The microtubule-associated protein TAU is a key driver of the neurodegeneration observed in Alzheimer’s disease (AD). Normally, TAU stabilizes neuronal microtubules (MT) and promotes essential MT-associated functions. Alternative splicing of the TAU-encoding MAPT gene results in the expression of six isoforms in the human brain. Models of AD and TAU pathology to date are mostly based on rodents, which differ in their TAU isoform expression and often rely on the overexpression of mutant human TAU to develop hallmarks of AD. Moreover, recent results from murine neurons highlight that TAU isoforms are differentially localized within neurons and may have isoform-specific functions, but human cellular data is scarce. In this study, we generated MAPT KO human induced pluripotent stem cells using CRISPR/Cas9 and induced neuronal differentiation using Ngn2. Differentiated TAU KO neurons show no major abnormalities or changes in neuronal activity but sightly decreased neurite outgrowth and AIS length. Yet, TAU-depleted neurons are protected from AD-like stress, e.g, Amyloid-beta oligomer (AβO)-induced reduction of neuronal activity. Re-expression of most individual TAU isoforms was sufficient to rescue the changes in neurite and AIS development. However, the 1N4R-TAU isoform alone was sufficient to restore neuronal vulnerability to AD-like stress. In sum, we describe here for the first time a human iPSC-based MAPT KO/TAU depletion model to study the function of TAU isoforms and their role in AD pathology. Our results suggest that 1N4R-TAU is involved in early TAU-mediated toxicity and a potential target for future therapeutic strategies for AD.

Background One hallmark of many neurodegenerative diseases, such as Alzheimer’s disease (AD), is the formation of neurofibrillary tangles by hyperphosphorylated Tau in the brain, resulting in neuronal death and cognitive decline. In the adult human brain, six Tau isoforms are expressed, originating from alternative splicing of exons 2, 3, and 10 of the MAPT gene. The isoforms differ in the number of N‐terminal inserts (0, 1, or 2N) and the C‐terminal repeat number (3 or 4R). Mutations affecting splicing of the MAPT gene can alter the Tau isoform ratio and ultimately cause neurodegeneration. Recent results from murine neurons highlight that the six human‐specific isoforms are differentially localized within neurons and influence microtubule dynamics in an isoform‐specific manner. Method To further investigate the Tau isoforms in a human neuronal model, we generated three Tau KO iPSC cell lines derived from Ngn2‐WTC11 cells using CRISPR/Cas9. Result Basic characterization of the Tau KO cell lines showed no difference in the morphology or pluripotency of iPSCs, the capability of neuronal differentiation or neuronal morphology compared to WT iPSCs and induced neurons. Initial results from re‐expressed TAU isoforms in Tau KO human neurons support the differential localization of the isoforms observed previously. Further experiments will investigate the isoform‐specific role of Tau regarding microtubule dynamics, axonal branching, and synapse formation under basal and pathological conditions. Conclusion Expected results will provide a major understanding of basic biological functions of Tau and help to identify potential therapeutic targets for the treatment of AD and related neurodegenerative diseases.