TrkB Agonist LM22A-4 Increases Oligodendroglial Populations During Myelin Repair in the Brain

The neurotrophin, brain-derived neurotrophic factor (BDNF) promotes central nervous system (CNS) myelination during development and after injury. This is achieved via activation of oligodendrocyte-expressed tropomyosin-related kinase (Trk) B receptors. However, while administration of BDNF has shown beneficial effects, BDNF itself has a poor pharmacokinetic profile. Here, we compare two TrkB-targeted BDNF-mimetics, the structural-mimetic, tricyclic dimeric peptide-6 (TDP6) and the non-peptide small molecule TrkB agonist LM22A-4 in the cuprizone model of central demyelination in female mice. Both mimetics promoted remyelination, increasing myelin sheath thickness and oligodendrocyte densities after one-week recovery. Importantly, LM22A-4 exerts these effects in an oligodendroglial TrkB-dependent manner. However, analysis of TrkB signaling by LM22A-4 suggests rather than direct activation of TrkB, LM22A-4 exerts its effects via indirect transactivation of Trk receptors. Overall, these studies support the therapeutic strategy to selectively targeting TrkB activation to promote remyelination in the brain.


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Introduction 25 The neurotrophin, brain-derived neurotrophic factor (BDNF) is an attractive therapeutic for many 26 neurodegenerative diseases due to its broad neuroprotective effects promoting neuronal survival, 27 synaptic plasticity and central nervous system (CNS) myelination (Chao, 2003;Fletcher et al., 2018b;28 Longo and Massa, 2013). Its action via oligodendrocyte expressed TrkB to potentiate and enhance 29 myelination (Du et  for remyelinating therapies to halt disease progression. However, BDNF itself has poor 32 pharmacokinetic properties; it is non-selective, also acting through the pan-neurotrophic receptor 33 p75 NTR , has a short-half life and has high molecular weight, limiting its ability to penetrate the blood-34 brain barrier (Longo and Massa, 2013;Poduslo and Curran, 1996). To overcome these limitations a 35 range of small molecule BDNF-mimetics that selectively target the TrkB receptor have been 36 developed (Longo and Massa, 2013). This includes tricyclic dimeric peptide-6 (TDP6) (O'Leary and 37 Hughes, 2003) and the partial TrkB agonist, LM22A-4 (Massa et al., 2010). 38 TDP6 is a structural peptide mimetic, designed to mimic the Loop 2 region of BDNF that is known to 39 interact with TrkB (Chao, 2003;O'Leary and Hughes, 2003). We have previously shown that TDP6 40 mimics BDNF in promoting neuronal survival (O'Leary and Hughes, 2003) and enhancing 41 myelination both in vitro (Wong et al., 2014) and during myelin repair following cuprizone 42 demyelination in vivo (Fletcher et al., 2018a). Similarly, LM22A-4 was identified during an in silico 43 screen to identify compounds with potential to mimic the Loop2 region of BDNF and is a non-44 peptide, partial TrkB agonist (Massa et al., 2010). It is 98% smaller than BDNF and has been shown 45 to have therapeutic potential in preventing neurodegeneration in animal models of traumatic brain 46 injury, stroke, Huntington's disease and Rhett syndrome (Gu et  Here, we compare the effect of intra-cerebroventricular (ICV) administration of these BDNF 50 mimetics following cuprizone demyelination in mice. Both mimetics promoted remyelination, in 51 particular myelin sheath thickness, after one-week recovery. Interestingly, LM22A-4 increased the 52 density of oligodendroglia in the corpus callosum more than TDP6. Importantly, these effects were 53 dependent on TrkB, as post-cuprizone treatment with LM22A-4 in mice with conditional deletion of 54 TrkB from oligodendrocytes abrogated the effects on both remyelination and oligodendroglial 55 density. While this indicates that LM22A-4 promotes myelin repair in a TrkB dependent manner, 56 assessment of TrkB phosphorylation and signaling in vitro suggests that LM22A-4 may not activate 57 TrkB directly, but rather result in delayed TrkB transactivation via a GPCR-mediated mechanism. 58 Collectively these data further verify that targeting TrkB activation is a cogent strategy to promote 59 myelin repair in the brain, and that alternate small molecule mimetic strategies are effective towards 60 this end. Further studies aimed at elucidating the precise mechanism of action are warranted to 61 optimize the therapeutic potential of this approach. 62 2 Materials and Methods 64

