丙戊酸是用來治療癲癇、雙極性情感性精神病以及神經病變性疼痛的藥物，然而目前對於丙戊酸的治療機轉尚不清楚。而之前的研究主要著重於丙戊酸對於神經元的直接作用，然而星狀神經膠細胞在丙戊酸治療疾病的過程中所扮演的角色仍需要更深入的研究。因此我的研究主題是探討丙戊酸對於神經膠細胞的直接作用，並且利用神經元-神經膠混合培養系統觀察丙戊酸對神經元的間接作用。
我們先前在微陣列分析中尋找初代星狀神經膠培養系統投予丙戊酸後有哪些與突觸成熟及突觸可塑性相關的基因受到丙戊酸調節。結果顯示neuroligin-1 (NL-1)、neuregulin-1 (NRG-1)、neuronal pentraxin-1 (NP-1)及Thrombospondin-3 (TSP-3)在星狀細胞的表現量會受到丙戊酸的調節而增加。而我接著利用即時聚合連鎖反應證實NL-1、NRG-1、NP-1及TSP-3 mRNA的表現量會隨著丙戊酸作用濃度與時間而增加，在作用24 小時候分別上升了1.9, 4, 10.8及2倍。接著利用免疫螢光染色在初代神經膠培養系統以及初代神經元-神經膠混合培養系統證實星狀細胞的確會表現這四個基因之產物。為了進一步了解丙戊酸如何調節這四個基因之表達，因此投予組蛋白去乙醯抑制劑及肝醣合成激-3β抑制劑觀察是否有類似丙戊酸之作用，結果顯示只有丁酸鈉具有類似丙戊酸的作用。此外，在初代神經元培養系統中投予丙戊酸，觀察到NL-1及TSP-3的表現量增加，但作用程度較弱。NRG-1的表現沒有明顯差異，而NP-1的表達明顯受到抑制。結果顯示丙戊酸在神經元及星狀細胞調控這四個基因作用不同。為進一步探討星狀細胞NL-1、NRG-1、NP-1及TSP-3轉譯之細胞黏附分子或細胞外間質分子產物是否影響神經元間突觸的功能，首先在初代神經元-神經膠細胞混合培養系統中投予丙戊酸後，利用西方點墨法觀察到丙戊酸作用24小時後興奮性突觸之突觸前蛋白EAAT2的表現有顯著的下降、而興奮性突觸之突觸後蛋白PSD-95的表現在投予丙戊酸72小時後有顯著上升。丙戊酸對抑制性突觸之突觸前蛋白VGAT蛋白表現沒有差異而抑制性突觸後之突觸後蛋白Gephyrin在丙戊酸作用72小時及120小時後蛋白顯著上升。
實驗結果顯示星狀神經膠細胞是丙戊酸重要的目標細胞，丙戊酸改變了星狀細胞上細胞黏附分子與細胞外間質分子的組成，並有可能能進一步調節突觸的架構。這樣的機制能讓我們進一步了解丙戊酸治療癲癇、精神病及神經病變性疼痛的過程中，星狀神經膠細胞可能扮演之重要角色。

Valproic acid (VPA) is a widely prescribed drug for generalized and partial epilepsies, bipolar disorders and neuropathic pain. However, the underling therapeutic mechanisms remain unclear. Most previous studies regarding the effects of VPA focused on neuron mechanisms only. The role of astrocytes under therapeutic mechanisms of VPA was not investigated. Because the effects of VPA on glial cells have seldom been investigated plus that valproate is known to regulate the cellular epigenetic status through histone modifications, we started to investigate the direct effect of valproate on glial cellular function and the indirect effects on neurons using primary mixed neuron-glial culture system.
