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.

A population of PC12 cells that is initiating apoptosis can be rescued by nerve growth factor.

Programmed cell death, or apoptosis, occurs asynchronously in neuronal cells. To overcome this asynchrony, rat pheochromocytoma (PC12) cells were separated at different stages of apoptosis on the basis of cell density. Live cells that exhibited no apoptotic features floated to the top of density gradients. The most dense cells showed extensive loss of cytochrome c from mitochondria, caspase activation, chromatin condensation, and DNA fragmentation. These cells were committed to apoptosis and could not be rescued by reculturing in with nerve growth factor (NGF). Cells of intermediate density displayed no DNA fragmentation, but had begun to show cytochrome c loss, caspase activation, and chromatin condensation. This population displayed upregulation of the prodeath factor, c-Jun, and downregulation of prosurvival kinase, Akt. Importantly, apoptosis was reversible by NGF in this population. These studies suggest that increased cell density correlates with an initial step in the apoptosis mechanism that precedes irreversible commitment to suicide.

CREB is cleaved by caspases during neural cell apoptosis.

Programmed cell death, or apoptosis, is a tightly regulated process mediated by selective cleavage of proteins by caspases, resulting in ordered destruction of the cell. In addition to structural proteins, proteins that mediate anti-apoptotic signal transduction are also substrates; their destruction eliminates potential futile attempts to escape execution. We asked whether cAMP response element binding protein (CREB), a transcription factor that mediates nerve growth factor (NGF) survival signals, is a target for caspases during apoptosis. CREB was specifically cleaved by caspases in neuroblastoma extracts, and in cells induced to undergo apoptosis by staurosporine. The destruction of CREB eliminates a key factor that could reverse apoptosis.

Phosphorylation-dependent Akt cleavage in neural cell in vitro reconstitution of apoptosis.

Neuronal apoptotic execution uses a cytochrome c-dependent caspase activation mechanism that is conserved in other cell types. Phosphatidylinositol 3-kinase and its downstream effector, Akt/protein kinase B, appear to control this mechanism and govern the life/death decision. We have developed a cell-free system using cytosol from human neuroblastoma (SY5Y) cells that reconstitutes biochemical features of neuronal apoptosis. In the presence of cytochrome c and ATP, caspase-9 and -3 were activated, which initiated chromatin condensation and DNA cleavage in rat pheochromocytoma (PC12) nuclei. Akt was cleaved in reactions where caspase-3 was activated and its cleavage was prevented by the caspase inhibitor DEVD-aldehyde. The phosphatase inhibitors orthovanadate and okadaic acid prevented catalytic processing and activation of caspase-3 and digestion of Akt and partially inhibited cleavage of caspase-9. Caspase-dependent destruction of Akt irreversibly inactivates this key mediator of survival signaling, ensuring that the execution pathway will prevail.

A signaling organelle containing the nerve growth factor-activated receptor tyrosine kinase, TrkA.

The topology of signal transduction is particularly important for neurons. Neurotrophic factors such as nerve growth factor (NGF) interact with receptors at distal axons and a signal is transduced by retrograde transport to the cell body to ensure survival of the neuron. We have discovered an organelle that may account for the retrograde transport of the neurotrophin signal. This organelle is derived from endocytosis of the receptor tyrosine kinase for NGF, TrkA. In vitro reactions containing semi-intact PC12 cells and ATP were used to enhance recovery of a novel organelle: small vesicles containing internalized NGF bound to activated TrkA. These vesicles were distinct from clathrin coated vesicles, uncoated primary endocytic vesicles, and synaptic vesicles, and resembled transport vesicles in their sedimentation velocity. They contained 10% of the total bound NGF and almost one-third of the total tyrosine phosphorylated TrkA. These small vesicles are compelling candidates for the organelles through which the neurotrophin signal is conveyed down the axon.

Endocytosis of activated TrkA: evidence that nerve growth factor induces formation of signaling endosomes.

The survival, differentiation, and maintenance of responsive neurons are regulated by nerve growth factor (NGF), which is secreted by the target and interacts with receptors on the axon tip. It is uncertain how the NGF signal is communicated retrogradely from distal axons to neuron cell bodies. Retrograde transport of activated receptors in endocytic vesicles could convey the signal. However, little is known about endocytosis of NGF receptors, and there is no evidence that NGF receptors continue to signal after endocytosis. We have examined early events in the membrane traffic of NGF and its receptor, gp140(TrkA) (TrkA), in PC12 cells. NGF induced rapid and extensive endocytosis of TrkA in these cells, and the receptor subsequently moved into small organelles located near the plasma membrane. Some of these organelles contained clathrin and alpha-adaptin, which implies that TrkA is internalized by clathrin-mediated endocytosis. Using mechanical permeabilization and fractionation, intracellular organelles derived from endocytosis were separated from the plasma membrane. After NGF treatment, NGF was bound to TrkA in endocytic organelles, and TrkA was tyrosine-phosphorylated and bound to PLC-gamma1, suggesting that these receptors were competent to initiate signal transduction. These studies raise the possibility that NGF induces formation of signaling endosomes containing activated TrkA. They are an important first step in elucidating the molecular mechanism of NGF retrograde signaling.

Multiple levels for regulation of TrkA in PC12 cells by nerve growth factor.

TrkA is a receptor tyrosine kinase for nerve growth factor (NGF). Recent studies indicate that NGF regulates not only activation of trkA kinase but also expression of the trkA gene. To further define NGF actions on trkA, we examined binding and signaling through trkA after both short and long intervals of NGF treatment. Induction of tyrosine phosphorylation on gp140trkA was rapidly followed by down-regulation of cell surface and total cellular gp140trkA. At later intervals, increased expression of trkA was evident in increased mRNA and protein levels. At 7 days, there was increased binding to gp140trkA and increased signaling through this receptor. NGF appears to regulate trkA at several levels. In neurons persistently exposed to NGF, maintenance of NGF signaling may require increased trkA gene expression.