國 立 成 功 大 學 補 助 優 秀
新 進 教 師 學 術 研 究 計 畫 結 案 報 告
情緒穩定劑於神經膠細胞之分子作用機轉
計畫主持人：陳柏熹
執行單位：醫學系精神學科
中文摘要:
關鍵詞： 神經膠細胞，情緒穩定劑、雙極性情感性精神病
近來之研究顯示神經膠細胞密切參與各種腦內訊息處理的過程。 瞭解神經膠細胞的功
能可能改變目前我們對腦與心智運作模式的了解。 而神經膠細胞功能失控也可能成為引發
多種疾病之原因。 研究發現神經病變性疼痛、癲癇與多種退化性神經疾病，甚至精神分裂
症、雙極性情感性精神病等精神疾病之病理機轉中神經膠細胞皆可能扮演重要角色。 因此
神經膠細胞也可能成為治療腦疾病的目標細胞。
雙極性情感性精神病又稱躁鬱症，是次於精神分裂症之第二常見精神病。目前雙極性
情感性精神病的藥物治療，以情緒穩定劑為主，包括 Lithium、Valproate (Depakote®)及
Carbamazepine (Tegretol®) 等。 然先前多數對情緒穩定劑作用機轉之研究均針對其對神經
元之作用為主。 而在神經元已知的眾多分子標的中，情緒穩定劑之間目前並未發現一共同
之作用標的可以解釋其共同的治療作用。
早期研究已經知道情緒穩定劑中之Valproate 於分子層次的作用包括調節細胞內訊息傳
導、神經傳導物質濃度以及離子通道等。 近來亦發現 Valproate 可藉抑制 HDAC 等蛋白之
乙烯化作用來調節基因表現。 但仍未確定這些作用與雙極性情感性精神病的臨床治療功效
有關。本研究之目的即是希望探討情緒穩定劑Valproate 對神經膠細胞之功能之影響與瞭解
其分子作用機轉。藉此將可進一步探討Valproate 對腦內神經膠細胞與神經元間訊息互動之
影響，進而驗證神經膠細胞在雙極性精神疾病治療與病理機轉中可能扮演之角色。
英文摘要:
Key word: Glia, mood stabilizer, bipolar disorder
Recent work indicates that glial cells are intimately involved in all aspects of our brain's information processing. Not only do glial cells talk with neurons, they communicate among themselves. Understanding more about how glia function may greatly alter our model of how the brain and mind work. The emerging realization of the importance of glia has given new life to an idea that glia may have key roles in central nervous system disorders from neuropathic pain and epilepsy to neurodegenerative diseases and may even contribute to schizophrenia, bipolar disorder, and other psychiatric disorders. There are also hints that glia may be promising therapeutic targets--a possibility that researchers have scarcely begun to explore.
Bipolar disorder, also known as manic-depressive illness, is a brain disorder that causes unusual shifts in a person's mood, energy, and ability to function. Most scientists now agree that there is no single cause for bipolar disorder—rather, many factors act together to produce the illness. Lithium, is the first mood-stabilizer approved by the U.S. Food and Drug Administration (FDA). Anticonvulsant medications, such as valproate (Depakote®) or carbamazepine (Tegretol®) also can have mood-stabilizing effects. However, most of the previous studies concerning the effects of mood stabilizers emphasize on the direct neuronal effects and thus might have excluded the role of glial cells.
Among them valproate is known to act on neuronal signal transduction, modulate neuronal transmission and ion channels. Recently valproate is noted to regulate gene expression through the inhibition of histone deacetylase (HDAC). However, the relationships between its pharmacological effects and therapeutic effects remain unclear. The main targets for this work will focus on the effects and molecular targets of valrpoate on glial cells. From there we can see how valproate modulates neuron-glail interactions and the role of glial cells in the brain pathology of bipolar disorder.