Our pioneering study using microarray analysis revealed that primary rat astroglial cultures treated with VPA altered the expression of specific genes associated with synaptic maturation and plasticity. The up-regulation of neuroligin-1 (NL-1), neuregulin-1 (NRG-1), neuronal pentraxin-1 (NP-1), and thrombospondin-3 (TSP-3) expressions had been confirmed by Real Time-PCR in a time and concentration dependent manner. Real-Time PCR showed a 1.9, 4, 10.8 and 2 fold increase of NL-1, NRG-1, NP-1 and TSP-3 respectively after 24 hours treatment. Immunocytochemical analysis revealed the colocalization of NL-1, NRG-1, NP-1 and TSP-3 in cells expressed GFAP in neuron-glial mixed cultures. To investigate how VPA regulate the expression of these astroglial genes, histone deacetylase and glycogen synthase kinase-3 inhibitors were used to test if they could mimic the effect of VPA on astrocyte. However, the results showed that inhibitions of HDAC and GSK-3β were not the direct targets to regulate these genes. Moreover, neuronal expressions of these genes were differentially regulated by VPA. Western blot showed that VPA decreased the level of excitatory presynaptic EAAT2 with 24hr VPA incubation, and increased the levels of excitatory postsynaptic PSD-95 with 72hr VPA incubation and inhibitory postsynaptic gephyrin with 72hr and 120hr VPA incubation. The results suggested that the astrocyte-derived factors might also have a role in the astrocyte-mediated modulation of synapses.
The results of the current study suggest glial cells are important targets for VPA, the drug for epilepsy, bipolar disorder and neuropathic pain. The disturbances of glial function may contribute to the pathology underling these brain disorders, and glial cells may serve as appropriate targets for CNS medications.

Allen, N.J., and Barres, B.A. 2009. Neuroscience: Glia - more than just brain glue. Nature 457, 675-677.
Barres, B.A.A.-m., et al. 2008. The mystery and magic of glia: a perspective on their roles in health and disease. UID - 18995817. Neuron 60, 430-440.
Bjartmar, L., et al. 2006. Neuronal pentraxins mediate synaptic refinement in the developing visual system. J Neurosci 26, 6269-6281.
Blackburn, D., et al. 2009. Astrocyte function and role in motor neuron disease: A future therapeutic target? Glia.
Boehler, M.D., et al. 2007. Added astroglia promote greater synapse density and higher activity in neuronal networks. UID - 18345351. Neuron glia biology 3, 127-140 PMC - PMC2267743 PST - ppublish.
Bolden, J.E., et al. 2006. Anticancer activities of histone deacetylase inhibitors. Nat Rev Drug Discov 5, 769-784.
Boroujerdi, A., et al. 2008. Injury discharges regulate calcium channel alpha-2-delta-1 subunit upregulation in the dorsal horn that contributes to initiation of neuropathic pain. Pain 139, 358-366.
Chen, C.J., and Liao, S.L. 2002. Oxidative stress involves in astrocytic alterations induced by manganese. Exp Neurol 175, 216-225.
Chen, G., et al. 1999a. Regulation of signal transduction pathways and gene expression by mood stabilizers and antidepressants. Psychosom Med 61, 599-617.
Chen, G., et al. 1999b. The mood-stabilizing agent valproate inhibits the activity of glycogen synthase kinase-3. J Neurochem 72, 1327-1330.
Chen, G., et al. 1999c. Valproate robustly enhances AP-1 mediated gene expression. Brain Res Mol Brain Res 64, 52-58.
Chen, P.S., et al. 2006. Valproate protects dopaminergic neurons in midbrain neuron/glia cultures by stimulating the release of neurotrophic factors from astrocytes. Mol Psychiatry 11, 1116-1125.
Christopherson, K.S., et al. 2005. Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis. Cell 120, 421-433.
Coyle Jt, M.H.K. 2002. Getting balance: drugs for bipolar disorder share target. Nat Med.
Craig, A.M., and Kang, Y. 2007. Neurexin-neuroligin signaling in synapse development. Curr Opin Neurobiol 17, 43-52.
Dean, C., and Dresbach, T. 2006. Neuroligins and neurexins: linking cell adhesion, synapse formation and cognitive function. Trends in neurosciences 29, 21-29.
Diamond, J.S. 2006. Astrocytes put down the broom and pick up the baton. Cell 125, 639-641.
Dityatev, A., and Schachner, M. 2003. Extracellular matrix molecules and synaptic plasticity. Nat Rev Neurosci 4, 456-468.
