Are TrkB receptor agonists the right tool to fulfill the promises for a therapeutic value of the brain-derived neurotrophic factor?

Abstract

Brain-derived neurotrophic factor signaling via its receptor tropomyosin receptor kinase B regulates several crucial physiological processes. It has been shown to act in the brain, promoting neuronal survival, growth, and plasticity as well as in the rest of the body where it is involved in regulating for instance aspects of the metabolism. Due to its crucial and very pleiotropic activity, reduction of brain-derived neurotrophic factor levels and alterations in the brain-derived neurotrophic factor/tropomyosin receptor kinase B signaling have been found to be associated with a wide spectrum of neurological diseases. However, because of its poor bioavailability and pharmacological properties, brain-derived neurotrophic factor itself has a very low therapeutic value. Moreover, the concomitant binding of exogenous brain-derived neurotrophic factor to the p75 neurotrophin receptor has the potential to elicit several unwanted and deleterious side effects. Therefore, developing tools and approaches to specifically promote tropomyosin receptor kinase B signaling has become an important goal of translational research. Among the newly developed tools are different categories of tropomyosin receptor kinase B receptor agonist molecules. In this review, we give a comprehensive description of the different tropomyosin receptor kinase B receptor agonist drugs developed so far and of the results of their application in animal models of several neurological diseases. Moreover, we discuss the main benefits of tropomyosin receptor kinase B receptor agonists, concentrating especially on the new tropomyosin receptor kinase B agonist antibodies. The benefits observed both in vitro and in vivo upon application of tropomyosin receptor kinase B receptor agonist drugs seem to predominantly depend on their general neuroprotective activity and their ability to promote neuronal plasticity. Moreover, tropomyosin receptor kinase B agonist antibodies have been shown to specifically bind the tropomyosin receptor kinase B receptor and not p75 neurotrophin receptor. Therefore, while, based on the current knowledge, the tropomyosin receptor kinase B receptor agonists do not seem to have the potential to reverse the disease pathology per se, promoting brain-derived neurotrophic factor/tropomyosin receptor kinase B signaling still has a very high therapeutic relevance.

Introduction

It is exactly 20 years ago that Sendtner and Thoenen (2002) wrote a review reporting on the success, or rather the lack of it in developing novel therapeutic approaches based on the existing research on neurotrophins. In their paper, they anticipated the development of improved methods and novel therapeutic procedures based on a future deeper understanding of the molecular and cellular mechanisms of neurotrophin signaling. Since then a significant amount of knowledge has indeed been accumulated both on the molecular and cellular mechanisms of neurotrophin signaling as well as on their role in the pathogenesis of several neurological diseases. In addition, new tools have been developed to modulate their signaling processes. It is therefore time to make a new assessment of the current state of affairs. Did we succeed in developing the “rationale therapeutic approaches” auspicated by Sendtner and Thoenen? What progresses have been made so far? What are the still unresolved limitations? In this review, we will address these questions looking specifically at the use of new tools to promote brain-derived neurotrophic factor (BDNF) signaling via its receptor tropomyosin receptor kinase B (TrkB); namely TrkB agonists.

Search Strategy and Selection Criteria

This review article was constructed using information collected from publications found using PubMed of the National Institute of Health, National Library of Medicine, and Google Scholar. Searches were performed until November 2022. The search strategy used different combinations of the following keywords: Neurotrophins, BDNF, TrkB, p75^NTR, signaling, CNS, plasticity, TrkB agonist, small-molecule TrkB agonist, 7,8-dihydroxyflavone, ZEB85, TrkB agonist antibody, therapy, neurological disorders neurodegeneration, neuroinflammation, Alzheimer’s disease, Parkinson’s disease, Major depression disorder. No limit was given to the year of publication. However, we made sure of citing the most recent review articles when possible. It was though sometimes necessary to also cite the original publications first describing specific molecules or findings, and this also a certain number of older citations were included.