Introduction
Bipolar disorder, also known as manic-depressive illness, is a brain disorder that causes unusual shifts in a person's mood, energy, and ability to function. Bipolar disorder afflicts approximately 1-3% of both men and women. Different from the normal ups and downs that everyone goes through, the symptoms of bipolar disorder are severe. They can result in damaged relationships, poor job or school performance, and even suicide. Episodes of mania and
depression typically recur across the life span. Between episodes, most people with bipolar disorder are free of symptoms, but as many as one-third of people have some residual symptoms.
A small percentage of people experience chronic unremitting symptoms despite treatment.
Treatment of bipolar disorders has progressed significantly in the last decade [1-2]. However, there has been no consensus on mechanisms underlying the therapeutic actions. The treatment of bipolar disorder depends on the current state of illness: manic phase, depressive phase, or
maintenance phase. During manic phase, Lithium (Li), valproate (VPA) and several second-generation antipsychotics (SGA), including olanzapine, risperidone, quetiapine, ziprasidone, and aripiprazole, are first-line treatments for acute mania (Anonymous 1994;
Solomon, Keitner et al. 1995; Scherk, Pajonk et al. 2007). With adequate dosing of medications and serum level, manic patients show appreciable drug effects on the 10th-14th days of treatment.
Figure 1.1 and table 1.1 show the treatment algorithm and the recommendation medications for mania episodes [3].
It is noteworthy that the most common mood stabilizers differ greater in their structures.
With the exception of antipsychotics, lithium and antiepileptics do not appear to specifically target cell surface receptors. Recent data has demonstrated that mood stabilizers exert major effects on intracellular signaling pathways that regulate cellular plasticity (Einat and Manji 2006;
Zhou, Zarate et al. 2006). The initial molecular targets modulated by the current mood stabilizers are listed the following tables (Gould, Quiroz et al. 2004; Quiroz, Singh et al. 2004). Glial cells are now been considered as an important therapeutic target for treatments [4]. Electroconvulsive seizures for treating mood disorders are noted to stimulate glial proliferation within the prefrontal
cortex of rats [5]. Strategies activated astrocytes could promote neuronal survival and rescue injured neurons by eliminating toxic substances, releasing neurotrophins and promoting tissue repair [6-7]. One neurotransmitter important for mood regulation, serotonin, was also found to regulate synthesis of the neurotrophic factor in astrocytes [8]. Glial cells may also play important roles in the neurogenesis process related to pharmacological treatments as well as the
non-pharmacological treatments for mood disorders [9-10].
We have previously shown that the mood stabilizer, valproic acid (VPA), increases the expression of GDNF and BDNF in astrocytes. This leads to indirect neurotrophic and protective effects on DA neurons in midbrain neuron-glia cultures [11]. Additionally, other studies also demonstrate that VPA increases GDNF and BDNF transcription in C6 glioma cells [12]. However, the mechanisms underlying effects of VPA remain unclear. In the current study, we explore
whether actions of VPA on BDNF and GDNF transcription in astrocytes, and DA neuronal survival are associated with HDAC inhibition and histone hyperacetylation.
Results
Fig. 1 Treatment with HDAC inhibitors preserves DA neuronal function in MPP+-treated neuron-glia cultures. Midbrain neuron-glia cultures were treated with vehicle, sodium butyrate (SB) (A) or trichostatin A (TSA) (B) at the indicated concentrations in the presence (black bars) or absence (white bars) of 0.5 μM MPP+. MPP+ was added 30 min after SB and TSA pretreatment. DA neuronal function was evaluated by the [3H]-dopamine uptake assay 7 d after the varying treatments. Results are expressed as percent of control and represent means ± S.E. of three separate experiments. *p< 0.05, **p< 0.01, compared with untreated control groups; #p<
0.05, ##p< 0.01, compared with MPP+ alone group.
Fig. 2. HDAC inhibitors prevent cell loss of DA neurons in MPP+-treated neuron-glia cultures.
