Vincristine and bortezomib use distinct upstream mechanisms to activate a common SARM1-dependent axon degeneration program
Stefanie Geisler, … , Jeffrey Milbrandt, Aaron DiAntonio
Stefanie Geisler, … , Jeffrey Milbrandt, Aaron DiAntonio
Published September 5, 2019
Citation Information: JCI Insight. 2019;4(17):e129920. https://doi.org/10.1172/jci.insight.129920.
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Research Article Cell biology Neuroscience

Vincristine and bortezomib use distinct upstream mechanisms to activate a common SARM1-dependent axon degeneration program

Abstract

Chemotherapy-induced peripheral neuropathy is one of the most prevalent dose-limiting toxicities of anticancer therapy. Development of effective therapies to prevent chemotherapy-induced neuropathies could be enabled by a mechanistic understanding of axonal breakdown following exposure to neuropathy-causing agents. Here, we reveal the molecular mechanisms underlying axon degeneration induced by 2 widely used chemotherapeutic agents with distinct mechanisms of action: vincristine and bortezomib. We showed previously that genetic deletion of SARM1 blocks vincristine-induced neuropathy and demonstrate here that it also prevents axon destruction following administration of bortezomib in vitro and in vivo. Using cultured neurons, we found that vincristine and bortezomib converge on a core axon degeneration program consisting of nicotinamide mononucleotide NMNAT2, SARM1, and loss of NAD+ but engage different upstream mechanisms that closely resemble Wallerian degeneration after vincristine and apoptosis after bortezomib. We could inhibit the final common axon destruction pathway by preserving axonal NAD+ levels or expressing a candidate gene therapeutic that inhibits SARM1 in vitro. We suggest that these approaches may lead to therapies for vincristine- and bortezomib-induced neuropathies and possibly other forms of peripheral neuropathy.

Authors

Stefanie Geisler, Ryan A. Doan, Galen C. Cheng, Aysel Cetinkaya-Fisgin, Shay X. Huang, Ahmet Höke, Jeffrey Milbrandt, Aaron DiAntonio

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Figure 3

Vincristine and BTZ induce SARM1-dependent depletion of axonal NAD+.

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Vincristine and BTZ induce SARM1-dependent depletion of axonal NAD+. (A)...
(A) HPLC was used to measure NAD+ levels in WT and SARM1-KO neurons from axon or cell body extracts and normalized to baseline. Two-way ANOVA shows main effect for group F (3, 8) = 52, P < 0.0001; time F (3, 24) = 39.77, P < 0.0001; and interaction F (9, 24) = 5.797, P = 0.0003. Tukey’s multiple-comparisons test, **P < 0.01, ***P < 0.001, ****P < 0.0001. Black asterisks, WT axon versus WT soma; red asterisks, WT axon versus SARM1-KO axon (n = 3). Representative bright-field images just before NAD+ extraction. Original magnification 200×. (B) NAD+ levels were measured as described in A. Two-way ANOVA showed main effects of group F (3, 46) = 30.23, P < 0.0001; time F (3, 46) = 88.83, P < 0.0001; and interaction F (9, 46) = 5, P = 0.0001. Tukey’s multiple-comparisons test, **P < 0.01, ****P < 0.0001; n = 3–5 per time point. Representative bright-field images just before NAD+ extraction. Original magnification 200×. (C) Diagram of selected mammalian NAD+ biosynthesis pathways. NMNAT, nicotinamide mononucleotide adenylyltransferase; NAMPT, nicotinamide phosphoribosyltrans¬ferase; NRK, nicotinamide riboside kinase. (D) WT DRG neurons expressing NRK1 were treated with 1 mM NR and vincristine, BTZ, or vehicle and NAD+ was measured from axon-only lysates . Axonal NAD+ concentration of DRG neurons not overexpressing NRK/NR is indicated by a dashed line (0.15 ± 0.05 μM NAD+; n = 8). In the presence of overexpressed NRK1 and NR, axonal NAD+ levels remained high 48 hours after vincristine administration. (E) WT DRG neurons expressing indicated constructs were treated with vincristine, and axon degeneration was determined. Two-way ANOVA showed main effects of group F (3, 15) = 32.82, P < 0.001; time F (3, 45) = 134.9, P < 0.0001; and interaction F (9, 45) = 44, P < 0.0001. Tukey’s multiple-comparisons test, ****P < 0.0001 (n = 5 for NRK/NR, n = 3 for NAMPT). (F) DRG neurons expressing indicated constructs were treated with BTZ. Two-way ANOVA showed main effects of group F (3, 17) = 21.13, P < 0.001; time F (3, 51) = 97.13, P < 0.0001; and interaction F (9, 51) = 35.95, P < 0.0001. Tukey’s multiple-comparisons test, ****P < 0.0001; *P < 0.05 (n = 5 NRK/NR, n = 3 NAMPT). (G and H) Representative bright-field photomicrographs of axons expressing the indicated constructs after vin¬cristine (G) or BTZ (H) administration. Original magnification 200×.