Learning and memory depend on the establishment, maintenance, strengthening or regulated elimination of synapses between neurons. These processes are mediated by neuronal activity and the activity-dependent secretion of factors that act at synapses. BDNF is one of the most potent mediators of synaptic plasticity as it is secreted during Long Term Potentiation (LTP) induction and is functionally essential for acute signaling cascades leading to LTP [reviewed in Lu et al. (2013); Park and Poo (2013). As a consequence, BDNF has a crucial role in cognitive functions (Bambah-Mukku et al., 2014; Panja et al., 2014). Critical insights into BDNF activities in humans have been provided by the identification of a single-nucleotide polymorphism (SNP) in the BDNF gene that converts a valine to methionine at codon 66 (Val66Me) (Egan et al., 2003). This polymorphism impairs BDNF trafficking and synaptic localization, causes a reduction in activity-dependent BDNF secretion and is associated with alterations in brain structure and function leading to several neurological and psychiatric disorders (Greenberg et al., 2009; Lu et al., 2013). Genetic or pharmacological manipulations of the levels or activity of the BDNF receptor Ntrk2 (TrkB) also result in impaired LTP and reduced synapse numbers causing deficits in the formation and consolidation of hippocampus-dependent memory (Minichiello, 2009). TrkB receptor signaling depends on the precise pattern of expression of the different isoforms generated by the Ntrk2 gene, including the full-length tyrosine kinase receptor (TrkB.FL) and truncated receptors lacking the kinase domain (TrkB.T1). Hypomorphic expression of the TrkB.FL causes hyperphagia-induced obesity due to reduced hypothalamic BDNF signaling, while genetic deletion of TrkB.T1 leads to increased anxiety related behavior associated with structural alterations in neurites of the amygdala (Carim-Todd et al., 2009; Xu et al., 2003). Moreover, up-regulation of TrkB.T1 levels in brains of a mouse model with a Chromosome 16 trisomia (Ts16) leads to an increased sensitivity of hippocampal neurons to BDNF deprivation due to a block in TrkB.FL functions (Dorsey et al., 2006). Importantly, alterations in NTRK2 isoforms receptor levels have also been associated with neuropsychiatric and neurodegenerative disorders (Dwivedi et al., 2003; Ernst et al., 2009a; Ferrer et al., 1999). Because loss and gain of function experiments have stressed the importance of proper TrkB.T1 expression regulation for normal brain development and function, we elected to investigate this mechanism. We took advantage of the observation that the Ts16 mouse model has a TrkB.T1 upregulation, despite having an intact Ntrk2 locus on Chromosome 13, to identify genes on Chromosome 16 responsible for the dysregulation of TrkB isoforms expression (Dorsey et al., 2006). We identified Rbfox1 as an RNA binding protein that regulates TrkB.T1 receptor levels. Rbfox1 is expressed only in neurons, heart and skeletal muscle, sites with notable TrkB expression. Moreover, RBFOX1 dysregulation has been associated with intellectual disability, autism, epilepsy and Parkinson disease, pathologies that have been associated with alterations in BDNF signaling as well (Chao et al., 2006; Conboy, 2017; Lu et al., 2013). We found that Rbfox1 upregulation impairs hippocampal BDNF-dependent LTP by specifically increasing TrkB.T1 receptor levels. Although Rbfox1 can regulate the splicing and abundance of many gene isoforms in the nervous system, we show that genetic reduction of the TrkB.T1 isoform in animals with up-regulated Rbfox1 is sufficient to restore hippocampal BDNF-dependent LTP, suggesting that Ntrk2 is a major target of Rbfox1 pathological dysregulation (Fogel et al., 2012; Gehman et al., 2011; Lee et al., 2016; Li et al., 2007; Underwood et al., 2005). Importantly, RNA-seq analysis of hippocampi with Rbfox1 upregulation validates the abnormal TrkB.T1 isoform levels, and also shows that the genes affected by increased Rbfox1 levels are different than those changed by its loss of function, thus suggesting that Rbfox1 has broader genetic targets than previously established.

