Tuning PAK Activity to Rescue Abnormal Myelin Permeability in HNPP

<div><p>Schwann cells in the peripheral nervous systems extend their membranes to wrap axons concentrically and form the insulating sheath, called myelin. The spaces between layers of myelin are sealed by myelin junctions. This tight insulation enables rapid conduction of electric impulses (action potentials) through axons. Demyelination (stripping off the insulating sheath) has been widely regarded as one of the most important mechanisms altering the action potential propagation in many neurological diseases. However, the effective nerve conduction is also thought to require a proper myelin seal through myelin junctions such as tight junctions and adherens junctions. In the present study, we have demonstrated the disruption of myelin junctions in a mouse model (<i>Pmp22</i>+/-) of hereditary neuropathy with liability to pressure palsies (HNPP) with heterozygous deletion of <i>Pmp22</i> gene. We observed a robust increase of F-actin in <i>Pmp22</i>+/- nerve regions where myelin junctions were disrupted, leading to increased myelin permeability. These abnormalities were present long before segmental demyelination at the late phase of <i>Pmp22</i>+/- mice. Moreover, the increase of F-actin levels correlated with an enhanced activity of p21-activated kinase (PAK1), a molecule known to regulate actin polymerization. Pharmacological inhibition of PAK normalized levels of F-actin, and completely prevented the progression of the myelin junction disruption and nerve conduction failure in <i>Pmp22</i>+/- mice. Our findings explain how abnormal myelin permeability is caused in HNPP, leading to impaired action potential propagation in the absence of demyelination. We call it “functional demyelination”, a novel mechanism upstream to the actual stripping of myelin that is relevant to many demyelinating diseases. This observation also provides a potential therapeutic approach for HNPP.</p></div

Schematic illustration of myelin junction disruption.

<p><b>(A)</b> Mechanism of functional demyelination (modified from Guo et al, Ann Neurol 2014): Myelin junctions in <i>Pmp22</i>+/+ nerve are in non-compact myelin regions, including paranodes, incisures and mesaxons. These junctions seal the spaces between myelin lamina. A <i>Pmp22</i>+/- nerve fiber is depicted and develops a tomaculae in the left paranode extending into juxtaparanode and internode, but there is no segmental demyelination. However, junction protein complexes are disrupted or disappeared in the non-compact myelin. These junction proteins may be found in aberrant locations, including perinuclear areas or tomaculous myelin. Abnormal junctions in <i>Pmp22</i>+/- nerves increase myelin permeability (or increase of capacitance). <b>(B)</b> Molecular architecture of junction protein complex: Transmembrane proteins establish "trans-adhesion" between opposing membranes. Through adaptor proteins, such as ZO1/2 or catenins, these junction protein complexes are stabilized by sub-membrane actin networks. Alteration of the actin network has been shown to disassemble junctions in epithelial cell models [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006290#pgen.1006290.ref029" target="_blank">29</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006290#pgen.1006290.ref030" target="_blank">30</a>].</p

PAK1 inhibitor PF-3758309 is therapeutic in the <i>Pmp22+/-</i> mice

<p>PAK1 inhibitor PF-3758309 is therapeutic in the <i>Pmp22+/-</i> mice</p

Disruption of myelin junctions in <i>Pmp22+/-</i> nerves during aging.

