AbstractAngiogenin (ANG) is a 14kDa single chain peptide member of the secretory pancreatic RNase A family. Mutations in ANG are associated with the fatal neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD). ANG is also upregulated in the developing nervous system and has recently been shown to have a role in haematopoietic stem cell maintenance. This apparent dual function of ANG in developing and adult tissue make it an attractive target for both regenerative and ameliorative approaches.
This thesis uses multiple in vivo and in vitro models to investigate the role of ANG in the nervous system and how disease-associated ANG variants disrupt this. It also shows ANG has a role in the developing nervous system and begins to explore the potential overlap between ANG and two other ALS-FTD associated proteins – C9orf72 and TDP43.
ANG must transit to the plasma membrane for secretion after synthesis. Small molecule inhibitors showed the trafficking of HA tagged ANG in human neuroblastoma cells was COPII and microtubule dependent. Blocking either secretion or the RNase activity of zebrafish ANG homologs resulted in fewer motor neurons (MNs), delayed pioneer axon outgrowth and branching defects in the trunk of developing zebrafish embryos.
No single receptor has been convincingly identified for the uptake of extracellular ANG in neurons. Perturbation of membrane dynamics, microtubules and macropinocytosis significantly reduced ANG uptake in a panel of neuronal and non-neuronal cell lines; and in primary mouse neuronal cultures. Exposure of differentiating pluripotent embryonic carcinoma cells to ALS-associated ANG mutant proteins led to decreased MN frequency and increased cell death demonstrating that neurotoxicity correlates with both reduced RNase activity and trafficking defects.
hiPSC derived cortical projection neurons carrying disease associated mutations show increased susceptibility to stress in organoids and impaired stress granule formation, particularly layer V neurons. ANG and ANG/TDP43 mutant neural progenitors also showed altered proliferation and disorganised neural rosette structure leading to subtle changes in subtype specification.
It is unclear whether the roles of ANG in the developing and mature nervous system are linked, i.e. whether subtle defects during development result in additional susceptibility later in life. It is also unclear whether other proteins associated with neurodegenerative disorders similarly play a role in neural development. Here I also characterise the developmental expression of C9orf72 in the mouse nervous system. Understanding how these factors interact has implications for any therapeutic approach to the disease.
|Date of Award||4 Nov 2020|
|Supervisor||Vasanta Subramanian (Supervisor)|
- Motor neuron disease
- Amyotrophic lateral sclerosis
- Neurodegenerative diseases
- Fronto-temporal dementia
- Stem cells
- Human induced pluripotent stem cells
- Cortical organoids
- Developmental biology