We have previously reported that the trisomic TS16 mouse model, which has an extra copy of Chromosome (Chr) 16, has a neuronal upregulation of the TrkB.T1 receptor isoform level (Dorsey et al., 2006). Since, the Ntrk2 gene is located in Chr 13 (Tessarollo et al., 1993), these data suggested that one or multiple genes present in Chr16 are responsible for this phenotype. A comparison between the genes isolated from brain immunoprecipitation of the spliceosome and bio-informatics analysis of the Ts16 unique region identified two RNA binding proteins, Tra2b (Sfrs10) and Rbfox1 (A2bp1), as potential candidates of TrkB.T1 expression regulation (Li et al., 2007). Tra2b is a ubiquitously expressed splicing factor that contributes to alternative splicing processes in a concentration dependent manner (Elliott et al., 2012). Therefore, we first tested whether Tra2b overexpression in neurons can change TrkB isoforms expression levels. However, infection of primary hippocampal neurons with a Tra2b-expressing lentivirus did not cause any change in TrkB isoforms expression (Figure 1—figure supplement 1). Next, we tested the RNA binding protein Rbfox1 that, compared to Tra2b, has a more restricted and overlapping pattern of expression with TrkB in organs such as brain, heart and muscle (Li et al., 2007; Stoilov et al., 2002). Western blot analysis of primary hippocampal neurons transfected with a lentiviral vector expressing Rbfox1 showed increased truncated TrkB receptor levels associated with Rbfox1 overexpression while the TrkB.FL isoform was unaffected (Figure 1A,B). Although TrkB.T1 is the dominant truncated TrkB isoform in mouse brain, the potential presence of other truncated isoforms prompted us to test the identity of the upregulated truncated isoform. As shown in Figure 1A (right panel) western blot analysis with an antibody recognizing the specific 11 amino acid intracellular domain of TrkB.T1 confirmed that TrkB.T1 is the isoform upregulated by Rbfox1. Moreover, QPCR analysis also revealed an increased level of TrkB.T1-specific mRNA while TrkB.FL was unchanged, paralleling the results from the western analysis (Figure 1C).

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To study the physiological significance of TrkB.T1 upregulation by Rbfox1 we generated a mouse model with an inducible gain of function Rbfox1 allele. By gene targeting of the Gt(ROSA)26Sor locus we introduced one copy of the Rbfox1 cDNA preceded by the CAG promoter to drive expression (R26-Fox1; Sakai and Miyazaki, 1997). A loxP-flanked stop cassette was inserted between the CAG promoter and the Rbfox1 c-DNA to allow for conditional activation of the gene (Figure 1—figure supplement 2A). Primary hippocampal neurons from heterozygous Rbfox1 transgenic mice (R26-Fox1) were isolated and transduced with a Cre-recombinase-expressing adenovirus (Ad-Cre) to delete the stop cassette and induce Rbfox1 expression. Compared to control neurons infected with the Ad-Cre, R26-Fox1 neurons showed increased Rbfox1 expression and subsequent TrkB.T1 upregulation (Figure 1D). These data suggest that this is an efficient model to up-regulate Rbfox1 and further validated our initial observation that lentiviral overexpression of Rbfox1 upregulates TrkB.T1 (Figure 1A–C).

Next, we tested whether Rbfox1 upregulation can regulate TrkB.T1 expression in vivo by crossing the R26-Fox1 model with the Nes-cre (N-cre) mouse (Tronche et al., 1999). First we tested Nes-cre expression in the hippocampus by crossing it with a Gt(ROSA)26Sor-LacZ (R26-LacZ) reporter mouse. β-Galactosidase staining (X-Gal) of the hippocampal area showed LacZ activity in granule cells of dentate gyrus (DG) and pyramidal cells of CA1 and CA3 regions, as previously reported (Chen et al., 2016; Schultz et al., 2011) (Figure 1—figure supplement 2B). Importantly, Nes-Cre (N-cre) crossing with the R26-Fox1 transgenic mouse (N-cre;Fox1) showed that hippocampal Rbfox1 expression is similar between N-cre and N-cre;Fox1 mice, and Rbfox1 expression does not co-localize with the glial marker, glial fibrillary acidic protein (GFAP) (Figure 1—figure supplement 2C,D). These data demonstrate the relevance of this model to study Rbfox1 neuronal up-regulation in vivo. N-cre;Fox1 animals showed a modest 25% increase of Rbfox1 mRNA level in the hippocampus (data not shown). At the protein level, similar to what observed in vitro, N-cre;Fox1 hippocampi had a significant 35% increase in TrkB.T1 associated with about 33% Rbfox1 protein upregulation, while TrkB.FL level was unaffected (Figure 1E,F). mRNA analysis also showed that TrkB.T1, but not TrkB.FL was significantly increased as a consequence of Rbfox1 upregulation (data not shown). Importantly, this model leads to a relatively modest up-regulation of Rbfox1 expression suggesting its validity to study the physiological consequences of Rbfox1 dysregulation.

To further test the specificity of this phenotype, we also investigated the protein levels of Ntrk3 (TrkC), another Ntrk family member also expressed in hippocampal neurons (Tessarollo et al., 1993), in the same N-cre;Fox1 hippocampi and found that the levels of TrkC isoform receptors were unaffected by in vivo Rbfox1 upregulation (Figure 1—figure supplement 3).