Midbrain neuron-glia cultures were treated with vehicle, 1.2 mM VPA, 1.2 mM SB, or 100 nM TSA in the presence (white bars) or absence (black bars) of 0.5 μM MPP+. MPP+ was added 30 min after SB and TSA pretreatment. DA neuronal cell loss was assessed by tyrosine hydroxylase (TH) immunostaining 7 d after treatment (A). Results are expressed as percent of control and are the means ± S.E. of three experiments. *p< 0.05, **p< 0.01, compared with untreated control groups; #p< 0.05, compared with MPP+ alone group. (B) Representative micrographs of morphological changes observed following treatment, as indicated in (A).
Fig. 3. HDAC inhibitors increase mRNA levels of GDNF in enriched astrocyte cultures. Enriched cortical astrocytes were treated with 1.2 mM VPA (A), 1.2 mM SB (B), or 100 nM TSA (C) and total RNA was harvested at 3, 6, 12, 24 or 48 h after treatment. First-strand cDNA was synthesized and then used for real-time PCR to determine relative amounts of GDNF mRNA.
Results are expressed as percent of untreated control and are the means ± S.E. of at least 3 separate experiments. *p< 0.05, * *p< 0.01, compared with untreated controls.
Fig. 4. HDAC inhibitors increase mRNA levels of BDNF in enriched astrocyte cultures. Enriched cortical astrocytes were treated with 1.2 mM VPA (A), 1.2 mM SB (B), or 100 nM TSA (C) and the total RNA was harvested at 3, 6, 12, 24 or 48 h after treatment. First-strand cDNA was synthesized and then used for real-time PCR to determine the amount of BDNF mRNA. Results are expressed as percent of untreated control and are the means ± S.E. of three to five independent experiments. * p< 0.05, * * p< 0.01, compared with control groups.
Fig. 5. HDAC inhibitors enhance GDNF promoter activity. C6 glioma cells were transfected with a pGL3-GDNF−1412/+24 reporter construct and treated with 1.2 mM VPA, 1.2 mM SB or 100 nM TSA for 24 h before the GDNF promoter activity was assayed in the luciferase reporter system. Results are the means ± S.E. of 3 independent experiments and expressed as relative luciferase activity (firefly luciferase relative to Renilla luciferase activity) compared to untreated cells. * p< 0.05, compared with control groups.
Fig. 6. GDNF promoter-associated histone H3 is hyperacetylated in astrocytes treated wih HDAC inhibitors. (A) Schematic representation of the rat GDNF promoter. The locations of GDNF promoter primers are indicated with arrowheads. (B) Enriched cortical astrocyte cultures were treated with 1.2 mM VPA, 1.2 mM SB, or 100 nM TSA for 5, 24 or 48 h. ChIP assay was performed using an anti-acetyl-histone H3 antibody. The amount of immunoprecipitated (ChIP DNA) and non-immunoprecipitated genome DNA (input DNA) following 24 h treatment was measured by PCR with GDNF Pa, GDNF Pb and GDNF Pc primer sets. PCR products were run on a 2% agarose gel and stained with ethidium bromide. Representative results from gels for each primer set are shown. (C) Real time PCR was performed with the GDNF Pc primer set to quantify the ChIP DNA and input DNA after 5, 24 or 48 h treatment using a relative standard curve method. The values of the ChIP DNA were normalized to the input DNA. Data are expressed as fold-change over the control and are the means ± S.E. of three independent experiments. * p< 0.05, * * p< 0.01, compared with control groups.
Discussion
Neurotrophic factors have been reported to protect neurons against noxious insults and promote neuron survival [13-16]. Thus, the identifcation of compounds that can induce endogenous secretion of neurotrophic factors would be of great significance in the treatment of various neurodegenerative diseases. Moreover, it has been hypothesized that a deficiency in the trophic support to neurons underlies the pathophysiology of mood disorders [17], implicating neurotrophic factors as a target of the treatment drugs. Indeed, evidence has shown that chronic treatment with antidepressants in animal models induces the expression of brain-derived neurotrophic factor (BDNF) and activation of its receptor signaling pathways in discrete brain areas, which are critical for their clinical efficacy [18].
