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jcb Home » 2016 Archive » 4 July » 214 (1): 103
Article

Facilitation of axon regeneration by enhancing mitochondrial transport and rescuing energy deficits

View ORCID ProfileBing Zhou, Panpan Yu, Mei-Yao Lin, View ORCID ProfileTao Sun, Yanmin Chen, Zu-Hang Sheng
Bing Zhou
Synaptic Functions Section, The Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892
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  • ORCID record for Bing Zhou
Panpan Yu
Guangdong–Hong Kong–Macau Institute of CNS Regeneration, Ministry of Education Joint International Research Laboratory of CNS Regeneration, Jinan University, Guangzhou 510632, China
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Mei-Yao Lin
Synaptic Functions Section, The Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892
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Tao Sun
Synaptic Functions Section, The Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892
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Yanmin Chen
Synaptic Functions Section, The Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892
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Zu-Hang Sheng
Synaptic Functions Section, The Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892
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DOI: 10.1083/jcb.201605101 | Published June 7, 2016
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  1. Figure 1.
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    Figure 1.

    snph KO cortical neurons display enhanced axon regrowth capacity. (A and B) A microfluidic chamber allows physical and fluidic separation of axons from cell bodies and dendrites. Cortical neurons were seeded in the soma chamber where cell bodies and dendrites are restricted to the soma chamber, whereas axons grow into the axon terminal chamber through the microgroove channels (MC; 450 µm in length). The device (a) allows quick and definitive detection of axons, (b) determines axon directionality, (c) reduces background signals from glial and neuronal cell bodies and dendrites, (d) permits axotomy along the edge of microgrooves by vacuum aspiration, and (e) measures axon regrowth. Neurons were stained with βIII-tubulin (A) or infected with lenti-YFP (B); axon removal in the terminal chamber by vacuum aspiration was confirmed by imaging live neurons expressing YFP before and after axotomy (B). (C–E) Images (C and D) and quantitative analysis (E) showing enhanced axon regrowth after axotomy in snph KO neurons. WT (C) and snph KO (D) cortical neurons were axotomized at DIV4 and allowed to regrow for 3 d. Axons were immunostained with βIII-tubulin at DIV7. Note that the normalized area of regenerating axons was significantly increased in snph KO neurons relative to controls (P < 0.001). (F and G) Images (F) and quantitative analysis (G) of WT and snph KO cortical neurons showing early phases of axon regrowth at 7 and 28 h after axotomy. (H and I) Representative images (H) and quantitative analysis (I) showing the formation of new growth cones after axotomy. Axonal terminals were costained with βIII-tubulin and Alexa Fluor 543 phalloidin 14 h after axotomy. Note that deleting snph facilitates the formation of growth cones (69.11 ± 2.90%, P = 0.0002) compared with WT (44.46 ± 1.92%) from injured axon tips. Imaging data were pooled from a total number of microgroove channels indicated within bars (E and G) taken from three pairs of littermates or a total number of axonal terminal chambers indicated within bars (I) and expressed as mean ± SE (Student’s t test).

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    Figure 2.

    SNPH-mediated mitochondrial anchoring contributes to reduced regrowth capacity in mature neurons. (A–C) Representative microfluidic images (A and B) and quantitative analysis (C) showing reduced capacity of axonal regrowth in mature cortical neurons after axotomy. Neurons were axotomized at DIV4 (A) or DIV12 (B) and imaged by staining of βIII-tubulin 3 (A) or 6 d (B) after injury. Although young neurons at DIV7 maintain some level of regrowth capacity, mature neurons at DIV18 show failed regeneration (P < 0.001, Mann–Whitney test). (D and E) Representative immunoblots (D) and quantitative analysis (E) showing progressive increase in SNPH expression with neuron maturation. Cortical neurons isolated from E18 mouse brains were cultured for 3, 7, 9, 12, 15, 18, and 22 d. Equal amounts (20 µg) of cell lysates were loaded and sequentially immunoblotted with various antibodies after stripping between applications of each antibody. Brain lysates from E18 WT, adult WT, and adult snph KO mice were used as controls. The intensity of SNPH bands were quantified from three repeats, calibrated with TOM20 levels, and then normalized to SNPH expression at DIV7. (F–H) Kymographs (F) and quantitative analysis (G and H) showing progressive decline of axonal mitochondrial motility with neuron maturation. Cortical neurons were transfected with DsRed-Mito or Rab7-YFP. Time-lapse images, obtained at DIV7, 9, 12, or 18, were recorded for 100 frames with 5-s intervals. In kymographs, vertical lines represent stationary organelles; oblique lines or curves to the right indicate anterograde transport toward distal terminals. Note that axonal mitochondria have progressively reduced motility, whereas late endosomes show no significant change in their motility in the same axons during maturation. Bars, 10 µm. Data were analyzed from the total number of chambers indicated within bars (C) or the total number of neurons indicated within bars (G and H) and expressed as mean ± SE and by one-way ANOVA test.

