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 synaptic 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 retraction 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 breakdown, whereas CC-3 is activated before any neurite loss in classical apoptosis. Third, a pan-caspase inhibitor and a p53 inhibitor 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 neurons. 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 proteins 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. SIGNIFICANCE STATEMENT Neuronal cell death can occur via a multitude of pathways and plays an important role in the developing nervous system as well as neurodegenerative diseases. One poorly understood pathway to neuronal cell death takes place on depletion of presynaptic SNARE proteins syntaxin-1, SNAP25, or MUNC18-1. The current study demonstrates that MUNC18-1 or syntaxin-1 depleted neurons show a new, atypical, staged cell death that does not resemble any of the established cell death pathways in neurons. Cell death on MUNC18-1 or syntaxin-1 depletion is characterized by consecutive neurite retraction, ultimately involving, but not driven by, classical apoptosis.