Role of the Brain-Derived Neurotrophic Factor/TrkB Signaling in the Central Nervous System in Health and Disease

BDNF is a versatile, pleiotropic molecule acting in the whole body, controlling processes that range from the development and function of the brain to metabolic processes. In particular, it regulates a plethora of different cellular processes in the brain involved in the development, plasticity, and maintenance of the structure and function of neuronal networks (Di Rosa et al., 2021). Furthermore, it promotes adult neurogenesis (Vilar and Mira, 2016) and activity-dependent synaptic plasticity (Korte et al., 1995; see also for reviews; Zagrebelsky and Korte, 2014; Zagrebelsky et al., 2020) and exerts powerful neuroprotective actions. The diversity of the effects is at least in part explained by the fact that both BDNF and its precursor proBDNF are both biologically active and act by binding to two transmembrane receptors, the TrkB, with a higher affinity for the mature form of BDNF and the p75 neurotrophin receptor (p75^NTR) preferentially binding proBDNF (Barbacid, 1993) resulting in very diverse, often opposite cellular outcomes (Figure 1A; Lu et al., 2005). Both BDNF and its receptors are widely expressed within the brain with their highest levels detected in the hippocampus, amygdala, cerebellum, and cerebral cortex in both rodents and humans (Hofer et al., 1990).

Figure 1.

BDNF-mediated effects on neuronal structure and function.

(A) BDNF and proBDNF are released at postsynaptic sites. BDNF binds TrkB with high affinity to induce its dimerization and phosphorylation at tyrosine residues in the cytoplasmatic domain, which serves as a docking site for several downstream signaling pathways. Especially the ERK pathways lead to activation of the transcription factor CREB mediating the transcription of a gene involved in growth and plasticity (e.g., cFOS). Both BDNF and proBDNF bind p75^NTR activating the Rho-ROCK pathway via the interaction with Rho-GDI as well as pro-apoptotic signaling via the interaction with Sortilin. While TrkB activation results in survival, growth, and facilitation of neuronal activity, signaling via the p75^NTR results in death and suppression of neuronal activity. ZEB85, as an example of TrkB receptor agonist antibodies, specifically activates the TrkB signaling. Created with BioRender.com. (B) Example tracings of DIV 7 neurons treated for 4 days with BDNF or ZEB85-Ab and their respective controls. Scale bar is 50 μm. (B’) Both BDNF and ZEB85-Ab significantly increase neurite complexity relative to the control conditions. Adapted from Tacke et al. (2022). (C) Application of ZEB85 rescues the deficit observed in long-term potentiation in organotypic hippocampal slices derived from BDNF heterozygous knockout mice. Adapted from Tacke et al. (2022). BDNF: Brain-derived neurotrophic factor; BDNFhet/het: BDNF heterozygous knockout mice; Ca²⁺: calcium ions; cFos: cFos proto-oncogene; c-Jun: Jun proto-oncogene; CREB: cAMP response element-binding protein; CTRL: control; CTRL-Ab: control antibody; DIV: days in vitro; ERK: extracellular signal-regulated kinase; JNK: c-Jun N-terminal kinase; MEK: mitogen-activated protein kinase kinase; p75^NTR: p75 neurotrophin receptor; proBDNF: BDNF precursor; Raf: RAF kinase; RAS-GTP: small GTPase from the rat sarcoma virus family; Rho-GDI: RHO protein GDP dissociation inhibitor; Shc: protein from the Src homology and collagen family; TrkB: tropomyosin receptor kinase B; ZEB85-Ab: ZEB85 antibody.

Mounting evidence suggests a bi-directional connection between BDNF expression, and regulation of inflammation. Increased production of BDNF from immune cells including infiltrating T-cells and macrophages contributes to the development of inflammation during allergic asthma thereby promoting neuronal changes leading to airway smooth muscle contraction and mucus hypersecretion (Braun et al., 2004). Moreover, the pathology of all neurodegenerative diseases as well as other neurological conditions is characterized by chronic neuroinflammation. Indeed, microglia, monocytes, and astrocytes secrete BDNF in response to tumor necrosis factor α and interleukin-6 (Saha et al., 2006; Gomes et al., 2013), and in turn BDNF levels modulate microglial proliferation and activation. While increased BDNF levels promote microglial proliferation and activation upon immune challenge (Zhang et al., 2014), BDNF secretion might also function as an inhibitory feedback to dampen microglia activation under very diverse pathological conditions ranging from spinal cord injury (Joosten and Houweling, 2004), age-dependent degeneration of the substantia nigra (Wu et al., 2020), diabetes (Han et al., 2019) and acute coronary disease (Kim et al., 2019). Indeed, BDNF^+/– knockout mice show an increased expression of proinflammatory cytokines associated with a depressive-like behavior upon peripheral immune challenge (Parrott et al., 2021). In addition, it is noteworthy, that the progressive decrease in BDNF-TrkB signaling during aging promotes the activation of microglia cells whereas, on the other hand, upregulation of BDNF signaling inhibits microglial activation via the TrkB-Erk-CREB pathway (Wu et al., 2020).

