The role of GDNF and its receptor GFRa1 in neuronal development and function

Neurotrophic factor glial cell line-derived neurotrophic factor (GDNF), its co-receptor GDNF family receptor alpha 1 (GFRa1), and signaling receptor RET tyrosine kinase are essential to enteric nervous system (ENS) development; mice knockout for Gdnf, Gfra1 or Ret lack the whole ENS distal to the stomach. These Gdnf/Gfra1/Ret knockout mice die at birth because of lack of ENS and kidneys hindering analysis of postnatal function of those proteins. Transgenic overexpression in animal models on the other hand relates to loss of physiological spatiotemporal regulation of gene expression. These two bottlenecks have hindered the understanding of the role and therapeutic potential of GDNF/GFRa1/RET signaling in congenital diseases, such as Hirschsprung’s disease, and degenerative neurological diseases, such as Parkinson’sdisease. To tackle at least some of these problems, we have generated and characterized new mouse models with either increased or decreased gene expression dose - from the gene’s endogenous locus and limited to naturally expressing cells. Novel mouse models with increased expression were generated by editing 3’ untranslated region (3’UTR) of the Gdnf gene in such a way that the edited 3’UTR lacks binding sites for negative regulators such as microRNAs. By preventing the posttranscriptional downregulation via the 3’UTR we were able to achieve Gdnf overexpression from the endogenous locus limited to the naturally Gdnf expressing cells. We showed that 3’UTR replacement or 3’UTR editing results in increased GDNF levels in the brain and kidneys, maintaining the spatiotemporal expression pattern with positive effects on the dopaminergic system and negative effects on the kidney size and urogenital tract development. We also found that 3’UTR regulates GDNF levels in the gastrointestinal tract and that 3’UTR controlled GDNF levels determine proportions of neuronal subtypes in the ENS. More specifically, inactivation of negative Gdnf 3’UTR regulation enhances nitrergic and cholinergic neuron numbers, and leads to increased gastrointestinal transit time, increased stool pellet size, and increased stool water content. In congenital Hirschsprung’s disease (HSCR) patients, on the other hand, lack of ENS ganglia in the distal gut leads to constipation and megacolon. Even though RET mutations are the most common cause of Hirschsprung’s disease, no causative mutations in GFRa1 are known. However, one study reported low GFRa1 mRNA levels in some HSCR patients, suggesting that perhaps instead of being caused by mutations some HSCR cases could be triggered by reduced GFRa1 levels. Complicating the establishment of disease etiology in GDNF/GFRa1/RET related HSCR, postnatal viable HSCR mouse models with a defect in GDNF/GFRa1/RET signaling are not available. Here, we generated GFRa1 hypomorphic mice by insertion of a selectable marker gene in opposite transcriptional direction after the Gfra1 exon 6. Insertion of an expression cassette in the opposite transcriptional direction often leads to under-expression from the other strand, resulting in hypomorph allele. We showed that a 70-80 % reduction in GFRa1 levels in mice resulted in congenital Hirschsprung’s disease and associated enterocolitis phenotype with 100 % penetrance. We were also able to shed light in the chronology of events in the pathogenesis of Hirschsprung’s disease associated enterocolitis: first goblet cell dysplasia accompanied by an abnormal mucin phenotype is proceeding into epithelial damage, later followed by microbial enterocyte adherence and bacterial tissue invasion which likely leads to death by sepsis. Previously all those features had been described in patients but the sequence of events had remained unclear. Our results suggest that dysregulation of GDNF or GFRa1 levels by epigenetic mechanisms may play a role in normal and pathogenic development of the enteric nervous system.

Grantor dc:publisher

Helsingin yliopisto

Year dc:date.issued

2020

Author dc:creator

• Porokuokka, L. Lauriina

• farmasia

Statement dc:rights

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Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/10138/314690

OAI record oai:identifier

https://helda.helsinki.fi/server/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Ahelda.helsinki.fi%3A10138%2F314690