Doble, B.W., and Woodgett, J.R. 2003. GSK-3: tricks of the trade for a multi-tasking kinase. J Cell Sci 116, 1175-1186.
Dodds, D.C., et al. 1997. Neuronal pentraxin receptor, a novel putative integral membrane pentraxin that interacts with neuronal pentraxin 1 and 2 and taipoxin-associated calcium-binding protein 49. J Biol Chem 272, 21488-21494.
Ehlers, M.D. 2005. Synapse formation: astrocytes spout off. Curr Biol 15, R134-137.
Fields, R.D., and Stevens-Graham, B. 2002. New insights into neuron-glia communication. Science 298, 556-562.
Freeman, M.R. 2005. Glial control of synaptogenesis. Cell 120, 292-293.
Fujii, K., et al. 2006. Serine racemase binds to PICK1: potential relevance to schizophrenia. Mol Psychiatry 11, 150-157.
Gould, T.D., et al. 2004. Emerging experimental therapeutics for bipolar disorder: insights from the molecular and cellular actions of current mood stabilizers. UID - 15136794. Molecular psychiatry 9, 734-755.
Gould, T.D., et al. 2006. Targeting glycogen synthase kinase-3 in the CNS: implications for the development of new treatments for mood disorders. Curr Drug Targets 7, 1399-1409.
Guo, S., et al. 2009. Lithium upregulates vascular endothelial growth factor in brain endothelial cells and astrocytes. Stroke 40, 652-655.
Hall, A.C., et al. 2002. Valproate regulates GSK-3-mediated axonal remodeling and synapsin I clustering in developing neurons. Mol Cell Neurosci 20, 257-270.
Huang, H.C., and Klein, P.S. 2006. Multiple roles for glycogen synthase kinase-3 as a drug target in Alzheimer's disease. Curr Drug Targets 7, 1389-1397.
Hughes, P., and Dragunow, M. 1995. Induction of immediate-early genes and the control of neurotransmitter-regulated gene expression within the nervous system. Pharmacol Rev 47, 133-178.
Johannessen, C.U. 2000. Mechanisms of action of valproate: a commentatory. Neurochem Int 37, 103-110.
Kida, Y., et al. 2006. Sodium butyrate up-regulates cathelicidin gene expression via activator protein-1 and histone acetylation at the promoter region in a human lung epithelial cell line, EBC-1. Mol Immunol 43, 1972-1981.
Kim, W.Y., et al. 2006. Essential roles for GSK-3s and GSK-3-primed substrates in neurotrophin-induced and hippocampal axon growth. Neuron 52, 981-996.
Lacroix-Fralish, M.L., et al. 2006. Differential regulation of neuregulin 1 expression by progesterone in astrocytes and neurons. Neuron Glia Biol 2, 227-234.
Li, B., et al. 2007. The neuregulin-1 receptor erbB4 controls glutamatergic synapse maturation and plasticity. Neuron 54, 583-597.
Manji, H.K., et al. 2001. The cellular neurobiology of depression. Nat Med 7, 541-547.
Markram, K., et al. 2008. Abnormal fear conditioning and amygdala processing in an animal model of autism. Neuropsychopharmacology 33, 901-912.
Mauch, D.H., et al. 2001. CNS synaptogenesis promoted by glia-derived cholesterol. Science 294, 1354-1357.
Mei, L., and Xiong, W.C. 2008. Neuregulin 1 in neural development, synaptic plasticity and schizophrenia. Nat Rev Neurosci 9, 437-452.
Milligan, E.D., and Watkins, L.R. 2009. Pathological and protective roles of glia in chronic pain. Nat Rev Neurosci 10, 23-36.
Muller, N., and Schwarz, M.J. 2007. The immune-mediated alteration of serotonin and glutamate: towards an integrated view of depression. Mol Psychiatry 12, 988-1000.
Nedergaard, M., et al. 2003. New roles for astrocytes: redefining the functional architecture of the brain. UID - 14522144. Trends in neurosciences 26, 523-530.
