Abstract
Alzheimer’s disease is a progressive neurodegenerative disease which is primarily associated with the accumulation of amyloid-β (Aβ) and tau to form extracellular Aβ plaques and intracellular neurofibrillary tangles (NFT). Recently there has been increasing focus on other pathological changes associated with AD, such as neuroinflammation which, along with the accumulation of tau, most strongly correlates with cognitive decline. One major player in neuroinflammation is astrocytes. In AD, reactive astrocytes accumulate around amyloid plaques. Molecular chaperones play an essential role in maintaining protein homeostasis and preventing protein aggregation by binding to misfolded proteins. One family of these chaperones are the small heat shock proteins (sHSPs). In addition to classical chaperone functions, sHSPs have also been implicated in other functions including cytoskeleton stabilization, anti-apoptotic and anti-inflammatory functions. Evidence suggests that sHSPs are upregulated in reactive astrocytes in neurodegenerative diseases, including AD, however their exact role has yet to be determined. Further to this, a number of studies have suggested that overexpressing astrocytic sHSPs in animal models of neurodegenerative disease may be protective in a non-cell autonomous manner. Therefore, the hypothesis for this thesis is that the upregulation of the ubiquitously expressed and stress-induced sHSP HSPB1, specifically in the astrocytes, is neuroprotective against AD-relevant pathology through a non-cell autonomous mechanism.To explore this hypothesis, the localisation and expression of HSPB1 was first characterized in human AD temporal cortex of different Braak stages by immunofluorescent analysis. In order to study the non-cell autonomous function of astrocytic HSPB1, organotypic brain slice culture models were used to replicate some of the pathological traits seen in AD, through treatment with cytokines known to induce reactive astrocytes in neurodegenerative disease and also with physiologically relevant levels of Aβ oligomers. A disease relevant tau pathology model was then set-up by transducing both primary mouse neurons and brain slices with tau adeno-associated virus (AAV) constructs. Finally, the potentially protective functions of HSPB1 were explored using recombinant HSPB1 treatment and by overexpression of astrocytic HSPB1 in the slices through AAVs.
Human data confirmed that HSPB1 is localised within reactive astrocytes in the AD brain and also showed that there were more HSPB1 positive reactive astrocytes in close proximity to amyloid plaques. Further to this, immunofluorescent analysis revealed that the majority of amyloid plaques found in the most severe AD cases were also HSPB1 positive, suggesting that HSPB1 is being extracellularly released in response to AD pathology. In those slices treated with cytokines, reactive astrocytes were induced and HSPB1 was found to co-localise with reactive astrocytes, reflecting what was seen in the human AD brain. Transduction with a pro-aggregatory mutant tau-AAV construct resulted in the accumulation of hyperphosphorylated tau in both the primary neurons and slice cultures. Interestingly, the accumulation of tau inclusions in the primary neurons was reduced in those neurons treated with recombinant HSPB1, highlighting the potentially protective chaperone function of extracellular HSPB1. Further studies will be necessary to conclude whether astrocytic HSPB1 can also reduce tau pathology in brain slices.
All in all, the organotypic brain slice cultures proved to be a good model for replicating some of the relevant AD pathological traits seen in the human disease, however further work is required to gain a deeper understanding behind the mechanisms by which HSPB1 may be protective in response to AD pathology. Overall this work contributes to current knowledge around the astrocytic sHSP
response in AD.
| Date of Award | 1 Dec 2022 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Maria Jimenez Sanchez (Supervisor) & Wendy Noble (Supervisor) |