Similar to results in mass cultures, NGF treatment of distal axons leads to a reduction (24% decrease) in phosphorylated dynamin1, in comparison to control treatment (Figure 5C and 5D). To test whether NGF regulates phosphorylation of dynamin1 in axons in vivo, we analyzed the levels of phospho-dynamin1 in a sympathetic target tissue, the salivary glands, in both wild-type and heterozygous NGF (NGF+/−) mice. Given that dynamin1 is neuron specific ( Urrutia et al., 1997), immunoblotting of salivary gland lysates with the phospho-dynamin1 antibody should reveal the status of dynamin1 phosphorylation FG-4592 solubility dmso locally in sympathetic nerve terminals that innervate

the target tissue. If target-derived NGF regulates dynamin1 phosphorylation

in vivo, then we would expect to see increased dynamin1 phosphorylation levels under conditions of reduced NGF signaling. We employed NGF+/− mice for this analysis because these mice display haploinsufficiency with reduced levels of NGF and sympathetic target innervation ( Brennan et al., 1999 and Ghasemlou et al., 2004), in contrast to homozygous selleck chemicals llc NGF null mice, which completely lack sympathetic innervation ( Glebova and Ginty, 2004). We found that NGF+/− mice have higher levels of phosphorylated dynamin1 on Ser-778 in sympathetic axons innervating the salivary glands, compared to wild-type animals (11.2% ± 2% increase;

Figures 5E and 5F). These findings provide in vivo evidence for NGF-dependent phosphoregulation of dynamin1 locally in sympathetic axons. To assess the role of dynamin1 dephosphorylation in supporting neurotrophin-dependent axon growth, sympathetic neurons were exposed for 24 hr to a cell-permeable peptide spanning the dynamin1 phospho-box (amino acids 769–784, incorporating Ser-774 and Ser-778) in which the two serines 774/778 were replaced with alanine (Ser774/778-Ala, dyn1769-784AA). The dyn1769-784AA peptide blocks dephosphosphorylation-dependent dynamin1 functions by binding and sequestering downstream effector molecules, such as syndapin1 (Anggono et al., 2006). Delivery of dyn1769-784AA (300 μM) into sympathetic neurons reduced NGF-mediated axon growth from an MYO10 average of 177 ± 14 μm/day to 90.6 ± 7.2 μm/day (Figures 5G, 5H, and 5M). In contrast, introduction of the phospho-mimetic peptide dyn1769-784EE (in which the serines 774/778 were substituted with glutamate) had no effect on NGF-mediated axon growth (Figures 5I and 5M). NT-3-mediated axon growth was not affected by delivery of either dyn1769-784AA or dyn1769-784EE (Figures 5J, 5K, 5L, and 5M). Together, these results provide evidence that calcineurin-mediated dephosphorylation of dynamin1 is a key signaling mechanism necessary for NGF-mediated, but not NT-3-mediated, axon growth.