科学研究費補助金 研究種目:一般研究(C) 課題番号:01570633 研究代表者:大磯 ユタカ 研究期間:1989-1990年度

The fetal lung epithelium secretes fluid into the potential pulmonary air-spaces by actively transporting chloride (Cl¯) into the lung lumen. This Cl¯-driven fluid secretion declines with the progression of lung development. Recent studies demonstrate that the A-type γ-aminobutyric acid receptor (GABAAR), a Cl¯ channel, and glutamic acid decarboxylase (GAD65/67), key GABA-synthesizing enzymes, are expressed in adult pulmonary epithelial cells (ECs), forming an autocrine GABAAR signaling system. My thesis study revealed that GABAAR π- and β2- subunits are expressed in high levels in the fetal rat lung epithelium and decline at birth, consistent with pattern of fluid secretion. Immunohistochemistry showed distinct profiles of expression for GABAAR subunits and GAD65/67. Treatment of alveolar ECs with dexamethasone reduced the GABAAR π-subunit expression. These results suggest that the GABAAR signaling in the fetal pulmonary epithelium is developmentally regulated and the GABAAR expression and GABAAR-mediated Cl¯ secretion in pulmonary ECs may be regulated by glucosteroids.

The fetal lung epithelium secretes fluid into the potential pulmonary air-spaces by actively transporting chloride (Cl¯) into the lung lumen. This Cl¯-driven fluid secretion declines with the progression of lung development. Recent studies demonstrate that the A-type γ-aminobutyric acid receptor (GABAAR), a Cl¯ channel, and glutamic acid decarboxylase (GAD65/67), key GABA-synthesizing enzymes, are expressed in adult pulmonary epithelial cells (ECs), forming an autocrine GABAAR signaling system. My thesis study revealed that GABAAR π- and β2- subunits are expressed in high levels in the fetal rat lung epithelium and decline at birth, consistent with pattern of fluid secretion. Immunohistochemistry showed distinct profiles of expression for GABAAR subunits and GAD65/67. Treatment of alveolar ECs with dexamethasone reduced the GABAAR π-subunit expression. These results suggest that the GABAAR signaling in the fetal pulmonary epithelium is developmentally regulated and the GABAAR expression and GABAAR-mediated Cl¯ secretion in pulmonary ECs may be regulated by glucosteroids.

Made available in DSpace on 2015-07-22T20:50:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-03-26. Added 1 bitstream(s) on 2015-08-11T03:25:44Z : No. of bitstreams: 1 Publico-10777.pdf: 584483 bytes, checksum: e11ab9839fe48602b080e48c338291cc (MD5) / Introdução: A cefaléia crônica diária é constituída por um grupo de cefaléias, dentre elas a enxaqueca crônica, comórbida com patologias como a depressão, o abuso de medicamentos, a obesidade e, mais recentemente, associada a casos de hipertensão intracraniana idiopática sem edema de papila. Objetivos: Determinar a prevalência de hipertensão intracraniana idiopática sem edema de papila e os níveis liquóricos de glutamato e ácido gama-aminobutírico em pacientes com enxaqueca crônica comparado a outros grupos de pacientes. Métodos: Foram estudados pacientes com enxaqueca crônica, mediante a realização do exame do líquido cefalorraquidiano com medida da pressão de abertura e dosagens dos níveis liquóricos dos aminoácidos glutamato e ácido gama-aminobutírico pela técnica de cromatografia líquida de alta resolução. Resultados: Dos pacientes submetidos a punção lombar, seis pacientes, em grupo de sessenta, tiveram elevação na pressão liquórica maior que 200 mm H20 sem acusar edema de papila à fundoscopia. Os pacientes que abusavam de triptanos mostraram níveis liquóricos de glutamato menores que aqueles com uso abusivo de outros tipos de medicações analgésicas e pacientes que não abusavam de nenhum tipo de medicação. Quanto aos níveis de ácido gamaaminobutírico no líquido cefalorraquidiano, esses foram menores nos pacientes com enxaqueca crônica e depressão quando comparados aos pacientes que tinham apenas enxaqueca crônica. Conclusões: A realização do estudo do líquido cefalorraquidiano foi importante em pacientes com enxaqueca crônica para a exclusão da hipertensão intracraniana idiopática sem papiledema, possibilitando perspectivas futuras para o entendimento da fisiopatogênese e desenvolvimento de novas terapias medicamentosas para a enxaqueca e suas comorbidades. / Introduction: Chronic daily headaches consist of a group of headaches, among them chronic migraine, that is comorbid with depression, overuse of medication, obesity and recently, cases of