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    Figure 3.

    Mature neurons regain regrowth capacity by enhancing mitochondrial transport. (A and B) Kymographs (A) and quantitative analysis (B) showing mitochondrial motility along microgrooves in microfluidic chambers. Time-lapse imaging was recorded in cortical neurons at DIV12 for a total of 100 frames with 5-s intervals. In kymographs, vertical lines represent stationary organelles; oblique lines or curves to the right indicate anterograde transport toward distal terminals. Note that the relative motility in control neurons expressing HA is significantly higher than the motility in neurons overexpressing HA-SNPH, but lower than the motility in neurons expressing HA-Miro1. (C and D) Representative microfluidic images (C) and quantitative analysis (D) showing regrowth capacity in mature cortical neurons. Neurons infected with lentivirus encoding SNPH, SNPH-dMTB, or Miro1 were grown on microfluidic chambers for 12 d before axotomy. Axon regeneration was evaluated 6 d after axotomy (DIV18). Note that abolishing mitochondrial transport by expressing SNPH shows failed axon regrowth, whereas enhancing mitochondrial transport by expressing Miro1 robustly increases axon regrowth capacity. (E) Partial recovery of regrowth capacity in the SNPH-expressing neurons by ATP application. The electroporated neurons were immediately plated on a microfluidic chamber with medium containing 200 µM ATP. (F) Recovery of regrowth capacity 14 h after axotomy in snph KO neurons is largely abolished by blocking mitochondrial ATP generation with 2 µM oligomycin (Oligo). The axonal chambers were briefly treated with 2 µM oligomycin for 4 h after axotomy. (G and H) Representative images (G) and quantitative analysis (H) showing enhanced axonal regrowth in snph KO adult DRG neurons. DRG neurons isolated from adult (P60) WT or snph KO mice were immunostained with βIII-tubulin at DIV1. Axon regrowth was quantified by Sholl analysis. The snph-deficient adult DRG neurons display increased axon branching as indicated by the total number of axonal intersections. Mitochondrial motility data were analyzed from the total number of microgrooves (B); axonal regrowth data were analyzed in terminal chambers where new axons grow from a total number of microgrooves (E and F), total number of terminal chambers (D), or total number of DRG neurons (H) indicated within bars or in parentheses and expressed as mean ± SE and by one-way ANOVA test (B and D), Student’s t test (F and H), or Mann–Whitney U test (E). Bars: (A) 20 µm; (C and G) 100 µm.

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    Figure 4.

    Mitochondrial transport impacts terminal mitochondrial recruitment and growth cone size. (A and B) Representative images (A) and quantitative analysis (B) showing correlation of mitochondrial density in the axonal terminals and growth cone size of adult DRG neurons after dissociation axotomy. Adult DRG neurons isolated from P60 mice were cotransfected with DsRed-Mito, GFP, and control vector, SNPH, SNPH-dMTB, or Mrio1, followed by immunostaining with Tau at DIV2. Note that expressing SNPH, but not its loss-of-function mutant SNPH-dMTB, blocks the delivery of mitochondria into growth tips and reduces average size of growth cones, whereas expressing Miro1 robustly increases terminal mitochondrial density and size of growth cones. (C and D) Images (C) and quantitative analysis (D) of distal mitochondrial distribution and growth cone size in cortical neurons coexpressing DsRed-Mito with GFP or together with SNPH, SNPH-dMTB, or Miro1. Neurons at DIV5 were immunostained with Tau. Note that both mitochondrial density in terminals and the average size of growth cones are decreased in neurons expressing SNPH (P < 0.01 and P < 0.001, respectively), but increased in neurons expressing Miro1 (P < 0.05) relative to control neurons. Arrows indicate the most distal mitochondrion in the SNPH-expressing axons (C). Data were analyzed from the total number of axons indicated within bars from more than three experiments and expressed as mean ± SE and by one-way ANOVA test. Bars, 10 µm.

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    Figure 5.

