Agnes W. Wong

Brain-Derived Neurotrophic Factor Promotes Central Nervous System Myelination via a Direct Effect upon Oligodendrocytes

The extracellular factors that are responsible for inducing myelination in the central nervous system (CNS) remain elusive. We investigated whether brain-derived neurotrophic factor (BDNF) is implicated, by first confirming that BDNF heterozygous mice exhibit delayed CNS myelination during early postnatal development. We next established that the influence of BDNF upon myelination was direct, by acting on oligodendrocytes, using co-cultures of dorsal root ganglia neurons and oligodendrocyte precursor cells. Importantly, we found that BDNF retains its capacity to enhance myelination of neurons or by oligodendrocytes derived from p75NTR knockout mice, indicating the expression of p75NTR is not necessary for BDNF-induced myelination. Conversely, we observed that phosphorylation of TrkB correlated with myelination, and that inhibiting TrkB signalling also inhibited the promyelinating effect of BDNF, suggesting that BDNF enhances CNS myelination via activating oligodendroglial TrkB-FL receptors. Together, our data reveal a previously unknown role for BDNF in potentiating the normal development of CNS myelination, via signalling within oligodendrocytes.

Oligodendroglial expression of TrkB independently regulates myelination and progenitor cell proliferation.

The neurotrophin brain-derived neurotrophic factor (BDNF) has been implicated in regulating CNS myelination. BDNF mutant mice exhibit a hypomyelinating phenotype, and BDNF exerts distinct effects upon oligodendroglial proliferation, differentiation, and myelination in vitro. To investigate the precise influence that BDNF exerts in regulating CNS myelination in vivo, we have generated conditional knock-out mice in which TrkB has been deleted specifically in oligodendrocytes. Deletion of TrkB disrupted normal oligodendrocyte myelination, resulting in a significant reduction in myelin protein expression and myelination of CNS white matter tracts during development. Importantly, conditional knock-out mice exhibited normal numbers of mature oligodendrocytes and normal numbers of myelinated axons; however, myelin thickness was significantly reduced during development. These data indicate that while TrkB expression in oligodendrocytes plays no role in the initial contact with axons, it exerts an important influence in subsequent stages to promote myelin ensheathment. The conditional knock-out mice also exhibited an increased density of oligodendrocyte progenitor cells (OPCs) in CNS white matter tracts. Concordant with these results, in vitro analyses using OPCs subjected to TrkB knockdown also revealed increased OPC proliferation. Our data suggested this effect was dependent upon TrkC and p75 expression. Thus, our data demonstrate that TrkB expression in oligodendroglia exerts a direct effect on oligodendrocytes to promote myelination and an indirect effect upon the OPC population, modifying their proliferative potential.

Extracellular signal-regulated kinase 1/2 signaling promotes oligodendrocyte myelination in vitro

J. Neurochem. (2012) 122, 1167–1180.

BDNF Exerts Contrasting Effects on Peripheral Myelination of NGF-Dependent and BDNF-Dependent DRG Neurons

Although brain-derived neurotrophic factor (BDNF) has been shown to promote peripheral myelination during development and remyelination after injury, the precise mechanisms mediating this effect remain unknown. Here, we determine that BDNF promotes myelination of nerve growth factor-dependent neurons, an effect dependent on neuronal expression of the p75 neurotrophin receptor, whereas BDNF inhibits myelination of BDNF-dependent neurons via the full-length TrkB receptor. Thus, BDNF exerts contrasting effects on Schwann cell myelination, depending on the complement of BDNF receptors that are expressed by different subpopulations of dorsal root ganglion neurons. These results demonstrate that BDNF exerts contrasting modulatory roles in peripheral nervous system myelination, and that its mechanism of action is acutely regulated and specifically targeted to neurons.