We have previously shown that the mood stabilizer valproic acid (VPA) increases the expression of glial cell line-derived neurotrophic factor (GDNF) and BDNF in astrocytes. This leads to indirect neurotrophic and protective effects on dopaminergic (DA) neurons in midbrain neuron-glia cultures [11]. Additionally, another study also demonstrated that VPA increases GDNF and BDNF transcription in C6 glioma cells [12]. Further, C6 glioma cells treated with various antidepressants show an increased expression of GDNF mRNA and protein [19].
Together, these studies strongly suggest that neurotrophic factors induced from astrocytes may mediate some therapeutic effects of drugs for mood disorders.
VPA was recently shown to act as a direct inhibitor of histone deacetylase (HDAC) [20-21]. Increasing evidence supports that HDAC inhibitors are neuroprotective against multiple insults. For instance, suberoyl anilide hydroxamic acid (SAHA) and sodium butyrate (SB) have been shown to ameliorate neurodegenerative phenotypes in several models of polyglutamine-associated neurodegenerative diseases [22-25]. Additionally, HDAC inhibitors are also neuroprotective in models of ischemia, oxidative stress [26-27], and neuroinflammation [28-29]. In the current study, we explored whether the effects of VPA on BDNF and GDNF transcription in astrocytes, and the survival of DA neurons are related to HDAC inhibition and
epilepsy, bipolar disorder and neuropathic pain. As we hypothesized, the disturbances of glial function could contribute to psychopathology, and glial cells may serve as appropriate targets for psychotrophic medications. Recent glial researches have further clarified the possible role of glia in psychiatric disorders. [30]. Moreover, a series genetic and pharmacological studies suggested that D-serine released from astrocytes could modulate the tensor of synaptic plasticity through NMDA receptors, which are known to be involved in the pathogenesis of schizophrenia [31-34].
D-serine had also been tried to be an add-on treatment for acute exacerbation of schizophrenia [35-37]. Evidences also supported glial cells as important targets for psychotrophic medications [38]. Minocycline showed possible antipsychotic effects on patients with schizophrenia [39].
After demonstrated glial cells are important cellular targets for VPA, we also showed that HDAC inhibition by VPA is the key molecular mechanisms for regulating glial function. There are cumulating evidences indicate VPA is a potent epigenetic modulator for brain function [40].
VPA can indirectly regulate methylation of CpG islands through blocking HDAC activity [41].
Treatment with VPA may lead to reactivation of methylation-silenced genes that are implicated in complex neuropsychiatric disorders [42-44]. Research also showed that treatment strategies which target the epigenetic machinery can restore normal gene activity in complex psychiatric disorders [45]. Sodium butyrate, another HDAC inhibitor investigated in the current study, was just noted to show antidepressant-like effects recently [46]. A newly published report showed lithium regulates survival by modulating histone methylation and chromatin structure [47].
Overall, the current study suggests VPA may achieve its therapeutic effects through epigenetic modulation.
參考文獻
1. Eldar-Finkelman, H., Glycogen synthase kinase 3: an emerging therapeutic target. Trends in Molecular Medicine, 2002. 8(3): p. 126-32.
2. Manji, H.K. and C.A. Zarate, Molecular and cellular mechanisms underlying mood stabilization in bipolar disorder: implications for the development of improved therapeutics. Molecular Psychiatry, 2002. 7(1).
3. Yatham, L.N., et al., Canadian Network for Mood and Anxiety Treatments (CANMAT) guidelines for the management of patients with bipolar disorder: update 2007. Bipolar Disorders, 2006. 8(6): p. 721-39.
4. Halassa, M.M., T. Fellin, and P.G. Haydon, The tripartite synapse: roles for
gliotransmission in health and disease. Trends Mol Med, 2007. 13(2): p. 54-63.
5. Ongur, D., et al., Electroconvulsive seizures stimulate glial proliferation and reduce expression of Sprouty2 within the prefrontal cortex of rats. Biol Psychiatry, 2007. 62(5): p.
