Date of Award

Winter 2017

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Professor Jochen H. M. Prehn

Second Supervisor

Dr Marion C. Hogg

Funder/Sponsor

Thierry Latran Fondation

Keywords

Amyotrophic Lateral Sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder affecting motoneurons. Mutations in ANG, encoding a member of the pancreatic RNase A superfamily, segregate with ALS. Angiogenin is an acute-phase protein, which is secreted from motoneurons under stress conditions. Our group demonstrated that it is taken up by astrocytes where it induces RNA cleavage and mediates neuroprotection in paracrine. It has been shown that angiogenin preferably cleaves tRNAs within the anticodon loop to produce tRNA-derived, stress-induced RNAs (tiRNAs).The functions of tiRNAs are unclear they have been shown to modulate various pathways, including apoptosis, gene expression and protein translation.

This study was performed to gain insights into the functions of angiogenin with focus on ribonucleolytic activity and to investigate whether specific tiRNA are responsible for the function of angiogenin. Stable SH-SY5Y cells overexpressing wild-type angiogenin and ALS-associated mutants ANG K40I and R31K were generated to investigate the effect of these mutations on angiogenin activity, tiRNA generation, and secretion. We demonstrated for the first time that ANG R31K exhibits an aberrant secretion, which is a novel function and adds new insights into R31K-missense associated ALS pathology.

Overexpressed wild-type angiogenin and R31K mutant were ribonucleolytic active and generated specific tiRNAs, such as 5`Val, whereas other tiRNAs (5`Ala) were not detected. We illustrated that 5`Val tiRNA was increased in the supernatant of the ANG WT and R31K cell lines compared to ribonucleolytic-inactive K40I mutant and control cell lines. Our findings reveal that tiRNAs are secreted, and may exert functions similar to miRNAs, which can be delivered to neighbouring cells where they regulate multiple targets. Furthermore, secreted tiRNAs might be employed as biomarkers to identify early stages of stress-induced neuronal degradation.

Functional analysis of tiRNAs demonstrated that 5`Val tiRNA protected SH-SY5Y cells against proteasomal stress. However, this protection was not mediated by SG formation. Investigations into whether angiogenin functions are mediated by tiRNAs illustrated that neither protection against hypoxia in primary motoneurons nor enhanced protein translation in primary astrocytes was mediated by synthetic tiRNAs. These results suggest that either tiRNA modifications are absent in synthetic tiRNAs or that a pool of tiRNAs are essential for mimicking the effect of angiogenin.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License.

File Size

8.16 MB

Comments

A thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2017.

Share

COinS