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Cited by 131Nerve injury and regenerationHereditary Neurological DisordersMitochondrial Function and Pathology

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Mitochondrial impairment activates the Wallerian pathway through depletion of NMNAT2 leading to SARM1-dependent axon degeneration

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Loreto, Andrea, Hill, Ciaran S., Hewitt, Victoria L., Orsomando, Giuseppe, Angeletti, Carlo, Gilley, Jonathan, Lucci, Cristiano, Sanchez-Martinez, Alvaro, Whitworth, Alexander J., Conforti, Laura, Dajas-Bailador, Federico, Coleman, Michael P.·2020·Neurobiology of Disease
DOI 10.1016/j.nbd.2019.104678PMC PMC7611775

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Abstract

Wallerian degeneration of physically injured axons involves a well-defined molecular pathway linking loss of axonal survival factor NMNAT2 to activation of pro-degenerative protein SARM1. Manipulating the pathway through these proteins led to the identification of non-axotomy insults causing axon degeneration by a Wallerian-like mechanism, including several involving mitochondrial impairment. Mitochondrial dysfunction is heavily implicated in Parkinson's disease, Charcot-Marie-Tooth disease, hereditary spastic paraplegia and other axonal disorders. However, whether and how mitochondrial impairment activates Wallerian degeneration has remained unclear. Here, we show that disruption of mitochondrial membrane potential leads to axonal NMNAT2 depletion in mouse sympathetic neurons, increasing the substrate-to-product ratio (NMN/NAD) of this NAD-synthesising enzyme, a metabolic fingerprint of Wallerian degeneration. The mechanism appears to involve both impaired NMNAT2 synthesis and reduced axonal transport. Expression of WLDS and Sarm1 deletion both protect axons after mitochondrial uncoupling. Blocking the pathway also confers neuroprotection and increases the lifespan of flies with Pink1 loss-of-function mutation, which causes severe mitochondrial defects. These data indicate that mitochondrial impairment replicates all the major steps of Wallerian degeneration, placing it upstream of NMNAT2 loss, with the potential to contribute to axon pathology in mitochondrial disorders.

