Ret is a multifunctional coreceptor that integrates diffusible- and contact-axon guidance signals.

Growing axons encounter multiple guidance cues, but it is unclear how separate signals are resolved and integrated into coherent instructions for growth cone navigation. We report that glycosylphosphatidylinositol (GPI)-anchored ephrin-As function as "reverse" signaling receptors for motor axons when contacted by transmembrane EphAs present in the dorsal limb. Ephrin-A receptors are thought to depend on transmembrane coreceptors for transmitting signals intracellularly. We show that the receptor tyrosine kinase Ret is required for motor axon attraction mediated by ephrin-A reverse signaling. Ret also mediates GPI-anchored GFRα1 signaling in response to GDNF, a diffusible chemoattractant in the limb, indicating that Ret is a multifunctional coreceptor for guidance molecules. Axons respond synergistically to coactivation by GDNF and EphA ligands, and these cooperative interactions are gated by GFRα1 levels. Our studies uncover a hierarchical GPI-receptor signaling network that is constructed from combinatorial components and integrated through Ret using ligand coincidence detection.

EGR4 is critical for cell-fate determination and phenotypic maintenance of geniculate ganglion neurons underlying sweet and umami taste.

The sense of taste starts with activation of receptor cells in taste buds by chemical stimuli which then communicate this signal via innervating oral sensory neurons to the CNS. The cell bodies of oral sensory neurons reside in the geniculate ganglion (GG) and nodose/petrosal/jugular ganglion. The geniculate ganglion contains two main neuronal populations: BRN3A+ somatosensory neurons that innervate the pinna and PHOX2B+ sensory neurons that innervate the oral cavity. While much is known about the different taste bud cell subtypes, considerably less is known about the molecular identities of PHOX2B+ sensory subpopulations. In the GG, as many as 12 different subpopulations have been predicted from electrophysiological studies, while transcriptional identities exist for only 3 to 6. Importantly, the cell fate pathways that diversify PHOX2B+ oral sensory neurons into these subpopulations are unknown. The transcription factor EGR4 was identified as being highly expressed in GG neurons. EGR4 deletion causes GG oral sensory neurons to lose their expression of PHOX2B and other oral sensory genes and up-regulate BRN3A. This is followed by a loss of chemosensory innervation of taste buds, a loss of type II taste cells responsive to bitter, sweet, and umami stimuli, and a concomitant increase in type I glial-like taste bud cells. These deficits culminate in a loss of nerve responses to sweet and umami taste qualities. Taken together, we identify a critical role of EGR4 in cell fate specification and maintenance of subpopulations of GG neurons, which in turn maintain the appropriate sweet and umami taste receptor cells.

Probing the multimodal fungiform papilla: complex peripheral nerve endings of chorda tympani taste and mechanosensitive fibers before and after Hedgehog pathway inhibition.

The fungiform papilla (FP) is a gustatory and somatosensory structure incorporating chorda tympani (CT) nerve fibers that innervate taste buds (TB) and also contain somatosensory endings for touch and temperature. Hedgehog (HH) pathway inhibition eliminates TB, but CT innervation remains in the FP. Importantly, after HH inhibition, CT neurophysiological responses to taste stimuli are eliminated, but tactile responses remain. To examine CT fibers that respond to tactile stimuli in the absence of TB, we used Phox2b-Cre; Rosa26LSL-TdTomato reporter mice to selectively label CT fibers with TdTomato. Normally CT fibers project in a compact bundle directly into TB, but after HH pathway inhibition, CT fibers reorganize and expand just under the FP epithelium where TB were. This widened expanse of CT fibers coexpresses Synapsin-1, ß-tubulin, S100, and neurofilaments. Further, GAP43 expression in these fibers suggests they are actively remodeling. Interestingly, CT fibers have complex terminals within the apical FP epithelium and in perigemmal locations in the FP apex. These extragemmal fibers remain after HH pathway inhibition. To identify tactile end organs in FP, we used a K20 antibody to label Merkel cells. In control mice, K20 was expressed in TB cells and at the base of epithelial ridges outside of FP. After HH pathway inhibition, K20 + cells remained in epithelial ridges but were eliminated in the apical FP without TB. These data suggest that the complex, extragemmal nerve endings within and disbursed under the apical FP are the mechanosensitive nerve endings of the CT that remain after HH pathway inhibition.

Biphasic functions for the GDNF-Ret signaling pathway in chemosensory neuron development and diversification.