Newton, S.S., and Duman, R.S. 2006. Chromatin remodeling: a novel mechanism of psychotropic drug action. Mol Pharmacol 70, 440-443.
Newton, S.S., et al. 2006. Chromatin remodeling: a novel mechanism of psychotropic drug action. Molecular pharmacology 70, 440-443.
Panatier, A., et al. 2006. Glia-derived D-serine controls NMDA receptor activity and synaptic memory. Cell 125, 775-784.
Pascual, O., et al. 2005. Astrocytic purinergic signaling coordinates synaptic networks. Science (New York, N.Y.) 310, 113-116.
Rosenberg, G. 2007. The mechanisms of action of valproate in neuropsychiatric disorders: can we see the forest for the trees? Cell Mol Life Sci 64, 2090-2103.
Rowe, M.K., et al. 2007. GSK-3 is a viable potential target for therapeutic intervention in bipolar disorder. Neurosci Biobehav Rev 31, 920-931.
Saijo, K., et al. 2009. A Nurr1/CoREST pathway in microglia and astrocytes protects dopaminergic neurons from inflammation-induced death. Cell 137, 47-59.
Sakisaka, T., and Takai, Y. 2005. Cell adhesion molecules in the CNS. J Cell Sci 118, 5407-5410.
Schena, M., et al. 1995. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467-470.
Schloesser, R.J., et al. 2008. Cellular plasticity cascades in the pathophysiology and treatment of bipolar disorder. UID - 17912251. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 33, 110-133.
Seifert, G., et al. 2006. Astrocyte dysfunction in neurological disorders: a molecular perspective. Nat Rev Neurosci 7, 194-206.
Shaltiel, G., et al. 2004. Valproate decreases inositol biosynthesis. Biol Psychiatry 56, 868-874.
Shaltiel, G., et al. 2007. Neurotrophic signaling cascades in the pathophysiology and treatment of bipolar disorder. Current opinion in pharmacology 7, 22-26.
Sia, G.M., et al. 2007. Interaction of the N-terminal domain of the AMPA receptor GluR4 subunit with the neuronal pentraxin NP1 mediates GluR4 synaptic recruitment. Neuron 55, 87-102.
Sudhof, T.C. 2008. Neuroligins and neurexins link synaptic function to cognitive disease. Nature 455, 903-911.
Sullivan, N.R., et al. 2004. Effect of valproic acid on serotonin-2A receptor signaling in C6 glioma cells. Journal of neurochemistry 90, 1269-1275.
Ullian, E.M., et al. 2001. Control of synapse number by glia. Science 291, 657-661.
Van Lint, C., et al. 1996. The expression of a small fraction of cellular genes is changed in response to histone hyperacetylation. Gene Expr 5, 245-253.
Verkhratsky, A., et al. 2006. Neuronal-glial networks as substrate for CNS integration. UID - 17125587. Journal of cellular and molecular medicine 10, 826-836.
Vincan, E., et al. 2000. Sodium butyrate-induced differentiation of human LIM2537 colon cancer cells decreases GSK-3beta activity and increases levels of both membrane-bound and Apc/axin/GSK-3beta complex-associated pools of beta-catenin. Oncol Res 12, 193-201.
Wolosker, H. 2007. NMDA receptor regulation by D-serine: new findings and perspectives. Mol Neurobiol 36, 152-164.
Wu, X., et al. 2008. Histone deacetylase inhibitors up-regulate astrocyte GDNF and BDNF gene transcription and protect dopaminergic neurons. Int J Neuropsychopharmacol 11, 1123-1134.
Xu, D., et al. 2003. Narp and NP1 form heterocomplexes that function in developmental and activity-dependent synaptic plasticity. Neuron 39, 513-528.
Yasuda, S., et al. 2009. The mood stabilizers lithium and valproate selectively activate the promoter IV of brain-derived neurotrophic factor in neurons. Mol Psychiatry 14, 51-59.
Yoo, C.B., and Jones, P.A. 2006. Epigenetic therapy of cancer: past, present and future. Nat Rev Drug Discov 5, 37-50.