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First published online November 26, 2007
doi: 10.1242/10.1242/dev.008979

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

¹ Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, M5S 1A8, Canada.
² Molecular and Medical Genetics, Faculty of Medicine, University of Toronto, Toronto, M5S 1A8, Canada.
³ Mount Sinai Hospital, Toronto, Canada.

View larger version (46K): [in this window] [in a new window]   Fig. 1. An NCS-1-related protein is expressed in L. stagnalis neurons. (A) Protein sequence alignment of the full NCS-1 clone from L. stagnalis (DQ099793) and the homologous proteins from A. californica (Q16981), Rattus norvegicus (P62168), Drosophila melanogaster (NP_996502) and Caenorhabditis elegans (P36608). Alignment was performed in ClustalW and the plot made in GeneDoc. (B) Western blot analysis of whole L. stagnalis and rat brains. Polyclonal anti-rat NCS-1 antibody identified a single band migrating at 23 kDa in both rat and L. stagnalis ganglia preparations. (C,D) Confocal imaging of anti-NCS-1 reactivity in a L. stagnalis PeA neuron cultured for 3 days in CM. Epifluorescent image of PeA cell stained with the anti-rat NCS-1 antibody show immunoreactivity throughout the neuron (C). Analysis of cellular distribution of NCS-1 reactivity in the different regions of the cell (D, n=5 cells). The data are presented as mean±s.e.m. Asterisk indicates a significant difference (P<0.05) compared with that in the primary neurites. Relativity intensities compared to primary neurites are 2.95±0.39 for soma, 1.91±0.31 for branch points, 1.27±0.29 for secondary neurites and 1.85±0.10 for growth cones.

View larger version (24K): [in this window] [in a new window]   Fig. 2. 27-mer dsRNA knockdown of NCS-1 in L. stagnalis. (A,B) Western blot analysis of ganglia protein extracts isolated 2 days after the snails were injected with either NCS-1 dsRNA (NCS dsRNA), control dsRNA (Ctrl dsRNA) or water (Ctrl), showing that NCS-1 dsRNA reduced the protein level of NCS-1. (A) Representative semiquantitative immunoblots of NCS-1 and β-actin expression in the central ganglia with indicated pretreatments. (B) A summary of normalized intensity of NCS-1 to β-actin ratio from dsRNA-treated groups compared with that from the control group. The solid line indicates the NCS-1 to β-actin ratio of the control group. NCS-1 dsRNA treatment (2 µl of 20 µM) resulted in an average 35.7±5.1% (n=4) reduction in the normalized relative protein expression of NCS-1 to β-actin, whereas the control dsRNA treatment did not affect the relative expression level of NCS-1 (2.7±2.5% reduction). (C,D) Confocal fluorescence images obtained from double immunocytochemical staining of β-actin and NCS-1, under different dsRNA treatments (5 nM) for 24 hours. (C) Representative β-actin and NCS-1 immunofluorescence images of PeA neurons in culture for 24 hours. (D) Summary of the NCS-1 levels from various regions of the culture PeA neurons. Relative fluorescence intensities of NCS to β-actin ratio (FNCS/FActin) for the control dsRNA group: soma 1.48±0.08; neurite 1.51±0.12; branch point 1.44±0.05; and growth cone 1.60±0.12. The relative ratio for NCS dsRNA group: soma 0.84±0.06; neurite 0.94±0.13; branch point 0.97±0.08; growth cone 1.10±0.16. The data are presented as mean±s.e.m. and are obtained from a total of 10 control and 15 NCS-1-specific dsRNA-treated cells. Asterisk indicates significant difference (P<0.05) between the control and NCS-1 dsRNA-treated conditions.

