06-574 Sigma-AldrichAnti-TrkA Antibody

Steroids and growth factors control neuronal development through their receptors under physiological and pathological conditions. We show that PC12 cells harbor endogenous androgen receptor (AR), whose inhibition or silencing strongly interferes with neuritogenesis stimulated by the nonaromatizable synthetic androgen R1881 or NGF. This implies a role for AR not only in androgen signaling, but also in NGF signaling. In turn, a pharmacological TrkA inhibitor interferes with NGF- or androgen-induced neuritogenesis. In addition, androgen or NGF triggers AR association with TrkA, TrkA interaction with PI3-K δ, and downstream activation of PI3-K δ and Rac in PC12 cells. Once associated with AR, filamin A (FlnA) contributes to androgen or NGF neuritogenesis, likely through its interaction with signaling effectors, such as Rac. This study thus identifies a previously unrecognized reciprocal cross-talk between AR and TrkA, which is controlled by β1 integrin. The contribution of FlnA/AR complex and PI3-K δ to neuronal differentiation by androgens and NGF is also novel. This is the first description of AR function in PC12 cells.

RET can be activated in cis or trans by its co-receptors and ligands in vitro, but the physiological roles of trans signaling are unclear. Rapidly adapting (RA) mechanoreceptors in dorsal root ganglia (DRGs) express Ret and the co-receptor Gfrα2 and depend on Ret for survival and central projection growth. Here, we show that Ret and Gfrα2 null mice display comparable early central projection deficits, but Gfrα2 null RA mechanoreceptors recover later. Loss of Gfrα1, the co-receptor implicated in activating RET in trans, causes no significant central projection or cell survival deficit, but Gfrα1;Gfrα2 double nulls phenocopy Ret nulls. Finally, we demonstrate that GFRα1 produced by neighboring DRG neurons activates RET in RA mechanoreceptors. Taken together, our results suggest that trans and cis RET signaling could function in the same developmental process and that the availability of both forms of activation likely enhances but not diversifies outcomes of RET signaling.

Depressive symptoms during pregnancy are common and may have impact on the developing child. Selective serotonin reuptake inhibitors (SSRIs) are the most prescribed antidepressant treatment, but unfortunately, these treatments can also negatively affect the behavioral development and health of a child during pregnancy. In addition, serotonin (5-HT) exerts neurotrophic actions with thus far not fully known effects in the offspring. The neurotrophic growth factor (NGF) is involved in neuronal cell survival and differentiation, and altered placenta levels have been found to increase the risk for pregnancy complications, similar to those found in women treated with SSRIs. We therefore investigated whether the NGF signaling pathway was altered in the placenta from women treated with SSRIs (n = 12) and compared them with placenta from depressed (n = 12) and healthy mothers (n = 12). Results from immunohistochemical stainings revealed that placental NGF protein levels of SSRI-treated women were increased in both trophoblasts and endothelial cells compared with depressed and control women. In addition, downstream of the NGF receptor TrkA, increased levels of the signaling proteins ROCK2 and phosphorylated Raf-1 were found in stromal cells and a tendency towards increased levels of ROCK2 in trophoblasts and endothelial cells in SSRI-treated women when compared to healthy controls. SSRI-treated women also displayed increased levels of phosphorylated ROCK2 in all placental cell types studied in comparison with depressed and control women. Interestingly, in placental endothelial cells from depressed women, NGF levels were significantly lower compared to control women, but ROCK2 levels were increased compared with control and SSRI-treated women. Taken together, these results show that the NGF signaling and downstream pathways in the placenta are affected by SSRI treatment and/or antenatal depression. This might lead to an altered placental function, although the clinical relevance of our findings still needs to be investigated.