idiopathic intracranial hypertension without papilloedema. Objectives: To establish idiopathic intracranial hypertension without papilloedema prevalence and glutamate and gamma-aminobutyric acid levels in cerebrospinal fluid from patients with chronic migraine compared to other groups of patients. Methods: We studied patients with chronic migraine, who underwent lumbar puncture to rule out idiopathic intracranial hypertension without papilloedema. Amino acids glutamate and gamma-aminobutyric acid levels were measured by high performance liquid chromatography in cerebrospinal fluid. Results: Six patients, among sixty, had CSF open pressure higher than 200 mm H20 without papilloedema on fundoscopy. Patients who overused triptans had glutamate levels lower than those with abuse of other analgesic types and nonoverusers. The gamma-aminobutyric acid levels in cerebrospinal fluid were lower in depressed patients when compared to patients without depression and controls. Conclusions: The study of the cerebrospinal fluid was important in patients with chronic migraine for the exclusion of idiopathic intracranial hypertension without papilloedema, opening perspectives for the understanding of the physiopathology and development of new drug therapies for migraine and its comorbidities. / TEDE / BV UNIFESP: Teses e dissertações

Peripheral nerve injuries occur with high incidence and often result in profound and permanent impact on the life of patients and on healthcare expenditure. Schwann cells (SC) play a promoting role in peripheral nerve regeneration providing physical and neurotrophic support that aids axon re-growth. However, these beneficial properties are not exploitable in nerve tissue engineering due to the difficulties in SC harvesting and expansion in culture. Adult stem cells derived from bone marrow (BM-MSC) and from adipose tissue (ASC) can be differentiated in SC-like cells and be used as SC substitutes in bioengineered nerve conduits for the improvement of peripheral nerve regeneration. Pharmacological intervention approaches for the treatment of nerve injury are still not clinically available. Nevertheless, γ-Aminobutyric acid (GABA) receptors have been recently suggested as a putative target for such purpose. GABA is the main inhibitory neurotransmitter of the adult brain and interacts with two different receptor types. However, both GABA-A and GABA-B receptor types are functionally expressed also in SC, where they are involved in the regulation of SC physiology and in the development of the peripheral nervous system (PNS).The aim of this thesis was to characterise the GABAergic system of BM-MSC and ASC differentiated into a SC-like phenotype and to evaluate changes in the expression levels following differentiation. Moreover, the effect of specific GABA receptor ligands on cell proliferation and neurotrophic potential of differentiated stem cells were assessed. Using reverse transcriptase polymerase chain reaction, western blot analysis and immunohistochemistry we demonstrated that adult stem cells express several subunits of both GABA-A and GABA-B receptor systems such as GABA-B1a, GABA-B1b and GABA-B2, as well as GABA-A α2 and GABA-A β3. Expression levels and cellular localisation were comparable with adult and neonatal SC cultures used as positive controls, and protein expression levels for some of the subunits changed following glial differentiation. Interestingly, stimulation of GABA receptors with specific agonists influenced stem cell proliferation in two opposite ways. Baclofen, a GABA-B receptor agonist decreased proliferation of SC and differentiated ASC (dASC), but not of SC-like BM-MSC (dBM-MSC). By contrast, muscimol, a GABA-A receptor agonist, increased proliferation in SC and in both dASC and dBM-MSC. This suggests that GABAergic signalling could be a potential player in the mechanisms regulating stem cell differentiation and proliferation as reported in SC. Finally, baclofen treatments on SC and dASC modulated the expression levels and the release of the neurotrophins BDNF and NGF, which are key actors in the processes involved with peripheral nerve regeneration. Although further studies will be needed to clarify the role of GABA receptors in the PNS, the presence of functional GABA receptors on SC-like adult stem cells could represent an exploitable pharmacological target to modulate stem cell physiology and improve their neurotrophic potential for peripheral nerve regeneration.

