Browsing by Subject "RET"

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  • Hakosalo, Vili (Helsingin yliopisto, 2021)
    Parkinson’s disease (PD) is the second most common neurogenerative disease. There are no drugs available to halt the progression of PD. The glial cell line-derived neurotrophic factor (GDNF) has been identified as a potential drug candidate against PD because of its protective properties on dopaminergic neurons, which are an especially vulnerable cell population in PD. It has been recently shown that GDNF can also attenuate aggregation of phosphorylated α-synuclein in dopaminergic neurons, which is one of the most important pathologies of PD. Phosphorylated α-synuclein is a primary component of Lewy bodies, which in turn, are vastly studied intracellular inclusions with a high correlation towards neurodegenerative diseases. GDNF signals through its main receptor RET and activates downstream signalling cascades. RET is indispensable for the effect of GDNF against α-synuclein aggregation. Importance of the downstream molecules Src, AKT and PI3K have been also pharmacologically demonstrated. However, complete mechanism of GNDF’s action and individual importance of downstream signalling molecules has been yet to establish. CRISPR/Cas9 gene editing tool has revolutionized the gene manipulation in biological research. In this thesis work, CRISPR/Cas9 guides were designed to target and mutate the c-Src, Akt1 and NURR1, which are important proteins of the GDNF/RET pathway. As a delivery system for the Cas9 enzyme and individual guides, lentiviral vectors were produced according to the protocols previously established in our laboratory and proved to be high efficiency. Modelling of α-synuclein aggregation in neurons was performed with pre-formed fibrils of α-synuclein, which induce the formation of intracellular Lewy body-like inclusions with the phosphorylation of α-synuclein at serine 129. In this study, primary dopaminergic neuron cultures from E13.5 mouse embryos were cultured in 96-well plates. For each of the target genes, I designed two guide variants, cloned them in lentiviral transfer vectors and produced lentiviral particles for neuronal transduction. My data shows that targeting Akt1 and c-Src impaired the protective mechanism of GDNF against Lewy body-like inclusions. For the importance of NURR1 more studies are needed for coherent conclusions. I also showed that targeting of NURR1 impaired the GDNF/RET signalling at least in one guide construct. The 15-day long cultivation did not affect to the dopaminergic cell numbers in any of the groups. Still the confirmation of successful CRISPR-induced genetic mutations by sequencing as well as the detailed mechanism of how GDNF prevents the formation of Lewy body-like inclusions will be a subject of future studies. This thesis provides important information for the molecular mechanism of attenuation of α-synuclein aggregation by GDNF through its main receptor RET.
  • Runeberg-Roos, Pia; Piccinini, Elisa; Penttinen, Anna-Maija; Matlik, Kert; Heikkinen, Hanna; Kuure, Satu; Bespalov, Maxim M.; Peranen, Johan; Garea-Rodriguez, Enrique; Fuchs, Eberhard; Airavaara, Mikko; Kalkkinen, Nisse; Penn, Richard; Saarma, Mart (2016)
    In Parkinson's disease midbrain dopaminergic neurons degenerate and die. Oral medications and deep brain stimulation can relieve the initial symptoms, but the disease continues to progress. Growth factors that might support the survival, enhance the activity, or even regenerate degenerating dopamine neurons have been tried with mixed results in patients. As growth factors do not pass the blood-brain barrier, they have to be delivered intracranially. Therefore their efficient diffusion in brain tissue is of crucial importance. To improve the diffusion of the growth factor neurturin (NRTN), we modified its capacity to attach to heparan sulfates in the extracellular matrix. We present four new, biologically fully active variants with reduced heparin binding. Two of these variants are more stable than WT NRTN in vitro and diffuse better in rat brains. We also show that one of the NRTN variants diffuses better than its close homolog GDNF in monkey brains. The variant with the highest stability and widest diffusion regenerates dopamine fibers and improves the conditions of rats in a 6-hydroxydopamine model of Parkinson's disease more potently than GDNF, which previously showed modest efficacy in clinical trials. The new NRTN variants may help solve the major problem of inadequate distribution of NRTN in human brain tissue. (C) 2016 Elsevier Inc. All rights reserved.
