TrkB and TrkC agonist antibodies improve function, electrophysiologic and pathologic features in TremblerJ mice

Experimental Neurology 224 (2010) 495–506

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Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r

TrkB and TrkC agonist antibodies improve function, electrophysiologic and pathologic features in TremblerJ mice Zarife Sahenk a,⁎, Gloria Galloway b, Chris Edwards a, Vinod Malik a, Brian K. Kaspar a, Amy Eagle a, Brent Yetter a, Alison Forgie c, David Tsao c, John C. Lin c a b c

Research Institute at Nationwide Children's Hospital, The Ohio State University, Columbus, OH, USA Division of Neurology, Department of Pediatrics at Nationwide Children's Hospital, The Ohio State University, Columbus, OH, USA Rinat, Pfizer Inc., South San Francisco, CA, USA

a r t i c l e

i n f o

Article history: Received 27 January 2010 Revised 26 April 2010 Accepted 18 May 2010 Available online 27 May 2010

Keywords: Charcot–Marie–Tooth neuropathy Therapeutic agonistic antibody TrkB, TrkC receptors Neurotrophin-3 Peripheral nerve regeneration TrJ mice

a b s t r a c t Neurotrophic factors have been considered as potential therapeutics for peripheral neuropathies. Previously, we showed that neurotrophin-3 (NT-3) promotes nerve regeneration in TremblerJ (TrJ) mice and in sural nerves from patients with Charcot–Marie–Tooth 1A (CMT1A). The relatively short plasma half-life of NT-3 and other neurotrophins, however, pose a practical difficulty in their clinical application. Therapeutic agonist antibodies (AAb) targeting the neurotrophic receptors may circumvent this obstacle due to their high specificity and long half-life. Using morphological, electrophysiological studies and functional motor testing, we assessed the efficacy of monoclonal TrkC AAb and TrkB AAb in the TrJ mice. Treatments of these AAbs individually or in combination over 20 weeks increased compound muscle action potential (CMAP) amplitude, which correlated with improved grip strength, as compared to the PBS control group. Improvements in CMAP amplitude were most prominent with TrkC AAb treatment. In all treatment groups, distal to the crush site of the sciatic nerves exhibited a significantly greater number of myelinated fibers (MFs) indicating improved regenerative response to injury. In the contralateral intact sciatic nerves, the number of MFs as well as the myelin thickness was also increased significantly by the AAb treatments, suggesting that the hypomyelination/ amyelination state of the peripheral nerves in TrJ improved. Therapeutic response to AAb combination was often, albeit not always, the most prominent, indicating a non-redundant effect of TrkB and TrkC AAbs. An early functional recovery and the correlative morphological changes of enhanced regeneration were seen with TrkC AAb treatment. These results provide evidence for potential therapeutic use of monoclonal agonist antibodies for neurotrophin receptors in CMT1A and other neuropathies. © 2010 Elsevier Inc. All rights reserved.

Introduction Charcot–Marie–Tooth (CMT) neuropathies are the most common inherited neurologic conditions with progressive symptoms usually manifesting in the first two decades of life and causing significant disabilities in some individuals (Skre, 1974). Clinical severity varies, even among affected family members. In general, most CMT patients show progression requiring ambulatory aids. Less frequently, severe childhood cases may be wheelchair- or ventilator-dependent. CMT is genetically heterogeneous. The vast majority show autosomal dominant inheritance and can be divided into demyelinating (CMT1) and axonal (CMT2) forms, based on electrophysiological criteria. CMT1 results from mutations in genes contributing to myelin formation. Even though the

⁎ Corresponding author. Departments of Pediatrics and Neurology, The Ohio State University, Center for Gene Therapy, Neuromuscular Program, Director, Neuromuscular Pathology, The Research Institute at Nationwide Children's Hospital, 700 Childrens Drive, WA 3024, Columbus, OH 43205. USA. Fax: + 1 614 355 5247. E-mail address: [email protected] (Z. Sahenk). 0014-4886/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2010.05.013

genetic defect primarily involves Schwann cells (SCs), the clinical disability correlates more accurately with axonal degeneration and reduced regeneration resulting from impaired SC–axon interactions (Sahenk, 1999). Approximately 40% of all CMT cases involve mutations in the PMP22 gene and are classified as CMT1A, making it the most prevalent form of the disease (Nelis et al., 2007, 1996; Skre, 1974). Gene duplication leading to PMP22 over-expression is the major cause of CMT1A; the less common dominant point mutations produce the most severe forms of the disease (Nelis et al., 2007). The treatment option for CMT neuropathies is very limited. Ankle foot orthoses and corrective surgery are palliative treatment that sometimes improve function and relieve discomfort. In rodent models, genetic manipulations have shown promise. The repertoire of approaches includes modulation of the PMP22 promoter, improving trafficking of mis-folded PMP22 protein and providing trophic support to nerves (Kaya et al., 2007; Khajavi et al., 2007; Sahenk et al., 2005; Sereda et al., 2003). Previously, we have shown that neurotrophin-3 (NT-3) promotes nerve regeneration in TremblerJ (TrJ) mice and in sural nerves from patients with CMT1A (Sahenk et

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al., 2005). The relatively short half-life of NT-3 and other neurotrophins, however, pose practical difficulty in their clinical application (Poduslo and Curran, 1996). Continuous, therapeutic blood levels of neurotrophins can be achieved using gene therapy. Another approach is to use therapeutic agonistic antibodies (AAb) which target their receptors with high specificity and long half-life. TrkC is the cognate receptor of NT-3, and NT-3 also binds to TrkB and TrkA with a lower affinity (Bothwell, 1995; Schweigreiter, 2006). Thus agonistic antibodies targeting TrkC as well as other Trk receptors may provide an alternative to neurotrophin therapy. In the present study we evaluate the potential efficacy of two agonistic monoclonal antibodies for tyrosine kinase receptors, C (TrkC) and B (TrkB), individually and in combination in TrJ mice. The TrJ is a CMT mouse model, occurred spontaneously and carries a PMP22 missense mutation (L16P), identical to one found in human patients, and recapitulates many aspects of pathology observed in CMT1A. TrJ mice have severe hypomyelination, decreased rate of slow axonal transport, small axon caliber, gait abnormalities and reduced motor nerve conduction (de Waegh and Brady, 1990, 1991; Meekins et al., 2007, 2004). Moreover, in TrJ mice, the hallmark of the axonal cytoskeletal alteration, the increase in neurofilament (NF) density, is associated with reduced NF phosphorylation, suggesting that axonal signal transduction mechanism(s) regulating kinase/phosphatase systems are affected (de Waegh et al., 1992; Starr et al., 1996). Furthermore, the TrJ axonal microtubule cytoskeleton is unstable, and there is alteration in the composition and phosphorylation of microtubule associated proteins (Kirkpatrick and Brady, 1994). These findings are consistent with our observations showing impaired nerve regeneration in this model (Sahenk et al., 2005). Similarly, regeneration is impaired in CMT1A nerves and this defect is more pronounced for large myelinated axons (Sahenk et al., 2003). In this study, using functional motor testing, sciatic nerve conduction and morphologic studies, we demonstrated improvements in electrophysiological parameters correlating with grip strength and morphometric data from both regenerating and intact sciatic nerves in all agonist antibody treatment groups compared to PBS-treated TrJ mice. These results support the therapeutic use of agonistic monoclonal antibodies for tyrosine kinase receptors, TrkC and TrkB in CMT1A. Materials and methods Trk receptor-specific agonist monoclonal antibody generation and characterization The two compounds tested in these studies are as follows: Mab38B8 is a mouse monoclonal antibody against TrkB, of the subtype IgG1. MabA5 is a high affinity, recombinant chimeric antibody against TrkC with a human variable region fused to a rat IgG1 constant region. The generation and activity of monoclonal antibodies against TrkB and TrkC were previously described (Lin et al., 2008; Ruiz et al., 2005). In brief, for Mab38B8 (Lin et al., 2008), murine monoclonal antibodies against the extracellular domain of human TrkB were generated, screened for cross-reactivity to mouse TrkB ECD, with no binding to other neurotrophin receptors TrkA, TrkC or p75NTR. Mab38B8 was identified as a TrkB receptor agonist based on that it can increase TrkB receptor phosphorylation in a CHO cell line stably expressing human TrkB and that it can support the survival of embryonic day 15 mouse nodose ganglion neurons over 48 h in vitro. Likewise, monoclonal antibodies against the human TrkC extracellular domain were generated and screened for cross-reactivity to mouse TrkC, lack of binding to TrkA, TrkB or p75NTR, and agonist activity. A murine Mab, 2256 (Ruiz et al., 2005) was selected based on its ability to induce phosphorylation of TrkC in a cell based assay, and to support the survival of embryonic day 12 rat trigeminal ganglion neurons over 48 h in vitro. To improve the affinity of this antibody to the TrkC receptor in preparation for the drug development in humans,

the Mab2256 was humanized and affinity matured using CDR grafting techniques. To facilitate long-term dosing in rodent models of CMT1A, the human constant regions were further replaced with a rat IgG1 heavy chain and rat kappa light chain constant domain, respectively. This humanized rat chimeric antibody was termed MabA5. To confirm this antibody retained the agonist characteristics of its murine parent, MabA5 was tested for its ability to induce TrkC phosphorylation in stable TrkC expressing CHO cells (Sadick et al., 1997) (an assay previously described by Sadick et al., 1997), and to support the survival of embryonic rat trigeminal neurons in culture as previously described in a dose-dependent fashion (Davies et al., 1993) (an assay previously described by Davies et al., 1993). All antibody preparations were endotoxin tested prior to administration to animals. Animals and treatment groups TrJ mice (pmp22Tr–J) and age-matched C57BL/6J control littermates (Jackson Laboratories, ME) were used in this study. All treatment protocols and animal surgeries were conducted under the protocols approved by the Nationwide Children's Hospital and The Ohio State University Animal Care and Use Committee. Investigators remained blind to the treatment agents until the end of each study. The design of the major therapeutic studies using TrkB and TrkC AAb's is outlined below: (A) For the nerve regeneration study three treatments (TrkB, TrkC and TrkB+ TrkC AAbs) and one sham (PBS) treated group, each containing 8 TrJ mice, 8 to 12 weeks old, were entered into the study. Control littermates, 6 in each group received TrkB or PBS treatment. Mice received weekly subcutaneous injections of Trk receptor-specific AAbs at 2 mg/kg dosage, for 21 weeks, starting 1 week prior to the induction of sciatic nerve crush. The morphological assessment of nerve regeneration were the primary endpoint of this study. (B) Next we investigated the effects of these Trk receptor-specific AAbs on the intact (i.e. uncrushed) sciatic nerve motor conduction parameters and on the motor functions. One cohort of TrJ mice at 10–12 weeks of age underwent baseline bilateral hind limb grip strength and electrophysiology, followed by weekly subcutaneous injections of Trk receptor-specific agonist antibodies (2 mg/kg) or PBS (Group 1; 13–18 mice in each treatment). At 20 weeks post treatment, endpoint electrophysiological and bilateral hind limb grip strength studies were performed. (C) A second cohort of TrJ mice underwent early-onset long-term treatment (Group 2), starting treatment at 5 to 7 weeks of age. To reduce the risk of any potential untoward effect in juvenile animals, all three Trk receptor AAb treatment (TrkC, TrkB and TrkC+ TrkB) groups and one PBS-treated group, each containing 13 mice, received weekly 0.5 mg/kg to 1 mg/kg of the therapeutic dose until they reached the body weight of 10– 12 week old mice at which time they underwent baseline hind limb grip strength testing and sciatic nerve conduction velocity determinations and continued on with weekly subcutaneous injections of the full dose of 2 mg/kg Trk receptor-specific AAbs for an additional 20 weeks. At 20 weeks post treatment with the standard dose, endpoint electrophysiological and ipsilateral grip strength studies were performed. At the end of each study mice were euthanized for tissue and serum collection; in selected animals, lumbosacral dorsal root ganglions (DRGs) and roots and right sciatic nerves, were removed and snap frozen for western blot analysis as described below. Determination of monoclonal antibody concentrations in mouse sera The serum levels of mouse monoclonal antibody Mab38B8 and rat chimeric monoclonal antibody MabA5 were determined by standard sandwich ELISA assays. For Mab38B8, a 96-well Maxisorp plate (Nunc)

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was preabsorbed overnight at 4 °C with 1.0 mg/ml of a protein A column-purified, recombinant mouse TrkB extracellular domain (R&D Systems, Minneapolis, MN). The TrkB coated plate was then blocked for 1 h at 25 °C, with PBS with 0.5% BSA and 0.05% Tween-20. The plate was washed three times with PBS with 0.05% Tween-20. A standard dilution series of Mab38B8, as well as appropriately diluted serum samples were incubated in the plate for 1 h at 25 °C. After washing, a horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (Jackson ImmunoResearch) was applied at 1:1000, incubated for 1 h at 25 °C, to detect bound mouse monoclonal antibody. Finally, the signals were detected by the colorimetric reaction of HRP with ABTS substrate (KPL Inc.). A similar ELISA assay was used to measure MabA5 with the following substitutions: the plate was coated with recombinant mouse TrkC extracellular domain (R&D Systems), a standard dilution of MabA5 was used, and an HRP-conjugated rabbit anti-rat IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) was used for detection. Pharmacokinetic study Adult male TrJ mice (n = 3) received a single subcutaneous injection of MabA5 at 2 mg/kg. A second cohort of adult male C57BL/6 mice (n = 2) received a single subcutaneous injection of Mab38B8 at 1 mg/kg. Blood samples were collected retro-orbitally at various time points after injection, and the serum concentration of each antibody at the different time points was determined by the respective ELISA assay described above. Surgical procedures, tissue allocation for morphological studies Under isoflurane anesthesia left sciatic nerves were exposed and crushed with a fine forceps at a level 5 mm distal to the sciatic notch to generate a regeneration paradigm on one side. The crush site was marked by a 10-0 nylon suture tie passed through the epineurium. Mice were killed quickly by an over-dosage of xylazine/ketamine anesthesia and the sciatic nerves from crushed and intact sites were removed under a dissecting microscope at 20 weeks post-crush. Approximately 2 mm in length tissue blocks immediately distal to the crush site and the subsequent three segments, all marked for proximo-distal orientation as well as the mid-sciatic segments from the contralateral uncrushed nerves were processed for plastic embedding for light microscope thick sections and electron microscopy using standard methods established in our laboratory. Myelinated fiber density determinations Quantitative analysis at the light microscopic level was performed on 1 µm-thick cross sections from regenerating and intact uncrushed sciatic nerves using a microscope-mounted video camera at 1600× magnification and image analysis soft ware (Bioquant TCW98 image analysis software, R&M Biometrics Inc., Nashville, TN) as previously described (Sahenk et al., 2003). Data assessing regeneration response were obtained from the second segment distal to the crush and from the mid-sciatic nerve segments of intact nerves in all cases. Composites of fiber size distribution histograms and mean myelinated fiber (MF) densities (number per mm2 of fascicular area) were generated by combining data from six mice. G ratio of the myelinated fibers The g ratio refers to the ratio of axonal diameter/fiber diameter and lower g ratios represent axons with thicker myelin (Beuche and Friede, 1985; Friede and Beuche, 1985). For each animal, measurements from all fibers in 3 randomly selected representative unit areas were included and the g ratio distribution histograms were generated as a percent of total fibers analyzed. Measurements from 554 fibers in

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PBS-treated, 639 in combination, 706 in TrkB AAb and 817 in TrkC AAb groups were included in each group. Ultrastructural morphometric analysis for neurofilament density determinations Ultrastructural morphometric studies were performed using cross sectional images at ×52,000 final magnification from tissue blocks, about 0.5 mm distal to crush injury site on day five following a short treatment period and on the intact mid-sciatic nerve segments, 20 weeks post treatment. NF density histograms were generated by determining the number of NFs per unit hexagonal area in randomly selected TrJ mice axons from treated and untreated groups as previously described (Sahenk and Mendell, 1992). Fifteen axons at promyelination or those exhibiting a few turns of surrounding uncompacted myelin with diameters ranging from 2.8 to 3.5 µm at 5 days post-crush were included. Ten randomly selected MFs with axon diameters ranging from 4.2 to 5.0 µm at 20 weeks post treatment were analyzed. Histological analysis of muscle The ipsilateral gastrocnemius muscle from three randomly selected animals was removed for studies assessing the extent of denervation and reinnervation induced changes. Twelve micron thick cross cryostatsections from left gastrocnemius muscles were stained for succinic dehydrogenase (HDS) for analysis of fiber diameters. For muscle fiber size morphometry, images from 3 representative areas, composed of predominantly dark, intermediate or light fibers were captured at ×20 magnification, and the shortest axial lengths as fiber diameters were recorded with a calibrated micrometer, using the AxioVision, 4.2 software (Zeiss). Fast twitch oxidative (FTO/intermediate) or glycolytic (FTG/light), and slow twitch oxidative (STO/dark) fiber diameters were determined separately and composite histograms were generated by combining data from each animal as percent of total fibers counted. On average, 700 fibers were analyzed in each group. Early regeneration studies To assess the state of regeneration in response to a specific Trk receptor AAb treatment in TrJ mice, densities of SC nuclei, SC apoptosis, axon diameter distribution and NF density at promyelination were determined at 5 day post-crush; the earliest time point we chose to study. Using 6 randomly selected and photographed unit areas, each representing 0.011750 mm2 of endoneurium from 1-µm thick cross sections (n = 3 in each group), the SC nuclei densities were determined by adding the number of SC nuclei belonging to the clusters of unmyelinated fibers to those at the promyelinating stage with 1:1 axon-SC relationship and expressed as number per unit area. Promyelinated axon diameters were obtained from the same images and the diameter distribution is expressed as percent of total. Using the TUNEL assay on cross sections of fresh frozen sciatic nerves, SC apoptosis was assessed following a short-term treatment with TrkC or TrkB AAbs or PBS on day 5 after sciatic nerve crush according to the instructions of the manufacturer (In Situ Cell Death Detection Kit, Roche Diagnostics, IA, USA). The number of apoptotic nuclei was quantitated in 6 randomly selected areas, obtained from 3 mice in each group and normalized per unit area of 91.176 µm2. In addition, intact contralateral sciatic nerves, DRGs and roots were removed and snap frozen immediately for western blot studies (see below). Further quantitative studies were carried out at day 14 post-crush to assess the spatiotemporal distribution of regeneration-associated myelination in TrkC AAb, TrkB AAb and PBS-treated TrJ mice. The number of MFs, individual unmyelinated axons at 1:1 relationship with their promyelinating SCs (UnMFs) and SC nuclei in association with clusters of unmyelinated fibers was determined in cross sections from 3

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consecutive nerve segments distal to crush site, each measuring ∼ 2 mm in length (labeled as D1, D2 and D3 and marked for proximodistal orientation, n = 3). The number of MFs and UnMFs were expressed as percent of total number of axons engulfed by myelinating SCs (MFs + UnMFs) in each segment. In addition, densities (mean ± SEM per mm2 of endoneurial area) of MFs, UnMFs and SC nuclei in D3 segment were calculated. Neurofilament cytoskeleton and phosphorylation following short and long-term treatment Proximal and mid-sciatic nerves, roots and DRGs from treated and untreated TrJ and age-matched control mice were used for quantitative western blot analysis of NF proteins with NF-H specific antibodies. Briefly, the tissues were harvested and immediately frozen over dry ice. Tissues were homogenized in RIPA buffer [50 Mm Tris–HCl pH 8.0, 1% NP-40, 150 mM NaCl, 0.5% sodium deoxycholate , 1% SDS, 1 mM EGTA, 1 mM Na3VO4, 1 mM NaF, PMSF (1:250), Complete protease inhibitor (1:25), and 25.5 mM sodium pyrophosphate] using blue tip and Kontes pestle. Protein concentrations were determined using RC/DC method (BioRad Laboratories). For SDS-PAGE, 5 µg of protein was run on 3–8% Tris–acetate NuPage gels (Invitrogen) and transferred to PVDF membrane (Amersham Biosciences). After blocking for 1 h in 5% nonfat dry milk in TBST (100 mM Tris–HCl, pH 8.0, 167 mM NaCl, 0.1% Tween), the western blots were incubated with diluted primary antibodies against total NF-H (AB1989, COOH-terminal antibody from Chemicon; diluted 1:500), hyperphosphorylated NF-H (SMI-31 from Sternberger; diluted 1:20,000) and hypophosphorylated NF-H (SMI-35 from Sternberger; diluted 1:10,000). Blots were washed and incubated in appropriate horseradish peroxidase–conjugated secondary antibodies at a dilution of 1:2000. GAPDH was used as loading control (Millipore Inc. diluted 1:500). Immunoreactive bands were visualized with the use of ECL Plus Western blotting detection system (GE Healthcare) and Hyperfilm ECL (Amersham Biosciences). Signal intensities were measured with ImageQuant software (GE Healthcare).

mice under isofluorane anesthesia. Studies were repeated in all TrJ mice at the end of 20 weeks of full dose treatment, 2 mg/kg. Temperatures were recorded with an infrared thermometer (Fisher Scientific) and body temperature was maintained between 32 and 36 °C using a heating pad. Following body temperature equilibration, left sciatic nerve conduction studies were obtained using a XLTEK NeuroMaX 1002 electromyograph (Ontario, Canada) and Rhythmlink disposable subdermal needle recording electrodes (for both stimulation and recording) in the following manner: the stimulating electrodes were placed at the proximal and distal stimulation sites (i.e., the left sciatic notch and just above the ankle, respectively), and a third pair of recording electrodes was positioned in the foot pad between the second and third digits of the left foot. The technique of near nerve stimulation was utilized to determine the minimal amount of current required to generate a compound muscle action potential and the motor nerve conduction studies were performed utilizing a current value equivalent to 150% of that amount. Several trials were performed in each case for reproducibility of the results. The latency, duration, negative area under curve, and conduction velocity values of the recorded sciatic motor responses were determined. A caliper was utilized to measure the inter-electrode distances and these distances were used in calculations of intersegmental conduction velocity. In addition, measurements of onset latency, duration and amplitude were also obtained. All animals were studied in a blinded fashion.

Statistical analysis Statistical analysis were performed in Graph pad Prism 4 software, using one- and two way ANOVA followed by Bonferroni or Dunnett's multiple post hoc comparisons. Unpaired Student's t test was performed when applicable. Significant level was set at 0.05. Summary statistics were reported as mean ± SEM.

Results

Motor function testing

Agonistic and pharmacokinetic properties of the monoclonal antibodies

For studies assessing regeneration, mice were tested for baseline motor function within 1 week prior to onset of treatment with specific antibodies. Motor function tests included bilateral simultaneous hind limb grip power and that of the left hind paw (sciatic nerve crush site) using a grip strength meter (Chatillon Digital Meter, Model DFIS-2, Columbus Instruments, Columbus, OH) which we have used extensively in many of our studies (Haidet et al., 2008). Bilateral or unilateral grip strength was assessed by allowing the animals to grasp a platform followed by pulling the animal until it releases the platform; the force measurements were recorded in four separate trials. Measurements were performed on the same day and time of each week. In the electrophysiology Groups 1 and 2, baseline measurements were done prior to the onset of weekly treatments of 2 mg/kg and obtained every other week during the treatment period. In addition, endpoint ipsilateral correlative grip strength was done in the left hind limb, prior to obtaining the electrophysiological measurements. For this, the left hind paw grip strength measurements were done in two sessions (AM, and PM), three trials in each, per day for three consecutive days. The mean of these measurements was used to correlate with nerve conduction studies obtained from the same limb. The observer was kept blinded to the treatment groups of the mice.

To investigate the therapeutic potential of TrkB and TrkC signaling systems in the chronic animal disease model, we generated the agonistic monoclonal antibodies (Materials and methods). As previously described, Mab38B8 is a mouse monoclonal TrkB agonist antibody in that it can increase TrkB receptor phosphorylation in a CHO cell line stably expressing human TrkB (Tsao et al., 2008) and that it can support the survival of embryonic day 15 mouse nodose ganglion neurons over 48 h in vitro with EC50 of 0.1 pM (Lin et al., 2008). MabA5, on the other hand, is a humanized rat chimeric agonist antibody for the TrkC receptor such that, in a stable CHO cell line expressing TrkC, MabA5 induces TrkC phosphorylation (Fig. S1). MabA5 also supports the survival of embryonic day12 rat trigeminal neurons in a dose-dependent fashion with an EC50 of 2.78 pM (Fig. S1). Furthermore, saturating concentrations of MabA5 can support the same total number of neurons as those grown in the presence of 2.5 nM NT3 (Fig. S1). The pharmacokinetic properties of both antibodies, determined by following a single dose administration and serial serum sampling up to 11 days, revealed a long circulating half-life of both antibodies (∼ 264 h for MabA5 and ∼ 150 h for Mab38B8). Accordingly, in the sera of mice treated for 20 weeks or longer with either MabA5 (TrkC AAb) or Mab38B8 (TrkB AAb), significant levels of therapeutic antibody were detected in their serum 11 days after the last dose (2.8 nM for MabA5 and 13.3 nM for Mab38B8). Serum concentrations several orders of magnitude above the EC50 for each AAb ensures the dose delivered was sufficient to saturate TrkC and TrkB receptors in vivo.

Nerve conduction studies The nerve conduction studies were performed on a total of 108 TrJ mice at baseline age of 10 to 12 weeks and in 7 age-matched wild type

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Morphological and functional improvements with TrkC and TrkB agonist antibodies in regenerating and intact TrJ sciatic nerves Twenty weeks after sciatic nerve crush, microscopic examination of cross sections of sciatic nerve segments (∼3 mm distal to the crush site) showed an increase of MF population in each of the agonist antibody treated groups (2 mg/kg once a week of TrkB AAb alone, TrkC AAb alone or the two antibodies combined) compared to that in the PBS-treated TrJ controls. An increase of MFs was noted in the contralateral uncrushed intact sciatic nerves as well. In fact, in all agonist antibody treated groups, quantitative studies confirmed a significant increase in MF densities not only in the regenerating nerves but also in the contralateral uncrushed/intact side compared to those from PBS-treated TrJ nerves (Table 1). These observations indicate that TrkB and TrkC AAbs, individually or in combination, have the capacity to improve the regenerative response to nerve crush and partially repair TrJ pathology. The MF densities between TrkC AAb alone, TrkB AAb alone or the combination treatment groups were not statistically different, however, suggesting that the effects of these two antibodies on the total MF density were not additive. Fig. 1 shows the composite histograms generated from each treatment group and PBS-treated control TrJ mice. Both in regenerating and intact nerves with all treatment groups, the increase in MF density is most prominent for those fibers with axonal diameter less than 4 µm. In the regenerating nerves this increase is associated with a shift to larger diameter axons (2–4 µm) , and was most pronounced in the combination group (8855 ± 280 vs. 6699 ± 293 in PBS, p b 0.001; 8245 ± 279 in TrkB AAb, p b 0.01 vs. PBS; 8395 ± 330 in TrkC AAb, p b 0.01 vs. PBS). Moreover, g ratio (axon diameter/fiber diameter) determinations of the MFs in intact sciatic nerves showed an increase in myelin thickness in all treatment groups, partially improving the hypomyelination/ amyelination state of the peripheral nerves, the hallmark of trembler pathology. The mean g ratio in the PBS-treated TrJ is 0.77 ± 0.003, significantly greater than that obtained from wild type (0.66 ± 0.002, p b 0.0001), reflecting the hypomyelination state in this model. g ratios were significantly reduced in all treatment groups, and the effect was the most prominent with combination and TrkB AAb treatments, showing a shift of distribution of g ratio to the left, indicative of an increased myelin thickness in comparison to the PBS-treated control TrJ as seen in Fig. 2 (p b 0.0001 for PBS vs. all treatment groups, TrkB vs. TrkC or combination, and combination vs. TrkC). The percent of fibers within a g ratio range of 0.4–0.7 constituted about 29% of total fibers in the combination group, 22% in the TrkB AAb group and 14% in the TrkC AAb group compared to that of only 6.5% in the PBS-treated control TrJ nerves. Furthermore, ultrastructural morphometric studies revealed improvements in NF cytoarchitecture in response to TrkB and TrkC AAb treatments by illustrating a shift in the NF packing density toward normal. This effect was more prominent with TrkC than TrkB AAb treatment (Fig. 3). Remarkably, the functional studies showed improvements in hind limb grip strength, which correlated with the morphological observa-

Table 1 Myelinated fiber density (number/mm2) in the sciatic nerves from Trembler mice. Treatment groups

No. of animals

Intact

Regenerating

mean ± SEM

mean ± SEM

PBS control TrkB AAb TrkC AAb TrkB + TrkC AAb Wild type

6 6 6 6 3

12,691 ± 398 15,527 ± 347⁎⁎⁎ 15,164 ± 516⁎⁎ 15,021 ± 369⁎⁎ 21,071 ± 327

14,382 ± 532 17,557 ± 409⁎⁎ 16,847 ± 578⁎ 17,089 ± 501⁎⁎ 27,710 ± 596

⁎ vs. PBS control, p b 0.05. ⁎⁎ vs. PBS control, p b 0.01. ⁎⁎⁎ vs. PBS control, p b 0.001.

Fig. 1. Myelinated axon size distribution histograms from regenerating (A) and contralateral uncrushed sciatic nerves (B) from TrJ mice treated with PBS, TrkC, TrkB or the combination of TrkB and TrkC receptor agonistic antibodies at 20 weeks post-crush. A histogram generated from an age-matched wild type (WT) sciatic nerve is included for comparison in B. Error bars = standard deviation of the mean.

tions (Fig. S2). By 3 weeks post-crush in the TrkC AAb alone and the combination groups, grip strength in limbs harboring the regenerating sciatic nerves improved significantly compared to the PBS-treated control TrJ mice. The group treated with TrkB AAb alone showed better performance later, by 11 to 13 weeks post-crush, compared to the PBS group. Overall with all treatment groups, trends toward improvement in the right hind limb grip strength harboring the uncrushed sciatic nerves were also observed and became prominent after 13 to 14 weeks of treatment (12–13 weeks after crushing the left sciatic nerve). In the wild type control mice, neither hind limb showed changes in performance in response to TrkB receptor AAbs. TrJ mice receiving TrkB AAb and TrkB + TrkC AAb combination treatments and wild type mice receiving TrkB AAb failed to gain weight compared to those receiving PBS. TrkC AAb treatment alone did not affect weight; therefore, lack of weight gain in the combination treatment group is attributed to the TrkB AAb. This is consistent with our previous finding that TrkB agonist antibody can suppress food intake and cause weight loss in mice and rats (Tsao et al., 2008). Electrophysiological findings with correlative grip strength and morphology Next we conducted additional studies (Group 1 and Group 2, see Materials and methods for details of study design) to assess the effects of Trk receptor AAbs on sciatic nerve conduction parameters and to obtain correlative ipsilateral grip strength data for endpoint electrophysiology.

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Fig. 2. G ratio distribution of fibers in the intact sciatic nerves from TrJ mice treated with PBS, TrkC, TrkB or the combination of TrkB and TrkC receptor agonistic antibodies for 21 weeks (A). A shift towards increased percent of fibers with smaller g ratio (thicker myelin) is more pronounced with combination and TrkB AAb treatment. One micrometer thick, toluidine blue stained cross section from mid-sciatic nerve of a TrJ mouse treated with combination AAbs (B) illustrate a notable increase in myelin thickness (arrows), compared to PBS-treated control trembler nerve with hypomyelinated and amyelinated (arrows) axons (C). Bar = 0.5 µm for B and C.

Electrophysiological assessment of the effects of these antibodies on the uncrushed TrJ nerves will be relevant to human peripheral neuropathies. Representative tracings of recorded compound muscle action potentials (CMAPs) from wild type and TrJ mice at 12 weeks of age are shown in Fig. 4, illustrating a marked reduction of the CMAP amplitude, prolongation of the distal motor latency and polyphasic prolonged duration of CMAP in the mutant relative to the wild type. Compared with wild type (mean ± SEM), TrJ demonstrated 78% reduction of CMAP amplitude (0.48 ± 0.05 vs. 2.19± 0.35 mV) and 64% reduction of area (0.88 ± 0.12 vs. 2.43 ± 0.43 mV ms), 293% prolongation of distal latency (2.44 ± 0.30 vs. 0.83± 0.09 ms) and 71% reduction of conduction velocity (10.74 ± 0.60 vs. 36.51 ± 1.18 m/s). All comparisons were highly significant (p b 0.0001). The mean distances between distal and proximal stimulation sites in the TrJ mice (18.1 ± 0.42 mm) and wild type mice (17.7± 0.2 mm) were not statistically different. These results are comparable to published values (Meekins et al., 2004). At the end of 20 weeks, there was an increase of CMAP amplitudes in all treatment groups compared to the PBS-treated TrJ controls (Table 2). The CMAP amplitude, a parameter proportional to the number of conducting motor axons in the nerve, showed significant increase for the combination (0.59 ± 0.06, n = 23 vs. 0.39 ± 0.03 mV, n = 25; p b 0.05) and the TrkC AAb groups (0.59± 0.05 mV, n = 31; p b 0.01), corresponding to a 51% increase in each in comparison with the PBStreated TrJ mice. In the TrkB AAb group the CMAP amplitude exhibited 28% increase (0.50 ± 0.02 mV, n = 29;) over that obtained with the PBStreated TrJ control group without reaching statistical significance. A significant increase in CMAP amplitude following proximal stimulation was also present in all three treatment groups (not shown), and was most pronounced with TrkC AAb treatment (0.44± 0.05 vs. 0.29 ± 0.03 mV; p b 0.01). The area of the CMAP, the most accurate measure reflecting the number of myelinated axons in the nerve, increased in all three treatment groups, but reached a statistically significant level only in the combination group compared to PBS-TrJ controls (0.88 ± 0.15 vs. 0.55 ± 0.06 mV.ms; p b 0.05). Similarly, increases in CMAP duration was observed with all three treatments and reached a statistical significance in the combination group (5.93 ± 0.61 vs. 4.43 ± 0.33 ms; p b 0.05). This

suggests that a combined treatment with TrkB and TrkC AAbs leads to an increase in regeneration-associated myelination, thereby creating a heterogeneous population of myelinated fibers. No statistically significant changes were seen in onset latency, the measure of the distal conduction velocity of the fastest motor fibers, for any treatment group vs. PBS control TrJ group. The nerve conduction velocity increases by AAb treatments were modest without reaching statistical significance. In the PBS-treated TrJ mice, over a period of 20 weeks, there was a decline in the CMAP amplitude and area suggesting a correlation with the natural progression of the neuropathic process in this model. Baseline CMAP amplitude of 0.48 ± 0.05 mV (n = 19) decreased down to 0.39 ± 0.03 mV (n = 25) at 20 weeks, which was not statistically significant. A 37.5% reduction of CMAP area from baseline to endpoint, however, was significant (0.88 ± 0.12 vs. 0.55 ± 0.06 mV ms; p = 0.012). Prior low dose treatment of Group 2 mice improved baseline CMAP amplitude, area and conduction velocities in all treatment groups compared to Group 1 without reaching statistical significance. At 20 weeks post treatment, endpoint CMAP amplitudes were higher than baseline values in all treatment groups but the changes did not reach statistical significance. Endpoint ipsilateral hind limb grip strength increased significantly in all treatment groups compared to PBS-treated TrJ mice and correlated positively with CMAP amplitude, area and conduction velocity. Compared with the PBS-treated TrJ mice, the combination group demonstrated a 55% increase of ipsilateral grip strength (40.7 ± 0.9, n = 21 vs. 26.2 ± 0.5 in BPS group, n = 31), while this increase was about 41% for TrkB AAb (37.1 ± 1.1, n = 30) and 28% for TrkC AAb group (33.6 ± 1.0, n = 29). p values were highly significant for all treatment groups vs. PBS control. There was no statistical difference among the AAb treatment groups. The early-onset low dose treatment (Group 2) did not result in a significant increase of ipsilateral grip strength in TrkC or combination groups, although the mean values from these mice in Group 2 were higher than in Group 1. When Group 1 and Group 2 were analyzed separately, the p values were also highly significant for all three AAb treatments vs. PBS. In addition, mice in all treatment groups performed better in bilateral hind limb grip performance and remained stable during the course of the study

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Fig. 3. Neurofilament density (number/unit hexagonal area of 0.05 µm2) distribution in the sciatic nerve axons from TrJ mice treated with TrkC or TrkB receptor agonistic antibodies for 21 weeks shows a shift to the left toward normalization compared to PBS-treated group. A histogram from wild type (WT) mice is included for comparison (A). Same magnification representative cross sectional areas of neurofilament cytoskeleton from PBS-TrJ control (B), WT (C), TrkC AAb (D) and TrkB AAb (E) treated TrJ sciatic nerves are shown. The neurofilament density decrease appear more pronounced with TrkC AAb treatment. Bar = 0.5 µm.

compared to the PBS control group, however the difference was significant only for the combination group. Alterations in muscle fiber types with TrkC and TrkB agonist antibodies Muscle fiber size and fiber type distribution can be useful in assessing the status of functional reinnervation. SDH histochemical stain was applied to the ipsilateral gastrocnemius muscle at 20 weeks post treatment. In all AAb treatment groups, the gastrocnemius muscle showed an increase in fiber diameter and percentage of fast twitch fibers, and type grouping as histologic evidence of reinnervation, correlating with CMAP parameters and increased hind limb grip strength (Fig. S3). The increase in fiber diameter resulted from increases

in the fast twitch glycolytic (FTG) muscle and to a lesser degree from fast twitch oxidative (FTO) muscles, and this was most remarkable in the combination treatment group. FTG fiber size increase owing to the combination treatment was highly significant in comparison to TrkB or TrkC AAb alone. Slow twitch oxidative (STO) fiber size showed no change in response to any treatment. In terms of fiber type distribution, there was a shift in all AAb treatment groups towards fast twitch fibers compared to the PBS-treated group. The percent of fast twitch fibers increased to between 74 and 79% (74% in combination, 78% in TrkC and 79% in TrkB AAb) from 68% in the PBS-treated control TrJ mice. In wild type mice gastrocnemius muscle analyzed similarly, we found the ratio of fast twitch to slow twitch fibers to be about 1.6 (61% vs. 39%, n = 1972). The increased ratio of fast to slow twitch in the PBS-TrJ (2.1),

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Fig. 4. Representative tracings of the sciatic nerve motor nerve conduction from a wild type (WT) and a TrJ mouse at baseline (A) and at the endpoint, following 21 weeks of treatment with TrkB + TrkC AAbs (B). The mutant at baseline demonstrates a marked reduction of the compound muscle action potential (CMAP) amplitude, prolonged distal latency, and polyphasic, prolonged duration of CMAP relative to the wild type. The calculated conduction velocity for the mutant is about 31% of the wild type. The mean ± SEM of distal latency, CMAP duration and amplitude, area and conduction velocities for wild type and TrJ mice is summarized in Table 2. The endpoint tracing in the TrJ mouse (B) illustrates a 230% increase in CMAP amplitude and a 125% increase in the CMAP area.

in comparison to wild type, is likely due to partial reinnervation initiated by the neuropathic process. These observations indicate that TrkB and TrkC AAb treatment resulted in a dramatic slow-to-fast transition in muscle fiber type, a process previously known to occur with reinnervation (Mendler et al., 2008).

Short-term treatment with TrkC AAb improves Schwann cell survival, regeneration-associated myelination and the axonal NF cytoskeleton An early recovery response in grip strength performance of limbs harboring regenerating sciatic nerves was observed with TrkC AAb treatment alone, or in combination with TrkB AAb. Lack of a similar response with TrkB AAb suggests a TrkC specific role in the early recovery process. In order to explore this concept, we compared the effects of TrkC and TrkB AAbs on early stages of regeneration following a short treatment course. We focused on two early time points: day 5 post-crush, prior to myelin compaction, and day 14 post-crush, when regeneration-associated myelination is already in progress in a proximo-distal manner. Both groups received a total of three (once per week) antibody injections, and the final injection was given 1 day prior to termination of the experiments. On day 5 post-crush, densities of SC nuclei, SC apoptosis and axon diameter distribution at promyelination stage were determined. The density of SC nuclei (mean ± SEM/mm2) in the TrkC AAb treated regenerating nerves was significantly increased compared to PBS or TrkB AAb treated regenerating TrJ nerves (8780 ± 141 vs. 7376 ± 291 in PBS control TrJ; p = 0.020, and 7476 ± 370 in TrkB AAb group; p = 0.032). TrkB AAb treatment did not alter the number of SC nuclei significantly compared to the PBS-treated TrJ group. At the same 5-day post-crush time point, we assessed SC apoptosis via a TUNEL assay of fresh frozen sections

Table 2 Endpoint Electrophysiology following Trk receptor agonist antibody treatments for 20 weeks (2 mg/kg/week). CMAP and conduction velocity in the sciatic nerve G1, G2 No Latency (ms) TrkB AAb TrkC AAb TrkB + TrkC PBS Wild type†

Duration (ms)

Amp (mV)

Area (mVms)

CV (m/s)

0.61 ± 0.26

11.90 ± 5.24 11.41 ± 2.94

29

2.28 ± 0.166 4.95 ± 1.56 0.50 ± 0.22

31

2.43 ± 0.38

4.54 ± 1.85 0.59 ± 0.27** 0.71 ± 0.37

23

2.30 ± 0.41

5.93 ± 2.92* 0.59 ± 0.29*

0.88 ± 0.71* 12.48 ± 4.70

25 7

2.41 ± 0.29 0.83 ± 0.09

4.43 ± .66 3.24 ± .46

0.55 ± 0.29 2.43 ± 0.43

* vs. PBS control, p b 0.05. **vs. PBS control, p b 0.01. † vs. TrJ, see text for statistics.

0.39 ± 0.15 2.19 ± 0.35

9.83 ± 2.50 36.51 ± 1.18

from TrJ sciatic nerve segments distal to the crush site (Fig. S4). The number of apoptotic nuclei, obtained from 3 mice in each group and normalized per unit area of 91,176 µm2 in the TrkC AAb treated nerves was lower (37.8 ± 5.9) compared to PBS (58.4 ± 4.1) or TrkB AAb treated group (57.8 ± 3.6), which correlated with the increased SC density in response to TrkC AAb treatment. Collectively, these results indicate that TrkC AAb, but not TrkB AAb, exerts a robust effect on promoting SC survival, during the early regenerative response in TrJ nerves. In the TrkC AAb group, as early as day 5 post-crush, axons at the promyelination stage showed evidence of radial growth, and a shift to larger diameters compared to TrkB AAb and PBS-treated TrJ groups, indicating a more advanced state of regeneration (Fig. 5). As seen in the size distribution histograms of axons at promyelination, 33% of TrkC AAb treated axons possess diameters of 2–3 µm, compared to 19.9% in the TrkB AAb, and 15% in the PBS group. Furthermore, in the TrkC AAb treated group, axons greater than 3 µm represent 13% of the total, while in TrkB and PBS groups this population is only 1%. Moreover, larger diameter axons are associated with improvements in NF packing density as shown by shift towards normal densities (i.e. less dense), which is more pronounced in the TrkC AAb group than those from TrkB AAb (not shown). Collectively, these findings suggest that axons in the TrkC AAb treated TrJ mice regenerated faster with an increase in the radial growth during the first 5 days of regeneration (Fig. 5). We hypothesized that increased SC survival, early axonal sprouting with changes in NF cytoskeletal properties and axonal radial growth induced by the TrkC AAb group should result in an enhancement of regeneration-associated myelination. To assess this possibility, we first studied the spatiotemporal progression of regeneration-associated myelination on day 14 post-crush in TrkC AAb, TrkB AAb and PBStreated TrJ mice, with cross sections from 3 consecutive nerve segments distal to the crush site. The number of MFs, the individual unmyelinated axons at 1:1 relationship with their promyelinating SCs (UnMFs), and the number of SC nuclei associated with unmyelinated axon clusters were determined. The highest percentage of MFs and the lowest percentage of UnMFs were found in the TrkC AAb group for each segment, which is significantly different from those in the PBS control treated group. As a consequence, the ratio of MFs to UnMFs was highest in the D1 segment, which gradually decreased in D2 and reached the lowest value in D3 (Fig. 6). These observations support a proximo-distal progression of myelination, best accomplished with TrkC AAb treatment. The difference between TrkC AAb treated and control treated TrJ mice was most pronounced in the D3 segments. To further evaluate the effects of these antibodies on the regeneration-associated myelination process, we compared the densities (mean ± SEM per unite fascicular area of 11,750 µm2) of the MFs, UnMFs and the SC nuclei associated with unmyelinated axon clusters in the distal third (D3) segment (Fig. S5). The MF density in the TrkC AAb treated nerves were significantly increased in comparison to

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Fig. 5. Axon size at promyelination on day 5 post-crush from TrJ mice treated with PBS, TrkC or TrkB receptor agonistic antibodies. One micrometer-thick, toluidine blue stained cross sections were obtained approximately 0.5 mm immediately distal to the crush site. Larger diameter axons at promyelination are present (arrows) in TrkC AAb treated nerves (A) compared to TrkB AAb (B) or PBS group. Axon size distribution histogram shows a shift to larger diameter axons in both treatment groups, but more prominent with TrkC AAb treatment (D).

those in the PBS group (86.6 ± 2.4 vs. 60.3 ± 0.8; p = 0.010). The increase in SC nuclei associated with unmyelinated axon clusters was not statistically significant (44.0 ± 2.1 vs. 33.3 ± 2.6; p = NS) while promyelinated axons (UnMFs) were decreased significantly (30.6 ± 2.3 vs.56.3 ± 0.3; p = 0.006). Compared to PBS controls, the TrkB AAb treatment also improved proximo-distal progression of the regeneration-associated myelination process, reflected as an increase of MF density (89.6 ± 10.4 vs. 60.3 ± 0.8) and a decrease of promyelinated axon density (46.0 ± 8.5 vs. 56.3 ± 0.3). While the overall spatiotemporal progression of myelination in TrkB AAb treated nerves appeared behind that in the TrkC AAb group, changes in densities of MF or promyelinated axons were not statistically significant, which is likely due to small sample size. The density of SC nuclei associated with unmyelinated axon clusters, representing a mixed SC population composed of those associated with nonmyelinated axons (Remak bundles) and with axon clusters at premyelination, was increased in the TrkC AAb treated group (44.0 ± 2.1 vs. 34.6 ± 0.8 in TrkB AAb, or vs. 33.3 ± 2.6 in PBS), in further support of the observations made on day 5 post-crush that SC survival is improved with TrkC AAb, but not with TrkB AAb. Trk specific AAb treatment and neurofilament phosphorylation Morphologic observations following a short-term treatment with TrkB and TrkC AAbs coupled with an early recovery in grip strength from limbs harboring regenerating sciatic nerves suggest important biological effects on SC survival and regeneration for these Trk receptor agonist AAbs in a cell specific manner. In order to determine

whether NF phosphorylation status correlates with an early regenerative response, we carried out western blot analyses to assess the phosphorylated status of NF-H following a short treatment period (once a week for 3 weeks), with TrkB or TrkC AAb. We observed a general decrease in the NF-H phosphorylation state in DRG, roots and the sciatic nerves with either AAb treatment group compared to PBStreated control TrJ mice (Fig. 7). This effect was more pronounced with TrkB AAb treatment suggesting a cell-type specific response. In addition, with TrkC AAb treatment, there was an increase in the hypophosphorylated NF-H and the total NF-H protein in the midsciatic nerve segments compared to the PBS-treated group (not shown). In comparison to wild type, a further decrease in the phosphorylated NF-H in the TrJ DRG, roots and sciatic nerves as shown here, suggests that TrkC and TrkB AAbs are capable of increasing the intrinsic growth state of axons similar to that seen in the re-injured axons which received a prior conditioning lesion, there by pushing them toward an enhanced regeneration mode (Smith and Skene, 1997). On the other hand, after 20 weeks of TrkC AAb treatment the phosphorylated NF-H was significantly increased in the proximal and mid-sciatic nerves while it remained unchanged in the DRG cell bodies and proximal roots (Fig. 7B). This correlates with a shift towards normalization of the NF packing density in the axon. Discussion In this study using histological, functional and electrophysiological parameters, we have tested the efficacy of TrkB and TrkC AAbs individually and in combination, in the TrJ mouse model for CMT. We

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Fig. 6. Spatiotemporal progression of regeneration-associated myelination on day 14 post-crush in the 3 consecutive regenerating sciatic nerve segments (d1, d2 and d3) from TrkC AAb, TrkB AAb and PBS-treated TrJ mice. In relation to the crush site, d1 depicts the location of sections for morphometric analysis, approximately 0.5 mm immediately distal to the crush. d2 and d3 are approximately 2 and 6 mm respectively distal to the crush site (see also Fig. S5 for the schematic diagram illustrating the method of tissue marking used during dissection to follow the proximo-distal orientation of each nerve segment). Dark and light color coding is used to designate myelinated (MF) and unmyelinated axons at promyelination (UnMF). In each segment, TrkC AAb treatment resulted in the highest percentage of MFs and the lowest percentage of UnMFs followed by TrkB AAb and PBS control groups.

show that these Trk receptor agonist antibodies can promote peripheral nerve regeneration following a nerve crush, and increase the number of MFs as well as the thickness of myelin in the uncrushed contralateral TrJ sciatic nerves. Furthermore, significant improvements in grip strength are associated with increases in CMAP amplitude, the most reliable measure of the number of conducting motor axons in the nerve, correlating with functional clinical improvement. The cell type(s) targeted by these Trk receptor-specific agonist antibodies that yield important biological effects in vivo may be neurons or SCs preferentially, or both concurrently — overall our data supports the latter possibility. The biological properties of the AAb appear to mimic those of their cognate natural ligands, BDNF and NT-3. In particular BDNF and NT-3, as well as their receptors, are expressed in SCs and play an essential role in nerve regeneration (Hall, 1986, 1989; Terenghi, 1999). Furthermore, NT-3 is an important component of the SC autocrine survival loop, which ensures SC survival and differentiation in adult nerves (Meier et al., 1999; Mirsky et al., 2002). In early time point regeneration studies with TrkC AAb treatment, we observed an increase in density of SC nuclei and a decrease in SC apoptosis, which indicated increased SC survival and proliferation compared to TrkB AAb or PBS-treated nerves. Previous studies have shown that developmental loss of NT-3 in vivo results in reduced levels of myelinspecific proteins, reduced extent of myelination, and increased apoptosis in SCs (Woolley et al., 2008). Our own observations in NT3 +/− heterozygotes and TrJ mice suggest that mutant SCs are highly susceptible to apoptotic cell death when they return to immature/ premyelinating mode in regenerating nerves. Exogenous NT-3 treatment of TrJ mice resulted in a significant SC density increase in regenerating as well as uncrushed contralateral sciatic nerves (Sahenk et al., 2005, 2008). Therefore, our results show that a short course of TrkC AAb treatment achieves the same biological effect as NT-3 by protecting SCs from apoptosis in nerve segments distal to the crush injury and imply that SCs are the target cell for this specific effect. Our regeneration studies have also revealed that a short course pretreatment with TrkC AAb resulted in a more advanced state of regeneration-associated myelination than the TrkB AAb treated group. As early as day 5 post-crush, TrkC AAb treated axons at the

promyelination stage showed evidence of radial growth by shifting to larger diameters. Moreover, on day 14, an advanced spatiotemporal progression of myelination in consecutive nerve segments was noted compared to TrkB AAb and PBS-treated TrJ nerves. These observations indicate a neuronal response, and suggest that the TrkC AAb promotes an earlier/faster axonal regeneration, thereby allowing axons to increase in diameter so that the onset of myelin compaction could be achieved. In agreement, the early recovery in grip strength performance of limbs harboring regenerating sciatic nerves observed with TrkC AAb alone highlights a motor neuron specific role for NT-3 and its receptor TrkC in the early recovery process. In TrkB AAb treated TrJ nerves, spatiotemporal progression of regeneration-associated myelination, that is the percent of myelinated and promyelinated axons (UnMFs) in nerve segments distal to the crush, is slightly behind those treated with TrkC AAb. However, the MF density in the last distal segment (D3) with TrkB AAb is comparable to TrkC AAb treatment while the density of promyelinated axons is greater. The SC nuclei associated with unmyelinated axon clusters are reduced in number compared to those from the TrkC AAb suggesting that the myelination, once started, is rather efficient with TrkB AAb. Moreover, the myelin thickness increase in the intact/uncrushed sciatic nerves treated with TrkB AAb was significantly greater than those with TrkC AAb suggesting that TrkB AAb promotes myelination. This feature is in agreement with the previous studies illustrating an enhancement of active myelin formation with BDNF in SC/DRG neuronal co-cultures (Chan et al., 2001). It is not surprising that these different biological effects individually induced by TrkB and TrkC AAbs, when in

Fig. 7. A short treatment period, once a week for 3 weeks with TrkB or TrkC AAb results in a decrease in the phosphorylated NF-H protein (A) in the lumbar DRG, roots and sciatic nerve compared to the PBS-treated control TrJ mice. L4-L6 DRG, roots and proximal and mid-sciatic nerve segments were subjected to quantitative western blotting. Data are normalized to PBS-treated TrJ and are means ± SEM (n = 3–4). Results from a long treatment period for 20 weeks are shown from TrkC AAb and PBS-treated control TrJ mice in B, showing increased amounts of phosphorylated NF-H protein in the proximal and mid-sciatic nerve segments.

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combination lead to the ultimate improvement in myelin thickness as shown in this report. Our studies assessing NF phosphorylation status in TrJ peripheral nerves provide additional correlative data indicating that TrkB and TrkC AAbs induce a cell specific early regenerative response. Previous studies of the TrJ sciatic nerve have shown a decrease in the NF content and NF-phosphorylation state, along with decreased axon caliber and NF packing density, compared to wild type mice (de Waegh et al., 1992). Our studies here show that in addition to the sciatic nerves in this mutant, the phosphorylated NF-H in the DRG cell bodies and roots is also decreased compared to the wild type mice. In the presence of an ongoing, progressive and preferentially distal axonal loss in this model, it is likely that TrJ nerves are in a perpetual regeneration mode, compatible with the NF hypophosphorylation state of the entire peripheral nerve, cell body and axon, during regeneration (Hoffman and Cleveland, 1988; Hoffman et al., 1987; Tetzlaff et al., 1988). Following axotomy, in contrast to tubulin and actin, which are upregulated, there is a strong down-regulation of NF mRNAs and proteins in the PNS, leading to reduced levels of axonally transported NFs, which remain suppressed until successful reinnervation occurs (Goldstein et al., 1988; Hoffman and Cleveland, 1988; Oblinger and Lasek, 1988; Tetzlaff et al., 1988; Wong and Oblinger, 1987). Interestingly, following the short-term treatment period, we found a further decrease in the phosphorylated NF-H in DRG, roots and the sciatic nerves with both treatment groups compared to PBS-treated control TrJ mice. This further decrease in the phosphorylated NF-H may suggest that TrkC and TrkB AAbs are capable of inducing a cell body response, similar to those seen with the conditioning lesion paradigm (Tetzlaff et al., 1996) . Earlier studies have shown that axons regenerate more rapidly after a test lesion if they received a prior conditioning lesion, and that transcription-dependent changes in gene expression and axonal transport, resulting in a further downregulation of NF synthesis in neurons are responsible for this conditioning lesion effect (McQuarrie, 1978; Richardson and Verge, 1987; Smith and Skene, 1997; Tetzlaff et al., 1996). Furthermore, it has been shown that hypophosphorylated NF-H and NF-M undergo more rapid axonal transport and form more stable complexes with NF transport motor kinesin, KIF-5A (Jung et al., 2000; Yabe et al., 2000). The early motor recovery response with TrkC AAb suggests that motor neurons are targeted to induce an appropriate cell body response similar to those seen with the conditioning lesion paradigm. We do not have functional data assessing sensory recovery, specific for the TrkB positive DRG neurons, to directly correlate with decreased phosphorylated NF-H levels. Additional studies are needed to elucidate the quantitative and cell-type specific differences in the phosphorylated NF-H levels and transcriptional changes in other cytoskeletal markers of regeneration, induced by these agonistic antibodies. Results from TrJ mice subjected to a long-term treatment show opposite changes in the phosphorylated NF-H levels in the sciatic nerves over a time period of 20 weeks. No change in phosphorylated NF-H was found in the DRG cell bodies and proximal roots while it was significantly increased in the proximal and mid-sciatic nerves in the TrkC AAb treated mice compared to the PBS group, correlating with a shift towards normalization of the NF packing density in the axon. Compared to TrkC AAb, a less prominent shift towards normalization of NF spacing is noted with TrkB AAb treatment. Collectively, these observations provide evidence for an important biological function of these Trk receptor AAbs in promoting regeneration by augmentation of the intrinsic growth state of the neuron and regulating NF cytoskeletal properties in the axon. Reports showing alterations in the NF expression and/or phosphorylation induced by NT-3 treatment of streptozotocin-induced diabetic rats have direct relevance to our studies by emphasizing the similarities to the findings that we obtained using the TrkC AAb (Sayers et al., 2003). NT-3 prevented proximal axon accumulation of NF-H and NF-M in this model; and also normalized the phosphory-

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lation status of these subunits. Similarly, NT-3 treatment was shown to ameliorate the abnormal accumulation of NF-H in DRG neurons and the MF loss associated with experimental cisplatin-induced sensory neuropathy (Gao et al., 1995). In addition, there is in vitro evidence for the ability of both NT-3 and BDNF to phosphorylate NF-H (Tokuoka et al., 2000). It has been postulated that highly phosphorylated tail domains of NF-M and NF-H are involved in establishing cross bridges between NFs, implicated in both organization and stabilization of the NF cytoskeleton (Yuan et al., 2006). The results of these preclinical studies unequivocally, and for the first time, show that TrkB and TrkC AAbs have the capacity to induce significant improvements in grip strength that correlate with increases in the sciatic nerve CMAP amplitude in the TrJ mouse model for CMT neuropathy. Previous studies in rats after nerve injuries have shown that forelimb grip strength and median nerve CMAP amplitude correlated positively and that the CMAP is a valid parameter that shows the typical time course of nerve regeneration and reinnervation (Wang et al., 2008). In our study we have provided evidence to support a multifactorial basis for increase CMAP amplitude. On the one hand we found increase in myelinated fiber density and improved myelin thickness in the sciatic nerve. In addition, we found an increase in muscle fiber size and type grouping consistent with reinnervation (collateral sprouting) of the gastrocnemius muscle from treatment. We can only infer that the combination of these factors had a positive effect on CMAP amplitude recorded from the intrinsic foot muscles. Increases in sciatic CMAP amplitude as a valid assessment of successful nerve regeneration is supported with our muscle histology data. In all treatment groups, ipsilateral gastrocnemius muscle showed increases in fiber diameter and percentage of fast twitch fibers as histological evidence correlating with CMAP amplitude increase. We believe that the summation of improvements in the nerve and muscle cited above translates into a functional improvement in grip strength as well as electrophysiological improvement in CMAP amplitude. It is significant that mice which received the combined treatment performed the best in motor testing and displayed not only a significant increase in CMAP amplitude but also CMAP area and duration, in full agreement with results from the morphological studies of nerve and muscle. It should be noted that in the PBS-treated TrJ mice, over a period of 20 weeks, there was a decline in the CMAP amplitude and area suggesting a correlation with the natural progression of the neuropathic process in this model. A significant, 37% reduction of CMAP area from baseline to endpoint reflects a loss of MFs over 20 weeks, in agreement with previous studies demonstrating a progressive axon loss by using stimulated single-fiber electromyography in this model (Meekins et al., 2007). In contrast, endpoint CMAP amplitudes were higher than baseline values in all treatment groups, without reaching statistical significance. Considering the severity of the large fiber loss and hypomyelination in this model, it is expected that these treatments did not produce statistically significant changes in onset latency, the measure of the distal conduction velocity of the fastest motor fibers, and the conduction velocity increases were meager. Our proof of principle studies provide evidence for potential therapeutic use of monoclonal agonist antibodies for TrkB and TrkC in patients with CMT1A. It is plausible that they may also be beneficial for other neuropathies. The strong correlation between the electrophysiological and functional data will be useful for designing future clinical trials, positioning CMAP amplitude as the primary outcome measure that will correlate with functional improvement in motor strength. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.expneurol.2010.05.013.

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