Comparison of nerve terminal events in vivo effecting retrograde transport of vesicles containing neurotrophins or synaptic vesicle components.

Although vesicular retrograde transport of neurotrophins in vivo is well established, relatively little is known about the mechanisms that underlie vesicle endocytosis and formation before transport. We demonstrate that in vivo not all retrograde transport vesicles are alike, nor are they all formed using identical mechanisms. As characterized by density, there are at least two populations of vesicles present in the synaptic terminal that are retrogradely transported along the axon: those containing neurotrophins (NTs) and those resulting from synaptic vesicle recycling. Vesicles containing nerve growth factor (NGF), NT-3, or NT-4 had similar densities with peak values at about 1.05 g/ml. Synaptic-derived vesicles, labeled with anti-dopamine beta-hydroxylase (DBH), had densities with peak values at about 1.16 g/ml. We assayed the effects of pharmacologic agents in vivo on retrograde transport from the anterior eye chamber to the superior cervical ganglion. Inhibitors of phosphatidylinositol-3-OH (PI-3) kinase and actin function blocked transport of both anti-DBH and NGF, demonstrating an essential role for these molecules in retrograde transport of both vesicle types. Dynamin, a key element in synaptic vesicle recycling, was axonally transported in retrograde and anterograde directions, and compounds able to interfere with dynamin function had a differential effect on retrograde transport of NTs and anti-DBH. Okadaic acid significantly decreased retrograde axonal transport of anti-DBH and increased NGF retrograde transport. We conclude that there are both different and common proteins involved in endocytosis and targeting of retrograde transport of these two populations of vesicles.

What is the importance of multivesicular bodies in retrograde axonal transport in vivo?

Neurons with long axons have a unique problem in generating signaling cascades that are able to reach the nucleus after receptor activation by neurotrophins at the nerve terminal. The straightforward concept of receptor binding and local generation of 2nd second messenger cascades is too simplistic. In this review we will outline a mechanism that would enable the complex signals generated at the nerve terminal to be conveyed intact to the cell body. There are three different sites in the neuron where 2nd messenger proteins can interact with the signaling complex and be activated. Signaling cascades are initiated both at the nerve terminal and at the cell body when 2nd messengers are recruited to the plasma membrane by activated receptors. After receptor-mediated endocytosis, 2nd messenger molecules continue to be recruited to the internalized vesicle; however, the mix of proteins differs in the nerve terminal and in the cell body. At the nerve terminal the activated pathways result in the formation of the neurotrophin signaling endosome, which includes molecules to be retrogradely transported to the cell body. When the retrograde neurotrophin signaling endosome reaches the cell body, it can recruit additional 2nd messenger molecules to finally generate the unique signal derived from the nerve terminal. We propose that the multivesicular body observed in vivo functions as an endosome carrier vehicle or retrosome. This retrosome enables the mix of signaling molecules recruited at the terminal to be transported intact to the cell body. This will allow the cell body to receive a snapshot of the events occurring at the nerve terminal at the time the retrosome is formed.

Transport of endosomal early antigen 1 in the rat sciatic nerve and location in cultured neurons.

Early endosomal antigen 1 (EEA1) is known to be a marker of early endosomes and in cultured hippocampal neurons it preferentially localizes to the dendritic but not the axonal compartment. We show in cultured dorsal root ganglia and superior cervical ganglia neurons that EEA1 localizes to the cell bodies and the neurites of both sensory and sympathetic neurons. We then show in vivo using a ligated rat sciatic nerve that EEA1 significantly accumulates on the proximal side and not on the distal side of the ligation. This suggests that EEA1 is transported in the anterograde direction in axons either as part of the homeostatic process or to the nerve ligation site in response to nerve injury.

Prolonged recycling of internalized neurotrophins in the nerve terminal.

BACKGROUND: Neurons require contact with their target tissue in order to survive and make correct connections. The retrograde axonal transport of neurotrophins occurs after receptor-mediated endocytosis into vesicles at the nerve terminal. However, the mechanism by which the neurotrophin signal is propagated from axon terminal to cell body remains unclear. METHODS: Retrograde axonal transport was examined using the transport of I(125)-labeled neurotrophins from the eye to sympathetic and sensory ganglia. The phenomena was further studied by adding rhodamine-labeled nerve growth factor (NGF) to cultures of dissociated sympathetic ganglia and the movement of organelles followed with the aid of video microscopy. RESULTS: I(125)-labeled neurotrophins were transported from the eye to the sympathetic and sensory ganglia. A 100-fold excess of unlabeled neurotrophin, administered up to 4 h after the labeled material, completely prevented accumulation of labeled neurotrophin in the ganglia. The effect was specific for the labeled neurotrophin as administration of a high concentration of a different neurotrophin failed to inhibit the transport. In dissociated cultures, we found rapid binding of label, to surface membrane receptors, followed by an accumulation of labeled vesicles in the growth cone. Incubation of these cultures with unlabeled NGF led to a rapid loss of label in the growth cones. CONCLUSIONS: These results suggest that there is a pool of internalized neurotrophin, in vesicles in the nerve terminal, which is in rapid equilibrium with the external environment. It is from this pool that a small fraction of the neurotrophin-containing vesicles is targeted for retrograde transport. Potential models for this system are presented.

