Li PP and Peng HB (2012), Regulation of axonal growth and neuromuscular j...

XB-ART-45901

Mol Biol Cell 2012 Oct 01;2320:4109-17. doi: 10.1091/mbc.E12-05-0367.

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Regulation of axonal growth and neuromuscular junction formation by neuronal phosphatase and tensin homologue signaling.

Li PP , Peng HB .

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During the development of the vertebrate neuromuscular junction (NMJ), motor axon tips stop growing after contacting muscle and transform into presynaptic terminals that secrete the neurotransmitter acetylcholine and activate postsynaptic ACh receptors (AChRs) to trigger muscle contraction. The neuron-intrinsic signaling that retards axonal growth to facilitate stable nerve-muscle interaction and synaptogenesis is poorly understood. In this paper, we report a novel function of presynaptic signaling by phosphatase and tensin homologue (PTEN) in mediating a growth-to-synaptogenesis transition in neurons. In Xenopus nerve-muscle cocultures, axonal growth speed was halved after contact with muscle, when compared with before contact, but when cultures were exposed to the PTEN blocker bisperoxo (1,10-phenanthroline) oxovanadate, axons touching muscle grew ~50% faster than their counterparts in control cultures. Suppression of neuronal PTEN expression using morpholinos or the forced expression of catalytically inactive PTEN in neurons also resulted in faster than normal axonal advance after contact with muscle cells. Significantly, interference with PTEN by each of these methods also led to reduced AChR clustering at innervation sites in muscle, indicating that disruption of neuronal PTEN signaling inhibited NMJ assembly. We thus propose that PTEN-dependent slowing of axonal growth enables the establishment of stable nerve-muscle contacts that develop into NMJs.

