Upregulation of synaptotagmin IV inhibits transmitter release in PC12 cells with targeted synaptotagmin I knockdown

Abstract

Background: The function of synaptotagmins (syt) in Ca2+-dependent transmitter release has been attributed primarily to Ca2+-dependent isoforms such as syt I. Recently, syt IV, an inducible Ca2+-independent isoform has been implicated in transmitter release. We postulated that the effects of syt IV on transmitter release are dependent on the expression of syt I.

Results: To test this, we increased syt IV expression in PC12 cells by either upregulation with forskolin treatment or overexpression with transfection. Two separately generated stable PC12 cell lines with syt I expression abolished by RNAi targeting were used and compared to control cells. We measured catecholamine release from single vesicles by amperometry and neuropeptide Y release from populations of cells by an immunoassay. In syt I targeted cells with forskolin-induced syt IV upregulation, amperometry measurements showed a reduction in the number of release events and the total amount of transmitter molecules released per cell. In cells with syt IV overexpressed, similar amperometry results were obtained, except that the rate of expansion for full fusion was slowed. Neuropeptide Y (NPY) release from syt I knockdown cells was decreased, and overexpression of syt IV did not rescue this effect.

Conclusions: These data support an inhibitory effect of syt IV on release of vesicles and their transmitter content. The effect became more pronounced when syt I expression was abolished.

Figure 1

Synaptotagmin IV (syt IV) expression is increased by forskolin. Control and syt I knockdown cells were treated with 50 μM forskolin (fsk) for 0, 2, 4 and 6 hours. (A) Immunoblot analysis shows that syt IV expression is upregulated after continuous fsk treatment for 4 hours in control PC12 cells. β-actin expression remained constant, and was used as a loading control. All lanes were loaded at 20 μg/lane. Syt IV expression was normalized to arbitrary densitometry units of β-actin measured in control PC12 cells (B), n = 7, and in shRNA-syt I knockdown cells (C), n = 4. Syt IV expressed peaked at 4 hours in both cell types (*p < 0.05).

Figure 2

Syt IV expression in syt I knockdown cells reduced evoked secretion. Representative amperometry recordings are shown from an untreated control cell (A), forskolin-treated control cell (B), untreated shRNA-syt I knockdown cell (C), and a forskolin-treated shRNA-syt I knockdown cell (D). The arrows depict where the continuously perfused solution was changed from HBSS to 50 mM K⁺ stimulating solution. (E) The average number of events per cell group is shown in the plot for forskolin-treated control (n = 19), shRNA-syt I knockdown (n = 16), and control transfected, CT (n = 12) cells, compared to untreated control (n = 24), shRNA-syt I knockdown (n = 24) and CT (n = 15) cells (**p < 0.01).

Figure 3

Overexpression of syt IV has similar effects on secretion from the cells as upregulation of syt IV. (A) Confocal images of cells are shown stained with fluorescently labeled antibodies to syt IV (red), EGFP (green), and nuclei (blue). The merge of all three images is shown on the right. The top group of images shows endogenous staining for syt IV in control cells that have not been transfected with the plasmids that express syt IV and EGFP. The lower group of images shows cells cotransfected with syt IV/EGFP plasmids in a 10:1 ratio. The images are representative of 3 independent experiments (scale bar = 10 μm). (B) Amperometry recordings are shown in a representative control cell transfected with the plasmid that expressed syt IV (pSyt IV) (C) and in an shRNA-syt I knockdown cell transfected with the pSyt IV (B). The arrows depict where the continuously perfused solution was changed from HBSS to 50 mM K⁺ stimulating solution. (D) The average number of events is plotted for control cells (n = 16) and two independent stable syt I knockdown cell lines (syt Ia, n = 16, and syt Ib, n = 20) compared to cells cotransfected with human syt IV/EGFP (control, n = 13 cells; syt Ia, n = 17 cells; and syt Ib, n = 17 cells; *p < 0.05 and **p < 0.01). (E) Basal and stimulated norepinephrine (NE) release were analyzed from 9-21 independent experiments. NE release is expressed as total NE (ng) per 50 μl sample (**p < 0.01).

Figure 4

The rate of the fusion pore opening is slowed in syt I knockdown cells that overexpress syt IV, but neither the amplitude nor quantal content of the individual release events are altered following syt IV upregulation. The 'double' means of the amplitude (A) and quantal content (B) values are shown in bar histograms for forskolin-treated and untreated control, shRNA-syt I knockdown, and CT cells. (C) The mean rate of rise (pA/ms) for each group of cells is plotted for the control and shRNA-syt I knockdown cells with control, endogenous levels of syt IV expressed, upregulated syt IV expression by forskolin treatment, or overexpression of syt IV by transfection with pSyt IV (*p < 0.05, **p < 0.01).

Figure 5

The overall number of events are reduced by syt IV upregulated in syt I targeted cells, but without any loss of the small or large diameter vesicle populations. Cumulative distribution histograms of the cubed root of the quantal content (pC)^1/3 are shown for untreated and forskolin-treated control (A, C) and shRNA-syt I knockdown (B, D) cells. The mean of each histogram is displayed on the individual graph generated with 48 bins.

Figure 6

LDCVs are synthesized and NPY release remains reduced in shRNA-syt I knockdown cells when syt IV is upregulated by forskolin treatment. (A) Large dense core vesicles are apparent in both control and fsk-treated cells. Electron micrographs reveal that in the control PC12 cells (top panels) and in the syt I knockdown cells (lower panels), both untreated (left side) and fsk-treated (right side) have large dense core vesicles (example shown in each panel by arrowhead). Scale bar = 500 nm. (B) The average stimulated NPY release from 9-12 independent experiments is plotted. Untreated and forskolin-treated control cells, and two independent syt I knockdown cell lines (shRNA-syt Ia and shRNA-syt Ic) were stimulated for 15 min with high K⁺ to evoke NPY release (**p < 0.01).