Small-molecule inhibition of STOML3 oligomerization reverses pathological mechanical hypersensitivity

Journal name:
Nature Neuroscience
Volume:
20,
Pages:
209–218
Year published:
DOI:
doi:10.1038/nn.4454
Received
Accepted
Published online
Corrected online

Abstract

The skin is equipped with specialized mechanoreceptors that allow the perception of the slightest brush. Indeed, some mechanoreceptors can detect even nanometer-scale movements. Movement is transformed into electrical signals via the gating of mechanically activated ion channels at sensory endings in the skin. The sensitivity of Piezo mechanically gated ion channels is controlled by stomatin-like protein-3 (STOML3), which is required for normal mechanoreceptor function. Here we identify small-molecule inhibitors of STOML3 oligomerization that reversibly reduce the sensitivity of mechanically gated currents in sensory neurons and silence mechanoreceptors in vivo. STOML3 inhibitors in the skin also reversibly attenuate fine touch perception in normal mice. Under pathophysiological conditions following nerve injury or diabetic neuropathy, the slightest touch can produce pain, and here STOML3 inhibitors can reverse mechanical hypersensitivity. Thus, small molecules applied locally to the skin can be used to modulate touch and may represent peripherally available drugs to treat tactile-driven pain following neuropathy.

At a glance

Figures

  1. Screening for small molecules that modulate STOML3 oligomerization.
    Figure 1: Screening for small molecules that modulate STOML3 oligomerization.

    (a) Schematic representation of BiFC analysis of protein–protein interactions used for small molecule screen. (b) Signal development observed when STOML3-VC (the C-terminal fragment of split Venus fluorescent protein) was used as prey and VN-tagged (N-terminal fragment of split Venus fluorescent protein) STOML3 variants that do not properly oligomerize were used as bait. Numbers indicate replicate reads; data are displayed as means ± s.e.m. (c) The normalized slope of BiFC signal development was used as a measure of oligomerization. Two-tailed unpaired t-test: STOML3 vs. STOML3-V190P, P = 0.0046 (t10 = 3.629); STOML3 vs. STOML3-LR89,90EE, P < 0.0001 (t10 = 9.265); STOML3 vs. nontransfected cells, P < 0.0001 (t10 = 12.15); numbers indicate replicate TECAN experiments derived from 4–6 independent transfections; data are shown as individual slopes and mean ± s.e.m. (d) Structures of hit compounds, the oligomerization blockers, OB-1 and OB-2. (e,f) Normalized slope of BiFC signal development in cells overexpressing Mus musculus (mm) or Homo sapiens (hs) STOML3 in the presence of OB-1 and OB-2 is shown. (e) Two-tailed unpaired t-test: mmSTOML3 vs. mmSTOML3 + OB-1, P = 0.0002 (t8 = 6.594); mmSTOML3 vs. mmSTOML3 + OB-2, P = 0.0064 (t9 = 3.527); numbers indicate replicate TECAN experiments derived from 4–6 independent transfections; data are displayed as individual slopes and mean ± s.e.m. (f) Mann-Whitney two-tailed U-test: hsSTOML3 vs. hsSTOML3 + OB-1, P = 0.0002); numbers indicate replicate TECAN experiments derived from two independent transfections with 4 replicates each; data are shown as individual slopes and mean ± s.e.m. (g) Representative reconstructed dSTORM images of STOML3-FLAG overexpressed in N2a cells. (h) Distribution of STOML3-FLAG domain size as detected by dSTORM imaging. Two-tailed unpaired t-test: STOML3 vs. STOML3-V190P, P = 0.0023 (t20 = 3.496); STOML3 vs. STOML3 + OB-1, P < 0.0001 (t26 = 6.533) STOML3 vs. STOML3 + OB-2, P = 0.0006 (t23 = 3.994); numbers indicate N2a cells derived from at least three transfections. Each data point represents a single cell; the FHWM of 100 randomly chosen domains was measured for each cell. **P < 0.01; ***P < 0.001.

  2. Quantitative analysis of the effect of hit compounds on mechanotransduction.
    Figure 2: Quantitative analysis of the effect of hit compounds on mechanotransduction.

    (a) Schematic of pillar array analysis of mechanotransduction in N2a cells. RE, recording electrode; NS, nanostimulator. (b) Stimulus–response curves for N2a cells treated with either OB-1 or OB-2. Two-way ANOVA (stimulus–response relationship): vehicle vs. OB-1, P = 0.0044, F1,131 = 8.390; vehicle vs. OB-2, P = 0.0388, F1,108 = 4.375; numbers indicate curves for N2a cells from >5 independent experiments. Mann-Whitney two-tailed U-test for 250–500 nm displacement: vehicle vs. OB-1, P = 0.0146); Vehicle vs. OB-2, P = 0.0363; numbers indicate cells stimulated in this range. Data are displayed as mean current amplitude ± s.e.m. (c) Hill plot of the concentration dependence of the OB-1 effect on the Piezo1 current in N2a cells. Mann-Whitney U-test: vehicle vs. 0.002 μM OB-1, P = 0.9266; vehicle vs. 0.02 μM OB-1, P = 0.1236; vehicle vs. 2 μM OB-1, P = 0.1112; vehicle vs. 20 μM OB-1, P = 0.0105; numbers indicate N2a cells recorded in 2–4 independent experiments; data are displayed as mean current amplitude of individual bins ± s.e.m. (d) Schematic of analysis of mechanotransduction in DRG neurons. (e) Stimulus–response curves for mechanoreceptors treated with either OB-1 or OB-2. Two-way ANOVA: vehicle vs. OB-1, P = 0.0007, F1,80 = 12.56; vehicle vs. OB-2, P = 0.0017, F1,78 = 10.59; numbers indicate recorded curves from at least three DRG preps. Two-tailed unpaired t-test for 0–10 nm displacement: vehicle vs. OB-1, P = 0.0462 (t11 = 2.246); vehicle vs. OB-2, P = 0.0291 (t12 = 2.477). Mann-Whitney two-tailed U-test for 10–50 nm displacement: vehicle vs. OB-1, P = 0.0053; vehicle vs. OB-2, P = 0.0068; for 50–100 nm displacement: vehicle vs. OB-1, P = 0.0224; for 100–500 nm displacement: vehicle vs. OB-1, P = 0.0239. (f) Stimulus–response curves for nociceptors treated with either OB-1 or OB-2. Two-way ANOVA: vehicle vs. OB-1, P = 0.0263, F1,61 = 5.185; vehicle vs. OB-2, not significant, F1,57 = 3.725. Mann-Whitney U-test for 100–250 nm displacement: vehicle vs. OB-1, P = 0.0388; for 250–500 nm displacement: vehicle vs. OB-2, P = 0.0087. For e and f, numbers indicate currents measured in DRG nociceptors from at least three DRG preparations derived from 5- to 7-week-old mice; data are displayed as current amplitude with each bin displayed as mean of cell averages ± s.e.m. (gi) In the presence of OB-1 we detected no difference in action potentials generated by current injection in either (h) mechanoreceptors or (i) nociceptors. Mann-Whitney U-test of mechanoreceptors, P = 0.215; Student's two-tailed t-test of nociceptors, P = 0.4743, F11,12 = 2.682; numbers indicate cultivated neurons recorded in 3 independent experiments; data are displayed as current amplitudes and mean of individual bins ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ns, nonsignificant.