    Axotomy depolarizes mitochondria in the vicinity of injured sites. (A) Representative images showing axonal mitochondria in adult DRG neurons before and after laser-based axotomy. Adult DRG neurons isolated from P60 mice were cotransfected with DsRed-Mito and GFP. Note that mitochondria at the axotomized site were immediately lost during axotomy (white arrows). (B and C) Kymographs showing axonal mitochondria labeled by DsRed-Mito (B) or colabeled by GFP-Mito and TMRE (C) before and after axotomy. The white arrows indicate laser-scanning sites (injured site), and the black arrows in the y axis show the laser execution time. Time-lapse images were captured to show that mitochondria in the vicinity suddenly shrunk (B) and lost the Δψm (TMRE staining; C). The images were first recorded at 5-s intervals for a total of 50 frames; the consecutive post-axotomy recording was collected at 5-s intervals for a total of 50 frames. (D and E) Representative images showing axotomy-induced depolarization of axonal mitochondria. Neurons were infected with pLenti-GFP (D) or GFP-Mito (E), and axons in the terminal chambers were loaded with 25 nM TMRE dye at DIV12, followed by laser-based axotomy and time-lapse imaging. Note that in neurons expressing GFP (D), axons were quickly broken up upon axotomy (white dashed lines), and a majority of mitochondria in the vicinity suddenly lost their TMRE staining (bottom right). In neurons expressing GFP-Mito (E), axotomy triggered a sudden loss of mitochondria staining by TMRE near the axotomy site (white dashed lines), whereas those depolarized mitochondria maintained GFP-Mito signals. MC, microgroove channels. (F and G) Representative images (F) and ratiometric kymograph (G) showing axotomy-induced ATP depletion. Cultured adult DRG neurons from 2-mo-old mice were transfected with red-shifted ATP probe GO-ATeam2, in which GFP and OFP were used as a FRET pair to monitor intracellular ATP levels with an affinity Kd of 2.3 mM at 37°C. Laser-based axotomy was applied (white bars in F, white arrow in G) along a distal axon. The green color is the cp173-mEGFP channel, and the red color is the OFP channel (mKOk). The ratiometric kymograph was generated by time-lapse imaging of a total of 100 frames with 5-s intervals. Axotomy was applied at the 51th frame (G). Note that axotomy triggers acute ATP depletion at millimolar levels in the vicinity of the injured site. Bars: (A–C) 10 µm; (D–G) 20 µm.

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    Figure 6.

    Enhanced mitochondrial transport recovers mitochondrial integrity and rescues energy deficits in injured axons. (A and B) Kymographs (A) and quantitative analysis (B) showing axonal mitochondrial flux of WT and snph KO cortical neurons along microgrooves. Neurons were infected with pLenti-GFP-Mito. Time-lapse imaging was recorded for 30 frames with a 5-s interval for a total of 2.5 min. In kymographs, vertical lines represent stationary organelles; oblique lines or curves to the right indicate anterograde transport toward terminals. The mitochondrial flux in axonal bundles was measured by the total events of bidirectional transport through microgrooves. The images were acquired by 40× lens. Note that deleting snph significantly increases axonal mitochondrial flux before and after axotomy when compared with WT neurons. Data were analyzed from the total number of microgrooves indicated within bars from more than three independent experiments and expressed as mean ± SE and by Student’s t test. (C and D) Images (C) and quantitative analysis (D) showing the recovery of TMRE staining on axonal mitochondria in the microgrooves adjacent to the axotomized site. Cortical neurons were infected with pLenti-GFP-Mito, and axon bundles were loaded with 20 nM TMRE dye before or 1 or 5 h after axotomy. Live images were acquired at 12 bit below the saturate setting for quantitative analysis of Δψm of individual GFP-Mito–labeled mitochondria in axon bundles. Note that axotomy impairs mitochondrial integrity by depolarizing Δψm (reduced TMRE staining, arrowheads) in the distal axons of WT neurons; the phenotype can be effectively reversed by enhanced mitochondrial transport in snph KO axons. Data were analyzed from the total number of mitochondria (n > 1,000) for each condition indicated within bars. (E and F) Pseudo-color images (E) and quantitative analysis (F) showing ATP maintenance in the growing tips 6 h after axotomy in WT and snph KO neurons infected with pLenti-PercevalHR. The F488nm/F405nm ratiometric mean intensity of PercevalHR reflects the relative ATP/ADP ratio. Note that enhanced mitochondrial transport in snph KO axons increases the ATP/ADP ratio in the growing tips (white arrows). (G and H) Pseudo-color ratiometric images (G) and the F560nm/F510nm ratiometric integrated intensity (H) of ATP probe GO-ATeam2. Both WT and snph KO cortical neurons were transfected with ATP-sensitive GO-ATeam2 probe, followed by imaging distal 150-µm microgrooves before or 1, 3, or 5 h after axotomy. Note that WT neurons display reduced F560nm/F510nm integrated intensity after axotomy, suggesting ATP depletion. Enhancing mitochondrial transport in snph KO neurons reverses energy deficits. (I) Overexpressing Miro1 rescues axotomy-induced energy deficits. Quantitative analysis shows the F560nm/F510nm ratiometric integrated intensity (left) and normalized F560nm/F510nm integrated intensity (right) of ATP probe GO-ATeam2. WT neurons were coinfected with lenti–GO-ATeam2 and lenti-control vector or lenti-Miro1 at DIV0, followed by axotomy at DIV8 and imaging of distal 150-µm microgrooves before or 1, 3, or 5 h after axotomy. Note that enhancing mitochondrial transport in WT neurons expressing Miro1 recovers energy deficits. Data were analyzed from the total number of microgrooves indicated within bars or in parentheses and expressed as mean ± SE and Mann–Whitney U test (F) or Student’s t test (D, H, and I). Bars, 20 µm.

