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, but its constitutive activity prevents spatiotemporal precision. To address this, we 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. Here we show that LATeNT works across multiple brain regions and at long-range axonal projections, with its effects reversible in 24 h. LATeNT enabled us to discover a hippocampal interneuron population that regulates anxiety-like behaviors and demonstrate the importance of postsynaptic endocannabinoid exocytosis for depolarization-induced suppression of inhibition in vivo. Beyond neuroscience, 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.