Light-activated tetanus neurotoxin for conditional proteolysis and inducible synaptic inhibition in vivo

The light chain of tetanus neurotoxin (TeNT) is a metalloprotease that potently inhibits synaptic transmission by cleaving the endogenous vesicle fusion protein VAMP2. However, its constitutive activity prevents spatiotemporal precision. To address this, researchers engineered light-activated TeNT (LATeNT) by inserting the light-sensitive LOV domain into an allosteric site and optimizing dynamic range via directed evolution. LATeNT’s activity is undetectable in the dark, but the protease turns on after 10–20 min of weak blue-light exposure to potently inhibit synapses in vivo. This study demonstrates that LATeNT works across multiple brain regions and at long-range axonal projections, with its effects reversible in 24 h. LATeNT enabled the identification of a hippocampal interneuron population that regulates anxiety-like behaviors and highlights the importance of postsynaptic endocannabinoid exocytosis for depolarization-induced suppression of inhibition in vivo. Additionally, LATeNT regulated endogenous insulin secretion from pancreatic beta cells and converted drug exposure, elevated Ca2+ or receptor activation into transgene expression or reporter secretion in HEK293T cells. With a large dynamic range, high light sensitivity and sustained effect, LATeNT enables versatile, spatiotemporally resolved proteolysis across diverse biological systems.