臺灣博碩士論文加值系統

Disrupted-in-Schizophrenia 1 (DISC1)是一種與精神疾病相關的基因，它的缺損與許多精神疾病有關，例如:思覺失調症(schizophrenia)、重度憂鬱症(major depression)、躁鬱症(bipolar disorder)及自閉症(autisum)。我們使用Disc1基因缺損的小鼠，作為精神疾病的動物模式。這種C57BL/6背景的Disc1基因缺損小鼠，帶有從129S6/SvEv背景小鼠來的變異，在第六個exon中缺少了25bp，並在第七的exon中出現stop codon。先前的實驗中已經發現， Disc1基因缺損的異型合子 (heterozygote, Het) 小鼠腦中，內側前額葉皮質(medial prefrontal cortex, mPFC)細胞的樹突棘密度比起野生型 (wildtype, WT) 小鼠，有明顯的降低，而這些小鼠也有工作記憶(working memory)的功能異常。
本實驗中，我們使用8至12週齡之雄性WT以及Disc1 Het小鼠。首先觀察在mPFC中，神經膠細胞 (microglia) 的分布情況、密度及細胞形態在兩種基因型的老鼠上是否有差異。我們利用Iba1組織免疫染色 (Immunohistochemistry)來標定神經膠細胞，並使用neurolucida軟體來三維重建神經膠細胞的細胞體與分枝結構。與WT小鼠比較，我們發現到Het小鼠內側前額葉皮質中的神經膠細胞分枝較少，複雜度較低而總分枝長度也比較短。這種形態上的不同，意味著Het小鼠腦中的神經膠細胞可能處於較為活化的狀態。
接下來，我們對WT和Het小鼠給予腹腔注射0.5 mg/kg的細菌內毒素lipopolysaccharide (LPS) 或生理食鹽水。24小時之後，觀察Het小鼠在受到LPS的感染(發炎)刺激下，其行為、神經膠細胞及生化表現，是否與WT小鼠有所不同？行為實驗方面，在曠野實驗 (open field test)中，我們發現不論是WT還是Het小鼠，在施打LPS的組別中，其活動力都比施打生理食鹽水組有大幅的下降。仔細分析後也發現，施打LPS的WT小鼠相較於打生理食鹽水的WT小鼠，在探索中間區域(central area) 的時間顯著的下降。在架高十字迷宮 (elevated plus maze) 中，施打LPS的WT小鼠探索開放區域 (open arm area) 的時間，比打生理食鹽水的WT小鼠更短。這些結果顯示，施打LPS的WT小鼠正處於一種焦慮的狀態。然而特別的是，Het小鼠在曠野實驗探索中間區域的時間，以及在架高十字迷宮開放區域的時間，在施打LPS或生理食鹽水的組別中，並沒有看到差異性，這顯示LPS的施打，並不會使Het小鼠產生焦慮的行為表現。在神經膠細胞方面，不論是WT還是Het組，小鼠腦中的神經膠細胞密度都會因為施打LPS而上升；除了Het小鼠的杏仁基底外側核 (basolateral amygdala, BLA)之外。在Het小鼠杏仁基底外側核，這個跟情緒非常相關的腦區，其神經膠細胞密度，並不會因施打LPS而改變。
在形態方面，與施打生理食鹽水組別比較，施打LPS組動物內側前額葉皮質中神經膠細胞的分支變少，分枝長度及整體分枝的總長度都明顯的減少，而複雜度也明顯的下降。在WT小鼠中，神經膠細胞在施打完LPS後有非常明顯的變化，然而在Het小鼠中，變化並沒有像WT小鼠明顯。
最後我們觀察細胞激素 (cytokines) 在內側前額葉皮質的表現量。發現施打完LPS後，TNF-α, IL1-β, TLR4的表現量有明顯的上升，但在兩個基因型之間並沒有太大的差異性。綜合我們的結果可以發現Het小鼠神經膠細胞處於比較活化的狀態，因此在施打LPS後的行為表現比起正常WT小鼠會有不同的表現。

Disrupted-in-Schizophrenia 1 (DISC1) is a susceptibility gene related to various mental disorders including schizophrenia, major depression, bipolar disorder and autism. In our Disc1 mutant mice, the normal Disc1 gene was replaced by 129S6/SvEv 25bp deletion variant. Mice carried one copy of mutant Disc1 gene (Het) resulted in reduced spine density in the mPFC and impaired working memory. In the present study, we first examined the status of microglia in the mPFC of wildtype (WT) and Disc1 Het mice. The morphology of microglia was revealed by Iba1 immunohistochemistry and three-dimensionally reconstructed. Compared with those in WT mice, Iba1-positive microglia in Het Disc1 mice had less branches and shorter branch length, signifying an activated condition. Secondly, we challenged the mice with lipopolysaccharide (LPS; 0.5 mg/kg). One day after LPS or saline injection, both WT and Disc1 Het mice exhibited reduced locomotor activity. The LPS-treated WT mice spent significantly less amount of time in the central region of the open field and less exploration time in the open arms of the elevated plus maze, indicating a LPS-induced anxiety-like behavior. However, in Het mice the time spent in the central region and in the open arms were not altered by LPS treatment. In WT mice the densities of microglia increased in the various forebrain areas after LPS encounter. However, in Disc1 Het mice, the density of microglia failed to increase in the basolateral amygdala (BLA).