Finally, neurotrophins and BDNF in particular have been emerging as mediators of energy homeostasis and metabolic processes (Podyma et al., 2021). Indeed, loss-of-function for TrkB is associated in humans with severe obesity due to hyperphagia (Yeo et al., 2004) while BDNF signaling plays a role in weight loss success (Primo et al., 2021). Indeed, both BDNF and its receptors are highly expressed in different nuclei of the hypothalamus where they are specifically involved in controlling feeding behavior (for a review see, Podyma et al., 2021). Moreover, BDNF and TrkB are involved in other aspects of metabolisms such as in regulating glucose tolerance and insulin secretion as well as energy expenditure and thermogenesis (Podyma et al., 2021).

Because of the very broad spectrum of BDNF actions and of its widespread expression pattern, deficits in its signaling have been involved in the pathophysiology of several very diverse brain-associated illnesses from schizophrenia, depression to Rett syndrome, Huntington’s disease, obesity, anorexia and also to neurodegenerative diseases. Indeed, BDNF is one of the neuroprotective, growth-promoting molecules released by neurons upon different physiological and pathophysiological conditions and interventions such as exercise, hypoxia, stress, epileptic seizures, and ischemia (Brigadski and Leßmann, 2020). Several brain pathologies are associated with significant alterations in BDNF expression and release. Specifically, while in a few neurological conditions, BDNF levels increase (e.g., chronic stress; Wook Koo et al., 2016), more often they are found to be reduced both in the brain and in serum (Azman and Zakaria, 2022). Hence BDNF has been suggested as a biomarker for different diseases and for the efficacy of their therapy (Azman and Zakaria, 2022). Most currently used treatments are accompanied by significant changes in BDNF expression and release levels. Among the neurological conditions associated with alterations in the BDNF/TrkB signaling are the most common neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease, and amyotrophic lateral sclerosis (for a recent exhaustive review see: Azman and Zakaria, 2022). All these conditions are characterized by the progressive loss of neuronal structure and function, leading to the region or type-specific death of neurons and impairment in synaptic plasticity, with severe consequences for memory formation, retention, or recall. Several studies have now convincingly underlined a link between these neurodegenerative diseases and alterations in BDNF signaling (for a review see Azman and Zakaria, 2022). Indeed, all of them have been associated with a reduction in BDNF protein and mRNA levels in the brain as well as in the peripheral blood of patients. This is especially well described for AD (Hock et al., 2000; Pláteník et al., 2014) where depletion of BDNF is strictly associated with the expression of the typical pathological hallmarks of AD including Aβ accumulation, TAU phosphorylation, and neuroinflammation (Wang et al., 2019). Also, the ratio between BDNF and proBDNF has been shown to shift toward an increase in proBDNF promoting TAU phosphorylation Abeta deposition and neurodegeneration (Bharani et al., 2020). Moreover, the levels of BDNF-promoter methylation in peripheral blood have been suggested as an epigenetic biomarker predicting the onset of AD (Chang et al., 2014) and the Val66Met polymorphisms have been associated with a higher risk for AD progression (Bessi et al., 2020). The Val66Met polymorphism occurs naturally producing a valine to methionine substitution at codon 66 of the BDNF gene and resulting in impaired BDNF secretion, and hippocampal plasticity episodic memory (Egan et al., 2003). Similarly, in PD low levels of circulating BDNF have been correlated with nigro-striatal degeneration (Hernández-Vara et al., 2020), cognitive impairment (Khalil et al., 2016), depression (Wang et al., 2017), as well as motor impairment (Scalzo et al., 2010) and the Val66Met polymorphisms, seems to be associated with a less favorable progression of the disease (Białecka et al., 2014).

BDNF action has been recently also involved in other neurological conditions including the consequences of an ischemic stroke and multiple sclerosis. Indeed, circulating BDNF levels are proposed as a potential biomarker in stroke (Mojtabavi et al., 2022) since its levels acutely decrease upon stroke in different brain areas involved in cognition and motor control. Low levels of circulating BDNF have been correlated with poor long-term functional outcomes after an ischemic stroke possibly due to its reduced plasticity-promoting activity supporting the network reorganization (Stanne et al., 2016). In multiple sclerosis, the percentage of BDNF gene methylation can be used as a predictive marker for the progression of the disease toward severe disability (Nociti et al., 2018). Interestingly, the BDNF Val66Met polymorphism has also been shown to protect against cognitive impairment and improve motor recovery in MS patients (Giordano et al., 2022).

