Retrograde transport of neurotrophins: fact and function

Retrograde signals generated by nerve growth factor (NGF) and other neurotrophins promote the survival of appropriately connected neurons during development, and failure to obtain sufficient retrograde signals may contribute to neuronal death occurring in many neurodegenerative diseases. The discovery over 25 years ago that NGF supplied to the axon terminals is retrogradely transported to the cell bodies suggested that NGF must reach the cell body to promote neuronal survival. Research during the intervening decades has produced a refinement of this hypothesis. The current hypothesis is that NGF bound to TrkA at the axon terminal is internalized into signaling endosomes, with NGF in their lumens bound to phosphorylated TrkA in their membranes, which are retrogradely transported to the cell bodies, where TrkA activates downstream signaling molecules that promote neuronal survival and regulate many aspects of neuronal gene expression. This model has been extrapolated to retrograde signaling by all neurotrophins. We consider the evidence for this model, focusing on results of experiments with neurons in compartmented cultures. Results to date indicate that while the transport of signaling endosomes containing NGF bound to TrkA may carry retrograde signals, retrograde survival signals can be carried by another mechanism that is activated by NGF at the axon terminal surface and travels to the cell body unaccompanied by the NGF that initiated it. It is hypothesized that multiple mechanisms of retrograde signaling exist and function under different circumstances. The newly discovered potential for redundancy in retrograde signaling mechanisms can complicate the interpretation of experimental results.     

Snapin recruits dynein to BDNF-TrkB signaling endosomes for retrograde axonal transport and is essential for dendrite growth of cortical neurons

Neurotrophin signaling is crucial for neuron growth. While the "signaling endosomes" hypothesis is one of the accepted models, the molecular machinery that drives retrograde axonal transport of TrkB signaling endosomes is largely unknown. In particular, mechanisms recruiting dynein to TrkB signaling endosomes have not been elucidated. Here, using snapin deficient mice and gene rescue experiments combined with compartmentalized cultures of live cortical neurons, we reveal that Snapin, as a dynein adaptor, mediates retrograde axonal transport of TrkB signaling endosomes. Such a role is essential for dendritic growth of cortical neurons. Deleting snapin or disrupting Snapin-dynein interaction abolishes TrkB retrograde transport, impairs BDNF-induced retrograde signaling from axonal terminals to the nucleus, and decreases dendritic growth. Such defects were rescued by reintroducing the snapin gene. Our study indicates that Snapin-dynein coupling is one of the primary mechanisms driving BDNF-TrkB retrograde transport, thus providing mechanistic insights into the regulation of neuronal growth and survival.     

Endosome-mediated retrograde axonal transport of P2X3 receptor signals in primary sensory neurons

Neurotrophins and their receptors adopt signaling endosomes to transmit retrograde signals. However, the mechanisms of retrograde signaling for other ligand/receptor systems are poorly understood. Here, we report that the signals of the purinergic (P)2X(3) receptor, an ATP-gated ion channel, are retrogradely transported in dorsal root ganglion (DRG) neuron axons. We found that Rab5, a small GTPase, controls the early sorting of P2X(3) receptors into endosomes, while Rab7 mediates the fast retrograde transport of P2X(3) receptors. Intraplantar injection and axonal application into the microfluidic chamber of α, β-methylene-ATP (α, β-MeATP), a P2X selective agonist, enhanced the endocytosis and retrograde transport of P2X(3) receptors. The α, β-MeATP-induced Ca(2+) influx activated a pathway comprised of protein kinase C, rat sarcoma viral oncogene and extracellular signal-regulated protein kinase (ERK), which associated with endocytic P2X(3) receptors to form signaling endosomes. Disruption of the lipid rafts abolished the α, β-MeATP-induced ERK phosphorylation, endocytosis and retrograde transport of P2X(3) receptors. Furthermore, treatment of peripheral axons with α, β-MeATP increased the activation level of ERK and cAMP response element-binding protein in the cell bodies of DRG neurons and enhanced neuronal excitability. Impairment of either microtubule-based axonal transport in vivo or dynein function in vitro blocked α, β-MeATP-induced retrograde signals. These results indicate that P2X(3) receptor-activated signals are transmitted via retrogradely transported endosomes in primary sensory neurons and provide a novel signaling mechanism for ligand-gated channels.     