The microglial morphology was greatly altered in LPS-treated mice of both genotypes. Compared with saline group, less branch, shorter branch length and lower complexity were found in LPS-treated mice. In WT mice, the changes of microglial morphology was dramatic. However, in Het mice the changes of microglial morphology were minimal.
Finally, we measured the levels of various cytokines in the mPFC. After LPS treatment TNF-α, IL-1β and TLR4 expression were significant increased. However, we did not find any difference between WT and Het mice. Our result indicated an altered microglial status which might contribute to the differential responses following LPS treatment.

中文摘要………………………………………………………………………………....I
Abstract……………………………………………………………………………......III
Contents…………………………………………………………………………….......V
List of Figures………………………………………………………………………...VII

Chapter 1 Introduction……………………………………………………………………………..1
1.1 Psychiatricdisorders…………………………..…………………………………...1
1.2 Neuroinflammation and Psychiatric disorders…………………………………......2
1.3 Disc1 and Psychiatric disorders…………………………………………………....3

Chapter 2 Materials and Methods………………………………………..………..6
2.1 Animals………………………………………………………….....………………6
2.2 Drug treatment……………………………………………….…………………….6
2.3 Behavior tests……………………………………………………………..…….....7
2.3.1 Open field test (OFT) ……………………………………..……………...7
2.3.2 Elevated plus maze (EPM) ……………………..………………………...7
2.3.3 Object exploration test………………………………………...…………..8
2.3.4 Acoustic startle responses and prepulse inhibition. (PPI) …………..........8
2.4 Immunohistochemistry…………………………...………...……………………..9
2.5 Estimation of microglia density in the forebrain. ………………………...……...10
2.6 Morphometric analysis of microglia…………………………...…………………10
2.7 qPCR……………………………………………...………………………………11
2.8 Statistical analysis…………………………………………...……………………12
Chapter 3 Results …………………………………………………………….……..13
3.1 Density and morphology of microglia in the mPFC…………...……….………...13
3.2 Behavioral responses to LPS treatment ……...…………………………….…….15
3.3 Microglial responses to LPS treatment……...…………………………….……...17
3.4 Levels of cytokines in the mPFC………………………..………………….….....18

Chapter4 Discussion…………………………………………….………………………………..21

Reference………………………………..……………………………………………..51

d''Avila JC, Lam TI, Bingham D, Shi J, Won SJ, Kauppinen TM, Massa S, Liu J, SwansonRA.Microglial activation induced by brain trauma is suppressed by post-injury treatmentwith a PARP inhibitor.J Neuroinflammation. 2012 9:31.