<p>Paraffin sections of mouse sciatic nerves were stained with antibodies. Percentages of abnormal paranodes or incisures were manually counted [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006290#pgen.1006290.ref005" target="_blank">5</a>]. An abnormal paranode or incisurae was defined as the staining was absent in more than a half of normally stained paranodal or incisure territory. <b>(A)</b> E-cadherin antibody stained paranodes (red-color stained areas adjacent to the node marked by an arrowhead in <b>A1</b>) in 3-month-old <i>Pmp22</i>+/+ mouse nerve fiber but showed no signal in <i>Pmp22</i>+/- paranodes (<b>A2</b>). White dots outline the margin of the nerve fiber, based on its phase-contrast image. A strong E-cadherin band at the <i>Pmp22</i>+/- node (arrowhead in <b>A2</b>) was presumably due to an ectopic expression in Schwann cell microvilli. E-cadherin antibodies also stained <i>Pmp22</i>+/+ incisures (arrow in <b>A3</b>) but showed minimal signals in <i>Pmp22</i>+/- incisures (arrow in <b>A4</b>). Scale bars = 10μm. <b>(B)</b> There was a significant increase of abnormal E-cadherin-stained paranodes and incisures from 1 month of age onward (n = 140–340 paranodes and 800–1,700 incisures from either 3 <i>Pmp22+/+</i> or 3 <i>Pmp22+/-</i> mice at each age group). *<i>P</i> < 0.01, ** <i>P</i> < 0.0001. <b>(C)</b> Mag staining was present in the paranodes (<b>C1</b>) and incisures (arrow in <b>C3</b>) of 5-month-old <i>Pmp22+/+</i> nerves but decreased in <i>Pmp22</i>+/- paranodes (<b>C2</b>) or incisures (arrow in <b>C4</b>). Scale bars = 10μm. <b>(D)</b> There was a significant increase of abnormal Mag-stained paranodes or incisures from 5 month of age onward (n = 160–300 paranodes and 900–1,500 incisures from 3 <i>Pmp22+/+</i> and 3 <i>Pmp22+/-</i> mice at each age group). *<i>P</i> < 0.01, ** <i>P</i> < 0.0001. <b>(E)</b> Teased sciatic nerve fibers of <i>Pmp22</i>+/+ mice at the 3 months of age were stained with antibodies against claudin-19 to show paranodes (<b>E1)</b> and incisures (arrow in <b>E3</b>). The staining was reduced in <i>Pmp22</i>+/- paranodes (<b>E2</b>) and incisures <b>(E4</b>). Scale bars = 10μm. <b>(F)</b> There was a significant difference found in paranodes from 3 months of age onward and in incisures from 1 month of age onward (n = 280–380 paranodes and 800–1,200 incisures from 3 <i>Pmp22+/+</i> and 3 <i>Pmp22+/-</i> mice at each age group). *<i>P</i> < 0.01, ** <i>P</i> < 0.0001.</p

A proposed mechanism for junction disruption in HNPP.

<p><u>Initiation</u>—PMP22 and E-cadheren travel via the secretory pathway from endoplasmic reticulum (ER)/Golgi apparatus to cytoplasmic membrane [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006290#pgen.1006290.ref036" target="_blank">36</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006290#pgen.1006290.ref037" target="_blank">37</a>]. PMP22 has been reported to regulate the endocytosis of E-cadheren via Arf6 (an ATPase) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006290#pgen.1006290.ref038" target="_blank">38</a>]. PMP22 may also form a protein complex with E-cadherin during the secretory pathway. Deficiency of PMP22 would affect the transport processes of E-cadherin or other junction proteins, thereby leading to the abnormal formation of junction complex. <u>Perpetuation</u>—β-catenin in adherens junction protein complex has been shown to interact with PAK1 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006290#pgen.1006290.ref023" target="_blank">23</a>]. Abnormal formation of junction complex activates PAK1 in <i>Pmp22</i>+/- Schwann cells (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006290#pgen.1006290.g006" target="_blank">Fig 6B</a>), which further promotes disruption of junction protein complexes. PAK1 is known to regulate actin polymerization [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006290#pgen.1006290.ref017" target="_blank">17</a>]. Thus, activation of PAK1 may disrupt the junctions via actin polymerization.</p

PAK1 inhibitor is therapeutic in <i>Pmp22+/-</i> mice.

<p><b>(A)</b> NCS on mouse sciatic nerves showed significantly higher CMAP amplitude in 3-month-old <i>Pmp22+/-</i> mice treated with 0.25–1.0 mg/kg PF-3758309 (n = 42) for 11 weeks, compared with that in the vehicle group (n = 24). There was a trend of dose-dependent change. *<i>P</i> < 0.05. <b>(B)</b> This difference of CMAP amplitudes was also found between 7-12-month-old <i>Pmp22+/-</i> mice treated with 0.25 mg/kg PF-3758309 and the vehicle group. CMAP was measured every 10 days. By the end of one month, CMAP amplitudes were already significantly different between the treated and vehicle groups. Thus, the treatment was stopped at this point. The baseline CMAP amplitudes prior to the treatment were not different between the two groups but decreased over the course of treatment in the vehicle group and unchanged in the PF-3758309 group. ** <i>P</i> < 0.01. <b>(C)</b> Teased nerve fibers from 3-month-old <i>Pmp22+/-</i> mice were stained with the fluorescence-phalloidin to reveal F-actin. A nerve fiber from a PF-3758309-treated mouse showed a lower intensity of F-actin fluorescence when compared with that in a nerve fiber from a vehicle-treated mouse. Arrowheads point to the node of Ranvier, which are flanked by paranodes on each side. Scale bars = 10μm. <b>(D)</b> Fluorescence intensity of F-actin staining was quantified by placing 2.5μm x 2.5μm interest box 10μm away from the node of Ranvier. The intensity was compared between the PF-3758309 treated group and the vehicle group (n = 65–92 analyzed paranodes from 3 vehicle mice and 3 PF-3758309 treated mice). Note that a high level of F-actin in 3-month-old mice correlates well with a high level of PAK1 activity in the mice at the same age (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006290#pgen.1006290.g005" target="_blank">Fig 5A</a>). ** <i>P</i> < 0.01, *** <i>P</i> < 0.001. <b>(E)</b> Sciatic nerve fascicles from 3-month-old <i>Pmp22+/-</i> mice were incubated with 3kDa Dextran, as described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006290#pgen.1006290.ref005" target="_blank">5</a>], to evaluate the myelin permeability. Individual teased nerve fibers were imaged. Arrowheads point to the node of Ranvier. Notice that a nerve fiber from a mouse treated with vehicle showed higher fluorescence intensity than that in a nerve fiber from a mouse treated with PF-3758309. Scale bars = 10μm. <b>(F)</b> Fluorescence intensity was quantified by placing a 2.5μm x 2.5μm interest box 10μm away from the middle point of the node of Ranvier. The intensity was significantly decreased in 3-month-old PF-3758309-treated nerve fibers, compared to those from the vehicle group (n = 495–521 analyzed paranodes from 3 vehicle mice and 3 PF-3758309-treated mice). *** <i>P</i> < 0.001.</p

