Essential role of Ret for defining non-peptidergic nociceptor phenotypes and functions in the adult mouse.

Transduction of pain following noxious stimuli is mediated by the activation of specialized ion channels and receptors expressed by nociceptive sensory neurons. A common early nociceptive sublineage expressing the nerve growth factor receptor TrkA diversifies into peptidergic and non-peptidergic nociceptors around birth. In this process, peptidergic neurons maintain TrkA expression, while non-peptidergic neurons downregulate TrkA and upregulate the common glial-derived neurotrophic factor family ligand receptor Ret and bind the isolectin B4 (IB4). Although Ret can have profound impacts on the molecular and physiological properties of nociceptive neurons, its role is not fully understood. Here we have deleted Ret in small- and medium-size sensory neurons, bypassing the early lethality of the full Ret knockout. We identify that Ret is expressed in two distinct populations of small-medium sized non-peptidergic neurons, an IB4(+) and an IB4(-) population. In these neurons, Ret is a critical regulator of several ion channels and receptors, including Nav1.8, Nav1.9, ASIC2a, P2X3, TrpC3, TrpM8, TrpA1, delta opioid receptor, MrgD, MrgA1 and MrgB4. Ret-deficient mice fail to respond to mustard oil-induced neurogenic inflammation, have elevated basal responses and a failure to terminate injury-induced sensitization to cold stimuli, hypersensitivity to basal but not injury-induced mechanical stimuli, while heat sensation is largely intact. We propose that elevated pain responses could be contributed by GPR35, which is dysregulated in adult Ret-deficient mice. Our results show that Ret is critical for expression of several molecular substrates participating in the detection and transduction of sensory stimuli, resulting in altered physiology following Ret deficiency.

Differential membrane compartmentalization of Ret by PTB-adaptor engagement.

Glial cell line-derived neurotrophic factor family ligands act through the receptor tyrosine kinase Ret, which plays important roles during embryonic development for cell differentiation, survival, and migration. Ret signaling is markedly affected by compartmentalization of receptor complexes into membrane subdomains. Ret can propagate biochemical signaling from within concentrates in cholesterol-rich membrane microdomains or lipid rafts, or outside such regions, but the mechanisms for, and consequences of, Ret translocation between these membrane compartments remain largely unclear. Here we investigate the interaction of Shc and Frs2 phosphotyrosine-binding domain-containing adaptor molecules with Ret and their function in redistributing Ret to specialized membrane compartments. We found that engagement of Ret with the Frs2 adaptor results in an enrichment of Ret in lipid rafts and that signal transduction pathways and chemotaxis responses depend on the integrity of such rafts. The competing Shc adaptor did not promote Ret translocation to equivalent domains, and Shc-mediated effects were less affected by disruption of lipid rafts. However, by expressing a chimeric Shc protein that localizes to lipid rafts, we showed that biochemical signaling downstream of Ret resembled that of Ret signaling via Frs2. We have identified a previously unknown mechanism in which phosphotyrosine-binding domain-containing adaptors, by means of relocating Ret receptor complexes to lipid rafts, segregate diverse signaling and cellular functions mediated by Ret. These results reveal the existence of a novel mechanism that could, by subcellular relocation of Ret, work to amplify ligand gradients during chemotaxis.

Subcellular receptor redistribution and enhanced microspike formation by a Ret receptor preferentially recruiting Dok.

Ret is a receptor tyrosine kinase for the GDNF family of ligands and plays important roles during nervous system development for cell proliferation, cell migration and neurite growth. Signaling initiated from intracellular tyrosine 1062, by recruitment of several different phosphotyrosine binding (PTB) proteins (i.e. Shc, Frs2 and Dok), is important for these biological effects. By a single amino acid substitution in the PTB domain binding sequence of Ret, we have rewired the receptor such that it preferentially recruits Dok (Ret(Dok+)) with little or no remaining interactions with Shc and Frs2. Ret(Dok+) displays a sustained MAP kinase activation and a loss of Akt signaling compared to Ret(WT). We show that early events after ligand stimulation of Ret(Dok+) include massive formation of fine microspikes that are believed to be priming structures for neurite growth from the cell soma. The Ret(Dok+) receptors relocated in the membrane compartment into focal clusters at the tip of the microspikes, which was associated with Cdc42 activation. These results suggest that engagement of different adaptor proteins by Ret results in very different downstream signaling and functions within neurons and that Dok recruitment leads to a rapid receptor relocation and formation of microspikes.

