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

Measurement of photon interaction parameters of high-performance polymers and their composites

WOS: 000438143300012In the present study, commercially important high-performance polymers and their composites have been investigated with respect to photon interactions as means of mass attenuation coefficient (/), mean free path (MFP), half-value layer (HVL), effective atomic number (Z(eff)), effective electron density (N-eff), and energy absorption and exposure buildup factors (EABF and EBF) at different photon energies. For this purpose, sample plates were prepared by extrusion and injection techniques using polyethersulfone, polyetherimide, acrylonitrile butadiene styrene copolymer, polyamide 66, polyphthalamide, and polypropylene copolymers as high-performance polymers and glass and carbon fibers as reinforcement. The (/) s of the materials were measured at 81 and 356keV photon energies to determine MFP, HVL, Z(eff), and N-eff. The theoretical values of these parameters were calculated via ZXCOM, WinXCom and Monte Carlo N-Particle simulation code (MCNP), and a good agreement was obtained between WinXCom-MCNP and MCNP-Exp. Finally, EABFs and EBFs of the samples were calculated up to around 40 MFP in the energy region 0.015-15MeV and significant variations were observed in the continuous energy and MFP regions

Reduced folate carrier 1 is present in retinal microvessels and crucial for the inner blood retinal barrier integrity

BackgroundReduced folate carrier 1 (RFC1; SLC19a1) is the main responsible transporter for the B9 family of vitamins named folates, which are essential for normal tissue growth and development. While folate deficiency resulted in retinal vasculopathy, the expression and the role of RFC1 in blood-retinal barrier (BRB) are not well known.MethodsWe used whole mount retinas and trypsin digested microvessel samples of adult mice. To knockdown RFC1, we delivered RFC1-targeted short interfering RNA (RFC1-siRNA) intravitreally; while, to upregulate RFC1 we delivered lentiviral vector overexpressing RFC1. Retinal ischemia was induced 1-h by applying FeCl3 to central retinal artery. We used RT-qPCR and Western blotting to determine RFC1. Endothelium (CD31), pericytes (PDGFR-beta, CD13, NG2), tight-junctions (Occludin, Claudin-5 and ZO-1), main basal membrane protein (Collagen-4), endogenous IgG and RFC1 were determined immunohistochemically.ResultsOur analyses on whole mount retinas and trypsin digested microvessel samples of adult mice revealed the presence of RFC1 in the inner BRB and colocalization with endothelial cells and pericytes. Knocking down RFC1 expression via siRNA delivery resulted in the disintegration of tight junction proteins and collagen-4 in twenty-four hours, which was accompanied by significant endogenous IgG extravasation. This indicated the impairment of BRB integrity after an abrupt RFC1 decrease. Furthermore, lentiviral vector-mediated RFC1 overexpression resulted in increased tight junction proteins and collagen-4, confirming the structural role of RFC1 in the inner BRB. Acute retinal ischemia decreased collagen-4 and occludin levels and led to an increase in RFC1. Besides, the pre-ischemic overexpression of RFC1 partially rescued collagen-4 and occludin levels which would be decreased after ischemia.ConclusionIn conclusion, our study clarifies the presence of RFC1 protein in the inner BRB, which has recently been defined as hypoxia-immune-related gene in other tissues and offers a novel perspective of retinal RFC1. Hence, other than being a folate carrier, RFC1 is an acute regulator of the inner BRB in healthy and ischemic retinas.Hacettepe Universit

The role of circadian rhythm in the regulation of cellular protein profiles in the brain