Abnormally increased actin polymerization in the regions where myelin junctions reside.

<p><b>(A)</b> Teased nerve fibers of mouse sciatic nerves were stained with fluorescent phalloidin, which was localized at nodes (arrowheads), incisures (arrows) and mesaxons (asterisks). F-actin was strongly expressed in <i>Pmp22+/-</i> nerves. Images in the 3^rd row were taken under confocal microscopy. The maximal projection of z-stack images was presented to show the mesaxon changes of F-actin at different layers. Scale bars = 10μm. <b>(B-C)</b> Fluorescence intensity was quantified by placing 2.5μm x 2.5μm interest box 10μm away from the node of Ranvier and by including the entire area of every incisures. The intensity of F-actin staining was increased in <i>Pmp22</i>+/- paranodes and incisures from 3 months of age onward (n = 40–50 paranodes, 60–70 incisures from 3 <i>Pmp22+/+</i> and 3 <i>Pmp22+/-</i> mice at each age group). ** <i>P</i> < 0.0001; M = month. <b>(D</b>). The mesaxons with clearly visible F-actin-staining (asterisk in <b>A</b>) were counted in teased nerve fibers of <i>Pmp22+/+</i> and <i>Pmp22+/-</i> mice. The F-actin stained mesaxons in <i>Pmp22</i>+/- mice were increased from 3 month of age onward (n = 75 mesaxons from 3 <i>Pmp22+/+</i> and 3 <i>Pmp22+/-</i> mice at each age group). ** <i>P</i> < 0.0001; M = month. <b>(E)</b> Western blot analysis of F-actin was performed in the sciatic nerves of 3 month-old <i>Pmp22+/+</i> and <i>Pmp22+/-</i> mice. <b>(F)</b> The levels of F-actin were significantly increased in <i>Pmp22+/-</i> nerves, compared with those in <i>Pmp22+/+</i> nerves. *<i>P</i> < 0.05. <b>(G)</b> <i>Pmp22+/+</i> and <i>Pmp22+/-</i> sciatic nerve explants were cultured for 3 hours in the presence of jasplakinolide (Jas) and double-stained with fluorescent phalloidin and an anti-Pan-Neurofascin antibody to label incisures. A group of explants was washed following Jas treatment and cultured for another 6 hours in jasplakinolide-free medium. The newly formed F-actin was strongly increased in <b>G6</b>. Scale bars = 10μm. <b>(H)</b> Fluorescence intensity was quantified by including the entire area of each incisures. The intensity of new F-actin was increased in 3 month-old <i>Pmp22</i>+/- incisures, compared with those in <i>Pmp22</i>+/+ nerve fibers (n = 120 incisures from 3 <i>Pmp22+/+</i> and 3 <i>Pmp22+/-</i> mice; Scale bars = 5μm). ** <i>P</i> < 0.0001.</p

PAK1 activity is increased in <i>Pmp2</i>2+/- nerves.