  • Li, Hao; Jakobson, Madis; Ola, Roxana; Gui, Yujuan; Kumar, Anmol; Sipilä, Petra; Sariola, Hannu; Kuure, Satu; Andressoo, Jaan-Olle (2019)
    Mechanisms controlling ureter lenght and the position of the kidney are poorly understood. Glial cellline derived neurotrophic factor (GDNF) induced RET signaling is critical for ureteric bud outgrowth, but the function of endogenous GDNF in further renal differentiation and urogenital system development remains discursive. Here we analyzed mice where 3' untranslated region (UTR) of GDNF is replaced with sequence less responsive to microRNA-mediated regulation, leading to increased GDNF expression specifically in cells naturally transcribing Gdnf. We demonstrate that increased Gdnf leads to short ureters in kidneys located in an abnormally caudal position thus resembling human pelvic kidneys. High GDNF levels expand collecting ductal progenitors at the expense of ureteric trunk elongation and result in expanded tip and short trunk phenotype due to changes in cell cycle length and progenitor motility. MEK-inhibition rescues these defects suggesting that MAPK-activity mediates GDNF's effects on progenitors. Moreover, Gdnf(hyper) mice are infertile likely due to effects of excess GDNF on distal ureter remodeling. Our findings suggest that dysregulation of GDNF levels, for example via alterations in 3' UTR, may account for a subset of congenital anomalies of the kidney and urinary tract (CAKUT) and/or congenital infertility cases in humans and pave way to future studies.
  • Chmielarz, Piotr; Er, Safak; Konovalova, Julia; Bandres, Laura; Hlushchuk, Irena; Albert, Katrina; Panhelainen, Anne; Luk, Kelvin; Airavaara, Mikko; Domanskyi, Andrii (2020)
    Background Parkinson's disease (PD) is associated with proteostasis disturbances and accumulation of misfolded alpha-synuclein (alpha-syn), a cytosolic protein present in high concentrations at pre-synaptic neuronal terminals. It is a primary constituent of intracellular protein aggregates known as Lewy neurites or Lewy bodies. Progression of Lewy pathology caused by the prion-like self-templating properties of misfolded alpha-syn is a characteristic feature in the brains of PD patients. Glial cell line-derived neurotrophic factor (GDNF) promotes survival of mature dopamine (DA) neurons in vitro and in vivo. However, the data on its effect on Lewy pathology is controversial. Objectives We studied the effects of GDNF on misfolded alpha-syn accumulation in DA neurons. Methods Lewy pathology progression was modeled by the application of alpha-syn preformed fibrils in cultured DA neurons and in the adult mice. Results We discovered that GDNF prevented accumulation of misfolded alpha-syn in DA neurons in culture and in vivo. These effects were abolished by deletion of receptor tyrosine kinase rearranged during transfection (RET) or by inhibitors of corresponding signaling pathway. Expression of constitutively active RET protected DA neurons from fibril-induced alpha-syn accumulation. Conclusions For the first time, we have shown the neurotrophic factor-mediated protection against the misfolded alpha-syn propagation in DA neurons, uncovered underlying receptors, and investigated the involved signaling pathways. These results demonstrate that activation of GDNF/RET signaling can be an effective therapeutic approach to prevent Lewy pathology spread at early stages of PD. (c) 2020 International Parkinson and Movement Disorder Society
  • Ukkola, Iiris; Nummela, Pirjo; Kero, Mia; Tammio, Hanna; Tuominen, Jenni; Kairisto, Veli; Kallajoki, Markku; Haglund, Caj; Peltomäki, Päivi; Kytölä, Soili; Ristimäki, Ari (2022)
