Disentangling the signaling complexity of nerve growth factor receptors by CRISPR/Cas9

The binding of nerve growth factor (NGF) to the tropomyosin–related kinase A (TrkA) and p75NTR receptors activates a large variety of pathways regulating critical processes as diverse as proliferation, differentiation, membrane potential, synaptic plasticity, and pain. To ascertain the details of TrkA‐p75NTR interaction and cooperation, a plethora of experiments, mostly based on receptor overexpression or downregulation, have been performed. Among the heterogeneous cellular systems used for studying NGF signaling, the PC12 pheochromocytoma‐derived cell line is a widely used model. By means of CRISPR/Cas9 genome editing, we created PC12 cells lacking TrkA, p75NTR, or both. We found that TrkA‐null cells become unresponsive to NGF. Conversely, the absence of p75NTR enhances the phosphorylation of TrkA and its effectors. Using a patch‐clamp, we demonstrated that the individual activation of TrkA and p75NTR by NGF results in antagonizing effects on the membrane potential. These newly developed PC12 cell lines can be used to investigate the specific roles of TrkA and p75NTR in a genetically defined cellular model, thus providing a useful platform for future studies and further gene editing.

pain-related disorders, [11][12][13] and neurodegenerative diseases. 14,15 The biological action of NGF depends on the cellular context and is elicited through binding and activation of its receptors, namely TrkA (Tropomyosin-Related Kinase receptor type I) [16][17][18][19] and p75 NTR (a member of the Tumor necrosis factor receptor superfamily). [20][21][22][23] TrkA has the highest affinity for NGF, and responds to ligand binding with dimerization and transphosphorylation, leading to the activation of key signaling pathways, such as extracellular signal-regulated kinase (ERK), phospholipase C-γ (PLC-γ) and phosphatidyl-inositol 3 kinase (PI3K). 21 These intracellular pathways promote NGF-mediated survival, differentiation, and synaptic plasticity. 24 p75 NTR is a single-pass transmembrane receptor with significant binding affinity for all neurotrophins and their immature forms (i.e., pro-neurotrophins 25 ). The polarity of p75 NTR effects depends on its interacting partners: with sortilin, p75 NTR causes apoptosis mediated by proneurotrophins 26 ; with LINGO-1 and Nogo-A, it participates in myelin-dependent inhibition of axonal growth 27 ; with Trks, it promotes survival, axonal growth, and differentiation. 28 Regarding this latter cooperation, several works showed that interaction with p75 NTR increases the NGF binding affinity of TrkA, 29 and potentiates signaling activation. 30 In addition, NGF regulates the ubiquitination of TrkA, 31,32 along with its increased endocytosis and retrograde transport. 33 Thus, the physical or functional cooperation of p75 NTR with TrkA is recognized to be a cellular process of paramount importance. On the contrary, the interaction mode and stoichiometry of TrkA and p75 NTR in the absence or presence of NGF stimulation are still a matter of hot debate. 30,34,35,36 Through the years, several studies on this cooperation were carried out using PC12 cells. [37][38][39] This cell line, which was established from a rat pheochromocytoma, is a gold standard model for assessing the biological activity of NGF in vitro, 40 owing to the expression of both NGF receptors. Indeed, PC12 cells are able to acquire the phenotype of sympathetic neurons (an NGF-dependent population) when exposed to NGF. 41 To analyze the individual contribution of NGF receptors to downstream signaling and the consequent cellular responses, several PC12 clones have been generated. For instance, (i) the PC12nnr5 clone, selected by chemically mutagenized cultures and notably lacking a genomic characterization, does not express TrkA 42 ; (ii) trk-PC12 cells stably overexpress TrkA, and have been used to investigate the role of TrkA in NGF-induced differentiation 43 ; (iii) the PC12-27 clone has wild type-like levels of TrkA, while the expression of p75 NTR is negligible, due to the repressive effect exerted by the REST (RE-1 silencer of transcription). 44 However, all these PC12 cell variants are genetically ill-defined.
Here, in order to dissect the contributions of TrkA and p75 NTR in mediating NGF-dependent signaling effects in the context of a genetically controlled background, we exploited the CRISPR/Cas9 gene editing technology. 45 Using this approach, we generated and characterized three PC12 clones: (i) TrkA knockout; (ii) p75 NTR knockout; (iii) TrkA-p75 NTR double knockout. Upon comparison with wild type PC12 cells, these three gene-edited cellular models gave us the opportunity to assess the contribution and fine tuning of the individual and combined receptor effects to intracellular signaling, without any possible confounding source of variability caused by, e.g., the use of antisense oligonucleotides, chemical inhibitors, drugs, or protein overexpression.

