Anna J. Reynolds

Molecular mechanisms regulating the retrograde axonal transport of neurotrophins.

Neurotrophins are released from target tissues following neural innervation and bind to specific receptors situated on the nerve terminal plasma membrane. The neurotrophin-receptor complex undergoes retrograde axonal transport towards the cell soma, where it signals to the nucleus. This process allows neurotrophins to perform their numerous functions, which include the promotion of neuronal survival and the outgrowth of axons towards certain target tissues. The molecular events controlling each of the components of retrograde axonal transport are beginning to become defined. There is good evidence for the participation of phosphatidylinositol 3-kinase, phosphatidylinositol 4-kinase and the actin cytoskeleton in neurotrophin retrograde axonal transport in vivo. It also appears that the retrograde motor protein dynein mediates the retrograde axonal transport in vivo of neurotrophins such as nerve growth factor. This review discusses the role of the neurotrophin receptors in binding and axonal transport, the endocytic processes required for neurotrophin internalization, the targeting and trafficking of neurotrophins, and the propagation of neurotrophin-induced signals along the axon.

Hypertolerance to morphine in Gzα-deficient mice

Abstract Our laboratory has generated a mouse deficient in the alpha (α) subunit of the G protein, Gz, (Gzα) gene and we have examined the involvement of Gzα in spinal and supraspinal analgesia and tolerance mechanisms. Spinal analgesia was tested by the response times to heat or cold tail flick times in a water bath at 50°C or −5°C and supraspinal analgesia was tested by the times for paw licking and jumping from a plate at 52°C or 0.5°C. Tolerance to morphine was induced in wild type and Gzα-deficient mice over a 5 day period and the behavioral tests were performed daily. The tail flick reaction times to both hot and cold stimuli did not differ between the wild type and Gzα-deficient mice. Analysis of the reaction times from the hot and cold plate tests showed the Gzα-deficient mice developed tolerance to morphine to a greater degree and at a faster rate than wild type mice. Opioid binding assays were performed on synaptic membranes prepared from naive and morphine tolerant wild type and Gzα-deficient brains. No changes in the affinity of morphine for its receptor or in the density of μ and δ opioid receptors were found between the two groups of mice in the naive or morphine tolerant state. This indicates that the absence of Gzα does not affect opioid receptor affinity or receptor up or down regulation. Our results suggest that the presence of Gzα delays the development of morphine tolerance and represents a possible therapeutic target for improving the clinical use of morphine.

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

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.

Signalling events regulating the retrograde axonal transport of 125I−βNerve growth factor in vivo

The molecular mechanisms regulating the retrograde axonal transport of nerve growth factor (NGF) are currently unknown. This study identifies some of the signalling events involved. The phosphoinositide 3-kinase (PI3-kinase) inhibitor wortmannin (1 nmol/eye) irreversibly inhibits the amount of 125I-betaNGF retrogradely transported in both sensory and sympathetic neurons. Another PI3-kinase inhibitor LY294002 (100 nmol/eye) also inhibited 125I-betaNGF retrograde transport in sensory neurons. The pp70S6K inhibitor rapamycin (1 micromol/eye) had the same effect, inhibiting 125I-betaNGF transport only in sensory neurons. The cPLA2 inhibitor AACOCF3 (10 nmol/eye) had no effect on 125I-betaNGF transport in either sensory or sympathetic neurons. The TrkA receptor tyrosine kinase inhibitor AG-879 (10 nmol/eye) reduced 125I-betaNGF transport by approximately 50% in both sensory and sympathetic neurons. Cytochalasin D (2 nmol/eye), a disruptor of actin filaments and the dynein ATPase inhibitor erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) both inhibited 125I-betaNGF retrograde transport. These results demonstrate that in vivo TrkA tyrosine kinase activity, actin filaments and dynein are involved in the retrograde transport of NGF. In addition, different PI3-kinase isoforms may be recruited within different neuronal populations to regulate the retrograde transport of NGF. Potentially, these isoforms could activate alternative signalling pathways, such as pp70S6K in sensory neurons.