  3. Mechanoreceptors can be silenced with local OB-1 treatment.
    Figure 3: Mechanoreceptors can be silenced with local OB-1 treatment.

    (a) Inset: electrical search protocol schema. A microelectrode (~1 M) was used to deliver electrical stimuli at two distant points of the saphenous nerve trunk in order to trace electrically identified units to their receptive fields. Proportions of non-mechanosensitive fibers are shown. We observed an increase in mechanically sensitive Aβ fibers 3 h after local OB-1 treatment (250–500 pmol OB-1 per paw); mechanosensitivity recovered 24 h after injection. Fisher's exact test for Aβ fibers: vehicle vs. OB-1 (male and female mice), P < 0.0001; vehicle vs. OB-1 (male), P < 0.0001; vehicle vs. OB-1 (female), P = 0.0033; OB-1 (male) vs. OB-1 (female), P = 1.0; OB-1 vs. OB-1 wash-out, P = 0.0183. For Aδ fibers: vehicle vs. OB-1, P = 0.2465. For C fibers: vehicle vs. OB-1, P = 1.0; data are displayed as percentage of individual fibers. (b) Stimulus response function of C-mechanoheat (C-MH) fibers shown using a series of ascending displacements (32–1,024 μm). C-MH fibers were significantly less responsive in OB-1 treated mice compared to vehicle treated controls. Two-way ANOVA: vehicle vs. OB-1, P = 0.0412 (F1,257 = 4.208); data are displayed as mean number of action potentials ± s.e.m. (c) Mean force thresholds for C-MH fiber discharge show a significant elevation of mechanical thresholds. Mann-Whitney U-test, P = 0.0233; data are displayed as individual thresholds and mean threshold ± s.e.m. (d) For C-mechanonociceptors (C-M fibers) there was no significant difference between vehicle-treated and OB-1 treated stimulus response functions. Two-way ANOVA, P = 0.3563 (F1,115 = 0.8579); data are displayed as mean number of action potentials ± s.e.m. (e) Mean force thresholds for C-M fiber were also not different between vehicle and OB-1 treatments. Two-tailed unpaired t-test, P = 0.7860, t18 = 0.2756; data are displayed as individual thresholds and mean threshold ± s.e.m. In ae, numbers indicate single sensory fiber recordings derived from 10–20 independent experiments using adult mice. *P < 0.05; **P < 0.01; ***P < 0.001; ns, nonsignificant.

  4. OB-1 reduces touch perception in mice, as shown by performance of head-restrained mice on a tactile perception task.
    Figure 4: OB-1 reduces touch perception in mice, as shown by performance of head-restrained mice on a tactile perception task.

    (a) Mice were trained to report a single tactile pulse stimulus (inset shows stimulus voltage command pulse for all 8 amplitudes). Trial structure: mice were trained to (1) hold the rest sensor and wait for a stimulus; (2) on detection of the stimulus reach and press the target sensor within 500 ms from stimulus onset; (3) obtain water reward by licking on successful trials. (b) Psychometric curves to different amplitude tactile stimuli are affected by injection of OB-1 into forepaw. Curves were constructed with a sigmoid fitting of the mean (n = 5 mice) hit rates to 7 different amplitudes of tactile stimuli and to a no stimulus trial (false alarm). Three conditions are displayed: injection of the vehicle (black), injection of OB-1 (magenta) and a recovery session with no prior injection (gray). (c) OB-1 application to forepaw attenuates perception of near threshold tactile stimuli. Grouped hit rates to three threshold amplitude values (125, 175 and 275 μm) from 5 mice were significantly reduced after OB-1 injection as compared to hit rates after vehicle injection or on recovery session without prior injection. Statistical tests were made on hit rates after subtraction of the corresponding false alarm rates. Wilcoxon signed-rank test: vehicle vs. OB-1, P = 0.026; OB-1 vs. OB-1 wash-out, P = 0.0043; vehicle vs. OB-1 wash-out, P = 0.12; numbers indicate mice treated, and data are displayed as average of individual hit rates of each mouse, grouped for 3 amplitudes ± s.e.m. *P < 0.05; **P < 0.01; ns, nonsignificant.