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Figures

Fig. 1 Regulators of the Wallerian pathway rescue axon degeneration caused by mitochondrial depolarisation. (A) ATP levels in wild-type SCG dissociated cultures after treatment with CCCP. Data are normalised to DMSO control at each time point (Mean ± SEM; n = 4; twoway ANOVA followed by Sidak post-hoc test; ****, p < 0.0001). (B) Representative phase contrast images of neurites from wild-type SCG explant cultures treated with increasing concentrations of CCCP. (C) Quantification of the degeneration index in experiments described in (B) from 3 fields per sample in 2 independent experiments (Mean ± SEM; n = 2). (D) Representative phase contrast images of neurites from wild-type and Sarm1 −/− SCG explant cultures at the indicated time points after CCCP treatment. (E) Quantification of the degeneration index in experiments described in (D) from 3 fields per sample in 4 independent experiments (Mean ± SEM; n = 4; two-way ANOVA followed by Tukey post-hoc test; ****, p < 0.0001. Statistical significance shown relative to +50 μM CCCP). (F) Representative phase contrast images of neurites from wild-type and Wlds SCG explant cultures at the indicated time points after CCCP treatment. (G) Quantification of the degeneration index in experiments described in (F) from 3 fields per sample in 4 independent experiments (Mean ± SEM; n = 4; two-way ANOVA followed by Tukey post-hoc test; ****, p < 0.0001. Statistical significance shown relative to +50 μM CCCP).
Fig. 1 Regulators of the Wallerian pathway rescue axon degeneration caused by mitochondrial depolarisation. (A) ATP levels in wild-type SCG dissociated cultures after treatment with CCCP. Data are normalised to DMSO control at each time point (Mean ± SEM; n = 4; twoway ANOVA followed by Sidak post-hoc test; ****, p < 0.0001). (B) Representative phase contrast images of neurites from wild-type SCG explant cultures treated with increasing concentrations of CCCP. (C) Quantification of the degeneration index in experiments described in (B) from 3 fields per sample in 2 independent experiments (Mean ± SEM; n = 2). (D) Representative phase contrast images of neurites from wild-type and Sarm1 −/− SCG explant cultures at the indicated time points after CCCP treatment. (E) Quantification of the degeneration index in experiments described in (D) from 3 fields per sample in 4 independent experiments (Mean ± SEM; n = 4; two-way ANOVA followed by Tukey post-hoc test; ****, p < 0.0001. Statistical significance shown relative to +50 μM CCCP). (F) Representative phase contrast images of neurites from wild-type and Wlds SCG explant cultures at the indicated time points after CCCP treatment. (G) Quantification of the degeneration index in experiments described in (F) from 3 fields per sample in 4 independent experiments (Mean ± SEM; n = 4; two-way ANOVA followed by Tukey post-hoc test; ****, p < 0.0001. Statistical significance shown relative to +50 μM CCCP).CC BY · PubMed Central
Fig. 2 Mitochondrial depolarisation leads to depletion of axonal NMNAT2. (A) Representative immunoblots of wild-type SCG neurite extracts probed for NMNAT2 and β-actin (loading control) at the indicated time points after CCCP treatment. (B) Quantification of normalised NMNAT2 levels (to β-actin) is shown, with data presented relative to DMSO control (Mean ± SEM; n = 3; one-way ANOVA followed by Bonferroni post-hoc test; ***, p < 0.001; **, p < 0.01). (C) Representative phase contrast images showing morphologically intact neurites at the time points used in (A). (D) Representative phase contrast images of neurites from wild-type-Nmnat2+/+, Nmnat2+/gtE (~60% expression), Nmnat2gtBay/gtE (~30% expression) SCG explant cultures at the indicated time points after CCCP treatment. (E) Quantification of the degeneration index in experiments described in (D) from 3 fields per sample in 4 independent experiments (Mean ± SEM; n = 4; two-way ANOVA followed by Tukey post-hoc test; ****, p < 0.0001; ***, p < 0.001; **, p < 0.01. Statistical significance shown relative to Nmnat2+/+ +50 μM CCCP).
Fig. 2 Mitochondrial depolarisation leads to depletion of axonal NMNAT2. (A) Representative immunoblots of wild-type SCG neurite extracts probed for NMNAT2 and β-actin (loading control) at the indicated time points after CCCP treatment. (B) Quantification of normalised NMNAT2 levels (to β-actin) is shown, with data presented relative to DMSO control (Mean ± SEM; n = 3; one-way ANOVA followed by Bonferroni post-hoc test; ***, p < 0.001; **, p < 0.01). (C) Representative phase contrast images showing morphologically intact neurites at the time points used in (A). (D) Representative phase contrast images of neurites from wild-type-Nmnat2+/+, Nmnat2+/gtE (~60% expression), Nmnat2gtBay/gtE (~30% expression) SCG explant cultures at the indicated time points after CCCP treatment. (E) Quantification of the degeneration index in experiments described in (D) from 3 fields per sample in 4 independent experiments (Mean ± SEM; n = 4; two-way ANOVA followed by Tukey post-hoc test; ****, p < 0.0001; ***, p < 0.001; **, p < 0.01. Statistical significance shown relative to Nmnat2+/+ +50 μM CCCP).CC BY · PubMed Central
Fig. 3 NMNAT2 depletion reflects impairment of both axonal transport and synthesis. (A) Representative immunoblot of wild-type SCG cell bodies/ganglia extracts probed for NMNAT2 and β-actin (loading control) at the indicated time points after CCCP treatment. (B) Quantification of normalised NMNAT2 levels (to β-actin) is shown, with data presented relative to DMSO control (Mean ± SEM; n = 4; oneway ANOVA followed by Bonferroni post-hoc test; ****, p < 0.0001; **, p < 0.01; *, p < 0.05). (C) Representative kymographs of wild-type SCG dissociated cultures expressing NMNAT2-EGFP. (D) Quantification of the % of motile NMNAT2 at the indicated time points after CCCP treatment from 3 neurites per condition in 4 independent experiments (Mean ± SEM; n = 4; two-way ANOVA followed by Sidak post-hoc test; *, p < 0.05. NS, non-significant). (E) Quantification of the % of motile bidirectional, anterograde and retrograde NMNAT2 at the indicated time points after CCCP treatment from 3 neurites per condition in 4 independent experiments (Mean ± SEM; n = 4; two-way ANOVA followed by Sidak post-hoc test; NS, non-significant).
Fig. 3 NMNAT2 depletion reflects impairment of both axonal transport and synthesis. (A) Representative immunoblot of wild-type SCG cell bodies/ganglia extracts probed for NMNAT2 and β-actin (loading control) at the indicated time points after CCCP treatment. (B) Quantification of normalised NMNAT2 levels (to β-actin) is shown, with data presented relative to DMSO control (Mean ± SEM; n = 4; oneway ANOVA followed by Bonferroni post-hoc test; ****, p < 0.0001; **, p < 0.01; *, p < 0.05). (C) Representative kymographs of wild-type SCG dissociated cultures expressing NMNAT2-EGFP. (D) Quantification of the % of motile NMNAT2 at the indicated time points after CCCP treatment from 3 neurites per condition in 4 independent experiments (Mean ± SEM; n = 4; two-way ANOVA followed by Sidak post-hoc test; *, p < 0.05. NS, non-significant). (E) Quantification of the % of motile bidirectional, anterograde and retrograde NMNAT2 at the indicated time points after CCCP treatment from 3 neurites per condition in 4 independent experiments (Mean ± SEM; n = 4; two-way ANOVA followed by Sidak post-hoc test; NS, non-significant).CC BY · PubMed Central
