Basic Science and Pathogenesis.

Background: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the deposition of amyloid-beta and hyperphosphorylated tau (P-tau) proteins in the brain. P-tau accumulates in neurons and is strongly associated with AD severity and affected brain regions. However, only a subset of neurons in AD exhibit tau pathology. The molecular mechanisms behind heterogeneous tau pathology and how it contributes to AD are not well understood.

Method: We developed a fluorescence-activated nuclear sorting (FANS) method to separate P-tau+ neuronal nuclei from P-tau- neuronal nuclei from the same brain tissue. To validate the specificity of P-tau+ neurons, we mixed non-AD control tissue with AD tissue and examined the origin of neuronal nuclei based on their genotype. We also subjected nuclei with P-tau signal from FANS to immunofluorescence microscopy to examine the morphology and localization of P-tau aggregates.

Results: We observed disease-specific P-tau signal for nuclei in advanced AD (Braak stage V-VI) cases. These P-tau+ nuclei are highly distinguished from the P-tau-free nuclei. Confocal immunofluorescence microscopy showed P-tau adherent to the outside of nuclei that exhibited P-tau signal by FANS. We demonstrated that the nuclei sorting method based on P-tau levels can highly enrich for P-tau+ nuclei (>380 fold) and has a high accuracy of 98% based on the mixing experiments. In addition, we were able to obtain high-quality single-cell genome amplification for P-tau-sorted nuclei using primary template-directed amplification (PTA), permitting single-nucleus genome interrogation.

Conclusion: We developed a highly efficient method using fluorescence-activated nuclear sorting (FANS) to separate the population of P-tau+ neuronal nuclei in AD brains. This method allows interrogation of human neuronal nuclei based on single-cell tau pathology and enables single-cell genomic studies of heterogeneous tau pathology in AD, toward insight into disease pathogenesis and therapeutic targets.