  5. Tactile-evoked pain can be treated with OB-1.
    Figure 5: Tactile-evoked pain can be treated with OB-1.

    (a) Development of tactile-evoked pain after traumatic nerve injury is shown. Paw withdrawal thresholds (PWT) to varying forces of von Frey filaments before and after unilateral CCI were measured. After nerve injury, Stoml3−/− mice developed significantly less tactile-evoked pain compared to wild-type (WT) animals. Two-way ANOVA: WT vs. Stoml3−/−, P < 0.0001 (F1,159 = 107.65). (b) Paw withdrawal latencies (PWLs) to a standard radiant heat source applied to the ipsilateral hind paw of WT and Stoml3−/− mice before and after CCI were not different between the genotypes. Mann-Whitney U-test: WT naive vs. WT CCI, P = 0.0065; Stoml3−/− naive vs. Stoml3−/− CCI, P = 0.0325; WT CCI vs. Stoml3−/− CCI, P = 0.2532; numbers indicate mice treated (one cohort). (c) Treatment of naive mice with OB-1 does not alter PWTs. Mann-Whitney U-test: naive vs. OB-1 treated, P = 0.2042; OB-1 treated vs. vehicle-treated (veh) P = 0.4545. (d) PWTs measured before and after nerve injury shows clear hypersensitivity, which is not reversed by injection of vehicle. Wilcoxon signed-rank test: CCI male & female vs. CCI + OB-1 male & female, P = 0.0002; CCI female vs. CCI + OB-1 female, P = 0.0156; Paired t-test: CCI male vs. CCI + OB-1 male, P = 0.0066 (t7 = 3.811). (e) Local ipsilateral (ipsi) treatment of the neuropathic paw with OB-1 effectively normalizes PWT but treatment of the contralateral (co) paw does not. Wilcoxon matched-pairs signed-rank test: CCI ipsi vs. CCI + OB-1 (contralateral injection measured on ipsilateral paw), P = 0.25. (f) Alleviation of hypersensitivity with OB-1 treatment is indistinguishable from gabapentin treatment. Wilcoxon matched-pairs signed-rank test: CCI vs. CCI + gabapentin, P = 0.0313 (t5 = 6.518). (g) Dose–response relationship of OB-1 is shown, ED50 = 4.42 μM or approximately 20 pmol. Mann-Whitney U-test: vehicle vs. 0.5 μM OB-1, P = 0.7178; vehicle vs. 5 μM OB-1, P = 0.0730; vehicle vs. 50 μM OB-1, P < 0.0001; vehicle vs. 100 μM OB-1, P = 0.0028. (h) Measurement of PWTs over time; the maximal analgesic efficacy developed between 3 and 9 h after local OB-1 injection. (i) No significant change in PWT was measured in Stoml3−/− mice with CCI after local administration of OB-1, Wilcoxon matched-pairs signed-rank test: CCI, Stoml3−/− vs. Stoml3−/− + OB-1, P = 0.125. In ai numbers indicate adult mice examined from all together more than 15 cohorts tested independently; data are displayed as individual PWTs (cf,i) or individual PWLs (b) and mean of individual median PWTs (a,g,i); error bars indicate s.e.m. (j) Stoml3 copy number derived from lumbar dorsal root ganglia (DRG L4–L6) determined using real-time PCR showing an ipsilateral upregulation of Stoml3 mRNA. The last two bars represent data from Stoml3−/− mice. Mann-Whitney U-test: CCI ipsi vs. co, P = 0.0079; naive ipsi vs. CCI ipsi, P = 0.0357; naive ipsi vs. co, P = 0.7000. Numbers indicate RNA preparations with L4–6 of two adult mice pooled for one RNA preparation; data represent the mean copy number ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ns, nonsignificant.

  6. Regulation of STOML3 in painful neuropathy.
    Figure 6: Regulation of STOML3 in painful neuropathy.

    (a) Cytochemistry of lumbar DRGs from Stoml3+/lacZ mice that had received a nerve injury (CCI). (b) The number of β-galactosidase (β-gal)-positive neurons increased after a unilateral CCI, predominantly in large cells. ***P < 0.001; Fisher's exact test, P < 0.0001; 17 images obtained from 6 adult Stoml3+/lacZ mice. (c) Schematic of the modified locus of StrepII knock-in mice. E, exon; nt, nucleotides. (d) Western blots of protein extracts taken from the sciatic nerve of 2 adult Stoml3StrepII/StrepII mice per protein preparation subjected to unilateral CCI. Extracts were made from 2 mice per time point; a specific StrepII-STOML3 band was detected ipsilateral (i) and contralateral (c) to the injury at all time points (bands are not detected in protein extracts from sciatic nerves of 2 adult Stoml3−/− mice per protein preparation). At Day 2, Day 6 and to a lesser extent Day 13 after injury, we observed much more protein on the injured side compared to the uninjured sciatic nerve. The same protein extracts were probed with antibodies against PGP9.5, a neuronal marker, which decreased dramatically on the injured side, a finding consistent with the known loss and atrophy of axons in the CCI model; for uncropped western blots see Supplementary Figure 9.

  7. Inhibition of STOML3 alleviates painful diabetic neuropathy.
    Figure 7: Inhibition of STOML3 alleviates painful diabetic neuropathy.

    (a) Diabetic peripheral neuropathy was induced using STZ. After development of peripheral neuropathy, diabetic mice received a single injection of OB-1 or vehicle respectively into the plantar surface of the hind paw. (a) Three hours after injection, OB-1 treated mice showed attenuated mechanical sensitivity displayed as percentage of withdrawal to increasing von Frey filaments. Two-way ANOVA: STZ + OB-1 vs. STZ, P > 0.0001, F1,132 = 28.07. Numbers indicate adult mice treated; data are displayed as mean of individual PWTs; error bars indicate s.e.m. (b) Mechanical thresholds required to elicit 60% withdrawal frequency. Wilcoxon signed-rank test: naive vs. STZ, P = 0.0005; two-tailed paired t-test: STZ vs. STZ + OB1, P = 0.0013, t11 = 4.287; STZ + OB1 vs. OB-1 wash-out, P = 0.0004, t11 = 4.939. (c,d) Diabetic mice in the vehicle treated group showed no reversal of mechanical hypersensitivity. Ordinary two-way ANOVA: STZ vs. STZ + vehicle, P = 0.0765, F1,144 = 3.184. Wilcoxon signed-rank test: naive vs. STZ, P = 0.0005. Paired t-test: STZ vs. STZ + vehicle, P = 0.3125, t12 = 1.054; STZ + vehicle vs. vehicle wash-out, P = 0.0859, t12 = 1.871. Numbers indicate mice treated from two cohorts tested independently; data are displayed as mean of individual PWTs; error bars indicate s.e.m. **P < 0.01, ***P < 0.001; ns, nonsignificant.

