Neuronal calcium sensor-1 modulation of optimal calcium level for neurite outgrowth

Abstract

Neurite extension and branching are affected by activity-dependent modulation of intracellular Ca 2+ , such that an optimal window of [Ca 2+ ] i is required for outgrowth. Our understanding of the molecular mechanisms regulating this optimal [Ca 2+ ] i remains unclear. Taking advantage of the large growth cone size of cultured primary neurons from pond snail Lymnaea stagnalis combined with dsRNA knockdown, we show that neuronal calcium sensor-1 (NCS-1) regulates neurite extension and branching, and activitydependent Ca 2+ signals in growth cones. An NCS-1 C-terminal peptide enhances only neurite branching and moderately reduces the Ca 2+ signal in growth cones compared with dsRNA knockdown. Our findings suggest that at least two separate structural domains in NCS-1 independently regulate Ca 2+ influx and neurite outgrowth, with the C-terminus specifically affecting branching. We describe a model in which NCS-1 regulates cytosolic Ca 2+ around the optimal window level to differentially control neurite extension and branching.

Caltubin, a Novel Molluscan Tubulin-Interacting Protein, Promotes Axonal Growth and Attenuates Axonal Degeneration of Rodent Neurons

“…Freshwater snails, L. stagnalis (a hermaphrodite), were kept in water at 20°C under a 12 h light/dark cycle and fed green leaf lettuce twice a week (Gardzinski et al, 2007;Hui et al, 2007). Adult snails with shell lengths of 15-20 mm were used.…”

“…Snails were anesthetized with 10% (v/v) Listerine for 10 min as previously described (Syed et al, 1990;Hui et al, 2007). Central ganglia were dissected in snail saline containing the following (in mM): 51.3 NaCl, 1.7 KCl, 4.1 CaCl 2 , 1.5 MgCl 2 , pH was adjusted to 7.9 with 1 M HEPES/NaOH, and incubated with 3 mg/ml trypsin (type III; Sigma-Aldrich) for 20 min.…”

“…For whole-animal RNA knockdown experiments, snails were anesthetized with 10% (v/v) Listerine, and 2 l of 20 M control siRNA or caltubin siRNA was injected into the head, above the central ganglia using a microliter syringe (Hamilton Company) Hui et al, 2007;Lu and Feng, 2011). Ganglia were removed 48 h after injection, and RNA or protein was extracted.…”

“…Antibodies against caltubin were raised in rat against synthetic peptides corresponding to the N-terminal (MERAFEDVRSQHRD) and C-terminal (DG-KLSLDEFKTLYSP) regions of the protein. Cultured PeA neurons were fixed, permeabilized, and incubated with specific primary antibodies (NCS-1: 1:300, BIOMOL, catalog #BML-NL3750-0100; ␤-tubulin: 1:300, Sigma-Aldrich, catalog #T0198; ␣-tubulin: 1:300, Sigma-Aldrich, catalog #T6074; ␤-actin: 1:300, Sigma-Aldrich, catalog #A1978; caltubin: 1:200), as described previously (Gardzinski et al, 2007;Hui et al, 2007). Cells were then incubated with their respective secondary antibody (488 goat anti-rat, Millipore Bioscience Research Reagents; Alexa Fluor 568 goat anti-mouse, Invitrogen; Alexa Fluor 633 goat anti-rabbit, Invitrogen) for 2 h at room temperature.…”

“…Confocal images were acquired using a TCS SL laser confocal microscope (Leica confocal software, version 2.5; build 1347; Leica Microsystems), as described previously (Gardzinski et al, 2007;Hui et al, 2007). Each Z-plane was 0.5 m. Background fluorescence was determined in three regions not containing cells throughout the entire z-stack.…”