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The microtubule-stabilizing chemotherapy drug paclitaxel (PTX) causes dose-limiting chemotherapy-induced peripheral neuropathy (CIPN), which is often accompanied by pain. Among the multifaceted effects of PTX is an increased expression of sodium channel Nav1.7 in rat and human sensory neurons, enhancing their excitability. However, the mechanisms underlying this increased Nav1.7 expression have not been explored, and the effects of PTX treatment on the dynamics of trafficking and localization of Nav1.7 channels in sensory axons have not been possible to investigate to date. In this study we used a recently developed live imaging approach that allows visualization of Nav1.7 surface channels and long-distance axonal vesicular transport in sensory neurons to fill this basic knowledge gap. We demonstrate concentration and time-dependent effects of PTX on vesicular trafficking and membrane localization of Nav1.7 in real-time in sensory axons. Low concentrations of PTX increase surface channel expression and vesicular flux (number of vesicles per axon). By contrast, treatment with a higher concentration of PTX decreases vesicular flux. Interestingly, vesicular velocity is increased for both concentrations of PTX. Treatment with PTX increased levels of endogenous Nav1.7 mRNA and current density in dorsal root ganglion neurons. However, the current produced by transfection of dorsal root ganglion neurons with Halo-tag Nav1.7 was not increased after exposure to PTX. Taken together, this suggests that the increased trafficking and surface localization of Halo-Nav1.7 that we observed by live imaging in transfected dorsal root ganglion neurons after treatment with PTX might be independent of an increased pool of Nav1.7 channels. After exposure to inflammatory mediators to mimic the inflammatory condition seen during chemotherapy, both Nav1.7 surface levels and vesicular transport are increased for both low and high concentrations of PTX. Overall, our results show that PTX treatment increases levels of functional endogenous Nav1.7 channels in dorsal root ganglion neurons and enhances trafficking and surface distribution of Nav1.7 in sensory axons, with outcomes that depend on the presence of an inflammatory milieu, providing a mechanistic explanation for increased excitability of primary afferents and pain in CIPN.

The peripheral nervous system responds to a wide variety of sensory stimuli, a process that requires great neuronal diversity. These diverse peripheral sensory neurons are closely associated with glial cells that originate from the neural crest (NC). However, the molecular nature and origins of diversity among peripheral glia is not understood. Here we used single cell RNA sequencing to profile and compare developing and mature glia from somatosensory lumbar dorsal root ganglia (DRG) and auditory spiral ganglia (SG). We found that the glial precursors (GPs) differ in their transcriptional profile and prevalence in these two systems. Despite their unique features, somatosensory and auditory GPs undergo convergent differentiation to generate myelinating and non-myelinating Schwann cells that are molecularly uniform. By contrast, although satellite glia surround the neuronal cell bodies in both ganglia, we found that those in the SG express multiple myelination-associated genes, while DRG satellite cells express components that suppress myelination. Lastly, we identified a set of glial signature genes that are also expressed by placode-derived supporting cells, providing new insights into commonalities among glia across the nervous system. This comprehensive survey of gene expression in peripheral glia constitutes a valuable resource for understanding how glia acquire specialized functions and how their roles differ across sensory modalities.

The peripheral nervous system responds to a wide variety of sensory stimuli, a process that requires great neuronal diversity. These diverse neurons are closely associated with glial cells originating from the neural crest. However, the molecular nature and diversity among peripheral glia are not understood. Here, we used single-cell RNA sequencing to profile developing and mature glia from somatosensory dorsal root ganglia and auditory spiral ganglia. We found that glial precursors (GPs) in these two systems differ in their transcriptional profiles. Despite their unique features, somatosensory and auditory GPs undergo convergent differentiation to generate molecularly uniform myelinating and non-myelinating Schwann cells. By contrast, somatosensory and auditory satellite glial cells retain system-specific features. Lastly, we identified a glial signature gene set, providing new insights into commonalities among glia across the nervous system. This survey of gene expression in peripheral glia constitutes a resource for understanding functions of glia across different sensory modalities.

Background. Astrocytomas are the most common primary human brain tumors. Receptor tyrosine kinases (RTKs), including tyrosine receptor kinase B (TrkB, also known as tropomyosin-related kinase B; encoded by neurotrophic tyrosine kinase receptor type 2 [NTRK2]), are frequently mutated by rearrangement/fusion in high-grade and low-grade astrocytomas. We found that activated TrkB can contribute to the development of astrocytoma and might serve as a therapeutic target in this tumor type. Methods. To identify RTKs capable of inducing astrocytoma formation, a library of human tyrosine kinases was screened for the ability to transform murine Ink4a−/−/Arf−/− astrocytes. Orthotopic allograft studies were conducted to evaluate the effects of RTKs on the development of astrocytoma. Since TrkB was identified as a driver of astrocytoma formation, the effect of the Trk inhibitors AZD1480 and RXDX-101 was assessed in astrocytoma cells expressing activated TrkB. RNA sequencing, real-time PCR, western blotting, and enzyme-linked immunosorbent assays were conducted to characterize NTRK2 in astrocytomas. Results. Activated TrkB cooperated with Ink4a/Arf loss to induce the formation of astrocytomas through a mechanism mediated by activation of signal transducer and activator of transcription 3 (STAT3). TrkB activation positively correlated with Ccl2 expression. TrkB-induced astrocytomas remained dependent on TrkB signaling for survival, highlighting a role of NTRK2 as an addictive oncogene. Furthermore, the QKI-NTRK2 fusion associated with human astrocytoma transformed Ink4a−/−/Arf−/− astrocytes, and this process was also mediated via STAT3 signaling. Conclusions. Our findings provide evidence that constitutively activated NTRK2 alleles, notably the human tumor-associated QKI-NTRK2 fusion, can cooperate with Ink4a/Arf loss to drive astrocytoma formation. Therefore, we propose NTRK2 as a potential therapeutic target in the subset of astrocytoma patients defined by QKI-NTRK2 fusion.