  8. Effects of STOML3-modulating molecules on other stomatin-domain proteins
    Supplementary Fig. 1: Effects of STOML3-modulating molecules on other stomatin-domain proteins

    (a) BiFC signal development observed when cells were transfected with stomatin –VC/-VN, STOML1-VC/-VN, STOML2-VC/-VN, STOML3-VC/-VN or podocin-VC/-VN expression constructs respectively. The slope of signal development was used as a measure of oligomerization. Note that OB-1 significantly inhibits oligomerization of stomatin, STOML1, STOML2 and STOML3 but does not affect podocin oligomerization. Unpaired t-test: stomatin vs. stomatin + OB-1, P < 0.0001; STOML1 vs. STOML1 + OB-1, P < 0.0001; STOML2 vs. STOML2 + OB-1, P < 0.0001; STOML3 vs. STOML3+ OB-1, P < 0.0001; Mann-Whitney U-test: podocin vs. podocin + OB-1, P = 0.0584; numbers indicate replicates derived from 2 independent cell transfections; data are shown as individual slopes and mean ± s.e.m.. (b) OB-1 was tested at two different concentrations (2 μM and 20 μM) using the BiFC assay. The normalized slope was significantly reduced when OB-1 was present. Unpaired t-test: vehicle vs. 20 μm OB-1, P < 0.0001; vehicle vs. 2 μm OB-1, P < 0.0001; numbers indicate replicates derived from two independent cell transfections; data are displayed as individual slopes and mean ± s.e.m. *** P < 0.001; ns nonsignificant.

  9. Stoml3 mRNA levels after inhibitor treatment and quantitative analysis of mechanically gated currents.
    Supplementary Fig. 2: Stoml3 mRNA levels after inhibitor treatment and quantitative analysis of mechanically gated currents.

    (a) N2a cells and acutely isolated DRGs were treated with either vehicle or OB-1 (20 μM) for 3 hours before mRNA was isolated, reverse transcribed and analyzed using qPCR. There were no detectable differences in Stoml3 transcript levels; numbers indicate independent RNA preparations derived from 3 N2a cell preparations and from two mice per DRG preparation; data are displayed as individual data points and mean ± s.e.m.. (b) Time course analysis of OB-1 activity in N2a cells. Cells were treated with 20 μM OB-1 for the indicated time and mechanotransduction was monitored using pillar arrays. Treatment of at least 3 hours was required for maximum inhibition of mechanically-gated currents. Mann-Whitney U-test: 0-1h vs. > 3h OB-1, P = 0.0003. Unpaired t-test: 1-2h vs. > 3h OB-1, P = 0.0033; 2-3h vs. >3h OB-1, P = 0.0132; numbers indicate currents measured from N2a cells derived from more than 6 independent experiments; data are shown as current amplitude, each bin is displayed as mean of cell averages ± s.e.m.. (c) Representative current trace with latency (magenta) activation time constant (τ1, blue) and inactivation time constant (τ2, green) indicated by dashed lines. The inactivation time constant of mechanically gated currents in DRG mechanoreceptors (mec) shown here was measured using elastomeric pillar arrays. There is a trend for longer inactivation time constants that is not significant. Mann-Whitney U-test: mec vs. mec + OB-1, P = 0.1966; mec vs. mec + OB-2, P = 0.7930; data are displayed as box and whisker plot, fit of inactivation of individual curve, displayed as median with 5-95 interquartiles. (d) In nociceptors (noci) treatment with OB-2 led to significantly longer latencies and significantly slower activation time constants compared to non-treated noci. Mann-Whitney U-test: vehicle vs. OB-2 (Latency), P = 0.0315; vehicle vs. OB-2 (activation time constant), P = 0.001; data are displayed as box and whisker plot, fit of inactivation of individual curve, displayed as median with 5-95 interquartiles. * P < 0.05; ** P < 0.01; *** P < 0.001.

  10. Effects of OB-1 on mechanically-activated currents in mouse sensory neurons.
    Supplementary Fig. 3: Effects of OB-1 on mechanically-activated currents in mouse sensory neurons.

    (a) Schematic diagram of a large diameter mouse sensory neuron with recording electrode (RE) and nanostimulator (NS). (b) Typical RA-mechanosensitive current evoked from mouse sensory neurons by poking the cell soma. (c) Typical RA-mechanosensitive currents evoked from neurons pre-incubated with OB-1 or vehicle. Red lines indicate the exponential fit of the current inactivation to determine τ2. (d) Distribution of cells found with and without a mechanically-activated current (MA current) in both groups. Note the significant loss of mechanically-activated currents in OB-1 treated neurons. Fisher's exact test: vehicle vs. OB-1, P = 0.009; numbers indicate neurons recorded, data are displayed as percentage of neurons. (e) The mean of inactivation time constants (τ2) of mechanically-gated currents is shown; note that τ2 is slower in OB-1 treated neurons when compared to controls. Mann-Whitney U-test: vehicle vs. OB-1, P = 0.0056; numbers indicate individual recordings; data are displayed as individual τ2 and mean ± s.e.m and were recorded from DRG neurons in at least 9 independent experiments. ** P < 0.01.

  11. OB-1 has no effect upon ASIC3-mediated currents nor on stomatin inhibition of ASIC3.
    Supplementary Fig. 4: OB-1 has no effect upon ASIC3-mediated currents nor on stomatin inhibition of ASIC3.

    (a) Neither the transient (T), nor sustained (S) phases of pH-evoked currents in CHO cells expressing ASIC3 were inhibited by OB-1 (20 μM). Student’s t-test: P = 0.2452, P = 0.7493; numbers indicate cells recorded (9 Vehicle, 8 OB-1), data are displayed as individual data points and mean peak current density ± s.e.m.. (b) Stomatin inhibits ASIC3-mediated pH gated currents, which is not modulated by OB-1. Student’s t-test, P = 0.4392, P = 0.8650; numbers indicate cells recorded (7 Vehicle, 7 OB-1); data are displayed as individual data points and mean peak current density ± s.e.m..

  12. Non silenced mechanoreceptors are functional after OB-1 treatment.
    Supplementary Fig. 5: Non silenced mechanoreceptors are functional after OB-1 treatment.