Delusions of parasitosis are a rare psychiatric disorder in which the patient has a fixed, false belief of being infested with parasites. The disorder is classified as primary if no cause is identified or secondary if associated with general organic conditions, psychiatric illnesses, and drugs (substance induced). Several medications have been reported in association with delusions of parasitosis, including anti-parkinsonian medications, ciprofloxacin, cetirizine, doxepin, and others. Delusions of parasitosis have not been previously reported to be associated with gabapentin use. We present the case of a patient who developed delusions of parasitosis after been initiated on gabapentin treatment for neuropathic pain and complete disappearance of symptoms after the medication was discontinued.

Microglial activation and behavioral abnormalities occur before neuronal loss in experimental murine prion disease; the behavioral changes coincide with a reduction in synaptic plasticity. Because synaptic plasticity depends on an intact perineuronal net (PN), a specialized extracellular matrix that surrounds parvalbumin (PV)-positive GABAergic (gamma-aminobutyric acid [GABA]) inhibitory interneurons, we investigated the temporal relationships between microglial activation and loss of PN and PV-positive neurons in ME7 murine prion disease. Anesthetized C57Bl/6J mice received bilateral intracerebral microinjections of ME7-infected or normal brain homogenate into the dorsal hippocampus. Microglial activation, PrP accumulation, the number of PV-positive interneurons, and Wisteria floribunda agglutinin-positive neurons (i.e. those with an intact PN) were assessed in the ventral CA1 and subiculum at 4, 8, 12, 16, and 20 weeks postinjection. Hippocampal areas and total neuron numbers in the ventral CA1 and subiculum were also determined. Loss of PN coincided with early microglial activation and with a reduction in synaptic plasticity. No significant loss of PV-positive interneurons was observed. Our findings suggest that the substrate of the earliest synaptic and behavioral abnormalities in murine prion disease may be inflammatory microglia-mediated degradation of the PN.

L’acide γ-aminobutyrique (GABA) est le principal neurotransmetteur inhibiteur du système nerveux central et est impliqué dans diverses pathologies incluant l’épilepsie, l’anxiété, la dépression et la dépendance aux drogues. Le GABA agit sur l’activité neuronale par l’activation de deux types de récepteurs; le canal chlorique pentamérique GABAA et l’hétérodimère obligatoire de récepteurs couplés aux protéines G (RCPG) GABAB. Chacun des récepteurs est responsable de phases distinctes de la réponse cellulaire au GABA. Lors d’une stimulation par le GABA, il est essentiel pour la cellule de pouvoir contrôler le niveau d’activité des récepteurs et au besoin, de limiter leur activation par des mécanismes de désensibilisation et de régulation négative. La désensibilisation nécessite le découplage du récepteur de ses effecteurs, ainsi que sa compartimentation hors de la membrane plasmique dans le but de diminuer la réponse cellulaire à l’agoniste. Les mécanismes de contrôle de l’activité de GABAB semblent anormaux pour un RCPG et sont encore mal moléculairement caractérisés. L’objet de cette thèse est d’étudier la régulation du récepteur GABAB et de sa signalisation par la caractérisation de nouvelles protéines d’interactions étant impliquées dans la désensibilisation, l’internalisation et la dégradation du récepteur. Une première étude nous a permis d’identifier la protéine NSF (N-ethylmaleimide sensitive factor) comme interagissant avec le récepteur hétérodimérique. Nous avons caractérisé le site d’interaction au niveau du domaine coiled-coil de chacune des deux sous-unités de GABAB et constaté la dépendance de cette interaction au statut de l’activité ATPasique de NSF. Nous avons observé que cette interaction pouvait être dissociée par l’activation de GABAB, induisant la phosphorylation du récepteur par la protéine kinase C (PKC) parallèlement à la désensibilisation du récepteur. L’activation de PKC par le récepteur est dépendante de l’interaction NSF-GABAB, ce qui suggère une boucle de rétroaction entre NSF et PKC. Nous proposons donc un modèle où, à l’état basal, le récepteur interagit avec NSF, lui permettant d’activer PKC en réponse à la stimulation par un agoniste, et où