505-12.
6. Batchelor, P.E., et al., Activated macrophages and microglia induce dopaminergic
sprouting in the injured striatum and express brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor. Journal of Neuroscience, 1999. 19(5): p. 1708-16.
7. Dong, Y. and E.N. Benveniste, Immune function of astrocytes. Glia, 2001. 36(2): p.
180-90.
8. Krzan, M., V.W. Wu, and J.P. Schwartz, Serotonin regulation of nerve growth factor synthesis in neonatal and adult astrocytes: comparison to the beta-adrenergic agonist isoproterenol. Journal of Neuroscience Research, 2001. 64(3): p. 261-7.
9. Gould, E. and P. Tanapat, Stress and hippocampal neurogenesis. Biological Psychiatry, 1999. 46(11): p. 1472-9.
10. Mehler, M.F., et al., Cytokine regulation of neuronal differentiation of hippocampal progenitor cells. Nature, 1993. 362(6415): p. 62-5.
11. Chen, P.S., et al., Valproate protects dopaminergic neurons in midbrain neuron/glia
cultures by stimulating the release of neurotrophic factors from astrocytes. Mol Psychiatry, 2006. 11(12): p. 1116-25.
12. Castro, L.M., M. Gallant, and L.P. Niles, Novel targets for valproic acid: up-regulation of melatonin receptors and neurotrophic factors in C6 glioma cells. J Neurochem, 2005.
95(5): p. 1227-36.
13. Lin, L.F., et al., GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science, 1993. 260(5111): p. 1130-2.
14. Son, J.H., et al., Neuroprotection and neuronal differentiation studies using substantia nigra dopaminergic cells derived from transgenic mouse embryos. J Neurosci, 1999. 19(1):
p. 10-20.
15. Grondin, R. and D.M. Gash, Glial cell line-derived neurotrophic factor (GDNF): a drug candidate for the treatment of Parkinson's disease. J Neurol, 1998. 245(11 Suppl 3): p.
P35-42.
16. Lapchak, P.A., et al., Glial cell line-derived neurotrophic factor: a novel therapeutic approach to treat motor dysfunction in Parkinson's disease. Exp Neurol, 1997. 144(1): p.
29-34.
17. Manji, H.K., W.C. Drevets, and D.S. Charney, The cellular neurobiology of depression.
Nat Med, 2001. 7(5): p. 541-7.
18. Duman, R.S., Role of neurotrophic factors in the etiology and treatment of mood disorders.
Neuromolecular Med, 2004. 5(1): p. 11-25.
19. Hisaoka, K., et al., Antidepressants Increase Glial Cell Line-Derived Neurotrophic Factor Production through Monoamine-Independent Activation of Protein Tyrosine Kinase and Extracellular Signal-Regulated Kinase in Glial Cells. J Pharmacol Exp Ther, 2007. 321(1):
21. Gottlicher, M., et al., Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells. Embo J, 2001. 20(24): p. 6969-78.
22. Steffan, J.S., et al., Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila. Nature, 2001. 413(6857): p. 739-43.
23. Ryu, H., et al., Histone deacetylase inhibitors prevent oxidative neuronal death
independent of expanded polyglutamine repeats via an Sp1-dependent pathway. Proc Natl Acad Sci U S A, 2003. 100(7): p. 4281-6.
24. Gardian, G., et al., Neuroprotective effects of phenylbutyrate against MPTP neurotoxicity, in Neuromolecular Med. 2004. p. 235-41.
25. Hockly, E., et al., Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, ameliorates motor deficits in a mouse model of Huntington's disease. Proc Natl Acad Sci U S A, 2003. 100(4): p. 2041-6.
26. Ren, M., et al., Valproic acid reduces brain damage induced by transient focal cerebral ischemia in rats: potential roles of histone deacetylase inhibition and heat shock protein induction. J Neurochem, 2004. 89(6): p. 1358-67.