The development of the taste system relies on the coordinated regulation of cues that direct the simultaneous development of both peripheral taste organs and innervating sensory ganglia, but the underlying mechanisms remain poorly understood. In this study, we describe a novel, biphasic function for glial cell line-derived neurotrophic factor (GDNF) in the development and subsequent diversification of chemosensory neurons within the geniculate ganglion (GG). GDNF, acting through the receptor tyrosine kinase Ret, regulates the expression of the chemosensory fate determinant Phox2b early in GG development. Ret-/- mice, but not Retfx/fx ; Phox2b-Cre mice, display a profound loss of Phox2b expression with subsequent chemosensory innervation deficits, indicating that Ret is required for the initial amplification of Phox2b expression but not its maintenance. Ret expression is extinguished perinatally but reemerges postnatally in a subpopulation of large-diameter GG neurons expressing the mechanoreceptor marker NF200 and the GDNF coreceptor GFRα1. Intriguingly, we observed that ablation of these neurons in adult Ret-Cre/ERT2; Rosa26LSL-DTA mice caused a specific loss of tactile, but not chemical or thermal, electrophysiological responses. Overall, the GDNF-Ret pathway exerts two critical and distinct functions in the peripheral taste system: embryonic chemosensory cell fate determination and the specification of lingual mechanoreceptors.

Plasma membrane localization of the GFL receptor components: a nexus for receptor crosstalk.

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) comprise a group of four homologous and potent growth factors that includes GDNF, neurturin (NRTN), artemin (ARTN), and persephin (PSPN). The survival, growth, and mitotic activities of the GFLs are conveyed by a single receptor tyrosine kinase, Ret. The GFLs do not bind directly to Ret in order to activate it, and instead bind with high affinity to glycerophosphatidylinositol (GPI)-anchored coreceptors called the GDNF family receptor-αs (GFRαs). Several mechanisms have recently been identified that influence the trafficking of Ret and GFRαs in and out of the plasma membrane, thereby affecting their availability for ligand binding, as well as their levels by targeting to degradative pathways. This review describes these mechanisms and their powerful effects on GFL signaling and function. We also describe the recent discovery that p75 and Ret form a signaling complex, also regulated by plasma membrane shuttling, that either enhances GFL survival signals or p75 pro-apoptotic signals, dependent on the cellular context.

Semaphorin 3A is a retrograde cell death signal in developing sympathetic neurons.

During development of the peripheral nervous system, excess neurons are generated, most of which will be lost by programmed cell death due to a limited supply of neurotrophic factors from their targets. Other environmental factors, such as 'competition factors' produced by neurons themselves, and axon guidance molecules have also been implicated in developmental cell death. Semaphorin 3A (Sema3A), in addition to its function as a chemorepulsive guidance cue, can also induce death of sensory neurons in vitro The extent to which Sema3A regulates developmental cell death in vivo, however, is debated. We show that in compartmentalized cultures of rat sympathetic neurons, a Sema3A-initiated apoptosis signal is retrogradely transported from axon terminals to cell bodies to induce cell death. Sema3A-mediated apoptosis utilizes the extrinsic pathway and requires both neuropilin 1 and plexin A3. Sema3A is not retrogradely transported in older, survival factor-independent sympathetic neurons, and is much less effective at inducing apoptosis in these neurons. Importantly, deletion of either neuropilin 1 or plexin A3 significantly reduces developmental cell death in the superior cervical ganglia. Taken together, a Sema3A-initiated apoptotic signaling complex regulates the apoptosis of sympathetic neurons during the period of naturally occurring cell death.

Exon Skipping in the RET Gene Encodes Novel Isoforms That Differentially Regulate RET Protein Signal Transduction.

