dSarm/Sarm1 is required for activation of an injury-induced axon death pathway

Abstract

Axonal and synaptic degeneration is a hallmark of peripheral neuropathy, brain injury, and neurodegenerative disease. Axonal degeneration has been proposed to be mediated by an active autodestruction program, akin to apoptotic cell death; however, loss-of-function mutations capable of potently blocking axon self-destruction have not been described. Here, we show that loss of the Drosophila Toll receptor adaptor dSarm (sterile α/Armadillo/Toll-Interleukin receptor homology domain protein) cell-autonomously suppresses Wallerian degeneration for weeks after axotomy. Severed mouse Sarm1 null axons exhibit remarkable long-term survival both in vivo and in vitro, indicating that Sarm1 prodegenerative signaling is conserved in mammals. Our results provide direct evidence that axons actively promote their own destruction after injury and identify dSarm/Sarm1 as a member of an ancient axon death signaling pathway.

Fig. 1

Identification of mutations that suppress Wallerian degeneration. (A) ORN MARCM clones in control, l(3)896, l(3)4621, and l(3)4705. Right, axotomy; left, uninjured control. n ≥ 15 brains. (B) Control and l(3)896 brains 30 days after injury. n ≥ 10 brains. (C) l(3)896 clones 50 days after injury. n = 11 brains. (D) MARCM clones in mushroom body (MB) γ neurons in control and l(3)896 backgrounds at the indicated developmental stages. dorsal (d) and medial (m) axonal branches (arrows), and dendrites (circled). n ≥ 15 brains for all. (E) dMP2 neurons with GFP. Ventral views (anterior up) of stage 16 embryos (left) and first-instar larvae. dMP2 neurons before (arrows) and after (arrowheads) segment-specific apoptosis. n ≥ 20 flies at each time point. (F) Wild-type and l(3)896 mutant clones with (right) or without (left) ectopic expression of hid. n ≥ 20 flies.

Fig. 2

Mutations in dsarm block Wallerian degeneration. (A) The lethality of l(3)896, l(3)4621, and l(3)4705 was mapped to region 66B using Exel or Bloomington Stock Center deficiencies. (B) Dsarm protein domains, positions, and effect of predicted point mutations. (C) UAS-dsarm in l(3)896 mutant clones or dsarm⁺ bacterial artificial chromosome rescue axonal degeneration defects in l(3)896/l(3)4621 flies. n = 12 flies.

Fig. 3

Sarm1^−/− neurons in primary culture are protected from Wallerian degeneration. (A) Phase contrast images of SCG explant cultures from wild-type (top), Sarm^−/− (middle), and Wld^S-expressing (bottom) animals at the indicated time after axotomy. (B) Quantification from (A). Mean T SEM, *P < 0.01. (C) Axon preservation at the indicated time points in cortical neuron cultures from E16.5 mouse embryos. α-Tau, red. (D) Quantification from (C). Mean T SEM, *P < 0.01. (E) Axon preservation at the indicated time points in DRG cultures from E13.5 mouse embryos. α-TUJI, green. (F) Quantification from (E). Mean T SEM, *P < 0.01.

Fig. 4

Sarm1 is required for Wallerian degeneration in mice in vivo. (A) Sciatic nerve distal to the injury site stained with Toludine blue. Time points and genotypes as indicated. (B) Ultrastructural analysis of Sarm^+/− and Sarm^−/− axons before or 14 days after axotomy. Higher magnification image (lower right) is shown in its entirety in fig. S4. my, myelin; nf, neurofilaments; m, mitochondrion. (C) Neuromuscular junction (NMJ) preservation at tibialis anterior muscles. red, AChR (postsynapse/muscle); green, NF-M/synpatophysin (presynapse). Arrows, motor end plates. (D) Immunoblot analysis of distal injured nerve segment. n = 4 mice at each time point and genotype.