A conserved function in phosphatidylinositol metabolism for mammalian Vps13 family proteins.

The Vps13 protein family is highly conserved in eukaryotic cells. In humans, mutations in the gene encoding the family member VPS13A lead to the neurodegenerative disorder chorea-acanthocytosis. In the yeast Saccharomyces cerevisiae, there is just a single version of VPS13, thereby simplifying the task of unraveling its molecular function(s). While VPS13 was originally identified in yeast by its role in vacuolar sorting, recent studies have revealed a completely different function for VPS13 in sporulation, where VPS13 regulates phosphatidylinositol-4-phosphate (PtdIns(4)P) levels in the prospore membrane. This discovery raises the possibility that the disease phenotype associated with vps13A mutants in humans is due to misregulation of PtdIns(4)P in membranes. To determine whether VPS13A affects PtdIns(4)P in membranes from mammalian neuronal cells, phosphatidylinositol phosphate pools were compared in PC12 tissue culture cells in the absence or presence of VPS13A. Consistent with the yeast results, the localization of PtdIns(4)P is specifically altered in VPS13A knockdown cells while other phosphatidylinositol phosphates appear unaffected. In addition, VPS13A is necessary to prevent the premature degeneration of neurites that develop in response to Nerve Growth Factor. The regulation of PtdIns(4)P is therefore a conserved function of the Vps13 family and may play a role in the maintenance of neuronal processes in mammals.

Recruitment of actin modifiers to TrkA endosomes governs retrograde NGF signaling and survival.

The neurotrophins NGF and NT3 collaborate to support development of sympathetic neurons. Although both promote axonal extension via the TrkA receptor, only NGF activates retrograde transport of TrkA endosomes to support neuronal survival. Here, we report that actin depolymerization is essential for initiation of NGF/TrkA endosome trafficking and that a Rac1-cofilin signaling module associated with TrkA early endosomes supports their maturation to retrograde transport-competent endosomes. These actin-regulatory endosomal components are absent from NT3/TrkA endosomes, explaining the failure of NT3 to support retrograde TrkA transport and survival. The inability of NT3 to activate Rac1-GTP-cofilin signaling is likely due to the labile nature of NT3/TrkA complexes within the acidic environment of TrkA early endosomes. Thus, TrkA endosomes associate with actin-modulatory proteins to promote F-actin disassembly, enabling their maturation into transport-competent signaling endosomes. Differential control of this process explains how NGF but not NT3 supports retrograde survival of sympathetic neurons.

Trk retrograde signaling requires persistent, Pincher-directed endosomes.

Target-derived neurotrophins use retrogradely transported Trk-signaling endosomes to promote survival and neuronal phenotype at the soma. Despite their critical role in neurotrophin signaling, the nature and molecular composition of these endosomes remain largely unknown, the result of an inability to specifically identify the retrograde signaling entity. Using EGF-bound nanoparticles and chimeric, EGF-binding TrkB receptors, we elucidate Trk-endosomal events involving their formation, processing, retrograde transport, and somal signaling in sympathetic neurons. By comparing retrograde endosomal signaling by Trk to the related but poorly neuromodulatory EGF-receptor, we find that Trk and EGF-receptor endosomes are formed and processed by distinct mechanisms. Surprisingly, Trk and EGF-receptors are both retrogradely transported to the soma in multivesicular bodies. However, only the Trk-multivesicular bodies rely on Pincher-dependent macroendocytosis and processing. Retrograde signaling through Pincher-generated Trk-multivesicular bodies is distinctively refractory to signal termination by lysosomal processing, resulting in sustained somal signaling and neuronal gene expression.

Trk activation in the secretory pathway promotes Golgi fragmentation.

