article: 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 (Pmp22+/-) of hereditary neuropathy with liability to pressure palsies (HNPP) with heterozygous deletion of Pmp22 gene. We observed a robust increase of F-actin in Pmp22+/- 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 Pmp22+/- 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 Pmp22+/- 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.
  Depolarizing current at the node of Ranvier is typically five times higher than the minimum required to trigger the action potential. This surplus is called the “safety factor” [1]. The safety factor is secured by the wrapping of the glial cell membrane around axons to produce the insulating sheath, called myelin. Schwann cells in the peripheral nervous system form the myelin during early development. However, the maturation process may extend over the first 5 years of human life when the conduction speed of action potential reaches the level of adulthood. The safety factor resulted from the insulation of myelin may be impaired in a variety of neurological diseases, usually as a result of the removal of myelin (demyelination). Denuded axons shunt the depolarizing current out of nerve fibers, leading to either a reduction of conduction velocity or complete failure of action potential propagation. The latter is called “conduction block” and produces focal sensory loss and/or limb paralysis [2,3]. Although demyelination is widely regarded as one of the most important mechanisms altering the safety factor, effective nerve conduction is also thought to require a proper myelin seal through myelin junctions such as tight junctions and adherens junctions. These junctions seal the spaces between adjacent myelin lamellae and between the myelin and axolemma [4]. We have observed excessively permeable myelin (i. e. , an increase of capacitance) in a mouse model of hereditary neuropathy with liability to pressure palsies (HNPP) due to disruption of these myelin junctions. This novel mechanism impairs action potential propagation in the absence of demyelination [5]. We call it “functional demyelination” (Fig 1A). Thus, this mechanism denotes pathological processes that may alter the insulating quality of myelin without physically stripping off the myelin sheath. HNPP is caused by a heterozygous deletion of PMP22 gene in human chromosome 17p12. PMP22 encodes a tetra-span membrane protein primarily expressed in peripheral nerve myelin [6–8]. Mice with heterozygous knockout of Pmp22 recapitulate the pathology of humans with HNPP, including tomacula with excessive myelin decompaction that extends from paranodes to juxtaparanodes and internodes [9]. Application of mechanical compression on Pmp22+/- mouse nerves induced conduction block (i. e. , failure of action potential propagation) more rapidly than that in Pmp22+/+ nerves. This finding is consistent with the key clinical features in patients with HNPP—focal sensory loss and weakness when nerves are exposed to mild mechanical stress [10,11]. Therefore, these mice have become an authentic model of HNPP. There are three types of junctions in myelin: tight junctions, adherens junctions, and septate junctions [4]. All are mainly in non-compact myelin regions (Fig 1A): paranodal loops, Schmidt-Lanterman incisures (SLI), and inner/outer mesaxons [12]. Although each type of junctions has distinct protein constituents, they share similar molecular architectures (Fig 1B). For instance, tight junctions are formed by polymerization of claudins, a family of tetraspan membrane proteins. C-terminals of claudins interact with a group of cytoplasmic adaptors such as ZO1 or ZO2 [13]. These PDZ-containing proteins directly interact with actins and link the tight junctions to the cytoskeleton for stabilization [14]. Adherens junctions employ a similar organization. E-cadherin has a glycosylated extracellular domain, a single transmembrane domain, and a cytoplasmic c-terminal tail that interacts with adaptor catenins (α-catenin, β-catenin and p120/σ-catenin). α-catenin directly interacts with actin filaments. The actin network is subject to the regulation of small GTPases (Cdc42 or Rac1) and their effectors such as p21-activated kinase (PAK1) [14]. Furthermore, all junctions are strengthened by a group of Ig-domain proteins, such as JAM-C in myelin, that form transmembrane dimers juxtaposed to the junctions to seal the space between the opposing membranes [15]. Because actin networks are involved in the stabilization of all junctions, we test a hypothesis that PMP22 deficiency disrupts myelin junctions by altering actin polymerization.
