Evaluating dynamic autophagic responses to tau inclusions

AbstractBackgroundAlzheimer’s disease and other tauopathies are characterised by the accumulation of misfolded microtubule‐associated protein tau in intracellular inclusions. We recently showed that newly formed tau inclusions are dynamic structures with a slower, but constant turnover of aggregated tau in murine organotypic brain slice cultures (BSCs) (Croft et al., 2021). Clearance by autophagy or the proteasomal machinery are indicated to be the key mechanisms involved in pathogenic protein clearance. However, the current tools to measure these processes have received mixed review, and in particular struggle to capture these processes in a continuous manner.MethodsTo dynamically assess autophagy in the presence of tau inclusions we have repurposed the autophagic flux sensor (GFP‐LC3‐RFP∆G‐LC3) to be delivered by rAAV to specific central nervous system (CNS) cell types in BSCs. This probe delivers a cytosolic internal control so autophagic flux can be estimated by calculating the GFP/RFP ratio. When utilized in conjunction with live imaging, the rate of autophagic flux can be measured over time in the same CNS cells expressing pathological or physiological tau. The rate of autophagic flux can also be measured in response to established/novel compounds or genetic manipulation.ResultsWe have successfully developed and characterized the use of rAAV‐GFP‐LC3‐RFP∆G‐LC3 specifically in neurons and astrocytes in BSCs and are expanding its use to other CNS cell types. We have validated its use to sense autophagic flux whilst pharmacologically enhancing and inhibiting autophagy in BSCs. We are now using this sensor to determine dynamic autophagic processes in the presence of soluble tau and aggregated tau and its response to other manipulations.ConclusionsMethods to understand autophagic processes have traditionally only provided snapshots over time. With this tool we can now dynamically assess autophagy in ex vivo BSCs and characterize changes in response to tau and other pathologies. Overall, understanding the mechanisms that enable tau turnover may guide therapies for Alzheimer’s disease and other neurodegenerative conditions where tau plays a role. Importantly, we will also begin to understand whether enhancing tau inclusion clearance may be beneficial or detrimental to cell health.