    Gene fusions can act as oncogenic drivers and offer targets for cancer therapy. Since fusions are rare in colorectal cancer (CRC), their universal screening seems impractical. Our aim was to investigate gene fusions in 62 CRC cases with deficient MLH1 (dMLH1) and BRAFV600E wild-type (wt) status from a consecutive real-life series of 2079 CRCs. First, gene fusions were analysed using a novel FusionPlex Lung v2 RNA-based next-generation sequencing (NGS) panel, and these results were compared to a novel Idylla GeneFusion assay and pan-TRK immunohistochemistry (IHC). NGS detected seven (7/62, 11%) NTRK1 fusions (TPM3::NTRK1, PLEKHA6::NTRK1 and LMNA::NTRK1, each in two cases, and IRF2BP2::NTRK1 in one case). In addition, two ALK, four RET and seven BRAF fusions were identified. Idylla detected seven NTRK1 expression imbalances, in line with the NGS results (overall agreement 100%). Furthermore, Idylla detected the two NGS-identified ALK rearrangements as one specific ALK fusion and one ALK expression imbalance, whilst only two of the four RET fusions were discovered. However, Idylla detected several expression imbalances of ALK (n = 7) and RET (n = 1) that were found to be fusion negative with the NGS. Pan-TRK IHC showed clearly detectable, fusion partner-dependent staining patterns in the seven NTRK1 fusion cases. Overall agreement for pan-TRK antibody clone EPR17341 was 98% and for A7H6R 100% when compared to the NGS. Of the 62 CRCs, 43 were MLH1 promoter hypermethylated (MLH1ph) and 39 were RASwt. All fusion cases were both MLH1ph and RASwt. Our results show that kinase fusions (20/30, 67%) and most importantly targetable NTRK1 fusions (7/30, 23%) are frequent in CRCs with dMLH1/BRAFV600Ewt/MLH1ph/RASwt. NGS was the most comprehensive method in finding the fusions, of which a subset can be screened by Idylla or IHC, provided that the result is confirmed by NGS.
  • Porokuokka, L. Lauriina; Virtanen, Heikki T.; Linden, Jere; Sidorova, Yulia; Danilova, Tatiana; Lindahl, Maria; Saarma, Mart; Andressoo, Jaan-Olle (2019)
    BACKGROUND & AIMS: RET, the receptor for the glial cell line-derived neurotrophic factor (GDNF) family ligands, is the most frequently mutated gene in congenital aganglionic megacolon or Hirschsprung's disease (HSCR). The leading cause of mortality in HSCR is HSCR-associated enterocolitis (HAEC), which is characterized by altered mucin composition, mucin retention, bacterial adhesion to enterocytes, and epithelial damage, although the order of these events is obscure. In mice, loss of GDNF signaling leads to a severely underdeveloped enteric nervous system and neonatally fatal kidney agenesis, thereby precluding the use of these mice for modeling postnatal HSCR and HAEC. Our aim was to generate a postnatally viable mouse model for HSCR/HAEC and analyze HAEC etiology. METHODS: GDNF family receptor alpha-1 (GFRa1) hypomorphic mice were generated by placing a selectable marker gene in the sixth intron of the Gfra1 locus using gene targeting in mouse embryonic stem cells. RESULTS: We report that 70%-80% reduction in GDNF co-receptor GFRa1 expression levels in mice results in HSCR and HAEC, leading to death within the first 25 postnatal days. These mice mirror the disease progression and histopathologic findings in children with untreated HSCR/HAEC. CONCLUSIONS: In GFRa1 hypomorphic mice, HAEC proceeds from goblet cell dysplasia, with abnormal mucin production and retention, to epithelial damage. Microbial enterocyte adherence and tissue invasion are late events and therefore unlikely to be the primary cause of HAEC. These results suggest that goblet cells may be a potential target for preventative treatment and that reduced expression of GFRa1 may contribute to HSCR susceptibility.
  • Runeberg-Roos, Pia; Penn, Richard D. (2020)
    The last decade has been a frustrating time for investigators who had envisioned major advances in the treatment of Parkinson's disease using neurotrophic factors. The first trials of glial cell line-derived neurotrophic factor for treating Parkinson's disease were very promising. Later blinded control trials were disappointing, not reaching the predetermined outcomes for improvement in motor function. Consideration of the problems in the studies as well as the biology of the neurotrophins used can potentially lead to more effective therapies. Parkinson's disease presents a multitude of opportunities for the cell biologist wanting to understand its pathology and to find possible new avenues for treatment.
