Small Molecule SARM1 Inhibitors Recapitulate the SARM1-/- Phenotype and Allow Recovery of a Metastable Pool of Axons Fated to Degenerate

Abstract

Axonal degeneration is responsible for disease progression and accumulation of disability in many neurodegenerative conditions. The axonal degenerative process can generate a metastable pool of damaged axons that remain structurally and functionally viable but fated to degenerate in the absence of external intervention. SARM1, an NADase that depletes axonal energy stores upon activation, is the central driver of an evolutionarily conserved program of axonal degeneration. We identify a potent and selective small molecule isoquinoline inhibitor of SARM1 NADase that recapitulates the SARM1^-/- phenotype and protects axons from degeneration induced by axotomy or mitochondrial dysfunction. SARM1 inhibition post-mitochondrial injury with rotenone allows recovery and rescues axons that already entered the metastable state. We conclude that SARM1 inhibition with small molecules has the potential to treat axonopathies of the central and peripheral nervous systems by preventing axonal degeneration and by allowing functional recovery of a metastable pool of damaged, but viable, axons.

Keywords: ALS; CIPN; SARM1; Wallerian; WldS; axonal degeneration; axonopathy; multiple sclerosis; neurodegeneration; neuropathy.

Figure 1.. Identification of Isoquinoline Inhibitors of SARM1 NADase

(A) Enzymatic assay for NAD+ hydrolysis, monitored by production of the SARM1 product ADPR, using constitutively active SARM1 constructs containing the SAM-TIR domain. The curve shows inhibition of ADPR production by isoquinolines compounds 1–6. Values represent mean ± SEM; compounds 1 n= 4, 2 n = 6, 3 n = 10, 4 n = 8, 5 n = 10, 6 n = 10. (B) Isoquinoline series showing compound 1 identified as a weak inhibitor of ADPR production by NAD+ hydrolysis. Incorporation of substituents in position 5 of the isoquinoline ring improved potency and specificity, leading to compound 6, subsequently referred as DSRM-3716. See also Tables S1 and S2.

Figure 2.. Isoquinolines Inhibit SARM1 Activity Caused by Axotomy

DRG neurons were treated with the isoquinoline SARM1 inhibitor DSRM-3716 at the concentrations indicated and subjected to axotomy. Metabolites from axonal lysates were prepared 4 h after axotomy and analyzed by mass spectrometry as described in STAR Methods. (A) cADPR increase 4 h after axotomy was inhibited in a dose-dependent manner by DSRM-3716. F(5,12) = 23.35, p < 0.0001. Values represent mean ± SEM, n = 3/condition. (B) Dose-dependent inhibition of NAD+ consumption after axotomy. F(5,12) = 19.35, p < 0.0001. Mean ± SEM, n = 3/condition. One-way ANOVA with Holm-Sidak post hoc; *p < 0.05, **p < 0.01, ****p < 0.0001, n.s. = not significant; representative of 2 independent experiments with similar results.

Figure 3.. Isoquinoline SARM1 Inhibitor Reproduces the Axonal Protective SARM1^−/− Phenotype

(A) 10 μM SARM1 inhibitor DSRM-3716 protected axons in mouse DRG neurons subject to axotomy (right panels). TMRM fluorescence was used to assess mitochondrial viability. Cut axons lost TMRM fluorescence, whereas axons treated with DSRM-3716 at 10 μM preserved TMRM fluorescence. Scale bar, 50 μm. (B) Quantification of fragmentation showed that axonal protection with isoquinolines was dose dependent. Values represent mean ± SEM; compounds 3–5 n= 3, DSRM-3716 n = 7; representative of at least 10 independent experiments with similar results. (C) Axotomized SARM1^−/− and WT mouse DRG neurons treated with 10 μM DSRM-3716 showed similar axonal protection. One-way ANOVA with Holm-Sidak post hoc, F(4,14) = 22.11, p < 0.0001. Values represent mean ± SEM; WT + DSRM-3716 n = 3, all others n = 4; ****p < 0.0001, n.s. = not significant. (D) Axonal damage was assessed by release of NfL to culture supernatants. Cultures treated with DSRM-3716 showed dose-dependent inhibition of NfL release with EC₅₀ = 1.9 μM. Mean ± SEM, n = 4, representative of 5 independent experiments with similar results. (E) Human iPSC-derived motor neurons were treated with 10 μM SARM1 inhibitor DSRM-3716 and subject to axotomy (right panel). Scale bar, 50 μm. (F) Quantification of fragmentation in human motor neuron iPSCs showed dose-dependent protection by DSRM-3716. One-way ANOVA with Holm-Sidak post hoc, F(6,28) = 49.07, p < 0.0001. Mean ± SEM; control n = 8, cut n = 7, all doses n = 4; *p < 0.05, ****p < 0.0001; representative of 4 independent experiments with similar results. See also Figures S1–S5.