To achieve accurate spatiotemporal patterns of gene expression, RNA-binding proteins (RBPs) guide nuclear processing, intracellular trafficking, and local translation of target mRNAs. In neurons, RBPs direct transport of target mRNAs to sites of translation in remote axons and dendrites. However, it is not known whether an individual RBP coordinately regulates multiple mRNAs within these morphologically complex cells. Here we identify SFPQ (Splicing factor, proline-glutamine rich) as an RBP that binds and regulates multiple mRNAs in dorsal root ganglion (DRG) sensory neurons and thereby promotes neurotrophin-dependent axonal viability. SFPQ acts within nuclei, cytoplasm and axons to regulate multiple functionally related mRNAs essential for axon survival. Notably SFPQ is required for co-assembly of laminb2 and bclw within RNA granules and for axonal trafficking of these mRNAs. Together these data demonstrate that SFPQ orchestrates spatial gene expression of a newly identified RNA regulon essential for axonal viability.

Establishment of neuronal circuitry depends on both formation and refinement of neural connections. During this process, target-derived neurotrophins regulate both transcription and translation to enable selective axon survival or elimination. However, it is not known whether retrograde signaling pathways that control transcription are coordinated with neurotrophin-regulated actions that transpire in the axon. Here we report that target-derived neurotrophins coordinate transcription of the antiapoptotic genebclwwith transport ofbclwmRNA to the axon, and thereby prevent axonal degeneration in rat and mouse sensory neurons. We show that neurotrophin stimulation of nerve terminals elicits newbclwtranscripts that are immediately transported to the axons and translated into protein. Bclw interacts with Bax and suppresses the caspase6 apoptotic cascade that fosters axonal degeneration. The scope ofbclwregulation at the levels of transcription, transport, and translation provides a mechanism whereby sustained neurotrophin stimulation can be integrated over time, so that axonal survival is restricted to neurons connected within a stable circuit.

Neurons are highly polarized cells with axonal and dendritic projections that extend over long distances. Target-derived neurotrophins provide local axonal cues that function in developing neurons, while physical or chemical injuries to long axons initiate local environmental cues in mature neurons. In both instances initial responses at the location of stimulation or injury must be coordinated with changes in the transcriptional program and subsequent changes in axonal protein content. To achieve this coordination, intracellular signals move 'there and back again' between axons and the nucleus. Here, we review new findings on neuronal responses to growth factors and injury and highlight the coordination of transcription, translation and transport required to mediate communication between axons and cell bodies.

How do neurons integrate intracellular communication from synapse to nucleus and back? Here we briefly summarize aspects of this topic covered by a symposium at Neuroscience 2011. A rich repertoire of signaling mechanisms link both dendritic terminals and axon tips with neuronal soma and nucleus, using motor-dependent transport machineries to traverse the long intracellular distances along neuronal processes. Activation mechanisms at terminals include localized translation of dendritic or axonal RNA, proteolytic cleavage of receptors or second messengers, and differential phosphorylation of signaling moieties. Signaling complexes may be transported in endosomes, or as non-endosomal complexes associated with importins and dynein. Anterograde transport of RNA granules from the soma to neuronal processes, coupled with retrograde transport of proteins translated locally at terminals or within processes, may fuel ongoing bidirectional communication between soma and synapse to modulate synaptic plasticity as well as neuronal growth and survival decisions.

SummaryCell polarization is essential throughout development for proliferation, migration, and differentiation. However, it is not known how extracellular cues correctly orient cell polarity at distinct stages of development. Here, we show that the endocytic adaptor protein Numb, previously characterized for its role in cell proliferation, subsequently plays an important role in cell migration. In neural precursors stimulated with the chemotactic factor BDNF, Numb binds to activated TrkB, the BDNF receptor, and functions both as an endocytic regulator for TrkB and as a scaffold for aPKC (aPKC). Thus, Numb promotes BDNF-dependent aPKC activation. Interestingly, Numb is also a substrate of aPKC. When phosphorylated, Numb exhibits increased efficacy in binding TrkB and in promoting a chemotactic response to BDNF. Therefore, Numb functions in a feed-forward loop to promote chemotaxis of neural precursors, linking BDNF, an extracellular cue, to aPKC, a critical component of the intrinsic polarity machinery.

