Downregulation of Yap1 during limb regeneration results in defective bone formation in axolotl

The Hippo pathway plays an imperative role in cellular processes such as differentiation, regeneration, cell migration, organ growth, apoptosis, and cell cycle. Transcription coregulator component of Hippo pathway, YAP1, promotes transcription of genes involved in cell proliferation, migration, differentiation, and suppressing apoptosis. However, its role in epimorphic regeneration has not been fully explored. The axolotl is a well-established model organism for developmental biology and regeneration studies. By exploiting its remarkable regenerative capacity, we investigated the role of Yap1 in the early blastema stage of limb regeneration. Depleting Yap1 using gene-specific morpholinos attenuated the competence of axolotl limb regeneration evident in bone formation defects. To explore the affected downstream pathways from Yap1 down-regulation, the gene expression profile was examined by employing LC-MS/MS technology. Based on the generated data, we provided a new layer of evidence on the putative roles of increased protease inhibition and immune system activities and altered ECM composition in diminished bone formation capacity during axolotl limb regeneration upon Yap1 deficiency. We believe that new insights into the roles of the Hippo pathway in complex structure regeneration were granted in this study.Science Academ

Active shrinkage protects neurons following axonal transection

Trauma, vascular events, or neurodegenerative processes can lead to axonal injury and eventual transection (axotomy). Neurons can survive axotomy, yet the underlying mechanisms are not fully understood. Excessive water entry into injured neurons poses a particular risk due to swelling and subsequent death. Using in vitro and in vivo neurotrauma model systems based on laser transection and surgical nerve cut, we demonstrated that axotomy triggers actomyosin contraction coupled with calpain activity. As a consequence, neurons shrink acutely to force water out through aquaporin channels preventing swelling and bursting. Inhibiting shrinkage increased the probability of neuronal cell death by about 3-fold. These studies reveal a previously unrecognized cytoprotective response mechanism to neurotrauma and offer a fresh perspective on pathophysiological processes in the nervous system.Yüzüncü Yıl Universit

