Identification of PDE10A related proteins via proteomic analysis

Aim: Phosphodiesterase 10A (PDE10A) regulates the expression of secondary messengers of cyclic adenosine monophosphate and cyclic guanosine monophosphate, which control several intracellular signaling pathways. Recently, deactivation of PDE10A has been a notable target for the treatment of neurodegenerative diseases. Herein, we identified the effects of PDE10A inhibition on protein profile using TAK-063 under physiological condi- tions in mice. Materials and Methods: In this study, 8-12 weeks old male C57BL6/J mice were divided into vehicle or 3 mg/kg TAK-063 groups. Thirty minutes after oral delivery of vehicle or TAK-063, animals were sacrificed and liquid chromatography-mass spectrome- try/mass spectrometry (LC-MS/MS) mediated proteomic analyses were performed from tissue samples taken from the striatum region of mice. After the LC-MS/MS analysis, identified proteins were classified based on biological activity, molecular function, and signal transduction pathways using PANTHER (protein annotation through evolutionary relationship, http://www.pantherdb.org/) program. Results: As a result of proteomic analyses, 1873 different proteins were identified. Sixty- one different proteins changed significantly depending on the administration of TAK-063. According to PANTHER classification, a significant part of the identified proteins found to be in the metabolite interconversion enzyme, transporter, and protein modifying enzyme category. The molecular function classification includes the catalytic activity, transporter activity, and binding functions. The signal transduction pathway analysis demonstrated that PDE10A affects ATP synthesis, FGF signaling, EGF receptor signaling, Huntington’s Disease, Parkinson’s Disease, pyrimidine metabolism, and ubiquitin-proteasome signal transduction pathways. Conclusion: TAK-063 mediated PDE10 deactivation is an essential target in the mech- anism of energy metabolism and neurodegenerative diseases

Separation Of Lanthanides Ions By Capillary Electrophoresis With Contactless Conductivity Dedection

