Human co-culture models of tau pathology

Tauopathies are neurodegenerative diseases marked by the accumulation of aggregated tau protein, leading to disruptions in neuronal function. Human induced pluripotent stem cell (iPSC) technology has revolutionized the study of tau pathology by facilitating research on human neurons. It is now acknowledged that the effects of intraneuronal tau aggregation extend beyond neurons to influence surrounding glial cells, particularly astrocytes, although the mechanisms involved remain unclear due to a lack of suitable co-culture models. This thesis addresses this gap by developing two- and three-dimensional (2/3D) human in vitro neuron/astrocyte co-culture models of tau pathology. In Chapter 2, a protocol for generating microwell co-cultures of iPSC-derived neurons and primary human astrocytes to model intraneuronal tau aggregation was established. Results demonstrated successful tau aggregation via overexpression of P301L tau and subsequent treatment with tau preformed fibrils (PFFs). In addition, for the first time in human neurons spontaneous aggregation of tau in the absence of PFFs was demonstrated by overexpression of double-mutated tau containing the P301L and S320F mutations. The detailed culture procedures provided serve as a valuable resource for future research. Chapter 3 utilized this co-culture model to explore the cell (non-) autonomous effects of intraneuronal tau aggregation. A high-content, automated microscopy approach was employed to quantify tau aggregation, cellular stress markers, and neuronal morphology. While neurons were not overtly affected by tau accumulation, astrocytes exhibited increased oxidative stress and activation of stress response pathways, independent of extracellular tau, which could be blocked by tau-targeting antisense therapy. In Chapter 4 a 3D human neuron/astrocyte co-culture model was developed to better simulate neuron/astrocyte interactions in the presence of intraneuronal tau pathology. This model displayed synapse formation and neuronal activity, and confirmed spontaneous tau aggregation without the use of PFFs, further validating its utility for studying tau pathogenesis in a physiologically relevant environment. Chapter 5 investigated the impact of the extracellular matrix (ECM) in 3D cultures on the diffusion and uptake of extracellular tau PFFs. Results revealed that unlike in 2D culture, extracellular tau PFFs did not induce tau aggregation in 3D culture, but this was not due to limited diffusion. This suggests that the biological ECM limits tau seeding in 3D culture, prompting the need for innovative (synthetic) ECMs to better mimic extracellular tau seeding. In summary, this thesis contributes to understanding the complex mechanisms underlying tauopathies by developing advanced in vitro models that recapitulate intraneuronal tau aggregation and its effects on surrounding astrocytes. These models provide valuable tools for investigating potential therapeutic interventions targeting tau pathology.