Retrograde communication from axonal targets to neuronal cell bodies is critical for both the development and function of the nervous system. Much progress has been made in recent years linking long-distance, retrograde signaling to a signaling endosome, yet the mechanisms governing the trafficking and signaling of these endosomes remain mostly uncharacterized. Here we report that in mouse sympathetic neurons, the target-derived nerve growth factor (NGF)-tropomyosin-related kinase type 1 (TrkA, also called Ntrk1) signaling endosome, on arrival at the cell body, induces the expression and recruitment of a new effector protein known as Coronin-1 (also called Coro1a). In the absence of Coronin-1, the NGF-TrkA signaling endosome fuses to lysosomes sixfold to tenfold faster than when Coronin-1 is intact. We also define a new Coronin-1-dependent trafficking event in which signaling endosomes recycle and re-internalize on arrival at the cell body. Beyond influencing endosomal trafficking, Coronin-1 is also required for several NGF-TrkA-dependent signaling events, including calcium release, calcineurin activation and phosphorylation of cAMP responsive element binding protein (CREB). These results establish Coronin-1 as an essential component of a feedback loop that mediates NGF-TrkA endosome stability, recycling and signaling as a critical mechanism governing developmental competition for survival.

The boundary cap is a transient group of neural crest-derived cells located at the presumptive dorsal root transitional zone (DRTZ) when sensory axons enter the spinal cord during development. Later, these cells migrate to dorsal root ganglia and differentiate into subtypes of sensory neurons and glia. After birth when the DRTZ is established, sensory axons are no longer able to enter the spinal cord. Here we explored the fate of mouse boundary cap neural crest stem cells (bNCSCs) implanted to the injured DRTZ after dorsal root avulsion for their potential to assist sensory axon regeneration.Grafted cells showed extensive survival and differentiation after transplantation to the avulsed DRTZ. Transplanted cells located outside the spinal cord organized elongated tubes of Sox2/GFAP expressing cells closely associated with regenerating sensory axons or appeared as small clusters on the surface of the spinal cord. Other cells, migrating into the host spinal cord as single cells, differentiated to spinal cord neurons with different neurotransmitter characteristics, extensive fiber organization, and in some cases surrounded by glutamatergic terminal-like profiles.These findings demonstrate that bNCSCs implanted at the site of dorsal root avulsion injury display remarkable differentiation plasticity inside the spinal cord and in the peripheral compartment where they organize tubes associated with regenerating sensory fibers. These properties offer a basis for exploring the ability of bNCSCs to assist regeneration of sensory axons into the spinal cord and replace lost neurons in the injured spinal cord.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder leading to paralysis and early death due to reduced SMN protein. It is unclear why there is such a profound motor neuron loss, but recent evidence from fly and mouse studies indicate that cells comprising the whole sensory-motor circuit may contribute to motor neuron dysfunction and loss. Here, we used induced pluripotent stem cells derived from SMA patients to test whether sensory neurons directly contribute to motor neuron loss. We generated sensory neurons from SMA induced pluripotent stem cells and found no difference in neuron generation or survival, although there was a reduced calcium response to depolarizing stimuli. Using co-culture of SMA induced pluripotent stem cell derived sensory neurons with control induced pluripotent stem cell derived motor neurons, we found no significant reduction in motor neuron number or glutamate transporter boutons on motor neuron cell bodies or neurites. We conclude that SMA sensory neurons do not overtly contribute to motor neuron loss in this human stem cell system.

Tyro3, Axl and Mertk (TAM) receptor tyrosine kinases play multiple functional roles by either providing intrinsic trophic support for cell growth or regulating the expression of target genes that are important in the homeostatic regulation of immune responses. TAM receptors have been shown to regulate adult hippocampal neurogenesis by negatively regulation of glial cell activation in central nervous system (CNS). In the present study, we further demonstrated that all three TAM receptors were expressed by cultured primary neural stem cells (NSCs) and played a direct growth trophic role in NSCs proliferation, neuronal differentiation and survival. The cultured primary NSCs lacking TAM receptors exhibited slower growth, reduced proliferation and increased apoptosis as shown by decreased BrdU incorporation and increased TUNEL labeling, than those from the WT NSCs. In addition, the neuronal differentiation and maturation of the mutant NSCs were impeded, as characterized by less neuronal differentiation (β-tubulin III+) and neurite outgrowth than their WT counterparts. To elucidate the underlying mechanism that the TAM receptors play on the differentiating NSCs, we examined the expression profile of neurotrophins and their receptors by real-time qPCR on the total RNAs from hippocampus and primary NSCs; and found that the TKO NSC showed a significant reduction in the expression of both nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), but accompanied by compensational increases in the expression of the TrkA, TrkB, TrkC and p75 receptors. These results suggest that TAM receptors support NSCs survival, proliferation and differentiation by regulating expression of neurotrophins, especially the NGF.

