The Mechanosensitive Ion Channel Piezo Inhibits Axon Regeneration

Abstract

Neurons exhibit a limited ability of repair. Given that mechanical forces affect neuronal outgrowth, it is important to investigate whether mechanosensitive ion channels may regulate axon regeneration. Here, we show that DmPiezo, a Ca²⁺-permeable non-selective cation channel, functions as an intrinsic inhibitor for axon regeneration in Drosophila. DmPiezo activation during axon regeneration induces local Ca²⁺ transients at the growth cone, leading to activation of nitric oxide synthase and the downstream cGMP kinase Foraging or PKG to restrict axon regrowth. Loss of DmPiezo enhances axon regeneration of sensory neurons in the peripheral and CNS. Conditional knockout of its mammalian homolog Piezo1 in vivo accelerates regeneration, while its pharmacological activation in vitro modestly reduces regeneration, suggesting the role of Piezo in inhibiting regeneration may be evolutionarily conserved. These findings provide a precedent for the involvement of mechanosensitive channels in axon regeneration and add a potential target for modulating nervous system repair.

Keywords: Drosophila; Piezo; axon regeneration; corneal sensory nerve; dendritic arborization neurons; ion channels; mammalian injury model; mechanosensitive; nitric oxide synthase.

Figure 1.. DmPiezo Inhibits Axon Regeneration in da Sensory Neurons

(A) Class III da neuron axons fail to regenerate in WT. DmPiezo removal as in DmPiezoKO and class III da neuron-specific RNAi leads to increased axon regeneration. Class III da neuron-specific expression of DmPiezo suppressed the enhanced regeneration in DmPiezoKO. The injury site is demarcated by the dashed circle. Arrow marks axon stalling, while arrowheads show the regrowing axon tips. (B–D) Quantification of class III da neuron axon regeneration with regeneration percentage (B), regeneration index (C), and regeneration length (D). Class III da neuron expression of DmPiezo, mPiezo1, or hPiezo1, but not mPiezo1–2336-Myc rescues the regeneration phenotype in DmPiezoKO. n = 22–49 neurons from 6 to 16 larvae. (E–G) Quantification of class IV da neuron axon regeneration with regeneration percentage (E), regeneration index (F), and regeneration length (G). Over-expression of the over-activating mPiezo1-TriM reduces class IV da neuron axon regeneration. n = 15–29 neurons from 4 to 7 larvae. (H) GFP-DmPiezo is present diffusely in the cell body and axon of uninjured class III da neurons. 24 hr AI, GFP-DmPiezo is enriched in the tip of the growth cone (dashed circle). Data are expressed as mean ± SEM in bar graphs in all figures. *p < 0.05, **p < 0.01, ***p < 0.001 by Fisher’s exact test (B and E), one-way ANOVA followed by Holm-Sidak’s test (C) or Dunnett’s test (D, F, and G). Scale bar, 20 μm. See also Figure S1.

Figure 2.. Removal of DmPiezo Enhances Axon Regeneration in the CNS but Does Not Improve Dendrite Regeneration

(A) DmPiezo removal increases class IV da neuron axon regrowth in the VNC. The injury site is demarcated by the dashed circle. The regenerating axons are illustrated in schematic diagrams with terminal branching marked in red, commissure regrowth in blue, and other regrowing axons in black. (B) Compared to WT, which shows limited regrowth, the regeneration percentage is increased in DmPiezoKO. n = 29–52 injured segments from 13 to 32 larvae. (C) DmPiezoKO increases terminal branching and commissure regrowth of class IV da neurons after injury in the VNC. n = 13–32 larvae. (D) Both WT and DmPiezoKO regrow dendrites after balding. The new dendrites are traced and marked in pink. The soma of the balded neuron is marked by arrows. (E) Regeneration percentage is similar between WT and DmPiezoKO. (F and G) There is no difference in the increase of dendritic branches between WT and DmPiezoKO, after single dendrite lesion (F) or balding (G). n = 11–18 neurons from 4 to 8 larvae for single-dendrite injury, and 3–6 neurons from 2 to 5 larvae for balding. *p < 0.05 by Fisher’s exact test (B and E) and two-tailed unpaired Student’s t test (C, F, and G). Scale bar, 20 μm. See also Figure S2.

Figure 3.. Knocking Down DmPiezo Specifically after Injury Is Sufficient to Promote Axon Regeneration

(A) DmPiezo is knocked down in class III da neurons with DmPiezo RNAi using the temperature-sensitive Gal80^ts, which is expressed in all cells by the tubulin (tub) promoter. Larvae were raised at the permissive temperature at 25°C until injury and then kept at the restrictive temperature at 30°C to allow DmPiezo RNAi expression. Knockdown DmPiezo specifically after injury is sufficient to promote axon regeneration. The injury site is demarcated by the dashed circle. Arrow marks axon stalling while arrowheads show the regrowing axon tips. (B–D) Quantification of class III da neuron axon regeneration with regeneration percentage (B), regeneration index (C), and regeneration length (D). (E) The genotypes of the animals and the temperature shift paradigm. n = 26–32 neurons from 4 to 6 larvae. *p < 0.05, **p < 0.01 by Fisher’s exact test (B) and two-tailed unpaired Student’s t test (C and D). Scale bar, 20 μm. See also Figure S3.

