Targeting GDNF receptors with small molecules for the treatment of Parkinson’s disease

Motor symptoms of Parkinson’s disease (PD) are caused by the degeneration of dopamine (DA) neurons. Currently, no disease-modifying therapies against PD are available. Therefore, the major challenge is developing a drug to protect and restore DA neurons. Glial cell line-derived neurotrophic factor (GDNF) has been identified and characterized for its ability to promote the survival of cultured nigrostriatal dopamine neurons and protect and repair them in animal models of PD. However, the outcomes of the clinical trials with GDNF in PD patients remain inconclusive. GDNF is a promising protein that halts neurodegeneration. GDNF does not cross through the blood-brain barrier (BBB) and has poor diffusion properties, complicating clinical use in PD patients. This thesis aimed to characterize the biological activity of GDNF family ligand (GFL) receptor agonists in both in vitro and in vivo preclinical models of PD. We have developed small molecular weight compounds acting similarly to GDNF in the dopamine system (GFL receptor agonist) with improved pharmacological characteristics. The first-generation GFL receptor agonist, a compound named BT13, potently activated the GDNF receptor REarranged during transfection (RET) and its downstream targets. It protected cultured dopamine neurons from neurotoxin-induced cell death and stimulated dopamine release in the mouse striatum. We further optimized BT13 and developed the second-generation GFL receptor agonist BT44 with ten-fold improved potency. BT44 activated RET, supported cultured DA neuron's survival and protected them from neurotoxin lesions, induced functional recovery, and protected striatal dopaminergic fibers in rats with experimentally induced PD. We also studied the selectivity of both BT13 and BT44 using different functional assays. Our results demonstrate that both BT13 and BT44 are selective towards RET. The compounds promote survival and protect the cultured dopamine neurons only in the presence of RET. BT44 is a promising lead compound that could be potentially developed as a drug for PD treatment. However, BT44 suffers from intrinsic poor aqueous solubility like many other hydrophobic drugs, making its preclinical development and clinical translation difficult. Therefore, to improve BT44 solubility, we developed nanoformulation of BT44 with Pox-based ABA triblock copolymers. We found that nanoformulated BT44 retains its biological activity and has improved absorption and blood-brain barrier (BBB) penetration compared to the pristine BT44. We also demonstrate that nanoformulated BT44 binds to the GFL receptors and competes with GDNF for binding to the RET receptor. The receptor binding studies were not possible with pristine BT44 due to its poor solubility. Taken together, our results indicate that both BT13 and BT44 can activate the RET receptor and its downstream signaling cascade necessary for neuronal survival and protection from neurotoxin insults. Further, our results show that in vitro BT44 is active in 10 nM concentrations, which is a realistic target concentration for actual drugs. Moreover, we developed a nanoformulation of BT44 to improve the solubility of the pristine compound and showed that this nanoformulation retains biological activity. With further optimization and detailed studies, BT44 could be developed as a drug-like candidate for disease-modifying treatment for PD.Tiivistelmä ei saatavilla

Glial cell line-derived neurotrophic factors (GFLs) and small molecules targeting RET receptor for the treatment of pain and Parkinson's disease

Rearranged during transfection (RET), in complex with glial cell line-derived (GDNF) family receptor alpha (GFR alpha), is the canonical signaling receptor for GDNF family ligands (GFLs) expressed in both central and peripheral parts of the nervous system and also in non-neuronal tissues. RET-dependent signaling elicited by GFLs has an important role in the development, maintenance and survival of dopamine and sensory neurons. Both Parkinson's disease and neuropathic pain are devastating disorders without an available cure, and at the moment are only treated symptomatically. GFLs have been studied extensively in animal models of Parkinson's disease and neuropathic pain with remarkable outcomes. However, clinical trials with recombinant or viral vector-encoded GFL proteins have produced inconclusive results. GFL proteins are not drug-like; they have poor pharmacokinetic properties and activate multiple receptors. Targeting RET and/or GFR alpha with small molecules may resolve the problems associated with using GFLs as drugs and can result in the development of therapeutics for disease-modifying treatments against Parkinson's disease and neuropathic pain.Peer reviewe

Ethnomedicinal practices in the arid zone of India: A study in urban and semi-urban areas of Bhuj, Gujarat