    (a) The proportion of each class of mechanosensitive afferents recorded three hours after local OB-1 or vehicle application is displayed. After OB-1 treatment no significant differences were observed in the receptor proportions among Aβ-, Aδ- or C-fibers compared to vehicle treated skin. Fisher's exact test: Aβ-fibers OB-1 vs. vehicle, P = 0.1722; Aδ-fibers OB-1 vs. vehicle, P = 0.8199; C-fibers OB-1 vs. vehicle, P = 1.0000; numbers indicate single sensory fiber recordings derived from more than 10 independent experiments using adult mice; data are displayed as percentage of individual fibers. (b-e) Receptive field properties of single sensory afferents recorded from vehicle-treated or OB-1 treated skin are shown. (b-d) A series of ramp and hold stimuli with increasing velocities (0.075, 0.15, 0.45, 1.5 and 15 mm/s at 92 μm displacements) was applied to low threshold mechanoreceptors, i.e. slowly adapting mechanoreceptors (SAM) (b), rapidly adapting mechanoreceptors (RAM) (c) and D-hair receptors (d). Mean firing frequencies during the ramp phase are plotted as function of stimulus velocity; numbers indicate fibers recorded; data are displayed as mean number of action potentials ± s.e.m.. (e) An ascending series of displacement stimuli (32 – 1024 μm) using a constant stimulus velocity was applied to A-mechanonociceptors (A-M). Mean firing frequencies were plotted as function of displacement amplitudes showing no changes in mechanosenitivity in A-Ms in OB-1 treated compared to vehicle-treated skin. Two-way ANOVA: SAM vehicle vs. OB-1, P = 0.8924; RAM vehicle vs. OB-1, P = 0.565; D-hair vehicle vs. OB-1, P = 0.8437; A-M vehicle vs. OB-1, P = 0.0912; numbers indicate single sensory fiber recordings derived from more than 5 independent experiments using adult mice; data are displayed as mean number of action potentials ± s.e.m..

  13. Ultrastructure of the sciatic nerve after unilateral CCI.
    Supplementary Fig. 6: Ultrastructure of the sciatic nerve after unilateral CCI.

    (a,d) Schematic drawings of the section plane. (b,c,e,f) High magnification of electron micrograph images of the ipsilateral (b,e) or contralateral side (c,f) proximal and distal to the ligation. (b,c) Proximal to the injury normal myelinated and unmyelinated nerve fibers with typical ultrastructure and intact myelin sheaths or Remak bundles are seen. (e,f) Distal to the injury strong demyelination and axonal degeneration were observed only ipsilateral to the injury. Scale bar = 2 μm, SC spinal cord, SN sciatic nerve.

  14. OB-1 treatment in additional pain models.
    Supplementary Fig. 7: OB-1 treatment in additional pain models.

    (a) Development of tactile-evoked pain using the spared nerve injury (SNI) model. Paw withdrawal thresholds (PWTs) are displayed; note that wild type mice develop a prolonged tactile-evoked pain ipsilateral to the injury. Two-way ANOVA: SNI, ipsi vs. Sham, ipsi, P < 0.0001; numbers indicate adult mice treated (two cohorts); data are displayed as mean of individual median PWTs ± s.e.m.. (b) PWTs are displayed, showing no alleviation of tactile-evoked pain behavior after local OB-1 treatment. Mann-Whitney U-test: SNI vs. OB-1, P > 0.9999; numbers indicate adult mice treated (two cohorts); data are displayed as mean of individual median PWTs ± s.e.m.. (c) Stoml3 copy number derived from lumbar DRG L4 - L6 (two mice per preparation) determined using real-time PCR showing no up-regulation of Stoml3 mRNA after SNI. Mann-Whitney U-test: naïve, ipsi vs. SNI, ipsi, P > 0.9999; numbers indicate RNA preparations, data are displayed as mean copy number ± s.e.m.. (d-g) A single dose of NGF (1 mg/kg body weight) was injected i.p. into adult mice to induce hyperalgesia in wild type and Stoml3-/- mice. (d,e) PWT or paw withdrawal latencies (PWL) are displayed before and after NGF-induced hyperalgesia in wild type and Stoml3-/- mice showing that prominent symptoms of thermal and mechanical hyperalgesia were not different between the genotypes. (f,g) Both, PWTs and PWLs, were measured in response to OB-1 before and after systemic NGF-injection, note that OB-1 does not alleviate NGF-induced mechanical (g) or thermal (h) hyperalgesia. Two-way ANOVA: PWT WT vs. Stoml3-/- P = 0.6067 (g); PWL WT vs. Stoml3-/- (e) P = 0.1078. Mann-Whitney U-test: PWT vehicle paw naïve vs. OB-1 paw naive, P = 0.3615; PWL Vehicle paw naïve vs. OB-1 paw naïve, P = 0.699. Wilcoxon matched-pairs signed rank test: PWT syst. NGF vs. syst. NGF + OB-1 (f), P = 0.125; PWL syst. NGF vs. syst. NGF + OB-1, P = 0.1563; numbers indicate adult mice examined; data are displayed as mean of individual median PWTs (d,f) or PWLs (e,g); error bars indicate s.e.m.. *** P < 0.001 ns nonsignificant.

  15. Generation of the Stoml3lacZ and Stoml3StrepII mouse strains
    Supplementary Fig. 8: Generation of the Stoml3lacZ and Stoml3StrepII mouse strains

    (a) Schematic representation of the targeting vector, the wild type Stoml3 locus, and the mutated Stoml3lacZ allele, before and after removal of the self-excision neomycin (cre, neo) cassette. A 12 kb genomic region of Stoml3 locus containing exon 1 (E1, black), NLS-lacZ (blue), DTA (yellow), the self-excision neomycin cassette, loxP (red arrowhead), and SpeI (S) restriction sites are depicted. Green lines indicate the predicted fragment sizes obtained after SpeI digestion of genomic DNA. A green bar shows the 5’ sequence used as a probe for Southern blot analyses shown in b. Blue lines indicate the predicted fragment sizes obtained by genotyping the tail genomic DNA as shown in c. (b) Southern blot analysis of SpeI digested tail genomic DNA from Stoml3+/lacZ and wild type mice. (c) Genotyping analysis of tail genomic DNA from Stoml3+/lacZ, wild type, and Stoml3lacZ/lacZ mice. Stoml3-LacZ F and LacZ int R primers amplified a 649 bp fragment from the Stoml3lacZ mutant allele. Stoml3-LacZ F and Stoml3-LacZ R primers amplified a 875 bp fragment from the Stoml3 wild type allele, M: 100 bp ladder (Invitrogen). (d) Schematic representation of the targeting vector, the wild-type Stoml3 locus, and the mutated Stoml3StrepII allele, before and after removal of the neomycin (neo) cassette. A 12 kb genomic region of Stoml3 locus containing exon 1 (E1, black), Strep-TagII (red), DTA (yellow), the neomycin cassette (neo), loxP (blue arrowhead), and SpeI (S) restriction sites are depicted. Green lines indicate the predicted fragment sizes obtained after SpeI digestion of genomic DNA. A green bar shows the 5’ sequence used as a probe for Southern blot analyses shown in e. Blue lines indicate the predicted fragment sizes obtained by genotyping the tail genomic DNA as shown in f. (e) Southern blot analysis of SpeI digested tail genomic DNA from wild type, Stoml3+/StrepII and Stoml3StrepII/StrepII mice. (f) Genotyping analysis of tail genomic DNA from wild type, Stoml3+/StrepII, and Stoml3StrepII/StrepII mice. Stoml3-Strep F and Stoml3-Strep R2 primers amplified a 321 bp fragment from the Stoml3StrepII mutant allele. Stoml3-Strep F and Stoml3-Strep R1 primers amplified a 438 bp fragment from the Stoml3 wild type allele, M: 100 bp ladder (Invitrogen).

