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. 2016 Nov 23;92(4):845-856.
doi: 10.1016/j.neuron.2016.09.049. Epub 2016 Oct 20.

Branch-Specific Microtubule Destabilization Mediates Axon Branch Loss During Neuromuscular Synapse Elimination

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Free PMC article

Branch-Specific Microtubule Destabilization Mediates Axon Branch Loss During Neuromuscular Synapse Elimination

Monika S Brill et al. Neuron. .
Free PMC article

Abstract

Developmental axon remodeling is characterized by the selective removal of branches from axon arbors. The mechanisms that underlie such branch loss are largely unknown. Additionally, how neuronal resources are specifically assigned to the branches of remodeling arbors is not understood. Here we show that axon branch loss at the developing mouse neuromuscular junction is mediated by branch-specific microtubule severing, which results in local disassembly of the microtubule cytoskeleton and loss of axonal transport in branches that will subsequently dismantle. Accordingly, pharmacological microtubule stabilization delays neuromuscular synapse elimination. This branch-specific disassembly of the cytoskeleton appears to be mediated by the microtubule-severing enzyme spastin, which is dysfunctional in some forms of upper motor neuron disease. Our results demonstrate a physiological role for a neurodegeneration-associated modulator of the cytoskeleton, reveal unexpected cell biology of branch-specific axon plasticity and underscore the mechanistic similarities of axon loss in development and disease.

Keywords: axonal transport; cytoskeleton; microtubule; neuromuscular junction; synapse elimination.