View larger version (44K): [in this window] [in a new window]   Fig. 3. NCS-1 knockdown enhances neurite outgrowth in L. stagnalis. (A) Phase-contrast images of a single PeA neuron before and 24 hours after addition of water (right), 5 nM control (middle) or NCS-1-specific (left) dsRNA. Cells were isolated and cultured in CM for 24 hours before treatment. (B,C) Neurite outgrowth was examined for the degree of branching (B) and the rate of extension (C). Both the number of new neurites (the degree of branching) and the rate of neurite extension were significantly higher after the NCS-1-specific dsRNA treatment compared with controls. The number of new neurites: mock control 1.50±0.39; control dsRNA (ctrl dsRNA) 1.04±0.39; and NCS-1 dsRNA 3.93±0.65. The rate of the extension (µm/hour): mock control 3.77±0.67; control dsRNA 3.47±0.49; NCS-1 dsRNA 6.44±0.49. The data are presented as mean±s.e.m. and obtained from 24 control, 43 control dsRNA and 57 NCS-1-specific dsRNA-treated cells. Asterisk indicates significant difference (P<0.05) from control condition.

View larger version (41K): [in this window] [in a new window]   Fig. 4. NCS-1 knockdown affects the action potential-induced fura-2 Ca2+ signal in growth cones in an activity-dependent manner. (A) Representative epifluorescent images of Ca2+ signals in L. stagnalis neurites treated with the control dsRNA or NCS-1 dsRNA. The Ca2+ ratiometric signals excited at 340 and 380 nm (F340/380) were obtained at rest (basal) or elicited by two, four or six depolarization-induced action potentials. Action potentials were evoked by 1 second depolarization using an intracellular sharp electrode impaled into the soma. (B) Representative examples showing that the Ca2+ signal (F340/380) was as a linear function of the number of action potentials. The Ca2+ signal from a single growth cone region was plotted against the number of action potentials used to evoke the response. The line represents a linear best fit of the data, which shows no apparent saturation of the signal within the range of action potentials used. The slopes of the fits are 0.037±0.004 (control), 0.031±0.003 (control dsRNA) or 0.011±0.001 (NCS-1 dsRNA). The slope was used to quantify the dynamics of Ca2+ change resulting from action potential stimulation. (C) Summary of the slope of the fit described in B from three to five cells with treatments as indicated. The mean slope dependencies are 0.037±0.006 (control, n=4), 0.027±0.003 (control dsRNA, n=3), and 0.010±0.004 (NCS-1 dsRNA, n=5). The data are presented as mean±s.e.m. Asterisk indicates significant difference (P<0.05) from the controls. AP, action potentials.

View larger version (29K): [in this window] [in a new window]   Fig. 5. CTN reduces Ca2+ currents from growth cones of isolated PeA neurons in L. stagnalis. (A) Representative Ca2+ (ruptured) whole-cell currents from a single transected growth cone (including adjacent neurite) without or with 50 µM scrambled CTN (srCTN) or CTN treatment. The currents were evoked from a holding potential of -100 mV and stepped from -60 mV to +80 mV at 10 mV increments, using a P/4 protocol. Currents were normalized by capacitance (pF). A phase-contrast imaging of a transected growth cone is shown in the insert at the bottom left. (B) Current density-voltage (I-V) plots from the traces in A. Peak current densities are smaller after CTN treatment compared to srCTN treatment and control conditions. The line represents a modified Boltzmann fit of the data, as described in Materials and methods and listed as G(pS/pF), Vrev (mV) and Vh (mV): control 0.34, 79 and 23; srCTN 0.27, 78 and 20; CTN 0.18, 80 and 40, respectively. (C) Average peak current density suggests that CTN significantly (P<0.05) reduces the peak current density in growth cones of PeA neurons. Data were obtained from five control (10.3±3.0 pA/pF), six srCTN (9.0±1.3 pA/pF) and five CTN (2.1±0.9 pA/pF) experiments. The data are presented as mean±s.e.m. Asterisk indicates significant difference (P<0.05) from the control condition.