Small fiber sensory neuropathy is a common disorder in which progressive degeneration of small-diameter nociceptors causes decreased sensitivity to thermal stimuli and painful sensations in the extremities. In the majority of patients, the cause of small fiber sensory neuropathy is unknown, and treatment options are limited. Here, we show that Bcl-w (Bcl-2l2) is required for the viability of small fiber nociceptive sensory neurons.Bcl-w−/−mice demonstrate an adult-onset progressive decline in thermosensation and a decrease in nociceptor innervation of the epidermis. This denervation occurs without cell body loss, indicating that lack of Bcl-w results in a primary axonopathy. Consistent with this phenotype, we show that Bcl-w, in contrast to the closely related Bcl-2 and Bcl-xL, is enriched in axons of sensory neurons and that Bcl-w prevents the dying back of axons.Bcl-w−/−sensory neurons exhibit mitochondrial abnormalities, including alterations in axonal mitochondrial size, axonal mitochondrial membrane potential, and cellular ATP levels. Collectively, these data establishbcl-w−/−mice as an animal model of small fiber sensory neuropathy and provide new insight regarding the role of Bcl-w and of mitochondria in preventing axonal degeneration.

Survival and maturation of dorsal root ganglia sensory neurons during development depend on target-derived neurotrophins. These target-derived signals must be transmitted across long distances to alter gene expression. Here, we address the possibility that long-range retrograde signals initiated by target-derived neurotrophins activate a specialized transcriptional program. The transcription factor MEF2D is expressed in sensory neurons; we show that expression of this factor is induced in response to target-derived neurotrophins that stimulate the distal axons. We demonstrate that MEF2D regulates expression of an anti-apoptoticbcl-2family member,bcl-w. Expression ofmef2dandbcl-wis stimulated in response to activation of a Trk-dependent ERK5/MEF2 pathway, and our data indicate that this pathway promotes sensory neuron survival. We find thatmef2dandbcl-ware members of a larger set of retrograde response genes, which are preferentially induced by neurotrophin stimulation of distal axons. Thus, activation of an ERK5/MEF2D transcriptional program establishes and maintains the cellular constituents of functional sensory circuits.

Cytoplasmic dynein is the multisubunit motor protein for retrograde movement of diverse cargoes to microtubule minus ends. Here, we investigate the function of dynein variants, defined by different intermediate chain (IC) isoforms, by expressing fluorescent ICs in neuronal cells. Green fluorescent protein (GFP)–IC incorporates into functional dynein complexes that copurify with membranous organelles. In living PC12 cell neurites, GFP–dynein puncta travel in both the anterograde and retrograde directions. In cultured hippocampal neurons, neurotrophin receptor tyrosine kinase B (TrkB) signaling endosomes are transported by cytoplasmic dynein containing the neuron-specific IC-1B isoform and not by dynein containing the ubiquitous IC-2C isoform. Similarly, organelles containing TrkB isolated from brain by immunoaffinity purification also contain dynein with IC-1 but not IC-2 isoforms. These data demonstrate that the IC isoforms define dynein populations that are selectively recruited to transport distinct cargoes.

SummaryDuring development, neural precursors migrate in response to positional cues such as growth factor gradients. However, the mechanisms that enable precursors to sense and respond to such gradients are poorly understood. Here we show that cerebellar granule cell precursors (GCPs) migrate along a gradient of brain-derived neurotrophic factor (BDNF), and we demonstrate that vesicle trafficking is critical for this chemotactic process. Activation of TrkB, the BDNF receptor, stimulates GCPs to secrete BDNF, thereby amplifying the ambient gradient. The BDNF gradient stimulates endocytosis of TrkB and associated signaling molecules, causing asymmetric accumulation of signaling endosomes at the subcellular location where BDNF concentration is maximal. Thus, regulated BDNF exocytosis and TrkB endocytosis enable precursors to polarize and migrate in a directed fashion along a shallow BDNF gradient.

It is generally believed that cerebellar granule neurons originate exclusively from granule neuron precursors (GNPs) in the external germinal layer (EGL). Here we identify a rare population of neuronal progenitors in mouse developing cerebellum that expresses Nestin. Although Nestin is widely considered a marker for multipotent stem cells, these Nestin-expressing progenitors (NEPs) are committed to the granule neuron lineage. Unlike conventional GNPs, which reside in the outer EGL and proliferate extensively, NEPs reside in the deep part of the EGL and are quiescent. Expression profiling reveals that NEPs are distinct from GNPs, and in particular, express markedly reduced levels of genes associated with DNA repair. Consistent with this, upon aberrant activation of Sonic hedgehog (Shh) signaling, NEPs exhibit more severe genomic instability and give rise to tumors more efficiently than GNPs. These studies identify a novel progenitor for cerebellar granule neurons and a novel cell of origin for medulloblastoma.

Neurotrophins were initially identified as critical regulators of neuronal survival. However, these factors have many additional functions. In the developing cerebellum the roles of the neurotrophins BDNF and NT3 include a surprising effect on patterning, as revealed by changes in foliation in neurotrophin-deficient mice. Here we examine the potential role of p75NTR in cerebellar development and patterning. We show that p75NTR is expressed at highest levels in the region of the cerebellum where foliation is altered in BDNF and NT3 mutants. Although the cerebellar phenotype of p75NTR mutant animals is indistinguishable from wild type, mutation of p75NTR in BDNF heterozygotes results in defects in foliation and in Purkinje cell morphologic development. Taken together, these data suggest that p75NTR activity is critical for cerebellar development under pathologic circumstances where neurotrophin levels are reduced.