  While we have described the disruption of myelin junctions in Pmp22+/- nerves [5], the disruption had yet been evaluated during aging. In this study, we found dislocation of E-cadherin (marker for adherens junction), Mag (marker for transmembrane protein of paranodal loop), and claudin-19 (marker for tight junction) in Pmp22+/- paranodes and incisures from 2 weeks to 10 months of age (Fig 2). Under electron microscopy, junction abnormalities with paranodal lamina splitting were qualitatively observed in Pmp22+/- nerves (S1 Fig). In line with our previous studies [5,10], localization of Caspr and neurofascin at septate junctions was unchanged (S2 Fig). Note that the total amounts of these junction proteins were not altered in Pmp22+/- nerves by Western blot [5]. Taken together, myelin junctions in Pmp22+/- nerves were abnormally formed during development and disrupted in adulthood. This abnormality was observable as early as 2 weeks, which was months ahead of segmental demyelination seen only after 10–12 months of age [5,10]. Results above predict action potential propagation failure in a subset of Pmp22+/- nerve fibers with no segmental demyelination but severely increased myelin permeability [1]. We performed nerve conduction studies (NCS) in mice at ages of 2,6, and 12 months. There was a significant reduction of compound muscle action potential (CMAP) amplitudes in all age-groups of Pmp22+/- mice compared with those in Pmp22+/+ mice (S1 Table). In contrast, conduction velocities were not altered in Pmp22+/- nerves. To determine whether the decrease of CMAP was due to axonal loss, we performed sciatic nerve morphometric analysis at ages of 1,3 and 6 months. The numbers of myelinated nerve fibers were not significantly different between Pmp22+/+ and Pmp22+/- mice (S2 Table). These findings do not support axonal loss. To directly evaluate conduction block, sciatic nerves were surgically exposed to eliminate technical variations in NCS (Fig 3A, 3B and 3C). Conduction block (Fig 3D and 3E) was detected in 12 out of 17 studied Pmp22+/- mice but not found in any Pmp22+/+ mice. The remaining 5 Pmp22+/- mice had at least one of the three abnormalities—prolongation of distal latency (Fig 3F), temporal dispersion (Fig 3G), or both. Therefore, conduction block was present in Pmp22+/- nerves. This explains the decrease of CMAP amplitudes in Pmp22+/- mice. F-actin is a common “denominator” in all types of junction complexes (Fig 1B) for junction stabilization [14]. We speculated an altered actin polymerization in PMP22 deficiency. Teased mouse nerve fibers were stained with rhodamine-conjugated phalloidin known to specifically label F-actin [16]. F-actin was localized in non-compact myelin regions (Fig 4A) where myelin junctions also reside (Figs 1 and 2). Quantification of F-actin fluorescence intensity showed a significant difference between Pmp22+/+ and Pmp22+/- nerves from 3 to 10 months of age (Fig 4B, 4C and 4D). Moreover, Western blot confirmed the increase of F-actin in Pmp22+/- nerves (Fig 4E and 4F). We tested dynamics of F-actin formation as described [16]. Jasplakinolide is a membrane permeable cyclo-depsipeptide that competes with phalloidin for F-actin binding. After saturating the existing F-actin with jasplakinolide, phalloidin only labeled newly formed F-actin. Pmp22+/- nerves showed a higher level of new F-actins than that in Pmp22+/+ nerves (Fig 4G and 4H). Alteration of actin polymerization prompted us to examine changes of F-actin’s regulators such as Cdc42, Rac1, and PAK1 [17]. Both Cdc42 and Rac1 are functionally essential. Removal of either Cdc42 or Rac1 results in severe dysmyelination [18], which makes the two molecules unfavorable targets of intervention. In contrast, constitutive knockout of PAK1 (Pak1-/-) produces negligible phenotype in mice [19]. We have also confirmed normal morphology, electrophysiology and functions in Pak1-/- peripheral nerves (S3 Fig). Yet, this kinase has been shown to play roles in actin polymerization and cellular focal adhesion [17]. In mouse sciatic nerves, we detected PAK1 (Fig 5A) and PAK2 but not PAK3 (S4 Fig). Levels of total PAK1 (Fig 5A and 5B) or PAK2 (S4 Fig) were not different between Pmp22+/+ and Pmp22+/- nerves. Immunostaining with antibodies against total PAK1 showed diffuse distribution in the sciatic nerves similarly between Pmp22+/+ and Pmp22+/- mice (Fig 5G). PAK1-3 activation involves autophosphorylation at multiple amino acid residues, including S144, S199, and/or