An Atypical, Staged Cell Death Pathway Induced by Depletion of SNARE-Proteins MUNC18-1 or Syntaxin-1

The presynaptic proteins MUNC18-1, syntaxin-1, and SNAP25 drive SNARE-mediated synaptic vesicle fusion and are also required for neuronal viability. Their absence triggers rapid, cell-autonomous, neuron-specific degeneration, unrelated to syn-aptic vesicle deficits. The underlying cell death pathways remain poorly understood. Here, we show that hippocampi of munc18-1 null mice (unknown sex) express apoptosis hallmarks cleaved caspase 3 (CC-3) and phosphorylated p53, and have condensed nuclei. However, side-by-side in vitro comparison with classical apoptosis induced by camptothecin uncovered striking differences to syntaxin-1 and MUNC18-1 depleted neurons. First, live-cell imaging revealed consecutive neurite re-traction hours before cell death in MUNC18-1 or syntaxin-1 depleted neurons, whereas all neurites retracted at once, directly before cell death in classical apoptosis. Second, CC-3 activation was observed only after loss of all neurites and cellular break-down, whereas CC-3 is activated before any neurite loss in classical apoptosis. Third, a pan-caspase inhibitor and a p53 inhib-itor both arrested classical apoptosis, as expected, but not cell death in MUNC18-1 or syntaxin-1 depleted neurons. Neuron -specific cell death, consecutive neurite retraction, and late CC-3 activation were conserved in syntaxin-1 depleted human neu-rons. Finally, no indications were observed for involvement of other established cell death pathways, including necroptosis, Wallerian degeneration, autophagic cell death, and pyroptosis. Together, these data show that depletion of presynaptic pro-teins MUNC18-1 or syntaxin-1 triggers an atypical, staged cell death pathway characterized by consecutive neurite retraction, ultimately leading to, but not driven by, apoptosis.