Target-derived neurotrophins are required for the growth and survival of innervating neurons. When released by postsynaptic targets, neurotrophins bind receptors (Trks) on nerve terminals. Activated Trks signal locally within distal axons and retrogradely through long axons to distant cell bodies in order to promote gene expression and survival. Although the mechanism of retrograde neurotrophin signaling is not fully elucidated, considerable evidence supports a model in which the vesicular transport of neurotrophin-Trk complexes transmits a survival signal that involves PI3K and Erk5. Other, non-vesicular modes of retrograde signaling are likely to function in parallel. Recent studies highlight the importance of the location of stimulation as a determinant of Trk signaling. Defects in signaling from distal axons to cell bodies may be causally related to neurodegenerative disorders.

During development of the nervous system, neural progenitors arise in proliferative zones, then exit the cell cycle and migrate away from these zones. Here we show that migration of cerebellar granule cells out of their proliferative zone, the external granule cell layer (EGL), is impaired in Bdnf–/– mice. The reason for impaired migration is that BDNF directly and acutely stimulates granule cell migration. Purified Bdnf–/– granule cells show defects in initiation of migration along glial fibers and in Boyden chamber assays. This phenotype can be rescued by exogenous BDNF. Using time-lapse video microscopy we find that BDNF is acutely motogenic as it stimulates migration of individual granule cells immediately after addition. The stimulation of migration reflects both a chemokinetic and chemotactic effect of BDNF. Collectively, these data demonstrate that BDNF is directly motogenic for granule cells and provides a directional cue promoting migration from the EGL to the internal granule cell layer (IGL).Movies available on-line

Neurotrophins were originally identified as target-derived factors that regulate the survival and differentiation of innervating neurons. However, neurotrophins can also be released by presynaptic cells to stimulate postsynaptic neurons. Recent studies indicate that differences exist between the signaling pathways activated by neurotrophin stimulation of nerve terminals (retrograde signaling) and neurotrophin stimulation of cell bodies. Retrograde signaling relies on the formation of signaling endosomes, vesicles containing activated Trk receptors and their ligands. Signaling endosomes travel from the nerve terminals to remote cell bodies, where they selectively activate a novel MAP kinase, Erk5, as well as PI3 kinase, and thereby stimulate neuronal survival. The differences in the signaling pathways activated by neurotrophins, which depends on the location of stimulation, provide a mechanism by which neurons can interpret the 'where' as well as the 'what' of growth factor stimulation.

Neurotrophins are key regulators of neuronal survival and function. Here we show that TrkB, the receptor for brain-derived neurotrophic factor (BDNF), is located at parallel fiber to Purkinje cell (PF/PC) synapses of the cerebellum. To determine the effects of TrkB receptor activation on synapse formation and function, we examined the parallel fiber to Purkinje cell synapses of mice with a targeted deletion of the BDNF gene. Although Purkinje cell dendrites are abnormal in BDNF −/− mice, PF/PC synapses are still able to form. Immunohistochemical analysis of mutant animals revealed the formation of numerous PF/PC synapses with the appropriate apposition of presynaptic and postsynaptic proteins. These synapses are functional, and no differences were detected in the waveform of evoked EPSCs, the amplitude of spontaneous mini-EPSCs, or the response to prolonged 10 Hz stimulus trains. However, paired-pulse facilitation, a form of short-term plasticity, is significantly decreased in BDNF −/− mice. Detailed ultrastructural analysis of the presynaptic terminals demonstrated that this change in synaptic function is accompanied by an increase in the total number of synaptic vesicles in mutant mice and a decrease in the proportion of vesicles that are docked. These data suggest that BDNF regulates both the mechanisms that underlie short-term synaptic plasticity and the steady-state relationship between different vesicle pools within the terminal.

During development, neural circuits are initially generated by exuberant innervation and are rapidly refined by selective preservation and elimination of axons. The establishment and maintenance of functional circuits therefore requires coordination of axon survival and degeneration pathways. Both developing and mature circuits rely on interdependent mitochondrial and cytoskeletal components to maintain axonal health and homeostasis; injury or diseases that impinge on these components frequently cause pathologic axon loss. Here, we review recent findings that identify mechanisms of axonal preservation in the contexts of development, injury, and disease.

The distinctive morphology of neurons, with complex dendritic arbors and extensive axons, presents spatial challenges for intracellular signal transduction. The endosomal system provides mechanisms that enable signaling molecules initiated by extracellular cues to be trafficked throughout the expanse of the neuron, allowing intracellular signals to be sustained over long distances. Therefore endosomes are critical for many aspects of neuronal signaling that regulate cell survival, axonal growth and guidance, dendritic branching, and cell migration. An intriguing characteristic of neuronal signal transduction is that endosomal trafficking enables physiological responses that vary based on the subcellular location of signal initiation. In this review, we will discuss the specialized mechanisms and the functional significance of endosomal signaling in neurons, both during normal development and in disease.