Activation of nascent receptor tyrosine kinases within the secretory pathway has been reported, yet the consequences of intracellular activation are largely unexplored. We report that overexpression of the Trk neurotrophin receptors causes accumulation of autoactivated receptors in the ER-Golgi intermediate compartment. Autoactivated receptors exhibit inhibited Golgi-mediated processing and they inhibit Golgi-mediated processing of other co-expressed transmembrane proteins, apparently by inducing fragmentation of the Golgi apparatus. Signaling from G protein-coupled receptors is known to induce Trk transactivation. Transactivation of nascent TrkB in hippocampal neurons resulting from exposure to the neuropeptide PACAP caused Golgi fragmentation, whereas BDNF-dependent activation of TrkB did not. TrkB-mediated Golgi fragmentation employs a MEK-dependent signaling pathway resembling that implicated in regulation of Golgi fragmentation in mitotic cells. Neuronal Golgi fragments, in the form of dendritically localized Golgi outposts, are important determinants of dendritic growth and branching. The capacity of transactivated TrkB to enhance neuronal Golgi fragmentation may represent a novel mechanism regulating neural plasticity.

Pincher-generated Nogo-A endosomes mediate growth cone collapse and retrograde signaling.

Nogo-A is one of the most potent myelin-associated inhibitors for axonal growth, regeneration, and plasticity in the adult central nervous system. The Nogo-A-specific fragment NogoDelta20 induces growth cone collapse, and inhibits neurite outgrowth and cell spreading by activating RhoA. Here, we show that NogoDelta20 is internalized into neuronal cells by a Pincher- and rac-dependent, but clathrin- and dynamin-independent, mechanism. Pincher-mediated macroendocytosis results in the formation of NogoDelta20-containing signalosomes that direct RhoA activation and growth cone collapse. In compartmentalized chamber cultures, NogoDelta20 is endocytosed into neurites and retrogradely transported to the cell bodies of dorsal root ganglion neurons, triggering RhoA activation en route and decreasing phosphorylated cAMP response element binding levels in cell bodies. Thus, Pincher-dependent macroendocytosis leads to the formation of Nogo-A signaling endosomes, which act both within growth cones and after retrograde transport in the cell body to negatively regulate the neuronal growth program.

Identification of a developmentally regulated pathway of membrane retrieval in neuronal growth cones.

The growth-cone plasma membrane constantly reconfigures during axon navigation and upon target recognition. The identity and regulation of the membrane pathway(s) participating in remodeling of the growth-cone surface remain elusive. Here, we identify a constitutive, high-capacity plasma-membrane-recycling activity in the axonal growth cones, which is mediated by a novel bulk endocytic pathway that is mechanistically related to macropinocytosis. This pathway generates large compartments at sites of intense actin-based membrane ruffling through the actions of phosphatidylinositol 3-kinase, the small GTPase Rac1 and the pinocytic chaperone Pincher. At early developmental stages, bulk endocytosis is the primary endocytic pathway for rapid retrieval of the growth-cone plasma membrane. At later stages, during the onset of synaptogenesis, an intrinsic program of maturation leads to downregulation of basal bulk endocytosis and the emergence of depolarization-induced synaptic-vesicle exo-endocytosis. We propose that the control of bulk membrane retrieval contributes to the homeostatic regulation of the axonal plasma membrane and to growth-cone remodeling during axonal outgrowth. In addition, we suggest that the downregulation of bulk endocytosis during synaptogenesis might contribute to the preservation of synaptic-vesicle specificity.

Trk-signaling endosomes are generated by Rac-dependent macroendocytosis.

Why neurotrophins and their Trk receptors promote neuronal differentiation and survival whereas receptor tyrosine kinases for other growth factors, such as EGF, do not, has been a long-standing question in neurobiology. We provide evidence that one difference lies in the selective ability of Trk to generate long-lived signaling endosomes. We show that Trk endocytosis is distinguished from the classical clathrin-based endocytosis of EGF receptor (EGFR). Although Trk and EGFR each stimulate membrane ruffling, only Trk undergoes both selective and specific macroendocytosis at ruffles, which uniquely requires the Rho-GTPase, Rac, and the trafficking protein, Pincher. This process leads to Trk-signaling endosomes, which are immature multivesicular bodies that retain Rab5. In contrast, EGFR endosomes rapidly exchange Rab5 for Rab7, thereby transiting into late-endosomes/lysosomes for degradation. Sustained endosomal signaling by Trk does not reflect intrinsic differences between Trk and EGFR, because each elicits long-term Erk-kinase activation from the cell surface. Thus, a population of stable Trk endosomes, formed by specialized macroendocytosis in neurons, provides a privileged endosome-based system for propagation of signals to the nucleus.