Tez (Yüksek Lisans) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2014Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2014Kapiler elektroforez, yüksek ayırma gücü, yüksek etkinlik, hızlı analiz, düşük miktarda elektrolit çözeltisi ve numune ile çalışılabilme gibi avantajları ile inorganik iyonlar için tercih edilen güçlü bir ayrım tekniğidir. Son yıllarda, temassız iletkenlik dedektörü (C4D) elektroforetik yöntemler için alternatif bir teknik olarak kullanılmaya başlanmıştır. Temaslı iletkenlik yöntemiyle kıyaslandığında, C4D bazı belirgin avantajlara sahiptir. C4D, çözelti ile temas etmediğinden elektrot kirlenmelerinden ve yüksek ayrım voltajından etkilenmez. Ayrıca dedektör iyi bir dayanıklılığa sahiptir. Genel bir dedeksiyon tekniği olmaya yakındır ve metal iyonları için yüksek hassasiyet ve seçiciliği olması nedeniyle C4D metal iyonlarının belirlenmesi için uygun bir yöntemdir. Dedeksiyon limiti de metal iyonlarının belirlenmesinde en çok kullanılan indirekt fotometrik dedeksiyona göre daha düşüktür. Lantanitler, nadir toprak elementleri olarak da bilinen ve periyodik tablonun f bloğunda bulunan metal çeşitleridir. Jeoloji ve nükleer endüstri gibi birçok kullanım alanı bulunmaktadır. Fiziksel ve kimyasal özelliklerinin birbirine çok benzemesi nedeniyle, lantanit iyonlarının ayrımlarını gerçekleştirmek güçtür. Kapiler zon elektroforezde (CZE) metal iyonlarının ayrımı elektroforetik mobilite farklarına göre gerçekleşir. CZE ile metal iyonlarının analizindeki sorunlardan biri türlerin benzer görünür elektroforetik mobilitelere sahip olmalarıdır. Bu yüzden metal iyonlarının tam bir ayrımını sağlamak için genellikle kompleksleştirici maddelere ihtiyaç vardır. Kompleksleştiriciler türlerin mobilitelerini farklandırmak amacıyla kullanılır. Lantanit iyonları benzer elektroforetik mobilitelere sahiptirler. Aynı şekilde bu iyonların birbirine çok yakın iyonik yarı çapları ve eşit yükleri nedeniyle belli bir kompleksleştici ile oluşturdukları komplekslerin stabilite sabitleri de birbirine çok yakındır. Bu iyonların tam bir ayrımını sağlamak için çalışma tamponuna uygun kompleksleşticilerin eklenmesi ile kompleksleştirme derecelerinin ve aynı zamanda elektroforetik mobilitelerinin birbirlerinden farklı olduğu uygun kompleksleştici konsantrasyonunun belirlenmesi gerekir. Bu çalışmada, CZE-C4D yöntemiyle iki adet kompleksleştirici madde, α-hidroksiizobütirik asit (HIBA) ve asetik asit (HAc), kullanılarak lantanitlerin ayrımı gerçekleştirilmiştir. 14 lantanitin tam bir ayrımı 1,0 mM HAc ile 4,5 mM HIBA içeren pH değeri 4,5 olan tampon çözeltide gerçekleştirilmiştir. Tekrarlanırlık işlemleri, optimum ayrım koşullarında ard arda 7 enjeksiyon yapılarak hesaplanmıştır. İnorganik anyonların göç zamanlarına ait RSD değerleri % 1,62-3,19 ve alanlarına ait değerler ise % 1,13 - 4,49 olarak bulunmuştur. Dedeksiyon limiti ise sinyal / gürültü oranının 3 katı alınarak 0,125-0,586 mM olarak tespit edilmiştir.Capillary electrophoresis (CE) is a powerful and useful separation technique for inorganic ions due to advantages of high efficiency, high resolution, low electrolyte, fast analysis and sample consumption. System of capillary electrophoresis is very simple. Column is filled up with a buffer solution and sample is injected inside this column, then high voltage is applied. Due to high voltage, analytes start move and separated from each other. All of the analytes are separated based on differentiation in their electrophoretic mobilities. Capillary zone electrophoresis (CZE) is applied often due to being the most simple capillary electorphoresis method. Separation realizes in respect of the charge-size ratio and the differences in electrophoretic mobilities of the separated ions. One of the most important terms about CE is electro osmotic flow. Analytes move through the electro osmotic flow, which occurs via applying high voltage and the analytes dependent on their charges move from anode to cathode. With CE method, various detectors can be used, which are UV-Vis, fluorescent and conductivity etc. Absorbance measurements work well for UV-absorbing organic species but the sensitivity is limited by the short optical path length available. Metal ions can only be analyzed by indirect UV detection which is lack of sensitivity and has a restricted linear range. Fluorescence detection is mainly used for the sensitive analysis of biomolecules, which can be added fluorescent for derivatization. But, most other groups of substances are not suitable to this technique. Recently, conductivity detection has become alternative detection technique for electrophoretic methods. Day by day, some kinds exist like contactless conductivity or capacitively coupled contactless conductivity detections (C4D). As a conductivity detection method, C4D is a good alternative method for metal ions. The detection in C4D is based on the difference of conductivity between the sample plug and the running buffer. Compared with the contact conductivity method, C4D has gained increasing importance due to its obvious advantages such as effective isolation from the high voltage and electrode contamination. Additionally it has better durability because of simplified production process of the detector. Detection cell in C4D is formed by two tubular metal electrodes that are placed around the capillary. They are separated by a gap of several mm. In order to avoid direct capacitive coupling between the electrodes a grounded Faraday shield is used. After an AC voltage is applied to the first electrode, the current passes through the cell, the detection gap between the electrodes inside the capillary, and back to the pick-up at the second electrode. The signal is then further amplified and processed. By using this basic principle, the detection is based on the difference in conductivity between the background electrolyte (BGE) and a zone of the analyte. The separated species are detected in a gap between two electrodes along of the capillary. Any analytical separation technique has two important features. First is the ability of resolution the samples, which is given by the selectivity and separation efficiency. Second is the detector sensitivity. CE has been a high efficiency technique with separation efficiencies often approaching over a million of theoretical plates. Such a high separation efficiency can lead to a good resolution of the analyzed species even if optimization of the separation conditions is not performed. On the other hand ions with very close ionic mobilities can be separated only in a suitable running electrolyte increasing the difference in their effective electrophoretic mobilities. Separations of similar species can be achieved with running electrolyte containing a constituent forming complexing equilibria with the analytes. In the separation pH and acid dissociation constants (pKa) has important influence. Lanthanides are 14 elements (Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu) whose properties are similar to lanthanum, so they are called lanthanides. Moreover, thay are known as the rare earth elements. Lanthanides can be analyzed by capillary electrophoresis, except Pm. Because Pm has no stabile structure like other lanthanides have. They have the similar physical and chemical properties, so their analysis cannot be realized easily. They are found in various contents, which are colloidal organic structures in nature, inorganic ligands and the earth Shell etc. The most found contents are geological area. These analysis is applied to miscellaneous instrumental techniques which are not only capillary electrophoresis techniques but also some chromatographic and another analysis techniques. Separation of lanthanides with capillary electrophoresis can be performed with applying on column complexation method. A complexing agent have to choose for differing the electrophoretic mobilities of lanthanides. The electrophoretic mobilities of lanthanide ions differ only unimportantly because of their similarities in their chemical and physical properties. Due to their equal charge and resembling ionic radii, the separation of lanthanide ions is not very simple process. Moreover, the stability constants of these metal ions with a given complexing agent occur only slight differences. A little bit differences in complex-forming of lanthanides are employed to achieve a successful separation. However, complexing agent has to be chosen to amplify the differences in the effective mobilities of lanthanides and make easy their separation. For this aim, complexation with organic ligands are generally used. In principle there are two main strategies for using the complexing agents in CE – on-capillary and precapillary complexation – which correspond approximately to the formation of partial (labil) and complete (inert) complexation. Both approaches have been successfully applied to separate lanthanide ions, although complete complexation seems to be more effective. Complete complexation has been widely employed for improvement of concentration sensitivity for having strong absorbance in the UV-Vis region. On-line or off-line reactions, using a number of complexing agents, have already been examined. With wise choice of either weak or strong complexing agents, the separation selectivity of metal ions having similar electrophoretic mobilities, have been enhanced. The nature of the complexing agent, its concentration and the pH of the running buffer are the main parameters governing the migration of lanthanide ions and affecting their separation. 2-hydroxyisobutyric acid (HIBA) is the most common complexing agent in separations of lanthanides; complexing agents such as lactic acid (LAc), acetic acid (HAc) were tested for separation of the lanthanide ions. In this study, the detection of lanthanide ions with C4D was investigated. The separation of lanthanide ions was achieved in the presence of two competitive ligands, HIBA and HAc. HIBA can insufficient for separation of lanthanide ions, so it needs a complexation co-agent for achieving completely separation. The resolution of partially complexed positively charged complexes is improved by using HIBA as main complexing agent and HAc an assistant complexing agent. Acids used in this study, gives partial complexation with the lanthanides with medium stability, and provide the electrophoretic mobility differences that a sufficient for a satisfactory detection of all 14 lanthanides. The detection limits of lanthanide ions are comparable to or better than that in the CE of lanthanides with indirect UV detection. The electrophoretic mobility of lanthanide ions differentiated by the degree of complexation with HIBA can be further differentiated by the addition of HAc. An on-column separation of 14 lanthanides was achieved in only 6 min using 1 mM HAc ve 4.5 mM HIBA at pH 4.5. Determination of lanthanide complexes was performed by C4D. The detection limits (signal-to-noise ratio=3) were determined to range to from about 0.125 to 0.586 µM. The repeatability for the lanthanide ions was investigated under the optimal conditions. The RSD values (n=7) for migration times was 1.62-3.19 % . Similarly, the RSD values for peak area were 1.13-4.49 %.Yüksek LisansM.Sc

