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DLK Activation Synergizes with Mitochondrial Dysfunction to Downregulate Axon Survival Factors and Promote SARM1-Dependent Axon Degeneration - PubMed

DLK Activation Synergizes with Mitochondrial Dysfunction to Downregulate Axon Survival Factors and Promote SARM1-Dependent Axon Degeneration

Daniel W Summers et al. Mol Neurobiol. 2020 Feb.

Abstract

Axon degeneration is a prominent component of many neurological disorders. Identifying cellular pathways that contribute to axon vulnerability may identify new therapeutic strategies for maintenance of neural circuits. Dual leucine zipper kinase (DLK) is an axonal stress response MAP3K that is chronically activated in several neurodegenerative diseases. Activated DLK transmits an axon injury signal to the neuronal cell body to provoke transcriptional adaptations. However, the consequence of enhanced DLK signaling to axon vulnerability is unknown. We find that stimulating DLK activity predisposes axons to SARM1-dependent degeneration. Activating DLK reduces levels of the axon survival factors NMNAT2 and SCG10, accelerating their loss from severed axons. Moreover, mitochondrial dysfunction independently decreases the levels of NMNAT2 and SCG10 in axons, and in conjunction with DLK activation, leads to a dramatic loss of axonal NMNAT2 and SCG10 and evokes spontaneous axon degeneration. Hence, enhanced DLK activity reduces axon survival factor abundance and renders axons more susceptible to trauma and metabolic insult.

Keywords: Axon; DLK; Mitochondria; NMNAT2; SARM1; STMN2.

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Conflict of interest statement

Competing Interests: A.D and J.M are co-founders of Disarm Therapeutics and members of the Scientific Advisory Board. The authors have no additional competing financial interests.

Figures

Figure 1.
Figure 1.. Acute DLK stimulation accelerates injury-induced axon degeneration.

(A) Phase contrast images of neurons treated as indicated. Scale bar = 5μm. (B) Quantification of axon degeneration at indicated time points after injury (N=4; p<0.0001, 2-way ANOVA for repeated measures). Forskolin pretreatment (30μM) accelerates axon degeneration after axotomy. (C) Neurons pre-treated with the cell permeable cAMP analog (8-cpt-cAMP; 250μM) show accelerated axon degeneration after axotomy (N=4, p=0.003, 2-way ANOVA for repeated measures). (D) Forskolin application 5 min after axotomy accelerates axon degeneration (N=3, p=0.0012, 2-way ANOVA for repeated measures). (E) Cas9-expressing neurons were transduced with sgRNAs to DLK and LZK or negative control (sgCtrl). Loss of DLK/LZK suppresses forskolin-enhanced axon degeneration (N=3, p<0.0001, 2-way ANOVA for repeated measures). (F) Axon degeneration is suppressed in SARM1−/− neurons pre-treated with forskolin (N=3, p<0.0001, 2-way ANOVA for repeated measures). Error bars represents +/-1 SEM.

Figure 2.
Figure 2.. Forskolin treatment promotes loss of NMNAT2 from DRG sensory axons.

(A,B) Forskolin treatment accelerates loss of NMNAT2 from severed axons. Forskolin (30 μM) was applied to DRG cultures five min prior to axon transection. Western blots are from axon-only extracts collected at the indicated time post axotomy and quantification shown in (B) (N=4 p=0.0093; unpaired t-test). Forskolin treatment stimulates a reduction in axonal steady-state levels of (C) NMNAT2 and (D) SCG10 levels with quantification of protein loss shown on the right (N=3). (E,F) DLK/LZK were inactivated in DRG neurons by CRISPR (sgDLK/LZK). DRG cultures were treated with forskolin for 2 hr and NMNAT2 levels detected from axon-only extracts. In (E), a short and long exposure (exp.) are shown from a representative western blot. (F) Quantification of endogenous, axonal NMNAT2 in the presence of control sgRNA (left panel) or sgDLK/LZK (right panel) after 2 hour treatment with forskolin (N=3; p=0.009 unpaired T-test, n.s. not significant). Since steady state levels of NMNAT2 are elevated in the absence of DLK/LZK the levels NMNAT2 are normalized internally in the left and right panels. Error bars represent +/- 1 SEM.

Figure 3.
Figure 3.. Activating DLK sensitizes axons to degeneration in response to mitochondrial dysfunction.