Neurotrophin receptor homolog-2 regulates nerve growth factor signaling

The neurotrophin receptor homolog (NRH2) is closely related to the p75 neurotrophin receptor (p75NTR); however, its function and role in neurotrophin signaling are unclear. NRH2 does not bind to nerve growth factor (NGF), however, is able to form a receptor complex with tropomyosin‐related kinase receptor A (TrkA) and to generate high‐affinity NGF binding sites. Despite this, the mechanisms underpinning the interaction between NRH2 and TrkA remain unknown. Here, we identify that the intracellular domain of NRH2 is required to form an association with TrkA. Our data suggest extensive intracellular interaction between NRH2 and TrkA, as either the juxtamembrane or death domain regions of NRH2 are sufficient for interaction with TrkA. In addition, we demonstrate that TrkA signaling is dramatically influenced by the co‐expression of NRH2. Importantly, NRH2 did not influence all downstream TrkA signaling pathways, but rather exerted a specific effect, enhancing src homology 2 domain‐containing transforming protein (Shc) activation. Moreover, downstream of Shc, the co‐expression of NRH2 resulted in TrkA specifically modulating mitogen‐activated protein kinase pathway activation, but not the phosphatidylinositol 3‐kinase/Akt pathway. These results indicate that NRH2 utilizes intracellular mechanisms to not only regulate NGF binding to TrkA, but also specifically modulate TrkA receptor signaling, thus adding further layers of complexity and specificity to neurotrophin signaling.

A small peptide mimetic of brain‐derived neurotrophic factor promotes peripheral myelination

The expression of the neurotrophins and their receptors is essential for peripheral nervous system development and myelination. We have previously demonstrated that brain‐derived neurotrophic factor (BDNF) exerts contrasting influences upon Schwann cell myelination in vitro – promoting myelination via neuronally expressed p75NTR, but inhibiting myelination via neuronally expressed TrkB. We have generated a small peptide called cyclo‐dPAKKR that structurally mimics the region of BDNF that binds p75NTR. Here, we have investigated whether utilizing cyclo‐dPAKKR to selectively target p75NTR is an approach that could exert a unified promyelinating response. Like BDNF, cyclo‐dPAKKR promoted myelination of nerve growth factor‐dependent neurons in vitro, an effect dependent on the neuronal expression of p75NTR. Importantly, cyclo‐dPAKKR also significantly promoted the myelination of tropomyosin‐related kinase receptor B‐expressing neurons in vitro, whereas BDNF exerted a significant inhibitory effect. This indicated that while BDNF exerted a contrasting influence upon the myelination of distinct subsets of dorsal root ganglion (DRG) neurons in vitro, cyclo‐dPAKKR uniformly promoted their myelination. Local injection of cyclo‐dPAKKR adjacent to the developing sciatic nerve in vivo significantly enhanced myelin protein expression and significantly increased the number of myelinated axons. These results demonstrate that cyclo‐dPAKKR promotes peripheral myelination in vitro and in vivo, suggesting it is a strategy worthy of further investigation for the treatment of peripheral demyelinating diseases.

TDP6, a brain-derived neurotrophic factor-based trkB peptide mimetic, promotes oligodendrocyte myelination.

Brain-derived neurotrophic factor (BDNF) plays critical roles in the development and maintenance of the central (CNS) and peripheral nervous systems (PNS). BDNF exerts its biological effects via tropomyosin-related kinase B (TrkB) and the p75 neurotrophin receptor (p75NTR). We have recently identified that BDNF promotes CNS myelination via oligodendroglial TrkB receptors. In order to selectively target TrkB to promote CNS myelination, we have used a putative TrkB agonist, a small multicyclic peptide (tricyclic dimeric peptide 6, TDP6) previously described by us that structurally mimics a region of BDNF that binds TrkB. We confirmed that TDP6 acts as a TrkB agonist as it provoked autophosphorylation of TrkB and its downstream signalling effector extracellular related-kinase 1 and 2 (Erk1/2) in primary oligodendrocytes. Using an in vitro myelination assay, we show that TDP6 significantly promotes myelination by oligodendrocytes in vitro, as evidenced by enhanced myelin protein expression and an increased number of myelinated axonal segments. In contrast, a second, structurally distinct BDNF mimetic (cyclo-dPAKKR) that targets p75NTR had no effect upon oligodendrocyte myelination in vitro, despite the fact that cyclo-dPAKKR is a very effective promoter of peripheral (Schwann cell) myelination. The selectivity of TDP6 was further verified by using TrkB-deficient oligodendrocytes, in which TDP6 failed to promote myelination, indicating that the pro-myelinating effect of TDP6 is oligodendroglial TrkB-dependent. Together, our results demonstrate that TDP6 is a novel BDNF mimetic that promotes oligodendrocyte myelination in vitro via targeting TrkB.