Ballinger MD, Saito A, Abazyan B, Taniguchi Y, Huang CH, Ito K, Zhu X, Segal H, Jaaro-Peled H, Sawa A, Mackie K, Pletnikov MV, Kamiya A. Adolescent cannabis exposure interacts with mutant DISC1 to produce impaired adult emotional memory.Neurobiol Dis. 2015 82: 176-184.
Biesmans S, Meert TF, Bouwknecht JA, Acton PD, Davoodi N, De Haes P, Kuijlaars J, Langlois X, Matthews LJ, VerDonck L, Hellings N, Nuydens R. Systemic immune activation leads to neuroinflammation and sickness behavior in mice.Mediators Inflamm. 2013: 271359.
Bodea LG, Wang Y, Linnartz-Gerlach B, Kopatz J, Sinkkonen L, Musgrove R, KaomaT,Muller A, Vallar L, Di Monte DA, Balling R, Neumann H.Neurodegeneration byactivation of the microglial complement-phagosome pathway.J Neurosci. 2014:8546-8456.
Carola V, D''Olimpio F, Brunamonti E, Mangia F, Renzi P. Evaluation of the elevated plus-maze and open-field tests for the assessment of anxiety-related behaviour in inbred mice. Behav Brain Res. 2002 134:49-57.
Chung DW, Yoo KY, Hwang IK, Kim DW, Chung JY, Lee CH, Choi JH, Choi SY, Youn HY, Lee IS, Won MH. Systemic administration of lipopolysaccharide induces cyclooxygenase-2 immunoreactivity in endothelium and increases microglia in the mouse hippocampus. Cell MolNeurobiol. 2010 3:531-541
Cherry JD, Olschowka JA, O''Banion MK. Neuroinflammation and M2 microglia: the good, the bad, and the inflamed.J Neuroinflammation. 2014 11:98.
Chen Z1, Jalabi W, Shpargel KB, Farabaugh KT, Dutta R, Yin X, Kidd GJ, Bergmann CC, Stohlman SA, Trapp BD.Lipopolysaccharide-induced microglial activation and neuroprotection against experimental brain injury is independent of hematogenous TLR4.J Neurosci. 2012 32:11706-11715.
Crystal C. Watkins, Sarah Ramsay Andrews. Clinical studies of neuroinflammatory mechanisms in schizophrenia. Schizophr Res. 2016:14-22.
CharllyanySabinoCustodio, Bruna Stefania Ferreira Mello. Time course of the effects of lipopolysaccharide on prepulse inhibition and brain nitrite content in mice. Eur J Pharmacol. 2013 713:31-38.
Calcia MA, Bonsall DR, Bloomfield PS, Selvaraj S, Barichello T, Howes OD.Stress and neuroinflammation: a systematic review of the effects of stress on microglia and the implications for mental illness.Psychopharmacology (Berl). 2016 233:1637-1650.
DellaGioia N, Hannestad J. A critical review of human endotoxin administration as an experimental paradigm of depression.NeurosciBiobehav Rev. 2010 34:130-143.
Eack SM, Wojtalik JA, Barb SM, Newhill CE, Keshavan MS, Phillips ML. Fronto-Limbic Brain Dysfunction during the Regulation of Emotion in Schizophrenia.PLoS One. 2016 11: e0149297.
Elsayed M, Magistretti PJ. A new outlook on mental illnesses: Glial involvement beyond the glue.Front Cell Neurosci. 2015 9: 468.
Fernandez-Lizarbe S, Pascual M, Guerri C. Critical role of TLR4 response in the activation of microglia induced by ethanol. J Immunol. 2009 183: 4733-4744.
Frick LR, Williams K, Pittenger C. Microglial dysregulation in psychiatric disease. Clin Dev Immunol. 2013: 608-654.
Greter M, Lelios I, Croxford AL.Microglia versus myeloid cell nomenclature during brain inflammation.Front Immunol. 2015 6:249.
García Bueno B, Caso JR, Madrigal JL, Leza JC. Innate immune receptor Toll-like receptor 4 signalling in neuropsychiatric diseases.NeurosciBiobehav Rev. 2016 64:134-147.