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Species referenced: Xenopus
Genes referenced: pten tns1
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	FIGURE 1:. Expression of PTEN in embryonic Xenopus spinal neurons. (A) PTEN expression in Xenopus tissues was assessed by immunoblotting. Extracts of Xenopus neural tubes (N), myotomal muscle (M), and whole embryos (E) were stained with an anti-PTEN antibody (top blot) and with anti-tubulin antibody to compare protein loading (bottom blot). Molecular weight marker positions are indicated on the right. (B–E) PTEN expression in neurons was examined by immunolabeling. Fixed and permeabilized embryonic spinal neurons were labeled with anti-PTEN and FITC-conjugated secondary antibodies (PTEN; B and C) or secondary antibodies alone (Ctl; D and E). Labeling for PTEN was detected along axons and also in growth cones (C).
	FIGURE 2:. Enhancement of axonal growth by the PTEN-inhibitor bpV. Elongating axons in Xenopus nerve–muscle cocultures were examined by time-lapse recordings. The sample images here show axonal growth over 30 min in control cocultures before contact with muscle (Ctl; A and B) and after (Ctl; C and D), and growth after muscle contact in cocultures treated with a PTEN-inhibitor (bpV, 100 nM; E and F). In both control and bpV-treated cultures, axonal growth slowed after touching muscle, but axons exposed to bpV advanced ∼50% faster than control axons. The axon–growth cone position is marked by a white arrowhead at time zero (0′) and by a black arrowhead at 30 min (30′). In 30 min, the control axon advanced 25 μm before target contact (A and B) but <5 μm after contact (C and D). In contrast, addition of bpV caused the axon to grow 23 μm in 30 min after muscle contact (E and F). (G) Quantification of axonal growth speeds in μm/min using distances axons advanced in control and bpV-treated cultures before and after contact with muscle. Mean ± SEM shown; number of axons examined by time-lapse recording: 10 (Ctl-before contact), 12 (Ctl-after contact), 22 (bpV-before contact), 39 (bpV-after contact); t test: *, p = 0.01, relative to control axons before contact with muscle; ^, p = 0.03 compared with control axons after muscle contact; n.s., not significant. For the difference between Ctl-before contact and bpV-before contact, p = 0.17.
	FIGURE 3:. Effect of bpV on axonal growth after NMJ formation. Spinal neurons were seeded on muscle cultures to allow NMJ formation. After 12–16 h, control or 100 nM bpV-containing medium was added to the cocultures. (A and B) In control cultures, little growth of the axon was seen at nerve–muscle contact sites within 30 min, but in bpV-treated cultures (C and D), axons were observed to resume active growth during the 30-min recording period. (E) Quantification of axonal growth speeds in untreated (Ctl) and bpV-treated cocultures. Mean ± SEM; t test: *, p = 0.05 compared with Ctl. Arrowheads indicate initial and final positions of the terminal ends of axonal shafts (A–D). Number of axons examined by time-lapse recording: 16 (Ctl-after contact), 11 (bpV-after contact).
	FIGURE 4:. Increase in axonal growth speed following the reduction of PTEN expression in neurons. Spinal neurons were isolated from embryos injected with control MO (Ctl-MO; A–C) or PTEN-specific MO(PTEN-MO; D–F) and cocultured with muscle cells obtained from uninjected embryos. Axons with Ctl-MO and PTEN-MO both slowed after contact with muscle, but after touching muscle, PTEN-MO-axons grew nearly as fast as control axons did before muscle contact. Arrowheads indicate initial and final positions of the terminal ends of axons (A, B, D, and E), and the presence of MOs tagged with fluorescein is shown by the fluorescence of the axons (C and F). (G) Immunoblots showing that PTEN expression was knocked down in Xenopus embryos injected with PTEN-MO but not Ctl-MO (top blot); anti-tubulin staining shows protein loading (bottom blot). (H) Quantification of growth speeds of axons with Ctl-MO and PTEN-MO. Mean ± SEM; t test: *, p = 0.02 compared with Ctl-MO-axons before contact with muscle; ^^, p < 0.005 relative to growth speed of Ctl-MO-axons after muscle contact; number of axons examined by time-lapse recording: 8 (Ctl-MO before contact), 30 (Ctl-MO after contact), 15 (PTEN-MO before contact), 33 (PTEN-MO after contact).
	FIGURE 5:. Regulation of axonal advance by exogenous PTEN proteins. Spinal neurons were cultured from Xenopus embryos injected with mRNAs encoding GFP (A–C) and GFP-tagged wild-type PTEN (GFP-WT-PTEN; D–F) or catalytically inactive PTEN (GFP-C124S-PTEN; G–I). These neurons were seeded on muscle cells cultured from uninjected embryos, and axonal growth was monitored by time-lapse imaging. After contact with muscle cells, axons overexpressing wild-type PTEN grew more slowly than control GFP axons, whereas axons expressing inactive PTEN grew faster. The advance of axons can be compared using the arrowheads that mark the terminal ends of growth cones; the expression of exogenous proteins in the axons is shown by GFP fluorescence (C, F, and I). (J) The average growth speeds of axons (expressing exogenous proteins) before and after muscle contact were quantified and are shown as mean ± SEM. t test: *, p = 0.04 relative to GFP axons before muscle contact; ^, p = 0.02 compared with GFP axons after contact with muscle. Number of axons examined by time-lapse recording: 7 (GFP-before contact), 11 (GFP-after contact), 12 (GFP-WT-PTEN-before contact), 17 (GFP-WT-PTEN-after contact), 18 (GFP-C124S-PTEN-before contact), 25 (GFP-C124S-PTEN-after contact).
	FIGURE 6:. Inhibition of NMJ formation by the PTEN-blocker bpV. NMJ formation in 1-d-old nerve–muscle cocultures (A and C) was examined by labeling AChR clusters with R-BTX (B and D). In control cocultures, AChRs were aggregated in muscle cells at innervation sites (B), but in bpV-treated cultures, nerve-induced AChR clustering was significantly reduced (D) and spontaneously formed AChR clusters or hot spots (h.s.) persisted; arrows point to axon–muscle contacts, arrowheads to nerve-induced AChR clusters. (E) NMJ assembly was quantified as percentages of nerve–muscle contacts with AChR clusters; mean ± SEM shown; t test: **, p < 0.01. Number of nerve–muscle pairs examined: 105 (Ctl), 104 (bpV).
	FIGURE 7:. Suppression of NMJ assembly by the down-regulation of neuronal PTEN expression. Spinal neurons from embryos injected with Ctl-MO (A and B) or PTEN-MO (D and E) were cocultured with muscle cells from uninjected embryos; nerve-induced clustering of AChRs was examined by R-BTX labeling (C and F). AChR aggregation at innervation sites was reduced in cocultures using neurons with PTEN-MO compared with those with Ctl-MO, and AChR hot spots (h.s.) were retained in muscle cells innervated by the PTEN-MO neurons (F). Arrows indicate axon–muscle contacts (A and D); green fluorescence shows the presence of fluorescein-tagged MOs in neurons (B and E); and arrowheads mark nerve-induced AChR clusters (C). Quantification of muscle AChR clustering at innervation sites (G) showed that NMJ formation was reduced by ∼40% in cocultures using PTEN-MO neurons compared with those with Ctl-MO neurons; mean ± SEM; t test: *, p = 0.01. Number of nerve–muscle pairs examined: 53 (Ctl-MO), 68 (PTEN-MO).
	FIGURE 8:. Reduction in NMJ development following the expression of inactive PTEN in neurons. Spinal neurons expressing GFP (A and B), GFP-WT-PTEN (D and E), or GFP-C124S-PTEN (G and H) were cocultured with normal muscle cells. R-BTX labeling showed that neurons expressing GFP (C) or WT-PTEN (F) induced AChR clustering better than those expressing C124S-PTEN (I). Arrows mark nerve tracks (A, D, and G); arrowheads point to AChR clusters at innervation sites in muscle (C and F); h.s. indicates an AChR hot spot (I); and GFP fluorescence shows the expression of exogenous proteins in neurons (B, E, and H). Quantification of NMJ formation (J) showed that GFP- and WT-PTEN neurons induced AChR clusters at 70–75% of innervation sites, whereas neurons with inactive PTEN triggered AChR aggregation at <50% of the sites at which they contacted muscle cells; mean ± SEM; t test: **, p < 0.01 relative to AChR cluster induction by GFP neurons. Number of nerve–muscle pairs examined: 53 (GFP), 45 (GFP-WT-PTEN), 87 (GFP-C124S-PTEN).

References [+] :

Anderson, Effects of innervation on the distribution of acetylcholine receptors on cultured muscle cells. 1977, Pubmed, Xenbase