Doctor of Philosophy
School of Biological Sciences
Roberts, Kate, Elucidating the effect of aggregated SOD1 on microglia and astrocytes in ALS, Doctor of Philosophy thesis, School of Biological Sciences, University of Wollongong, 2016. http://ro.uow.edu.au/theses/4840
A large body of literature suggests that amyotrophic lateral sclerosis (ALS) pathology is intimately linked with both aggregated proteins and neuroinflammation; specifically activation, recruitment and dysfunction of microglia and astrocytes. However, there has been little work performed that aimed to understand the impact that aggregates of Cu/Zn superoxide dismutase (SOD1) have on microglia and/or astrocytes.
Mutant SOD1 associated with ALS has recently been shown to activate microglia in a CD14 dependant mechanism when found in the extracellular space, providing one potential explanation of glial activation during disease. Recent work demonstrates a strong link between protein inclusions and cell loss and as a result neuroinflammation in ALS and although they may be made up of different proteins, inclusions are associated with all forms of ALS. With this in mind, the first aim of this project sought to determine if aggregated SOD1 would activate microglia. Recombinant SOD1 was aggregated and this, or soluble non-aggregated forms of SOD1 were then added to EOC.13 microglial cells or primary microglial cells in culture. Although soluble nonaggregated mutant SOD1 has been shown to promote microglial activation in the past, we found that aggregated SOD1 was able to much more efficiently activate microglia in culture when compared with the unaggregated form of mutant SOD1. In addition to CD14 dependant pathways, aggregated SOD1 also bound to the surface of glial cells and was internalized in a lipid raft and scavenger receptor dependent manner. Here, for the first time, we have shown that aggregated mutant SOD1 potently activates microglia.
There is growing evidence to suggest astrocyte dysfunction in ALS. Previous evidence has suggested that accumulation of mutant Cu/Zn superoxide dismutase (SOD1) initiates activation of ER stress in motor neurons. As protein inclusions have been observed in astrocytes in ALS, the second aim of this project sought to determine if exogenous aggregated mutant SOD1 would affect activation or viability of primary astrocytes. Recombinant mutant SOD1 was aggregated and subsequently added to primary astrocytes in culture. Exogenously added aggregated mutant SOD1 was taken up into the cytosol of astrocytes, remained there for at least 7 days, causing endoplasmic reticulum stress, astrocyte senescence and ultimately cell death. With the use of a panel of endocytosis inhibitors we were able to show that aggregated SOD1 was internalized by primary astrocytes via macropinocytosis. This work, for the first time, links uptake of aggregated mutant SOD1 to dysfunction and toxicity in primary astrocytes.
While the first two chapters showed dramatic effects this was performed in 2 D cultures. To demonstrate glial dysfunction in a biological system proteins were infused or injected in to the nervous system of mice. Here we report that soluble SOD1G93A infused over 5 weeks into the brains ventricles of wild type mice, resulted in no significant impact on anterior horn microglia and astrocytes, when compared to soluble SOD1WT infusion. Whereas, 72 hours after injection aggregated SOD1 caused increased microglial activity and caused astrocyte senescence around murine brain ventricles. These results suggest that the aggregated form of SOD1 has a significant impact on the activation state of microglia and astrocyte dysfunction in vivo, and may directly affect disease progression in ALS. As a result, future therapeutics that work simultaneously to prevent SOD1 aggregate induced microglial activation and astrocyte dysfunction may be effective in slowing ALS progression.