Background/aim: Circadian rhythm plays a significant role in the regulation of almost all kinds of physiological processes. In addition, it may also have a direct or indirect effect on the neurodegenerative processes, including Alzheimer's disease, Parkinson's disease, and ischemic stroke. Therefore, the identification of circadian rhythm-related proteins is crucial to be able to understand the molecular mechanism of the circadian rhythm and to define new therapeutic target for the treatment of degenerative disorders. Materials and methods: To identify the light and dark regulated proteins, 8-12 weeks, male Balb/C mice were used at two different time points (morning (Zeitgeber time-0 (ZT0)) and midnight (ZT18)) under physiological conditions. Therefore, brain tissues were analyzed via liquid chromatography tandem mass spectrometry. Results: A total of 1621 different proteins were identified between ZT0 and ZT18 mice. Among these proteins, 23 proteins were differentially expressed (p < 0.05 and fold change 1.4) in ZT18 mice, 11 upregulated (AKAP10, ALDOC, BLK, NCALD, NFL, PDE10A, PICAL, PSMB6, RL10, SH3L3, and SYNJ1), and 12 downregulated (AT2A2, AT2B1, CPNE5, KAP3, MAON, NPM, PI51C, PPR1B, SAM50, TOM70, TY3H, and VAPA) as compared with ZT0 mice. Conclusion: Taken together, here we identified circadian rhythm-related proteins, and our further analysis revealed that these proteins play significant roles in molecular function, membrane trafficking, biogenesis, cellular process, metabolic process, and neurodegenerative disorders such as Parkinson's disease

The neuroprotective role of melatonin and normobaric oxygen therapy after cerebral ischemia

Dünya Sağlık Örgütü'nün verilerine göre beyin felci dünyada ölüm sebepleri bakımından 2. sırada yer alan bir hastalıktır. Her üç beyin felci vakasından biri ölüm ile sonuçlanırken beyin felci sonrası hayatta kalanlar ise yaşamlarının geri kalanını ciddi nörolojik eksiklikler ile sürdürmek zorunda kalmaktadır. Klinik açıdan beyin felci sonrası yüksek konsantrasyonda normobarik oksijen (NBO) tedavisinin uygulanması reperfüzyon hasarını, serbest oksijen radikallerinin (ROS) üretimi üzerinden arttırması nedeniyle yıllardır büyük tartışmalara neden olmaktadır. Aynı zamanda penumbra bölgesindeki elektriksel dalgalanmaların da kan damarlarında daralmaya neden olduğu patofizyolojik süreçler düşünüldüğünde NBO tedavisinin tekrar değerlendirilmesi gerekliliğini ortaya koymaktadır. Beyin felci sonrasında özellikle iskemik alanda artan ROS'ların NBO tedavisi ile beraber daha fazla artacağı düşünüldüğünde bu tedavinin serbest radikal yakalayıcı bir molekül olan melatonin ile kombinasyonunun tedavinin etkinliği arttıracağı düşünülmektedir. Bunun için beyin felci sonrası %21, %70 veya %100 NBO tedavilerinin tek başlarına veya melatonin ile kombinasyonun etkisini araştırabilmek için 8-12 haftalık erkek Balb/c farelere 30 veya 90 dakikalık orta serebral arter tıkanması sonrası sırasıyla 72 veya 24 saat reperfüzyon gerçekleştirilmiştir. Reperfüzyonun hemen başlangıcında farelere %21, %70 veya %100 NBO tedavileri uygulanmış ve 4 mg/kg melatonin veya taşıyıcı intraperitonel olarak uygulanmıştır. Bu tez kapsamında orta serebral arter tıkanması metodu kullanarak gerçekleştirilen beyin felci sonrasında uygulanan %100 NBO tedavisi ve bu tedavinin melatonin ile kombinasyonu DNA kırıklarının oluşumunu, hasar alanını, beyin ödemini ve kan beyin bariyeri geçirgenliğini azaltırken, nörolojik iyileşmeyi ve iskemik bölgedeki beyin kan akımını arttırdığı görülmüştür. Elde edilen bulguların özellikle beyin felci gibi nörodejeneratif hastalıkların tedavisine yönelik yeni strateji ve hedef moleküllerin bulunmasına katkı sağlaması beklenmektedir.Stroke is the second leading cause of death worldwide according to World Health Organization data. One third of all stroke cases results in death, while the survivors suffer from serious neurological deficits for the rest of their lives. Use of high concentration normobaric oxygen (NBO) therapy following stroke in the clinic is under substantial debate for many years since it results in increased production of reactive oxygen species (ROS). In addition, considering the pathophysiological processes which result in vasoconstriction due to the electrical fluctuations in the penumbra, it is important to re-evaluate the use of NBO therapy. It is believed that the efficacy of combinatory treatment consisting NBO therapy with free radical scavenger melatonin is going to be enhanced, as the increased ROS following cerebral ischemia are further promoted in the ischemic area by NBO. To this end, to investigate the effects of 21%, 70% or 100% NBO treatment alone or in combination with melatonin after cerebral ischemia, 8-12 weeks-old male Balb/c mice were subjected to 30 or 90 minutes of middle cerebral artery occlusion followed by 72 or 24 hours of reperfusion. Immediately at the onset of reperfusion, 21%, 70% or 100% NBO treatments and 4 mg/kg melatonin or vehicle control were administered intraperitoneally. In this thesis, 100% NBO treatment and combination of this treatment with melatonin, which was applied after cerebral ischemia that was carried out using middle cerebral artery occlusion method was demonstrated to decrease DNA fragmentation, infarct area, brain edema and blood brain barrier permeability, while increasing neurological recovery and cerebral blood flow in the ischemic region. The obtained results are expected to contribute to the identification of new strategies and target molecules intended for treatment of neurodegenerative disorders, especially such as brain ischemia

