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. 2006 Apr 26;26(17):4630-7.
doi: 10.1523/JNEUROSCI.0009-06.2006.

A role for synaptotagmin VII-regulated exocytosis of lysosomes in neurite outgrowth from primary sympathetic neurons

Rosa M E Arantes  1 , Norma W Andrews
Affiliations

A role for synaptotagmin VII-regulated exocytosis of lysosomes in neurite outgrowth from primary sympathetic neurons

Rosa M E Arantes et al. J Neurosci. .

Abstract

Neurite outgrowth is mediated by the exocytosis of intracellular vesicles at the tips of elongating neuronal processes. The lysosomal vesicle-associated soluble N-ethylmaleimide-sensitive factor attachment protein receptor tetanus neurotoxin insensitive vesicle-associated membrane protein (TI-VAMP)/VAMP7 was previously implicated in membrane fusion events mediating neurite outgrowth, but the participation of lysosomes in this exocytic process has remained unclear. Here, we show that VAMP7 and the lysosomal glycoprotein Lamp1 extensively colocalize in vesicles present throughout the soma and neurite outgrowths of primary sympathetic neurons. Synaptotagmin VII (Syt VII), a Ca(2+)-sensing synaptotagmin isoform previously shown to interact with VAMP7 during lysosomal exocytosis in fibroblasts, was detected on a subset of these lysosomal glycoprotein 1 (Lamp1)/VAMP7-positive neuronal vesicles. Ionophore-stimulated exocytosis triggered exposure of the luminal domains of both Lamp1 and Syt VII at overlapping sites on the neuronal surface, indicating that the Syt VII-containing lysosomal compartments fuse with the plasma membrane in response to [Ca2+]i elevation. To determine whether Syt VII was required for the exocytic events mediating neurite extension, we followed the development of superior cervical ganglion neurons explanted from Syt VII-deficient mice. The results revealed a marked defect in neurite outgrowth and arborization, suggesting that Ca(2+)-dependent, Syt VII-regulated exocytosis of late endosomes/lysosomes plays a role in the addition of new membrane to developing neurite extensions.

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Figures

Figure 1.
Figure 1.
Lamp1-containing vesicles are present in the cell body and throughout neuritic outgrowths of SCG neurons. A, C, DIC images of SCG neurons at 2.5 h (A) or 4 h (C) after plating, showing the typical pyramidal, globular shape of the cells with thin extensions originating from the soma (arrows). B, D, E, Immunofluorescence of saponin-permeabilized SCG neurons with anti-Lamp1 mAbs (green) and nuclear DAPI staining (blue) after 1 h (B) and 4 h (D, E) in culture. Arrows point to distal regions and branching points of growing neurites, where Lamp1-positive vesicles were detected (D, E).
Figure 2.
Figure 2.
Lamp1 colocalizes with VAMP7 but not VAMP2 in developing SCG neurons. A, Immunofluorescence of permeabilized SCG neurons with mAbs against Lamp1 (green in merged image) and VAMP7 (red in merged image) after 1 h (top) or 4 h (bottom) in culture. Arrows point to vesicles containing both Lamp1 and VAMP7. B, Immunofluorescence of permeabilized SCG neurons with mAbs against Lamp1 (red in merged image) and VAMP2 (green in merged image) after 4 h in culture. Arrowheads point to Lamp1-positive vesicle clusters at distal regions of the growing neurite, which do not colocalize with VAMP2-positive synaptic vesicles.
Figure 3.
Figure 3.
Syt VII is present in Lamp1-positive compartments of developing SCG neurons. Immunofluorescence was performed on saponin-permeabilized SCG neurons after 2.5 h in culture with anti-Lamp1 mAbs and anti-Syt VII affinity-purified polyclonal antibodies. Arrows point to compartments containing both Lamp1 (red in merged image) and Syt VII (green in merged image). Nuclei in merged image were stained with DAPI (blue).
Figure 4.
Figure 4.
The luminal domain of Lamp1 and Syt VII colocalize on the surface of SCG neurons after Ca2+-triggered exocytosis. Intact SCG neurons cultured for 2.5 h were exposed or not to ionomycin and surface labeled at 4°C with antibodies against luminal epitopes of Lamp1 (red in merged image) and Syt VII (green in merged image). Arrows point to regions of coexposure of the luminal regions of Lamp1 and Syt VII on the neuronal surface. The DIC images on the right side of the bottom panel correspond to the same neurons shown on the left.
Figure 5.
Figure 5.
Neurite outgrowth is impaired in SCG explants from Syt VII-deficient mice. A, Average neurite length after increasing periods of time in culture. The data are expressed as the mean ± SD of neurite length average values from independent experiments (n = 3–5). B, Phase contrast images of SCG explants after 24 and 120 h in culture, illustrating the reduced neurite outgrowth observed in Syt VII−/− neurons (right panels).
Figure 6.
Figure 6.
Neurite growth is impaired in neurons isolated from SCG explants from Syt VII-deficient mice. Phase contrast images of isolated SCG neurons after increasing periods of time in culture show the reduced complexity of the outgrowing neuritic trees in Syt VII−/− neurons.
Figure 7.
Figure 7.
Neurite extension and arborization is impaired in neurons isolated from SCG explants from Syt VII-deficient mice. A, Average neurite length after increasing periods of time in culture. B, Average number of primary neurites after increasing periods of time in culture. C, Average number of branching points after increasing periods of time in culture. D, Average neurite density after 24 h in culture. The data are expressed as the mean ± SD of average values from independent experiments (n = 3).
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