In mammals, the neurotrophin family is comprised of four members: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and neurotrophin-4/5 (NT4/5). Two types of receptors mediate their actions, the Trk family of tyrosine kinase receptors and the p75 NGF receptor, a member of the tumor necrosis factor receptor superfamily. Binding experiments with cell lines were used to determine the ligand-receptor relationship between neurotrophins and their receptors. NGF binds to TrkA, whereas BDNF and NT4/5 bind to TrkB. NT-3 signals mainly through TrkC but can also bind with lower affinity to TrkA and TrkB (Bothwell, 1995; Chao and Hempstead, 1995; Friedman and Greene, 1999; Kaplan and Miller, 2000; Huang and Reichardt, 2003). The phenotypes observed in mice with targeted mutations in neurotrophins and their Trk receptors have demonstrated the specificity of these interactions, particularly in the developing peripheral nervous system (PNS; Snider, 1994; Tessarollo, 1998). Trk receptors, upon ligand binding, activate well-known intracellular signaling cascades such as the Ras/MAPK pathway, phosphoinositide 3 kinase and phospholipase Cγ (PLCγ) pathways. In the early experiments of gene targeting of the Trk genes in mice, the mutations were directed toward the tyrosine kinase domain region of the full-length receptors because of the established biological activities of these isoforms. However, Trk genes by alternative splicing can produce a wide array of other isoforms. For many of these isoforms the pattern of expression, the conservation among species, and their physiological abundance has not yet been elucidated (Tsoulfas et al., 1993; Luberg et al., 2010, 2015). However, the TrkB and TrkC genes, by alternative splicing generate isoforms that lack tyrosine kinase activity, are conserved among species and are expressed at high levels (Klein et al., 1990; Middlemas et al., 1991; Tsoulfas et al., 1993; Valenzuela et al., 1993; Garner and Large, 1994; Luberg et al., 2010, 2015). Though they were first discovered about 30 years ago, only recently we have begun to learn about the function of these types of receptors. This was in part due to their lack of tyrosine kinase activity, unclear signaling and no obvious pro-survival effect on neurons. The lack of pro-survival functions on different neuronal populations was confirmed in vivo with the second generation of mouse models targeting all Trk genes isoforms showing only minimal phenotypic differences compared to the initial mouse models targeting the kinase domain (Klein et al., 1993, 1994; Smeyne et al., 1994; Tessarollo et al., 1997; Liebl et al., 2000; Luikart et al., 2005). Thus, for a long time, the main functions attributed to these truncated receptor isoforms were of acting as dominant-negative inhibitors of the full-length receptors or limiting ligand availability (Biffo et al., 1995; Eide et al., 1996; Palko et al., 1999). In 2003 (Rose et al., 2003), the report that TrkB.T1 can signal independently by inducing calcium release from the intracellular stores, and in 2006, the generation of a new mouse model targeting specifically the exon encoding the TrkB.T1 isoform without affecting the spatio-temporal pattern of expression of TrkB full-length (TrkB.FL; Dorsey et al., 2006) started a new research effort into the functional role of these enigmatic receptors. As outlined below this research has led to new exciting and unanticipated functions for TrkB.T1 in mammalian physiology. Truncated TrkC isoforms signal by binding the scaffold protein Tamalin which in turn activates the Rac1 GTPase through the adenosine diphosphate-ribosylation factor 6 (Esteban et al., 2006). Moreover, it has also been shown that TrkC.T1 can lead to neural differentiation in collaboration with p75NTR (Hapner et al., 1998). However, the generation of a suitable mouse model to study TrkC truncated receptors in vivo has been hampered by the fact that targeting of the exons encoding specifically the truncated TrkC.T1 (also known as TrkC.NC2) leads to dysregulation of the TrkC.FL receptor expression (Bai et al., 2010). Therefore, the lack of a proper mouse model has limited our ability to study the precise function of TrkC.T1 in vivo. In this review, we focus on the current knowledge on truncated TrkB.T1, the most studied truncated Trk receptor to date. We discuss the many in vivo functions that have been uncovered so far and its relevance in the pathophysiology of neuronal disorders in both animal models and human diseases.