In contrast to conventional signaling by growth factors that requires their continual presence, a 1-min pulse of nerve growth factor (NGF) is sufficient to induce electrical excitability in PC12 cells due to induction of the peripheral nerve type 1 (PN1) sodium channel gene. We have investigated the mechanism for this triggered signaling pathway by NGF in PC12 cells. Mutation of TrkA at key autophosphorylation sites indicates an essential role for the phospholipase C-gamma (PLC-gamma) binding site, but not the Shc binding site, for NGF-triggered induction of PN1. In concordance with results with Trk mutants, drug-mediated inhibition of PLC-gamma activity also blocks PN1 induction by NGF. Examination of the kinetics of TrkA autophosphorylation indicates that triggered signaling does not result from sustained activation and autophosphorylation of the TrkA receptor kinase, whose phosphorylation state declines rapidly after NGF removal. Rather, TrkA triggers an unexpectedly prolonged phosphorylation and activation of PLC-gamma signaling that is sustained for up to 2 h. Prevention of the elevation of intracellular Ca2+ levels using BAPTA-AM results in a block of PN1 induction by NGF. Sustained signaling by PLC-gamma provides a means for differential neuronal gene induction after transient exposure to NGF.

A new localization element in the 3′ untranslated region of the IMPA1 mRNA enables its NGF-dependent targeting to sympathetic axons, suggesting that high local inositol levels are required for axon growth and maintenance.

Target-derived neurotrophins initiate signals that begin at nerve terminals and cross long distances to reach the cell bodies and regulate gene expression. Neurotrophin receptors, Trks, themselves serve as retrograde signal carriers. However, it is not yet known whether the retrograde propagation of Trk activation reflects movement of Trk receptors from neurites to cell bodies or reflects serial activation of stationary Trk molecules. Here, we show that neurotrophins selectively applied to distal neurites of sensory neurons rapidly induce phosphorylation of the transcription factor cAMP response element-binding protein (CREB) and also cause a slower increase in Fos protein expression. Both nuclear responses require activation of neurotrophin receptors (Trks) at distal nerve endings and retrograde propagation of Trk activation to the nerve cell bodies. Using photobleach and recovery techniques to follow biologically active, green fluorescent protein (GFP)-tagged BDNF receptors (TrkB-GFP) in live cells during retrograde signaling, we show that TrkB-GFP moves rapidly from neurites to the cell bodies. This rapid movement requires ligand binding, Trk kinase activity, and intact axonal microtubules. When they reach the cell bodies, the activated TrkB receptors are in a complex with ligand. Thus, the retrograde propagation of activated TrkB from neurites to cell bodies, although rapid, reflects microtubule-dependent transport of phosphorylated Trk-ligand complexes. Moreover, the relocation of activated Trk receptors from nerve endings to cell bodies is required for nuclear signaling responses. Together, these data support a model of retrograde signaling whereby rapid vesicular transport of ligand-receptor complex from the neurites to the cell bodies mediates the nuclear responses.

The human nerve growth factor receptor (TrkA) contains four potential N-glycosylation sites that are highly conserved within the Trk family of neurotrophin receptors, and nine additional sites that are less well conserved. Using a microscale deglycosylation assay, we show here that both conserved and variable N-glycosylation sites are used during maturation of TrkA. Glycosylation at these sites serves two distinct functions. First, glycosylation is necessary to prevent ligand-independent activation of TrkA. Unglycosylated TrkA core protein is phosphorylated even in the absence of ligand stimulation and displays constitutive kinase activity as well as constitutive interaction with the signaling molecules Shc and PLC-γ. Second, glycosylation is required to localize TrkA to the cell surface, where it can trigger the Ras/Raf/MAP kinase cascade. Using confocal microscopy, we show that unglycosylated active Trk receptors are trapped intracellularly. Furthermore, the unglycosylated active TrkA receptors are unable to activate kinases in the Ras-MAP kinase pathway, MEK and Erk. Consistent with these biochemical observations, unglycosylated TrkA core protein does not promote neuronal differentiation in Trk PC12 cells even at high levels of constitutive catalytic activity. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 323–336, 1999

During development target-derived neurotrophins promote the survival of neurons. However, mature neurons no longer depend on the target for survival. Do target-derived neurotrophins retain retrograde signaling functions in mature neurons, and, if so, how are they executed? We addressed this question by using a phosphotyrosine-directed antibody to locate activated Trk receptors in adult rat sciatic nerve. We show that catalytically active Trk receptors are located within the axon of adult rat sciatic nerve and that they are distributed throughout the length of the axons. These catalytically active receptors are phosphorylated on tyrosine at a position that couples them to the signal-generating proteins Ras and PI3 kinase. Neurotrophin applied at sciatic nerve terminals increases both catalytic activity and phosphorylation state of Trk receptors at distant points within the axons. Trk activation initiated at the nerve terminals propagates through the axon toward the nerve cell body at an initial rate that exceeds that of conventional vesicular transport. However, our data suggest that this rapid signal is nevertheless vesicle-associated. Thus, in mature nerves, activated Trk receptors function as rapid retrograde signal carriers to execute remote responses to target-derived neurotrophins.