Henry CJ, Huang Y, Wynne A, Hanke M, Himler J, Bailey MT, Sheridan JF, Godbout JP. Minocycline attenuates lipopolysaccharide (LPS)-induced neuroinflammation, sickness behavior, and anhedonia. J Neuroinflammation. 2008:15.
Hikida T, Gamo NJ, Sawa A. DISC1 as a therapeutic target for mental illnesses.ExpertOpinTher Targets. 2012 16:1151-1160.
Hung YY, Kang HY, Huang KW, Huang TL. Association between toll-like receptors expression and major depressive disorder.Psychiatry Res. 2014 220:283-286.
Hiroyuki Nawa, Nori Takei. Recent progress in animal modeling of immune inflammatory processes in schizophrenia: Implication of specific cytokines. Neurosci Res. 2006 56:2-13.
Inge C.M. Hoogland, Carin Houbolt, David J. van Westerloo, Willem A. van Gool, and Diederik van de Beek. Systemic inflammation and microglial activation: systematic review of animal experiments. J Neuroinflammation. 2015 12: 114.
Jangra A, Sriram CS, Lahkar M. Lipopolysaccharide-Induced Behavioral Alterations Are Alleviated by Sodium Phenylbutyrate via Attenuation of Oxidative Stress and Neuroinflammatory Cascade. Inflammation. 2016 39:1441-1452.
Ji K, Miyauchi J, Tsirka SE. Microglia: an active player in the regulation of synaptic activity.Neural Plast. 2013:627325.
Johnson AW, Jaaro-Peled H, Shahani N, Sedlak TW, Zoubovsky S, Burruss D, Emiliani F, Sawa A, Gallagher M. Cognitive and motivational deficits together with prefrontal oxidative stress in a mouse model for neuropsychiatric illness. Proc Natl AcadSci U S A. 2013 110:12462-12467.
Jones KA, Thomsen C. The role of the innate immune system in psychiatric disorders.Mol CellNeurosci. 201353:52-62.
Juan LW, Liao CC, Lai WS, Chang CY, Pei JC, Wong WR, Liu CM, Hwu HG, Lee LJ.Phenotypic characterization of C57BL/6J mice carrying the Disc1 gene from the 129S6/SvEv strain. Brain StructFunct. 2014 219:1417-1431.
Jurczyk A, Nowosielska A, Przewozniak N, Aryee KE, DiIorio P, Blodgett D, Yang C, Campbell-Thompson M, Atkinson M, Shultz L, Rittenhouse A, Harlan D, Greiner D, Bortell R. Beyond the brain: disrupted in schizophrenia 1 regulates pancreatic β-cell function via glycogen synthase kinase-3β. FASEB J. 2016 30: 983–993.
Ji KA, Yang MS, Jeong HK, Min KJ, Kang SH, Jou I, Joe EH.Resident microglia die and infiltrated neutrophils and monocytes become major inflammatory cells in lipopolysaccharide-injected brain.Glia. 2007 55:1577-1588.
Kelly TM, Daley DC. Integrated treatment of substance use and psychiatric disorders.Soc Work Public Health. 2013: 388–406.
Kettenmann H, Kirchhoff F, Verkhratsky A. Microglia: new roles for the synaptic stripper.Neuron. 2013 77:10-8.
Kilpinen H, Ylisaukko-Oja T, Hennah W, Palo OM, Varilo T, Vanhala R, Nieminen-von Wendt T, von Wendt L, Paunio T, Peltonen L. Association of DISC1 with autism and Asperger syndrome. Mol Psychiatry. 2008 13:187-196.
Kirkpatrick B, Miller BJ. Inflammation and schizophrenia.Schizophr Bull. 2013 39: 1174-1179.
Kondo S, Kohsaka S, Okabe S. Long-term changes of spine dynamics and microglia after transient peripheral immune response triggered by LPS in vivo. Mol Brain. 2011 4:27.