The role of hi̇ppo pathway i̇n axolotl arm regenerati̇on

Hippo yolağı farklılaşma, yenilenme, hücre göçü, organ büyümesi, apoptoz, hücre döngüsü gibi birçok hücresel olayda etkin rol oynamaktadır. Aksolotl canlılarda yenilenmeyi aydınlatmak için sık kullanılan bir modeldir. Bu çalışmada neotenik ve metamorfik aksolotlarda kol yenilenmesinde Hippo yolağında bulunan YAP ve LATS proteinlerinin durumu incelenmiştir. Çalışmada aksoltl kol yenilenmesinde metamorfik ve neotonik aksolotlarda kol hasarı yapılmış ve hasarın 10. gününde doku örnekleri toplanmıştır. Bu doku örneklerinde kantitatif gerçek zamanlı polimeraz zincir reaksiyonu (KGZ-PZR) ve immunohistokimya yapılmıştır. Gen inhibisyonu yapılan grupta ise, metamorfik ve neotonik hayvanlarda kol hasarının 10. gününde morfolino sistemi kullanılarak YAP ve LATS proteinlerinin inhibisyonu incelenmiştir. 6 gün beklenildikten sonra dokular izole edilip: KGZ-PZR, immunohistokimya ve proteomik çalışması ile sonuçlar değerlendirilmiştir. Kol hasarının 10. gününde, neotonik hayvanlarda YAP mRNA miktarı metamorfik hayvanlara göre 72 kat fazlayken, neotonik hayvanlarda LATS mRNA miktarı metamorfik hayvanlara göre 87 kat fazla olmuştur. Bu değerler hasarın 16. gününde neotonik hayvanlarda YAP mRNA miktarı metamorfik hayvanlara göre 227 kat fazlayken, neotonik hayvanlarda LATS mRNA miktarı metamorfik hayvanlara göre çok daha fazla artış (849 kat) göstermiştir. İnhibisyon yapılan metamorfik hayvanlarda, inhibisyon sonrası YAP ve LATS miktarları kontrol grubuna göre artmıştır. İnhibisyon yapılan neotonik hayvanlarda LATS inhibisyonu sonrası YAP ve LATS mRNA miktarları kontrol grubuna göre azalırken, YAP inhibisyonu sonrası YAP ve LATS mRNA miktarları kontrol grubuna göre artmıştır. İmmunohistokimya sonuçları ise pozitif hücreler sayılarak elde edilmiştir. Yenilenme tamamlanınca bilgisayarlı tomografi kullanılarak kemik fotoğrafları çekilmiştir. Proteomiks sonuçlarına göre neotonik hayvanda YAP inhibisyonu sonrası yara iyileşmesi proteinleri anlamlı artış göstermiştir. Sonuç olarak Hippo yolağı aksolotl kol yenilenmesinde elzem yolaklardan biri olmaya adaydır. Bu tez TÜBİTAK (1002 – 119Z976) tarafından desteklenmiştir.Axolotl is a unique model organism in terms of regeneration capacity. Considering the tasks of the Hippo pathway known in the literature, this pathway is expected to have an effective role in axolotl arm regeneration. Morpholino is mRNA inhibition system. It is one of the most frequently used techniques to illuminate regeneration in living things like axolotl. In this study, arm damage was performed for both metamorphic and neotenic axolotls. Tissue samples were collected on the 10th day after arm injury. Quantitative real-time Polymerase Chain Reaction (qRT-PCR) and immunohistochemistry were performed on these samples. In the gene-inhibited group, 10 days were waited after arm injury in metamorphic and neotenic animals, and inhibition of YAP and LATS proteins were performed on the 10th day using morpholino system. At 16 days after arm injury, tissues were isolated and qRT-PCR, immunohistochemistry and proteomics were performed. Yap decreased about 4 times and LATS decreased approximately 2 times in qRT-PCR analysis that shows the effectiveness of morpholino. On the 10th day after arm cut, while the amount of YAP mRNA in neotenic animals was 72 times higher than metamorphic animals; the amount of LATS mRNA in neotenic animals is 87 times higher than in metamorphic animals. These rates are on the 16th day; while the amount of YAP mRNA in neotenic animals was 227 times higher than metamorphic animals; the amount of LATS mRNA in neotenic animals is 849 times higher than in metamorphic animals. In inhibited neotenic animals, after LATS inhibition, the amount of YAP and LATS mRNA decreased compared to the control group. After YAP inhibition, the amount of YAP and LATS mRNA increased compared to control group. Immunohistochemistry results were obtained by counting positive cells. After the regeneration was completed, bones were visualized using computed tomography. According to proteomics results wound healing proteins after YAP inhibition showed significant increase in neotonic animal. As a result, Hippo pathway is essential pathway in axolotl arm regeneration. This thesis was supported by TUBITAK (1002 – 119Z976

The effects of axotomy injury on membrane tension of dorsal root ganglion neurons