T423 [17]. In addition, PAK1, not PAK2-3, can be phosphorylated at T212 to activate PAK1 independently of small GTPases and regulate F-actin formation [20]. Western blot of sciatic nerve lysates showed a significantly increased level of T212 (but not S144) in Pmp22+/- nerves compared with that in Pmp22+/+ nerves (Fig 5A, 5B, 5C and 5D). The activity of pPAK1 reached its peak at age of 3-month-old, which correlated with the time when tomacula are actively formed [10]. We verified specificity of total PAK1, S144, and T212 antibodies using Pak1+/+ and Pak1-/- nerves. All antibodies detected PAK1 in Pak1+/+ nerves but not in Pak1-/- nerves (Fig 5A and 5C). PAK1 has been shown to phosphorylate MAPK kinase-1 (MEK1) at its S298 residue [21]. Western blot revealed an increase of phosphorylated MEK1 in Pmp22+/- nerves compared with that in Pmp22+/+ nerves (Fig 5E and 5F). Together, these data support an increase of PAK1 activation in PMP22 deficient nerves. Residues in other PAKs could be phosphorylated but cannot be substantiated due to the lack of specific antibodies. Both T212 and S144 antibodies failed to stain mouse nerves in our immunofluorescence experiments. To determine whether PAK1 is associated with junction protein complexes, we first performed co-immunoprecipitation (co-IP) in mouse sciatic nerve lysates and verified interactions between E-cadherin, β-catenin and σ-catenin (protein elements of adherens junction) (Fig 6C, 6D and 6E) [22]. Next, by using β-catenin antibodies, we were able to pull down PAK1 from the sciatic nerve lysates (Fig 6E). Because β-catenin is known to reside in the non-compact myelin regions and is an element of myelin adherens junction complex [22], PAK1 would associate with adherens junction protein complex. This finding is also consistent with a previously published study showing interactions between PAK1 and β-catenin [23]. To understand how PAK1 is activated in Pmp22+/- myelin, we tested whether the disruption of adherens junction protein complex activates PAK1. We first verified the presence of adherens junction protein complex in culture Schwann cells, including β-catenin, σ-catenin and E-cadherin (S4 Fig). By using siRNA, β-catenin or σ-catenin was knocked down in culture Schwann cells. PAK1 activity indexed by T212 was increased in either β-catenin or σ-catenin knock-down cells compared with that in control cells treated with scramble siRNA (Fig 6F1,6F2,6G1 and 6G2). Together, these findings suggest that PAK1 is present in the junction protein complexes. PAK1 may be activated when the junction protein complex is not formed normally. To determine how PMP22 deficiency affects adherens junctions, we speculated an interaction between PMP22 and E-cadherin. This speculation was based on our previous study showing interactions between PMP22 and other junction proteins with Ig or Ig-like extracellular domains [5]. HA- or GFP-tagged PMP22 and E-cadherin were co-expressed in 293a cells. The co-IP showed an interaction between PMP22 and the extracellular domain of E-cadherin (Fig 6A and 6B). When co-IP was done in mouse sciatic nerve lysates, interactions between endogenous PMP22 and E-cadherin were only detectable in mice younger than postnatal day 15 but not in adult nerves (Fig 6C). This is in agreement with our immunostaining showing that PMP22 was localized into non-compact myelin regions in developing nerves but confined to internodal compact myelin and separated from myelin junctions in adult nerves [5]. These findings suggest that PMP22 might affect junction protein complex formation through its interactions with junction proteins during development. Abnormally formed junction complex would thereby activate PAK1. Heterozygous deletion of PMP22 in patients with HNPP still leaves an intact allele of PMP22. The allele of PMP22 results in a partial production of PMP22 proteins [8], which would allow a portion of normal myelin junctions formed. We reasoned that the activated PAK1 would further disrupt those normally formed junctions, presumably via alterations of actin polymerization. PF3758309 is a commercially available PAK inhibitor. Chow et al have tested this compound (25mg/kg) in a skin cancer mouse model with a 7–10 day course of intraperitoneal (i. p.) injection [24]. The compound penetrated into the nervous system [25]. We first tested mouse tolerance to PF3758309 (i. p. daily). The dose of 2. 