Figure 4.. DmPiezo Regulates Calcium Transients during Axon Regeneration

(A) DmPiezoKO does not change the action potential (AP) firing of class III da neurons induced by displacements of the larval body wall. This mechanical response is largely abolished one day after injury (D1 AI) in both WT and DmPiezoKO. n = 5–10 neurons from 3 to 5 larvae. (B) Axon injury triggers action potential firing in class III da neurons, and the firing frequency is comparable between WT and DmPiezoKO. n = 5–6 neurons from 4 to 5 larvae. (C and D) At 48 hr AI, spontaneous calcium transients are present in the growth cones of WT larvae, but they are significantly reduced in DmPiezoKO. Calcium imaging was performed using membrane targeting myr-GCaMP6(s) in 3^rd-instar larvae in vivo. For both WT (C) and DmPiezoKO (D), the neuronal cell body and the injured axon are shown on the left. The growth cone region (pink square) is zoomed in and six frames from the time-lapse are shown. The red circle indicates the injury site and the blue line marks the region of interest (ROI) used for measuring fluorescence intensity. The raw calcium trace is shown with the six time points corresponding with the frames presented above. (E–G) The amplitude of calcium transients in the growth cone is reduced in DmPiezoKO at 48 hr AI. The GCaMP fluorescence signal in each frame was subtracted and divided by the average fluorescence intensity of ROI in the 100 frames for each neuron, to normalize the baseline to 0. The fast Fourier transform (FFT) algorithm in MATLAB was then applied to decompose the fluorescence function of time into frequency components. The frequency domain representation of the original signal is shown by magnitude spectrum. The y axis indicates the relative amplitude of fluorescence vibration after FFT at specific frequency of the x axis. A statistical difference was observed only at 48 hr AI (G), but not pre-injury (E) or 24 hr AI (F). In particular, at 48 h AI, WT shows significantly higher amplitude at some low-frequency groups (0.002, 0.01, 0.014, 0.02 Hz) than those of DmPiezoKO. n = 17, 19, 17 neurons from 9, 10, 9 larvae for WT, and 22, 22, 16 neurons from 11, 11, 8 larvae for DmPiezoKO at pre-injury, 24 and 48 hr AI, respectively. *p < 0.05, **p < 0.01, ***p < 0.001 by two-way ANOVA followed by Sidak’s multiple comparisons test. Scale bar, 20 μm. See also Figure S4.

Figure 5.. DmPiezo Functions through Calcium Signaling to Inhibit Axon Regeneration

(A) Inhibiting CamKII function with class III da neuron-specific expression of CamKII-I.Ala or CamKII RNAi promotes axon regeneration. CamKII-I.Ala over-expression in the DmPiezoKO background does not further enhance axon regeneration. Overexpression of the constitutively active CamKII-CamKII.T287D in DmPiezoKO attenuates the enhanced regeneration phenotype. While axon regeneration is slightly improved in Cam^n339/+ (a calmodulin-null allele) heterozygotes, transheterozygotes of Cam^n339/+ and DmPiezoKO/⁺ significantly promote axon regeneration. The injury site is demarcated by the dashed circle, and arrowheads mark the regrowing axon tips. (B–G) Quantification of class III da neuron axon regeneration with regeneration percentage (B and E), regeneration index (C and F), and regeneration length (D and G). n = 20–49 neurons from 5 to 16 larvae. (H) In uninjured control, phosphor-CamKII (Thr³⁰⁵) staining (p-αCamKII) is present diffusely in the class III da neuron cell body, axon, and dendrites, both in WT and DmPiezoKO. After injury, WT class III da neurons show enriched p-aCamKII staining in the growth cone tip (yellow dashed circle), which is not present in DmPiezoKO. (I) CamKII RNAi in class III da neurons specifically abolishes the p-aCamKII staining in the cell body (magenta dashed circle), dendrites and axon. (J) At 24 hr AI, a small fraction (14%) of the injured class III da neurons in WT or DmPiezoKO show p-aCamKII staining in the growth cone tip. However, at 48 h AI, whereas 50% of the WT class III da neurons show obvious staining in the growth cone tip, it is not present in DmPiezoKO. n = 4–8 neurons from 3 to 5 larvae. *p < 0.05, **p < 0.01, ***p < 0.001 by Fisher’s exact test (B and E), one-way ANOVA followed by Dunnett’s test (C, F, and G) or Dunn’s test (D). Scale bar, 20 μm. See also Figure S5.