Effects of bmal1 on akt signal pathway after oxygen glucose deprivation

WOS: 000453220100004

Investigation of the role of PDE10A inhibition after traumatic brain injury

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Effects of pde10a and bmal1 on circadian rhythm and brain injury

...Türkiye Bilimsel ve Teknolojik Araştırma Kurumu (TUBITAK

Role of circadian rhythm on focal cerebral ischemia

Yüksek Lisans TeziSirkadiyen ritim, günlük fizyolojik fonksiyonları ayarlamakla kalmayıp aynı zamanda patofizyolojik süreçleri de etkileyen biyolojik bir saattir. Daha önceki çalışmalara bağlı olarak serebral iskemi ve sirkadiyen ritim arasında kuvvetli bir ilişki olduğu düşünülmektedir. Günümüze kadar sirkadiyen ritim ile ilgili yapılan çalışmaların büyük bir çoğunluğu klinik gözlemlere dayandığından çalışmalar kısıtlı bir alanda kalmıştır. Bununla birlikte, iskemi sonrası patofizyolojik olaylara karşı sirkadiyen ritmin göstereceği hücresel ve moleküler mekanizmalar büyük ölçüde bilinmemektedir. Bu amaçla 8-12 haftalık erkek Balb/ C farelerine 6 saatlik aralıklarla (ZT0, ZT6, ZT12 ve ZT18) dört farklı zaman noktasında 30 dk orta serebral arter tıkanması takiben 72 saatlik reperfüzyon gerçekleştirildi. Nöronal sağkalım ve apopitotik hücre ölümü immuno-histokimyasal metotlarla incelendi. Biyolojik saat proteinleri; Period-1, Period-2, CLOCK ve Bmal1 protein ifadeleri Western Blot ile değerlendirildi. Ek olarak, PI3K sinyal yolağı Pathscan metoduyla değerlendirildi. Sirkadiyen ritim ile ilişkili proteinler sıvı kromatografi-kütle spektrometresi yöntemleri ile analiz edildi. Yapılan bu çalışmada zamana bağlı olarak değişen sirkadiyen ritim proteinleri beyin felci sonrası gelişen beyin hasarını doğrudan etkilediğinin kanıtlarını sunulmaktadır. Gece yarısı (ZT18; 24:00) beyin felci geçiren farelerde sabah (ZT0, 06:00) beyin felci geçirenlere göre sirkadiyen ritim proteinleri Bmal1, Per1 ve CLOCK proteinlerinin seviyeleri arttığı ve bunlarla beraber nöronal sağkalımın arttığı, apopitotik hücre sayısının ise azaldığı gösterildi. Ayrıca yapılan protein çalışmaları ile bu etkileri sağkalım kinazlarından p-AKT ve p-Erk-1/2 üzerinden yaptığı gösterildi. Bu bilgilere ek olarak yapılan geniş ölçekli proteomik analizi sonucu sabah ZT0'a kıyasla; ZT18 grubunda GNAZ, NEGR1, IMPCT ve PDE1B seviyesi artarken, CSKP, HBB1, HBB2 ve HBA düzeyleri anlamlı ölçüde düştüğü gözlemlendi. Elde edilen sonuçlar, gece beyin felci geçirenlerde nöronal hasarın daha az olduğu ve bunu sağkalım kinazları ve sirkadiyen ritim ile alakalı proteinlerin ifadesinin artmasıyla sağladığı gösterildi.Circadian clocks are endogenous timers not only adjusting daily physiological functions but also affect pathophysiological processes. Depends on previous studies, there have been strong relation between cerebral ischemia and circadian rhythm. In addition, occurance of stroke cases displays a time of day variation. To date, most of the circadian expression studies remained restricted with clinical observations. Nevertheless, the molecular and cellular mechanism linking circadian rhythm to the internal response mechanisms against pathophysiological