Among psychiatric diseases, the pathogenesis of the highly debilitating major depressive disorder has also been linked to alterations in BDNF/TrkB signaling (for a review see: Colucci-D’Amato et al., 2020). Indeed, BDNF mRNA and protein are significantly reduced in postmortem brains of suicidal patients (Sonal and Raghavan, 2018) and several meta-analysis data indicate an association between the Val66Met polymorphism and the susceptibility to develop major depressive disorder (Youssef et al., 2018). Significantly lower BDNF levels have also been shown in schizophrenic patients in particular for those with lower cognitive scores suggesting that BDNF is involved in the pathophysiology of schizophrenia, and its associated cognitive impairment (Zhang et al., 2012).

Taken together, while the pathogenesis of most of the neurological diseases described above is still largely unclear, in all of them a strict correlation has been established between the symptoms and pathological alterations and the expression levels and signaling of BDNF.

Evidence for a Neuroprotective, Therapeutic Effect of Promoting Brain-Derived Neurotrophic Factor/TrkB Signaling

To what extent the alterations in BDNF signaling are a mere consequence of the diseases rather than the underlying cause starting it or promoting its progression is mostly not yet known. However, due to its general neuroprotective effects increasing BDNF signaling is likely to succeed in reducing the disease symptoms or in preventing its progression, even without curing it. In support of this observation are several examples of positive outcomes of therapeutic interventions in which BDNF levels or its signaling were increased in mouse models for different neurological diseases. Due to space constrictions in this review, we will report only on a few selected examples (for recent reviews see: Zuccato and Cattaneo, 2009; Gupta et al., 2013; Castrén and Monteggia, 2021). Accumulating evidence strongly suggests that increased BDNF signaling may positively influence cognition in AD. Indeed, BDNF administration was shown to have positive effects on learning and memory in a mouse model of dementia (Ando et al., 2002) and exert neuroprotective effects against Aβ peptide toxicity (Arancibia et al., 2008). Moreover, adeno-associated virus-mediated expression of human BDNF in a mouse model for AD reestablished the reduced BDNF levels and attenuated behavioral deficits, prevented neuron loss, alleviated synaptic degeneration, and reduced neuronal abnormality (Jiao et al., 2016). Particularly interesting is a recent study examining the positive effects of physical activity on AD. The results show that physical exercise requires neurogenesis to protect the brain from AD and that BDNF is essential for this protection (Choi et al., 2018). Along this line, intermittent fasting has been shown to have several beneficial BDNF-mediated cognitive effects (Sleiman et al., 2016).

Several studies attempted to establish the therapeutic effects of different approaches to increase BDNF levels in different mouse models for PD with controversial results (for a review see: Palasz et al., 2020). However, many studies revealed at least a partial prevention of neuronal cell loss and the increase in dopaminergic neurons in the substantia nigra especially if BDNF was administrated before inducing Parkinsonism (Kim et al., 2012). Moreover, increasing BDNF ameliorated the motor behavior of PD-affected monkeys and rats (Tsukahara et al., 1995; Hernandez-Chan et al., 2015).

Signaling of BDNF via the TrkB receptor has been shown to be crucial for the action of drugs commonly used for treatment both in patients and animal models of depression (Umemori et al., 2018). Indeed, chronic administration of antidepressants results in increased BDNF expression (Chen et al., 2001) as well as in TrkB activation (Saarelainen et al., 2003). Moreover, the effects of antidepressants are prevented in BDNF (Adachi et al., 2008) knockout mice and upon TrkB loss-of-function (Saarelainen et al., 2003), while the overexpression of TrkB within the dentate gyrus results in antidepressants-like behavioral effects (Koponen et al., 2005). In addition, it has recently been shown that several antidepressant drugs directly bind to the transmembrane domain of TrkB thereby promoting its localization at the membrane and its activation upon BDNF binding (Casarotto et al., 2021).

Taken together, several preclinical studies show significant benefits from approaches increasing the BDNF/TrkB signaling in different neurological disease animal models.