| Molecular biology for gene editing
The human codon-optimized Cas9 and chimeric guide RNA expression plasmid (pX459) developed by the Zhang lab 46 were obtained from Addgene (Waterton, MA, USA). To generate gRNA plasmids, a pair of annealed oligonucleotides (20 base pairs) were cloned with the BbsI restriction enzyme into the single guide RNA scaffold of the pX459 plasmid.

| Cell culture and transfection
Rat pheochromocytoma PC12 cells line were maintained at 37°C and 5% CO 2 in DMEM medium (Invitrogen, Monza, Italy) supplemented with 10% horse serum, 5% fetal bovine serum, 1% penicillin/streptomycin, 1% L-glutamine (Gibco-ThermoFisher, Monza, Italy), and grown as monolayer cultures according to ATCC standard protocols. Cells were plated at 80%-90% confluence in 3 cm diameter Petri dishes and transfected using Lipofectamine 2000 (Thermo-Fisher Scientific, Monza, Italy; 11 668-027) according to the manufacturer's instructions. Individual PC12 clones were obtained by treating the transfected PC12 cells with 6-9 μg/ml of puromycin for 24/36 h after transfection before replating transfected cells in 96-well plates at limiting dilution, in order to achieve single-cell seeding and subsequent monoclonal expansion. Then, the selected PC12 cell clones were routinely grown and frozen as described in ATCC protocols.

| Western blot
PC12 cells and mutant PC12 clones were cultured in 3 cm diameter petri dishes. PC12 cells were stimulated with wild type NGF (5 or 100 ng/ml) or maintained in basal conditions, then harvested at different post-stimulation times. Cells were lysed in RIPA buffer containing the following (in mM): NaCl (150), EDTA, 5

| Cell differentiation
PC12 cells were maintained as described above. For differentiation assays, cells were plated into 12-well plates coated with 20 μg/ml Poly-L-Lysine (P4707 Sigma-Aldrich) at low density (1 × 10 4 cells/cm 2 ). Differentiation was induced by treatment with serum-free medium supplemented with wild type NGF at different concentrations: 5, 10, 20, 50, and 100 ng/ml. Exposure to serum-free medium alone was used as a control. Cells were imaged after 5 days of treatment using an AxioObserver microscope (Zeiss, Jena, Germany) at 40× magnification. Morphological analysis of differentiation was performed on imaged cells using ImageJ (NIH), and the average length of neurites of differentiated cells was measured, with the operator being blind to the genotype of cells.

| Patch-clamp recordings
Recordings were performed by adapting the procedure described in. 47 Briefly, cells were cultured on poly-Llysine-coated glass coverslips, then transferred to a submerged recording chamber, continuously perfused with oxygenated Tyrode's solution containing (in mM): NaCl (150), KCl, 4 MgCl 2, 1 CaCl 2, 4 Glucose, 10 HEPES, 10 pH 7.4 with NaOH. Borosilicate glass pipettes (1B150F-4, WPI, Sarasota, FL, USA) were pulled with a P-97 puller (Sutter Instruments, Novato, CA, USA) to a resistance of 5-6 MΩ when filled with an internal solution containing (in mM): K-Gluconate (145), MgCl 2 2, HEPES, 10 EGTA (0.1), Mg 2+ -ATP (2.5), Na + -GTP (0.25), phosphocreatine, 5 pH 7.35 with KOH. After achieving whole-cell configuration and allowing at least 3 min for complete equilibration between cytosol and internal solution, the membrane potential was recorded with the amplifier in the I = 0 configuration. NGF (100 ng/ml) was delivered via bath application. To analyze the dependency of NGF-induced variations in the membrane potential on K + and Na + currents, 5 mM tetraethylammonium (TEA) was added to the bath, or NaCl was substituted with 135 mM N-methyl-D-glucamine (NMDG), respectively. 48 Recordings were performed at 32°C. Access resistance and membrane capacitance were monitored during each recording, which was discarded if series resistance varied more than 20% of the initial value. Data were acquired using a MultiClamp 700A amplifier, connected to a Digidata 1550A digitizer (Molecular Devices, San Jose, CA, USA), and sampled at 10 kHz. Analysis was done with Clampfit 11.1 (Molecular Devices).