Figure 4.. Pharmacological SARM1 Inhibition Protects Axons after Injury

(A) Mouse DRG neurons were subject to axotomy, and 30 μM SARM1 inhibitor was added at the times indicated in the figure. Scale bar, 50 μm. (B) Quantification of axonal protection adding compound at the times indicated after injury at 10 and 30 μM shows that addition of SARM1 inhibitor after injury provided complete protection up to 3 h after injury and partial protection thereafter. Extent of protection was assessed at 16 h after axotomy. Two-way ANOVA with Tukey post hoc. For time of compound addition, F(4,30) = 15.73, p < 0.0001. Mean ± SEM; n = 4; *p < 0.05, **p < 0.01, ***p < 0.001, n.s. = not significant; representative of 2 independent experiments with similar results.

Figure 5.. Pharmacological SARM1 Inhibition Protects from Rotenone-Induced Axonal Degeneration

(A) WT mouse DRGs exposed to 25 μM rotenone showed complete axonal degeneration after 48 h (second panel). Axons in SARM1^−/− neurons (third panel) and WT neurons treated with 30 μM DSRM-3716 (right panel) did not fragment. Scale bar, 50 μm. (B) Quantification of axonal degeneration in cells treated with 25 μM rotenone + 30 μM DSRM-3716 show robust protection comparable to protection from 25 μM rotenone observed in axons from SARM1^−/−. One-way ANOVA with Holm-Sidak post hoc, F(3,16) = 51.45, p < 0.0001. Mean ± SEM; untreated n = 8, WT n = 3, SARM1^−/− n = 3, WT + DSRM-3716 n = 6; ****p < 0.0001, n.s. = not significant, representative of 6 independent experiments with similar results. (C) NfL is released over time into the culture supernatant in response to 25 μM rotenone and can be prevented by DSRM-3716. Mean ± SEM; n = 3. (D) Mouse DRG neurons were exposed to rotenone for the times indicated and examined for axonal morphology (top panels) and TMRM fluorescence (bottom panels). Yellow arrowheads indicate axonal blebs induced by rotenone, appearing 15 min after rotenone (earliest time examined), and maintained for 3 h. TMRM fluorescence was maintained during the 3-h period. Representative images of n = 4. Scale bar, 50 μm. (E) Close examination of axons treated with 25 μM rotenone for 3 h shows accumulation of βIII-tubulin, NfM, dephosphorylated NfH/SMI32, and β-APP in axonal blebs (arrows) compared with non-swollen areas of the same axon and to control axons. Using TMRM fluorescence, we found no specific association between mitochondria and axonal blebs. White arrowheads: blebs with few mitochondria; white arrows: blebs with several mitochondria; yellow arrows: mitochondria outside blebbed portions of compromised axons. Scale bar, 10 μm. See also Figures S6 and S7.

Figure 6.. Pharmacological SARM1 Inhibition Protects Axons Fated to Degenerate

Mouse DRG neurons were exposed to a 3-h pulse of 25 μM rotenone and rinsed 3×. Immediately after rotenone removal, a subset of cultures received 30 μM DSRM-3716. All cells were examined 48 h after initial exposure to rotenone. (A) Cells treated as indicated were examined for axonal morphology (top panels) and mitochondrial function by TMRM (bottom panels) as in Figure 5D. A 3-h pulse of rotenone induced complete axonal degeneration similar to continuous exposure to rotenone. In both instances, axons lost TMRM fluorescence. Treatment with 30 μM DSRM-3716 immediately after rotenone removal protected axons from fragmentation and preserved mitochondrial function assessed by TMRM fluorescence. Representative images of n = 4. Scale bar, 50 μm. (B) Quantification of axonal fragmentation after 1- or 3-h pulses of 25 μM rotenone compared with continuous exposure in the experiment described in (A). One-way ANOVA with Sidak post hoc, F(5,36) = 126.6, p < 0.0001. Untreated n = 12, treated n = 6; representative of 5 experiments with similar results. (C) Quantification of NfL released into the culture media also showed comparable levels of axonal damage by rotenone pulses compared with continuous exposure, which was protected by 30 μM DSRM-3716. SARM1 inhibitor was added at the time of treatment for continuous exposure to rotenone or immediately after rotenone removal. One-way ANOVA with Sidak post hoc, F(6,23) = 43.05, p < 0.0001. Mean ± SEM; untreated n = 12, all other groups n = 3; ****p < 0.0001, n.s. = not significant. See also Figures S6C and S7C.

Figure 7.. Pharmacological SARM1 Inhibition Allows Recovery from an Intermediate Metastable Stage of Axonal Damage

Mouse DRG neurons were exposed to a 3-h pulse of 25 μM rotenone, and immediately after rotenone removal, a subset of cultures received 30 μM SARM1 inhibitor DSRM-3716. (A) Example of an axonal bleb formed by exposure to rotenone (yellow arrow), which recovered after treatment with 30 μM DSRM-3716. Scale bar, 10 μm. (B) Kaplan-Meir survival curves in cultures from which rotenone was removed show that axonal blebs induced by rotenone fragmented over time until reaching 100% by 48 h. (C) Kaplan-Meir survival curves in cultures to which 30 μM SARM1 inhibitor was added immediately after rotenone removal show that by 48 h, 60% of axonal blebs induced by rotenone reverted over time, 30% remained, and 10% fragmented. Green, healthy axons with no blebs; yellow, intermediate stage of axonal damage consisting of axonal blebs; red, individual blebs that underwent axonal transection. See also Videos S1 and S2.