Periferik sinir hasarından sonra, arka kök gangliyonundaki nöronlar ve omurilik ve beyin sapındaki motor nöronlar büyük morfolojik ve moleküler değişimlere uğrarlar. Hasar sonucu aksonal dejenerasyon ve ağrı oluşmaktadır. Periferik sinir hasarına tedavi geliştirmek için nöronlardaki morfolojik ve moleküler değişimlerin tespit edilmesi gerekmektedir. Aksonal hasar in vitro ortamda yapılabilmektedir (aksotomi). Aksotomiye bağlı olarak hücre boyutunda küçülme meydana gelmektedir. Aksonal hasarda kalpainler ve kalsiyum önemli işlevler yerine getirmektedir. Hasarda hücre iskeleti ile ilişkili proteinler de önemlidir. Kaspaz-3 bağımlı aktin ve tübülinin kesimi, yaralanmalardaki aksonal hasarlarda önemli bir göstergedir. Bu çalışma ile, literatürde tam olarak nasıl ölçüleceği belirlenmemiş, arka kök gangliyonu nöronlarında hücre zarı gerginliklerin (lateral gerginlik ve kortikal gerginlik) ayrı ayrı ölçülmesi, değerlendirilmesi ve hasarın altında yatan moleküler mekanizmaların aydınlatılması amaçlanmaktadır. Çalışmamızda fareden elde edilen arka kök gangliyonu nöronlarında lateral ve kortikal kuvvet ölçümü optik cımbızlar (optical tweezers) sistemi ile yapılmıştır. Aksotomi öncesi-sonrası kuvvet ölçümüne ek olarak, moleküler mekanizmayı bulmak için, canlı Kaspaz-3/7 boyası parlaklık ölçümleri hızlandırılımış (time-lapse) floresan mikroskobu ile yapılmıştır. Çalışmamızın sonuçlarına göre, aksotomiden sonra hücre boyutunda anlamlı küçülme meydana gelmiştir. Aksotomi öncesi lateral kuvvet, aksotomi öncesi kortikal kuvvet ve aksotomi sonrası kortikal kuvvet birbirleri ile anlamlı olarak ilişkilidir. Ayrıca aksotomi ile gerçekleşen mutlak alan değişikliği ile aksotomi sonrası lateral kuvvet, aksotomi sonrası kortikal kuvvet ve Kaspaz-3/7 parlaklık değişikliği anlamlı koreledir. Bu çalışmanın sonuçları, periferik sinir hasarının altında yatan mekanik değişikliklerin aydınlatılmasına yönelik kaynaklara katkı sağlayacaktır.After peripheral nerve injury, neurons in dorsal root ganglia and motor neurons of spinal cord and brain stem undergo fundamental morphological and molecular changes. Axonal degeneration and pain are arised from results of nerve injury. For improving treatment of peripheral nerve injury, these morphological and molecular changes must be detected. Axonal degeneration can be done in vitro (axotomy). Volume of cell bodies decreases with axotomy. Calcium influx into the cell body and calcium dependent molecules like calpeins are important in axonal injury. Cytoskeletal-related proteins are important in nerve injury. Caspase-3 dependent cleavage of actin and tubulin is a marker for axonal degeneration. There are less informations about mechanical changes of neurons in nerve injury. With this study, measurements and analysis of cellular tensions like lateral and cortical tensions in dorsal root ganglion neurons seperately and molecular mechanisms of peripheral nerve injury have been aimed. Cortical and lateral tensions were measured in dorsal root ganglion neurons from Balb-c mice using optical tweezer system. For understanding molecular mechanisms of nerve injury, addition to lateral and cortical measurements before and after axotomy, intensities of Caspase-3/7 were measured using time-lapse flourescence microscope. According to results of our study, Volumes of cells are significantly changed. Lateral tension before axotomy, cortical tension before axotomy and cortical tension after axotomy were correlated each other significantly. Furthermore, absolute area changes which are derived from axotomy were correlated with lateral forces after axotomy, cortical forces after axotomy and changes in Caspase-3/7 intensity significantly. Results of our study can contribute to further studies on the mechanical changes underlying peripheral nerve injury

Neuroprotective effects of curcumin-loaded emulsomes in a laser axotomy-induced CNS injury model

