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.

The microtubule-associated protein Tau is highly enriched in axons of brain neurons where it regulates axonal outgrowth, plasticity, and transport. Efficient axonal Tau sorting is critical since somatodendritic Tau missorting is a major hallmark of Alzheimer’s disease and other tauopathies. However, the molecular mechanisms of axonal Tau sorting are still not fully understood. In this study, we aimed to unravel to which extent anterograde protein transport contributes to axonal Tau sorting. We developed a laser-based axotomy approach with single-cell resolution and combined it with spinning disk confocal microscopy enabling multi live-cell monitoring. We cultivated human iPSC-derived cortical neurons and mouse primary forebrain neurons in specialized chambers allowing reliable post-fixation identification and Tau analysis. Using this approach, we achieved high post-axotomy survival rates and observed axonal regrowth in a subset of neurons. When we assessed somatic missorting and phosphorylation levels of endogenous human or murine Tau at different time points after axotomy, we surprisingly did not observe somatic Tau accumulation or hyperphosphorylation, regardless of their regrowing activity, consistent for both models. These results indicate that impairment of anterograde transit of Tau protein and acute axonal damage may not play a role for the development of somatic Tau pathology. In sum, we developed a laser-based axotomy model suitable for studying the impact of different Tau sorting mechanisms in a highly controllable and reproducible setting, and we provide evidence that acute axon loss does not induce somatic Tau accumulation and AT8 Tau phosphorylation. Graphical Abstract UV laser-induced axotomy of human iPSC-derived and mouse primary neurons results in decreased somatic levels of endogenous Tau and AT8 Tau phosphorylation.

Primary murine neurons have proved to be an essential tool for the general investigation of neuronal polarity, polarized TAU distribution, and TAU-based neuronal dysfunction in disease paradigms. However, mature primary neurons are notoriously difficult to transfect with non-viral approaches and are very sensitive to cytoskeletal manipulation and imaging. Furthermore, standard non-viral transfection techniques require the use of a supportive glia monolayer or high-density cultures, both of which interfere with microscopy. Here we provide a simple non-viral liposome-based transfection method that enables transfection of TAU in low levels comparable to endogenous TAU. This allows the investigation of e.g., distribution and trafficking of TAU, without affecting other cytoskeleton-based parameters such as microtubule density or microtubule-based transport. Using this protocol, we achieve a profound transfection efficiency, but avoid high overexpression rates. Importantly, this transfection method can be applied to neurons at different ages and is also suitable for very old cultures (up to 18 days in vitro). In addition, the protocol can be used in cultures without glial support and at suitable cell densities for microscopy-based single-cell analysis. In sum, this protocol has proven a reliable tool suitable for most microscopy-based approaches in our laboratory.

TAU pathology is a major hallmark of many neurodegenerative diseases summarized under the term tauopathies. In most of these diseases, e.g., Alzheimer’s Disease, the neuronal axonal microtubule-binding TAU protein becomes mislocalized to the somatodendritic compartment. In human disease, this missorting of TAU is accompanied by an abnormally high phosphorylation state of the TAU protein, and several downstream pathological consequences (e.g. loss of microtubules, degradation of postsynaptic spines, impaired synaptic transmission, neuronal death). While some mechanisms of TAU sorting, missorting and associated pathologies have been addressed in rodent models, few studies have addressed human TAU in physiological disease-relevant human neurons. Suitable human-derived in vitro models are necessary. This protocol provides a simple step-by-step protocol for generating homogeneous cultures of cortical glutamatergic neurons using an engineered Ngn2 transgene carrying WTC11 iPSC line. We further demonstrate strategies to improve neuronal maturity, i.e., synapse formation, TAU isoform expression, and neuronal activity by co-culturing hiPSC-derived glutamatergic neurons with mouse-derived astrocytes. Finally, we explain a simple way for high-efficiency lentiviral transduction of hiPSC-derived neurons at almost all stages of differentiation.

Primary murine neurons are a well-established tool for investigating TAU in the context of neuronal development and neurodegeneration. However, culturing primary neurons is usually time-consuming and requires multiple feeding steps, media exchanges, proprietary media supplements, and/or preparation of complex media. Here we describe i) a relatively cheap and easy cell culture procedure for the cultivation of forebrain neurons from embryonic mice (E13.5) based on a commercially available neuronal supplement (NS21), ii) a protocol for the cultivation of hippocampal and cortical neurons from postnatal (P0-P3) animals, as well as iii) basic fixation and immunofluorescence techniques for the staining of neuronal markers and endogenous TAU. We demonstrate a staining technique, which minimizes antibody consumption and allows for fast and convenient processing of samples for immunofluorescence microscopy of endogenous TAU in primary neurons. We also provide a protocol that enables cryopreservation of fixed cells for years without measurable loss of TAU signal. In sum, we provide reliable protocols enabling microscopy-based studies of TAU in primary murine neurons.