Browsing by Subject "Ret"

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  • Montonen, Heidi (Helsingfors universitet, 2013)
    Literature review: The plasma membrane DA transporter (DAT) belongs to the family of Na+/ClÙÄÉ≠ dependent neurotransmitter transporters. DAT is the primary mechanism for clearance of dopamine from the extracellular space and transporting it back to the presynaptic nerve terminals. There's a great interest in the DAT and its regulation as its substrate, dopamine, mediates a wide array of physiological functions e.g. locomotor activity, cognition and the control of motivated behaviors. With selective transport DAT limits the intensity and the duration of dopaminergic signal. Its function is regulated by several kinases, phosphatase and protein-protein interactions. The altered expression of DAT may be related to several neurological diseases such as Parkinson's disease, addiction and ADHD. To study DAT's function, several genetically modified mouse lines including DAT knockout mice, DAT knockdown mice and DAT knock in mice with elevated DAT levels have been generated. Experimental part: Glial cell line-derived neurotrophic factor (GDNF) plays important role in the survival and function of dopaminergic neurons, learning, memory and synaptic plasticity. More recently, several studies have shown that GDNF can also negatively regulate the actions of abused drugs. The aim of this study was to investigate GDNF's role and mechanism of action in plasticity and function of the dopaminergic neurons projecting to striatum. For that purpose, we used in vivo microdialysis in freely moving mice. We chose two different mouse lines: MEN2B mice with constitutive active Ret-signaling and elevated striatal dopamine concentrations, and GDND-cKO mice that lack GDND in the central nervous system. Microdialysis guide cannula was implanted in the dorsal striatum in the stereotaxic surgery and the mice were allowed to recover for 5-7 days. The concentrations of dopamine and its metabolites DOPAC and HVA and also 5-HIAA were determined from the samples by highperformance liquid chromatography. Microdialysis was performed twice for every mouse on days 1 and 4. Between microdialysis days, the mice were given amphetamine 1 mg/kg i.p. on days 2 and 3. In the microdialysis experiment, the mice received amphetamine stimulation (100 µM/60 min) via microdialysis probe. The placements of microdialysis probes were verified from fixed brain sections after the experiments. Amphetamine increased the dopamine output in both mouse lines, but there were no statistically significant differences in striatal dopamine concentrations between genotypes neither after acute nor chronic administration. However, there was a difference between the dopamine outputs in days 1 and 4 in both MEN2B and GDNF-cKO mice: The striatal dopamine concentrations were significantly lower on the second microdialysis day. This may be a sing from tolerance to the drug. However, without more research, it is not possible, by this experiment, to draw direct conclusions of GDNF's role in addiction and in plasticity in striatum. It is possible that the differences between genotypes are too small to be seen with microdialysis. Development of compensatory mechanisms in mice cannot be ruled out either. Effects may also vary between different brain areas.
  • Perea, Daniel; Guiu, Jordi; Hudry, Bruno; Konstantinidou, Chrysoula; Milona, Alexandra; Hadjieconomou, Dafni; Carroll, Thomas; Hoyer, Nina; Natarajan, Dipa; Kallijärvi, Jukka; Walker, James A.; Soba, Peter; Thapar, Nikhil; Burns, Alan J.; Jensen, Kim B.; Miguel-Aliaga, Irene (2017)
    Expression of the Ret receptor tyrosine kinase is a defining feature of enteric neurons. Its importance is underscored by the effects of its mutation in Hirschsprung disease, leading to absence of gut innervation and severe gastrointestinal symptoms. We report a new and physiologically significant site of Ret expression in the intestine: the intestinal epithelium. Experiments in Drosophila indicate that Ret is expressed both by enteric neurons and adult intestinal epithelial progenitors, which require Ret to sustain their proliferation. Mechanistically, Ret is engaged in a positive feedback loop with Wnt/Wingless signalling, modulated by Src and Fak kinases. We find that Ret is also expressed by the developing intestinal epithelium of mice, where its expression is maintained into the adult stage in a subset of enteroendocrine/enterochromaffin cells. Mouse organoid experiments point to an intrinsic role for Ret in promoting epithelial maturation and regulating Wnt signalling. Our findings reveal evolutionary conservation of the positive Ret/Wnt signalling feedback in both developmental and homeostatic contexts. They also suggest an epithelial contribution to Ret loss-of-function disorders such as Hirschsprung disease.
  • Immonen, Anastasia (Helsingfors universitet, 2010)
    Tutkimuksen tavoitteena oli ensisijaisesti oppia kanan alkion manipuloinnin tekniikka ja tutkia sen avulla hermostopienan solujen vaellusta kehittyvässä alkiossa. Toinen tavoite oli tarkastella Ret-geenin mutaation (C634R) vaikutusta hermostopienan migraatiokaavaan. Tutkittu Ret-geenin mutaatio aikaansaa geenin tuotteen uudenlaisen toiminnankuvan, jolloin tuloksena voi olla syöpäsairaus. Tutkimus suoritettiin käyttämällä kanan alkiota. Kananmunia haudottiin inkubaattorissa sopivaan kehitysvaiheeseen, minkä jälkeen alkiota käsiteltiin kuoreen tehdyn aukon kautta. Mikroskoopin avulla alkion kehittyvään hermostoputkeen ruiskutettiin plasmidi-Dna:ta, joka sisälsi gfp-geenin sekä joko tutkittavan geenimutaation tai villityypin geenin. Plasmidin siirron jälkeen alkio elektroporoitiin, jotta siirretty perimäaines pääsisi alkion solujen sisään proteiinisynteesin alkamiseksi. Gfp:n fluoresoivan ominaisuuden avulla voitiin jälkitarkastelussa seurata plasmidin geenien ilmentymistä. Tutkimustulosten perusteella voidaan todeta tarkastellun Ret-geenin mutaation aiheuttavan hermostopienan solujen vaelluskaavioon häiriöitä verrattuna kontrolliin. Häiriöitä olivat mm. solujen rykelmäinen ryhmittyminen sekä hermostopienan solujen vaelluksen paikoittainen katkeaminen ja hajanaisuus. Tutkimusaineiston suppeuden vuoksi, mitään varmoja johtopäätöksiä suoritetun tutkimuksen perusteella ei voida vielä tehdä, vaan asia vaatii lisää tutkimuksia.