Doctor of Philosophy
School of Biological Sciences
Zeineddine, Rafaa, The role of the prion-like protein SOD1 and macropinocytosis in the propagation of disease in ALS; an infectious idea, Doctor of Philosophy thesis, School of Biological Sciences, University of Wollongong, 2016. http://ro.uow.edu.au/theses/4879
With the onset of the rapidly increasing population, the impact of age related neurodegenerative diseases including Amyotrophic lateral sclerosis, Alzheimer’s disease, Creutzfeldt-Jakob disease, Parkinson’s disease, Huntington’s disease and frontotemporal dementia is becoming a predominant health and economic concern. Amyotrophic lateral sclerosis (ALS) is a devastating neuromuscular degenerative disease that currently has no effective treatment or therapeutics. ALS is characterised by a focal onset of motor neuron loss, followed by contiguous outward spreading of pathology throughout the nervous system, resulting in paralysis and death generally within a few years after diagnosis. The mechanisms underlying neurodegeneration of motor neurons and disease progression are currently unknown; however, current evidence implicates a range of cellular mechanisms. These mechanisms include, deficient protein quality control, aberrant RNA metabolism, oxidative stress, endoplasmic reticulum stress, glutamate excitotoxicity, mitochondrial dysfunction, fragmentation of the Golgi apparatus, activated glia, axonal transport defects and neuroinflammation. These dysfunctional cellular pathways may be associated with the protein aggregates that are hallmarks of ALS pathology. However, the dysfunction in several cellular processes does not explain the spreading of pathology, here the aberrant release and uptake of toxic proteins including SOD1 and TDP-43 and their subsequent accumulation and deposition in motor neurons may contribute. Given this hypothesis, the work presented in this thesis aimed to further examine the role of SOD1 and TDP-43 in the propagation of neurodegeneration in ALS, and investigate whether the proteins exhibit prion-like properties. The term “Prion-like” as used here refers to the misfolding and aggregation of a disease specific protein that subsequently escapes the cellular environment and seeds aggregation in a naïve cell.
The main aims of this thesis were to: investigate the mechanisms underpinning the uptake of SOD1 aggregates into murine NSC-34 cells (Chapter 2); Examine the subsequent release of SOD1 into the cytosol, detect released extracellular SOD1, and observe for secreted SOD1 internalisation into NSC‐34 motor neurons, then identify and quantify seeding activity in recipient cells expressing SOD1 and characterise this interaction using a novel flow cytometry method to quantify protein aggregation; Flow cytometric characterisation of inclusions and trafficking (FloIT) (Chapter 3); determine whether exogenous recombinant SOD1 protein aggregates can induce and/or contribute to TDP-43 pathology (Chapter 4); determine if SOD1 aggregates can enter humanised models of motor neurons via the same mechanism of action using both iPSC derived motor neurons and primary neurons (Chapter 5).