Since Rbfox1 upregulation increases TrkB.T1 expression in vivo (Figure 1E,F) we also tested whether lower levels of Rbfox1 cause a reduction of TrkB.T1 expression. Western blot analysis of hippocampi dissected from Rbfox1 heterozygous (Gehman et al., 2011) animals showed a significant 50% reduction in Rbfox1 protein levels. However, there was no change in TrkB.T1 or TrkB.FL receptor isoforms expression (Figure 2A,B). Therefore, we tested whether other Rbfox family members could compensate for the partial loss of Rbfox1. Hippocampal lysates from Rbfox1 heterozygous animals showed a significant 35% upregulation of Rbfox2 whereas Rbfox3 expression was unchanged [Figure 2A,B; (Vuong et al., 2018). This result strongly suggests that Rbfox1 is haploinsufficient. Moreover, it suggests that Rbfox2 is upregulated by Rbfox1 reduction providing a possible explanation for why TrkB receptor isoform expression is not altered in the hippocampus of Rbfox1 heterozygous mice. To further test compensatory mechanisms by other Rbfox genes, we overexpressed Rbfox2 and Rbfox3 in primary hippocampal neurons by lentiviral transduction. Interestingly, overexpression of Rbfox2 but not Rbfox3 caused TrkB.T1 upregulation as observed in N-cre-Rbfox1 transgenic mice (Figure 2C,D) suggesting functional redundancy at least in the context of TrkB.T1 receptor regulation. However, contrary to the upregulation of Rbfox2 caused by loss of one Rbfox1 allele, Rbfox2 expression was not downregulated in response to increased Rbfox1 expression (Figure 1—figure supplement 3).

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The finding that overexpression of Rbfox1 causes a specific increase in the amount of TrkB.T1 mRNA without affecting the full-length receptor (TrkB.FL) suggests that Rbfox1 may act by increasing TrkB.T1 mRNA stability or expression and not as an alternative splicing factor. Therefore, we first analyzed Rbfox1 iCLIP datasets in brain to investigate the presence of Ntrk2 transcripts among the Rbfox1 targets (Figure 3A; Damianov et al., 2016). iCLIP experiments were performed from two distinct mouse brain nuclear fractions including a high molecular weight fraction (HMW) which is more intimately associated with chromatin and enriched in pre-mRNA species, and a more soluble nuclear fraction which excludes chromatin associated protein like histone H3 and is enriched in mature mRNAs (Damianov et al., 2016). Analysis of both HMW and soluble nuclear fractions showed several iCLIP-hits along the Ntrk2 sequence covering both intronic as well as 3’-UTR regions of the gene, suggesting direct binding of Rbfox1 to Ntrk2 transcripts (TrkB.FL and TrkB.T1) (Figure 3A).

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To further validate Rbfox1 binding to Ntrk2 mRNAs we performed RNA immunoprecipitation (RIP) experiments (Figure 3C) (Jayaseelan et al., 2011). Lysates from wild type hippocampal neurons were subjected to immunoprecipitation (IP) with a Rbfox1 specific monoclonal antibody (1D10, a kind gift of Dr. Douglas Black) followed by RT-PCR analysis to detect bound RNA. While immunoprecipitation with control IgG failed to pull down any of the tested mRNA, IP with the anti-Rbfox1 antibody pulled down both the TrkB.FL and TrkB.T1 mRNA as well as a proximal intronic region upstream of the specific TrkB.T1 exon (Figure 3C), confirming the association found in the iCLIP experiments. Calmodulin-binding transcription activator 1 (Camta1) mRNA, used as positive control (Gehman et al., 2011; Lee et al., 2016), was also pulled down whereas Sirtuin 1 (Sirt1), used as a negative control, was not, even though it is present in the neuron lysates like all the other mRNAs (Figure 3C, Input lanes).

The iCLIP analysis also showed the presence of hit-clusters in the TrkB.T1 3’-UTR region of both TrkB.FL and TrkB.T1 (Figure 3A) suggesting a possible role for Rbfox1 in stabilizing TrkB mRNAs by antagonizing specific miRNAs binding to the 3' UTR (Lee et al., 2016). Therefore, we generated a HEK293 cell line with doxycycline-inducible Rbfox1 expression and transfected it with TrkB.T1 cDNA containing the 3’UTR sequence (Figure 3—figure supplement 1A,B). Surprisingly, despite the presence in the 3’UTR of six ‘GCATG’ Rbfox1 binding sites, expression of Rbfox1 caused no change in the level of expression of TrkB.T1 suggesting that the TrkB.T1 mRNA isoform is regulated by Rbfox1 through a different mechanism not involving the 3’UTR region. Similar experiments done in a mouse neuroblastoma cell line (Neuro-2a) confirmed that this result is independent of the cell type (Figure 3—figure supplement 1C,D).

