Recapitulation of endogenous 4R tau expression and formation of insoluble tau in directly reprogrammed human neurons

Tau is a microtubule-binding protein expressed in neurons and the equal ratio between 4-repeat (4R) and 3-repeat (3R) isoforms are maintained in normal adult brain function. Dysregulation of 3R:4R ratio causes tauopathy and human neurons that recapitulate tau isoforms in health and disease will provide a platform for elucidating pathogenic processes involving tau pathology. We carried out extensive characterizations of tau isoforms expressed in human neurons derived by microRNA-induced neuronal reprogramming of adult fibroblasts. Transcript and protein analyses showed miR-neurons expressed all six isoforms with the 3R:4R isoform ratio equivalent to that detected in human adult brains. Also, miR-neurons derived from familial tauopathypatients with a 3R:4R ratio altering mutation showed increased 4R tau and the formation of insoluble tau with seeding activity. Our results collectively demonstrate the utility of miRNA-induced neuronal reprogramming to recapitulate endogenous tau regulation comparable to the adult brain in health and disease. De-identified human adult dermal fibroblasts from healthy and IVS10+16 patients were transduced with lentivirus to express the brain enriched microRNAs, miR-9/9* and miR-124, cortical-enriched transcription factor MYT1L, and the small molecules ISX9 (in induce NeuroD family of transcription factors) and DAPT (to enhance neuronal reprogramming) to directly convert fibroblasts into miNs as previously established (Yoo et al., Nature 2011; Richner et al., Nat. Prot. 2015; Abernathy, Kim, and McCoy et al., 2017). En masse, cells were collected for both bulk RNA-seq and single cell RNA-seq and prepared for analysis as described in the Protocols.