cette activation permet à PKC de phosphoryler le récepteur, induisant sa dissociation de NSF et sa désensibilisation. Nous avons par la suite étudié la dégradation et l’ubiquitination constitutive de GABAB et la régulation de celles-ci par PKC et l’enzyme de déubiquitination USP14 (ubiquitin-specific protease 14). Au niveau basal, le récepteur est ubiquitiné, et présente une internalisation et une dégradation rapide. L’activation de PKC augmente l’ubiquitination à la surface cellulaire et l’internalisation, et accélère la dégradation du récepteur. USP14 est en mesure de déubiquitiner le récepteur suite à l’internalisation, mais accélère aussi la dégradation par un mécanisme indépendant de son activité enzymatique. Nos résultats suggèrent un mécanisme où l’ubiquitination promeut l’internalisation et où USP14 cible le récepteur ubiquitiné vers un processus de dégradation lysosomale. La troisième étude porte sur la régulation de la densité de récepteurs à la membrane plasmique par la protéine Grb2 (growth factor receptor-bound protein 2). Nous avons déterminé que Grb2 interagit avec GABAB1 au niveau de la séquence PEST (riche en proline, glutamate, sérine et thréonine) du domaine carboxyl-terminal, et que cette interaction module l’expression à la surface du récepteur hétérodimérique en diminuant l’internalisation constitutive par un mécanisme encore inconnu. Cette inhibition de l’internalisation pourrait provenir d’une compétition pour le site de liaison de Grb2 à GABAB1, ce site étant dans une région interagissant avec plusieurs protéines impliquées dans le trafic du récepteur, tels le complexe COPI et la sous-unité γ2S du récepteur GABAA (1, 2). En proposant de nouveaux mécanismes moléculaires contrôlant l’activité et l’expression à la membrane du récepteur GABAB par les protéines NSF, PKC, USP14 et Grb2, les études présentées dans cette thèse permettent de mieux comprendre les processus d’internalisation et de dégradation, ainsi que du contrôle de l’activité de GABAB par la désensibilisation, ouvrant la porte à une meilleure compréhension de la signalisation GABAergique. / γ-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter of the central nervous system and is involved in diverse pathologies such as epilepsy, anxiety, depression and drug addiction. GABAergic modulation of neuronal activity involves two different subsets of receptors: the GABAA receptor chlorine channel and the heterodimer of G protein coupled receptors (GPCR) GABAB. Each of these receptors is responsible for mediating distinct parts of the GABA-induced signaling. Upon stimulation, it is vital for the cell to control the signaling input and prevent overstimulation, using mechanisms such as functional desensitization and down-regulation to achieve this. The processes controlling GABAB receptor activity are atypical for a GPCR and have yet to be fully characterized. The aim of this thesis is to elucidate the mechanisms controlling GABAB activity by discovering novel proteins interactions mediating receptor desensitization, internalization and ubiquitination. In the first study, we identified the N-ethylmaleimide sensitive factor (NSF) as a GABAB interacting protein and characterized its interaction site as the coiled-coil structure on both GABAB sub-units. We also showed that this interaction is sensitive to the ATPase state of NSF and that agonist treatment of GABAB led to dissociation of NSF from the receptor in a protein kinase C (PKC) dependent manner. Interestingly, GABA-induced PKC activation was dependent on the NSF-GABAB interaction, suggesting a feedback mechanism for PKC. Both PKC and NSF were involved in mediating receptor desensitization, suggesting a novel role of NSF in receptor signaling regulation. In the proposed model, NSF interacts with GABAB at the basal state, and upon agonist stimulation, PKC is activated and can phosphorylate the receptor, promoting NSF dissociation and GABAB desensitization. We then studied constitutive GABAB ubiquitination and degradation and its regulation by PKC and the deubiquitinating enzyme USP14 (Ubiquitin-specific protease 14). GABAB shows a high constitutive ubiquitination and internalization level. Activation of PKC promotes both phenomena and accelerates the rate of lysosomal receptor degradation. In contrast, USP14 promotes post-endocytic deubiquitination of the receptor, but also accelerates receptor