Neurodegenerative diseases are associated with protein misfolding and amyloid aggregation. The work presented in this thesis involves the use of fluorescence lifetime imaging microscopy (FLIM) and other biophysical techniques to elucidate the molecular mechanisms that underlie different amyloid-associated neurodegenerative dis- eases. Currently, there is no prevailing therapeutic option for the latter. Hence, the work presented aims to develop physiologically relevant assays with the potential for diagnostics and drug-screening platforms, as well as to identify different therapeutic targets to alleviate disease progression.

In Alzheimer’s disease (AD), there is the characteristic formation of intracellular aggregates of amyloid-β 42 (Aβ42), which have an associated free energy barrier and exothermic elongation kinetics; these aggregates then diffuse out into the extracellular matrix between neuronal cells, triggering neuronal death. By performing intracellular thermometry in live mammalian cells using fluorescent polymeric thermometers (FPT), Aβ42 aggregation is revealed to lead to cellular thermogenesis, which is rescued upon treatment with an amyloid-β (Aβ) elongation-inhibiting, small compound drug. The latter validates the potential use of intracellular thermometry as a diagnostic platform to test inhibitory drugs in live cells. It is further shown that the thermogenesis effect primarily stems from the exothermic elongation of Aβ42, which overrides any contribution from mitochondrial dysfunction. For a better understanding of heat dissipation from Aβ peptides, classical molecular dynamic simulations are performed. It is shown that heat retention by an Aβ peptide is promoted under intracellular-mimicking conditions due to fewer water-protein hydrogen bonding interactions, which highlights the importance of incorporating physiologically relevant parameters for modelling biological and intracellular environments.

Fused-in sarcoma (FUS) is a phase-separating, DNA/RNA binding protein that localises to cell nuclei under physiological conditions, where it mediates DNA repair. Mutations to its nuclear localisation signal (NLS) result in cytoplasmic mislocalisation associated with the onset of amyotrophic lateral sclerosis (ALS). Through the combined use of FLIM and single particle tracking (SPT), a tool to infer intracellular FUS viscosity in live cells, without the introduction of an external sensor, is created. Moreover, it is identified that ALS- associated mutants, P525L and R495X exhibit loss of cytoskeletal integrity and enhanced euchromatin expression. Furthermore, they experience greater dysregulation of their autophagy-lysosomal pathway, as modulated by transcription factor EB (TFEB). The latter is triggered by increased de-acidification of lysosomes, which also leads to a collapse in tubular endoplasmic reticulum (ER) and mitochondrial fission. Macroautophagy needed for degradation and clearance of aberrant FUS aggregates is inhibited despite an up-regulation of TFEB and increased lysosomal biogenesis. Although the sequence of events leading to macroautophagy inhibition which perpetuates aberrant FUS aggregation remains unknown, it is believed that regulating TFEB and lysosomal function could be a potential therapeutic option for ALS/FTD.

The rich β-sheets that comprise an amyloid result in electron delocalisation leading to intrinsic amyloid fluorescence in the visible range, upon ultraviolet (UV) excitation. This phenomenon is independent of intrinsic aromatic fluorescence, the excitation and emission of which occur solely in the UV range. A two-photon (2P) titanium sapphire (Ti:S) laser is used to achieve UV excitation to characterise different amyloids by their unique intrinsic fluorescence lifetimes in a label-free manner. It is further shown that intrinsic amyloid fluorescence lifetime can be used to distinguish between different polymorphic populations of alpha-synuclein (αS) (known to influence disease pathology) formed under intracellular- and extracellular-mimicking buffers, as well as disaggregated fibrils. 2P-FLIM gives a medium-throughput assay which avoids the use of extrinsic fluorescent markers that may otherwise affect aggregation behaviour and the polymorphic species formed.