5mg/kg or 25mg/kg killed over 50% of 20 Pmp22+/- mice within 15 days with drastic reduction of body weight, but no death was found in 21 vehicle-treated Pmp22+/- mice. Thus, Pmp22+/- mice were treated with 0. 25,0. 5,1. 0mg/kg PF3758309 or vehicle—saline (Fig 7 and Table 1). Animals tolerated these lower doses well with no change of body weight or increase of death. To give a sufficient time for recovery of myelin permeability, we injected the compound for 11 weeks. Injection started at age of postnatal day 7. The treatment prevented the decline of CMAP amplitudes with all three dosages (Fig 7A), but did not restore the CMAP amplitudes to the levels in Pmp22+/+ mice (3. 2±0. 6mV in ten 3-month-old Pmp22+/+ mice versus 1. 5±0. 8mV in eight 3-month-old Pmp22+/- mice treated with 1. 00mg/kg PF-3758309). The remaining outcomes were collected only from mice treated with the lowest dose of 0. 25mg/kg. PF3758309 suppressed levels of T212, phosphorylated MEK1 (S298) and F-actin (Table 1, Fig 7C and 7D), as well as improved abnormal claudin-19 distributions (i. e. , tight junctions; Table 1) and myelin permeability (Fig 7E and 7F) compared with those in the vehicle group. Using teased nerve fibers, we quantified, as described [10], the percentages of nerve fibers with tomacula, a key pathology of HNPP. Tomacula were fewer in the treated group (Table 1). It is important to determine whether the treatment is still effective after the developmental stage. Moreover, in human clinical trials, a range of ages, instead of a single age point, are usually included. Mice were enrolled at ages of 6–11 months. Again, F-actin levels were significantly lower in the PF3758309 (0. 25mg/kg) group (Fig 7D). In Fig 7B, we measured CMAP amplitudes prior to the treatment. After treatment, CMAP amplitudes decreased about a half (from 1. 6±0. 7mV to 0. 7±0. 2mV) in the vehicle group, but the decrease was completely prevented over the course of PF3758309 treatment. The measurement of baseline CMAP was not possible in the 1st set of experiment since the injection started at age of 1 week when mouse paws were too small for any reliable recording. The percentages of tomacula in the 2nd set of experiment were not significantly different between PF3758309 and vehicle groups. However, we noticed one outlier with the highest percentage of tomacula (39. 7%) in PF3758309 group. We then counted another 14 Pmp22+/- mice (un-injected). None of them showed tomacula above 39. 7%. When the outlier was removed, the difference of tomacula between PF3758309 and vehicle group was significant (the 6-7th column in Table 1). Finally, by Western blot, PAK1 activity indexed by T212 was decreased in PF3758309 groups compared with vehicle groups. The claudin-19 distribution was also improved in the treated group (Table 1). Two additional PAK inhibitors (FRAX597 and FRAX486) were commercially available. However, they failed to inhibit PAK1 activity in mouse peripheral nerves (Ratio of T212/PAK1: vehicle 1. 07± 0. 23 versus FRAX597 1. 30±0. 62, n = 4 vehicle mice and 5 FRAX597 mice, p = 0. 502; vehicle 0. 97± 0. 13 versus FRAX486 0. 93±0. 13, n = 4 vehicle mice and 5 FRAX486 mice, p = 0. 601). They were not suitable for the treatment. This failure was presumably due to the poor penetrance into the peripheral nerve system. Suppression of F-actin formation by PF3758309 may not be due to PAK1 but an off-target effect. One may test this issue by crossing Pak1-/- into Pmp22+/- mice to remove Pak1 gene in Pmp22+/- mice. However, a compensation effect of PAK2 activity in Pak1-/- mice has been observed [26]. We have confirmed an increase of phosphorylated PAK2 (S20) in Pak1-/- Schwann cells (row 6 in Fig 8). Thus, this approach does not help. When Schwann cells were treated with either vehicle or PF3758309, F-actin became hardly detectable in Pak1+/+ cells. However, PF3758309 failed to change the levels of F-actin in Pak1-/- cells (row 1 in Fig 8). This finding suggests that PF3758309 suppresses F-actin formation mainly via PAK1. Note that F-actin formation may be regulated by many different signaling pathways, not just PAK1. Thus, deletion of PAK1 alone in cells does not necessarily decrease the level of F-actin, which could be even increased by unidentified compensating mechanisms, such as PAK2 (row 1 in Fig 8). This notion was in line with the fact that PF3758309 inhibited all levels of F-actin, phosphorylated PAK1 (T212 in Table 1) and phosphorylated PAK2 (S20) (row 6 in Fig 8). This also suggests that PF3758309 is not a specific inhibitor for PAK1, but affects other PAKs.