degradation in a catalytically-independent manner. Our results suggest a mechanism where PKC-induced cell surface ubiquitination promotes GABAB endocytosis and USP14 interaction promotes endosomal sorting toward lysosomal degradation. In the third study, we identified the growth factor receptor-bound protein 2 (Grb2) as a protein interacting with the PEST (proline, glutamate, serine, threonine rich) sequence of GABAB1 through a SH3-domain interaction and forming a ternary complex with the functional GABAB heterodimer. We showed that Grb2 can regulate cell surface density of GABAB by decreasing constitutive endocytosis, suggesting that this interaction can compete for binding of the PEST sequence with proteins such as the GABAA γ2S sub-unit or the COPI complex (1, 2), promoting higher cell surface stability. In proposing novel molecular mechanisms controlling GABAB signaling and cell surface expression through NSF, PKC, USP14 and Grb2, this thesis highlights the complex regulation of GABAB activity by its functional desensitization, ubiquitination, endocytosis and degradation.

L’acide γ-aminobutyrique (GABA) est le principal neurotransmetteur inhibiteur du système nerveux central et est impliqué dans diverses pathologies incluant l’épilepsie, l’anxiété, la dépression et la dépendance aux drogues. Le GABA agit sur l’activité neuronale par l’activation de deux types de récepteurs; le canal chlorique pentamérique GABAA et l’hétérodimère obligatoire de récepteurs couplés aux protéines G (RCPG) GABAB. Chacun des récepteurs est responsable de phases distinctes de la réponse cellulaire au GABA. Lors d’une stimulation par le GABA, il est essentiel pour la cellule de pouvoir contrôler le niveau d’activité des récepteurs et au besoin, de limiter leur activation par des mécanismes de désensibilisation et de régulation négative. La désensibilisation nécessite le découplage du récepteur de ses effecteurs, ainsi que sa compartimentation hors de la membrane plasmique dans le but de diminuer la réponse cellulaire à l’agoniste. Les mécanismes de contrôle de l’activité de GABAB semblent anormaux pour un RCPG et sont encore mal moléculairement caractérisés. L’objet de cette thèse est d’étudier la régulation du récepteur GABAB et de sa signalisation par la caractérisation de nouvelles protéines d’interactions étant impliquées dans la désensibilisation, l’internalisation et la dégradation du récepteur. Une première étude nous a permis d’identifier la protéine NSF (N-ethylmaleimide sensitive factor) comme interagissant avec le récepteur hétérodimérique. Nous avons caractérisé le site d’interaction au niveau du domaine coiled-coil de chacune des deux sous-unités de GABAB et constaté la dépendance de cette interaction au statut de l’activité ATPasique de NSF. Nous avons observé que cette interaction pouvait être dissociée par l’activation de GABAB, induisant la phosphorylation du récepteur par la protéine kinase C (PKC) parallèlement à la désensibilisation du récepteur. L’activation de PKC par le récepteur est dépendante de l’interaction NSF-GABAB, ce qui suggère une boucle de rétroaction entre NSF et PKC. Nous proposons donc un modèle où, à l’état basal, le récepteur interagit avec NSF, lui permettant d’activer PKC en réponse à la stimulation par un agoniste, et où cette activation permet à PKC de phosphoryler le récepteur, induisant sa dissociation de NSF et sa désensibilisation. Nous avons par la suite étudié la dégradation et l’ubiquitination constitutive de GABAB et la régulation de celles-ci par PKC et l’enzyme de déubiquitination USP14 (ubiquitin-specific protease 14). Au niveau basal, le récepteur est ubiquitiné, et présente une internalisation et une dégradation rapide. L’activation de PKC augmente l’ubiquitination à la surface cellulaire et l’internalisation, et accélère la dégradation du récepteur. USP14 est en mesure de déubiquitiner le récepteur suite à l’internalisation, mais accélère aussi la dégradation par un mécanisme indépendant de son activité enzymatique. Nos résultats suggèrent un mécanisme où l’ubiquitination promeut l’internalisation et où USP14 cible le récepteur ubiquitiné vers un processus de dégradation lysosomale. La troisième étude porte sur la régulation de la densité de récepteurs à la membrane plasmique par la protéine Grb2 (growth factor receptor-bound protein 2). Nous avons déterminé que Grb2 interagit avec