Rearranged during transfection (RET), a receptor tyrosine kinase that is activated by the glial cell line-derived neurotrophic factor family ligands (GFLs), plays a crucial role in the development and function of the nervous system and additionally is required for kidney development and spermatogenesis. RET encodes a transmembrane receptor that is 20 exons long and produces two known protein isoforms differing in C-terminal amino acid composition, referred to as RET9 and RET51. Studies of human pheochromocytomas identified two additional novel transcripts involving the skipping of exon 3 or exons 3, 4, and 5 and are referred to as RET(Δ) (E3) and RET(Δ) (E345), respectively. Here we report the presence of Ret(Δ) (E3) and Ret(Δ) (E345) in zebrafish, mice, and rats and show that these transcripts are dynamically expressed throughout development of the CNS, peripheral nervous system, and kidneys. We further explore the biochemical properties of these isoforms, demonstrating that, like full-length RET, RET(ΔE3) and RET(ΔE345) are trafficked to the cell surface, interact with all four GFRα co-receptors, and have the ability to heterodimerize with full-length RET. Signaling experiments indicate that RET(ΔE3) is phosphorylated in a similar manner to full-length RET. RET(ΔE345), in contrast, displays higher baseline autophosphorylation, specifically on the catalytic tyrosine, Tyr(905), and also on one of the most important signaling residues, Tyr(1062) These data provide the first evidence for a physiologic role of these isoforms in RET pathway function.

Lipid Rafts Are Physiologic Membrane Microdomains Necessary for the Morphogenic and Developmental Functions of Glial Cell Line-Derived Neurotrophic Factor In Vivo.

Glial cell line-derived neurotrophic factor (GDNF) promotes PNS development and kidney morphogenesis via a receptor complex consisting of the glycerophosphatidylinositol (GPI)-anchored, ligand binding receptor GDNF family receptor α1 (GFRα1) and the receptor tyrosine kinase Ret. Although Ret signal transduction in vitro is augmented by translocation into lipid rafts via GFRα1, the existence and importance of lipid rafts in GDNF-Ret signaling under physiologic conditions is unresolved. A knock-in mouse was produced that replaced GFRα1 with GFRα1-TM, which contains a transmembrane (TM) domain instead of the GPI anchor. GFRα1-TM still binds GDNF and promotes Ret activation but does not translocate into rafts. In Gfrα1(TM/TM) mice, GFRα1-TM is expressed, trafficked, and processed at levels identical to GFRα1. Although Gfrα1(+/TM) mice are viable, Gfrα1(TM/TM) mice display bilateral renal agenesis, lack enteric neurons in the intestines, and have motor axon guidance deficits, similar to Gfrα1(-/-) mice. Therefore, the recruitment of Ret into lipid rafts by GFRα1 is required for the physiologic functions of GDNF in vertebrates. Significance statement: Membrane microdomains known as lipid rafts have been proposed to be unique subdomains in the plasma membrane that are critical for the signaling functions of multiple receptor complexes. Their existence and physiologic relevance has been debated. Based on in vitro studies, lipid rafts have been reported to be necessary for the function of the Glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors. The receptor for GDNF comprises the lipid raft-resident, glycerophosphatidylinositol-anchored receptor GDNF family receptor α1 (GFRα1) and the receptor tyrosine kinase Ret. Here we demonstrate, using a knock-in mouse model in which GFRα1 is no longer located in lipid rafts, that the developmental functions of GDNF in the periphery require the translocation of the GDNF receptor complex into lipid rafts.

CD2-associated protein (CD2AP) enhances casitas B lineage lymphoma-3/c (Cbl-3/c)-mediated Ret isoform-specific ubiquitination and degradation via its amino-terminal Src homology 3 domains.

Ret is the receptor tyrosine kinase for the glial cell line-derived neurotrophic factor (GDNF) family of neuronal growth factors. Upon activation by GDNF, Ret is rapidly polyubiquitinated and degraded. This degradation process is isoform-selective, with the longer Ret51 isoform exhibiting different degradation kinetics than the shorter isoform, Ret9. In sympathetic neurons, Ret degradation is induced, at least in part, by a complex consisting of the adaptor protein CD2AP and the E3-ligase Cbl-3/c. Knockdown of Cbl-3/c using siRNA reduced the GDNF-induced ubiquitination and degradation of Ret51 in neurons and podocytes, suggesting that Cbl-3/c was a predominant E3 ligase for Ret. Coexpression of CD2AP with Cbl-3/c augmented the ubiquitination of Ret51 as compared with the expression of Cbl-3/c alone. Ret51 ubiquitination by the CD2AP·Cbl-3/c complex required a functional ring finger and TKB domain in Cbl-3/c. The SH3 domains of CD2AP were sufficient to drive the Cbl-3/c-dependent ubiquitination of Ret51, whereas the carboxyl-terminal coiled-coil domain of CD2AP was dispensable. Interestingly, activated Ret induced the degradation of CD2AP, but not Cbl-3/c, suggesting a potential inhibitory feedback mechanism. There were only two major ubiquitination sites in Ret51, Lys(1060) and Lys(1107), and the combined mutation of these lysines almost completely eliminated both the ubiquitination and degradation of Ret51. Ret9 was not ubiquitinated by the CD2AP·Cbl-3/c complex, suggesting that Ret9 was down-regulated by a fundamentally different mechanism. Taken together, these results suggest that only the SH3 domains of CD2AP were necessary to enhance the E3 ligase activity of Cbl-3/c toward Ret51.