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    Figure 7.

    SNPH KO sciatic nerves display enhanced regenerative capacity after crush injury. Adult WT and snph KO mice (2 mo old) were subjected to a sciatic nerve crush injury and sacrificed at 3 d after injury. Regenerating axons through and beyond the lesion site were visualized by their expression of GAP-43 on sciatic nerve longitudinal sections. DAPI was used to label the cell nuclei. Sciatic nerves from the contralateral side were used as uninjured controls. (A) Although SNPH-labeled mitochondria are abundant along the axons (labeled by βIII-tubulin) of the uninjured WT sciatic nerve, SNPH staining was absent in the same region of snph KO mice. (B) Expression of GAP-43 was undetectable in the uninjured sciatic nerve axons of WT and snph KO mice. (C) Representative images of sciatic nerve longitudinal sections show GAP-43–positive regenerating axons. Note that at 3 d after the crush injury, snph KO mice display a marked increase in the number and growth distance of GAP-43–positive axons past the injury site as compared with the WT littermates. The crush site is indicated by asterisks. (D) Quantification of GAP-43–positive axons in the distal sciatic nerves reveals that regenerating axons grew significantly longer distances at 3 d after injury in snph KO mice than WT controls. The number of GAP-43–positive axons at various distances from the crush site was counted and normalized to the crush site. Note that snph KO mice display significantly more regenerating axons at 1.5 (***, P < 0.001), 2.0 (**, P < 0.01), 2.5 (**, P < 0.01), and 3.0 mm (***, P < 0.001) distal to the crush site (two-way ANOVA, followed by Bonferroni’s post-hoc test). (E) The regeneration index was measured as the distance away from the crush site in which the mean number of regenerating axons is half of that observed at the crush site. snph KO sciatic nerves show a higher regeneration index compared with WT (P = 0.01, Student’s t test). Data are mean ± SE (n = 6 mice per genotype and 3 longitudinal sections per animal).

  8. Figure 8.
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    Figure 8.

    Relative expression of GAP-43 after sciatic nerve injury. (A and B) Representative images (A) and quantitative analysis (B) show increased GAP-43 expression in snph KO DRG neurons. snph KO and WT littermates (9 wk old) were subjected to a left-sided sciatic nerve injury. Both injured ipsilateral and uninjured contralateral L5 DRGs of WT and snph KO mice were co-immunostained with antibodies against GAP-43, βIII-tubulin, and DAPI 3 d after crush injury. Note that in snph KO mice, a higher GAP-43 expression is detected in uninjured DRGs (contralateral) as compared with the WT mice (P = 0.0013). GAP-43 expression is further increased in response to the sciatic nerve injury in snph KO mice (P < 0.0001). Data were analyzed from the total number of neurons indicated within bars from four mice for each genotype and expressed as mean ± SE and by two-way ANOVA followed by Tukey’s post-hoc test. Bars, 50 µm.

  9. Figure 9.
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    Figure 9.

    Model illustration of enhanced mitochondrial transport critical for mature neurons to regain axonal regenerative capacity. (A) Although mitochondrial transport in WT axons progressively declines with maturation, axonal injury depolarizes local mitochondria, thus leading to energy deficits. Energy deficits may reflect the intrinsic restriction of mature axons to regenerate after injury. (B) Enhanced local ATP supply via healthy mitochondrial flux into injured axons is critical to meet metabolic requirements for axonal regeneration. Enhancing mitochondrial transport in snph KO axons not only helps remove those dysfunctional mitochondria, but also replenishes healthy ones to the injured axons, thus recovering mitochondrial integrity and rescuing energy deficits.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
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Facilitation of axon regeneration by enhancing mitochondrial transport and rescuing energy deficits
Bing Zhou, Panpan Yu, Mei-Yao Lin, Tao Sun, Yanmin Chen, Zu-Hang Sheng
J Cell Biol Jul 2016, 214 (1) 103-119; DOI: 10.1083/jcb.201605101

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The Journal of Cell Biology: 217 (4)

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