Since Rbfox1 binds both TrkB.T1 and TrkB.FL mRNAs, we next tested whether Rbfox1 upregulation differentially regulates the stability of the two mRNA isoforms by using ethynyl-uridine (EU) to label new nascent RNA followed by purification and QPCR analysis. Measurements of newly synthesized Ntrk2 mRNAs levels with basal or up-regulated expression of Rbfox1 showed that TrkB.T1, but not TrkB.FL mRNA was significantly stabilized (p = 0.03) in primary hippocampal neurons by the increased levels of Rbfox1 (Figure 3D). Testing of the same samples for Ntrk3 (TrkC) mRNA transcripts, showed that neither TrkC.FL nor the truncated TrkC.T1 mRNA stability was affected by Rbfox1 upregulation (Figure 3—figure supplement 2). These data suggest a specific role of this RBP in stabilizing the TrkB.T1 mRNA receptor isoform.

We next asked whether Rbfox1 RNA binding activity is required for increasing TrkB.T1 levels. Adenoviruses containing a wild type or a Rbfox1 mutant lacking RNA binding capability (F158A) were used to transduce primary hippocampal neurons (Hakim et al., 2010; Jin et al., 2003). As shown in Figure 3E–F, overexpression of Rbfox1-mutant failed to increase TrkB.T1 levels suggesting that Rbfox1 RNA binding activity is essential for this function.

The findings that loss of function of Rbfox1 causes an upregulation of Rbfox2 and does not influence TrkB.T1 expression (Figure 2A,B) whereas Rbfox1 gain of function does not change Rbfox2 levels and increases TrkB.T1 expression (Figure 1—figure supplement 3) suggest that up or down regulation of Rbfox1 may target different genes. To test this hypothesis, we performed whole transcriptome deep coverage RNA-seq analysis of hippocampi (with an average of 100 million reads) from N-cre;Fox1 and control N-cre animals. The RNA-seq confirmed our findings that Rbfox2 mRNA is not modulated in N-cre;Fox1 transgenic animals (Supplementary file 1). More importantly, the RNA-seq analysis confirmed that TrkB.T1 mRNA was the only upregulated Ntrk2 isoform (Figure 4). It also showed that among all the Ntrk2 isoforms annotated in the Ensembl database the TrkB.T1 (Ntrk2-202) and TrkB.FL (Ntrk2-201) isoforms are by far the most highly expressed in the hippocampus with approximately 15,000 and 12,000 reads respectively, whereas the other isoforms combined represent only about 5% of the total TrkB transcripts (Figure 4B). In western blot analysis, an almost complete lack of signal at the level of the truncated isoform in the hippocampus of TrkB.T1 knockout mice also confirmed that TrkB.T1 is the main truncated isoform expressed in this region and no other truncated isoforms are present (Figure 4C).

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Gene ontology (GO) term analysis of the RNA-seq data revealed that the major biological processes affected in the N-cre;Fox1 hippocampus are involved in important neuronal functions comprising synapse organization, neurotransmitter secretion, axonal development and many others (Figure 5A; Supplementary file 1). Interestingly, we found that the TrkB-receptor ligand Bdnf is upregulated in the N-cre;Fox1 mouse although it is not clear whether this is the result of a compensatory mechanism in response to the upregulation of TrkB.T1 or whether Bdnf is a direct target of Rbfox1 (Supplementary file 1). The most surprising result came from the parallel analysis, using identical parameters, of the Rbfox1-KO and the N-cre;Fox1 hippocampal RNA-seq raw data (Vuong et al., 2018). In fact, we found that the gene-isoforms that are differentially expressed in the hippocampus of N-cre;Fox1 mice are mostly different from the gene-isoforms affected by Rbfox1 deletion [Figure 5B; Supplementary file 1 and 2; (Vuong et al., 2018) (Gehman et al., 2011). These findings were further supported by the very limited or complete lack of overlap of genes that are dysregulated by hippocampal Rbfox1 gain of function (N-cre;Fox1) or Rbfox1 loss of function in brain (Rbfox1-KO) (Gehman et al., 2011) or hippocampal neurons lacking both Rbfox1 and Rbfox3 [(Figure 5—figure supplement 1) (Lee et al., 2016).