TrkB Agonists

Despite the wealth of promising preclinical data indicating a potential for BDNF for the treatment of several neurological conditions, the results of clinical trials using recombinant BDNF have been so far rather disappointing (Ochs et al., 2000). This is likely to be a consequence of the poor bioavailability of BDNF, due to its small molecular size and highly basic charge limiting its penetration through the blood-brain barrier and its diffusion within the central nervous system parenchyma (Palasz et al., 2020). Furthermore, the exogenous application of BDNF cannot reproduce the high degree of temporal and spatial specificity that characterizes its signaling in the healthy brain. Indeed, transport, synthesis, and secretion of BDNF occur in an activity-dependent manner (Tongiorgi, 2008) providing spatially and temporally precise actions specifically at synapses (Harward et al., 2016). Moreover, exogenous BDNF application may result in unwanted, in part adverse effects by binding the p75 neurotrophin receptor (p75^NTR), sortilin complex (Woo et al., 2005), which is known to mediate opposite cellular functions than TrkB (Chapleau and Pozzo-Miller, 2012).

Several attempts have been made to circumvent the limitations to the therapeutic use of recombinant BDNF and to develop drugs with a higher specificity for the BDNF/TrkB signaling pathway. These include the development of a series of low molecular weight drugs with more favorable pharmacokinetic properties and with a high degree of specificity for the TrkB receptor as well as TrkB agonist antibodies (Longo and Massa, 2013).

Small Molecule TrkB Agonists

Specifically, peptide ligands (Longo and Massa, 2013) as well as different non-peptide, small molecule ligands capable of activating TrkB signaling with high potency and specificity have been developed (Fletcher and Hughes, 2006). The synthetic peptide ligands bind to specific domains of the neurotrophin receptors resulting in their activation and neurotrophic activity in vitro (Cardenas-Aguayo Mdel et al., 2013) showing that peptidomimetics can indeed modulate neurotrophin receptor function. However, peptide compounds show several pharmacological limitations including low stability, reduced bioavailability, and little penetration of the blood-brain barrier (Longo and Massa, 2013). Therefore, the focus of recent research has rather been on the development of non-peptide small molecule TrkB agonists including 7,8-dihydroxyflavone (7,8-DHF), deoxygedunin, LM22A-4, and amitriptylione. Several of these have been reported to activate TrkB in living cells in vitro (Longo and Massa, 2013). The flavonoid 7,8-DHF (Jang et al., 2010) induces the phosphorylation of TrkB and its downstream targets AKT and ERK resulting in the inhibition of neuronal death (Jang et al., 2010; Liu et al., 2014) and prevents the age-related dendritic spine loss in vitro (Zeng et al., 2011). Similarly, LM22A-4 was shown to activate TrkB and its downstream targets in primary hippocampal neurons thereby preventing neuronal degeneration in in vitro models for neurodegenerative diseases (Massa et al., 2010). The effects of 7,8-DHF and LM22A-4 were blocked by the co-application of K252a supporting their specificity for the Trk receptors (Jang et al., 2010; Massa et al., 2010). Moreover, the application of 7,8-DHF to TrkB^F616A-derived cells further reinforced the requirement for TrkB of the 7,8-DHF effects (Jang et al., 2010).

Among the small molecule, TrkB agonist 7,8-DHF is the best characterized and its efficacy has been also assessed in vivo in several disease models. Interestingly, 7,8-DHF rescues spatial and fear memory defects and facilitates synaptic plasticity in cognitively impaired aged rats (Zeng et al., 2012a, b). Moreover, it has been shown to act as a neuroprotective factor in models of ischemia-, kainic acid- or MPTP-induced injury (Jang et al., 2010). 7,8-DHF has also been reported to be beneficial in mouse models for AD (Devi and Ohno, 2012), especially if the treatment is started in the pre-symptomatic phase of the disease (Aytan et al., 2018). On the other hand, administration of 7,8-DHF did not reduce the amyloid pathology and did not alleviate the cognitive impairment in the AD mouse model APP23/PS45 (Zhou et al., 2015) leaving its therapeutic value in this context still unclear. Furthermore, treatment with 7,8-DHF prevented the development of a depressive profile, promoted TrkB-Tyr816 phosphorylation in the dentate gyrus as well as the proliferation of neuronal precursors, and acted as an antidepressant as tested in the forced swim test in a mouse model for depression (Blugeot et al., 2011). Interestingly, 7,8-DHF has also been shown to have strong anti-inflammatory properties. Indeed, it was shown to suppress the release of pro-inflammatory mediators and cytokines in LPS-stimulated microglia cells by inhibiting the NF-κB and MAPK signaling pathways (Park et al., 2014). This is of special interest due to the crucial role that neuroinflammation seems to play in many different neurological conditions ranging from neurodegenerative diseases to traumatic injury and depression. 7,8-DHF (Garcia-Diaz Barriga et al., 2017), as well as LM22A-4 (Simmons et al., 2013), have also been shown to rescue TrkB phosphorylation and improve the motor symptoms and the respiratory functions in mouse models of Rett syndrome and Huntington’s disease respectively. More recently, 7,8-DHF was shown to decrease body weight by promoting lipid oxygenation and increasing energy expenditure, improving insulin blood concentrations, and lower blood glucose levels especially in female mice (Liu et al., 2016).