Anterograde and retrograde transport of active extracellular signal-related kinase 1 (ERK1) in the ligated rat sciatic nerve

Neurons are one of the most polarized cells and often the nerve terminals may be located long distances from the cell body, thus signal transduction in neurons unlike other cells may need to be conducted over large distances. The mitogen-activated protein/extracellular signal-regulated kinases (MAP kinases or ERKs) regulate a diverse array of functions and in neurons, the ERK signalling pathways appear to have an important role in activity-dependent regulation of neuronal function. Using the ligated rat sciatic nerve as an experimental model we previously showed that the ERK1/2, MAP/ERK kinase (MEK1/2) and the p110 catalytic subunit of PI3-kinase are transported in the rat sciatic nerve. We have extended these findings to determine if these proteins are transported in the active state using antibodies that specifically detect the active form of ERK1/2, MEK1/2 and AKT which is activated downstream of PI3-kinase. We show significant accumulation of active ERK1 on the proximal and distal sides of a nerve ligation after 16 h. Active ERK2 also appeared to be accumulating at the ligature, however this did not reach statistical significance. In contrast there was not any significant accumulation of active MEK1/2 or active AKT. A component of both active ERK1 and active ERK2 is present in between the two ligations suggesting they are also present in the surrounding Schwann cells and are activated in response to nerve injury. Taken together our results suggest that a component of the accumulation of active ERK1 on the distal and proximal side of the nerve ligations results from transport in the anterograde and retrograde direction in the rat sciatic nerve.

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.

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.

Retrograde axonal transport of neurotrophins: Differences between neuronal populations and implications for motor neuron disease

During development, neurons die if they do not receive neurotrophin support from the target cells they are innervating. Neurotrophins are delivered from the target to the cell bodies of the innervating neurons by interacting with specific receptors located on the nerve terminals and then together are retrogradely transported to the cell body. This process consists of a number of distinct events including endocytosis of neurotrophin and its receptor into coated vesicles; vesicle sorting followed by retrograde axonal transport to the cell body, where interaction of the activated receptor initiates a signalling cascade at the cell body that causes the survival response. It has recently been shown that the signalling molecules associated with retrograde transport differ between neuronal populations. In sympathetic but not sensory neurons, a wortmannin‐sensitive molecule (phosphatidylinositol kinase) is essential for the retrograde transport of neurotrophins. In sensory but not sympathetic neurons, a rapamycin‐sensitive molecule (pp70S6K) is associated with retrograde transport of neurotrophins. This is strong evidence that sympathetic and sensory neurons utilize different signalling pathways to perform the same cellular function; retrograde transport. These findings may provide clues to understanding neurological diseases, such as motor neuron disease, in which axonal transport is impaired specifically in motor neurons.

Differential mRNA Expression and Subcellular Locations of PI3-Kinase Isoforms in Sympathetic and Sensory Neurons

Phosphatidylinositol 3‐kinase (PI3‐kinase) enzymes are key signalling molecules in the PC12 and neuronal cell survival pathway and are also involved in the regulation of retrograde axonal transport of nerve growth factor (NGF), with sympathetic neurons more sensitive to the effects of wortmannin/LY294002 than sensory neurons (Bartlett et al. [1997]; Brain Res. 761:257–262; Reynolds et al. [1998] Brain Res. 798:67–74). In this article, we characterized the mRNA expression of PI3‐kinase isoforms in mouse sympathetic superior cervical ganglia (SCG) and sensory trigeminal ganglia (TGG) and examined the subcellular locations of immunoreactivity of the PI3‐kinase isoforms in mouse cultured SCG and dorsal root ganglion (DRG) neurons. Both the SCG and the TGG express mRNA for the p110α, β, γ, δ, and vps34p PI3‐kinase isoforms, but the TGG and not the SCG express mRNA for the p170 PI3‐kinase isoform. In cultured SCG and DRG neurons, p110α, β, and γ immunoreactivity is in the SCG and DRG growth cones, and predominantly in puncta throughout the growth cone varicosity. However, in the cell bodies immunoreactivity varied, p110α is localized predominantly at the plasma membrane, while p110β and γ is localized in the perinuclear region of the cells. In addition, unlike other cell types, wortmannin has little effect on actin filament polymerization in either mouse cultured SCG or DRG neurons. J. Neurosci. Res. 56:44–53, 1999. 

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

Abstract: 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 βNGF 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‐βNGF retrograde transport. PAO (20 nM to 200 μM) 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‐βNGF 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‐βNGF retrograde axonal transport in vivo.