Browsing by Subject "RET"

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.

As a genome editing tool, CRISPR-Cas9 has provided a robust way to generate mutations in the gene of interest, at a certain time point, and in selected cell populations. The impairment of dopaminergic neurons in the substantia nigra is addressed to be one of the main pathologies of Parkinson’s disease. The histopathology of Lewy Bodies, with an undetermined role, accompanies the demise of DA neurons. Development of strategies for the prevention the neurodegeneration has a potential to slow down the progression of Parkinson’s disease. In this study, a novel, neuron-specific CRISPR-Cas9 system was developed for the purpose of dissecting neuroprotective pathways in primary dopaminergic neurons. The optimization of the tool was done by targeting EGFP at TH-positive neurons obtained from transgenic animals expressing EGFP in dopaminergic neurons. Complete loss of EGFP was achieved at day 6 after the introduction of the CRISPR-Cas9 via lentiviral vectors. There were no survival or transduction efficiency differences. Two significant pathways for the survival of dopaminergic neurons, the microRNA biogenesis and GDNF/RET signaling were selected to collect the preliminary data. Dicer, Trbp, Translin, Ago-2 and Ret were targeted with single sgRNAs, which were specifically designed to create indel mutations in these genes, and specific lentivirus vectors were produced with each guide. After transduction with the lentivirus vectors, survival of the TH-positive neurons was unaffected. Data obtained from the quantitative PCR suggested that there was 50-70% decline in transcript levels of Trbp. However, the unchanged transcript levels of the other miRNA-related targets suggest the need for further optimization of the specific guides. Knockdown of Ret was validated by inhibition of pharmacological benefits of GDNF. Overall, this research has shown the further development of this CRISPR-Cas9 tool would be useful to dissect neuroprotective signaling pathways in dopaminergic neurons.

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.

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.

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