27. Leng, Y. and D.M. Chuang, Endogenous alpha-synuclein is induced by valproic acid through histone deacetylase inhibition and participates in neuroprotection against glutamate-induced excitotoxicity. J Neurosci, 2006. 26(28): p. 7502-12.
28. Qi, X., et al., Sodium 4-phenylbutyrate protects against cerebral ischemic injury. Mol Pharmacol, 2004. 66(4): p. 899-908.
29. Kim, H.J., et al., Histone deacetylase inhibitors exhibit anti-inflammatory and
neuroprotective effects in a rat permanent ischemic model of stroke: multiple mechanisms of action. J Pharmacol Exp Ther, 2007. 321(3): p. 892-901.
30. Muller, N. and M.J. Schwarz, The immune-mediated alteration of serotonin and glutamate:
towards an integrated view of depression. Mol Psychiatry, 2007.
31. Fujii, K., et al., Serine racemase binds to PICK1: potential relevance to schizophrenia.
Mol Psychiatry, 2006. 11(2): p. 150-7.
32. Diamond, J.S., Astrocytes put down the broom and pick up the baton. Cell, 2006. 125(4):
p. 639-41.
33. Panatier, A., et al., Glia-derived D-serine controls NMDA receptor activity and synaptic memory. Cell, 2006. 125(4): p. 775-84.
34. Wolosker, H., D-serine regulation of NMDA receptor activity. Sci STKE, 2006. 2006(356):
p. pe41.
35. Morita, Y., et al., A genetic variant of the serine racemase gene is associated with schizophrenia. Biol Psychiatry, 2007. 61(10): p. 1200-3.
36. Lane, H.Y., et al., Glycine transporter I inhibitor, N-methylglycine (sarcosine), added to clozapine for the treatment of schizophrenia. Biol Psychiatry, 2006. 60(6): p. 645-9.
37. Lane, H.Y., et al., Sarcosine or D-serine add-on treatment for acute exacerbation of schizophrenia: a randomized, double-blind, placebo-controlled study. Arch Gen Psychiatry, 2005. 62(11): p. 1196-204.
38. Labuzek, K., et al., Chlorpromazine and loxapine reduce interleukin-1beta and interleukin-2 release by rat mixed glial and microglial cell cultures. Eur
39. Miyaoka, T., et al., Possible antipsychotic effects of minocycline in patients with schizophrenia. Prog Neuropsychopharmacol Biol Psychiatry, 2007. 31(1): p. 304-7.
40. Fischer, A., et al., Recovery of learning and memory is associated with chromatin remodelling. Nature, 2007. 447(7141): p. 178-82.
41. Detich, N., V. Bovenzi, and M. Szyf, Valproate induces replication-independent active DNA demethylation. J Biol Chem, 2003. 278(30): p. 27586-92.
42. Dong, E., et al., Histone hyperacetylation induces demethylation of reelin and 67-kDa glutamic acid decarboxylase promoters. Proc Natl Acad Sci U S A, 2007. 104(11): p.
4676-81.
43. Mitchell, C.P., et al., Histone deacetylase inhibitors decrease reelin promoter methylation in vitro. J Neurochem, 2005. 93(2): p. 483-92.
44. Tremolizzo, L., et al., Valproate corrects the schizophrenia-like epigenetic behavioral modifications induced by methionine in mice. Biol Psychiatry, 2005. 57(5): p. 500-9.
45. Levenson, J.M., DNA (cytosine-5) methyltransferase inhibitors: a potential therapeutic agent for schizophrenia. Mol Pharmacol, 2007. 71(3): p. 635-7.
46. Schroeder, F.A., et al., Antidepressant-like effects of the histone deacetylase inhibitor, sodium butyrate, in the mouse. Biol Psychiatry, 2007. 62(1): p. 55-64.
47. McColl, G., et al., Pharmacogenetic analysis of lithium-induced delayed aging in Caenorhabditis elegans. J Biol Chem, 2008. 283(1): p. 350-7.