TrkA/p75 NTR double knock-outs
To disentangle the specific contributions of TrkA and p75 NTR to the effects of NGF, we used CRISPR/Cas9 gene editing to generate PC12 cell mutants lacking TrkA, p75 NTR , or both genes. Guide RNAs (gRNAs) were designed to avoid off-target editing (see Materials and Methods). To counteract possible phenotypic artifacts due to the selection of a single cell clone, we isolated and characterized at least 3 different independent PC12 clones for each genotype (TrkA −/− , p75 NTR−/− , TrkA −/− ; p75 NTR−/− ). The relevant genomic region from each PC12 clone was sequenced to verify the presence of a nonsense frameshift mutation near the protospacer adjacent motif (PAM) sequence ( Figure 1A). Western blot analysis confirmed the lack of expression of the corresponding proteins, namely TrkA and p75 NTR , in gene-edited clones ( Figure 1B and Supporting Information Figure S1A).

| Analyzing NGF-mediated signaling in TrkA −/− PC12 cells
After checking that TrkA knock-out results in the absence of the corresponding protein without affecting the levels of the other main NGF receptor, p75 NTR , we also wanted to verify that the corresponding signaling effectors were unresponsive to NGF. Therefore, we stimulated TrkA −/− PC12 cells with 100 ng/ml NGF. In accordance with the absence of phosphorylated TrkA ( Figure 1B), the phosphorylation of the main effectors of TrkA, i.e., ERK and Akt, was severely impaired in TrkA −/− clones (Figure 2A,B).
To demonstrate that the lack of responsiveness to NGF of TrkA −/− mutants do not affect the capability to respond to other stimuli, cells were treated with 100 ng/ ml Fibroblast Growth Factor (FGF). Indeed, FGF binds to a different tyrosine kinase receptor (FGFR), but converges on the same intracellular pathways as NGF, including Akt and ERK. 49 Stimulation with FGF resulted in normal ERK and Akt phosphorylation, demonstrating the presence of active signaling mediated by FGFR in TrkA −/− PC12 cells (Supporting Information Figure S2A).
Finally, we wanted to prove whether the re-expression of TrkA in TrkA −/− null PC12 cells is sufficient to restore NGF-induced neural differentiation. To this end, we transfected TrkA −/− cells with a construct carrying wild type TrkA, and we observed a recovery of neurite outgrowth in response to NGF. On the contrary, transfection with a "dead" TrkA mutant 50 did not rescue responsiveness to NGF of TrkA −/− cells (Supporting Information Figure S2B).
These data demonstrate that (i) TrkA is required to activate the ERK and Akt pathways in response to NGF; (ii) the absence of TrkA does not disrupt the responsiveness of Akt and ERK to extracellular stimuli different from NGF; (iii) re-expression of wild type TrkA in PC12 TrkA −/− cells recover their responsiveness to NGF in terms of neurite outgrowth.

| Analyzing NGF-mediated signaling
in p75 NTR−/− PC12 cells p75 NTR has been often referred to as a "co-receptor" collaborating with TrkA. 21,51,52,53 However, conflicting findings have been obtained from previous studies using antisense oligonucleotides, chemical inhibitors, or overexpression constructs. 54 Thus, we used our novel p75 NTR−/− PC12 cell clone to address this point.
Western blot analysis confirmed the absence of the p75 NTR protein, while endogenous TrkA was normally expressed ( Figure 1B). Mutated clones and wild type cells were then stimulated with 100 ng/ml NGF to evaluate the phosphorylation of TrkA, ERK, and Akt.
Interestingly, the absence of p75 NTR led to a significant increase in phospho-TrkA above the level displayed by control wild type PC12 cells subjected to the same treatment ( Figure 1B). In keeping with this, at a high concentration (100 ng/ml) of NGF, the activation of Akt via phosphorylation in p75 NTR−/− cells was also higher than in control cells (Figure 2A), whereas the phosphorylation of ERK was comparable between p75 NTR−/− and control cells ( Figure 2B). We also analyzed earlier time points, namely 5 and 15 min post-NGF application, and still detected a higher Akt phosphorylation in NGF-treated p75 NTR−/− cells compared to WT cells ( Figure 2C). Thus, the situation of 5-and 15-min NGF treatment matches that observed at 30 min, with a larger difference between p75 NTR−/− and WT cells (5 min, 545.23 ± 73.53%, 15 min, 518.64 ± 62.46% of WT + NGF-15 min level; 30 min, 204.00 ± 35.04% of WT + NGF levels). In contrast, we found an early enhancement of ERK phosphorylation at 5 min post-NGF treatment in p75 NTR−/− cells compared to WT cells, which disappeared at 15 min post-NGF treatment ( Figure 2C).
In cells treated with 5 ng/ml NGF, the lack of p75 NTR did not result in increased phosphorylation of TrkA ( Figure 3A) and Akt ( Figure 3B), while ERK phosphorylation was significantly lower than in control cells ( Figure 3C).
These data demonstrate that, at saturating concentrations of NGF (100 ng/ml), the absence of p75 NTR leads to increased NGF-TrkA signaling, suggesting that in normal PC12 cells, when TrkA and p75 NTR are coexpressed, the p75 NTR signaling stream sends an inhibitory signal to reduce the TrkA signaling stream. On the contrary, this mechanism of p75 NTR inhibition on TrkA signaling is not active at low concentrations of NGF (5 ng/ml). The level at which this inhibition is exerted remains to be ascertained.