PtdIns 4-kinasebeta and neuronal calcium sensor-1 co-localize but may not directly associate in mammalian neurons.

It was recently demonstrated that the yeast homologue of phosphatidylinositol 4-kinasebeta PIK1 is directly associated with frq1, the yeast homologue of mammalian neuronal calcium sensor-1 (NCS-1) (Hendricks et al., [1999] Nat. Cell Biol. 1:234- 241). This was a novel finding and suggests that a calcium binding protein activates and regulates PtdIns 4-kinasebeta. This finding had not been shown in mammalian cells and both PtdIns 4-kinasebeta and NCS-1 have been shown to have important roles in the regulation of exocytotic release associated with neurotransmission. The aims of this study were to determine if PtdIns 4-kinasebeta and NCS-1 directly associate in mammalian neural tissues. We show that the immunostaining pattern for PtdIns 4-kinasebeta and NCS-1 is co-localized throughout the neurites of newborn cultured dorsal root ganglia (DRG) neurons but not in E13 DRG neurons. We then provide biochemical evidence that PtdIns 4-kinasebeta may not be in physical association with NCS-1 in mammalian nervous tissue unlike that previously reported in yeast.

Signalling organelle for retrograde axonal transport of internalized neurotrophins from the nerve terminal.

The retrograde axonal transport of neurotrophins occurs after receptor-mediated endocytosis into vesicles at the nerve terminal. We have been investigating the process of targeting these vesicles for retrograde transport, by examining the transport of [125I]-labelled neurotrophins from the eye to sympathetic and sensory ganglia. With the aid of confocal microscopy, we examined the phenomena further in cultures of dissociated sympathetic ganglia to which rhodamine-labelled nerve growth factor (NGF) was added. We found the label in large vesicles in the growth cone and axons. Light microscopic examination of the sympathetic nerve trunk in vivo also showed the retrogradely transported material to be sporadically located in large structures in the axons. Ultrastructural examination of the sympathetic nerve trunk after the transport of NGF bound to gold particles showed the label to be concentrated in relatively few large organelles that consisted of accumulations of multivesicular bodies. These results suggest that in vivo NGF is transported in specialized organelles that require assembly in the nerve terminal.

In sympathetic but not sensory neurones, phosphoinositide-3 kinase is important for NGF-dependent survival and the retrograde transport of 125I-betaNGF.

The way in which the same ligands and receptors have different functional effects in different cell types must depend on subtle differences in the second messenger cascades. Sensory and sympathetic neurones both retrogradely transport nerve growth factor (NGF) and depend on NGF for their developmental survival. NGF binding to the high affinity tyrosine kinase (TrkA) receptors initiates second messenger signalling cascades, one of which includes the activation of phosphoinositide-3 kinase (PI3-kinase). We demonstrate that 100-fold higher concentrations of the PI3-kinase inhibitor, Wortmannin, are required to inhibit the survival effects and retrograde axonal transport of NGF in sensory neurones than in sympathetic neurones. Similarly, although less potently than Wortmannin, the PI3-kinase inhibitor LY294002 required a 10-fold higher concentration to inhibit the survival effects of NGF in sensory than in sympathetic neurones. Inhibitors of other second messengers, including staurosporine, pertussis and cholera toxins, failed to have an effect on the transport of the NGF receptor complex in both cell types. Also, Wortmannin did not affect the structural integrity of the sympathetic nerve terminals. As PI3-kinase is present in both neuronal populations, this suggests that the Wortmannin sensitive isoform of PI3-kinase (p110) is essential in sympathetic neurones both for survival and for NGF-TrkA receptor complex trafficking. As sensory neurones also depend on NGF for their developmental survival and endocytose and retrogradely transport the NGF-TrkA receptor complex, this population of neurones may either recruit a different isoform of PI3-kinase or utilize PI3-kinase independent signalling pathways for these cellular functions.