  • Huynh, Thi Le Hang (Helsingfors universitet, 2010)
    In the written part of my master -thesis I discuss about GDNF signalling and more specifically how the changes in the GDNF/GFRα1/Ret signaling affect the nigrostriatal dopaminergic neurons in different mutant mice. In the animal models of Parkinson's disease the neuroprotective and neurorestorative effects of exogenous GDNF are very clear which raises hope for use of GDNF in treatment of Parkinson's disease. In intact animals GDNF stimulates the function of nigrostriatal dopaminergic system. Revealing the role of GDNF/GFRα1/Ret signaling in development, maintenance and protection of nigrostriatal dopaminergic system will certainly help in search for treatment of neurodegeneration in Parkinson's disease. In knockout mouse models GDNF/GFRα1/Ret signaling is not crucial for prenatal nigrostriatal dopaminergic neuron development, but it has been shown that it plays an important role in the early postnatal development. Also, it was shown that reduced GDNF/GFRα1/Ret signaling compromises nigrotriatal dopaminergic system in heterozygous GDNF/GFRα1/Ret knockout mice. However the physiological roles of endogenous GDNF and its signalling in the nigrostriatal dopaminergic neurons are not very well understood. In the experimental part of my master -thesis I studied how reduced endogenous GDNF signaling affects the dopaminergic system after 6-OHDA induced neurotoxicity in the conventional heterozygous GDNF mice. Besides that I examined the effects of elevated endogenous GDNF on dopaminergic system of 7 days old so-called GDNF hypermorphs mice. The effects of reduced endogenous GFRα1 levels on dopaminergic system of 20 days old GFRα1 hypomorphs have also been studied. The obtained date showed that mice with the reduced levels of endogenous GDNF are not more susceptible to the 6-OHDA induced neurotoxicity than the wild type littermates. Elevated endogenous GDNF levels did not affect early postnatal development of the nigrostriatal dopaminergic system in GDNF hypermorphs mice as revealed by normal intensity of TH staining in striatum and normal number of TH-positive cells in the substantia nigra pars compacta. Reduced levels of endogenous GFRα1 levels did not affect monoamine levels in the striatum of GFRα1 hypomorph mice.
  • Raza, Shaffaq (Helsingin yliopisto, 2020)
    Growth differentiation factor 15 (GDF15), a member of TGF-β super family is a soluble cytokine that is associated with different pathological conditions including cancer, cardiac and renal failure and obesity. Its high serum levels are linked with symptoms like cachexia/anorexia in cancer patients and can be used as a marker for these diseases. Its crucial role in weight regulation and energy homeostasis has been demonstrated by treating obese mice with GDF15, which results in weight lose along with improved glucose metabolism and increased insulin tolerance. It is now known that GDF15 exerts its metabolic effect by binding to a GDNF receptor -α-Like (GFRAL) receptor along with co-receptor RET. Interestingly, these two receptors co-localize only in the brain stem area of mice and humans indicating involvement of a neuronal circuit in GDF15 mediated effects. Despite its implications in major health disorders, little is known about the interaction of GDF15 with its receptors and how this interaction in turn modulates different cellular signalling and functions. The aim of the thesis was to study the mechanism and factors involved in endocytosis of GDF15. I employed high content imaging and flow cytometry techniques to visualize and analyse the internalization of ligand-receptor complex and investigate the role of actin, dynamin and phosphoinositide 3 kinase in the process. The results suggest that similar to the internalization of other cellular growth factors, the uptake of GDF15 is affected by disruption of the actin cytoskeleton. The role of dynamin is still unclear. I also discovered that the internalization of GDF15 was inefficient even in cells that expressed the receptor GFRAL, with large cell-to-cell variation. By following the intracellular localization of the receptor GFRAL, my results revealed that the receptor GFRAL is not efficiently exported to the plasma membrane and most of the protein is retained in the Golgi compartment of cells. This phenomenon was stronger in murine fibroblast cells, where the receptor was almost exclusively trapped in the secretory compartment, explaining why the uptake of the ligand GDF15 is so inefficient in these cells. The system developed during this project will now be used to analyse different factors involved in the uptake of GDF15 and eventually uncover the possible endocytic pathway. Moreover, the Golgi retention of the receptor opens up new questions to investigate like whether the physiological function of GDF15 is regulated by receptor export signals. This will help deciphering the complex and mysterious interaction of GDF15 with its receptor GFRAL.