DLK signaling in axotomized neurons triggers complement activation and loss of upstream synapses.

Axotomized spinal motoneurons (MNs) lose presynaptic inputs following peripheral nerve injury; however, the cellular mechanisms that lead to this form of synapse loss are currently unknown. Here, we delineate a critical role for neuronal kinase dual leucine zipper kinase (DLK)/MAP3K12, which becomes activated in axotomized neurons. Studies with conditional knockout mice indicate that DLK signaling activation in injured MNs triggers the induction of phagocytic microglia and synapse loss. Aspects of the DLK-regulated response include expression of C1q first from the axotomized MN and then later in surrounding microglia, which subsequently phagocytose presynaptic components of upstream synapses. Pharmacological ablation of microglia inhibits the loss of cholinergic C boutons from axotomized MNs. Together, the observations implicate a neuronal mechanism, governed by the DLK, in the induction of inflammation and the removal of synapses.

Neurotrophic factors in the physiology of motor neurons and their role in the pathobiology and therapeutic approach to amyotrophic lateral sclerosis.

The discovery of the neurotrophins and their potent survival and trophic effects led to great enthusiasm about their therapeutic potential to rescue dying neurons in neurodegenerative diseases. The further discovery that brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and glial cell line-derived neurotrophic factor (GDNF) had potent survival-promoting activity on motor neurons led to the proposal for their use in motor neuron diseases such as amyotrophic lateral sclerosis (ALS). In this review we synthesize the literature pertaining to the role of NGF, BDNF, CNTF and GDNF on the development and physiology of spinal motor neurons, as well as the preclinical studies that evaluated their potential for the treatment of ALS. Results from the clinical trials of these molecules will also be described and, with the aid of decades of hindsight, we will discuss what can reasonably be concluded and how this information can inform future clinical development of neurotrophic factors for ALS.

Expression of axonal protein degradation machinery in sympathetic neurons is regulated by nerve growth factor.

Deficiencies in protein degradation and proteolytic function within neurons are linked to a number of neurodegenerative diseases and developmental disorders. Compartmentalized cultures of peripheral neurons were used to investigate the properties and relative abundance of the proteolytic machinery in the axons and cell bodies of sympathetic and sensory neurons. Immunoblotting of axonal proteins demonstrated that LAMP2, LC3, and PSMA2 were abundant in axons, suggesting that lysosomes, autophagosomes and proteasomes were located in axons. Interestingly, the expression of proteins associated with lysosomes and proteasomes were upregulated selectively in axons by NGF stimulation of the distal axons of sympathetic neurons, suggesting that axonal growth and maintenance requires local protein turnover. The regulation of the abundance of both proteasomes and lysosomes in axons by NGF provides a link between protein degradation and the trophic status of peripheral neurons. Inhibition of proteasomes located in axons resulted in an accumulation of ubiquitinated proteins in these axons. In contrast, lysosome inhibition in axons did not result in an accumulation of ubiquitinated proteins or the transferrin receptor, a transmembrane protein degraded by lysosomes. Interestingly, lysosomes were transported both retrogradely and anterogradely, so it is likely that ubiquitinated proteins that are normally destined for degradation by lysosomes in axons can be transported to the cell bodies for degradation. In summary, proteasomal degradation occurs locally, whereas proteins degraded by lysosomes can most likely either be degraded locally in axons or be transported to cell bodies for degradation.

Oral Sensory Neurons of the Geniculate Ganglion That Express Tyrosine Hydroxylase Comprise a Subpopulation That Contacts Type II and Type III Taste Bud Cells.