Fig. 4 Changes in the NMN/NAD ratio following CCCP administration. (A) Schematic representation of NAD salvage pathway from nicotinamide and points at which FK866 and bacterial NMN deamidase will act (NAM, nicotinamide; NaMN, nicotinic acid mononucleotide; NMN, nicotinamide mononucleotide; NAD, nicotinamide adenine dinucleotide; NAMPT, nicotinamide phosphoribosyltransferase; NMNAT, nicotinamide mononucleotide adenylyltransferase). (B,C) NMN and NAD levels and NMN/NAD ratios in neurite (B) and cell body/ganglia (C) fractions from Sarm1 −/− SCG explant cultures at the indicated time points after CCCP treatment (Mean ± SEM; n =5; oneway ANOVA followed by Bonferroni post-hoc test; ****, p < 0.0001; **, p < 0.01; *, p < 0.05. Statistical significance shown relative to 12 h DMSO).
Fig. 4 Changes in the NMN/NAD ratio following CCCP administration. (A) Schematic representation of NAD salvage pathway from nicotinamide and points at which FK866 and bacterial NMN deamidase will act (NAM, nicotinamide; NaMN, nicotinic acid mononucleotide; NMN, nicotinamide mononucleotide; NAD, nicotinamide adenine dinucleotide; NAMPT, nicotinamide phosphoribosyltransferase; NMNAT, nicotinamide mononucleotide adenylyltransferase). (B,C) NMN and NAD levels and NMN/NAD ratios in neurite (B) and cell body/ganglia (C) fractions from Sarm1 −/− SCG explant cultures at the indicated time points after CCCP treatment (Mean ± SEM; n =5; oneway ANOVA followed by Bonferroni post-hoc test; ****, p < 0.0001; **, p < 0.01; *, p < 0.05. Statistical significance shown relative to 12 h DMSO).CC BY · PubMed Central
Fig. 5 Inhibition of NMN synthesis protects neurites against CCCP toxicity. (A,B) Representative immunoblot of wild-type SCG neurite extracts probed for NAMPT and β-actin (loading control) at the indicated time points after CCCP treatment. Quantification of normalised NAMPT levels (to β-actin) is shown, with data presented relative to DMSO control (Mean ± SEM; n = 4; one-way ANOVA followed by Bonferroni post-hoc test; NS, non-significant). (C) Representative phase contrast images of neurites from wild-type SCG explant cultures at the indicated time points after CCCP, FK866 and NMN treatment. Where indicated, FK866 and NMN were added at the same time of CCCP. (D) Quantification of the degeneration index in experiments described in (C) from 3 fields per sample in 4 independent experiments (Mean ± SEM; n = 4; two-way ANOVA followed by Tukey post-hoc test; ****, p < 0.0001. Statistical significance shown relative to +50 μM CCCP). (E) Representative phase contrast images of neurites from wild-type SCG explant cultures at the indicated time points after CCCP and NMN treatment. (F) Quantification of the degeneration index in experiments described in (E) from 3 fields per sample in 4 independent experiments (Mean ± SEM; n = 4; two-way ANOVA followed by Tukey post-hoc test).
Fig. 5 Inhibition of NMN synthesis protects neurites against CCCP toxicity. (A,B) Representative immunoblot of wild-type SCG neurite extracts probed for NAMPT and β-actin (loading control) at the indicated time points after CCCP treatment. Quantification of normalised NAMPT levels (to β-actin) is shown, with data presented relative to DMSO control (Mean ± SEM; n = 4; one-way ANOVA followed by Bonferroni post-hoc test; NS, non-significant). (C) Representative phase contrast images of neurites from wild-type SCG explant cultures at the indicated time points after CCCP, FK866 and NMN treatment. Where indicated, FK866 and NMN were added at the same time of CCCP. (D) Quantification of the degeneration index in experiments described in (C) from 3 fields per sample in 4 independent experiments (Mean ± SEM; n = 4; two-way ANOVA followed by Tukey post-hoc test; ****, p < 0.0001. Statistical significance shown relative to +50 μM CCCP). (E) Representative phase contrast images of neurites from wild-type SCG explant cultures at the indicated time points after CCCP and NMN treatment. (F) Quantification of the degeneration index in experiments described in (E) from 3 fields per sample in 4 independent experiments (Mean ± SEM; n = 4; two-way ANOVA followed by Tukey post-hoc test).CC BY · PubMed Central
Fig. 6 Highwire deficiency rescues loss of a subpopulation of dopaminergic (PPL1) neurons in Pink1 Drosophila mutants. (A) Schematic image of a Drosophila brain with the PPL1 cluster of dopaminergic neurons shown in green (‘Created with BioRender’). (B) Representative images of adult Drosophila (20 days old) brains stained with anti-TH antibody. The PPL1 cluster of dopaminergic neurons is shown. (C) Quantification of the number of dopaminergic neurons per PPL1 cluster (Mean ± SEM; n = 16–25; one-way ANOVA followed by Tukey post-hoc test; **, p < 0.01). (D) Lifespan curves of wildtype, HiwΔN, Pink1B9, HiwΔN Pink1B9 flies (n > 130 flies per condition; log-rank (Mantel-Cox) test. ****, p < 0.0001). (E,F) Analysis of climbing and flying ability of 7 days old flies of the indicated genotypes (Mean ± SEM; n = 3 climbing, n = 9 flying; one-way ANOVA followed by Tukey post-hoc test; NS, non-significant). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6 Highwire deficiency rescues loss of a subpopulation of dopaminergic (PPL1) neurons in Pink1 Drosophila mutants. (A) Schematic image of a Drosophila brain with the PPL1 cluster of dopaminergic neurons shown in green (‘Created with BioRender’). (B) Representative images of adult Drosophila (20 days old) brains stained with anti-TH antibody. The PPL1 cluster of dopaminergic neurons is shown. (C) Quantification of the number of dopaminergic neurons per PPL1 cluster (Mean ± SEM; n = 16–25; one-way ANOVA followed by Tukey post-hoc test; **, p < 0.01). (D) Lifespan curves of wildtype, HiwΔN, Pink1B9, HiwΔN Pink1B9 flies (n > 130 flies per condition; log-rank (Mantel-Cox) test. ****, p < 0.0001). (E,F) Analysis of climbing and flying ability of 7 days old flies of the indicated genotypes (Mean ± SEM; n = 3 climbing, n = 9 flying; one-way ANOVA followed by Tukey post-hoc test; NS, non-significant). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)CC BY · PubMed Central
Fig. 7 Mitochondrial dysfunction as an upstream signal activating the Wallerian pathway. Schematic representation of the Wallerian pathway (‘Created with BioRender’). Injury and mitochondrial impairment act as two independent insults resulting in the activation of the Wallerian pathway.
Fig. 7 Mitochondrial dysfunction as an upstream signal activating the Wallerian pathway. Schematic representation of the Wallerian pathway (‘Created with BioRender’). Injury and mitochondrial impairment act as two independent insults resulting in the activation of the Wallerian pathway.CC BY · PubMed Central