  Our study revealed a novel mechanism—myelin junctions are disrupted through PAK1 activity in HNPP mouse model. This change results in conduction block in the Pmp22+/- nerves, thereby explaining focal sensory/motor deficits in HNPP. Moreover, myelin junction disruption occurred much earlier than segmental demyelination in Pmp22+/- mice [5,10,11]. Therefore, functional demyelination represents an upstream mechanism prior to the actual stripping of myelin, a key pathology shared by many demyelinating diseases. We believe that this is an important conceptual advance since this mechanism reveals molecular targets that may be intervened prior to the removal of myelin. The conclusion above is supported by several lines of evidence. First, PAK1 activity indexed by T212 is increased in Pmp22+/- nerves. The time course of the PAK1-increase mirrors the progression of tomacula [10]. Second, based on our co-IP data, PAK1 interacts with adherens junction protein complexes either directly or indirectly, as demonstrated previously [23]. Interactions between PMP22 and other myelin-junction-related proteins have also been found in our previous study [5]. Because all myelin junctions are localized to the non-compact myelin regions [5], activated PAK1 would be available to affect other types of junctions and F-actin. T212 phosphorylation in PAK1 has been shown to recruit PAK1 to submembrane actins [20], where PAK1 activity may be further promoted by certain lipids, such as sphingosine or phosphoinositides, independent of small GTPases [27,28]. Thus, an increase of T212 in Pmp22+/- nerves is highly relevant for F-actin formation locally around the myelin junctions. This finding is consistent with numerous studies in epithelial cells that demonstrated junction disruption after altering actin polymerization [29,30]. This finding is also in line with another study in transected mouse sciatic nerves; an increase of F-actin in myelin through the activation of a small GTPase, Rac1, promoted the removal of E-cadherin, a marker of adherens junctions. Inhibition of actin polymerization prevented the E-cadherin from being removed [16]. Interestingly, septate junctions are spared in Pmp22+/- nerves [5,10], which was replicated in the present study. Like epithelial cells, myelinating Schwann cell polarizes into “apical-like and basolateral-like” domains [31]. We speculate that septate junctions are involved in a domain and mechanisms distinct from other myelin junctions. Indeed, in our previous study [5], PMP22 was found in the paranodal regions during the early development but was not observed in the septate junction region. Third, PAK inhibitor (PF3758309) improved F-actin dysregulation, junction disruption, and abnormal myelin permeability in Pmp22+/- nerves. The decline of CMAP amplitudes was completely prevented by the PAK inhibitor. The inhibitor was even effective in the adult nerves with fully developed pathology (Fig 7 and Table 1). These results were from a large cohort of mice (total n = 82 mice) of two age groups with three different dosages. Effective dose of PF3758309 (0. 25mg/kg) was 100 times lower than the dose of 25mg/kg used in skin cancer mouse model [24]. Such low dose of 0. 25mg/kg PF3758309 makes its use safer. Indeed, we treated the mice for 11 weeks, which was a long duration rarely seen in literature and far longer than the duration of 7–10 days used in the skin cancer mouse model [24]. Yet, there was no increase of mouse mortality and observable side-effect. PAK inhibitor was effective in aged animals (6–11 months; Table 1). While pharmacological treatment is unlikely given to asymptomatic patients, the PAK inhibitor would still be effective in the symptomatic patients with the pathology fully developed. This is also in line with our previous observation of dynamic paranodal changes during action potential propagation in adult nerves [32]. Thus, PAK inhibitors may become a promising therapy for HNPP. Our data suggest that PF-3758309 also inhibits PAK2 in the peripheral nerves. Thus, the beneficial effect in