Medulloblastomas may be derived from granule cells of the developing cerebellum. Children with tumors expressing high levels of the neurotrophin-3 receptor, TrkC, have a more favorable outcome. During development, TrkC is expressed in the most mature granule cells. Favorable medulloblastomas may be derived from more highly differentiated granule cells.

A study in this issue describes the first multipotent stem cell identified in the postnatal cerebellum. These cells can generate inhibitory interneurons, astrocytes and oligodendrocytes. They may also be responsible for a class of childhood brain tumors.

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

SummaryThe neurotrophin receptors TrkA, TrkB, and TrkC are localized at the surface of the axon terminus and transmit key signals from brain-derived neurotrophic factor (BDNF) for diverse effects on neuronal survival, differentiation, and axon formation. Trk receptors are sorted into axons via the anterograde transport of vesicles and are then inserted into axonal plasma membranes. However, the transport mechanism remains largely unknown. Here, we show that the Slp1/Rab27B/CRMP-2 complex directly links TrkB to Kinesin-1, and that this association is required for the anterograde transport of TrkB-containing vesicles. The cytoplasmic tail of TrkB binds to Slp1 in a Rab27B-dependent manner, and CRMP-2 connects Slp1 to Kinesin-1. Knockdown of these molecules by siRNA reduces the anterograde transport and membrane targeting of TrkB, thereby inhibiting BDNF-induced ERK1/2 phosphorylation in axons. Our data reveal a molecular mechanism for the selective anterograde transport of TrkB in axons and show how the transport is coupled to BDNF signaling.

Neurons extend axonal processes over long distances, necessitating efficient transport mechanisms to convey target-derived neurotrophic survival signals from remote distal axons to cell bodies. Retrograde transport, powered by dynein motors, supplies cell bodies with survival signals in the form of “signaling endosomes”. In this review, we will discuss new advances in our understanding of the motor proteins that bind to and move signaling components in a retrograde direction, and discuss mechanisms that might specify distinct neuronal responses to spatially restricted neurotrophin signals. Disruption of retrograde transport leads to a variety of neurodegenerative diseases, highlighting the role of retrograde transport of signaling endosomes for axonal maintenance and the importance of efficient transport for neuronal survival and function.

Recent studies of neurotrophin knockout mice shed light on the distinct requirements for neurotrophins in the central as compared to the peripheral nervous system. These findings reveal pleiotropic roles for neurotrophins including roles in neuronal patterning and differentiation.

Trk signaling is of widespread importance during development as it regulates the differentiation, survival and migration of multiple cell types. Similar fanctions for Trks can be found in a wide variety of cancers. These activities not only determine the biology of these cancers but also offer a potentially unique target for the control of cancer cell growth and motility through the inhibition of the Trk kinase.

Mutations that alter dynein function are associated with neurodegenerative diseases, but it is not known why defects in dynein-dependent transport impair neuronal survival. Here we show that dynein function in axons is selectively required for the survival of neurons that depend on target-derived neurotrophins. Stimulation of axon terminals with neurotrophins causes internalization of neurotrophin receptors (Trks). Using real-time imaging of fluorescently tagged Trks, we show that dynein is required for rapid transport of internalized, activated receptors from axon terminals to remote cell bodies. When dynein-based transport is inhibited, neurotrophin stimulation of axon terminals does not support survival. These studies indicate that defects in dynein-based transport reduce trafficking of activated Trks and thereby obstruct the prosurvival effect of target-derived trophic factors, leading to degeneration of target-dependent neurons.

In inhibiting neurite outgrowth, several myelin components, including the extracellular domain of Nogo-A (Nogo-66)1, oligodendrocyte myelin glycoprotein (OMgp)2 and myelin-associated glycoprotein (MAG)3,4, exert their effects through the same Nogo receptor (NgR). The glycosyl phosphatidylinositol (GPI)-anchored nature of NgR indicates the requirement for additional transmembrane protein(s) to transduce the inhibitory signals into the interior of responding neurons. Here, we demonstrate that p75, a transmembrane protein known to be a receptor for the neurotrophin family of growth factors5,6, specifically interacts with NgR. p75 is required for NgR-mediated signalling, as neurons from p75 knockout mice are no longer responsive to myelin and to each of the known NgR ligands. Blocking the p75–NgR interaction also reduces the activities of these inhibitors. Moreover, a truncated p75 protein lacking the intracellular domain, when overexpressed in primary neurons, attenuates the same set of inhibitory activities, suggesting that p75 is a signal transducer of the NgR–p75 receptor complex. Thus, interfering with p75 and its downstream signalling pathways may allow lesioned axons to overcome most of the inhibitory activities associated with central nervous system myelin.

The classic view of the neurotrophin as a messenger produced by the postsynaptic cell that signals survival to the presynaptic cell has become expanded. Segal discusses the evidence that the neurotrophin brain-derived nerve growth factor (BDNF) can be also released from the presynaptic cell onto the dendritic spine of a postsynaptic cell to regulate the neuronal circuit in two directions: forward (from presynaptic cell to postsynaptic dendrite) and the classic backward direction (from postsynaptic cell to the presynaptic axon). These results remind us that in the brain, neurons exist in a complex circuit and one neuron's presynaptic cell is another neuron's postsynaptic cell. The evidence that a single growth factor may have different actions based on the localization of the receipt of the signal adds more complexity to the brain's neuronal circuitry.