Plants are a vital and life-sustaining natural resource as they provide many ecosystem services and supply food, fibre, fuel, medicine, etc. They are distributed heterogeneously in different ecosystems. With the increasing urbanization in all parts of the world, the vegetation overlapped with urban localities like parks, gardens, roadsides, wasteland and other human habitations. Among this vegetation, many of them are used in ethnomedicine or in ayurvedic medicine by people for a long period of time. These valuable plant resources are now under threat due to various anthropogenic activities in the urban landscape. Bhuj is a small historic city in the arid zone of India with a rich diversity of medicinal plants. With the increasing human population and associated developmental activities within the city, many of these plants are under threat of extinction. Therefore, an attempt was made to document these medicinal plants distributed in different parts of the city and their uses for ethnomedicinal purposes. The plants were intensively surveyed and documented using a questionnaire survey, discussion and cross-checked with available literature. A total of 123 species of ethnomedicinal plants were documented and analysed for their uses in curing different health problems and conservation purposes in the urban landscape

Zebrafish GDNF and its co-receptor GFR alpha 1 activate the human RET receptor and promote the survival of dopaminergic neurons in vitro

Glial cell line-derived neurotrophic factor ( GDNF) is a ligand that activates, through coreceptor GDNF family receptor alpha-1 (GFR alpha 1) and receptor tyrosine kinase "RET ", several signaling pathways crucial in the development and sustainment of multiple neuronal populations. We decided to study whether non-mammalian orthologs of these three proteins have conserved their function: can they activate the human counterparts? Using the baculovirus expression system, we expressed and purified Danio rerio RET, and its binding partners GFRa1 and GDNF, and Drosophila melanogaster RET and two isoforms of coreceptor GDNF receptor-like. Our results report high-level insect cell expression of posttranslationally modified and dimerized zebrafish RET and its binding partners. We also found that zebrafish GFRa1 and GDNF are comparably active as mammalian cell- produced ones. We also report the first measurements of the affinity of the complex to RET in solution: at least for zebrafish, the Kd for GFR alpha 1-GDNF binding RET is 5.9 mu M. Surprisingly, we also found that zebrafish GDNF as well as zebrafish GFRa1 robustly activated human RET signaling and promoted the survival of cultured mouse dopaminergic neurons with comparable efficiency to mammalian GDNF, unlike E. coli-produced human proteins. These results contradict previous studies suggesting that mammalian GFRa1 and GDNF cannot bind and activate non-mammalian RET and vice versa.Peer reviewe

Novel RET agonist for the treatment of experimental neuropathies

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) alleviate symptoms of experimental neuropathy, protect and stimulate regeneration of sensory neurons in animal models of neuropathic pain, and restore their functional activity. However, clinical development of GFL proteins is complicated by their poor pharmacokinetic properties and multiple effects mediated by several receptors. Previously, we have identified a small molecule that selectively activates the major signal transduction unit of the GFL receptor complex, receptor tyrosine kinase RET, as an alternative to GFLs, for the treatment of neuropathic pain. We then introduced a series of chemical changes to improve the biological activity of these compounds and tested an optimized compound named BT44 in a panel of biological assays. BT44 efficiently and selectively stimulated the GFL receptor RET and activated the intracellular mitogene-activated protein kinase/extracellular signal-regulated kinase pathway in immortalized cells. In cultured sensory neurons, BT44 stimulated neurite outgrowth with an efficacy comparable to that of GFLs. BT44 alleviated mechanical hypersensitivity in surgery- and diabetes-induced rat models of neuropathic pain. In addition, BT44 normalized, to a certain degree, the expression of nociception-related neuronal markers which were altered by spinal nerve ligation, the neuropathy model used in this study. Our results suggest that the GFL mimetic BT44 is a promising new lead for the development of novel disease-modifying agents for the treatment of neuropathy and neuropathic pain.Peer reviewe

Glial cell line-derived neurotrophic factor receptor REarranged during transfection agonist supports dopamine neurons in Vitro and enhances dopamine release In Vivo