  • Iherman-Hella, Anneliis; Lume, Maria; Miinalainen, Ilkka; Pirttiniemi, Anniina; Gui, Yujuan; Peränen, Johan; Charron, Jean; Saarma, Mart; Costantini, Frank; Kuure, Satu Helena (2014)
  • Virtanen, Valtter B.; Salo, Perttu P.; Gao, Jia; Löf-Granström, Anna; Milani, Lill; Metspalu, Andres; Rintala, Risto J.; Saarenpää-Heikkilä, Outi; Paunio, Tiina; Wester, Tomas; Nordenskjöld, Agneta; Perola, Markus; Pakarinen, Mikko P. (2019)
    The pathogenesis of Hirschsprung disease is complex. Although the RET proto-oncogene is the most frequently affected gene in Hirschsprung disease, rare coding sequence variants explain only a small part of Hirschsprung disease cases. We aimed to assess the genetic background of Hirschsprung disease using a genome-wide association analysis combined with sequencing all RET exons in samples from 105 Hirschsprung disease cases (30 familial and 75 sporadic) and 386 controls. As expected, variants in or near RET showed the strongest overall association with Hirschsprung disease and the most statistically significant association was observed when using a recessive genetic model (rs2435357, NC_000010.10:g.43582056T > C; genotype TT, OR = 17.31, P = 1.462 x 10(-21)). Previously published associations in variants in SEMA (rs11766001, NC_000007.13:g.84145202A > C; allele C, OR = 2.268, P = 0.009533) and NRG1 (rs4541858, NC_000008.10:g.32410309A > G; allele G, OR = 1.567, P = 0.015; rs7835688, NC_000008.10:g.32411499G > C; allele C, OR = 1.567, P = 0.015) were also replicated in the genome-wide association analysis. Sequencing revealed a total of 12 exonic RET rare variants. Of these, eight amino acid changing rare variants and two frameshift variants caused or possibly caused Hirschsprung disease. Only a minority of the Hirschsprung disease cases (9/30 familial; 7/75 sporadic) carried one of the rare variants. Excluding the rare variant carriers from the genome-wide association analysis did not appreciably change the association of rs2435357 with Hirschsprung disease. We estimate that approximately two thirds of the sporadic cases may be statistically attributed to the recessive action of the common non-coding RET variants. Thus, even though most cases do not carry rare RET variants, combinations of rare variants and the common non-coding RET variant cause the majority of the cases in our population.