Internalization and transport of a ligand-receptor complex are required to initiate cell body responses to target-derived neurotrophin. However, it is not known whether internalized receptors and cell surface receptors initiate the same signaling pathways and biological responses. Here we use a temperature-sensitive mutant of dynamin (G273D) to control the subcellular localization of activated NGF receptors (Trks). We show that dynamin function is required for ligand-dependent endocytosis of Trk receptors. In PC12 cells, nerve growth factor (NGF) stimulation promotes both survival and neuronal differentiation. These distinct biological responses to NGF are controlled by receptors signaling from different locations within the cell. Neuronal differentiation is promoted by catalytically active Trks within endosomes in the cell interior. In contrast, survival responses are initiated by activated receptors at the cell surface where they orchestrate prolonged activation of the kinase Akt. Thus, interactions between Trk receptor tyrosine kinases and intracellular signaling molecules are dictated both by phosphotyrosine motifs within the receptors and by the intracellular location of phosphorylated receptors.

TrkA receptors are well known for promoting neuronal cell survival. However, in some neuroblastic tumors, TrkA activation can instead induce apoptosis. In this issue of Neuron , Harel et al. identify CCM2 as a mediator of TrkA-dependent cell death, suggesting that CCM2 is a distinctive type of tumor suppressor that modulates tyrosine kinase signaling.

While target-derived neurotrophins are required for the survival of developing neurons in the PNS, the functions of neurotrophins in the CNS are unclear. Mice with a targeted gene deletion of brain-derived neurotrophic factor (BDNF) exhibit a wide-based gait. Consistent with this behavioral evidence of cerebellar dysfunction, there is increased death of granule cells, stunted growth of Purkinje cell dendrites, impaired formation of horizontal layers, and defects in the rostral–caudal foliation pattern. These abnormalities are accompanied by decreased Trk activation in granule and Purkinje cells of mutant animals, indicating that both cell types are direct targets for BDNF. These data suggest that BDNF acts as an anterograde or an autocrine–paracrine factor to regulate survival and morphologic differentiation of developing CNS neurons, and thereby affects neural patterning.

We have examined the hypothesis that the segregation of LGN axon terminals into ocular dominance (OD) patches in layer 4 of the visual cortex requires neurotrophins, acting as signals to modulate the pattern of synaptic connectivity. Neurotrophin receptor antagonists, composed of the extracellular domain of each member of the trk family of neurotrophin receptors fused to a human Fc domain, were infused directly into visual cortex during the peak phase of OD column formation. Infusion of trkB–IgG, which binds BDNF and NT-4/5, inhibited the formation of OD patches within layer 4, while trkA–IgG and trkC–IgG, which preferentially bind NGF and NT-3, respectively, had no effect. The autoradiographic labeling of LGN terminals in cortical layer 4 was reduced by trkB–IgG, in contrast with the increased labeling observed following NT-4/5 infusion. These data suggest that an endogenous ligand of trkB, normally present in limiting amounts within visual cortex, is necessary for the selective growth and remodeling of LGN axons into eye-specific patches.