Taken together, non-peptide small molecule TrkB agonists have been shown to exert several beneficial effects in different in vitro essays as well as in vivo in mouse models for neurological diseases. However, the cellular and that these positive effects directly derive from the specific activation of TrkB has not always been addressed satisfactorily. Indeed, in some studies, TrkB phosphorylation has not been analyzed or using assays that are not specific enough and the specificity for TrkB has been assessed exclusively by co-application of the general tyrosine protein kinase inhibitor K252a (Tapley et al., 1992) known to affect all Trk receptors. Indeed a recent study applying a series of reliable quantitative methods for direct measurement of TrkB phosphorylation and activation of downstream kinases was unable to reproduce the BDNF-induced robust and dose-dependent receptor activation using several of the TrkB agonist compounds (Boltaev et al., 2017). This report was confirmed by an independent study in which both 7,8-DHF and LM22A-4 failed to mimic the ability of BDNF to protect striatal neurons from mHTT-induced cell death as well as to induce TrkB phosphorylation (Todd et al., 2014). Therefore, the results obtained with several non-peptide small molecule TrkB agonists should be interpreted with caution keeping in mind that some of the beneficial effects observed could actually be due to TrkB-independent neuroprotective and effects as shown for 7,8-DHF due to its antioxidant activity (Chen et al., 2011). Thus, the development of specific TrkB agonists with robust activity in both in vitro and in vivo systems remains an important goal.

TrkB Agonist Antibodies

One possible reason for the failure of small-molecule compounds to activate TrkB is the small size that prevents them from being able to bridge two TrkB monomers thereby inducing the dimerization required for the activation. The development of TrkB agonist antibodies is especially interesting in this context since their structure is more similar to the BDNF dimer and therefore more likely to successfully dimerize TrkB. Indeed, several TrkB agonist antibodies have been developed and characterized until now (Qian et al., 2006; Todd et al., 2014; Traub et al., 2017; Merkouris et al., 2018). Via the generation of hybridoma clones, Qian et al. (2006) developed five mouse monoclonal antibodies (mAbs) showing a highly selective binding to TrkB followed by the activation of both proximal and secondary downstream signaling molecules (Figure 1A). While the binding affinity for TrkB and the functional efficacy of these compounds are comparable to BDNF, they do not bind the p75^NTR at all. Moreover, these TrkB mAbs promote neuronal survival and neurite outgrowth in vitro (Qian et al., 2006). Among them, 29D7 showed the strongest affinity toward TrkB, binding both the mouse and the human receptor and promoting its phosphorylation (Todd et al., 2014) and the survival and growth of cultivated cortical neurons. Furthermore, a pre-treatment with the mAb 29D7 was found to enhance neuronal survival in a mouse model of cerebral ischemia. This effect was long-lasting, significantly more than the one of BDNF itself, and correlated with the sensorimotor functional recovery underlying the therapeutic potential of TrkB agonist mAbs (Kim et al., 2014). The effects of two additional TrkB-specific agonist antibodies, AB2 and AB20 were compared to those of BDNF in human-induced pluripotent stem cell-derived neurons. While both antibodies were significantly less powerful than BDNF in inducing TrkB phosphorylation, they induced phosphorylation of the downstream ERK, AKT, and CREB with a higher potency as well as the transcription of the synaptic plasticity marker VGF (Traub et al., 2017). Interestingly, treatment with AB20 resulted in longer-lasting activation of TrkB and its downstream signaling when compared to BDNF. This is possibly due to the lack of internalization of the receptor upon binding AB20 making it available for a renewed stimulation (Traub et al., 2017). For this antibody, the possible binding to p75^NTR was not assessed. An additional TrkB agonist antibody, 1D7 (mAb 1D7) was tested for its ability to support the survival of retinal ganglion cells in two mouse models of retinal degeneration. Interestingly, while both BDNF and mAb1D7 induced comparable levels of TrkB phosphorylation only mAb 1D7 exerted a significant neuroprotective effect both upon transection of the optic nerve and in a model for glaucoma (Bai et al., 2010). This is possibly due to the ability of mAb1D7 to induce a much more long-lasting activation of TrkB in these assays than BDNF. Indeed, long-lasting Trk activation was shown to lead to long-lived physiological effects in neurons (Maliartchouk et al., 2000). Here the higher stability of the TrkB antibodies may be of advantage compared to the short half-life of BDNF and poor bioavailability underlying the therapeutic value of these compounds.