| Exploring the contribution of p75 NTR and TrkA to PC12 cell differentiation
NGF plays a key role in promoting the survival of PC12 cells in the absence of serum 40 and is essential for their differentiation. 38,40 In addition to the downstream signaling mediated by TrkA, p75 NTR is also involved in neurotrophin-induced differentiation. 21 In order to elucidate the individual contribution of p75 NTR and TrkA to PC12 cell differentiation, we measured neurite length in response to treatment with a saturating concentration of NGF (100 ng/ml) and found that only wild type and p75 NTR−/− cells (i.e., only cells with functional TrkA) showed neurite outgrowth in response to NGF. Under these conditions, the response to NGF of p75 NTR−/− cells was significantly higher than wild type cells. On the other hand, lack of TrkA completely abolished neurite elongation in response to NGF, an effect that was also observed in TrkA −/− ; p75 NTR−/− double knock-out cells ( Figure 4A).
Then, we tested different concentrations of NGF on wild type and p75 NTR−/− cells and found that a low concentration of NGF (5 ng/ml) was enough to induce differentiation of wild type cells, resulting in neurite outgrowth, whereas no effect was observed in p75 NTR−/− cells ( Figure 4B). However, at higher concentrations of NGF (i.e., 10 ng/ml, 20 ng/ml, 50 ng/ml), the lack of p75 NTR enhanced differentiation ( Figure 4B), which is consistent with our findings on TrkA signaling ( Figure 2).
We conclude that functional TrkA is required to induce PC12 cells differentiation and that p75 NTR modulates this action, by showing differential effects at low or high NGF concentrations.

TrkA to PC12 cell membrane potential
After characterizing the contribution of TrkA and p75 NTR to intracellular signaling and differentiation using classical, well-established assays, we decided to investigate a key aspect of neuronal function, namely the regulation of membrane potential by NGF.
To analyze the effect of TrkA and p75 NTR on the membrane potential, we employed patch-clamp recordings on the three different gene-edited cell lines that we generated and compared their responses to non-engineered control cells. We first looked for possible effects of receptor knockout on the resting membrane potential of our cell lines in the absence of any manipulation and did not find any significant difference ( Figure 5A). A strikingly different picture emerged when we treated cells with NGF (100 ng/ml) via bath perfusion. In line with previous literature, 48 control cells were quickly, but transiently, depolarized by NGF. This transient response was abolished by inactivation of the TrkA gene, which resulted in TrkA −/− cells being hyperpolarized by NGF. p75 NTR inactivation (p75 NTR−/− cells) had an opposite effect on the membrane potential, causing a strong membrane depolarization, which outlasted that observed in wild type cells. Finally, inactivation of both TrkA and p75 NTR (TrkA −/− ; p75 NTR−/− cells) abolished the response of PC12 cells to NGF, with only a mild and transient hyperpolarization being detected ( Figure 5B).
Then, we sought to determine the possible currents mediating the effects of NGF on the membrane potential.
This functional measure of the effect of NGF using single-cell electrophysiology agrees with our data on intracellular signaling and cell differentiation, further supporting an antagonistic role of TrkA and p75 NTR .