GABAB1 au niveau de la séquence PEST (riche en proline, glutamate, sérine et thréonine) du domaine carboxyl-terminal, et que cette interaction module l’expression à la surface du récepteur hétérodimérique en diminuant l’internalisation constitutive par un mécanisme encore inconnu. Cette inhibition de l’internalisation pourrait provenir d’une compétition pour le site de liaison de Grb2 à GABAB1, ce site étant dans une région interagissant avec plusieurs protéines impliquées dans le trafic du récepteur, tels le complexe COPI et la sous-unité γ2S du récepteur GABAA (1, 2). En proposant de nouveaux mécanismes moléculaires contrôlant l’activité et l’expression à la membrane du récepteur GABAB par les protéines NSF, PKC, USP14 et Grb2, les études présentées dans cette thèse permettent de mieux comprendre les processus d’internalisation et de dégradation, ainsi que du contrôle de l’activité de GABAB par la désensibilisation, ouvrant la porte à une meilleure compréhension de la signalisation GABAergique. / γ-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter of the central nervous system and is involved in diverse pathologies such as epilepsy, anxiety, depression and drug addiction. GABAergic modulation of neuronal activity involves two different subsets of receptors: the GABAA receptor chlorine channel and the heterodimer of G protein coupled receptors (GPCR) GABAB. Each of these receptors is responsible for mediating distinct parts of the GABA-induced signaling. Upon stimulation, it is vital for the cell to control the signaling input and prevent overstimulation, using mechanisms such as functional desensitization and down-regulation to achieve this. The processes controlling GABAB receptor activity are atypical for a GPCR and have yet to be fully characterized. The aim of this thesis is to elucidate the mechanisms controlling GABAB activity by discovering novel proteins interactions mediating receptor desensitization, internalization and ubiquitination. In the first study, we identified the N-ethylmaleimide sensitive factor (NSF) as a GABAB interacting protein and characterized its interaction site as the coiled-coil structure on both GABAB sub-units. We also showed that this interaction is sensitive to the ATPase state of NSF and that agonist treatment of GABAB led to dissociation of NSF from the receptor in a protein kinase C (PKC) dependent manner. Interestingly, GABA-induced PKC activation was dependent on the NSF-GABAB interaction, suggesting a feedback mechanism for PKC. Both PKC and NSF were involved in mediating receptor desensitization, suggesting a novel role of NSF in receptor signaling regulation. In the proposed model, NSF interacts with GABAB at the basal state, and upon agonist stimulation, PKC is activated and can phosphorylate the receptor, promoting NSF dissociation and GABAB desensitization. We then studied constitutive GABAB ubiquitination and degradation and its regulation by PKC and the deubiquitinating enzyme USP14 (Ubiquitin-specific protease 14). GABAB shows a high constitutive ubiquitination and internalization level. Activation of PKC promotes both phenomena and accelerates the rate of lysosomal receptor degradation. In contrast, USP14 promotes post-endocytic deubiquitination of the receptor, but also accelerates receptor degradation in a catalytically-independent manner. Our results suggest a mechanism where PKC-induced cell surface ubiquitination promotes GABAB endocytosis and USP14 interaction promotes endosomal sorting toward lysosomal degradation. In the third study, we identified the growth factor receptor-bound protein 2 (Grb2) as a protein interacting with the PEST (proline, glutamate, serine, threonine rich) sequence of GABAB1 through a SH3-domain interaction and forming a ternary complex with the functional GABAB heterodimer. We showed that Grb2 can regulate cell surface density of GABAB by decreasing constitutive endocytosis, suggesting that this interaction can compete for binding of the PEST sequence with proteins such as the GABAA γ2S sub-unit or the COPI complex (1, 2), promoting higher cell surface stability. In proposing novel molecular mechanisms controlling GABAB signaling and cell surface expression through NSF, PKC, USP14 and Grb2, this thesis highlights the complex regulation of GABAB activity by its functional desensitization, ubiquitination, endocytosis and degradation.

