamine ß-hydroxylase, which may increase norepinephrine synthesis. This chronic social defeat induced upregulation of DBH expression was mediated through corticosterone and corticosteroid receptors, with possible interference from antidepressants.

Chronic social defeat up-regulates expression of the serotonin transporter in rat dorsal raphe nucleus and projection regions in a glucocorticoid-dependent manner.

Chronic stress and dysfunction of the serotonergic system in the brain have been considered two of the major risks for development of depression. In this study, adult Fischer 344 rats were subjected to a regimen of chronic social defeat (CSD). To mimic stressful conditions, some rats were not exposed to CSD, but instead treated with corticosterone (CORT) in oral solution while maintained in their home cage. Protein levels of the serotonin transporter (SERT) in the dorsal raphe nucleus (DRN), hippocampus, frontal cortex, and amygdala were examined by Western blotting or immunofluorescence staining. The results showed that CSD up-regulated SERT protein levels in the DRN, hippocampus, frontal cortex, and amygdala regions. This up-regulation was abolished or prevented by adrenalectomy, or treatment with antagonists of corticosteroid receptors mifepristone and spironolactone, alone or in combination. Similarly, up-regulated SERT protein levels in these brain regions were also observed in rats treated with oral CORT ingestion, which was analogously prevented by treatment with mifepristone and spironolactone. Furthermore, both CSD- and CORT-induced up-regulation of SERT protein levels in the DRN and three brain regions were attenuated by simultaneous treatment with fluoxetine, an antidepressant that specifically inhibits serotonin reuptake. The results indicate that up-regulation in SERT protein levels in the DRN and forebrain limbic structures caused by CSD regimen was mainly motivated by CORT through corticosteroid receptors. The present findings demonstrate that chronic stress is closely correlated with the serotonergic system by acting on the regulation of the SERT expression in the DRN and its projection regions, which may contribute to the development of depression.