Experimental animals and cuprizone induced demyelination 65
Female C57BL/6 mice aged 8 weeks were fed 0.2% cuprizone in normal chow (Teklad Custom  66 Research Diets, USA) for 6 weeks to induce demyelination. Cuprizone feed was removed, and mice 67 were sacrificed or received intracerebroventricular osmotic pumps for 7 days. 68 For experiments in conditional knockout mice, female, 8-10 week-old CNPase +/x TrkB fl/fl ( Following stereotaxic surgery all mice were placed in a recovery chamber maintained at 32°C and 84 were monitored for adverse reactions immediately following surgery and then daily. After 7 days of 85 ICV infusion, mice were taken for necropsy and brain removed for immunostaining and electron 86 microscopy (EM). 87

Electron microscopy and analysis 109
Semi-thin (0.5-0.1µm) sections of caudal corpus callosum in a sagittal plane were collected on glass 110 slides and stained with 1% toluidine blue to select region of analysis. Ultrathin (0.1µm) sections were 111 subsequently collected on 3x3mm copper grids and specimens examined using a JEOL 1001 112 transmission electron microscope. Images were captured with MegaView III CCD cooled camera 113 operated with iTEM AnalySIS software (Olympus Soft Imaging Systems GmbH). A minimum of six 114 distinct fields of view were imaged at 5000 or 10000x magnification for each animal. The proportion 115 of myelinated axons, axon diameter and g-ratio were analysed manually using FIJI/ImageJ (National 116 Institutes of Health Protein concentrations were determined by Bradford assay and lysates stored at -80°C until use. 164

Statistical analyses 173
Data were analyzed by unpaired t-test, 1-way ANOVA or mixed effect models for repeated measures 174 (GraphPad Prism 8), to test the effect of TrkB agonist treatments with post-hoc multiple comparisons 175 as appropriate. Statistical significance was set as p<0.05. 176 3 Results 177

LM22A-4 and TDP6 increase myelin sheath thickness during remyelination 178
We have previously shown treatment with TDP6, a structural mimetic of BDNF, enhances the 179 number of axons remyelinated and increases myelin sheath thickness during recovery after 6-weeks 180 cuprizone challenge in an oligodendroglial-TrkB dependent manner (Fletcher et al., 2018a). Here, we 181 compared TDP6 with LM22A-4, a small molecule TrkB agonist reported to be a functional BDNF-182 mimetic (Massa et al., 2010). Demyelination by cuprizone feeding was confirmed by myelin basic 183 protein (MBP) immunostaining, with severely reduced levels of MBP expression observed in animals 184 taken at 6 weeks of cuprizone feeding (minimum = 2/cohort; Figure S2). Cuprizone feed was 185 withdrawn and remaining animals received ICV minipumps containing aCSF (artificial cerebrospinal 186 fliud), TDP6 (40µM) or LM22A-4 (500µM) for 7 days. 187 To examine the extent of remyelination, MBP-immunostaining in the caudal corpus callosum was 188 assessed. This revealed both TDP6 and LM22A-4 treatment increased (p<0.0001) the percentage area 189 of MBP + staining compared to treatment with the aCSF vehicle (Fig. 1A, quantified in Fig. 1B). EM 190 analysis indicated that mice treated with TDP6 exhibited a trend increase towards (p=0.09) more 191 remyelinated axons compared to those receiving aCSF, whereas for those receiving LM22A-4, no 192 increase was observed (p=0.46; Fig. 1C). Both TDP6 and LM22A-4 treatment resulted in a 193 significant (p=0.002) reduction in mean g-ratio indicative of increased myelin thickness (Fig. 1D). 194 Linear regression analysis of g-ratio against axon diameter (Fig. 1E) indicated that although both 195 TDP6 and LM22A-4 treatments increase myelin sheath thickness during remyelination (Fig. 1D), 196 TDP6 exerted a more consistent effect with a significant decrease in y-intercept (p=0.0032), but no 197 change in slope (p=0.35) indicating that g-ratio was reduced across all axonal diameters, whereas for 198 LM22A-4 there was a significant increase in slope (p=0.006), indicative of reduced g-ratio and 199 thicker myelin on smaller diameter axons. Collectively, these data are consistent with our previous 200 findings that BDNF-TrkB signaling increases myelin sheath thickness during remyelination in vivo 201 (Fletcher et al., 2018a). 202