Evidence in support of signaling endosome-based retrograde survival of sympathetic neurons

The mechanism by which target-derived Nerve Growth Factor (NGF) signaling is propagated retrogradely, over extremely long distances, to cell bodies to support survival of neurons is unclear. Here we show that survival of sympathetic neurons supported by NGF on distal axons requires the kinase activity of the NGF receptor, TrkA, in both distal axons and cell bodies. In contrast, disruption of TrkA activity exclusively in proximal axonal segments affects neither retrograde NGF-TrkA signaling in cell bodies nor neuronal survival. Ligand-receptor internalization is necessary for survival of neurons supported by NGF on distal axons. Furthermore, antibody neutralization experiments indicate that retrogradely transported NGF, within cell bodies, is critical for neuronal survival but not for growth of distal axons. Taken together, our results indicate that retrogradely transported NGF-TrkA complexes promote sympathetic neuron survival.     

Can Molecular Motors Drive Distance Measurements in Injured Neurons?

Injury to nerve axons induces diverse responses in neuronal cell bodies, some of which are influenced by the distance from the site of injury. This suggests that neurons have the capacity to estimate the distance of the injury site from their cell body. Recent work has shown that the molecular motor dynein transports importin-mediated retrograde signaling complexes from axonal lesion sites to cell bodies, raising the question whether dynein-based mechanisms enable axonal distance estimations in injured neurons? We used computer simulations to examine mechanisms that may provide nerve cells with dynein-dependent distance assessment capabilities. A multiple-signals model was postulated based on the time delay between the arrival of two or more signals produced at the site of injury–a rapid signal carried by action potentials or similar mechanisms and slower signals carried by dynein. The time delay between the arrivals of these two types of signals should reflect the distance traversed, and simulations of this model show that it can indeed provide a basis for distance measurements in the context of nerve injuries. The analyses indicate that the suggested mechanism can allow nerve cells to discriminate between distances differing by 10% or more of their total axon length, and suggest that dynein-based retrograde signaling in neurons can be utilized for this purpose over different scales of nerves and organisms. Moreover, such a mechanism might also function in synapse to nucleus signaling in uninjured neurons. This could potentially allow a neuron to dynamically sense the relative lengths of its processes on an ongoing basis, enabling appropriate metabolic output from cell body to processes.     

Retrograde axonal transport and motor neuron disease

Transport of material between extensive neuronal processes and the cell body is crucial for neuronal function and survival. Growing evidence shows that deficits in axonal transport contribute to the pathogenesis of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Here we review recent data indicating that defects in dynein-mediated retrograde axonal transport are involved in ALS etiology. We discuss how mutant copper-zinc superoxide dismutase (SOD1) and an aberrant interaction between mutant SOD1 and dynein could perturb retrograde transport of neurotrophic factors and mitochondria. A possible contribution of axonal transport to the aggregation and degradation processes of mutant SOD1 is also reviewed. We further consider how the interference with axonal transport and protein turnover by mutant SOD1 could influence the function and viability of motor neurons in ALS.     

Endocytosis and endosomes at the crossroads of regulating trafficking of axon outgrowth-modifying receptors

In neurons, many receptors must be localized correctly to axons or dendrites for proper function. During development, receptors for nerve growth and guidance are targeted to axons and localized to growth cones where receptor activation by ligands results in promotion or inhibition of axon growth. Signaling outcomes downstream of ligand binding are determined by the location, levels and residence times of receptors on the neuronal plasma membrane. Therefore, the mechanisms controlling the trafficking of these receptors are crucial to the proper wiring of circuits. Membrane proteins accumulate on the axonal surface by multiple routes, including polarized sorting in the trans Golgi network, sorting in endosomes and removal by endocytosis. Endosomes also play important roles in the signaling pathways for both growth-promoting and -inhibiting molecules: signaling endosomes derived from endocytosis are important for signaling from growth cones to cell bodies. Growth-promoting neurotrophins and growth-inhibiting Nogo-A can use EHD4/Pincher-dependent endocytosis at the growth cone for their respective retrograde signaling. In addition to retrograde transport of endosomes, anterograde transport to axons in endosomes also occurs for several receptors, including the axon outgrowth-promoting cell adhesion molecule L1/NgCAM and TrkA. L1/NgCAM also depends on EHD4/Pincher-dependent endocytosis for its axonal polarization. In this review, we will focus on receptors whose trafficking has been reported to be modulated by the EHD4/Pincher family of endosomal regulators, namely L1/NgCAM, Trk and Nogo-A. We will first summarize the pathways underlying the axonal transport of these proteins and then discuss the potential roles of EHD4/Pincher in mediating their endocytosis.     