In the developing cerebellum, young granule neurons in the external germinal layer respond preferentially to BDNF, while mature neurons within the inner portion of the cerebellum respond preferentially to NT3. Here we show that this anatomic distinction reflects a developmentally regulated switch at the level of neurotrophin receptor gene expression. The salient feature of the developmental switch is a change in the ration of mRNA transcripts encoding functional BDNF and NT3 receptor tyrosine kinases. The ratio of the BDNF receptor trkB to the NT3 receptor trkC reverses from 5:1 in neonatal cerebellum to 1:3 in adult cerebellum. TrkB and TrkC are closely related transmembrane tyrosine protein kinases. However, activation of BDNF and NT3 receptors in cerebellar granule neurons do not give equivalent biological responses. In aggregate cell culture and single cell assays, BDNF enhances axonal outgrowth of early granule cells by influencing neurite elongation. In contrast, NT3 alters the morphology of outgrowth. Collectively, these findings suggest that regulation of neurotrophin receptors during cerebellar development is important for the timing and morphology of axonal growth. In the developing cerebellum, young granule neurons in the external germinal layer respond preferentially to BDNF, while mature neurons within the inner portion of the cerebellum respond preferentially to NT3. Here we show that this anatomic distinction reflects a developmentally regulated switch at the level of neurotrophin receptor gene expression. The salient feature of the developmental switch is a change in the ration of mRNA transcripts encoding functional BDNF and NT3 receptor tyrosine kinases. The ratio of the BDNF receptor trkB to the NT3 receptor trkC reverses from 5:1 in neonatal cerebellum to 1:3 in adult cerebellum. TrkB and TrkC are closely related transmembrane tyrosine protein kinases. However, activation of BDNF and NT3 receptors in cerebellar granule neurons do not give equivalent biological responses. In aggregate cell culture and single cell assays, BDNF enhances axonal outgrowth of early granule cells by influencing neurite elongation. In contrast, NT3 alters the morphology of outgrowth. Collectively, these findings suggest that regulation of neurotrophin receptors during cerebellar development is important for the timing and morphology of axonal growth. In the developing cerebellum, young granule neurons in the external germinal layer respond preferentially to BDNF, while mature neurons within the inner portion of the cerebellum respond preferentially to NT3. Here we show that this anatomic distinction reflects a developmentally regulated switch at the level of neurotrophin receptor gene expression. The salient feature of the developmental switch is a change in the ration of mRNA transcripts encoding functional BDNF and NT3 receptor tyrosine kinases. The ratio of the BDNF receptor trkB to the NT3 receptor trkC reverses from 5:1 in neonatal cerebellum to 1:3 in adult cerebellum. TrkB and TrkC are closely related transmembrane tyrosine protein kinases. However, activation of BDNF and NT3 receptors in cerebellar granule neurons do not give equivalent biological responses. In aggregate cell culture and single cell assays, BDNF enhances axonal outgrowth of early granule cells by influencing neurite elongation. In contrast, NT3 alters the morphology of outgrowth. Collectively, these findings suggest that regulation of neurotrophin receptors during cerebellar development is important for the timing and morphology of axonal growth. In the developing cerebellum, young granule neurons in the external germinal layer respond preferentially to BDNF, while mature neurons within the inner portion of the cerebellum respond preferentially to NT3. Here we show that this anatomic distinction reflects a developmentally regulated switch at the level of neurotrophin receptor gene expression. The salient feature of the developmental switch is a change in the ration of mRNA transcripts encoding functional BDNF and NT3 receptor tyrosine kinases. The ratio of the BDNF receptor trkB to the NT3 receptor trkC reverses from 5:1 in neonatal cerebellum to 1:3 in adult cerebellum. TrkB and TrkC are closely related transmembrane tyrosine protein kinases. However, activation of BDNF and NT3 receptors in cerebellar granule neurons do not give equivalent biological responses. In aggregate cell culture and single cell assays, BDNF enhances axonal outgrowth of early granule cells by influencing neurite elongation. In contrast, NT3 alters the morphology of outgrowth. Collectively, these findings suggest that regulation of neurotrophin receptors during cerebellar development is important for the timing and morphology of axonal growth.

Medulloblastoma, the most common malignant brain tumor of childhood, has a variable prognosis. Although half of the children and young adults with the disease survive longer than 10 years after diagnosis, the others relapse and die despite identical therapy. We have examined the expression of neurotrophins and their receptors in medulloblastoma samples snap frozen in the operating room to preserve RNA integrity. All tumors (n = 12) were found to express mRNA encoding neurotrophin 3 and its receptor TrkC. The level of trkC expression was highly variable, with a more than 50-fold difference between the highest and lowest values. By Kaplan-Meier analysis, patients with tumors expressing high levels of trkC mRNA had significantly longer intervals without disease progression than those with low levels (log-rank, P = 0.03) and a more favorable overall survival (log-rank, P = 0.05). Thus, trkC expression is a prognostic indicator for patients with medulloblastoma.

Neurotrophins and their receptors are widespread in the developing and mature CNS. Identifying the differentiation state of neurotrophin-responsive cells provides a basis for understanding the developmental functions of these factors. Studies using dissociated and organotypic cultures of rat cerebellum demonstrated that the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) affect developing granule cells at distinct stages in differentiation. While early granule neurons in the external germinal layer responded to BDNF, more mature granule cells responded to NT-3. BDNF, but not NT-3, enhanced survival of granule cells in cultures of embryonic cerebella. Thus, BDNF and NT-3 have distinct sequential functions that are likely to be critical in the development of the cerebellum. BDNF may promote the initial commitment, while NT-3 may direct the subsequent maturation of granule cells.

Axon guidance relies on precise translation of the gradients of the extracellular signals into local changes of cytoskeletal dynamics, but the molecular mechanisms regulating dose-dependent responses of growth cones are still poorly understood. Here we show that during embryonic development in growing axons low level of Semaphorin3A stimulation is buffered by the prolyl isomerase Pin1. We demonstrate, that Pin1 stabilizes CDK5-phosphorylated CRMP2A, the major isoform of CRMP2 in distal axons. Consequently, Pin1 knockdown or knockout reduces CRMP2A level specifically in distal axons and inhibits axon growth, which can be fully rescued by Pin1 or CRMP2A expression. Moreover, Pin1 knockdown or knockout increases sensitivity to Sema3A-induced growth cone collapse in vitro and in vivo leading to developmental abnormalities in axon guidance. These results identify an important isoform-specific function and regulation of CRMP2A in controlling axon growth, and uncover Pin1-catalyzed prolyl isomerization as a regulatory mechanism in axon guidance.

Mounting evidence indicates that the nervous system plays a central role in cancer pathogenesis. In turn, cancers and cancer therapies can alter nervous system form and function. This Commentary seeks to describe the burgeoning field of "cancer neuroscience'' and encourage multidisciplinary collaboration for the study of cancer-nervous system interactions.