View larger version (40K): [in this window] [in a new window]   Fig. 6. The C-terminus of NCS-1 peptide (CTN) affects the action potential-induced fura-2 Ca2+ signal in growth cones in an activity-dependent manner. (A) Representative epifluorescent images of Ca2+ signals in L. stagnalis neurites before (control) and after CTN treatment (50 µM). The Ca2+ ratiometric signals (F340/380) were obtained at rest (basal) or elicited by one, four or six depolarization-induced action potentials. Action potentials were evoked by 1 second depolarization using an intracellular sharp electrode impaled into the soma. (B) Representative examples showing the Ca2+ signal (F340/380) were as a linear function of the number of action potentials in the neurites under different conditions. The Ca2+ signal from a single growth cone region was plotted against the number of action potentials used to evoke the response. The line represents a linear best fit of the data, which shows no apparent saturation of the signal within the range of action potentials used. The slopes of the plots are 0.037±0.004 (control), 0.041±0.002 (srCTN) and 0.019±0.002 (CTN). (C) Summary of the slope, as described in B, from three to eight cells with treatments as indicated. The mean slope dependencies are 0.037±0.006 (control, n=4), 0.042±0.004 (srCTN, n=3) and 0.021±0.003 (CTN, n=8). The data are presented as mean±s.e.m. Asterisk indicates significant difference (P<0.05) from the controls. AP, action potentials.

View larger version (66K): [in this window] [in a new window]   Fig. 7. CTN increases the rate of neurite branching but not of neurite growth in L. stagnalis. (A) Phase-contrast images of a single PeA neuron before and 24 hours after treatment with water (control, left), 50 µM srCTN (middle) or 50 µM CTN (right). Cells were isolated and cultured in CM 24 hours before treatment. (B,C) Neurite branching but not the rate of neurite extension was selectively affected by CTN treatment. (B) Branching (as indicated by the number of new neurites) increased in CTN-treated cells as soon as 3 hours after treatment (mock control 1.50±0.39; srCTN 0.83±0.25; CTN 4.57±0.81). (C) The rate of the neurite extension did not differ between the treatments (mock control 3.77±0.67 µm/hour; srCTN 3.79±1.02 µm/hour; CTN 3.43±0.38 µm/hour). The data are presented as mean±s.e.m. and were obtained from 24 control, 21 srCTN and 44 CTN cells. Asterisk indicates significant difference (P<0.05) from control condition.

View larger version (29K): [in this window] [in a new window]   Fig. 8. Model of NCS-1 in Ca2+-dependent neurite outgrowth pattern in L. stagnalis. (A) Neurite outgrowth processes, elongation and branching, are dependent on [Ca2+]. At basal Ca2+ (100 nM), both processes are negligible (Stage 1). Optimal electrical or chemical stimulation increases [Ca2+]i (200-300 nM) and causes both process to be activated (Stage 2). This level of Ca2+ would be sufficient to allow binding to the high-affinity sites in NCS-1, thus allowing further Ca2+ increases (400-500 nM) through a positive-feedback mechanism such as NCS-1 stimulation of TRPC5 (Stage 3). At this stage, neurite extension is inhibited without affecting branching; but as the morphological determination of branching cannot be observed without extension, the branching also appears stalled. If the levels of Ca2+ continue to elevate (micromolar quantities), the likelihood of binding to the low-affinity site in NCS-1 is increased, thereby facilitating voltage-dependent Ca2+ channels and consequently raising the levels higher (Stage 4). At this stage, neurite outgrowth processes are both limited. (B) Calibration curve of Fura-2 signal against free [Ca2+]i. From the Ca2+-imaging data (Figs 4 and 6), a calibration curve was constructed as described previously (Feng et al., 2002; Grynkiewicz et al., 1985). The arrows indicate the Ca2+ levels in PeA cells at either resting (basal) or electrically stimulated conditions with different treatments, and their corresponding stages as described in the model. (C,D) Structure of NCS-1 protein. (C) Tertiary structure of human NCS-1 (also known as FREQ - HUGO) protein (1G8I). Indicated are the N- and C-termini as well as the four EF-hand structures. Ca2+ ions are shown as yellow spheres bound to the three functional EF-hands. (D) Schematic structure of the protein showing the four EF-hands is shown below. Of the EF-hands, three are functional (coloured) and one is non-functional (grey). The high-affinity Ca2+-binding sites (red), EF3 and EF4, are adjacent to each other and the C-terminus. Binding of Ca2+ to EF3 and the low-affinity site EF2 (blue) exposes a hydrophobic crevasse and putative binding pocket for interacting proteins (Bourne et al., 2001).

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