Regulation of ras signaling dynamics by Sos-mediated positive feedback.

The RTK-Ras-ERK cascade is a central signaling module implicated in the control of diverse biological processes including cell proliferation, differentiation, and survival. The coupling of RTK to Ras is mediated by the Ras-specific nucleotide-exchange factor Son of Sevenless (Sos), which activates Ras by inducing the exchange of GDP for GTP . Considerable evidence indicates that the duration and amplitude of Ras signals are important determinants in controlling the biological outcome . However, the mechanisms that regulate the quantitative output of Ras signaling remain poorly understood. We define a previously unrecognized regulatory component of the machinery that specifies the kinetic properties of signals propagated through the RTK-Ras-ERK cascade. We demonstrate that the establishment of a positive feedback loop involving Ras.GTP and Sos leads to an increase in the amplitude and duration of Ras activation in response to EGF stimulation. This effect is propagated to downstream elements of the pathway as reflected by sustained EGF-induced ERK phosphorylation and enhanced SRE-dependent transcription. As a consequence, the physiological endpoint of EGF action is switched from proliferation to differentiation. We propose that the engagement of Ras/Sos positive feedback loop may contribute to the mechanism by which ligand stimulation is coupled to discrete biological responses.

Pincher-mediated macroendocytosis underlies retrograde signaling by neurotrophin receptors.

Retrograde signaling by neurotrophins is crucial for regulating neuronal phenotype and survival. The mechanism responsible for retrograde signaling has been elusive, because the molecular entities that propagate Trk receptor tyrosine kinase signals from the nerve terminal to the soma have not been defined. Here, we show that the membrane trafficking protein Pincher defines the primary pathway responsible for neurotrophin retrograde signaling in neurons. By both immunofluorescence confocal and immunoelectron microscopy, we find that Pincher mediates the formation of newly identified clathrin-independent macroendosomes for Trk receptors in soma, axons, and dendrites. Trk macroendosomes are derived from plasma membrane ruffles and subsequently processed to multivesicular bodies. Pincher similarly mediates macroendocytosis for NGF (TrkA) and BDNF (TrkB) in both peripheral (sympathetic) and central (hippocampal) neurons. A unique feature of Pincher-Trk endosomes is refractoriness to lysosomal degradation, which ensures persistent signaling through a critical effector of retrograde survival signaling, Erk5 (extracellular signal-regulated kinase 5). Using sympathetic neurons grown in chamber cultures, we find that block of Pincher function, which prevents Trk macroendosome formation, eliminates retrogradely signaled neuronal survival. Pincher is the first distinguishing molecular component of a novel mechanistic pathway for endosomal signaling in neurons.

Pincher, a pinocytic chaperone for nerve growth factor/TrkA signaling endosomes.

A central tenet of nerve growth factor (NGF) action that is poorly understood is its ability to mediate cytoplasmic signaling, through its receptor TrkA, that is initiated at the nerve terminal and conveyed to the soma. We identified an NGF-induced protein that we termed Pincher (pinocytic chaperone) that mediates endocytosis and trafficking of NGF and its receptor TrkA. In PC12 cells, overexpression of Pincher dramatically stimulated NGF-induced endocytosis of TrkA, unexpectedly at sites of clathrin-independent macropinocytosis within cell surface ruffles. Subsequently, a system of Pincher-containing tubules mediated the delivery of NGF/TrkA-containing vesicles to cytoplasmic accumulations. These vesicles selectively and persistently mediated TrkA-erk5 mitogen-activated protein kinase signaling. A dominant inhibitory mutant form of Pincher inhibited the NGF-induced endocytosis of TrkA, and selectively blocked TrkA-mediated cytoplasmic signaling of erk5, but not erk1/2, kinases. Our results indicate that Pincher mediates pinocytic endocytosis of functionally specialized NGF/TrkA endosomes with persistent signaling potential.