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Next, we decided to investigate the physiological consequences of TrkB.T1 changes of expression. Rbfox1 upregulation causes a specific increase in the levels of TrkB.T1 without changing the expression of TrkB full-length (TrkB.FL). Therefore, we created an in vitro system to mimic this situation and study BDNF/TrkB signaling. We first generated a cell line with stable expression of TrkB.FL (HEK293-TrkB.FL) followed by transfection with a plasmid to express increasing amounts of TrkB.T1. Stimulation with BDNF showed an inverse correlation between TrkB.T1 expression levels and TrkB.FL (p-TrkB.FL) and ERK (p-ERK) phosphorylation suggesting an impairment in TrkB.FL signaling when TrkB.T1 is upregulated (Figure 6A). Importantly, deletion of TrkB.T1 in primary hippocampal neurons increased both basal as well as BDNF-stimulated p-TrkB.FL and p-ERK compared to control neurons suggesting that TrkB.T1 expression levels are potent regulators of TrkB.FL signaling (Figure 6B,C,D).

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Since BDNF and TrkB are potent modulators of synaptic transmission and plasticity, we then tested whether TrkB.T1 upregulation would affect these brain activities [reviewed in Lu et al. (2013). Although we showed that TrkB.T1 is up-regulated in the hippocampus of N-cre;Fox1 mice, we investigated TrkB expression at the synaptic terminals by analyzing hippocampal synaptosomes (Figure 7). Immunoblot analysis of hippocampal synaptosomal fractions from controls and N-cre;Fox1 mice showed that Rbfox1 leads to a significant ~20% upregulation in TrkB.T1 but not TrkB.FL protein levels at the synaptic terminals. The synaptic protein PSD-95 used as control was enriched in the synaptosomal fraction but unaffected by Rbfox1 overexpression (Figure 7A,B).

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Therefore, we tested the physiological significance and net outcome of Rbfox1-induced upregulation of TrkB.T1 by studying hippocampal synaptic plasticity (Figure 8). Due to the fact that Rbfox1 affects several genes regulating synaptic plasticity [Figure 5A; Supplementary file 1; (Gehman et al., 2011) we focused on BDNF-induced long term potentiation (LTP) at the Schaffer collateral-CA1 neuron synapses to dissect the specific effect on LTP caused by alterations in BDNF/TrkB signaling. We applied extracellular BDNF (20 ng/ml) to hippocampal slices from control mice for 20 min while the field excitatory postsynaptic potentials (fEPSPs) were elicited at 20 s intervals. As previously reported, BDNF induced a strong, gradual increase in the fEPSP in control N-cre (Ctrl) hippocampi (Kang and Schuman, 1995). Surprisingly, N-cre;Fox1 animals had a blunted induction of LTP in response to BDNF (Figure 8) and at 50 min this response was only about 30% of Ctrl (Ctrl 218.14 ± 22.42% vs. N-cre;Fox1 72.95 ± 12.82%) (Figure 8A–C). To test whether the BDNF-induced LTP deficit in the mutant animals was caused by the augmented TrkB.T1 expression, we introduced a TrkB.T1 KO allele in N-cre;Fox1 (N-cre;Fox1;T1+/-) mouse model to reduce its levels. As shown in Figure 8—figure supplement 1, this strategy led to a significant, although partial rescue of the BDNF-induced LTP deficit caused by Rbfox1 upregulation. Interestingly, analysis of BDNF-induced LTP in TrkB.T1 heterozygous animals (T1+/-) was similar to that of controls, suggesting that TrkB.T1 is not haploinsufficient and its levels influence BDNF-induced LTP only in the situation of upregulation (Figure 8—figure supplement 2). To further test whether TrkB.T1 levels are most critical in neurons, where Rbfox1 is expressed, we introduced a conditional TrkB.T1-loxP allele into the N-cre;Fox1 model (N-cre;Fox1;T1-Flx). This strategy allows for the simultaneous induction of Rbfox1 activation, which causes TrkB.T1 upregulation, and deletion of one copy of TrkB.T1 in the same neurons where Nes-cre is active. Importantly, this strategy also led to a partial rescue of BDNF-induced LTP over time, which became significant at 50 min (187.97 ± 49.26%) strongly suggesting that the TrkB.T1 up-regulation in neurons caused by Rbfox1 is causing the impairment in BDNF-induced LTP (Figure 8A–C). Analysis of the Shaffer collateral-CA1 excitability (Input/Output relationship Figure 8E) and presynaptic function with paired pulse stimulation (Figure 8F) showed no differences between the controls and mutant animals suggesting that hippocampal circuitries overall are not affected by the genetic manipulations. Taken together, these data strongly suggest that Rbfox1 upregulation leads to a deficit in the BDNF-induced LTP caused, at least in part, by an increase in TrkB.T1 receptor expression.