(A) Experimental timeline and phase contrast images of DRGs neurons after the indicated treatment. DRG neurons were treated with oligomycin (250nM) for 18 hr, at which point Forskolin (30μM) was added to the media and then distal axons were imaged 8hr later. (A) Phase contrast images and (B) quantification of axon degeneration from DRG neurons pretreated with indicated doses of oligomycin followed by treatment with vehicle or forsklin (N=6. *p=0.0207, ***p=0.0007, and **p=0.0087, unpaired T-test within dosage).(C) Forskolin and oligomycin (500 nM) were applied to DRGs simultaneously and axon degeneration was monitored in distal axon segments (N=4, 8hr, *p=0.0434, 12hr, *p=0.0149; 2-way ANOVA repeated measures with post-hoc Bonferroni correction). (D) Neurons were treated with indicated concentration of CCCP. Axon degeneration was measured 8 hr post forskolin treatment (N=5, **p=0.0011 and p=0.003 for unpaired T-tests within doses 12.5μM and 50μM respectively). (E) Axon degeneration was measured in neurons treated with forskolin and oligomycin and expressing Cas9 with sgRNAs to DLK and LZK or scrambled sgRNA control (N=4 p=0.0025 unpaired T-test). (F) WT or SARM1−/− neurons were treated with vehicle, forskolin, oligomycin, or a combination of forksolin and oligomycin. Axon degeneration was quantified 8 hr later (N=3 p=0.0011 unpaired T-test). Scale Bar = 5μm. Error bars represent +/-1 SEM. (G) ATP is depleted from axons of DRG neurons 8 hr after treatment with koningic acid (KA) (5μM) or oligomycin (500 nM) (N=4, ****p<0.0001 unpaired T-test). (H) Neurons were pretreated with KA for 18 hr then forskolin or oligomycin was added. Treatment with KA and forskolin does not elicit degeneration. However, combined treatment with KA and oligomycin induces axon degeneration. Axon degeneration was measured 8 hr later (N=3, ***p=0.0003; unpaired T-test).

Figure 4.
Figure 4.. Local activation of DLK and mitochondrial dysfunction stimulates axon degeneration.

Assessment of cell death in DRG neurons in response to oligomycin and forskolin treatment. DRG neurons were first treated with oligomycin (500nM) or vehicle for 18 hr. Then, forskolin (30μM) was applied for eight hours and neuronal cell death was measured using (A) uptake of ethidium homodimer to assess membrane permeability or (B) generation of cleaved caspase 3. As a positive control, neurons were deprived of nerve growth factor for a 24-hour period to induce cell death (Membrane permeability: N=3 p<0.0001; Cleaved Caspase 3: N=3 ***p=0.001; Single Factor ANOVA with posthoc Turkey T-test). (C,D) DRG neurons were seeded in microfluidic chambers. Using the same conditions described above, oligomycin and forskolin were applied to chambers containing axons. DRG neurons were fixed and axon integrity assessed after immunostaining for TUJ1. (E) Quantification of degeneration was performed on TUJ1-stained axons to assess fragmentation in axon compartment (N=4 **p=0.0016 Single Factor ANOVA). Scale bar = 5μm. Error bars represent +/-1 SEM.

Figure 5.
Figure 5.. Oligomycin and forskolin promote loss of NMNAT2 from axons.

(A) Neurons were treated with forskolin, oligomycin, or a combination of forksolin and oligomycin for 2 hr and axonal NMNAT2 levels were assessed. Shown below is quantification of NMNAT2 levels as a ratio of vehicle control (N=4, ****p<0.0001, *p=.0147, 0.103 respectively with single factor ANOVA with Bonferroni post hoc T-test correction). (B) Neurons treated as in (A) were also assessed for axonal SCG10 levels with quantification below (N=3, ****p<0.0001, **p= 0.0076, and *p=0.0158 from single factor ANOVA with Bonferroni post hoc T-test correction). (C) Oligomycin reduces axonal NMNAT2-myc protein expressed via lentivirus from ubiquitin promoter. Neurons were treated with vehicle or oligomycin (500nM) for two hours. Quantification of NMNAT2-myc levels is shown to the right (N=4 *p=0.0144 unpaired t-test). (D, E) Axonal NMNAT2 protein is reduced by oligomycin in the presence of Cas9 and sgRNAs to DLK/ LZK (N=5, ***p=0.0011 and 0.0064 for unpaired T-tests within sgCtrl and sgDLK/LZK respectively). Error bars represent +/-1 SEM.

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References

    1. Gerdts J, Summers DW, Milbrandt J, DiAntonio A (2016) Axon Self-Destruction: New Links among SARM1, MAPKs, and NAD+ Metabolism. Neuron 89:449–460. 10.1016/j.neuron.2015.12.023 - DOI - PMC - PubMed
    1. DiAntonio A (2019) Axon degeneration: mechanistic insights lead to therapeutic opportunities for the prevention and treatment of peripheral neuropathy. Pain 160 Suppl 1 :S17–S22. 10.1097/j.pain.0000000000001528 - DOI - PMC - PubMed
    1. Le Pichon CE, Meilandt WJ, Dominguez S, et al. (2017) Loss of dual leucine zipper kinase signaling is protective in animal models of neurodegenerative disease. Sci Transl Med 9:eaag0394 10.1126/scitranslmed.aag0394 - DOI - PubMed
    1. Asghari Adib E, Smithson LJ, Collins CA (2018) An axonal stress response pathway: degenerative and regenerative signaling by DLK. Curr. Opin. Neurobiol 53:110–119. 10.1016/j.conb2018.07.002 - DOI - PMC - PubMed
    1. Farley MM, Watkins TA (2018) Intrinsic Neuronal Stress Response Pathways in Injury and Disease. Annu Rev Pathol Mech Dis. 10.1146/annurev-pathol-012414-040354 - DOI - PubMed

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