the treated Pmp22+/- mice could relate to other types of PAKs, in addition to PAK1. Loss-of-function of PAK3 has been shown to affect synaptic plasticity [33]. Although the effective dose of PF-3758309 was very low in our study, it remains to be determined whether the treatment results in any side-effect in cognitive functions. The severity of myelin permeability varied in different Pmp22+/- nerve fibers (Fig 1B in Guo et al Ann Neurol 2014) [5]. This variability would produce two different populations of myelinated nerve fibers in HNPP. Those in the first group have severely" leaky" myelin (i. e. , high capacitance), leading to failure of action potential propagation in the absence of demyelination (Fig 3). We call this" functional demyelination" . Inhibition of PAK1 is expected to restore the nerve conduction in the group of nerve fibers, which is reflected by the prevention of CMAP decline in PF3758309-treated Pmp22+/- mice (Fig 7 and Table 1). Those in the second group have mildly increased permeability of myelin, which still allows action potentials to propagate but would partially compromise the safety factor of action potential propagation. This compromised safety factor would put the PMP22-deficient nerve fibers at risk to conduction failure if the fiber is challenged by external factors, such as mechanical stress. Indeed, our previous study has demonstrated that mechanical compression induced conduction block in Pmp22+/- nerves faster than that in Pmp22+/+ nerves [10]. It is not until the very late stage (>10–12 months) when obvious segmental demyelination and axonal loss start in Pmp22+/- mice [10,34,35]. Note conduction velocities in NCS are determined by large myelinated nerve fibers [1]. As long as there are some large myelinated nerve fibers still conducting action potentials in Pmp22+/- nerves, conduction velocities would remain normal or minimally decreased. This is also consistent with the observations in patients with HNPP [11]. In summary, we propose a pathogenic mechanism of two steps—initiation and perpetuation (Fig 9). Initiation: PMP22 and E-cadherin (or other junction proteins) travel via the secretory pathway from endoplasmic reticulum (ER) /Golgi apparatus to cytoplasmic membrane [36,37]. Like the polarized epithelial cells, E-cadherin in developing Schwann cells has to be transported from apical domain (internodal membrane) to basolateral domain (paranode and incisures) through endocytosis [22,37]. PMP22 has been reported to regulate the endocytosis of E-cadherin via Arf6, an ATPase [38]. PMP22 may affect E-cadherin transport during development (Fig 6). In supporting this notion, our study has shown that PMP22 is transiently expressed in non-compact myelin regions of developing nerves but disappears in those compartments after maturation [5]. Because abnormal junction formation is upstream to the PAK1 activation, the PAK inhibitor would not affect this step. Perpetuation: HNPP with heterozygous deletion of PMP22 still have a normal allele that produces about a half of PMP22 proteins in normal controls [8]. These residual PMP22 proteins would permit some myelin junctions to form. However, after the activation of PAK1 by abnormally formed junction complex (Fig 6), PAK1 activity promotes further junction disruption in adulthood (Figs 2 and 6). Thus, this junction disruption, not the abnormal junction formation during early development, would be affected by the PAK inhibitor. It remains to be determined whether the junction disruption was through the increase of F-actin and/or via another unidentified pathway. However, studies in epithelial cells have demonstrated junction disruption by altering actin polymerization [29,30]. After all, inhibition of PAK1 activity did suppress the formation of F-actin and improved claudin-19 distribution (Figs 7C, 7D and 8; Table 1). Together, these findings not only provide a mechanistic explanation for abnormal myelin permeability and impaired action potentials propagation in HNPP, but also offer a promising therapeutic approach for this disease.