Figures

Figure 1
Figure 1
Retreating Axon Branches Lack Mitochondrial Transport and Dismantle Their Microtubular Cytoskeleton (A–C) Sequential photo-bleaching in transgenic mice, where all motor axons express a fluorescent protein, defines the synaptic territories of competing axon branches during synapse elimination. Confocal image of a small part of the synaptic field in a fixed triangularis sterni muscle at P9 (Thy1-YFP-16, white; α-bungarotoxin, orange) (A) with photo-bleaching steps for one NMJ (boxed in A and C; magnified in B). Schematic illustration of the photo-bleaching (B). Tracing of axons in (A) highlighting the branching pattern of motor units, with a retraction bulb (“rebu”), singly innervated (“sin”) and doubly innervated (territory in percentage) NMJs labeled (C). (D and E) Examples of photo-bleached NMJs representative of the bins used in graphs below, shown in pseudo-color (D) and as schematic of one input (E); overall NMJ outline, red. (F) Net delivery of fluorescently tagged mitochondria (anterograde minus retrograde flux) by an input to the NMJ versus synaptic territory of this input (n ≥ 24 axons, ≥24 Thy1-Mito-CFP-K × Thy1-YFP-16 mice per group). (G) Level of βIII-tubulin immunostaining normalized to cytoplasmic YFP versus synaptic territory (n ≥ 17 axons, ≥6 Thy1-YFP-16 mice per group). Scale bars, 20 μm in (A) (applies also to C); 10 μm in (D) (applies also to E). Data are mean ± SEM. Significance statements are given in main text. See also Figures S1–S3.
Figure 2
Figure 2
Microtubules Are Locally Fragmented in Retreating Axon Branches (A) EB3 comet density versus synaptic territory (n ≥ 10 axons, ≥6 Thy1-EB3-YFP × Thy1-CFP-5 mice per group). (B) Normalized ratio of EB3 comet density over βIII-tubulin levels (as a measure of microtubule length) at different stages of synapse elimination (calculated from data shown in Figures 1G and 2A). (C) Maximum intensity projection (left, 20 s) of a time-lapse sequence in a P9 Thy1-EB3-YFP explant showing a retraction bulb (outlined on the right) next to a singly innervated NMJ. Dashed boxes indicate the sites of distal and proximal measurements on the retraction bulb. (D) EB3 comet density at distal and proximal sites along retraction bulbs (n = 10 axons, 6 mice; points show individual measurements; values derived from the same branch are connected). (E) Maximum intensity projections (20 s) of time-lapse recordings from stem axons, giving rise to branches ending in a retraction bulb (rebu; left) or at a singly innervated NMJ (sin; right; outlines below; P10 Thy1-EB3-YFP). Dashed boxes indicate sites of measurement in stem or branch of the indicated type. (F) EB3 comet density in retraction bulbs and singly innervating branches compared to their respective stem axons (n ≥ 16 axons, ≥8 mice per group). Scale bars, 10 μm in (C); 5 μm in (E). Data are mean ± SEM; paired t test or Mann-Whitney test was used to determine significance in (D) and (F), respectively: ∗∗∗p < 0.001; n.s. p ≥ 0.05. See also Figure S3.
Figure 3
Figure 3
Ultrastructural Analysis of Microtubule Loss in Retreating Axon Branches (A–C) Correlated serial sectioning reconstruction of the ultrastructure of a doubly innervated P8 NMJ, which was first characterized by sequential photo-bleaching (A); Thy1-YFP-16; inputs pseudo-colored) and then reconstructed by EM and surface rendered in (B). Branch territories as determined by bleaching and EM are indicated in (A) and (B), respectively. Please note that the percentages of covered synaptic territory in (A) add to more than 100% due to branch overlaps that cannot be resolved in the light microscope, an issue first noted in Kasthuri and Lichtman (2003). Rendering of axonal microtubules (C) (red; displayed area indicated by dashed red box in B). (D) Single electron micrograph (pseudo-colored as in A and B and orientation of plane as indicated in B) shows individual microtubule segments (pseudo-colored red). (E and F) Higher magnification views of boxed areas in (D) (magenta input in E; white input in F) with individual microtubule segments marked by red arrowheads. (G) Microtubule length per volume versus synaptic territory based on EM reconstruction of eight inputs from four NMJs and four mice. Magenta and white colored circles represent the axons shown in (A)–(F). Scale bars/boxes, 5 μm in (A), 1 μm3 in (B) and (C), and 1 μm in (D). See also Figure S2.
Figure 4
Figure 4
Pharmacological Stabilization of Microtubules Delays Synapse Elimination (A) Percentage of poly-innervated synapses following epothilone B (0.5 μg/μL) or vehicle (polyethylene glycol, PEG) injection on P4 (n ≥ 7 Thy1-YFP-16 mice for P7; n ≥ 6 P9; n ≥ 5 P11; n ≥ 5 P13; n ≥ 3 P21). (B) Confocal image of a doubly innervated NMJ in a P21 Thy1-YFP-16 mouse (white) injected with epothilone B on P4 (α-bungarotoxin, orange). (C–E) βIII-tubulin levels (C); n ≥ 45 axons, ≥4 mice), EB3 comet density (D; n ≥ 13 axons, ≥5 mice) and EB3 comet to tubulin ratios (E) in retraction bulbs (rebu) and singly innervating (sin) branches of P6 Thy1-YFP-16 mice injected with epothilone B or PEG on P5. Scale bars, 5 μm in (B). Data are mean + SEM; Mann-Whitney test was used to determine significance: ∗∗∗p < 0.001; ∗∗p < 0.01; p < 0.05; n.s. p ≥ 0.05. See also Figures S4 and S5.
