Laser-Induced Axotomy of Human iPSC-Derived and Murine Primary Neurons Decreases Somatic Tau and AT8 Tau Phosphorylation: A Single-Cell Approach to Study Effects of Acute Axonal Damage

Abstract

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.

Scraping Assay as a Novel Strategy to Evaluate Axonal Regeneration Using Human-Induced Pluripotent Stem Cell-Derived Neurons

“…23) However, this model is used to evaluate cell viability and neurite morphology but not axonal regeneration. Although laser-induced axotomy has been established as a model for in vitro injury, 24) it has not been designed for high-throughput experiments. Therefore, we developed a scraping-assay-based screening system to evaluate axonal regeneration in hiPSC-derived neurons.…”

Tau here, tau there, tau almost everywhere: Clarifying the distribution of tau in the adult CNS

“…The other major body of work that demonstrates axonal enrichment of tau in cultured neurons utilizes exogenous expression of fluorescently labeled tau to visualize its subcellular localization (Bachmann et al, 2021;Bell & Zempel, 2022;Bell-Simons et al, 2023;Zempel et al, 2017). In these studies, fluorescently labeled tau is co-transfected with tdTomato, and tau fluorescence in distinct subcellular compartments is normalized to tdTomato in the same compartments.…”