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Genomic mutations affecting RBFOX1 expression have been associated with intellectual disability, epilepsy, autism and Parkinson’s disease (Bill et al., 2013; Conboy, 2017; Lin et al., 2016). Rbfox1 is an RBP that regulates the RNA metabolism of hundreds of genes expressed in neurons; thus, a major challenge has been to identify those genes that are in a nodal position downstream of Rbfox1 in transducing its normal and pathological functions (Gehman et al., 2011; Lee et al., 2016; Vuong et al., 2018; Weyn-Vanhentenryck et al., 2014). So far, the recognized experimental paradigms to identify and study genes targeted by Rbfox1 have been by downregulation or knockout experiments (Gehman et al., 2011; Lee et al., 2016). Here we show that Rbfox1 upregulation influences the neuronal expression level of the BDNF receptor TrkB.T1, a change not present in Rbfox1 deletion experiments, and this regulation has significant physiological consequences. Importantly, whole transcriptome RNA-seq analysis of hippocampi from mice with Rbfox1 upregulation revealed a completely different set of differentially expressed gene-isoforms when compared to those identified by Rbfox1 knock-out experiments. Although there are limitations associated with comparing data sets from different studies our conclusions are supported by the parallel analysis of raw data from all studies (Vuong et al., 2018) using uniform parameters and filtering, and similar findings from other independent models of Rbfox1 loss of function (Figure 5—figure supplement 1; Lee et al., 2016; Gehman et al., 2011).

These results suggest that upregulation or downregulation of a specific RBP can have different genetic outcomes and may be relevant to understanding how to approach pathologies caused by RBP dysregulation. A notable example is provided by Tra2b (Sfrs10), an RBP ubiquitously expressed but whose differences in cellular concentrations is believed to lead to tissue-specific pattern of splicing (Elliott et al., 2012). Moreover, in spinal muscular atrophy (SMA), a disease caused by deletion of the SMN1 gene, expression of the adjacent full-length SMN2 gene, that includes exon7, can ameliorate disease in some patients. Tra2b overexpression in transfected cells appears to increase splicing of SMN2 exon seven promoting the production of a full-length SMN2, thus suggesting that this strategy could ameliorate SMA disease (Hofmann et al., 2000). However, Tra2b deletion in mice (Sfrs10^−/−) does not influence splicing of SMN2 exon 7 (Mende et al., 2010).

Upregulation of Rbfox1 causes increased expression of TrkB.T1 which in turn blunts BDNF-induced LTP. RNA-seq analysis, besides validating TrkB.T1 up-regulation, identified more than five hundred differentially expressed gene-isoforms in the hippocampus of N-cre;Fox1 animals, including many involved in important neuronal functions (Figure 5; Supplementary file 1). However, decreasing only TrkB.T1 levels in the same cells where Rbfox1 is up-regulated, is sufficient to restore BDNF-induced LTP (Figure 8). This is important because BDNF-induced LTP is a potent modulator of brain synaptic plasticity (Lu et al., 2013). Although alterations in BDNF signaling caused by disruptions in its expression or changes in levels of Ntrk2 receptor isoforms have been associated with neurodegeneration, psychiatric disorders, intellectual disabilities and autism, most studies have focused on the mechanisms regulating Bdnf expression and not its receptor TrkB (Qin et al., 2016; Zheng et al., 2016). For example, it has been shown that at the transcription level Bdnf expression is regulated by epigenetic factors and via the use of multiple promoters. Different transcription factors can bind to these promoters to generate transcripts containing the unique BDNF protein encoding exon. In addition, the neuronal spatially and temporally regulated processing of the pro-BDNF peptide provides another level at which BDNF function can be modulated (Reviewed in Hing et al. (2018)). The significance of this second regulatory mechanism has been validated by the Val66Met BDNF polymorphism in humans which affects the processing and trafficking of BDNF and causes phenotypes including profound impairments in synaptic and cognitive functions (Chen et al., 2005; Egan et al., 2003; Soliman et al., 2010). However, almost nothing is known about the mechanisms regulating Ntrk2 expression or the mechanisms leading to its pathological dysregulation (Kemppainen et al., 2012; Wong et al., 2012). Contrary to the Bdnf gene that has multiple promoters activated by different stimuli, the Ntrk2 locus has only one promoter. The NTRK2 gene spans over 350 Kb of DNA sequence and allows for the generation, by alternative splicing of at least 36 potential transcripts from 24 exons (Luberg et al., 2010). However, gene targeting experiments have shown that two isoforms, namely TrkB.FL and TrkB.T1, are the most highly expressed and phenotypically important during development [Figure 4; (Dorsey et al., 2006; Fulgenzi et al., 2015; Klein et al., 1993). To date, no RBPs have been shown to regulate Ntrk2 splicing or mRNA transcripts levels. Thus, Rbfox1 is the first gene shown to regulate Ntrk2 expression at the mRNA level. Its direct binding to Ntrk2 RNA (Figure 3C) and its function in promoting TrkB.T1 RNA stability, rather than splicing (Figure 3D) are strongly supported by: 1) the in vivo and in vitro Rbfox1 overexpression leading to up-regulation of only TrkB.T1 and no changes in TrkB.FL; 2) the confirmation of the change in expression of only TrkB.T1 by the RNA-seq analysis in N-cre-Fox1 mouse hippocampus; 3) the analysis of new nascent RNA showing that TrkB.T1, but not TrkB.FL RNA is more stable upon Rbfox1 overexpression. Nevertheless, the details of this newly identified molecular mechanism by which RbFox1 expression influences TrkB.T1 mRNA levels needs to be further elucidated since it appears to be independent of an action on the 3’UTR region (Figure 3—figure supplement 1). Importantly, this mechanism does not appear to be limited to TrkB.T1 since it has also been reported that in the hippocampus of Rbfox1 knock-out mice some genes lacking iCLIP clusters or GCAUG binding elements in the 3’UTR region can still be differentially expressed (Vuong et al., 2018) and Rbfox1 can increase the mRNA concentration of genes that lack identified 3’UTR miRNA binding sites (Lee et al., 2016).