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Authors
Loreto, A., Hill, C. S., Hewitt, V. L., Orsomando, G., Angeletti, C., Gilley, J., Lucci, C., Sanchez-Martinez, A., Whitworth, A. J., Conforti, L., Dajas-Bailador, F., & Coleman, M. P.
Affiliations
  • University of Cambridge (GB)
  • Marche Polytechnic University (IT)
  • University of Nottingham (GB)
Corresponding author
  • Loreto, Andrea
  • Coleman, Michael P.
ORCID
Venue
Neurobiology of Disease
Publisher
Elsevier BV
Published
2020-02
Volume
134
Pages
104678
ISSN
0969-9961 (ISSN-L)
DOI
10.1016/j.nbd.2019.104678
PMC
PMC7611775
OpenAlex
W2984519822
Cited by
131
References
51
Citations by year
2026: 12 · 2025: 13 · 2024: 23 · 2023: 16 · 2022: 22 · 2021: 30 · 2020: 13
FWCI
7.84
Type
journal article
Language
en
Open access
Licence
Locations
  • Neurobiology of Disease (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion, other-oa)
  • PubMed Central (acceptedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • UCL Discovery (University College London) (submittedVersion)
  • Repository@Nottingham (University of Nottingham) (submittedVersion, cc-by)
  • Università Politecnica delle Marche (Università Politecnica delle Marche) (submittedVersion, cc-by-nc-nd)
  • Università Politecnica delle Marche (Università Politecnica delle Marche) (submittedVersion)
  • Apollo (University of Cambridge) (submittedVersion)
  • Apollo
Funders
  • Medical Research Council
  • Parkinson's UK
  • Wellcome Trust
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Nerve injury and regeneration
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Hereditary Neurological Disorders
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
Keywords
Wallerian degeneration, mitochondrial dysfunction, NMNAT2, SARM1, axonal degeneration, mitochondrial membrane potential, mitochondrial uncoupling, axonal transport, sympathetic neurons, NAD biosynthesis, PINK1, mitochondrial disease, neuroprotection
MeSH terms
Animals, Axons, Cytoskeletal Proteins, Drosophila, Male, Mice, Inbred C57BL, Mitochondria, Nicotinamide-Nucleotide Adenylyltransferase, Wallerian Degeneration, Armadillo Domain Proteins, Membrane Potential, Mitochondrial

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