Purpose: Curcumin, a polyphenol isolated from the rhizomes of turmeric, holds great potential as a neuroprotective agent in addition to its anti-inflammatory and antioxidant characteristics. The poor bioavailability and low stability of curcumin are the greatest barriers to its clinical use. This study aims to investigate the neuroprotective effect of curcumin on axonal injury, by delivering the lipophilic polyphenol to a primary hippocampal neuron culture by means of a lipid-based drug delivery system, named emulsomes.Methods: To study neuroregeneration ex vivo, an injury model was established through single-cell laser axotomy on hippocampal neurites. Upon treatment with curcumin-loaded emulsomes (CurcuEmulsomes), curcumin and CurcuEmulsome uptake into neurons was verified by three-dimensional Z-stack images acquired with confocal microscopy. Neuron survival after axonal injury was tracked by propidium iodide (PI) and Hoechst staining. Alterations in expression levels of physiological markers, such as anti-apoptotic marker Bcl2, apoptotic marker cleaved caspase 3, neuroprotective marker Wnt3a and the neuronal survival marker mTOR, were investigated by immunocytochemistry analyses.Results: The results indicated significant improvement in the survival rate of injured neurons upon CurcuEmulsome treatment. Bcl2 expression was significantly higher for injured neurons treated with curcumin or CurcuEmulsome. Reduction in caspase 3 expression was seen in both curcumin and CurcuEmulsome treatment, whereas there were no significant changes in Wnt3a and mTOR expression.Conclusion: The established laser-axotomy model was proven as a reliable methodology to study neurodegenerative models ex vivo. CurcuEmulsomes delivered curcumin to primary hippocampal neurons successfully. Treated with CurcuEmulsomes, injured hippocampal neurons benefit from the neuroprotective effects of curcumin, exhibiting a higher survival rate and increased anti-apoptotic marker levels

Nanobodies: Diagnostic and therapeutic antibody fragments

Nanokorlar, bilinen en küçük ve fonksiyonel antikor fragmanlarıdır. Yapısında sadece iki ağır zincir bulunduran antikorların (heavy chain only antibodies; HcAb) antijen bağlama bölgesi, “nanokor” (nanobody) olarak adlandırılmıştır. Böylelikle bütün bir antikor molekülü kadar fonksiyonel olan ve sadece ağır zincirden oluşan (variable domain of the heavy chain of HcAb; VHH) en küçük antikor fragmanı tanımlanmıştır. Nanokorlar, küçük boyutları sayesinde antikorların ulaşamadığı bölgelere dahi bağlanabilmektedir. Bunun yanında immün tepkimelere yol açmamaları ve görüntülemede diğer antikorlara nazaran daha iyi sonuç vermeleri sebebiyle son yıllarda kanser, enfeksiyon, enflamasyon ve nörodejeneratif hastalıklar başta olmak üzere pek çok hastalığa yönelik tanı ve tedavi amaçlı araştırmalarda kullanılmaya başlanmıştır. Vücut sıvılarında bulunmasının dışında prokaryotik ve ökaryotik canlılarda da kolayca üretilebilmesi ile nanokorlar, rekombinant proteinler olarak da elde edilebilmektedir. Bu sayede, nanokorlara entegre edilerek terapötik moleküller geliştirilmektedir ve bu çalışmalarla birlikte nanokorlar, hedefe yönelik tedavi yaklaşımları için yeni perspektifler sunmaktadır. Ayrıca nanokorlara özgün görüntüleme ajanları bağlanarak, tanı amaçlı moleküller de oluşturulmaktadır. Nanokorların biyosensör olarak kullanılmasına ilişkin sürdürülen çalışmalar ise in vivo görüntüleme için yeni araçlar geliştirilmesine olanak sağlayacaktır.Nanobodies are the smallest and functional antibody fragments ever known. Antigen-binding region of antibodies composed of two heavy chains only (heavy chain only antibodies; HcAb) is termed as “nanobody”. Thus, the smallest antibody fragment that is as functional as a whole antibody and comprises of only heavy chains has been identified. Recently, nanobodies have been used in the diagnosis and treatment of numerous diseases including especially cancer, infection, inflammation, and neurodegenerative diseases since they are the smallest functional antibody fragments, do not lead immunological reactions, have better imaging capacities than other antibodies. Beside residing in body fluids, nanobodies can be engineered as recombinant proteins since they can be easily produced in both prokaryotic and eukaryotic organisms. Furthermore, several therapeutic molecules integrated to nanobodies have been developed and in the light of these studies, nanobodies provide new perspectives to targeted therapy approaches. In addition to that, nanobodies can be served as diagnostic molecules upon modification with imaging agents. Ongoing research about the use of nanobodies in biosensor development is another promising area that allow the development of new tools for in vivo imaging