Background Motor symptoms of Parkinson's disease (PD) are caused by degeneration and progressive loss of nigrostriatal dopamine neurons. Currently, no cure for this disease is available. Existing drugs alleviate PD symptoms but fail to halt neurodegeneration. Glial cell line-derived neurotrophic factor (GDNF) is able to protect and repair dopamine neurons in vitro and in animal models of PD, but the clinical use of GDNF is complicated by its pharmacokinetic properties. The present study aimed to evaluate the neuronal effects of a blood-brain-barrier penetrating small molecule GDNF receptor Rearranged in Transfection agonist, BT13, in the dopamine system. Methods We characterized the ability of BT13 to activate RET in immortalized cells, to support the survival of cultured dopamine neurons, to protect cultured dopamine neurons against neurotoxin-induced cell death, to activate intracellular signaling pathways both in vitro and in vivo, and to regulate dopamine release in the mouse striatum as well as BT13's distribution in the brain. Results BT13 potently activates RET and downstream signaling cascades such as Extracellular Signal Regulated Kinase and AKT in immortalized cells. It supports the survival of cultured dopamine neurons from wild-type but not from RET-knockout mice. BT13 protects cultured dopamine neurons from 6-Hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP+)-induced cell death only if they express RET. In addition, BT13 is absorbed in the brain, activates intracellular signaling cascades in dopamine neurons both in vitro and in vivo, and also stimulates the release of dopamine in the mouse striatum. Conclusion The GDNF receptor RET agonist BT13 demonstrates the potential for further development of novel disease-modifying treatments against PD. (c) 2019 International Parkinson and Movement Disorder SocietyPeer reviewe

A Novel Small Molecule Supports the Survival of Cultured Dopamine Neurons and May Restore the Dopaminergic Innervation of the Brain in the MPTP Mouse Model of Parkinson's Disease

We previously showed that monoterpenoid (1R,2R,6S)-3-methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-diol 1 alleviates motor manifestations of Parkinson's disease in animal models. In the present study, we designed and synthesized monoepoxides of (1R,2R,6S)-3-methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-diol 1 and evaluated their biological activity in the MPTP mouse model of Parkinson's disease. We also assessed the ability of these compounds to penetrate the blood-brain barrier (BBB). According to these data, we chose epoxide 4, which potently restored the locomotor activity in MPTP-treated mice and efficiently penetrated the BBB, to further explore its potential mechanism of action. Epoxide 4 was found to robustly promote the survival of cultured dopamine neurons, protect dopamine neurons against toxin-induced degeneration, and trigger the mitogen-activated protein kinase (MAPK) signaling cascade in cells of neuronal origin. Meanwhile, neither the survival-promoting effect nor MAPK activation was observed in non-neuronal cells treated with epoxide 4. In the MPTP mouse model of Parkinson's disease, compound 4 increased the density of dopamine neuron fibers in the striatum, which can highlight its potential to stimulate striatal reinnervation and thus halt disease progression. Taken together, these data indicate that epoxide 4 can be a promising compound for further development, not only as a symptomatic but also as a neuroprotective and neurorestorative drug for Parkinson's disease.Peer reviewe

Overview Of Data Over Digital Subscrieber Line In Czech Republic.

Import 22/07/2015Tato bakalářská práce je zaměřena na rozbor technologie ADSL a to, jak je využívána a nabízena vybranými poskytovateli v České republice k užívání veřejným sektorem. V teoretické části pojednává o technologii ADSL a jejích vývojových verzích. Dále se zabývá řešením přípojky a vlivů rušení na vedení těchto přípojek. Hlavní částí práce je část praktická, ta se zabývá průzkumem poskytovatelů v České republice. Konkrétně využitím digitálních účastnických smyček poskytovateli, jejich nabídkou ADSL služeb pevného připojení k internetu a také doplňkových služeb a poplatků s pevným připojením spojených. Poslední částí práce je analýza možností připojení těchto služeb na konkrétní přípojku. Obsah této práce má napomoci běžným uživatelům k pochopení dané problematiky spojené se zřízením a výběrem ADSL služeb. Prostředky k řešení praktické části práce jsou především komunikace s poskytovateli ADSL služeb a získávání informací.This bachelor thesis is focused on the analysis of the technology ADSL as it is used in the public sector and offered by chosen providers in Czech Republic. In the theoretical part, it refers to the technology ADSL and its developmental versions. Furthermore, it examines the connection solution and the influence of interruption on conducting these connections. The main part of the thesis is the practical part which undertakes the research of providers in Czech Republic. Namely, it explores use of digital subscriber loops by providers, theirs ADSL service offers of the stable internet connection and its complementary services and charges. The final part of the thesis is the connection analysis of these complementary services on the specific connection. The content of this thesis should provide help to common users to understand given issues related to the selection and setting up ADSL services. Tools used for the practical part are particularly communication with ADSL service providers and acquiring appropriate information.440 - Katedra telekomunikační technikyvýborn

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe