Female C57BL/6 mice aged 8 weeks were fed 0.2% cuprizone in normal chow (Teklad Custom Research Diets, New Brunswick, NJ, USA) for 6 weeks to induce demyelination. Female mice were used due to the increased incidence of MS in females (Wallin et al., 2019). Cuprizone feed was removed, and mice were sacrificed or received intracerebroventricular osmotic pumps for 7 days.

For experiments in conditional knockout mice, female, 8–10 week-old CNPaseCre^+/– × TrkB^fl/fl (Lappe-Siefke et al., 2003; Luikart et al., 2005; Fletcher et al., 2018b) on C57BL/6 background underwent the procedures described above.

All mice were housed in specific pathogen-free conditions at the Melbourne Brain Centre Animal Facility. All animal procedures were approved by the Florey Institute for Neuroscience and Mental Health Animal Ethics Committee and followed the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.

Following cuprizone feeding, mice received either: 40 μM TDP6, 500 μM LM22A-4 or the artificial cerebrospinal fluid (aCSF) vehicle via ICV as described in Fletcher et al. (2018b). Briefly, cannulae of osmotic pumps (flow rate: 0.5 μL/h, Azlet) were stereotaxically inserted over the right lateral ventricle under isoflurane anesthesia. Infusion concentrations of TDP6 and LM22A-4 were determined based on previous in vitro studies characterizing their effective concentrations (Massa et al., 2010; Wong et al., 2014). TDP6 infused animals were the same animals from Fletcher et al. (2018b) and LM22A-4 infusion experiments were performed concurrently. Following stereotaxic surgery, all mice were placed in a recovery chamber maintained at 32°C and were monitored for adverse reactions immediately following surgery and then daily. After 7 days of ICV infusion, mice were taken for necropsy and brain removed for immunostaining and electron microscopy (EM).

Mice were anesthetized and transcardially perfused with 0.1 M sterile mouse isotonic phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA). Brains were collected and post-fixed overnight in 4% PFA. The first millimeter of the right hemisphere from the sagittal midline was selected for EM processing as previously described (Fletcher et al., 2018b). The remaining tissue and contralateral hemisphere were cryoprotected in 30% sucrose prior to embedding in OCT. Frozen sections were cut in the sagittal orientation at 10 μm thickness using a cryostat maintained between −20°C and −17°C and collected on SuperfrostPlus slides, air-dried and stored at −80°C until use. Approximately 70–100 μm separated adjacent sections on each slide. The sections beyond 2.64 μm Bregma laterally were excluded.

Immunofluorescent staining was performed as previously described (Fletcher et al., 2018b). Briefly, slides were washed in PBS before overnight incubation at room temperature with primary antibodies. Slides were then washed and incubated with the appropriate fluorophore-conjugated secondary antibody for 2 h at room temperature in the dark. Slides were washed, and counterstained with nuclear marker Hoescht33442 [omitted for myelin basic protein (MBP) and oligodendroglial stains] before mounting with aqueous mounting media (DAKO). All immunohistochemistry was performed in batches.

Antibodies used were: rat anti-MBP (1:200, MAB386, Millipore, MA, USA), rabbit anti-Olig2 (1:200, AB9610, Millipore, MA, USA), mouse anti-CC1 (1:200, APC, OP80, CalBioChem, CA, USA), goat anti-platelet derived growth factor receptor-α (PDGFRα, 1:200, AF1062. R&D Systems, MN, USA), goat anti-Iba1 (1:200, ab5076, Abcam, UK), mouse anti-glial fibrially acidic protein (GFAP, 1:100, MA360, Millipore, MA, USA) and rabbit anti-phosphorylated TrkB^S478 (1:200 R-1718-50, Biosensis).

Semi-thin (0.5–0.1 μm) sections of caudal corpus callosum in a sagittal plane were collected on glass slides and stained with 1% toluidine blue to select region of analysis. Ultrathin (0.1 μm) sections were subsequently collected on 3 × 3 mm copper grids and specimens examined using a JEOL 1001 transmission electron microscope. Images were captured with MegaView III CCD cooled camera operated with iTEM AnalySIS software (Olympus Soft Imaging Systems GmbH). A minimum of six distinct fields of view were imaged at 5,000 or 10,000× magnification for each animal. The proportion of myelinated axons, axon diameter and g-ratio were analyzed manually using FIJI/ImageJ (National Institutes of Health, Bethesda, MD, USA). For g-ratios at least 100 axons from three mice per group were measured. Resin embedding, sectioning and post-staining and EM imaging were performed at the Peter MacCallum Centre for Advanced Histology and Microscopy.

Imaging was performed blind to treatment group and restricted to the caudal corpus callosum approximately −1.1 to −3.0 mm from Bregma in the rostral-caudal direction. Tracts contributing to the dorsal hippocampal commissure were excluded from analysis. For each analysis, a minimum of three sections per animal was imaged.

To quantify the level of remyelination images of MBP stained sections were collected with an AxioVision Hr camera attached to a Zeiss Axioplan2 epi-fluorescence microscope under a 20× objective. Uniform exposure times were used. Remaining images were acquired with a Zeiss LSM780 or LSM880 confocal microscope with 405 nm, 488 nm, 561 nm and 633 nm laser lines. For each fluorescent stain uniform settings were used.

MBP staining was measured on single channel grayscale images using the default automated threshold function in FIJI/ImageJ and limited to a standard region of interest (ROI) of 625,000 μm² for each section. Data were expressed as a percentage area of positive staining in a single ROI.

The Flp-In 293 cell system (ThermoScientific) was used to generate isogenic TrkB expressing HEK293 cell lines where TrkB was integrated into the host cell genome. Flp-In cells express a hygromycin resistance gene, which enables the use of hygromycin (50 μg/μL) to exert selective pressure for cells carrying the Flp-In construct.

Briefly, to generate the TrkB expressing HEK293 cell line (Supplementary Figure S1A), Ntrk2 (NM_012731.2) was amplified by PCR from rat cDNA and recombined into pDONR201 entry vector by BP clonase II reaction (Invitrogen) according to the manufacturer’s instructions. DH5α bacteria (ThermoScientific) were transformed with the entry vector by heat shock and positive colonies expressing the pDONR201 plasmid were selected using kanamycin resistance. pDONR201 plasmid containing Ntrk2 was purified using a mini-prep kit (Promega) and recombined into pEF5/FRT/V5-DEST destination vector by LR clonase II reaction (ThermoScientific). Following transformation with the destination vector, DH5α bacterial colonies were placed under ampicillin selective pressure and plasmid DNA extracted. At each step, successful recombination of Ntrk2 into the entry and destination vectors was confirmed by restriction ligase digest with ApaI (NEB) and Sanger sequencing (Australian Genome Research Facility).

Once the Ntrk2 destination vector was generated, the Flp-In HEK293 host cells were transfected with the Ntrk2 destination vector and the Flp-recombinase vector pOG44 with 50 μg/μL hygromycin according to the manufacturer’s instructions. Flp-In HEK293 cells were maintained at 37°C with 5% CO₂ in Dulbecco’s modified eagle medium (DMEM) with 10% fetal bovine serum, 1% L-glutamine, 1% penicillin and 1% streptomyocin. TrkB expression by the isogenic TrkB Flp-In HEK293 cells was verified by Western blot (Supplementary Figures S1B–E).

To examine the capacity of LM22A-4 to mimic the BDNF-TrkB signaling cascade, isogenic TrkB FlP-In HEK293 cells were starved in serum-free media for 2 h before treatment with 4 nM BDNF or 500 nM LM22A-4 for 0, 5, 15, 30, 60 and 240 min. Concentrations were chosen based on previous work (Massa et al., 2010; Wong et al., 2014). Cells were lysed with TNE (Tris) buffer containing protease (Complete Mini) and phosphatase inhibitors (PhosStop Roche, 50 mM Sodium Fluoride). Protein concentrations were determined by Bradford assay and lysates stored at −80°C until use.

Lysates were separated by SDS-PAGE (4%–12% Bis-Tris, Invitrogen, Carlsbad, CA, USA) and transferred to PVDF membrane and probed with antibodies against TrkB (1:1,000, sc-8316, SantaCruz Biotechnology, Santa Cruz, CA, USA) and pTrkB^s478 (1:1,000, R-1718-50, Biosensis), p44/42 MAPK (ERK1/2, 1:1,000, #9102 Cell Signaling Technologies, Danvers, MA, USA) and phosphorylated ERK1/2 (pERK1/2, 1:1,000, #9101 Cell Signaling Technologies, Danvers, MA, USA). All blots shown are representative of at least three independent experiments. Optical density value for each band was determined using FIJI/ImageJ and corrected to loading control and normalized against the relevant control condition.

Data were analyzed by unpaired t-test, one-way analysis of variance (ANOVA) or mixed effect models for repeated measures (GraphPad Prism 8), to test the effect of TrkB agonist treatments with post hoc multiple comparisons as appropriate. Statistical significance was set as p < 0.05.

We have previously shown treatment with TDP6, a structural mimetic of BDNF, enhances the number of axons remyelinated and increases myelin sheath thickness during recovery after 6-weeks cuprizone challenge in an oligodendroglial-TrkB dependent manner (Fletcher et al., 2018b). Here, we compared TDP6 with LM22A-4, a small molecule TrkB agonist reported to be a functional BDNF-mimetic (Massa et al., 2010). Demyelination by cuprizone feeding was confirmed by MBP immunostaining, with severely reduced levels of MBP expression observed in animals taken at 6 weeks of cuprizone feeding (minimum = 2/cohort; Supplementary Figure S2). Cuprizone feed was withdrawn and remaining animals received ICV minipumps containing artificial cerebrospinal fluid (aCSF), TDP6 (40 μM) or LM22A-4 (500 μM) for 7 days.

To examine the extent of remyelination, MBP-immunostaining in the caudal corpus callosum was assessed. This revealed both TDP6 and LM22A-4 treatment increased (p < 0.0001) the percentage area of MBP⁺ staining compared to treatment with the aCSF vehicle (Figure 1A, quantified in Figure 1B). EM analysis indicated that mice treated with TDP6 exhibited a trend (p = 0.09) increase towards more remyelinated axons compared to those receiving aCSF, whereas for those receiving LM22A-4, no increase was observed (p = 0.46; Figure 1C). Both TDP6 and LM22A-4 treatment resulted in a significant (p = 0.002) reduction in mean g-ratio indicative of increased myelin thickness (Figure 1D). Linear regression analysis of g-ratio against axon diameter (Figure 1E) indicated that although both TDP6 and LM22A-4 treatments increase myelin sheath thickness during remyelination (Figure 1D), TDP6 exerted a more consistent effect with a significant decrease in y-intercept (p = 0.0032), but no change in slope (p = 0.35) indicating that g-ratio was reduced across all axonal diameters, whereas for LM22A-4 there was a significant increase in slope (p = 0.006), indicative of reduced g-ratio and thicker myelin on smaller diameter axons. Collectively, these data are consistent with our previous findings that BDNF-TrkB signaling increases myelin sheath thickness during remyelination in vivo (Fletcher et al., 2018b).

Next, we assessed oligodendroglial populations in the corpus callosum by co-immunostaining Olig2 with platelet-derived growth factor receptor-alpha (PDGFRα) and CC1 to identify Olig2⁺PDGFRα⁺ oligodendrocyte progenitor cells (OPCs), Olig2⁺CC1⁺ post-mitotic oligodendrocytes (CC1 the clone for APC, a cytoplasmic marker highly expressed in post-mitotic oligodendrocytes) and an Olig2⁺PDGFRα⁻CC1⁻ intermediate oligodendroglial population (Figure 2A). Counts in the caudal corpus callosum revealed TDP6 and LM22A-4 increased the total population of Olig2⁺ oligodendroglia compared to treatment with aCSF vehicle (Figure 2B, p < 0.0001). Interestingly, LM22A-4 treatment exerted a more profound effect, increasing the density of Olig2⁺ oligodendroglia above TDP6 (Figure 2B, p = 0.0001). Both TDP6 and LM22A-4 treatments increased the density of Olig2⁺CC1⁺ post-mitotic oligodendrocytes compared to aCSF vehicle (Figure 2D, p = 0.013), consistent with the pro-differentiation effect of TrkB activation on oligodendroglia. However, assessment of Olig2⁺PDGFRα⁺ OPCs indicated LM22A-4 also increased the density of OPCs compared to treatment with TDP6 or aCSF vehicle (Figure 2C, p = 0.011). Overall, these data suggest that selectively targeting TrkB during remyelination primarily enhances oligodendroglial differentiation.

To examine whether these effects of LM22A-4 and TDP6 were due to alterations in lineage progression during differentiation, the proportion of Olig2⁺PDGFRα⁺, Olig2⁺CC1⁺ and Olig2⁺ only cells out of the total Olig2⁺ population were assessed (Figure 2E). The proportion of cells that were OPCs or post-mitotic oligodendrocytes were unchanged between groups. However, LM22A-4 treatment significantly increased the proportion of Olig2⁺ only cells (26% ± 7%) compared to treatment with either TDP6 (6% ± 8%) or aCSF vehicle (10% ± 7%; Figure 2E, mean ± SD, p < 0.0001, χ² distribution test). These data suggest LM22A-4 treatment may exert a greater effect than TDP6 to increase the proliferation or survival of oligodendroglia during myelin repair.

To determine if TDP6 and LM22A-4 infusions stimulated TrkB phosphorylation on oligodendroglia we performed triple immunolabeling for pTrkB^S478 with PDGFRα and CC1 to identify OPCs and post-mitotic oligodendrocytes, respectively (Figure 3A). This revealed that TDP6 and LM22A-4 infusions were successful, with an increased proportion of pTrkB^S478+ cells in the corpus callosum during remyelination, compared to the aCSF vehicle (Figure 3B, p = 0.0022). Assessment of the proportion of pTrkB^S478+ cells positive for the OPC marker PDGFRα indicated treatment with TDP6 and LM22A-4 had no effect on TrkB activation on OPCs (Figure 3C, p = 0.21). However, the proportion of TrkB^S478+CC1⁺ post-mitotic oligodendrocytes increased with TDP6 treatment compared to treatment with LM22A-4 (Figure 3D, p = 0.046). These data suggest that LM22A-4 can signal via TrkB during remyelination in vivo and is consistent with previous findings that TDP6 stimulates TrkB phosphorylation on CC1⁺ oligodendrocytes.

To determine whether the effects of LM22A-4 on myelin sheath thickness and oligodendrocyte populations during myelin repair are dependent on oligodendroglial TrkB expression, we repeated the infusion experiment in CNPaseCre^+/– × TrkB^fl/fl mice in which TrkB is genetically deleted from maturing oligodendrocytes. These mice have a 3-fold reduction in TrkB⁺ oligodendroglia but adult myelination and oligodendrocyte populations are unaffected (Fletcher et al., 2018b). Cuprizone was administered for 6 weeks, and LM22A-4 or aCSF vehicle was infused via ICV minipumps for 7 days. Immunostaining for MBP revealed that LM22A-4 treatment in the oligodendroglial TrkB knockout mice had no effect on the percentage area of MBP⁺ immunostaining compared to the aCSF vehicle (Figure 4A, quantified in Figure 4B, p = 0.21). Similarly, EM analysis (Figure 4D) revealed there was no change in the proportion of axons myelinated with LM22A-4 treatment (Figure 4C, p = 0.85) or the mean g-ratio (aCSF: 0.77 ± 0.087; LM22A-4: 0.78 ± 0.088, p = 0.90, n = 3–4/group, unpaired t-test). These data are consistent with oligodendroglial TrkB expression being necessary for LM22A-4 to increase myelin sheath thickness during remyelination.

To determine if LM22A-4 treatment increased oligodendroglial populations during remyelination in oligodendroglial TrkB knockout mice, triple immunolabeling for Olig2-PDGFRα-CC1 was performed in the contralateral caudal corpus callosum (Figure 5A). Counts revealed that LM22A-4 treatment had exerted no change in the density of Olig2⁺ oligodendroglia (p = 0.91; Figure 5B), Olig2⁺PDGFRα⁺ OPCs (p = 0.38; Figure 5C) or Olig2⁺CC1⁺ post-mitotic oligodendrocytes (p = 0.94; Figure 5D) compared to the aCSF vehicle. Similarly, there was no difference in the proportion of Olig2⁺ only cells between aCSF vehicle and LM22A-4 treatment in the oligodendroglial TrkB knockout mice (Figure 5E, p = 0.33, χ² distribution test). Collectively, these data confirm that the action of LM22A-4 in increasing oligodendroglial populations is dependent on oligodendroglial expressed TrkB.

To examine the level of TrkB phosphorylation in LM22A-4 treated oligodendroglial TrkB knockout mice, immunohistochemistry for pTrkB^S478 with oligodendrocyte markers PDGFRα and CC1 was performed (Figure 6A). Analysis of the caudal corpus callosum revealed that in the oligodendroglial TrkB knockout mice LM22A-4 treatment did not increase the proportion of pTrkB^S478+ cells compared to the aCSF vehicle (Figure 6B, p = 0.24). This was also reflected with no change in the proportion of pTrkB^S478+ cells positive for oligodendroglial markers PDGFRα (p = 0.99; Figure 6C) or CC1 (p > 0.99; Figure 6D). These data indicate that LM22A-4 mediated TrkB phosphorylation during remyelination requires oligodendroglial TrkB expression.

To determine if LM22A-4 elicits a signaling cascade mimicking typical BDNF-TrkB signaling, we generated an isogenic stable TrkB expressing HEK293 (293-TrkB) cell line using the Flp-In system (Supplementary Figure S1A). TrkB expression in the 293-TrkB cells was confirmed by Western blot and compared to TrkB expression generated by transiently transfecting Flp-In HEK293 cells with the Ntrk2 expression vector. This revealed that transiently transfected cells overexpress both mature glycosylated and unprocessed TrkB receptors, whereas the 293-TrkB cells express only the fully mature glycosylated form (Supplementary Figure S1B). To confirm that the 293-TrkB cells responded to BDNF, cells were treated with BDNF (0.04 nM to 40 nM) for 15 min (Supplementary Figure S1C) which resulted in increasing levels of TrkB and ERK1/2 phosphorylation (Supplementary Figure S1D).

As determined in the original report characterizing LM22A-4 as functional BDNF mimetic (Massa et al., 2010), we used 500 nM as the standard concentration for our in vitro studies. The 293-TrkB cells were treated with 4 nM BDNF or 500 nM LM22A-4 for a time course of 5, 15, 30, 60 and 240 min and assessed for TrkB and ERK1/2 phosphorylation by Western blot (Figure 7A). Densitometric analysis (Figure 7B) revealed that compared to BDNF treatment, which increased TrkB phosphorylation within 5 min (p = 0.012), LM22A-4 did not significantly increase levels of phosphorylated TrkB until 240 min of treatment (p = 0.02). The effects of LM22A-4 treatment on ERK1/2 phosphorylation where levels peaked at 5 min of treatment, and significantly declined compared to BDNF from 15 to 240 min (Figure 7C). Collectively, these data indicate that LM22A-4 does not elicit a signaling cascade that mimics typical BDNF-TrkB signaling, suggesting that pERK1/2 is upstream of TrkB phosphorylation in the pathway stimulated by LM22A-4.

The animal study was reviewed and approved by The Florey Animal Ethics Committee, Florey Institute of Neuroscience and Mental Health.