We present a toolbox for the study of molecular interactions occurring between NGF and its receptors. By means of a suitable insertional mutagenesis method we show the insertion of an 8 amino acid tag (A4) into the sequence of NGF and of 12 amino acid tags (A1 and S6) into the sequence of TrkA and P75NTR NGF-receptors. These tags are shortened versions of the acyl and peptidyl carrier proteins; they are here covalently conjugated to the biotin-substituted arm of a coenzyme A (coA) substrate by phosphopantetheinyl transferase enzymes (PPTases). We demonstrate site-specific biotinylation of the purified recombinant tagged neurotrophin, in both the immature proNGF and mature NGF forms. The resulting tagged NGF is fully functional: it can signal and promote PC12 cells differentiation similarly to recombinant wild-type NGF. Furthermore, we show that the insertion of A1 and S6 tags into human TrkA and P75NTR sequences leads to the site-specific biotinylation of these receptors at the cell surface of living cells. Crucially, the two tags are labeled selectively by two different PPTases: this is exploited to reach orthogonal fluorolabeling of the two receptors co-expressed at low density in living cells. We describe the protocols to obtain the enzymatic, site-specific biotinylation of neurotrophins and their receptors as an alternative to their chemical, nonspecific biotinylation. The present strategy has three main advantages: i) it yields precise control of stoichiometry and site of biotin conjugation; ii) the tags used can be functionalized with virtually any small probe that can be carried by coA substrates, besides (and in addition to) biotin; iii) above all it makes possible to image and track interacting molecules at the single-molecule level in living systems.

Nerves and vessels often run parallel to one another, a phenomenon that reflects their functional interdependency. Previous studies have suggested that neurovascular congruency in planar tissues such as skin is established through a "one-patterns-the-other" model, in which either the nervous system or the vascular system precedes developmentally and then instructs the other system to form using its established architecture as a template. Here, we find that, in tissues with complex three-dimensional structures such as the mouse whisker system, neurovascular congruency does not follow the previous model but rather is established via a mechanism in which nerves and vessels are patterned independently. Given the diversity of neurovascular structures in different tissues, guidance signals emanating from a central organizer in the specific target tissue may act as an important mechanism to establish neurovascular congruency patterns that facilitate unique target tissue function.

Facilitation of nerve growth factor (NGF) signaling by the p75 neurotrophin receptor (p75(NTR)) is critical for neuronal survival and differentiation. However, the interaction between p75(NTR) and TrkA receptors required for this activity is not understood. Here, we report that a specific 29-amino acid peptide derived from the intracellular domain fragment of p75(NTR) interacts with and potentiates binding of NGF to TrkA-expressing cells, leading to increased neurite outgrowth in sympathetic neurons as a result of enhanced Erk1/2 and Akt signaling. An endogenous intracellular domain fragment of p75(NTR) (p75(ICD)) containing these 29 amino acids is produced by regulated proteolysis of the full-length receptor. We demonstrate that generation of this fragment is a requirement for p75(NTR) to facilitate TrkA signaling in neurons and propose that the juxtamembrane region of p75(ICD) acts to cause a conformational change within the extracellular domain of TrkA. This finding provides new insight into the mechanism by which p75(NTR) and TrkA interact to enhance neurotrophic signaling.