Evidence for Phosphatidylinositol 4-Kinase and Actin Involvement in the Regulation of 125I-β-Nerve Growth Factor Retrograde Axonal Transport

The signaling events regulating the retrograde axonal transport of neurotrophins are poorly understood, but a role for phosphatidylinositol kinases has been proposed. In this study, we used phenylarsine oxide (PAO) to examine the participation of phosphatidylinositol 4-kinases in nerve growth factor (NGF) retrograde axonal transport within sympathetic and sensory neurons. The retrograde transport of 125I-labeled betaNGF was inhibited by PAO (0.5-2 nmol/eye), and this effect was diminished by dilution. Coinjection of 2,3-dimercaptopropanol with PAO reduced its ability to inhibit 125I-betaNGF retrograde transport. PAO (20 nM to 200 microM) also inhibited NGF-dependent survival of both sympathetic and sensory neuronal populations. F-actin staining in sympathetic and sensory neuronal growth cones was disrupted by PAO at 10 and 2 nM, respectively, and occurred within 5 min of exposure to the drug. The actin inhibitor latrunculin A also rapidly affected F-actin staining in vitro and reduced 125I-betaNGF retrograde axonal transport in vivo to the same extent as PAO. These results suggest that both phosphatidylinositol 4-kinase isoforms and the actin cytoskeleton play significant roles in the regulation of 125I-betaNGF retrograde axonal transport in vivo.     

On Trk for retrograde signaling.

Target-derived neurotrophins like nerve growth factor (NGF) mediate biological effects by binding to and activating Trk neurotrophin receptors at nerve terminals. The activated Trk receptors then stimulate local effects at nerve terminals, and retrograde effects at neuronal cell bodies that often reside at considerable distances from the terminals. However, the nature of the retrograde signal has been mysterious. Recent experiments suggest that the major retrograde signal required for survival and gene expression consists of activated Trk itself. Remarkably, signaling by Trk may differ at the terminal versus the neuronal cell body as a consequence of the retrograde transport mechanism, thereby allowing NGF to not only promote growth locally, but to specifically support survival and gene expression retrogradely.     

Mechanism of uptake and retrograde axonal transport of noradrenaline in sympathetic neurons in culture: reserpine-resistant large dense-core vesicles as transport vehicles

The uptake and retrograde transport of noradrenaline (NA) within the axons of sympathetic neurons was investigated in an in vitro system. Dissociated neurons from the sympathetic ganglia of newborn rats were cultured for 3-6 wk in the absence of non-neuronal cells in a culture dish divided into three chambers. These allowed separate access to the axonal networks and to their cell bodies of origin. [3H]NA (0.5 X 10(-6) M), added to the axon chambers, was taken up by the desmethylimipramine- and cocaine-sensitive neuronal amine uptake mechanisms, and a substantial part was rapidly transported retrogradely along the axons to the nerve cell bodies. This transport was blocked by vinblastine or colchicine. In contrast with the storage of [3H]NA in the axonal varicosities, which was totally prevented by reserpine (a drug that selectively inactivates the uptake of NA into adrenergic storage vesicles), the retrograde transport of [3H]NA was only slightly diminished by reserpine pretreatment. Electron microscopic localization of the NA analogue 5-hydroxydopamine (5-OHDA) indicated that mainly large dense-core vesicles (700-1,200-A diam) are the transport compartment involved. Whereas the majority of small and large vesicles lost their amine dense-core and were resistant to this drug. It, therefore, seems that these vesicles maintained the amine uptake and storage mechanisms characteristic for adrenergic vesicles, but have lost the sensitivity of their amine carrier for reserpine. The retrograde transport of NA and 5-OHDA probably reflects the return of used synaptic vesicle membrane to the cell body in a form that is distinct from the membranous cisternae and prelysosomal structures involved in the retrograde axonal transport of extracellular tracers.     

Synapse-to-nucleus signaling

Signals generated in distal subcellular compartments of neurons must often travel long distances to the nucleus to trigger changes in gene expression. This retrograde signaling is critical to the development, function, and survival of neural circuits, and neurons have evolved multiple mechanisms to transmit signals over long distances. In this review, we briefly summarize the range of mechanisms whereby distally generated signals are transported to neuronal nuclei. We then focus on the transport of soluble signals from the synapse to the nucleus during neuronal plasticity.     

Long-distance retrograde neurotrophic signaling

The retrograde communication of neurotrophic signals from axon terminals to neuron cell bodies is crucial for neuron survival and plasticity. Several mechanisms have been proposed in the past, but recent evidence strongly supports the hypothesis that the retrograde propagation of self-regenerating signaling organelles, derived from the endocytosis of activated neurotrophin-bound receptor tyrosine kinases, is the primary mechanism responsible for this long-distance communication.     