Lee LJ, Chen WJ, Chuang YW, Wang YC. Neonatal whisker trimming causes long-lasting changes in structure and function of the somatosensory system. Exp Neurol. 2009 219:524-532.
Li WY, Chang YC, Lee LJ, Lee LJ. Prenatal infection affects the neuronal architecture and cognitive function in adult mice. Dev Neurosci. 201436:359-370.
Maes M, Kubera M, Leunis JC, Berk M. Increased IgA and IgM responses against gut commensals in chronic depression: further evidence for increased bacterial translocation or leaky gut. J Affect Disord. 2012 141:55-62.
Masuda T, Prinz M. Microglia: A unique versatile cell in the central nervous system. ACS ChemNeurosci.2016 7: 428-34.
McKernan DP, Dennison U, Gaszner G, Cryan JF, Dinan TG. Enhanced peripheral toll-like receptor responses in psychosis: further evidence of a pro-inflammatory phenotype.Transl Psychiatry. 2011 1:e36.
Merikangas KR, Ames M, Cui L, Stang PE, Ustun TB, Von Korff M, Kessler RC. The impact of comorbidity of mental and physical conditions on role disability in the US adult household population.Arch Gen Psychiatry. 2007 64:1180-1188.
Millar JK, Christie S, Anderson S, Lawson D, Hsiao-Wei Loh D, Devon RS, Arveiler B, Muir WJ,Blackwood DH, Porteous DJ. Genomic structure and localisation within a linkage hotspot of Disrupted In Schizophrenia 1, a gene disrupted by a translocation segregating with schizophrenia. Mol Psychiatry. 2001:173-178.
Möller HJ, Czobor P. Pharmacological treatment of negative symptoms in schizophrenia. Eur Arch Psychiatry ClinNeurosci. 2015265:567-578.
Morgan JT, Chana G, Abramson I, Semendeferi K, Courchesne E, Everall IP. Abnormal microglial-neuronal spatial organization in the dorsolateral prefrontal cortex in autism. Brain Res. 2012 1456: 72-81.
Najjar S, Pearlman DM, Alper K, Najjar A, Devinsky O. Neuroinflammation and psychiatric illness. J Neuroinflammation. 2013 10:43.
Nayak D, Roth TL, McGavern DB. Microglia development and function.Annu Rev Immunol. 2014:367-402.
R. Norkett, S. Modi, N. Birsa, T.A. Atkin, D. Ivankovic, M. Pathania, S.V. Trossbach, C. Korth, W.D. Hirst, J.T. Kittler. DISC1-dependent regulation of mitochondrial dynamics controls the morphogenesis of complex neuronal dendrites. J Biol Chem. 2016 291:613-629.
Oliveira J1, Busson M, Etain B, Jamain S, Hamdani N, Boukouaci W, Amokrane K, Bennabi M, Le Guen E, Dargél AA, Houenou J, Ivanova R, Bellivier F, Henry C, Kahn JP, Charron D, Krishnamoorthy R, Vervoitte L, Leboyer M, TamouzaR.Polymorphism of Toll-like receptor 4 gene in bipolar disorder. J Affect Disord. 2014 152-154:395-402.
Ormel J, VonKorff M, Ustun TB, Pini S, Korten A, Oldehinkel T. Common mental disorders and disability across cultures. Results from the WHO Collaborative Study on Psychological Problems in General Health Care.JAMA. 1994 272:1741-1748.
Prinz M, Priller J. Microglia and brain macrophages in the molecular age: from origin to neuropsychiatric disease.Nat Rev Neurosci. 201415:300-312.
Prut L, BelzungC.The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review.Eur J Pharmacol. 2003463:3-33.
E. Pinero-Martos, B. Ortega-Vila, J. Pol-Fuster, E. Cisneros-Barroso, L. Ruiz-Guerra, A. Medina-Dols, D. Heine-Suner, J. Llado, G. Olmos, C. Vives-Bauza. Disrupted in schizophrenia 1 (DISC1) is a constituent of the mammalian mitochondrial contact site and cristae organizing system (MICOS) complex, and is essential for oxidative phosphorylation. Hum Mol Genet. 2016 25:4157-4169.