Several fully human TrkB agonist antibodies were identified in a function-based cellular screening assay from a combinatorial human short-chain variable fragment antibody library (Merkouris et al., 2018). The most effective full agonist antibody, ZEB85 shows a potency, selectivity, and activity comparable to BDNF regarding TrkB phosphorylation and activation of the canonical downstream signaling (Merkouris et al., 2018; Tacke et al., 2022). Moreover, treatment with ZEB85 supported the preservation of the dendritic arbor of retinal ganglion cells in mouse retinal explants (Merkouris et al., 2018). The activity of ZEB85 has also been compared to the one of BDNF in a series of well-established BDNF-dependent essays including dendrite growth and dendritic spine density as well as neuronal activation as shown by the expression of different immediate early genes in hippocampal neurons and activity-dependent synaptic plasticity. The results showed that ZEB85 mimics the effects of BDNF in promoting neurite outgrowth (Figure 1B and B’ and Tacke et al., 2022), the expression of cFOS and the activation of pERK downstream of the TrkB phosphorylation (Tacke et al., 2022). Moreover, ZEB85 rescues the dendritic complexity in BDNF-deficient Parvalbumin-positive interneurons and the impairment in long-term potentiation observed in BDNF heterozygous knockout mice (Figure 1C; Korte et al., 1996; Zagrebelsky et al., 2018; Tacke et al., 2022). Finally, while both BDNF (Kellner et al., 2014) and ZEB85 do not increase dendritic spine density in healthy neurons, co-application of ZEB85 with oligomerized amyloid beta1–42 (Aβ_1–42) prevents dendritic spine loss in primary hippocampal neurons, especially regarding mushroom spines (Tacke et al., 2022).

A therapeutic potential for a TrkB agonistic antibody in AD was recently shown in vivo in APP/PS1 transgenic mice. The agonist antibody AS86 was developed by immunization-hybridoma technologies using the human extracellular domain of TrkB as the antigen and was shown to induce TrkB phosphorylation, attenuate Aβ_1–42-induced cell death, facilitate dendritic growth, and enhance synaptic function in vitro in a manner comparable to BDNF (Wang et al., 2020). Interestingly, after a single injection of AS86 through the tail vein the antibody could be detected both in the plasma with a half-life of days and in the brain up to 30 days post injection where it activated TrkB signaling. Moreover, peripheral administration of AS86 rescued the deficits in novel object recognition memory and reversed the spatial memory deficits typically observed in APP/PS1 transgenic mice (Wang et al., 2020). Importantly, the bi-weekly administration of AS86 for as long as 9 months did not induce any side effects. In addition, AS86 showed several advantages when compared to BDNF including a higher half-life in plasma and brain as well as a specific binding to TrkB and not to p75^NTR. Thus, the long-term administration of TrkB agonistic antibody could be indeed a feasible approach for AD therapy (Table 1).

Table 1.