CAR-Associated Vesicular Transport of an Adenovirus in Motor Neuron Axons

Axonal transport is responsible for the movement of signals and cargo between nerve termini and cell bodies. Pathogens also exploit this pathway to enter and exit the central nervous system. In this study, we characterised the binding, endocytosis and axonal transport of an adenovirus (CAV-2) that preferentially infects neurons. Using biochemical, cell biology, genetic, ultrastructural and live-cell imaging approaches, we show that interaction with the neuronal membrane correlates with coxsackievirus and adenovirus receptor (CAR) surface expression, followed by endocytosis involving clathrin. In axons, long-range CAV-2 motility was bidirectional with a bias for retrograde transport in nonacidic Rab7-positive organelles. Unexpectedly, we found that CAR was associated with CAV-2 vesicles that also transported cargo as functionally distinct as tetanus toxin, neurotrophins, and their receptors. These results suggest that a single axonal transport carrier is capable of transporting functionally distinct cargoes that target different membrane compartments in the soma. We propose that CAV-2 transport is dictated by an innate trafficking of CAR, suggesting an unsuspected function for this adhesion protein during neuronal homeostasis.     

Obligatory Activation of SRC and JNK by GDNF for Survival and Axonal Outgrowth of Postnatal Intestinal Neurons

The neurotrophin GDNF acts through its co-receptor RET to direct embryonic development of the intestinal nervous system. Since this continues in the post-natal intestine, co-cultures of rat enteric neurons and intestinal smooth muscle cells were used to examine how receptor activation mediates neuronal survival or axonal extension. GDNF-mediated activation of SRC was essential for neuronal survival and axon outgrowth and activated the major downstream signaling pathways. Selective inhibition of individual pathways had little effect on survival but JNK activation was required for axonal maintenance, extension or regeneration. This was localized to axonal endings and retrograde transport was needed for central JUN activation and subsequent axon extension. Collectively, GDNF signaling supports neuronal survival via SRC activation with multiple downstream events, with JNK signaling mediating structural plasticity. These pathways may limit neuron death and drive subsequent regeneration during challenges in vivo such as intestinal inflammation, where supportive strategies could preserve intestinal function.

     

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.     

Nuclear transport factors in neuronal function

Active nucleocytoplasmic transport of macromolecules requires soluble transport carriers of the importin/karyopherin superfamily. Although the nuclear transport machinery is essential in all eukaryotic cells, neurons must also mobilise importins and associated proteins to overcome unique spatiotemporal challenges. These include switches in importin α subtype expression during neuronal differentiation, localized axonal synthesis of importin β1 to coordinate a retrograde injury signaling complex on axonal dynein, and trafficking of regulatory and signaling molecules from synaptic terminals to cell bodies. Targeting of RNAs encoding critical components of the importins complex and the Ran system to axons allows sophisticated local regulation of the system for mobilization upon need. Finally, a number of importin family members have been associated with mental or neurodegenerative diseases. The extended roles recently discovered for importins in the nervous system might also be relevant in non-neuronal cells, and the localized modes of importin regulation in neurons offer new avenues to interrogate their cytoplasmic functions.     

Axonal involvement in the Wlds neuroprotective effect: analysis of pure motoneurons in a mouse model protected from motor neuron disease at a pre-symptomatic age

The identification of the Wlds gene that delays axonal degeneration in several models of neurodegenerative disease provides an interesting tool to study mechanisms of axonal loss. We showed that crossing a mouse mutant with a motoneuron disease (pmn for progressive motor neuronopathy) with mice that express the Wlds gene delayed axonal loss, increased the life span, partially rescued axonal transport deficit and prolonged the survival of the motoneuron cell bodies. To determine factors involved in the neuroprotective effect of Wlds, we combined laser capture microdissection and microarray analysis to identify genes that are differentially regulated at a pre-symptomatic age in motoneuron cell bodies in pmn/pmn,Wlds/Wlds mice as compared with pmn/pmn mice. Only 56 genes were de-regulated; none of the 'classical' genes implicated in apoptosis were de-regulated. Interestingly, a large proportion of these genes are related to axonal function and to retrograde and anterograde transport (i.e. members of the dynactin complex and kinesin family). These results were confirmed by real-time PCR, in situ hybridization and at protein level in sciatic nerves. Thus, genes related to axonal function and in particular to axonal transport may be involved at an early stage in the neuroprotective property of the Wlds gene and confirm the importance of axonal involvement in this model of motor neuron disease.