Oral sensory neurons of the geniculate ganglion (GG) innervate taste papillae and buds on the tongue and soft palate. Electrophysiological recordings of these neurons and fibers revealed complexity in the number of unique response profiles observed, suggesting there are several distinct neuronal subtypes. Molecular descriptions of these subpopulations are incomplete. We report here the identification of a subpopulation of GG oral sensory neurons in mice by expression of tyrosine hydroxylase (TH). TH-expressing geniculate neurons represent 10-20% of oral sensory neurons and these neurons innervate taste buds in fungiform and anterior foliate taste papillae on the surface of the tongue, as well as taste buds in the soft palate. While 35-50% of taste buds on the tongue are innervated by these TH+ neurons, 100% of soft palate taste buds are innervated. These neurons did not have extragemmal processes outside of taste buds and did not express the mechanosensory neuron-associated gene Ret, suggesting they are chemosensory and not somatosensory neurons. Within taste buds, TH-expressing fibers contacted both Type II and Type III cells, raising the possibility that they are responsive to more than one taste quality. During this analysis we also identified a rare TH+ taste receptor cell type that was found in only 12-25% of taste buds and co-expressed TRPM5, suggesting it was a Type II cell. Taken together, TH-expressing GG oral sensory neurons innervate taste buds preferentially in the soft palate and contact Type II and Type III taste bud receptor cells.

The differential axonal degradation of Ret accounts for cell-type-specific function of glial cell line-derived neurotrophic factor as a retrograde survival factor.

Glial cell line-derived neurotrophic factor (GDNF) is a neuronal growth factor critical for the development and maintenance of central and peripheral neurons. GDNF is expressed in targets of innervation and provides support to several populations of large, projection neurons. To determine whether GDNF promotes retrograde survival over long axonal distances to cell bodies, we used a compartmentalized culture system. GDNF supported only modest and transient survival of postnatal sympathetic neurons when applied to their distal axons, in contrast to dorsal root ganglion (DRG) sensory neurons in which GDNF promoted survival equally well from either distal axons or cell bodies. Ret, the receptor tyrosine kinase for GDNF, underwent rapid proteasomal degradation in the axons of sympathetic neurons. Interestingly, the level of activated Ret in DRG neurons was sustained in the axons and also appeared in the cell bodies, suggesting that Ret was not degraded in sensory axons and was retrogradely transported. Pharmacologic inhibition of proteasomes only in the distal axons of sympathetic neurons caused an accumulation of activated Ret in both the axons and cell bodies during GDNF stimulation. Furthermore, exposure of the distal axons of sympathetic neurons to both GDNF and proteasome inhibitors, but neither one alone, promoted robust survival, identical to GDNF applied directly to the cell bodies. This differential responsiveness of sympathetic and sensory neurons to target-derived GDNF was attributable to the differential expression and degradation of the Ret9 and Ret51 isoforms. Therefore, the local degradation of Ret in axons dictates whether GDNF family ligands act as retrograde survival factors.

Translatomic analysis of regenerating and degenerating spinal motor neurons in injury and ALS.

The neuromuscular junction is a synapse critical for muscle strength and coordinated motor function. Unlike CNS injuries, motor neurons mount robust regenerative responses after peripheral nerve injuries. Conversely, motor neurons selectively degenerate in diseases such as amyotrophic lateral sclerosis (ALS). To assess how these insults affect motor neurons in vivo, we performed ribosomal profiling of mouse motor neurons. Motor neuron-specific transcripts were isolated from spinal cords following sciatic nerve crush, a model of acute injury and regeneration, and in the SOD1G93A ALS model. Of the 267 transcripts upregulated after nerve crush, 38% were also upregulated in SOD1G93A motor neurons. However, most upregulated genes in injured and ALS motor neurons were context specific. Some of the most significantly upregulated transcripts in both paradigms were chemokines such as Ccl2 and Ccl7, suggesting an important role for neuroimmune modulation. Collectively these data will aid in defining pro-regenerative and pro-degenerative mechanisms in motor neurons.

Proteomic analysis of the slit diaphragm complex: CLIC5 is a protein critical for podocyte morphology and function.

Podocytes are morphologically complex cells, the junctions of which form critical elements of the final filtration barrier. Disruption of their foot processes and slit diaphragms occur early in the development of many glomerular diseases. Here, we biochemically purified fractions enriched with slit diaphragm proteins and performed a proteomic analysis to identify new components of this important structure. Several known slit diaphragm proteins were found, such as podocin and nephrin, confirming the validity of the purification scheme. However, proteins on the apical membrane such as podocalyxin were neither enriched nor identified in our analysis. The chloride intracellular channel protein 5 (CLIC5), predominantly expressed in podocytes, was enriched in these fractions and localized in the foot process apical and basal membranes. CLIC5 colocalized and associated with the ezrin/radixin/moesin complex and with podocalyxin in podocytes in vivo. It is important to note that CLIC5(-/-) mice were found to have significantly decreased foot process length, widespread foot process abnormalities, and developed proteinuria. The ezrin/radixin/moesin complex and podocalyxin were significantly decreased in podocytes from CLIC5(-/-) mice. Thus, our study identifies CLIC5 as a new component that is enriched in and necessary for foot process integrity and podocyte function in vivo.