  16. Uncropped western blots.
    Supplementary Fig. 9: Uncropped western blots.

    Uncropped pictures of the Western blot shown in the manuscript in Figure 6. * unknown bands; LO: left-over of 55-70kDa bands shown in the top panel after stripping the blot.

Change history

Corrected online 09 January 2017
When this article was first published online, a version of the reporting checklist supplementary file with information pertaining to a previous version of this manuscript, which does not match the information presented in the published article, was posted by mistake. As of 9 January 2017, this mismatched file has been replaced by a file that matches the information in the article.

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Author information

  1. Present address: Neurobiology Group, SISSA, International School for Advanced Studies, Trieste, Italy.

    • Simone Pifferi
  2. These authors contributed equally to this work.

    • Christiane Wetzel &
    • Kate Poole

Affiliations

  1. Department of Neuroscience, Max Delbrück Center for Molecular Medicine, Berlin, Germany.

    • Christiane Wetzel,
    • Simone Pifferi,
    • Cristina Picci,
    • Caglar Gök,
    • Diana Hoffmann,
    • Liudmila Lapatsina,
    • Raluca Fleischer,
    • Ewan St John Smith,
    • Valérie Bégay,
    • Mirko Moroni,
    • Luc Estebanez,
    • Johannes Kühnemund,
    • Jan Walcher,
    • James F A Poulet,
    • Kate Poole &
    • Gary R Lewin
  2. Department of Biomedical Sciences, Section of Cytomorphology, University of Cagliari, Monserrato (California), Italy.

    • Cristina Picci
  3. Neuroscience Research Center and Cluster of Excellence NeuroCure, Charité-Universitätsmedizin, Berlin, Germany.

    • Diana Hoffmann,
    • Luc Estebanez,
    • James F A Poulet &
    • Gary R Lewin
  4. Institute of Pharmacology, Heidelberg University, Heidelberg, Germany.

    • Kiran K Bali &
    • Rohini Kuner
  5. Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin, Germany.

    • André Lampe,
    • Edgar Specker,
    • Martin Neuenschwander,
    • Jens Peter von Kries &
    • Volker Haucke
  6. Department of Pharmacology, University of Cambridge, Cambridge, UK.

    • Ewan St John Smith
  7. Freie Universität Berlin, Berlin, Germany.

    • Jan Schmoranzer
  8. Department of Physiology and EMBL Australia Node for Single Molecule Science, School of Medical Sciences, UNSW, Sydney, Australia.

    • Kate Poole

Contributions

K.P. designed and carried out the screen and characterized small molecules with dSTORM and patch clamp electrophysiology. C.W. performed ex vivo skin electrophysiology and experiments in mice and behavioral experiments. S.P. screened OB-1 for effects on mechanosensitive currents in DRGs. C.P. performed behavioral and real-time PCR experiments and performed histochemical analysis of the Stoml3lacZ mice. C.G. determined IC50s using the pili method. D.H. performed touch perception assays with L.E., who established the methodology. K.K.B. and R.K. established the diabetic neuropathy model and performed and analyzed behavioral experiments. A.L. and K.P. performed and analyzed dSTORM experiments. L.L., V.B., K.P., C.P. and J.W. generated and characterized the Stoml3lacZ and Stoml3StrepII mice. L.L. and R.F. performed molecular cloning experiments. E.St.J.S. performed ASIC experiments. M.M. performed additional electrophysiological experiments. J.K. analyzed transmission electron microscopy data. E.S. synthesized molecules and managed compound libraries. M.N. performed statistical analyses of high-throughput screening data and helped in design and execution of the screen. J.P.v.K. supervised screening experiments. J.F.A.P. established touch-perception assays and supervised the acquisition and analysis of the data. V.H. and J.S. directed and supervised imaging experiments. K.P., C.W. and G.R.L. wrote the paper. K.P., C.W. and G.R.L. conceived and directed the project.

Competing financial interests

G.R.L., K.P., C.W. and L.L. are named as inventors on a patent application related to data in this paper.

Corresponding authors

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Author details

Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Effects of STOML3-modulating molecules on other stomatin-domain proteins (56 KB)

    (a) BiFC signal development observed when cells were transfected with stomatin –VC/-VN, STOML1-VC/-VN, STOML2-VC/-VN, STOML3-VC/-VN or podocin-VC/-VN expression constructs respectively. The slope of signal development was used as a measure of oligomerization. Note that OB-1 significantly inhibits oligomerization of stomatin, STOML1, STOML2 and STOML3 but does not affect podocin oligomerization. Unpaired t-test: stomatin vs. stomatin + OB-1, P < 0.0001; STOML1 vs. STOML1 + OB-1, P < 0.0001; STOML2 vs. STOML2 + OB-1, P < 0.0001; STOML3 vs. STOML3+ OB-1, P < 0.0001; Mann-Whitney U-test: podocin vs. podocin + OB-1, P = 0.0584; numbers indicate replicates derived from 2 independent cell transfections; data are shown as individual slopes and mean ± s.e.m.. (b) OB-1 was tested at two different concentrations (2 μM and 20 μM) using the BiFC assay. The normalized slope was significantly reduced when OB-1 was present. Unpaired t-test: vehicle vs. 20 μm OB-1, P < 0.0001; vehicle vs. 2 μm OB-1, P < 0.0001; numbers indicate replicates derived from two independent cell transfections; data are displayed as individual slopes and mean ± s.e.m. *** P < 0.001; ns nonsignificant.

