Apoptotic cells expose Phosphatidylserine (PS), that serves as an “eat me” signal for engulfing cells. Previous studies have shown that PS also marks degenerating axonsduring developmental pruning or in response to insults (Wallerian degeneration), but the pathways that control PS exposure on degenerating axons are largely unknown. Here, we used a series of in vitro assays to systematically explore the regulation of PS exposure during axonal degeneration. Our results show that PS exposure is regulated by the upstream activators of axonal pruning and Wallerian degeneration. However, our investigation of signaling further downstream revealed divergence between axon degeneration and PS exposure. Importantly, elevation of the axonal energetic status hindered PS exposure, while inhibition of mitochondrial activity caused PS exposure, without degeneration. Overall, our results suggest that the levels of PS on the outer axonal membrane can be dissociated from the degeneration process and that the axonal energetic status plays a key role in the regulation of PS exposure.

In recent years, there has been growing interest in SARM1 as a potential breakthrough drug target for treating various pathologies of axon degeneration. SARM1-mediated axon degeneration relies on its TIR domain NADase activity, but recent structural data suggest that the non-catalytic ARM domain could also serve as a pharmacological site as it has an allosteric inhibitory function. Here, we screened for synthetic small molecules that inhibit SARM1 by stabilizing the ARM-dependent inactive, compact octamer ring conformation, and tested a selected set of these compounds in a DRG axon degeneration assay. Using cryo-EM, we found that one of the newly discovered inhibitors, a Calmidazolium designated TK106, not only stabilizes the previously reported inhibited conformation of the octamer, but also promotes the formation of a meta-stable structure: a duplex of octamers (16 protomers), which we have now determined to 4.0 A resolution. In the duplex, each ARM domain protomer is not only engaged in lateral interactions with neighboring protomers but is further stabilized by contralateral contacts with the opposing octamer ring. Mutagenesis of the duplex contact sites leads to SARM1 activation in cultured cells. Based on our data we propose that the duplex assembly constitutes an additional auto-inhibition mechanism that tightly prevents pre-mature activation and axon degeneration.

The innervation of the mammary gland is controlled by brain-derived neurotrophic factor (BDNF), and sexually dimorphic sequestering of BDNF by the truncated form of TrkB (TrkB.T1) directs male-specific axonal pruning in mice. It is unknown whether other cues modulate these processes. We detected specific, non-dimorphic, expression of Semaphorin family members in the mouse mammary gland, which signal through PlexinA4. PlexinA4 deletion in both female and male embryos caused developmental hyperinnervation of the gland, which could be reduced by genetic co-reduction of BDNF. Moreover, in males, PlexinA4 ablation delayed axonal pruning, independently of the initial levels of innervation. In support of this, in vitro reduction of BDNF induced axonal hypersensitivity to PlexinA4 signaling. Overall, our study shows that precise sensory innervation of the mammary gland is regulated by the balance between trophic and repulsive signaling. Upon inhibition of trophic signaling, these repulsive factors may promote axonal pruning.

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