  All mice were housed in Tennessee Valley Healthcare System (TVHS) animal facility, a part of Vanderbilt University animal care system. Experimental procedures were approved by the IACUC of Vanderbilt University (M1500006-00). All mice in this study were euthanized by CO2 asphyxiation, according to the guidelines of the IACUC at the Vanderbilt University and the recommendations of the Panel on Euthanasia of the American Veterinary Association. Pmp22+/- mice were backcrossed with C57Bl6J mice (Jackson Lab) for more than 10 generations to reach congenic. Mice were genotyped as described [9]. The primers for genotyping are listed in S3 Table. The congenic Pmp22+/- mice have been extensively characterized [5]. They showed pathology and other features similar to those in Pmp22+/- mice with mixed background (C57Bl6J/129) [10]. Pak1-/- mice were from Dr. Jonathan Chernoff’s lab, Fox Chase Cancer Center, USA. The Pak1-/- mice were produced in C57Bl6J background and have been described with negligible phenotype [19]. For PAK1 inhibitor (PF3758309) injections, Pmp22+/- mice were randomized into vehicle and treated groups. Based on our power calculation of variations derived from mouse compound nerve action potentials, 7 mice for each group would have a 92% chance to detect a significant difference. PF-3758309 (Cat# CT-PF0375, ChemieTek) was dissolved in normal saline for intraperitoneal injection (i. p.) daily. Sciatic nerves or cells were lysed in immunoprecipitation buffer (Cat# 87788, Thermo scientific) with proteinase/phosphatase inhibitor cocktail and incubated with primary antibodies overnight at 4°C with rotation (70 rpm). Protein G agarose beads (Cat# 15920–010, Life technologies) were added for another 2 hour incubation at 4°C. Samples were eluted with Laemmli sample buffer (Cat# 161–0737, Bio-rad), resolved by SDS-PAGE, and analyzed by immunoblot. All antibodies and their titers in this study are listed in S4 Table. This technique has been validated [5]. In brief, 1 cm sciatic nerve fascicles were submerged in artificial CSF after epineurium removal and sealed at both ends with Vaseline. A 3kDa Dextran of fluorescence (2 mg/ml, Cat# D3329, Life technologies) was added for one hour incubation at room temperature without oxygenation. After washing, the nerve fascicles were fixed in 4% PFA for 10 minutes and teased into individual nerve fibers on glass slides for fluorescence microscopy. Fluorescence intensity was quantified by placing a 5μm x 5μm interest box 10μm away from the middle point of the node of Ranvier. Silencing of β-catenin and σ-catenin were carried out using Accell SMARTpool siRNAs (Cat# A-062106-13 and A-040628-15, Dharmacon). Schwann cells were transfected with 1μM siRNA. The efficiency of the knockdown was evaluated by Western blot 72 hours after the transfection. Accell non-targeting siRNA (Cat# D-001910, Dharmacon) was used as negative control. This method was modified from our published study [5,10]. In brief, sciatic nerves were fixed, embedded in paraffin, and cut into 5μm-thick slices. Sections were incubated overnight with primary antibodies at 4°C. After washing, sections were stained for 1 hour with secondary antibodies. The stained slides were examined under a Leica fluorescent microscope (Leica DM6000B). For teased nerve stains, sciatic nerves were fixed in 4% paraformaldehyde (PFA) overnight and teased into individual fibers on glass slides. The slides were dried overnight, reacted with primary antibodies, and followed by secondary antibodies. For newly formed F-actin staining, as described [16], the existing F-actin in sciatic nerve explants was saturated with a cell-permeable actin-binding compound, jasplakinolide (Cat# 420127, Millipore), at 1μM in culture media. After washing with PBS, the explants were incubated in a drug-free media for 6 hours at 37°C. The explants were fixed in 4% PFA and teased for F-actin staining using fluorescent phalloidin (1: 400, Cat# R415, Life Technologies). NCS was previously described [10]. In brief, mice were anesthetized with isoflurane (VetEquip Inc. Cat# 908106; 1. 7L/minute of oxygen at 1. 0 bar; 1. 5% of the total oxygen flow being vaporized with Isoflurane). This anesthetic drug has been tested in our laboratory. It does not affect nerve conduction if the procedure is completed within 25 minutes. A skilled technician in our lab gets NCS done in each mouse within 6 minutes. For the experiments of conduction block in Fig 3, Avertin (250mg/kg, i. p.) was used for anesthesia. Avertin did not affect CMAP over 2 hours [5]. CMAP was recorded from the intrinsic foot muscle using needle electrodes. Stimulation electrodes were positioned percutaneously at the sciatic notch and adjacent to the tibial nerve at the ankle. CMAP amplitudes were measured from baseline to the peak of negative deflection. This method has been described [39]. Epon sections (1μM thickness) of