  2. Supplementary Figure 2: Stoml3 mRNA levels after inhibitor treatment and quantitative analysis of mechanically gated currents. (89 KB)

    (a) N2a cells and acutely isolated DRGs were treated with either vehicle or OB-1 (20 μM) for 3 hours before mRNA was isolated, reverse transcribed and analyzed using qPCR. There were no detectable differences in Stoml3 transcript levels; numbers indicate independent RNA preparations derived from 3 N2a cell preparations and from two mice per DRG preparation; data are displayed as individual data points and mean ± s.e.m.. (b) Time course analysis of OB-1 activity in N2a cells. Cells were treated with 20 μM OB-1 for the indicated time and mechanotransduction was monitored using pillar arrays. Treatment of at least 3 hours was required for maximum inhibition of mechanically-gated currents. Mann-Whitney U-test: 0-1h vs. > 3h OB-1, P = 0.0003. Unpaired t-test: 1-2h vs. > 3h OB-1, P = 0.0033; 2-3h vs. >3h OB-1, P = 0.0132; numbers indicate currents measured from N2a cells derived from more than 6 independent experiments; data are shown as current amplitude, each bin is displayed as mean of cell averages ± s.e.m.. (c) Representative current trace with latency (magenta) activation time constant (τ1, blue) and inactivation time constant (τ2, green) indicated by dashed lines. The inactivation time constant of mechanically gated currents in DRG mechanoreceptors (mec) shown here was measured using elastomeric pillar arrays. There is a trend for longer inactivation time constants that is not significant. Mann-Whitney U-test: mec vs. mec + OB-1, P = 0.1966; mec vs. mec + OB-2, P = 0.7930; data are displayed as box and whisker plot, fit of inactivation of individual curve, displayed as median with 5-95 interquartiles. (d) In nociceptors (noci) treatment with OB-2 led to significantly longer latencies and significantly slower activation time constants compared to non-treated noci. Mann-Whitney U-test: vehicle vs. OB-2 (Latency), P = 0.0315; vehicle vs. OB-2 (activation time constant), P = 0.001; data are displayed as box and whisker plot, fit of inactivation of individual curve, displayed as median with 5-95 interquartiles. * P < 0.05; ** P < 0.01; *** P < 0.001.

  3. Supplementary Figure 3: Effects of OB-1 on mechanically-activated currents in mouse sensory neurons. (62 KB)

    (a) Schematic diagram of a large diameter mouse sensory neuron with recording electrode (RE) and nanostimulator (NS). (b) Typical RA-mechanosensitive current evoked from mouse sensory neurons by poking the cell soma. (c) Typical RA-mechanosensitive currents evoked from neurons pre-incubated with OB-1 or vehicle. Red lines indicate the exponential fit of the current inactivation to determine τ2. (d) Distribution of cells found with and without a mechanically-activated current (MA current) in both groups. Note the significant loss of mechanically-activated currents in OB-1 treated neurons. Fisher's exact test: vehicle vs. OB-1, P = 0.009; numbers indicate neurons recorded, data are displayed as percentage of neurons. (e) The mean of inactivation time constants (τ2) of mechanically-gated currents is shown; note that τ2 is slower in OB-1 treated neurons when compared to controls. Mann-Whitney U-test: vehicle vs. OB-1, P = 0.0056; numbers indicate individual recordings; data are displayed as individual τ2 and mean ± s.e.m and were recorded from DRG neurons in at least 9 independent experiments. ** P < 0.01.

  4. Supplementary Figure 4: OB-1 has no effect upon ASIC3-mediated currents nor on stomatin inhibition of ASIC3. (51 KB)

    (a) Neither the transient (T), nor sustained (S) phases of pH-evoked currents in CHO cells expressing ASIC3 were inhibited by OB-1 (20 μM). Student’s t-test: P = 0.2452, P = 0.7493; numbers indicate cells recorded (9 Vehicle, 8 OB-1), data are displayed as individual data points and mean peak current density ± s.e.m.. (b) Stomatin inhibits ASIC3-mediated pH gated currents, which is not modulated by OB-1. Student’s t-test, P = 0.4392, P = 0.8650; numbers indicate cells recorded (7 Vehicle, 7 OB-1); data are displayed as individual data points and mean peak current density ± s.e.m..

  5. Supplementary Figure 5: Non silenced mechanoreceptors are functional after OB-1 treatment. (121 KB)

    (a) The proportion of each class of mechanosensitive afferents recorded three hours after local OB-1 or vehicle application is displayed. After OB-1 treatment no significant differences were observed in the receptor proportions among Aβ-, Aδ- or C-fibers compared to vehicle treated skin. Fisher's exact test: Aβ-fibers OB-1 vs. vehicle, P = 0.1722; Aδ-fibers OB-1 vs. vehicle, P = 0.8199; C-fibers OB-1 vs. vehicle, P = 1.0000; numbers indicate single sensory fiber recordings derived from more than 10 independent experiments using adult mice; data are displayed as percentage of individual fibers. (b-e) Receptive field properties of single sensory afferents recorded from vehicle-treated or OB-1 treated skin are shown. (b-d) A series of ramp and hold stimuli with increasing velocities (0.075, 0.15, 0.45, 1.5 and 15 mm/s at 92 μm displacements) was applied to low threshold mechanoreceptors, i.e. slowly adapting mechanoreceptors (SAM) (b), rapidly adapting mechanoreceptors (RAM) (c) and D-hair receptors (d). Mean firing frequencies during the ramp phase are plotted as function of stimulus velocity; numbers indicate fibers recorded; data are displayed as mean number of action potentials ± s.e.m.. (e) An ascending series of displacement stimuli (32 – 1024 μm) using a constant stimulus velocity was applied to A-mechanonociceptors (A-M). Mean firing frequencies were plotted as function of displacement amplitudes showing no changes in mechanosenitivity in A-Ms in OB-1 treated compared to vehicle-treated skin. Two-way ANOVA: SAM vehicle vs. OB-1, P = 0.8924; RAM vehicle vs. OB-1, P = 0.565; D-hair vehicle vs. OB-1, P = 0.8437; A-M vehicle vs. OB-1, P = 0.0912; numbers indicate single sensory fiber recordings derived from more than 5 independent experiments using adult mice; data are displayed as mean number of action potentials ± s.e.m..