events after ischemia remained largely unknown. To this end, 8-12 weeks male Balb/c mice were exposed to min of MCAO following 72 h reperfusion at four different Zeitgeber time (ZT) points with 6-h intervals (ZT0, ZT6, ZT12, and ZT18). Disseminate neuronal injury and DNA fregmantation were examined using immunohistochemistry. Core clock proteins; Period-1, Period-2, CLOCK and Bmal1 protein expressions were evaluated by Western Blot. In addion, PI3K signaling pathway analysed via planar surface immune assay and circadian rhythm related proteins were evaluated by liquid chromatography– mass spectrometry tools. Here, we present evidence that activational changes of proteins by circadian rhythm regulations directly influence brain injury following cerebral ischemia. Midnight (ZT18; 24:00) I/R injury in mice resulted in significantly improved neuronal survival, and decreased apoptosis compared with ischemia induced at ZT0 (06:00), which were associated with increased expressions of circadian proteins Bmal1, PerI, and Clock proteins and survival kinases p-AKT and p-Erk-1/2. Furthermore, proteomics revealed significantly reduced CSKP, HBB1, HBB2, and HBA levels, while increased GNAZ, NEGR1, IMPCT, and PDE1B at midnight as compared with ZT0. Results showed that nighttime ischemic injury results in less severe neuronal damage, with increased neuronal survival, increased levels of survival kinases and circadian clock proteins, and also alters the circadian-related proteins

Physiological and pharmacological roles of melatonin in the pathophysiological components of cellular injury after ischemic stroke

Apart from its metabolic or physiological functions, melatonin has a potent cytoprotective activity in the physiological and pathological conditions. It is synthetized by the pineal gland and released into the blood circulation but particularly cerebrospinal fluid in a circadian manner. It can also easily diffuse through cellular membranes due its small size and lipophilic structure. Its cytoprotective activity has been linked to its potent free radical scavenger activity with the desirable characteristics of a clinically- reliable antioxidant. Melatonin detoxifies oxygen and nitrogen-based free radicals and oxidizing agents, including the highly toxic hydroxyl-and peroxynitrite radicals, initiating cellular damage. However, the cytoprotective activity of melatonin is complex and cannot be solely limited to its free radical scavenger activity. It regulates cellular signaling pathways through receptor– dependent and independent mechanisms. Most of these downstream molecules, such as PI3K/AKT pathway components, also contribute to the cytoprotective effects of melatonin. In this term, melatonin is a promising molecule for the treatment of neurodegenerative disorders, such as ischemic stroke, which melatonin reduces ischemic brain injury in animal models of ischemic stroke. It regulates also circadian rhythm proteins after ischemic stroke, playing roles in cellular survival. In this context, present article summarizes the possible role of melatonin in the pathophysiological events after ischemic stroke