Association of p75(NTR) with caveolin and localization of neurotrophin-induced sphingomyelin hydrolysis to caveolae.

Caveolae are plasma membrane microdomains that are enriched in caveolin, the structural protein of caveolae, sphingomyelin, and other signaling molecules. We previously suggested that neurotrophin-induced p75(NTR)-dependent sphingomyelin hydrolysis may be localized to the plasma membrane. Therefore, we examined if caveolae were a major site of p75(NTR)-dependent sphingomyelin hydrolysis in p75(NTR)-NIH 3T3 fibroblasts. Caveolin-enriched membranes (CEMs) were prepared by either detergent or detergent-free extraction and separated from noncaveolar membranes by centrifugation through sucrose gradients. Immunoblot analysis of the individual gradient fractions indicated that caveolin and p75(NTR) were enriched in CEMs. The localization of p75(NTR) to CEMs was not an artifact of receptor overexpression in the fibroblasts because a similar distribution of p75(NTR) was evident from PC12 cells, which endogenously express p75(NTR). In the p75(NTR) fibroblasts, nerve growth factor induced a time-dependent hydrolysis of sphingomyelin only in CEMs with no hydrolysis detected in noncaveolar membranes. Intriguingly, endogenous p75(NTR) was found to co-immunoprecipitate with caveolin, suggesting that p75(NTR) may associate with caveolin in vivo. This interaction was confirmed in vitro by the co-immunoprecipitation of a glutathione S-transferase fusion protein expressing the cytoplasmic domain of p75(NTR) with caveolin. Collectively, these results demonstrate that neurotrophin-induced p75(NTR)-dependent sphingomyelin hydrolysis localizes to CEMs and suggest that the interaction of p75(NTR) with caveolin may affect signaling through p75(NTR).

Phosphoinositide 3-kinase regulates crosstalk between Trk A tyrosine kinase and p75(NTR)-dependent sphingolipid signaling pathways.

The mechanism of crosstalk between signaling pathways coupled to the Trk A and p75(NTR) neurotrophin receptors in PC12 cells was examined. In response to nerve growth factor (NGF), Trk A activation inhibited p75(NTR)-dependent sphingomyelin (SM) hydrolysis. The phosphoinositide 3-kinase (PI 3-kinase) inhibitor, LY294002, reversed this inhibition suggesting that Trk A activation of PI 3-kinase is necessary to inhibit sphingolipid signaling by p75(NTR). In contrast, SM hydrolysis induced by neurotrophin-3 (NT-3), which did not activate PI-3 kinase, was uneffected by LY294002. However, transient expression of a constituitively active PI 3-kinase inhibited p75(NTR)-dependent SM hydrolysis by both NGF and NT-3. Intriguingly, NGF induced an association of activated PI 3-kinase with acid sphingomyelinase (SMase). This interaction localized to caveolae-related domains and correlated with a 50% decrease in immunoprecipitated acid SMase activity. NGF-stimulated PI 3-kinase activity was necessary for inhibition of acid SMase but was not required for ligand-induced association of the p85 subunit of PI 3-kinase with the phospholipase. Finally, this interaction was specific for NGF since EGF did not induce an association of PI 3-kinase with acid SMase. In summary, our data suggest that PI 3-kinase regulates the inhibitory crosstalk between Trk A tyrosine kinase and p75(NTR)-dependent sphingolipid signaling pathways and that this interaction localizes to caveolae-related domains.

Measurement of the rate of myelination using a fluorescent analogue of ceramide.

Fluorescence digital imaging microscopy was used to investigate the process of myelin formation by Schwann cells in neuronal cocultures. The uptake of the fluorescent ceramide analogue N-[5-(5,7-dimethyl BODIPY)-1-pentanoyl]D-erythro-sphingosine (C5-DMB-ceramide) and its return to the plasma membrane as the corresponding fluorescent sphingomyelin and galactocerebroside analogues were measured. Through observation of this process it was possible to determine the rate of lipid synthesis in myelin internodes. The highest rate of synthesis of fluorescent sphingomyelin and galactocerebroside analogues was observed between days 3 and 7 after induction of myelination. This rate was approximately 5-fold greater than the steady-state rate of synthesis in fully myelinated internodes and 10-fold higher than the rate observed prior to myelination. The internode diameter increased during the first 3 days of myelination, but this was followed by a reduction in diameter and then an increase until the myelin sheath formation was completed. Internodes were found to be heterogeneous in terms of lipid distribution, with fluorescence intensities ranging 5-fold in myelinating cultures. Additionally, the rate of lipid transport along the internode was slow since there was a quicker increase in fluorescence intensity near the cell body of the Schwann cell than near the nodes of Ranvier. The results show that fluorescence digital imaging microscopy can be used to study the process of myelin formation and to determine the rate of formation, lipid transport, and heterogeneity of the myelin membrane.

Caveolin interacts with Trk A and p75(NTR) and regulates neurotrophin signaling pathways.

Neurotrophins signal through Trk tyrosine kinase receptors and the low-affinity neurotrophin receptor p75(NTR). We have shown previously that activation of Trk A tyrosine kinase activity can inhibit p75(NTR)-dependent sphingomyelin hydrolysis, that caveolae are a localized site for p75(NTR) signaling, and that caveolin can directly interact with p75(NTR). The ability of caveolin to also interact with tyrosine kinase receptors and inhibit their activity led us to hypothesize that caveolin expression may modulate interactions between neurotrophin signaling pathways. PC12 cells were transfected with caveolin that was expressed efficiently and targeted to the appropriate membrane domains. Upon exposure to nerve growth factor (NGF), caveolin-PC12 cells were unable to develop extensive neuritic processes. Caveolin expression in PC12 cells was found to diminish the magnitude and duration of Trk A activation in vivo. This inhibition may be due to a direct interaction of caveolin with Trk A, because Trk A co-immunoprecipitated with caveolin from Cav-Trk A-PC12 cells, and a glutathione S-transferase-caveolin fusion protein bound to Trk A and inhibited NGF-induced autophosphorylation in vitro. Furthermore, the in vivo kinetics of the inhibition of Trk A tyrosine kinase activity by caveolin expression correlated with an increased ability of NGF to induce sphingomyelin hydrolysis through p75(NTR). In summary, our results suggest that the interaction of caveolin with neurotrophin receptors may have functional consequences in regulating signaling through p75(NTR) and Trk A in neuronal and glial cell populations.