Necroptosis is SARMful to your health.

Necroptosis is a cell death pathway involved in inflammation and disease. In this issue, Ko et al. (2020. J. Cell Biol.https://doi.org/10.1083/jcb.201912047) link SARM1, the executioner of Wallerian degeneration of axons, to necroptosis, revealing a unique form of axonal disassembly likely involved in neurodegenerative disorders.

Cell non-autonomous requirement of p75 in the development of geniculate oral sensory neurons.

During development of the peripheral taste system, oral sensory neurons of the geniculate ganglion project via the chorda tympani nerve to innervate taste buds in fungiform papillae. Germline deletion of the p75 neurotrophin receptor causes dramatic axon guidance and branching deficits, leading to a loss of geniculate neurons. To determine whether the developmental functions of p75 in geniculate neurons are cell autonomous, we deleted p75 specifically in Phox2b + oral sensory neurons (Phox2b-Cre; p75fx/fx) or in neural crest-derived cells (P0-Cre; p75fx/fx) and examined geniculate neuron development. In germline p75-/- mice half of all geniculate neurons were lost. The proportion of Phox2b + neurons, as compared to Phox2b-pinna-projecting neurons, was not altered, indicating that both populations were affected similarly. Chorda tympani nerve recordings demonstrated that p75-/- mice exhibit profound deficits in responses to taste and tactile stimuli. In contrast to p75-/- mice, there was no loss of geniculate neurons in either Phox2b-Cre; p75fx/fx or P0-Cre; p75fx/fx mice. Electrophysiological analyses demonstrated that Phox2b-Cre; p75fx/fx mice had normal taste and oral tactile responses. There was a modest but significant loss of fungiform taste buds in Phox2b-Cre; p75fx/fx mice, although there was not a loss of chemosensory innervation of the remaining fungiform taste buds. Overall, these data suggest that the developmental functions of p75 are largely cell non-autonomous and require p75 expression in other cell types of the chorda tympani circuit.

CD2AP and Cbl-3/Cbl-c constitute a critical checkpoint in the regulation of ret signal transduction.

The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are critical for nervous system development and maintenance. GFLs promote survival and growth via activation of the receptor tyrosine kinase (RTK) Ret. In sympathetic neurons, the duration of Ret signaling is governed by how rapidly Ret is degraded after its activation. In an effort to elucidate mechanisms that control the half-life of Ret, we have identified two novel Ret interactors, CD2-associated protein (CD2AP) and Cbl-3. CD2AP, an adaptor molecule involved in the internalization of ubiquitinated RTKs, is associated with Ret under basal, unstimulated conditions in neurons. After Ret activation by GDNF, CD2AP dissociates. Similarly, the E3-ligase Cbl-3 interacts with unphosphorylated Ret and dissociates from Ret after Ret activation. In contrast to their dissociation from autophosphorylated Ret, an interaction between CD2AP and Cbl-3 is induced by GDNF stimulation of sympathetic neurons, suggesting that CD2AP and Cbl-3 dissociate from Ret as a complex. In neurons, the overexpression of CD2AP enhances the degradation of Ret and inhibits GDNF-dependent survival, and gene silencing of CD2AP blocks Ret degradation and promotes GDNF-mediated survival. Surprisingly, Cbl-3 overexpression dramatically stabilizes activated Ret and enhances neuronal survival, even though Cbl-family E3 ligases normally function to trigger RTK downregulation. In combination with CD2AP, however, Cbl-3 promotes Ret degradation rapidly and almost completely blocks survival promotion by GDNF, suggesting that Cbl-3 acts as a switch that is triggered by CD2AP and oscillates between inhibition and promotion of Ret degradation. Consistent with the hypothesis, Cbl-3 silencing in neurons only inhibited Ret degradation and enhanced neuronal survival in combination with CD2AP silencing. CD2AP and Cbl-3, therefore, constitute a checkpoint that controls the extent of Ret downregulation and, thereby, the sensitivity of neurons to GFLs.