Abstract
Protein interactions of Tau are of interest in efforts to decipher pathogenesis in Alzheimer’s disease, a subset of frontotemporal dementias, and other tauopathies. We CRISPR-Cas9 edited two human cell lines to generate broadly adaptable models for neurodegeneration research. We applied the system to inducibly express balanced levels of 3-repeat and 4-repeat wild-type or P301L mutant Tau. Following 12-h induction, quantitative mass spectrometry revealed the Parkinson’s disease-causing protein DJ-1 and non-muscle myosins as Tau interactors whose binding to Tau was profoundly influenced by the presence or absence of the P301L mutation. The presence of wild-type Tau stabilized non-muscle myosins at higher steady-state levels. Strikingly, in human differentiated co-cultures of neuronal and glial cells, the preferential interaction of non-muscle myosins to wild-type Tau depended on myosin ATPase activity. Consistently, transgenic P301L Tau mice exhibited reduced phosphorylation of regulatory myosin light chains known to activate this ATPase. The direct link of Tau to non-muscle myosins corroborates independently proposed roles of Tau in maintaining dendritic spines and mitochondrial fission biology, two subcellular niches affected early in tauopathies.
Highlights
More than 30 years ago the Tau protein was first identified in cellular deposits that accumulate in Alzheimer disease (AD)[1]
We used CRISPR-Cas[9] gene editing technology to insert foundation cassettes harboring an antibiotic resistance gene flanked by lox sites, which are later used as acceptor sites to efficiently integrate large inducible constructs, into the human adeno-associated virus integration site 1 (AAVS1) genomic safe harbor of two cell lines with useful characteristics for Alzheimer disease and Tau-related research, namely IMR32 neuroblastoma cells[16,17,18] and a neuroprogenitor cell line derived from the ventral mesencephalon (ReN VM)[19], hereafter referred to as IMR and ReN cells
The similar sizes of the enhanced green fluorescent protein (EGFP) ligand and the GFP binding protein (GBP) bait are favourable for achieving a high density of ligand-bait pairings
Summary
More than 30 years ago the Tau protein was first identified in cellular deposits that accumulate in Alzheimer disease (AD)[1]. It would be desirable to be able to rapidly generate a human diploid cell model that can be fully differentiated into neurons, astrocytes or oligodendrocytes and can be induced to (1) express a protein-of-interest, (2) visualize its subcellular distribution, and (3) study its interactions and post-translational modifications To address these shortcomings, we used CRISPR-Cas[9] gene editing technology to insert foundation cassettes harboring an antibiotic resistance gene flanked by lox sites, which are later used as acceptor sites to efficiently integrate large inducible constructs, into the human adeno-associated virus integration site 1 (AAVS1) genomic safe harbor of two cell lines with useful characteristics for Alzheimer disease and Tau-related research, namely IMR32 neuroblastoma cells[16,17,18] and a neuroprogenitor cell line derived from the ventral mesencephalon (ReN VM)[19], hereafter referred to as IMR and ReN cells.
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