mouse sciatic nerves were examined under the 63X objective. The entire field of transverse sections of each nerve was imaged for analysis. Images were imported into software (ImagePro Plus). Areas of each field were counted to obtain the number of nerve fibers. Electron microscopy on mouse sciatic nerves was performed as described [40]. Briefly, sciatic nerves were cryofixed in a high-pressure freezer (HPM100; Leica) and freeze substitution was performed in an embedding system at low temperature (AFS; Leica) using the tannic acid–OsO4 protocol. Samples were embedded in Epon, sectioned (Ultracut S Ultramicrotome, Leica), and stained with an aqueous solution of 2% uranyl acetate followed by lead citrate. Samples were examined in a LEO EM 912AB electron microscope (Zeiss, Oberkochen, Germany). Pictures were taken with an on-axis 2048x2048-CCD-camera (TRS, Moorenweis, Germany). Chopped sciatic nerves were immediately dropped into RIPA buffer (Cat# R0278, Sigma) with proteinase/phosphatase inhibiter cocktail (Cat# 5872, Cell Signaling). Samples were homogenized for protein isolation. Protein concentration was determined by BCA assay (Prod#23225, Thermo Scientific). Samples were loaded into SDS-PAGE gels and transferred to a PVDF membrane. The membranes were blotted with 5% non-fat milk and incubated overnight at 4°C with primary antibodies, and followed by secondary antibodies. The immune complexes were detected by the enhanced chemoilluminescence (Cat# NEL103001, Perkin Elmer). In some cases, the blots were stripped and re-probed with additional antibodies. Quantification of band intensity was performed by the ImageJ software (http: //rsbweb. nih. gov/ij/). E-cadherin-GFP plasmid was purchased from Addgene (Cat# 28009). PMP22-HA was obtained from Genocopoia (Cat# EX-D0117-M06). The primers for E-cadherin pE-cad- (E+T) -GFP were, forward: 5’-CCCAAGCTTGCCACCATGGGCCCTTGGAGCCGC-3’, reverse: 5’-CCGCTCGAGAAACAGCAAGAGCAGCAGAATCAG-3’; pE-cad- (T+I) -GFP, forward: 5’-CGGGGTACCGCCACCATGATTCTGGGGATTCTTGGAGG-3’, reverse: 5’-CCGCTCGAGGTCGTCCTCGCCGCCT-3’. The accuracy of all plasmids was verified by DNA sequencing. The plasmids were transfected into HEK293a cells by using Effectene (Cat# 301425, Qiagen) according to the manufacturer’s instructions. Statistical analysis was performed using GraphPad Prism software version 6. 0 or SAS 9. 4. The data was represented as the mean ± SD. For normally distributed data, a Student t test was utilized. The Wilcoxon Rank-sum test was used when the data were not under normal distribution. Differences were considered significant when the P value was less than 0. 05.
summary: Demyelination has been widely regarded as one of the most important mechanisms impairing the nerve conduction of electrical impulses in many neurological diseases. Failure of the nerve conduction results in sensory and motor deficits. In the present study, we investigated a novel mechanism that also impairs nerve conduction without the removal of myelin, called functional demyelination. Deletion of one of two copies of the PMP22 gene causes hereditary neuropathy with liability to pressure palsies (HNPP). Our recent study reports that deficiency of PMP22 in HNPP disrupts myelin junctions of Schwann cells that seal the spaces between layers of myelin membrane. This disruption leads to “leaky” myelin that impairs the conduction of electrical impulses on the nerves. In the present study, using a HNPP mouse model (Pmp22+/-), we have identified a molecular pathway responsible for the disruption of myelin junctions. Our results showed an increase of actin polymerization in the areas where myelin junctions resided. This increase was associated with an up-regulation of PAK1 activity, a kinase function that is known to regulate actin polymerization. We thus treated the HNPP mice with PAK1 inhibitor. This treatment completely prevented the progression of nerve conduction failure and HNPP pathology. This work offers a promising therapeutic approach for HNPP. Moreover, myelin junction disruption takes place long before the actual stripping of myelin (demyelination) in the late phase of HNPP. Therefore, our finding provides a mechanism upstream to segmental demyelination, a pathological process relevant to many demyelinating diseases.
keywords: cell physiology
  medicine and health sciences
  action potentials
  nervous system
  membrane potential
  junctional complexes
  electrophysiology
  neuroscience
  macroglial cells
  animal models
  tight junctions
  model organisms
  schwann cells
  nerve fibers
  research and analysis methods
  animal cells
  proteins
  mouse models
  glial cells
  biochemistry
  cellular neuroscience
  protein complexes
  anatomy
  nerves
  cell biology
  physiology
  neurons
  biology and life sciences
  cellular types
  sciatic nerves
  neurophysiology
title: Tuning PAK Activity to Rescue Abnormal Myelin Permeability in HNPP