Figure 5
Figure 5
Post-translational Modifications of Microtubules in Axon Branches of Different Competition State (A)–(I) show stainings for post-translationally modified microtubules (red) superimposed on βIII-tubulin (white). Quantification of post-translational modifications normalized to βIII-tubulin in bulb-tipped retreating axon branches (rebu) and in singly innervating axon branches (sin; B: n ≥ 38 axons from 4 mice per group; D: n ≥ 41, 6; F: n ≥ 57, 4; H: n ≥ 41, 5). Levels of polyglutamylated tubulin (normalized to βIII-tubulin) versus synaptic territory of competing axon branches (n ≥ 161 axons from 10 mice) (I). The monochrome left-hand panels are adjusted with non-linear gamma to enhance visibility of the thin retreating axons; in the merged right-hand panels, both channels are linearly adjusted (A, C, E, and G). Scale bars, 5 μm throughout. Data are mean + SEM. Mann-Whitney test was used to determine significance: ∗∗∗p < 0.001; ∗∗p < 0.01; n.s. p ≥ 0.05.
Figure 6
Figure 6
Targeting of the Mouse Spastin Locus Using a “Knockout-First” Allele (A) Schematic representation (not to scale) of the mouse wild-type (WT; first drawing) and mutant Spast gene locus (bottom three drawings). The vector Spasttm1a(KOMP)Wtsi was used to generate spastin “knockout-first” mice (second drawing from top). A floxed spastin allele was generated by genome-wide deletion of lacZ and NeoR using ACTB-FLPe mice (third drawing from top). Constitutive spastin KO mice were generated by deleting the floxed exon 5 in the germline using CMV-Cre mice (bottom drawing). Note that exon 4 in the Spast gene is an alternatively spliced exon. (B) Southern blot following EcoRV digestion confirms correct insertion of the targeting cassette’s 5′ region (probe location is indicated in A). Wild-type, 18.5 kb. KO first, 14.8 kb. (C) Long-range PCR proves correct insertion of the targeting cassette’s 3′ region. PCR product of primers 1 (“P1”) and 2 (“P2”): 5,043 bp (primer locations are indicated in A). (D) PCR confirms deletion of exon 5 (green). Primer positions 3, 4, and 5 (“P3” to “P5”) are indicated in (A). (E and F) Schematic (E) and table (F) detailing the structure and expected size of the spastin gene products. (G) Western blot showing reduced spastin protein expression in P12 spinal cord from homozygous (−/−) and heterozygous (+/−) mice of the knockout-first allele compared to wild-type mice (+/+). Actin serves as loading control. Note the presence of spastin isoforms due to two translation initiation codons: the full-length M1 and the shorter isoform M87.
Figure 7
Figure 7
Genetic Stabilization of Microtubules Delays Synapse Elimination (A) Percentage of poly-innervated synapses in spastin KO mice compared to wild-type (WT) littermates (n ≥ 5 mice for P7; n ≥ 6 mice for P7; n ≥ 8 P9; n ≥ 6 P11; n ≥ 4 P13; n ≥ 6 P21; evaluation either based on the Thy1-YFP-16 transgene or βIII-tubulin staining). (B) Confocal image of a doubly innervated P21 NMJ in a spastin KO × Thy1-YFP-16 mouse (α-bungarotoxin, orange). (C–E) Spastin deletion delays retraction of “loser” branches. Single frames spaced by about 3 hr taken in explants derived from WT (left) and spastin KO (right) pups crossed to Thy1-YFP-16 or Thy1-CFP-5 (C). Average speed of retraction (D) and distribution histogram of individual recordings (E) (KO: n ≥ 65 axons from 7 mice; WT: n ≥ 62 axons from 7 mice). (F–H) βIII-tubulin levels (F); n ≥ 42 axons, ≥5 mice per genotype, P7–P9), EB3 comet density (G); n ≥ 20 axons, ≥8 mice, P8–P13) and EB3 to tubulin ratios (H) in retraction bulbs and singly innervating axon branches of spastin KO mice compared to wild-type littermates. (I and J) Selective deletion of spastin in cholinergic neurons. Triangularis sterni muscle of a ChAT-Cre × CAG-tdTomato reporter mouse at P9 demonstrating recombination in all motor neurons (100% ± 0%; n = 3 mice; tdTomato, red; α-bungarotoxin, orange; βIII-tubulin, white) (I). Inset shows higher magnification of boxed area. Percentage of poly-innervated synapses from P7–P21 in motor-neuron specific spastin KO mice (SpastinMN KO; ChAT-Cre × Spastinfl/fl) compared to littermate controls (SpastinMN WT; pooled ChAT-Cre-negative Spastin fl/fl, ChAT-Cre-negative Spastin fl/+, and ChAT-Cre-positive wild-type littermates; n ≥ 5 mice for P7; n ≥ 4 P9; n ≥ 8 P11; n ≥ 5 P13; n ≥ 5 P21) (J). Scale bars, 5 μm in (B), 3 μm in (C), and 50 μm in (I). Data are mean + SEM. Mann-Whitney test was used to determine significance in (A), (D), (F), and (J) and unpaired t test in (G): ∗∗∗p < 0.001; ∗∗p < 0.01; p < 0.05; n.s. p ≥ 0.05. See also Figure S4 and S6.

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