A novel role of suppressor of cytokine signaling-2 in the regulation of TrkA neurotrophin receptor biology

Suppressor of cytokine signaling‐2 (SOCS2) is a regulator of intracellular responses to growth factors and cytokines. Cultured dorsal root ganglia neurons from neonatal mice with increased or decreased SOCS2 expression were examined for altered responsiveness to nerve growth factor (NGF). In the presence of NGF, SOCS2 over‐expression increased neurite length and complexity, whereas loss of SOCS2 reduced neurite outgrowth. Neither loss nor gain of SOCS2 expression altered the relative survival of these cells, suggesting that SOCS2 can discriminate between the differentiation and survival responses to NGF. Interaction studies in 293T cells revealed that SOCS2 immunoprecipitates with TrkA and a juxtamembrane motif of TrkA was required for this interaction. SOCS2 also immunoprecipitated with endogenous TrkA in PC12 Tet‐On cells. Over‐expression of SOCS2 in PC12 Tet‐On cells increased total and surface TrkA expression. In contrast, dorsal root ganglion neurons which over‐expressed SOCS2 did not exhibit significant changes in total levels but an increase in surface TrkA was noted. SOCS2‐induced neurite outgrowth in PC12 Tet‐On cells correlated with increased and prolonged activation of pAKT and pErk1/2 and required an intact SOCS2 SH2 domain and SOCS box domain. This study highlights a novel role for SOCS2 in the regulation of TrkA signaling and biology.

Neurite outgrowth in normal and injured primary sensory neurons reveals different regulation by nerve growth factor (NGF) and artemin.

Neurotrophic factors have been intensively studied as potential therapeutic agents for promoting neural regeneration and functional recovery after nerve injury. Artemin is a member of the glial cell line-derived neurotrophic factor (GDNF) family of ligands (GFLs) that forms a signalling complex with GFRα3 and the tyrosine kinase Ret. Systemic administration of artemin in rodents is reported to facilitate regeneration of primary sensory neurons following axotomy, improve recovery of sensory function, and reduce sensory hypersensitivity that is a cause of pain. However, the biological mechanisms that underlie these effects are mostly unknown. This study has investigated the biological significance of the colocalisation of GFRα3 with TrkA (neurotrophin receptor for nerve growth factor [NGF]) in the peptidergic type of unmyelinated (C-fibre) sensory neurons in rat dorsal root ganglia (DRG). In vitro neurite outgrowth assays were used to study the effects of artemin and NGF by comparing DRG neurons that were previously uninjured, or were axotomised in vivo by transecting a visceral or somatic peripheral nerve. We found that artemin could facilitate neurite initiation but in comparison to NGF had low efficacy for facilitating neurite elongation and branching. This low efficacy was not increased when a preconditioning in vivo nerve injury was used to induce a pro-regenerative state. Neurite initiation was unaffected by artemin when PI3 kinase and Src family kinase signalling were blocked, but NGF had a reduced effect.

A Novel Small Molecule GDNF Receptor RET Agonist, BT13, Promotes Neurite Growth from Sensory Neurons in Vitro and Attenuates Experimental Neuropathy in the Rat

Neuropathic pain caused by nerve damage is a common and severe class of chronic pain. Disease-modifying clinical therapies are needed as current treatments typically provide only symptomatic relief; show varying clinical efficacy; and most have significant adverse effects. One approach is targeting either neurotrophic factors or their receptors that normalize sensory neuron function and stimulate regeneration after nerve damage. Two candidate targets are glial cell line-derived neurotrophic factor (GDNF) and artemin (ARTN), as these GDNF family ligands (GFLs) show efficacy in animal models of neuropathic pain (Boucher et al., 2000; Gardell et al., 2003; Wang et al., 2008, 2014). As these protein ligands have poor drug-like properties and are expensive to produce for clinical use, we screened 18,400 drug-like compounds to develop small molecules that act similarly to GFLs (GDNF mimetics). This screening identified BT13 as a compound that selectively targeted GFL receptor RET to activate downstream signaling cascades. BT13 was similar to NGF and ARTN in selectively promoting neurite outgrowth from the peptidergic class of adult sensory neurons in culture, but was opposite to ARTN in causing neurite elongation without affecting initiation. When administered after spinal nerve ligation in a rat model of neuropathic pain, 20 and 25 mg/kg of BT13 decreased mechanical hypersensitivity and normalized expression of sensory neuron markers in dorsal root ganglia. In control rats, BT13 had no effect on baseline mechanical or thermal sensitivity, motor coordination, or weight gain. Thus, small molecule BT13 selectively activates RET and offers opportunities for developing novel disease-modifying medications to treat neuropathic pain.