蛋白激酶CK2（Casine kinase 2）為四單體所構成，針對配受質蛋白之絲胺酸或蘇胺酸位置進行磷酸化，先前研究已經發現在紋狀體腦區之CK2的表現量與活性皆高於大腦中其餘腦區，而紋狀體腦區主要神經細胞為-氨基丁酸神經元（GABAergic neurons）的medium spiny neuron（MSN），會受到來自黑質多巴胺神經細胞（dopaminergic neurons）的調控。此外，DARPP-32（dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDA）蛋白亦被發現大量表現於在MSN細胞中，且為CK2之受質蛋白質。雖然CK2已被證實參與多巴胺神經元的神經保護機制，但是否參與MSN細胞對運動行為調控之生理機制仍未清楚。由於已有研究發現施予1-甲基-4-苯基-1,2,3,6-四氢吡啶（MPTP）藥物處理造成黑質-紋狀體腦區受損之老鼠腦內-氨基丁酸（GABA）的生合成酵素─麩胺酸脫羧酵素67（GAD67）表現量與正常老鼠不同，因此本論文研究的主題擬在大鼠實驗模式中利用MPP+造成投射至紋狀體之多巴胺神經細胞受損，探討當多巴胺調控紋狀體神經細胞能力缺失的狀態下，MSN細胞之CK2、DARPP-32和GAD蛋白表現與動物運動行為之相關性。 實驗結果發現，直接於紋狀體給予1-甲基-4-苯基碘化啶 （MPP+ Iodide）皆會造成CK2、DARPP-32以及GAD67之蛋白質含量的減少，多巴胺及其代謝物和GABA等神經化學傳遞物質亦有減少的現象；另外，在MPP+給予前分別操弄CK2或DARPP-32 胺基酸Ser102磷酸化的表現，皆會改變GAD67蛋白質含量與黑質酪胺酸羥化酶（Tyrosine Hydroxylase, TH）蛋白質含量，同時神經化學傳遞物質的含量或代謝亦有改變。由現有之結果推測CK2/DARPP-32/GAD67細胞訊息傳遞機制可能參與巴金森氏症運動行為失常之細胞層面的調控。 / Protein kinase CK2 is a heterotetrameric and serine/threonine protein kinase. Its protein levels and activity are found to be elevated in the striatum when compared to other brain areas. CK2 is known to involve in the neuroprotective effects of dopaminergic neurons, whether it also regulates the neuronal function relative to motor behaviors is still unclear. DARPP-32 protein is known as one of the substrates for CK2 and is highly expressed in the GABAergic medium spiny neurons (MSN) responsible for dopamine stimulation in the striatum. Furthermore, other studies have indicated that the expression of glutamic acid decarboxylase 67 (GAD67) mRNA and protein was different in the striatum of MPTP vs. naïve animals, which is one of the enzymes responsible for the synthesis of neurotransmitter GABA. In the present study, we observed that the parallel changes in protein levels of CK2, DARPP-32 and GAD67 in the striatum and TH in the substantia nigra of MPP+-treated. We also found that manipulation of CK2 or DARPP-32 gene expression aggravated the MPP+-induced neuropathological dificts. The present results suggest that CK2/DARPP-32/GAD67 signaling pathway might involve in the cellular mechanism of motor-deficit in Parkinson’s disease.