Cell migration by a FRS2-adaptor dependent membrane relocation of ret receptors.

During development neural progenitor cells migrate with extraordinary precision to inhabit tissues and organs far from their initial position. Little is known about the cellular basis for directional guidance by tyrosine kinase receptors (RTKs). RET is a RTK with important functions in guiding the migration of neuronal cells, and RET dysregulation leads to clinical disease such as agangliosis of the colon. We show here that RET migration in neuroepitheliomal and non-neuronal cells is elicited by the activation of specific signaling pathways initiated by the competitive recruitment of the FRS2 adaptor molecule to tyrosine 1062 (Y1062) in RET. FRS2 selectively recruited RET to focal complexes and led to activation of SRC family kinases and focal adhesion kinase (FAK). Activation of SRC depended on its direct interaction with RET at a different intracellular tyrosine (Y981) and activation of molecular signaling from these two separate sites in concert regulated migration. Our data suggest that an important function for FRS2 is to concentrate RET in membrane foci, leading to an engagement of specific signaling complexes localized in these membrane domains.

Genetic evidence for selective neurotrophin 3 signalling through TrkC but not TrkB in vivo.

Neurotrophins control neuronal survival in a target-derived manner during the period of naturally occurring cell death in development. The specificity of this mechanism has been attributed to a restricted spatio-temporal expression of neurotrophin ligands in target tissues, as well as a selective expression of their cognate tyrosine kinase (Trk) receptors in different neuronal subpopulations. However, several in vitro and in vivo studies of null mutant mice have suggested that neurotrophin 3 (NT 3) also signals through the non-preferred TrkB receptor. In this study, we have directly addressed the in vivo preference of NT 3 to signal through TrkB or TrkC, by crossing the NT 3 knock-in mice (BDNF(NT 3/NT 3) mice) with the TrkB- or TrkC-null mutant mice. We find that TrkB is dispensable, whereas TrkC is required for the neuronal rescue by the NT 3 allele in the brain-derived neurotrophic factor- and NT 3-dependent cochleovestibular system. Our results show that NT 3 maintains survival of cells as well as target innervation only through interactions with TrkC in vivo. TrkB and TrkC receptors are thus not functionally redundant for NT 3, even when coexpressed in neurons of the cochleovestibular system.

Experimental subarachnoid hemorrhage induces changes in the levels of hippocampal NMDA receptor subunit mRNA.

NMDA receptors may play a crucial role in nerve cell death following subarachnoid hemorrhage (SAH). Changes in NMDA receptor-mediated transmission appear before neuronal death in rodent models of transient ischemia, and NMDA receptor function is known to be dependent on subunit composition. Here, we have investigated whether mRNA expression of the NMDA receptor subunits is altered in the hippocampal formation 3-5 h following experimental SAH, and correlated these early alterations to subsequent delayed cell death. SAH was induced by intraluminal perforation of the internal carotid artery intracranially, and cerebral blood flow (CBF) was bilaterally monitored by laser-Doppler flowmetry. Early changes in NMDA receptor subunit mRNA and early nerve cell death were analyzed at 3-5 h after SAH, and delayed nerve cell death was analyzed at 2-7 days after SAH. Duration of ipsilateral CBF reduction below 30% of baseline (CBF30) was predictive of ipsilateral delayed nerve cell death in the CA1 2-7 days after SAH. At CBF30 > 9 min, we found downregulation of mRNA for NR2A, NR2B, and NR3B at 3-5 h after SAH, whereas the levels of NR1 mRNA were unaffected. The downregulation of NR2A and NR2B mRNA may result in a reduced NMDA receptor function. Such reduction may be sufficient to provide neuroprotection in the dentate gyrus, where no cell death appears, but insufficient to rescue neurons in the hippocampus proper following SAH.