Developing the Spatial Transcriptomics Method coppaFISH 3D to Study Selective Vulnerability in Tauopathies

The brain is composed of millions of diverse cells that can be divided into biologically relevant types. Tauopathies are neurodegenerative diseases that involve the intracellular accumulation of the hyperphosphorylated and aggregated tau protein. Frontotemporal dementia (FTD) is a primary tauopathy, while Alzheimer’s disease (AD) is a secondary tauopathy, it is accompanied by the aggregation of beta-amyloid in plaques. Tau accumulates in specific spatial patterns, but the most vulnerable cell types are still unknown. Spatially resolved transcriptomics can detect mRNA and use it to determine each cells type, while maintaining its spatial position and local environment. However, most methods are only compatible with 2D sections, which only capture a small fraction of the tissue and can drastically warp it by stretching or shrinking different structures by different amounts. In this thesis I describe the process of developing coppaFISH 3D, a method that uses 50μm, PFA-fixed tissue sections, and the software packages needed to analyse 3D data. I optimised the method to improve reliability, accuracy and throughput. coppaFISH 3D can also be combined with in vivo calcium imaging to link transcriptomic cell types to neuronal activity. I used coppaFISH 3D to compare the cell types most vulnerable to tau pathology in the hippocampus of control, FTD and AD model mice. I show that tau phosphorylated at different sites accumulates in different cell types, AT8 in oligodendrocytes and T231 primarily in excitatory neurons. I identify global and region-specific changes in gene expression in all major cell types. Oligodendrocytes show responses to tau pathology and upregulation of myelin genes specifically in AD model mice. I see heterogeneity in plaque-associated genes between individual plaques and identify a transcriptomic axis based on the expression of activated glial genes. This study establishes coppaFISH 3D as a cutting-edge method for studying selective vulnerability in neurodegenerative diseases and answering many other important biological questions.