The finding that the mouse and human genome contain fewer genes than previously thought have underlined the importance of alternative splicing as a mechanism to build more complex organisms (Lander et al., 2001; Venter et al., 2001; Waterston et al., 2002a,b). Importantly, alternative splicing in mammals (Clancy, 2008; Donaldson and Beazley-Long, 2016) is particularly prominent and highly conserved in the brain (Raj and Blencowe, 2015). Functionally, alternative splicing can result in protein isoforms that are inactive or exhibit similar, different, or even opposing actions. Moreover, alternatively spliced transcripts and their protein isoform products show dynamic changes in expression and function that are dependent on cell, tissue, age, and context (physiological vs. pathological). BDNF and its receptor TrkB are good examples of genes whose function is tightly associated with alternative splicing. The BDNF gene consists of at least 8 different promoters that can generate 18 separate transcripts. While all transcripts generate the same BDNF polypeptide, the presence of different promoters allows for the differential regulation of BDNF expression. For example, in the cortex, promoter IV-dependent transcription is responsible for activity-induced BDNF expression (Timmusk et al., 1993; Timmusk and Metsis, 1994; Tao et al., 1998; Aid et al., 2007; Hong et al., 2008). In addition, the different mRNA variants can be transported to differential subcellular locations for local translation of BDNF leading to selective morphological remodeling of dendrites (Baj et al., 2016). The human TrkB gene, spanning about 400 kbp, consists of 24 exons that through a complex pattern of alternative splicing can generate up to 30 TrkB isoforms (Stoilov et al., 2002; Luberg et al., 2010). However, despite the abundance of potential TrkB isoforms the most highly expressed isoforms in the mammalian brain are the TrkB.FL and TrkB.T1 receptors (Tomassoni-Ardori et al., 2019). These TrkB receptors are differentially expressed within the diverse areas of the nervous system in a spatial and temporal fashion (Stoilov et al., 2002; Luberg et al., 2010). Developmentally, TrkB.FL is predominantly expressed in the embryonic and early postnatal CNS whereas TrkB.T1 expression is very low in the embryo but increases gradually postnatally and peaks in adulthood (Escandon et al., 1994). At the cellular level, in the nervous system, expression of TrkB.FL is restricted to neurons whereas TrkB.T1 is expressed in both neurons and glia cells (Sommerfeld et al., 2000; Dorsey et al., 2006; Holt et al., 2019; Tomassoni-Ardori et al., 2019). Interestingly, outside the nervous system, TrkB.T1 appears to be the main isoform as it is expressed in the adult heart, kidney, lung, and pancreas (Stoilov et al., 2002; Fulgenzi et al., 2015, 2020).

As mentioned above, the TrkB locus produces many different TrkB receptor isoforms by alternative splicing. However, very little is known about the mechanisms regulating their spatial and temporal expression. TrkB.T1 is the major isoform expressed in the adult mammalian brain and the best characterized among all known truncated isoforms. It includes the exons encoding the extracellular, transmembrane and the juxtamembrane domain that are common to the TrkB.FL receptor up to exon 15. After exon 15, instead of including exon 17 which is the first exon specific to the TrkB.FL isoform, the splicing machinery uses exon 16 which encodes a short intracellular 11 amino acid tail (FVLFHKIPLDG) that is unique to the TrkB.T1 isoform (Figure 1). This sequence lacks obvious homology to any known protein motifs but is 100% conserved between rodents, human and chicken (Klein et al., 1990; Middlemas et al., 1991; Biffo et al., 1995; Luberg et al., 2010). In addition to the 11 aa tail, exon 16 includes a stop codon and has its own 3′UTR sequence with a number of polyadenylation sites. The mechanism underlying the coding of TrkB.T1 versus the TrkB.FL isoform by alternative splicing is unknown and deserves further study. So far, only mechanisms regulating the levels of expression of the different isoforms have been described. For example, TrkB.T1 downregulation in the cortex of suicide victims appears to result from combined epigenetic mechanisms, including methylation of the promoter and the 3′UTR DNA sequence, histone modifications, and microRNA binding (Ernst et al., 2009, 2011; Maussion et al., 2012). More recently, the RNA binding protein RbFox1 has been shown to increase the levels of TrkB.T1 in the hippocampus by direct binding to the TrkB.T1 mRNA. In turn, TrkB.T1 upregulation impairs BDNF-dependent LTP which can be rescued by genetically restoring TrkB.T1 levels strongly suggesting that TrkB.T1 regulates important brain functions (Tomassoni-Ardori et al., 2019).