Figure 6.. Nos Functions in Conjunction with DmPiezo to Inhibit Axon Regeneration

(A) Class III da neurons do not regrow in heterozygotes of DmPiezoKO/⁺ or Nos^Δ15/⁺, whereas their regeneration is enhanced in the double transheterozygotes of DmPiezoKO/⁺ Nos^Δ15/⁺, and in Nos^Δ15. Class III da neuron-specific overexpression of Nos suppresses the enhanced regeneration phenotype in DmPiezoKO. The injury site is demarcated by the dashed circle. Arrow marks axon stalling, while arrowheads show the regrowing axon tips. (B–D) Quantification of class III da neuron axon regeneration with regeneration percentage (B), regeneration index (C), and regeneration length (D). n = 26–43 neurons from 7 to 10 larvae. *p < 0.05, ***p < 0.001 by Fisher’s exact test (B), one-way ANOVA followed by Holm-Sidak’s test (C and D). Scale bar, 20 μm. See also Figure S6.

Figure 7.. for or PKG Inhibits Axon Regeneration Downstream of Nos

(A) Class III da neuron axon regeneration is enhanced in for^k04703, transheterozygotes of for^k04703/ for^DfED243, class III da neuron-specific expression of forRNAi, double transheterozygotes of Nos^Δ15/⁺, for^DfED243/+, but not in for^11.247, heterozygotes of Nos^Δ15/⁺ or for^k04703/⁺. Class III da neuron-specific overexpression of Nos fails to mitigate the regeneration enhancement seen in for^k047037, whereas FOR-T1 over-expression largely suppresses the phenotype in Nos^Δ15. (B–M) Quantification of class III da neuron axon regeneration with regeneration percentage (B, H, and K), regeneration index (C, I, and L), and regeneration length (D, J, and M). Quantification of class IV da neuron axon regeneration with regeneration percentage (E), regeneration index (F), and regeneration length (G). Whereas expressing a single copy of FOR-T1 with two independent alleles: for-T1 and dg2-P1 or FOR-T2 with dg2-P2A all fail to achieve significant inhibition in class IV da neurons, overexpressing two copies of FOR-T1 (for-T1X2) impedes axon regeneration. n = 17–37 neurons from 5 to 15 larvae. *p < 0.05, **p < 0.01, ***p < 0.001 by Fisher’s exact test (B, E, H, and K), one-way ANOVA followed by Holm-Sidak’s test (C and F), Dunn’s test (D), or Dunnett’s test (G, I, J, L, and M). Scale bar, 20 μm. See also Figure S7.

Figure 8.. Piezo Inhibits Axon Regeneration in Mammalian Injury Models

(A) Piezo1 agonist Yoda1 (30 μM) modestly reduces axon regeneration of rat hippocampal neurons cultured in a microfluidic chamber, when applied to the axon terminal chamber immediately after injury. The axons are labeled with α-Gap43 staining. (B) The axon coverage area is measured and normalized to the total width of the microgrooves. The value from the Yoda1 group is further normalized to the corresponding DMSO vehicle control group in the same experiment. Yoda1 treatment modestly reduces the coverage area of the regrown axons. n = 6 experiments. (C) The proposed Piezo-CamKII-Nos-for or PKG signaling cascade that responds to injury and inhibits axon regeneration. (D) To label corneal sensory axons and generate sensory neuron-specific Piezo1 conditional knockout (Piezo1 cKO), mice are bred with Advillin(Avil)-CreER; Rosa-stop-tdTomato; Piezo1^fl/fl alleles, and Cre-mediated recombination is induced by tamoxifen (TAM) injection. (E) A 2-photon microscopy based intravital imaging system is implemented to visualize and manipulate tdTomato-labeled corneal sensory nerves in live mice. Serial optical sections are collected to reconstitute a max-projection image of the entire cornea. The timeline describes the experimental paradigm, including tamoxifen injection, laser ablation, nerve ablation, and imaging. (F) The larger nerve trunks located in the stroma are ablated before they branch into thinner fibers (dashed circle). The degeneration of the nerve fibers downstream of the ablation sites both in the stroma and the subbasal nerve plexus is confirmed by imaging at 24 and 48 hr after ablation. (G) In the control group (normal siblings, Avil-CreER; Piezo1^fl/+ + TAM), corneal sensory axons show limited regeneration within 7 days post-ablation, leaving large areas deprived of sensory innervation. Piezo1 cKO (Avil-CreER; Piezo1^fl/fl + TAM) show accelerated axon regeneration, with more vacant space reinnervated by the regenerating nerve fibers. (H) The regenerating axons are manually traced and the length of regrown axons is measured. Piezo1 cKO exhibits significantly higher regeneration capacity as reflected by the increase of the total length of regenerating nerve fibers. The dataset for normalized regeneration B is shown. n = 4 mice for control and 5 mice for Piezo1 cKO. *p < 0.05, **p < 0.01 by two-tailed unpaired Student’s t test (B), two-way ANOVA followed by Sidak’s multiple comparisons test (H). Scale bar, 100 μm. See also Figure S8.