p75 NTR−/− PC12 cells to the analysis of a pathologically relevant NGF mutant
Alterations in the NGF-TrkA-p75 NTR axis are involved in a growing number of diseases. 14,15 Among these, mutations in NGF, including in the R100 residue, cause Hereditary Sensory and Autonomic Neuropathy type V. 12,55,56 Previous work has shown that the HSANVrelated NGF R100E mutant has an identical TrkA binding affinity as that of wild type NGF, and a 200-fold reduced affinity for p75 NTR,14,57 and can be therefore described as a TrkA-biased agonist.
To investigate the effect of R100-mutated NGF on signaling pathways specific to TrkA or p75 NTR , we treated PC12 cells with either wild type NGF (NGF WT ), or NGF R100E. 58 When treated with NGF R100E , wild type PC12 cells showed reduced phosphorylation of TrkA in comparison to NGF WT administration ( Figure 6A). This effect was even more pronounced in p75 NTR−/− cells subjected to the same treatments, while, as expected, no signal could be detected in protein extracts from TrkA −/− cells ( Figure 6A). Moreover, the absence of p75 NTR was associated with a higher Akt phosphorylation than in wild type cells in response to NGF WT (see above), but not to NGF R100E ( Figure 6B). The response of ERK to NGF R100E was unaffected in wild type cells in comparison to NGF WT , while a reduction was observed in the absence of p75 NTR ( Figure 6C).
In conclusion, these data demonstrate that by comparing the response of our gene-edited NGF receptor cells to ligands with a different receptor-engagement profile, a biochemical dissection of the contribution of TrkA and p75 NTR to specific aspects of signaling can be easily carried out.

| DISCUSSION
Signaling by the "NGF system" is a complex process, with the TrkA receptor being more specific (but with some degree of promiscuity, such as NT3 binding) and p75 NTR being common to all neurotrophins. 59 The pro-neurotrophin precursors add to the complexity. For instance, proNGF binds both TrkA 60 and p75 NTR,61 in addition to the binding to sortilin, which mediates pro-apoptotic signals. 26 Over the years, NGF signaling has been investigated in a large number of different cellular systems. This heterogeneous set of results does not allow disentangling the cell-type specific aspects, from other more fundamental technical issues, such as, for instance, the fact that many studies have been performed via receptor overexpression in heterologous systems.
In order to overcome these pitfalls, it would be very convenient to have a standardized cellular model system in which each signaling component can be genetically isolated and removed. In this respect, mouse models in which the receptors have been knocked out by homologous recombination could be helpful in investigating NGF signaling. Indeed, knockout mice for TrkA 62 and p75 NTR have been created. 63,64 However, homozygous TrkA knockout mice show an early lethal phenotype 62 and cannot be bred as homozygotes with other knockout lines to yield double knockouts. As for p75 NTR knockout mice, the two existing strains, carrying mutations in exon 3 63 or exon 4, 64 both display features that defy definitive conclusions. The p75 NTRΔExon3 knockout mouse 63 still encodes an alternatively spliced isoform that might be (partially) functional. The p75 NTRΔExon4 knockout mouse 64 still expresses an intracellular fragment of p75 NTR that has pro-apoptotic properties. 65 To overcome these problems, conditional knockout mice for TrkA 66 and p75 NTR67 have been generated, but their crossbreeding to derive single or double knockout neurons, of the same overall genetic background, has not been reported.
In this regard, a simpler, standardized, and wellvalidated cellular system would facilitate investigations on signaling mechanisms controlled by the NGF system. The PC12 cell line, introduced by Greene and Tischler in 1976 38 has offered the gold standard system to the neurotrophin community to analyze NGF-induced signaling through its receptors TrkA and p75 NTR. 21,68 In this report, we describe the generation and use of a set of new cell lines, based on the PC12 line, with genome editing inactivation of either TrkA or p75 NTR , or both. Notably, a serendipitously isolated TrkA knockout variant (i.e., PC12nnr5) has been the main tool for cellular studies requiring the loss of function of this receptor. 42 However, the PC12nnr5 clone is genetically poorly defined.
These novel cellular tools allowed us to demonstrate, in a controlled manner, that: (i) TrkA is necessary and sufficient for NGF sensing; (ii) the absence of p75 NTR enhances TrkA-mediated signaling at high concentration of NGF; (iii) TrkA and p75 NTR collaborate to promote cell differentiation at low NGF concentrations; (iv) TrkA and p75 NTR have opposing polarities on the regulation of the membrane potential by NGF.