Dysregulation of homeostatic BDNF signaling has been associated with a number of neuropsychiatric disorders. Most therapeutic strategies aimed at restoring or enhancing BDNF “trophic” support have focused on exogenous administration of BDNF (e.g., in ALS) and the development of BDNF agonists binding to the TrkB extracellular domain such as TrkB activating antibodies (Lin et al., 2008; Perreault et al., 2013; Guo et al., 2019). To date, the search for small molecule agonists has been mainly unsuccessful, or questionable at best (Todd et al., 2014; Boltaev et al., 2017; Pankiewicz et al., 2021). The delivery of BDNF has universally failed as a therapeutic agent probably due to its inability to cross the BBB, the short half-life, and the difficulties of delivery to specific brain areas in a precise spatio-temporal fashion (Miranda-Lourenco et al., 2020). A variety of approaches are currently being pursued to address the issue of BDNF delivery, including the use of biomaterials and nanoparticles, viral mediated gene delivery and transplantation of neurotrophin-producing cells (Houlton et al., 2019; Jarrin et al., 2021). Unfortunately, most of these approaches ignore the possibility that changes at the level of TrkB receptor, including TrkB.T1 upregulation, may influence the downstream BDNF signaling and therefore the therapeutic outcome. Indeed, upregulation of TrkB.T1 in a trisomic mouse model renders hippocampal neurons completely unresponsive to BDNF both in vitro and in vivo. Importantly, restoration of TrkB.T1 to physiological levels rescues neurons sensitivity to BDNF and neuronal cell death in vivo (Dorsey et al., 2002, 2006). These observations have critical implications for the therapeutic use of TrkB agonist antibodies that bind selectively to TrkB. While these antibodies do not engage p75NTR and have a longer half-life (days instead of minutes to hours seen with BDNF) allowing for better diffusion into the neural tissues (Guo et al., 2019; Han et al., 2019), they still bind to TrkB.T1 with the risk of being sequestered and neutralized. The data presented in this review therefore warrants a critical reexamination of these approaches because neurons may not suffer from a deficit of neurotrophin supply but rather an intrinsic block of signaling at the receptor level. Interestingly, transactivation of TrkB receptors seems to circumvent this issue. Use of adenosine A2A receptor agonists that transactivate TrkB intracellularly have been shown to enhance survival of lesioned facial motoneurons (Wiese et al., 2007) and lead to a delay in disease progression in a mouse model of ALS (Domeniconi and Chao, 2010; Yanpallewar et al., 2012). A similar transactivation of TrkB by glucocorticoids has been shown to promote downstream signaling and exert neurotrophic effect (Jeanneteau et al., 2008). Since, the TrkB transactivation mechanism occurs independently of BDNF and is not influenced by the presence of TrkB.T1 at the membrane, it is possible to increase the ability of diseased neurons to respond to TrkB signaling. One caveat is that TrkB.T1, as described earlier, can independently affect calcium signaling and imbalances in this isoform levels may influence neuronal survival by altering intracellular calcium levels. Indeed, dysregulation of calcium homeostasis in spinal motoneurons of SOD1 mutant mice has already been reported (Damiano et al., 2006; Guatteo et al., 2007). This scenario calls for the consideration of multiple strategies to identify the therapeutic potential of BDNF-TrkB signaling: one to reduce TrkB.T1 to correct its dominant-negative function and neuronal calcium levels and the other to increase activation of TrkB.FL by transactivation. An attractive approach to regulate aberrant TrkB isoforms expression is by targeting TrkB alternative splicing (AS). By targeting AS it is possible to regulate the alternate exon inclusion or block exon skipping to enhance read-through of full length isoform or even blocking a splice site to promote formation of a particular splice variant (Graziewicz et al., 2008; Geib and Hertel, 2009; Lin et al., 2015). This approach has now become reality as a therapeutic antisense oligonucleotide (ASO) that binds to the survival motor neuron 2 (SMN2) messenger RNA has been approved by the FDA for the treatment of spinal muscular atrophy (SMA). Treatment with the steric block ASO Nusinersen, that binds to the SMN2 mRNA, was found to promote exon 7 inclusion resulting in increased full length SMN protein. This ultimately leads to the increased survival and motor function in patients of SMA (Wood et al., 2017). A similar approach could be used to correct TrkB.T1 upregulation since even limited efficacy in correcting aberrant splicing levels of TrkB.T1 may lead to significant therapeutic benefits and may even be more desirable considering that TrkB.T1 is important for the normal function of other organs such as the heart and the endocrine pancreas.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

LT and SY wrote the manuscript. Both authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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