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Due to the importance of 4R tau in the pathogenicity of primary tauopathies, it has been challenging to model these diseases in iPSC-derived neurons, which express very low levels of 4R tau. To address this problem we have developed a panel of isogenic iPSC lines carrying the MAPT splice-site mutations S305S, S305I or S305N, derived from four different donors. All three mutations significantly increased the proportion of 4R tau expression in iPSC-neurons and astrocytes, with up to 80% 4R transcripts in S305N neurons from as early as 4 weeks of differentiation. Transcriptomic and functional analyses of S305 mutant neurons revealed shared disruption in glutamate signaling and synaptic maturity, but divergent effects on mitochondrial bioenergetics. In iPSC-astrocytes, S305 mutations induced lysosomal disruption and inflammation and exacerbated internalization of exogenous tau that may be a precursor to the glial pathologies observed in many tauopathies. In conclusion, we present a novel panel of human iPSC lines that express unprecedented levels of 4R tau in neurons and astrocytes. These lines recapitulate previously characterized tauopathy-relevant phenotypes, but also highlight functional differences between the wild type 4R and mutant 4R proteins. We also highlight the functional importance of MAPT expression in astrocytes. These lines will be highly beneficial to tauopathy researchers enabling a more complete understanding of the pathogenic mechanisms underlying 4R tauopathies across different cell types.

Understanding regulation of MAPT splicing is important to the etiology of many nerurodegenerative diseases, including Alzheimer disease (AD) and progressive supranuclear palsy (PSP), in which different tau isoforms accumulate in pathologic inclusions. MAPT, the gene encoding the tau protein, undergoes complex alternative pre-mRNA splicing to generate six isoforms. Tauopathies can be categorized by the presence of tau aggregates containing either 3 (3R) or 4 (4R) microtubule binding domain repeats (determined by inclusion/exclusion of exon 10), but the role of the N terminal domain of the protein, determined by inclusion/exclusion of exons 2 and 3 has been less well studied. Using an unbiased correlational screen in human brain tissue, we observed coordination of MAPT exons 2 and 10 splicing. Expression of exon 2 splicing regulators and subsequently exon 2 inclusion are differentially disrupted in PSP and AD brain, resulting in the accumulation of 1N4R isoforms in PSP and 0N isoforms in AD temporal cortex. Furthermore, we identified different N-terminal isoforms of tau present in neurofibrillary tangles, dystrophic neurites and tufted astrocytes, indicating a role for differential N-terminal splicing in the development of disparate tau neuropathologies. We conclude that N-terminal splicing and combinatorial regulation with exon 10 inclusion/exclusion is likely to be important to our understanding of tauopathies.

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Somatic stem cells have been used to treat a number of disorders for decades but are limited in their ability to differentiate and self‐renew. Induced pluripotent stem cells (iPSCs) not only have the ability to generate cells of all three germ layers, but bypass the ethical concerns related to embryonic stem cells. They also hold the prospect of utilizing a patient's own cells, thus avoiding host‐vs‐graft issues. In 2006, Takahashi and Yamanaka discovered that expression of only four genes were required to induce pluripotency: Oct4, Sox2, Klf4 and c‐Myc (OSKM). Since then, pluripotency has been induced most effectively by transfection or transduction of these genes. However, efficiencies are low, expression is difficult to control, and off‐target effects are a concern. Inducing pluripotency by exogenous delivery of OSKM proteins using cell‐penetrating peptides (CPPs), while promising, is also inefficient due to of poor penetration and entrapment in endosomes. Our novel CPP‐adaptor system overcomes entrapment by relying on the calcium flux during endosomal trafficking. The adaptor, TAT‐CaM, is a recombinant protein consisting of commonly used CPP, TAT, fused to calmodulin. The protein of interest (the ‘cargo’) has an engineered calmodulin binding site (CBS). TAT‐CaM binds purified CBS‐cargo outside the cell where the Ca2+ concentration is relatively high, but after CPP‐mediated endocytosis, cargo dissociates from TAT‐CaM inside endosomes as calcium concentrations drop, leaving the CPP‐adaptor trapped but cargo free to escape to the cytoplasm and beyond. We have used our adaptor system to deliver submicromolar doses of CBS‐Oct4 protein, inducing pluripotency as measured by expression of stem state markers and alkaline phosphatase staining. CBS‐Sox2 and CBS‐Klf4 have also been delivered and while insufficient to induce pluripotency, play an efficiency enhancing role. While not directly comparable to published results, CPP‐mediated efficiency as measured expression of stem‐state markers is higher than current transfection‐ and transduction‐mediated methods. Parameters such as concentration and timing of doses and molar ratios of OSK to each other and TAT‐CaM affect efficiency. iPSCs created via our CPP‐adaptor system can be cultured, expanded over multiple passages and differentiated into other cell types such as cardiomyocytes. Success in this work will lead to a virus‐ and nucleic acid‐free, nontoxic, tunable method for inducing pluripotency.Support or Funding InformationThis work was supported by NIH grant R15GM120691 and R15NS100632This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.