Phosphodiesterase 10A deactivation induces long-term neurological recovery, peri-infarct remodeling and pyramidal tract plasticity after transient focal cerebral ischemia in mice

The phosphodiesterase (PDE) superfamily comprises enzymes responsible for the cAMP and cGMP degradation to AMP and GMP. PDEs are abundant in the brain, where they are involved in several neuronal functions. High PDE10A abundance was previously observed in the striatum; however its consequences for stroke recovery were unknown. Herein, we evaluated the effects of PDE10A deactivation by TAK-063 (0.3 or 3 mg/kg, initiated 72 h post-stroke) in mice exposed to intraluminal middle cerebral artery occlusion. We found that PDE10A deactivation over up to eight weeks dose-dependently increased long-term neuronal survival, angiogenesis, and neurogenesis in the peri-infarct striatum, which represents the core of the middle cerebral artery territory, and reduced astroglial scar formation, whole brain atrophy and, more specifically, striatal atrophy. Functional motor-coordination recovery and the long-distance plasticity of pyramidal tract axons, which originate from the contralesional motor cortex and descend through the contralesional striatum to innervate the ipsilesional facial nucleus, were enhanced by PDE10A deactivation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed a set of dopamine receptor-related and neuronal plasticity-related PDE10A targets, which were elevated (e.g., protein phosphatase-1 regulatory subunit 1B) or reduced (e.g., serine/threonine protein phosphatase 1α, β-synuclein, proteasome subunit α2) by PDE10A deactivation. Our results identify PDE10A as a therapeutic target that critically controls post-ischemic brain tissue remodeling and plasticity