| A genome editing approach to dissect the individual components of the NGF receptor system
Classical approaches (e.g., antisense oligonucleotides, gene overexpression, in vivo gene targeting) and a number of different heterogeneous experimental systems have been largely employed by different groups, over the years, to shape our current understanding of the cellular and functional mechanisms of NGF signaling. 2,29,54,62 However, the lack of a simple, robust, and standardized model susceptible to easy genetic manipulation left somewhat unresolved the analytical dissection of the early and late events of NGF signaling and the relationships between NGF, its co-receptors and the cellular context. In this regard, we chose the PC12 cell line, not only to disentangle the herein discussed roles of TrkA and p75 NTR , but also as a base for the future study of additional components of the NGF multireceptor system, such as sortilin, or pathologically relevant mutations of TrkA 69 and NGF (e.g., 13,57,58,69 ). Indeed, the random mutagenesis approaches so far used to select the currently used mutant PC12 clones, showing absent or reduced expression of TrkA or p75 NTR,42,54 do not allow a precise control on the corresponding gene manipulation, nor are suitable for the specific manipulation of two genes in parallel, as we did in the present study.

TrkA-p75 NTR interplay
A critical and controversial question in NGF signaling is whether the TrkA and p75 NTR receptors combine in heteromeric complexes 30,34,36 and cooperate or compete, resulting in new features compared to the simple sum of pathways activated by either TrkA and p75 NTR receptor alone.
First, we confirmed that our CRISPR/Cas9-based approach abrogates NGF-induced phosphorylation of TrkA, along with abolishing the downstream phosphorylation of Akt and ERK. Then, we found that the absence of p75 NTR enhances TrkA phosphorylation. These results demonstrate that, at a high concentration (100 ng/ml) of NGF, p75 NTR antagonizes the effects of NGF-TrkA interaction, and that p75 NTR alone is not able to transduce binding of NGF signaling into the activation of ERK and Akt. Our findings are consistent and support the previously postulated functional antagonism between TrkA and p75 NTR. 70 Of note, TrkA-mediated activation of the two pathways depends on common (i.e., Shc and Grb2) 71 and specific interactors, such as SH2-B and CD2AP for Akt, 72,73 or Src for ERK. 74 Differential recruitment of these components of NGF receptor-associated signaling may contribute to explain the concentration-specific phosphorylation patterns of Akt and ERK, along with their different temporal kinetics.
Finally, we applied our cell platform to the study of the biochemical properties of NGF R100E , a mutated isoform with important implications for pain insensitivity diseases. 12,58 The key feature of R100-mutated NGF is its reduced ability to elicit hyperalgesia while maintaining an unaltered neurotrophic activity. 12,13 The absence of this heavy side effect has led to testing NGF R100E for the therapy of neurodegeneration. 14 Here, we show that NGF R100E application results in lower phosphorylation of TrkA in wild type PC12 cells. However, when both receptors are present (i.e., in wild type cells), this has no effect on both Akt and ERK phosphorylation, which are equivalently stimulated by the two NGF ligands. Interestingly, in the absence of p75 NTR , the phosphorylation of Akt was significantly higher in response to NGF WT, but not NGF R100E treatment, thus reproducing an analogous trend on TrkA phosphorylation. These data demonstrate that mutationspecific aspects of receptor engagement and signaling can be unmasked and dissected using our TrkA-and p75 NTRnull PC12 cells.