Summary of the main TrkB agonists analyzed including the results obtained both in vitro and in vivo

Tested agonist	Results in vitro	Results in rodents	Citations
7,8-Dihydroxyflavone	1) Prophosphorylation of TrkB, Akt, and ERK; 2) Inhibition of neuronal death; 3) Prevents age-related spine loss in rat slices; 4) Stimulates cytokine production from microglia; 5) Not protective for striatal neurons from mHTT-induced toxicity.	1) Rescues spatial/fear memory; facilitates synaptic plasticity in cognitively impaired aged rats; 2) Neuroprotective in models of ischemia, kainic acid, or MPTP lesions; 3) Beneficial in APP/PS1, but not in APP23/PS45 transgenic mice; 4) Prevents depressive profile and promotes TrkB phosphorylation; 5) Anti-inflammatory.	Jang et al., 2010, 2014; Liu et al., 2014; Zeng et al., 2011, 2012a, b; Devi and Ohno, 2012; Aytan et al., 2018; Liu et al., 2010; Blugeot et al., 2011; Park et al., 2012, 2014; Todd et al., 2014
LM22A-4	1) Activates TrkB and downstream signaling; 2) Prevents neuronal death; 3) Not protective for striatal neurons from mHTT-induced toxicity.	Improves motor symptoms and increases TrkB phosphorylation in mouse models of Rett syndrome and Huntington’s disease.	Massa et al., 2010; Schmid et al., 2012; Simmons et al., 2013; Todd et al., 2014
29D7 antibody	1) Binds both mouse and human TrkB, induces phosphorylation; 2) Promotes survival and growth of cortical neurons.	Enhances survival of neurons in a model of cerebral ischemia.	Qian et al., 2006; Todd et al., 2014; Kim et al., 2014
AB2 and AB20	Induce TrkB, ERK, AKT, CREB phosphorylation and transcription of VGF in human-induced pluripotent stem cell-derived neurons		Traub et al., 2017
mAb1D7 antibody	Induces TrkB phosphorylation comparable to BDNF.	Supports survival of retinal ganglion cells in two mouse models for retinal degeneration.	Bai et al., 2010
ZEB85 antibody	1) Selectively activates TrkB and downstream signaling 2) Supports dendritic development in retinal ganglion cells and hippocampal neurons 3) Promotes neuronal activation and plasticity in hippocampal slices 4) Prevents Abeta-induced dendritic spine loss		Merkouris et al., 2018; Tacke et al., 2022
AS86 antibody	1) Induces TrkB phosphorylation; 2) Attenuates Abeta-induced neuronal death; 3) Facilitates dendritic growth; 4) Enhances synaptic function.	1) Enters the central nervous system upon intra-venous injection; 2) Rescues memory deficits in APP/PS1 transgenic mice.	Wang et al., 2020

Concluding Remarks and Discussion

In the last twenty years, great progress has been made in elucidating the pathogenic mechanisms as well as the signaling pathways involved in several neurological diseases. Among these certainly, the best examples are AD and Huntington’s disease as well as major depressive disorder and the relevance of the underlying alterations in the BDNF/TrkB signaling pathway. Nevertheless, still today detailed knowledge of the pathogenic mechanisms did not lead to the development of successful therapeutic approaches, especially regarding the possibility to modulate BDNF signaling via TrkB. However, more than the small molecule TrkB agonists, still showing a series of pharmacological limitations, the TrkB agonist antibodies seem to fulfill several of the requirements for a successful therapy. Indeed, especially Wang and colleagues could provide proof for two crucial facts regarding TrkB agonistic antibodies: that when administered peripherally they do cross the blood-brain barrier and diffuse into the brain tissue and that they can be administered long-term without side effects. Furthermore, trans-blood-brain barrier systems have been developed and shown to successfully transport antibodies (Pardridge, 2012; Boado et al., 2013).

Among the supposed limitations of a therapy based on the exogenous administration of BDNF is the lack of spatial specificity. Indeed, the current understanding is that BDNF activity relies on locally regulated signaling, i.e. at synapses. The results reported above seem to contradict this view describing significant benefits from the peripheral administration of TrkB agonist antibodies (Wang et al., 2020). Furthermore, the long-term activation of TrkB receptors reported upon treatment with some agonist antibodies seems to support their beneficial activity without resulting in unwanted or negative side effects. This is probably also due to the specificity of all these antibodies for TrkB and especially the lack of binding to the p75^NTR.

While the beneficial effects of a treatment with TrkB agonist drugs are obvious for very diverse neurological diseases, whether these drugs have the potential to become a disease-modifying treatment is still open. Indeed, the results so far rather indicate that their main benefits derive from their general neuroprotective activity or their capacity to promote neuronal plasticity and not from reversing the disease pathology. On the other hand, whether the alterations in BDNF/TrkB signaling described are the cause of the disease or rather a correlation or even just a secondary consequence of the cell loss is still not clear.

Additional file: Open peer review report 1 ^{(80.7KB, pdf)} .