  • Rautanen, Pauliina Eeva Maria (Helsingin yliopisto, 2021)
    Growth Differentiation Factor-15 (GDF15) is a neurotrophic factor associated with anorexia and weight loss. It is elevated in obesity and various diseases. It signals by forming a tripartite complex with the coreceptor Glial cell line-derived neurotrophic factor (GDNF) family receptor alpha-like (GFRAL) and the receptor Rearranged during transfection (RET). Targeting this pathway has therapeutic potential for the treatment of obesity and anorexia-cachexia syndrome, but many aspects are still unclear. What is the affinity of binding between these proteins? Does GDF15 induce dose- and time-dependent RET phosphorylation and activate intracellular signaling pathways, and are there differences between GDF15 and GDNF signaling, as the different bend angles of their complexes suggest? Can soluble GDF15-GFRAL mediate the effects of GDF15 outside of the brainstem, and what is the function of the short cytoplasmic domain of GFRAL? Furthermore, how well is the pathway evolutionally conserved between species? Binding affinities were assessed with microscale thermophoresis, whereas RET phosphorylation and intracellular signaling assays were performed utilizing immunoprecipitation and western blotting. GFRAL-RET binding is low-affinity (350 nM ± 223) similarly to GFRα1-RET binding (GDNF family receptor alpha-1), whereas GDF15-RET binding without GFRAL does not occur. GDF15 appears to compete for binding to GFRAL or RET, differing from GDNF mechanisms, but noise in the data may have affected the results. The data provide ideas about the ligand-receptor complex formation. Furthermore, RET phosphorylation by GDF15 is dose- and time-dependent. Firstly, the strongest RET and ERK activation occur at GDF15 concentrations typical of disease states. Secondly, RET activation by GDF15 is rapid and sustained like by GDNF activation, whereas ERK activation by GDF15 is rapid and much more transient than by GDNF. Thirdly, AKT activation by GDF15 is much weaker than by GDNF. The differences may be caused by different conformations of binding surfaces for adaptor proteins being available on RET because the bend angles of the complexes are different. Moreover, soluble GDF15-GFRAL does not activate RET, although soluble GDNF-GFRα1 does. Also, the short cytoplasmic domain of GFRAL is not necessary for activating AKT and ERK pathways, but may be needed to activate RET. Furthermore, GDF15 from cynomolgus monkey, but not rat or mouse, activates RET with human GFRAL, indicating sequence similarity in the active site of GDF15. In conclusion, novel aspects of GDF15 signaling and differences between GDF15 and GDNF signaling were discovered.
  • Rosti, Katja; Goldman, Adrian; Kajander, Tommi (2015)
    Background: The protein growth arrest specific-1 (GAS1) was discovered based on its ability to stop the cell cycle. During development it is involved in embryonic patterning, inhibits cell proliferation and mediates cell death, and has therefore been considered as a tumor suppressor. GAS1 is known to signal through two different cell membrane receptors: Rearranged during transformation (RET), and the sonic hedgehog receptor Patched-1. Sonic Hedgehog signalling is important in stem cell renewal and RET mediated signalling in neuronal survival. Disorders in both sonic hedgehog and RET signalling are connected to cancer progression. The neuroprotective effect of RET is controlled by glial cell-derived neurotrophic factor family ligands and glial cell-derived neurotrophic factor receptor alphas (GFR alpha s). Human Growth arrest specific-1 is a distant homolog of the GFRas. Results: We have produced and purified recombinant human GAS1 protein, and confirmed that GAS1 is a monomer in solution by static light scattering and small angle X-ray scattering analysis. The low resolution solution structure reveals that GAS1 is more elongated and flexible than the GFRas, and the homology modelling of the individual domains show that they differ from GFR alpha s by lacking the amino acids for neurotrophic factor binding. In addition, GAS1 has an extended loop in the N-terminal domain that is conserved in vertebrates after the divergence of fishes and amphibians. Conclusions: We conclude that GAS1 most likely differs from GFRas functionally, based on comparative structural analysis, while it is able to bind the extracellular part of RET in a neurotrophic factor independent manner, although with low affinity in solution. Our structural characterization indicates that GAS1 differs from GFR alpha's significantly also in its conformation, which probably reflects the functional differences between GAS1 and the GFR alpha s.
  • Vähämurto, Pauli (Helsingfors universitet, 2009)
    Selvitin tutkimuksessani VEGF-C:n ja RET:n vaikutusta hiiren enterisen hermoston ja imusuoniston kehitykseen. Yhden ja kahden VEGF-C-alleelin puutos johti neuronien määrän vähenemiseen jejunumissa ja koolonissa. RET-alleelien puutos vähensi myös neuronien määrää ja kahden puutos esti neuronien kehittymisen. VEGF-C ja etenkin RET-muuntogeenisiä alkioita oli myös hiiripoikueissa vähemmän kuin Mendelistisen jakauman perusteella voisi olettaa. Tämä viittaa lisääntyneeseen kuolleisuuteen in utero. Myös ihon karvatuppia oli RET-homogeenisissä vähemmän kuin villityyppisissä. Lisäksi selvitin mitkä vasta-aineet soveltuvat käytettäväksi suolten erityyppisissä vastaainevärjäyksissä