Date of Award

2011

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Professor Jochen Prehn

Second Supervisor

Dr. Philippe Marin

Keywords

Amyotrophic Lateral Sclerosis, Ribonuclease, Pancreatic

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a progressive, fatal neurodegenerative disease of the motor system affecting both upper and lower motoneurons. The majority of ALS cases occur sporadically, however roughly 10 % show a hereditary component. Mutations in the hypoxiainducible factor angiogenin segregate with ALS pedigrees and the protein is expressed in motoneurons. Angiogenin shows potent neuroprotective properties in vitro and in vivo and application of recombinant human angiogenin to mixed spinal cord cultures revealed non-neuronal uptake of the protein. As the disease affects both motoneurons and non-neuronal neighbouring cells like astrocytes and microglia, a paracrine mechanism of neuroprotection has been suggested.

This study was performed to gain insights into the mechanism of angiogenininduced neuroprotection via surrounding glial cells focusing on astrocytes. The vesicular internalisation of recombinant human angiogenin by primary astrocytes was investigated in culture using pharmacological inhibitors, cell transfection and immunocytochernistry. This revealed that angiogenin endocytosis by astrocytes is clathrin-dependent and involves dynamin for vesicle scission. Vesicle budding and trafficking do not require a functional microtubule network. A fraction of the internalised angiogenin is targeted for lysosomal degradation, while the majority remains in uncharacterised sorting endosomes. The receptor for angiogenin on astrocytes was identified to be the heparansulfate proteoglycan syndecan 4 by colocalisation studies and protein knock down.

To further elucidate the role of angiogenin in paracrine neuroprotection, modification of the astrocyte secretome was investigated in response to angiogenin treatment by quantitative mass spectrometry. Metabolic protein labelling by Stable Isotope Labelling with Amino acids in Culture (SILAC) was optimised for primary astrocytes and proteins were identified by Fourier transform tandem mass spectrometry (nano-LC-FT-MSIMS). This screen identified over 1,500 proteins in the supernatant of primary astrocytes, many of which are known to exhibit extracellular location like components of the Extracellular Matrix (ECM), cytokines, growth factors and growth factor binding proteins. However, proteins with established intracellular function like splicing factors and ribosomal proteins were detected, too. Quantification using the MaxQuant software showed significant regulation of over 100 proteins in response to angiogenin treatment. The most strongly regulated proteins are involved in modifying the ECM or contribute to immune responses and might be responsible for the neuroprotective effects of angiogenin. These data also suggest the involvement of surrounding immune and endothelial cells in the biological activity of angiogenin. Additionally, local transfer of factors involved in protein translation from astrocytes to surrounding cells may be affected by angiogenin.

In conclusion, this study has shed new light on the role of angiogenin in the complex cellular interplay in the Central Nervous System (CNS) focusing on astrocytes and how they interact with neighbouring cells. Further studies will be necessary to elucidate the functional signalling outcome of angiogenin focusing on its neuroprotective activities, in particular regarding ALS pathology with the aim of finding new therapies for this fatal disease.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 License.

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Comments

A thesis submitted to the Royal College of Surgeons in Ireland for the degree of Doctor of Philosophy from the National University of Ireland in 2011.

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