Neurons from human mesenchymal stem cells display both spontaneous and stimuli responsive activity

Mesenchymal stem cells have the ability to transdifferentiate into neurons and therefore one of the potential adult stem cell source for neuronal tissue regeneration applications and understanding neurodevelopmental processes. In many studies on human mesenchymal stem cell (hMSC) derived neurons, success in neuronal differentiation was limited to neuronal protein expressions which is not statisfactory in terms of neuronal activity. Established neuronal networks seen in culture have to be investigated in terms of synaptic signal transmission ability to develop a culture model for human neurons and further studying the mechanism of neuronal differentiation and neurological pathologies. Accordingly, in this study, we analysed the functionality of bone marrow hMSCs differentiated into neurons by a single step cytokine-based induction protocol. Neurons from both primary hMSCs and hMSC cell line displayed spontaneous activity (>= 75%) as demonstrated by Ca++ imaging. Furthermore, when electrically stimulated, hMSC derived neurons (hMd-Neurons) matched the response of a typical neuron in the process of maturation. Our results reveal that a combination of neuronal inducers enhance differentiation capacity of bone marrow hMSCs into high yielding functional neurons with spontaneous activity and mature into electrophysiologically active state. Conceptually, we suggest these functional hMd-Neurons to be used as a tool for disease modelling of neuropathologies and neuronal differentiation studies.Istanbul Medipol University Scientific Research Projects Committee Yeditepe Universit

Effects of ginkgo biloba components and calcium channel blockers on cortical neuron excitability in wag/rij rats with absence epilepsy: In vitro calcium imaging study

...Türkiye Bilimsel ve Teknolojik Araştırma Kurumu (TUBITAK

Effect of ginkgo biloba extract egb 761 on intracellular calcium level and excitability in wag/rij rat cortical neuron culture

Türkiye Bilimsel ve Teknolojik Araştırma Kurumu (TUBITAK)..

Potential of novel methyl jasmonate analogs as anticancer agents to metabolically target HK-2 activity in glioblastoma cells

Change in the energy metabolism of cancer cells, which display significant differences compared to normal cells, is a rising phenomenon in developing new therapeutic approaches against cancers. One of the metabolic enzymes, hexokinase-II (HK-II) is involved in glycolysis, and inhibiting the HK-II activity may be a potential metabolic target for cancer therapy as most of the drugs in clinical use act on DNA damage. Methyl jasmonate (MJ) is one of the compounds blocking HK-II activity in cancer cells. In a previous study, we showed that the novel MJ analogs inhibit HK-II activity through VDAC detachment from the mitochondria. In this study, to evaluate the potential of targeting HK-2 activity, through patient cohort analysis, we first determined HK-2 expression levels and prognostic significance in highly lethal glioblastoma (GBM) brain tumor. We then examined the in vitro therapeutic effects of the novel analogs in the GBM cells. Here, we report that, among all, compound-10 (C-10) showed significant in vitro therapeutic efficacy as compared to MJ which is in use for preclinical and clinical studies. Afterward, we analyzed cell death triggered by C-10 in two different GBM cell lines. We found that C-10 treatment increased the apoptotic/necrotic cells and autophagy in GBM cells. The newly developed analog, C-10, was found to be lethal against GBM by the activation of cell death authorities, mostly in a necrotic and autophagic fashion at the early stages of the treatment. Considering that possibly decreased intracellular ATP levels by C-10 mediated inhibition of HK-2 activity and disabled VDAC interaction, a more detailed analysis of HK-2 inhibition-mediated cell death can provide a deep understanding of the mechanism of action on the oncosis/necroptosis axis. These findings provide an option to design clinically relevant and effective novel HK-II inhibitors and suggest novel MJ analogs to further study them as potential anticancer agents against GBM.European Cooperation in Science and Technology (COST