TrkB signaling dynamics initiated by LM22A-4 do not mimic BDNF 272
To determine if LM22A-4 elicits a signaling cascade mimicking typical BDNF-TrkB signaling, we 273 generated an isogenic stable TrkB expressing HEK293 (293-TrkB) cell line using the Flp-In system 274 ( Supplementary Fig. 1A). and assessed for TrkB and ERK1/2 phosphorylation by Western blot (Fig. 7A). Densitometric 285 analysis (Fig. 7B) revealed that compared to BDNF treatment, which increased TrkB 286 phosphorylation within 5mins (p=0.012), LM22A-4 did not significantly increase levels of 287 phosphorylated TrkB until 240mins of treatment (p=0.02). The effects of LM22A-4 treatment on 288 ERK1/2 phosphorylation where levels peaked at 5mins of treatment, and significantly declined 289 compared to BDNF from 15 to 240mins (Fig. 7C) . This 325 appears to contrast our observations, where exogenous BDNF or TDP6 exerted no effect on the 326 density or proliferative fraction of OPCs during remyelination after cuprizone (Fletcher et al., 2018a). 327 The different observations between these two distinct experimental approaches may ultimately reflect 328 context, wherein oligoendroglia subjected to a lifetime of BDNF haploinsufficiency simply behave 329 differently. However, it echoes our findings that TDP6 treatment in oligodendroglial TrkB knockout mice 352 resulted in increased myelination of small diameter axons during myelin repair (Fletcher et al., 353 2018a). It is tempting to speculate that small diameter axons are exerting a selective effect in both 354 instances, but it is critical to distinguish growth in myelin thickness as an oligodendrocyte-driven 355 function (Ishii et al., 2012). To date, a direct axonal signal that instructs oligodendrocytes to increase 356 myelin thickness has not been identified, although the number of myelin wraps is known to increase 357 as circuit activity increases with the maturing brain (Sturrock, 1980). In contrast, initiation of 358 myelination, particularly for small diameter axons is known to require axonally derived signals 359 (Bechler et al., 2017;Gautier et al., 2015), suggestive that TrkB expression by neurons may 360 potentially confer a pro-myelinating signal to oligodendrocytes. 361 Concerningly, a recent report indicates that LM22A-4 does not activate TrkB at all (Boltaev et al.,362 2017). The fact that LM22A-4 failed to promote remyelination and increase oligodendroglial density 363 in the oligodendrocyte TrkB knockout mice clearly indicates TrkB is necessary for the action of 364 LM22A-4 and this is the first in vivo genetic evidence that LM22A-4 requires TrkB for activity. 365 Although LM22A-4 may exert its effect through direct activity on TrkB, our data and findings by 366 Boltaev and is a pattern consistent with Trk-receptor transactivation. 380 The extended 4-hour timeframe required to detect TrkB phosphorylation following LM22A-4 381 treatment in vitro is consistent with Trk-receptor transactivation, where it can take up to 6 hours to 382 elicit detectable Trk receptor phosphorylation, and results in signal bias towards Akt (Lee and Chao, 383 2001). We propose that LM22A-4 is mediates its increase in OPC density during remyelination by 384 Trk-transactivation potentially via GPCRs (Fig. 8) phosphorylation of TrkB receptors confined in transport vesicles and not expressed at the cell surface 389 (Fig. 8). This could be tested in vitro with LM22A-4 treated TrkB-293 cells or primary 390 oligodendrocytes co-treated with Src inhibitors. Full understanding of the mode of action for 391 LM22A-4 is warranted in order to optimize its therapeutic potential. Overall, our hypothesised mode 392 of action for LM22A-4 is parsimonious with known roles of Src-family kinases / Fyn in Trk-393 transactivation (Rajagopal et al., 2004;Rajagopal and Chao, 2006)  The raw data supporting the conclusions of this manuscript will be made available by the authors, 446 without undue reservation, to any qualified researcher. remyelinated trended towards increasing with TDP6 treatment (p=0.09) compared to aCSF, but was 582 unchanged between aCSF and LM22A-4 (p=0.48) and LM22A-4 and TDP6 (p=0.31, n=3-4/group), 583 however (D) the mean g-ratio was decreased (p=0.002) in both TDP6 and LM22A-4 treated mice, 584 indicative of thicker myelin sheaths compared to those receiving aCSF vehicle (n=3/group). (E) 585 Scatter plot of g-ratio against axonal diameter. Linear regression revealed that TDP6 treatment 586 resulted in a decrease in y-intercept (p=0.0032) but no change in slope (p=0.