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. 2013 Aug 14;33(33):13569–13580. doi: 10.1523/JNEUROSCI.1197-13.2013

Figure 6.

Figure 6.

SAM-TIR expression induces axonal degeneration and nonapoptotic neuronal death. A, SAM-TIR expression induces pronounced blebbing (arrowheads) in HEK293 cells at 24 h post-transfection. B, SAM-TIR overexpression in HEK293 cells leads to a loss of cell viability measured by the metabolic resazurin assay at 36 h post-transfection. Cell viability is unaffected by expression of full-length SARM, SAM, or mutated SAM-TIR bearing an 8×Ala replacement mutation in the TIR domain that blocks SARM function in axon degeneration (SAM-TIR mut = [697-704A]). ***p < 0.001 unpaired t test with Bonferroni correction. C, Lentiviral packaging of SAM-TIR is made possible by a conditionally expressed DIO SAM-TIR: a lentivirus construct containing inverted SAM-TIR-GFP flanked by loxp and lox2722 sites is conditionally expressed in cells only upon Cre-dependent recombination. Ub, Human ubiquitin C promoter. D, SAM-TIR expression (DIO-SAM-TIR lentivirus plus Cre lentivirus) induces axon degeneration that is not blocked by the apoptosis-inhibiting protein Bcl-XL, pan-caspase inhibitor Z-VAD-FMK (ZVFMK; 10 μg/ml), or calpain inhibitor ALLN (25 μm). Chelation of extracellular Ca ions with EGTA (2.5 mm) provides partial suppression of axon degeneration. E, Representative α-tubulin-stained axons from experiment shown in D. F, SAM-TIR expression induces axon degeneration in both wild-type and SARM−/− axons measured 3 d postinfection; ***p < 0.001 unpaired t tests with Bonferroni correction. G, SAM-TIR expression induces large translucent neuronal swellings evident at 2 d postinfection. H, SAM-TIR expression induces neuronal death measured by ethidium homodimer staining at 3 d postinfection. Cell death is not blocked by the apoptosis-inhibiting protein Bcl-XL, pan-caspase inhibitor Z-VAD-FMK (ZVFMK; 10 μg/ml), calpain inhibitor ALLN (25 μm), or chelation of extracellular Ca ions with EGTA (2.5 mm). ***p < 0.001 unpaired t tests with Bonferroni correction. I, Proposed model, SARM forms SAM-mediated complexes that promote axon degeneration via intracomplex TIR interactions. Top, In uninjured cells, the N terminus of SARM suppresses the degeneration-promoting C terminus. Injury leads to release of this inhibition. Bottom, Incorporation of TIR-less molecules into SARM complexes renders them nonfunctional. Scale bars, 50 μm, error bars = SEM.