Réus GZ, Fries GR, Stertz L, Badawy M, Passos IC, Barichello T, Kapczinski F, Quevedo J. The role of inflammation and microglial activation in the pathophysiology of psychiatric disorders.Neuroscience. 2015 300:141-154.
Salim S. Oxidative stress and psychological disorders. CurrNeuropharmacol. 2014:140-147.
Sanacora G, Banasr M. From pathophysiology to novel antidepressant drugs: glial contributions to the pathology and treatment of mood disorders. Biol Psychiatry. 2013:1172-1179.
Schafer DP, Stevens B. Synapse elimination during development and disease: immune molecules take centre stage.BiochemSoc Trans. 2010 38:476-481.
Sulakhiya K, Keshavlal GP, Bezbaruah BB, Dwivedi S, Gurjar SS, Munde N, Jangra A, Lahkar M, Gogoi R. Lipopolysaccharide induced anxiety- and depressive-like behaviour in mice are prevented by chronic pre-treatment of esculetin. Neurosci Lett. 2016 611:106-111.
Takahashi Y, Yu Z, Sakai M,TomitaH.Linking Activation of microglia and peripheral monocyticcells to the pathophysiology of psychiatric disorders.Front Cell Neurosci. 201610:144.
Torres-Platas SG, Cruceanu C, Chen GG, Turecki G, Mechawar N. Evidence for increased microglial priming and macrophage recruitment in the dorsal anterior cingulate white matter of depressed suicides. Brain BehavImmun. 2014 42:50-59.
Trépanier MO, Hopperton KE, Mizrahi R, Mechawar N, Bazinet RP. Postmortem evidence of cerebral inflammation in schizophrenia: a systematic review.Mol Psychiatry. 2016 21:1009-1026.
Tang Y, Le W. Differential Roles of M1 and M2 Microglia in Neurodegenerative Diseases.MolNeurobiol. 2016 53:1181-1194.
VanOs J, Kapur S. Schizophrenia. Lancet. 2009 374:635-645.
Wake H, Moorhouse AJ, Miyamoto A, Nabekura J. Microglia: actively surveying and shaping neuronal circuit structure and function. Trends Neurosci. 2013 36:209-217.
Wang YC, Ho UC, Ko MC, Liao CC, Lee LJ. Differential neuronal changes in medial prefrontal cortex, basolateral amygdala and nucleus accumbens after postweaning social isolation. Brain StructFunct. 2012 217:337-351.
Watkins CC, Andrews SR. Clinical studies of neuroinflammatory mechanisms in schizophrenia. SchizophrRes. 2016 176:14-22.
Yao L, Kan EM, Lu J, Hao A, Dheen ST, Kaur C, Ling EA. Toll-like receptor 4 mediates microglial activation and production of inflammatory mediators in neonatal rat brain following hypoxia: role of TLR4 in hypoxic microglia. J Neuroinflammation. 2013 10:23.
Yirmiya R, Rimmerman N, Reshef R. Depression as a microglial disease. Trends Neurosci. 2015 38:637-658.
Yolanda Diz-Chaves, Mariana Astiz. Prenatal stress increases the expression of proinflammatory cytokines and exacerbates the inflammatory response to LPS in the hippocampal formation of adult male mice. Brain Behav Immun. 2013 28:196-206.
Couch Y, Xie Q, Lundberg L, Sharp T, Anthony DC. A model of post-infection fatigue is associated with increased TNF and 5-HT2A receptor expression in mice. PLoS One. 2015:e0130643.
Zhang X, Dong H, Zhang S, Lu S, Sun J, Qian Y. Enhancement of LPS-induced microglial inflammation response via TLR4 under high glucose conditions. Cell PhysiolBiochem. 2015 35: 1571-1581.
Zorumski CF, Izumi Y. NMDA receptors and metaplasticity: mechanisms and possible roles in neuropsychiatric disorders.NeurosciBiobehavRev. 2012 36: 989-1000.