<p><b>(A)</b> Western blot of phosphorylated PAK1 (T212) and total PAK1 (t-PAK1) in the sciatic nerves from 0.5–5 month-old <i>Pmp22+/+</i> and <i>Pmp22+/-</i> mice. Both T212 and t-PAK1 were not detectable in the sciatic nerves of <i>Pak1</i>-/- mice (line 9). <b>(B)</b> T212 level was normalized against t-PAK1 levels. T-PAK1 level was normalized against β-Tubulin levels. The levels of T212, but not t-PAK1 levels, were significantly increased in <i>Pmp22+/-</i> nerves, compared with those in <i>Pmp22+/+</i> nerves. *<i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001. <b>(C)</b> Western blot of S144 in the sciatic nerves of 3 month-old <i>Pmp22+/+</i> and <i>Pmp22+/-</i> mice. S144 were not detectable in the sciatic nerves of <i>Pak1</i>-/- mice (line 4). <b>(D)</b> S144 levels were normalized against t-PAK1 levels. S144 level was not significantly different between <i>Pmp22+/+</i> and <i>Pmp22+/-</i> nerves. <b>(E)</b> Western blot for phosphorylated MEK1 (S298) and total MEK1 (t-MEK1) in the sciatic nerves of 3 month-old <i>Pmp22+/+</i> and <i>Pmp22+/-</i> mice. <b>(F)</b> S298 levels were normalized against t-MEK1 levels. S298 levels were significantly increased in <i>Pmp22+/-</i> nerves, compared with those in <i>Pmp22+/+</i> nerves. *** <i>P</i> < 0.001. <b>(G)</b> Longitudinal (G1, G5) and transverse (G2, G6) sections of sciatic nerves were stained with antibodies against PAK1. The staining was superimposed with phase-contrast images (G3, G4), which showed PAK1 located in myelin and axons. PAK1 were not detectable in the sciatic nerves of <i>Pak1</i>-/- mice (G7). Scale bars = 10μm.</p

PAK inhibitor PF-3758309 blocks actin polymerization via PAK1.

<p>F-actin, phosphorylated PAK1 (S144, T212) and phosphorylated PAK2 (S20) were analyzed in <i>Pak1+/+</i> and <i>Pak1-/-</i> primary Schwann cell culture after the cells were treated with PF-3758309 (9μM) for 12 hour. PF-3758309 suppressed the levels of F-actin in <i>Pak1+/+</i> Schwann cells but failed to do so in <i>Pak1-/-</i> Schwann cells. The total actin and GAPDH were used as loading controls. Note that the specificity of S20 antibody has been demonstrated by Zhan et al [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006290#pgen.1006290.ref042" target="_blank">42</a>].</p

Conduction block was detected in naïve <i>Pmp22</i>+/- nerves.

<p><b>(A)</b> A diagram shows the setting for the experiments. A1-5 indicates the sites where the stimulation electrodes were placed on surgically exposed sciatic nerve. <b>(B)</b> In conventional NCS, <u>proximal</u> stimulation electrode is inserted blindly into the sciatic notch (white arrow in B). Variations of distances between the electrode and sciatic nerve (array of white asterisks) are not avoidable. This variation was eliminated by surgically exposing the sciatic nerves. Two black dots indicate the sites where <u>distal</u> stimulation electrodes were placed around ankle. <b>(C)</b> Area nearby ankle was dissected to reveal the tibial nerve (arrow in C). Due to the tiny space of this area, distance between the electrode and tibial nerve was highly consistent (two asterisks represent the sites of black dots in B). Thus, it did not require surgical exposure to place the distal stimulation electrodes. Note that needle electrode at the asterisk sites was inserted just through the dermis to avoid any nerve injury. <b>(D)</b> CMAP amplitudes were similar between A1 to A4. <b>(E)</b> CMAP in a <i>Pmp22</i>+/- mouse at A3-A5 showed a >50% reduction of the A2 amplitude. This finding demonstrated a conduction block that was defined as a ≥50% decrease of proximal CMAP amplitude over the distal CMAP amplitude, a stringent criterion used in human NCS [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006290#pgen.1006290.ref041" target="_blank">41</a>]. Conduction block was found in 12 out of 17 studied <i>Pmp22</i>+/- mice, but not in <i>Pmp22</i>+/+ mice. (<b>F</b>) CMAP was recorded from a different mouse and showed a distal latency (3.3ms) 2 times longer than that (1.2ms) in <i>Pmp22</i>+/+ nerve (A2 in D). The doubled distal latency was found in 2 mice out of the 17 <i>Pmp22</i>+/- mice, while the remaining 15 mice had variable degrees of prolonged distal latency. <b>(G)</b> CMAP in this mouse had a duration of 4ms (temporal dispersion) that was about twice longer than that in <i>Pmp22</i>+/+ nerve (A1 in D). In average, the CMAP duration in 17 <i>Pmp22</i>+/- mice (3.9±1.7ms) was significantly longer than that in 7 <i>Pmp22</i>+/+ mice (2.3±0.4ms; p = 0.001; 3–10 month old).</p