While upregulation of Rbfox1 changes TrkB.T1 expression, a reduction in its level does not have an effect. The upregulation of Rbfox2 supports a compensatory mechanism by this family member since both genes are expressed in neurons and like all Rbfox proteins they bind to the same (U)GCAUG motif. However, the fact that only overexpression of Rbfox2, but not Rbfox3, can upregulate TrkB.T1 in primary neurons (Figure 2C,D) suggest that different, yet unknown binding partners might be part of this mechanism. Mass spectrometry analysis of Rbfox protein complexes should help identifying which molecular players can differentiate Rbfox protein functions.

Upregulation of Rbfox1 has an inhibitory function on BDNF-induced synaptic plasticity most likely by reducing the TrkB kinase signaling through an increase of TrkB.T1 expression. This is in line with the observation that nervous system–specific deletion of Rbfox1 results in heightened susceptibility to spontaneous and kainic acid–induced seizures which suggest increased neuronal excitability (Gehman et al., 2011). The presence of such regulatory mechanisms on TrkB function is important considering the increased evidence supporting a key role for TrkB activation in epileptogenesis caused by status epilepticus (McNamara and Scharfman, 2012). Thus, having TrkB activity under the control of Rbfox1, which also regulates the splicing of several synaptic function genes including ion channels, neurotransmitter receptors, structural proteins of the synapse and vesicle fusion proteins, suggests a unifying biological system to regulate neuronal function and excitation (Gehman et al., 2011; McNamara and Scharfman, 2012).

In humans, reduced RBFOX1 expression has been associated with autism, epilepsy and heart disease although the mechanistic relationship between RBFOX1 downregulation and these disorders is still unknown (Bill et al., 2013; Gao et al., 2016). Impaired BDNF signaling has also been associated with these diseases raising the interesting possibility that RBFOX1 regulation of the expression of TrkB receptors might, at least in part, be part of the pathogenetic mechanism. Increasing or decreasing TrkB.T1-receptor levels indeed affects BDNF/TrkB signaling (Figure 6). Although, we do not see changes in TrkB.T1 expression in Rbfox1 heterozygous mice, it is conceivable that the compensatory mechanisms between mice and humans are different. For example, Nedd4-2 heterozygous mice have minimal developmental abnormalities such as hyperactivity, increased basal synaptic transmission and enhanced sensitivity to inflammatory pain (Yanpallewar et al., 2016). However, human heterozygous missense mutations cause major malformations including periventricular nodular heterotopia leading to hypotonia, intellectual disability, seizures, syndactyly and cleft palate (Broix et al., 2016; Kato et al., 2017). In addition, Ntrk2 heterozygous mice do not show major abnormalities while loss of one NTRK2 (TrkB) allele in human leads to hyperphagic obesity and severe impairment in memory, learning and nociception (Klein et al., 1993; Yeo et al., 2004).

