Tau Conformers in FTLD-MAPT  Undergo Liquid-liquid Phase Separation and Perturb the Nuclear Envelope

Background: Germline mutations in the MAPT gene cause some forms of frontotemporal lobar degeneration (FTLD). Recent studies show that a single mutation in MAPT can promote alternative tau misfolding pathways engendering divergent tau conforms and representing clinical heterogeneity, and that under conditions of cell-free molecular crowding the repertoire of tau forms can include liquid-liquid phase separation (LLPS). Methods: Neuronal nuclear morphologies in FTLD patients and TgTau P301L transgenic mice were analyzed by immunohistochemistry of nuclear lamina. Tau conformers associated with a common behavioral variant of frontotemporal dementia were cloned by endpoint dilution; the cells were assayed for viability and biochemical markers of cell death and were also assessed by video microscopy and photobleaching to determine dynamic aspects of aggregate formation. Results: Analysis of post-mortem tissues from aged neurologically normal controls and other neurodegenerative syndromes indicated that microtubule-associated nuclear clefts were associated with chronological aging and disruptions of the nuclear envelope with FTLD-MAPT. Tau conformers present in FTLD cases and transduced into reporter cells had a high propensity to condense on the nuclear envelope and to disrupt nuclear-cytoplasmic transport. Nuclear envelope fluorescent tau signals and small fluorescent inclusions in a stable clonal line behaved as a demixed liquid state under live cell conditions; indicative of LLPS effects, these droplets exhibited spherical morphology, fusion events and recovery from photobleaching. While pathogenic mutations in some proteins can interfere with physiological functions of membrane-less organelles, a disease-causing MAPT mutation perturbed nuclear-cytoplasmic transport by gain-of-function formation of LLPS on the nuclear envelope, this acting as a molecular cue to trigger regulated cell death. Thioflavin S-positive intracellular aggregates were prevalent in tau-derived inclusions with a size bigger than 3 µm 2 , inferring that a threshold of critical mass in the liquid state condensation may drive liquid-solid phase transitions. Conclusions: Our findings indicate that within a spectrum of alternative conformers, tau undergoing LLPS is a notably toxic species; demixed droplets on the nuclear envelope hindering nuclear-cytoplasmic transport can serve to trigger cytotoxic pathways and may act as nurseries for the abundant fibrillar structures present at end-stage disease.