  6. Supplementary Figure 6: Ultrastructure of the sciatic nerve after unilateral CCI. (158 KB)

    (a,d) Schematic drawings of the section plane. (b,c,e,f) High magnification of electron micrograph images of the ipsilateral (b,e) or contralateral side (c,f) proximal and distal to the ligation. (b,c) Proximal to the injury normal myelinated and unmyelinated nerve fibers with typical ultrastructure and intact myelin sheaths or Remak bundles are seen. (e,f) Distal to the injury strong demyelination and axonal degeneration were observed only ipsilateral to the injury. Scale bar = 2 μm, SC spinal cord, SN sciatic nerve.

  7. Supplementary Figure 7: OB-1 treatment in additional pain models. (135 KB)

    (a) Development of tactile-evoked pain using the spared nerve injury (SNI) model. Paw withdrawal thresholds (PWTs) are displayed; note that wild type mice develop a prolonged tactile-evoked pain ipsilateral to the injury. Two-way ANOVA: SNI, ipsi vs. Sham, ipsi, P < 0.0001; numbers indicate adult mice treated (two cohorts); data are displayed as mean of individual median PWTs ± s.e.m.. (b) PWTs are displayed, showing no alleviation of tactile-evoked pain behavior after local OB-1 treatment. Mann-Whitney U-test: SNI vs. OB-1, P > 0.9999; numbers indicate adult mice treated (two cohorts); data are displayed as mean of individual median PWTs ± s.e.m.. (c) Stoml3 copy number derived from lumbar DRG L4 - L6 (two mice per preparation) determined using real-time PCR showing no up-regulation of Stoml3 mRNA after SNI. Mann-Whitney U-test: naïve, ipsi vs. SNI, ipsi, P > 0.9999; numbers indicate RNA preparations, data are displayed as mean copy number ± s.e.m.. (d-g) A single dose of NGF (1 mg/kg body weight) was injected i.p. into adult mice to induce hyperalgesia in wild type and Stoml3-/- mice. (d,e) PWT or paw withdrawal latencies (PWL) are displayed before and after NGF-induced hyperalgesia in wild type and Stoml3-/- mice showing that prominent symptoms of thermal and mechanical hyperalgesia were not different between the genotypes. (f,g) Both, PWTs and PWLs, were measured in response to OB-1 before and after systemic NGF-injection, note that OB-1 does not alleviate NGF-induced mechanical (g) or thermal (h) hyperalgesia. Two-way ANOVA: PWT WT vs. Stoml3-/- P = 0.6067 (g); PWL WT vs. Stoml3-/- (e) P = 0.1078. Mann-Whitney U-test: PWT vehicle paw naïve vs. OB-1 paw naive, P = 0.3615; PWL Vehicle paw naïve vs. OB-1 paw naïve, P = 0.699. Wilcoxon matched-pairs signed rank test: PWT syst. NGF vs. syst. NGF + OB-1 (f), P = 0.125; PWL syst. NGF vs. syst. NGF + OB-1, P = 0.1563; numbers indicate adult mice examined; data are displayed as mean of individual median PWTs (d,f) or PWLs (e,g); error bars indicate s.e.m.. *** P < 0.001 ns nonsignificant.

  8. Supplementary Figure 8: Generation of the Stoml3lacZ and Stoml3StrepII mouse strains (107 KB)

    (a) Schematic representation of the targeting vector, the wild type Stoml3 locus, and the mutated Stoml3lacZ allele, before and after removal of the self-excision neomycin (cre, neo) cassette. A 12 kb genomic region of Stoml3 locus containing exon 1 (E1, black), NLS-lacZ (blue), DTA (yellow), the self-excision neomycin cassette, loxP (red arrowhead), and SpeI (S) restriction sites are depicted. Green lines indicate the predicted fragment sizes obtained after SpeI digestion of genomic DNA. A green bar shows the 5’ sequence used as a probe for Southern blot analyses shown in b. Blue lines indicate the predicted fragment sizes obtained by genotyping the tail genomic DNA as shown in c. (b) Southern blot analysis of SpeI digested tail genomic DNA from Stoml3+/lacZ and wild type mice. (c) Genotyping analysis of tail genomic DNA from Stoml3+/lacZ, wild type, and Stoml3lacZ/lacZ mice. Stoml3-LacZ F and LacZ int R primers amplified a 649 bp fragment from the Stoml3lacZ mutant allele. Stoml3-LacZ F and Stoml3-LacZ R primers amplified a 875 bp fragment from the Stoml3 wild type allele, M: 100 bp ladder (Invitrogen). (d) Schematic representation of the targeting vector, the wild-type Stoml3 locus, and the mutated Stoml3StrepII allele, before and after removal of the neomycin (neo) cassette. A 12 kb genomic region of Stoml3 locus containing exon 1 (E1, black), Strep-TagII (red), DTA (yellow), the neomycin cassette (neo), loxP (blue arrowhead), and SpeI (S) restriction sites are depicted. Green lines indicate the predicted fragment sizes obtained after SpeI digestion of genomic DNA. A green bar shows the 5’ sequence used as a probe for Southern blot analyses shown in e. Blue lines indicate the predicted fragment sizes obtained by genotyping the tail genomic DNA as shown in f. (e) Southern blot analysis of SpeI digested tail genomic DNA from wild type, Stoml3+/StrepII and Stoml3StrepII/StrepII mice. (f) Genotyping analysis of tail genomic DNA from wild type, Stoml3+/StrepII, and Stoml3StrepII/StrepII mice. Stoml3-Strep F and Stoml3-Strep R2 primers amplified a 321 bp fragment from the Stoml3StrepII mutant allele. Stoml3-Strep F and Stoml3-Strep R1 primers amplified a 438 bp fragment from the Stoml3 wild type allele, M: 100 bp ladder (Invitrogen).

  9. Supplementary Figure 9: Uncropped western blots. (175 KB)

    Uncropped pictures of the Western blot shown in the manuscript in Figure 6. * unknown bands; LO: left-over of 55-70kDa bands shown in the top panel after stripping the blot.

PDF files

  1. Supplementary Text and Figures (1,767 KB)

    Supplementary Figures 1–9 and Supplementary Tables 1 and 2

  2. Supplementary Methods Checklist (370 KB)

Excel files

  1. Supplementary Dataset 1 (48 KB)

    Excel data sheet of results of in vitro screen for pharmacological activity of OB1 on a panel of 79 receptors.

Additional data