Most epidemiological studies associate gene loss of function caused by deletions or point mutations with disease. Indeed, even for RBFOX1 most studies have looked at RBFOX1 downregulation. However, our findings suggest the need to investigate causal relationship between RBFOX1 gain of function to specific pathologies. For example, it has been reported that neurons derived from iPSCs of Parkinson's disease (PD) patients have elevated RBFOX1 levels (Lin et al., 2016). Interestingly, there is already abundant data showing a role for BDNF/TrkB signaling in the etiology of Parkinson's disease, at least from in vivo animal models. For example, ablation of the Bdnf gene impairs the survival and/or maturation of substantia nigra (SN) dopamine (DA) neurons during development (Baquet et al., 2005); loss of one copy of Ntrk2 leads to an age-dependent increase in the levels of α-synuclein in the SN (von Bohlen und Halbach et al., 2005), and in a mouse model with a chronic reduction in TrkB signaling (~30% of WT) there is an age-dependent and selective degeneration of SN DA neurons and increased vulnerability of these neurons to neurotoxins (Baydyuk and Xu, 2014). Because it has also been reported that neurons of PD patients have an increase in TrkB.T1 expression (Fenner et al., 2014), it will be of interest to test whether sporadic PD patients have RBFOX1 upregulation and, if that is the case, an association with increased TrkB.T1 receptor levels. In another study, it has been reported that in the human population there is an intronic SNP affecting an enhancer in the fourth intron of RBFOX1 that leads to a substantial increase in expression (Carter et al., 2017). While this SNP has been identified in the context of a study evaluating how inherited polymorphisms carried in the germline affect the somatic evolution of a tumor, it will be important to study whether this SNP has other effects on cognitive brain functions and leads to an increase in the incidence of neurodegenerative disorders where TrkB.T1 upregulation has also been reported (Cai et al., 2006; Dwivedi et al., 2003; Ernst et al., 2009b; Karege et al., 2005a; Karege et al., 2005b). Most importantly, the change in TrkB.T1 level caused by Rbfox1 upregulation is not dramatic, but it is occurring at the synaptic terminals (Figure 7) which in turn can compromise critical BDNF functions on synaptic plasticity (Figure 8). Examples where small changes in the expression of specific genes are very detrimental and relevant to human pathology have already been described. For instance, gain of function mutations that facilitate or enhance the activation of protein kinase Cγ (PKC) by only approximately 10% have been associated with Alzheimer’s disease (Alfonso et al., 2016). PKC is required for the synaptic depression caused by amyloid-β (Aβ) and it has been suggested that a lifetime of slightly enhanced signaling may sensitize individuals to the detrimental effects of Aβ leading to AD (Newton, 2018).

In all, our study lays the foundation to investigate whether upregulation of Rbfox1 leads to alteration of brain functions or neurodegenerative disorders that are ultimately associated with a dysregulation in BDNF/TrkB signaling, a pathway that has already been convincingly associated with normal development of the nervous system.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Table 1 and 2. The RNA-seq data generated for this study have been deposited in NCBI's Gene Expression Omnibus and is accessible through GEO Series accession number GSE136253.

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Author details

1. Francesco Tomassoni-Ardori

   Neural Development Section, Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, United States

   Contribution

   Conceptualization, Data curation, Investigation, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

2. Gianluca Fulgenzi

   Neural Development Section, Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, United States

   Contribution

   Conceptualization, Formal analysis, Investigation

   Competing interests

   No competing interests declared

3. Jodi Becker

   Neural Development Section, Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, United States

   Contribution

   Investigation, Performed experiments

   Competing interests

   No competing interests declared

4. Colleen Barrick

   Neural Development Section, Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, United States

   Contribution

   Investigation, Performed experiments

   Competing interests

   No competing interests declared

5. Mary Ellen Palko

   Neural Development Section, Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, United States

   Contribution

   Investigation, Performed experiments

   Competing interests

   No competing interests declared

6. Skyler Kuhn

   1. CCR Collaborative Bioinformatics Resource (CCBR), Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, United States
   2. Advanced Biomedical Computational Science, Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute, Frederick, United States

   Contribution

   Formal analysis, Assisted with preparing manuscript

   Competing interests

   No competing interests declared

7. Vishal Koparde

   1. CCR Collaborative Bioinformatics Resource (CCBR), Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, United States
   2. Advanced Biomedical Computational Science, Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute, Frederick, United States

   Contribution

   Formal analysis, Assisted with preparing manuscript

   Competing interests

   No competing interests declared

8. Maggie Cam

   1. CCR Collaborative Bioinformatics Resource (CCBR), Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, United States
   2. Advanced Biomedical Computational Science, Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute, Frederick, United States

   Contribution

   Resources, Supervision

   Competing interests

   No competing interests declared

9. Sudhirkumar Yanpallewar

   Neural Development Section, Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, United States

   Contribution

   Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

10. Shalini Oberdoerffer

    Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, United States

    Contribution

    Formal analysis, Provided intellectual contribution

    Competing interests

    No competing interests declared

11. Lino Tessarollo

    Neural Development Section, Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, United States

    Contribution

    Conceptualization, Data curation, Supervision, Funding acquisition, Writing—original draft, Writing—review and editing

    For correspondence

    tessarol@mail.nih.gov

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-6420-772X

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