| Interplay between Trka and p75 NTR in PC12 cell differentiation
Despite the controversy on the contribution of TrkA and p75 NTR to downstream signaling, previous reports suggest that both ERK and Akt play a role in NGF-induced neurite outgrowth. 75,76 Our gene-edited PC12 clones allowed us to directly demonstrate that the absence of p75 NTR , in keeping with the increased phosphorylation of TrkA and Akt, results in stronger neurite-like processes outgrowth at high NGF concentrations.
Interestingly, p75 NTR -null cells expressing only TrkA are less sensitive to low-concentration (i.e., 5 ng/ml) NGF, showing less differentiation than wild type cells, accompanied by lower ERK phosphorylation. Despite being nonstatistically significant, the decreased phosphorylation of TrkA observed in p75 NTR -null cells treated with 5 ng/ ml NGF can contribute to the lower phospho-ERK levels and can be linked to the fact that p75 NTR also acts as a coreceptor for TrkA to increase its affinity for NGF. 51 Thus, it can be hypothesized that, in the absence of p75 NTR , the concentration of NGF must cross a critical threshold to fully exert its effects on cell differentiation. Thus, p75 NTR has a dual role in the initial steps of NGF signaling: (i) facilitating the presentation of NGF to TrkA and increasing its effectiveness, at low concentrations and (ii) negatively regulating the outcome of signaling, at higher concentrations. Moreover, our data extend previous findings of enhanced growth of primary cultures of sympathetic neurons from p75 NTR−/− mice. 70 Thus, our results fit very well with the general idea in the literature, but it is remarkable that a few simple experiments exploiting the newly generated PC12 cell lines show this very clearly.
Our data also show a direct correlation between the concentration of NGF and the level of differentiation; at the signaling level, this can relate to the idea that both ERK and Akt play a role to elicit NGF-induced neurite outgrowth in PC12 cells. 54,76 4.4 | Interplay between TrkA and p75 NTR in regulating the membrane potential Our gene-editing approach also allowed a clean dissection of the role of the two receptors of NGF in regulating the membrane potential. The long-term development of electrical excitability in PC12 cells after 2 weeks of induction of differentiation with NGF was described in the early foundational study by Dichter et al. 77 Here, we investigated the receptor dependence of rapid effects of NGF on PC12 cell membrane potential.
First, in naïve PC12 cells, we found that NGF caused a transient depolarization, in agreement with Shimazu and colleagues. 48 Moreover, our findings showed that TrkA gene ablation abolishes the early depolarization and unmasks a late hyperpolarization. On the other hand, p75 NTR gene ablation had an opposite outcome and turned the early depolarization from transient to prolonged. Both effects were lost upon the knock out of both genes, thus pointing to opposite roles of TrkA and p75 NTR in controlling the membrane potential.
Of note, our system totally ablates the expression of either one or both receptors. This could explain why the presence of p75 NTR alone (i.e., in TrkA −/− cells) resulted in NGF inducing hyperpolarization, whereas overexpression of this receptor in 3 T3 cells, which also express TrkA, resulted in depolarization. 48 As a step toward the identification of the channels and conductances responsible for the effects of TrkA and p75 NTR activation on the membrane potential, we found that K + channel blockade and replacement of extracellular Na + prevented NGF from causing hyperpolarization in TrkA −/− cells and depolarization in p75 NTR−/− cells, respectively. These findings are in line with Shimazu et al., 48 who demonstrated that a Na + -free extracellular solution abolishes NGF-induced membrane depolarization, while blockade of K + channels with TEA eliminated NGF-induced membrane hyperpolarization in wild type PC12 cells. By performing TEA experiments on TrkA −/− cells, we did not need to combine TEA administration with Na + -free extracellular solution, as knockout of this receptor abolished NGFinduced depolarization per se.
These electrophysiological results provide a quantitative and robust experimental read-out for the early effects of NGF signaling and might form the basis for an experimental assay for the activity of small molecule NGF agonists or antagonists and a comparison of proNGF with NGF.

| CONCLUSIONS
In conclusion, we employed CRISPR/Cas9-based gene editing to generate new PC12 cell lines that can be used to disentangle the complexity of the NGF/TrkA/ p75 NTR system. Our data on intracellular signaling, cell differentiation, and membrane electrical potential point to an antagonistic role of TrkA and p75 NTR in transducing the binding of NGF at the cell surface. As a further expression of a widely used physiological building motif, this interaction creates a push-pull system, which expands the dynamic range of NGFassociated cellular responses. Identifying the molecular effectors supporting this system will be the focus of our next experiments.
The newly generated PC12 mutants will be very useful for genetic reconstitution experiments. For instance, the many TrkA mutants described as being responsible for congenital insensitivity to pain diseases, such as Hereditary Sensory and Autonomic Neuropathy type IV (HSAN IV; 77) can now be expressed on a genetically clean background. Similarly, the expression of a single NGF receptor, or the absence of both, can be exploited to detect differences in signaling elicited by, e.g., NGF mutants responsible for HSAN type V, 13,78,79 proNGF, 35,60 neurotrophin NT3, or by synthetic ligands or antagonists. 80,81 Finally, our three new PC12 clones, TrkA −/− , p75 NTR−/− , TrkA −/− /p75 NTR−/− will be available as a cellular platform for further gene-editing operations to dissect the downstream key components mediating the wide array of NGF effects on cell pathophysiology.