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NERVOUS SYSTEM CELL BIOLOGY

Stable gene silencing of synaptotagmin I in rat PC12 cells inhibits Ca²⁺-evoked release of catecholamine

¹Department of Pharmacological and Physiological Science, St. Louis University School of Medicine, St. Louis, Missouri; and ²Department of Neurobiology, Pharmacology and Physiology, University of Chicago, Chicago, Illinois

Submitted 15 October 2005 ; accepted in final form 31 January 2006

	ABSTRACT

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION GRANTS REFERENCES

 
Synaptotagmin (syt) I is a Ca²⁺-binding protein that is well accepted as a major sensor for Ca²⁺-regulated release of transmitter. However, controversy remains as to whether syt I is the only protein that can function in this role and whether the remaining syt family members also function as Ca²⁺ sensors. In this study, we generated a PC12 cell line that continuously expresses a short hairpin RNA (shRNA) to silence expression of syt I by RNA interference. Immunoblot and immunocytochemistry experiments demonstrate that expression of syt I was specifically silenced in cells that stably integrate the shRNA-syt I compared with control cells stably transfected with the empty shRNA vector. The other predominantly expressed syt isoform, syt IX, was not affected, nor was the expression of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins when syt I levels were knocked down. Resting Ca²⁺ and stimulated Ca²⁺ influx imaged with fura-2 were not altered in syt I knockdown cells. However, evoked release of catecholamine detected by carbon fiber amperometry and HPLC was significantly reduced, although not abolished. Human syt I rescued the release events in the syt I knockdown cells. The reduction of stimulated catecholamine release in the syt I knockdown cells strongly suggests that although syt I is clearly involved in catecholamine release, it is not the only protein to regulate stimulated release in PC12 cells, and another protein likely has a role as a Ca²⁺ sensor for regulated release of transmitter.

RNA interference; amperometry; exocytosis

Although a number of proteins are involved in the release of vesicles and are reported to be critical for release, one vesicle protein, synaptotagmin (syt), has been established as a Ca²⁺ sensor and regulator of both exo- and endocytosis (35, 40). The family of syt proteins is composed of at least 16 different genes, syt I–XVI (9). Syt I, the best studied isoform, is composed of a short intravesicular NH₂ terminus followed by a single transmembrane domain and two extravesicular Ca²⁺-binding domains, or C2 domains, that are highly conserved among species (36, 37). Subtle differences among Syt isoforms potentially lead to a large number of possible functions within this protein family (for review, see Ref. 45). Although many of the isoforms are integral vesicle membrane proteins found in neuronal and neuroendocrine vesicles (36), the isoforms vary in their ability to bind Ca²⁺ and to bind phospholipids as a function of Ca²⁺ through their two C2 domains (1, 10, 26, 40, 42, 48, 52, 54). Syt isoforms also can form homo- and heterooligomers that lead to further complexity of the syt family (25, 56).

Evidence from a number of systems indicates that syt I is involved in vesicle docking, fusion, and recycling (for reviews, see Refs. 43, 45). For example, fast synchronous release was abolished in mice and Drosophila that lack syt I, but slow asynchronous release remained (19, 57) and even compensated for the loss of fast synchronous release (33). Early evidence that syt I plays a role in secretion came from results with antibodies, recombinant fragments, and peptides designed against specific regions of syt I protein that interfered with vesicle release (12). Furthermore, because mutations in the syt I gene disrupted the fourth-order Ca²⁺ dependence of synchronous release, it was proposed that syt I is a Ca²⁺ sensor for regulated release of transmitter (15, 28, 42, 57). However, alternative explanations may explain the observations, as syt I may act to control the expression and trafficking of other proteins, or it may physically act as a link between the vesicle release machinery and the pore of the Ca²⁺ channel. Syt I also is abundantly localized in endocrine vesicle membranes of adrenal chromaffin cells and pheochromocytoma (PC12) cells. Both of these cells have been well utilized to study the mechanism of Ca²⁺-dependent transmitter release from vesicles. In this study, we investigated whether syt I is essential for Ca²⁺-evoked release of catecholamines in the model neuroendocrine PC12 cells by using plasmid-based RNA interference (RNAi) to establish a cell line in which syt I levels are stably and specifically reduced. Although viability, phenotype, and resting Ca²⁺ levels are not altered compared with control cells, syt I knockdown cells exhibit significantly reduced, but not eliminated, release of catecholamines.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION GRANTS REFERENCES

 
Antibodies. The anti-syt I and anti--actin mouse monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA). The anti-syt II and anti-syt VII goat antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-syt III rabbit antibody was a generous gift from Dr. Bryan A. Wolf (University of Pennsylvania, Philadelphia, PA). The anti-syt IX mouse antibody was obtained from BD Biosciences-Pharmingen (termed syt V; San Diego, CA) and recognizes the isoform whose amino acid sequence begins with MFPEP. Anti-SNAP-25 and anti-VAMP-2 rabbit antibodies were obtained from Alomone Labs (Jerusalem, Israel); anti-syntaxin I mouse antibody was obtained from Medical and Biological Laboratories (Nagoya, Japan); and anti-synaptophysin I mouse monoclonal antibody was obtained from Chemicon (Temecula, CA). For immunoblots, horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology and also recognize the Cruz molecular weight standards. For immunocytochemistry, secondary goat-anti-mouse FITC-conjugated antibody was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).

Plasmids. We constructed a plasmid designed to express short hairpin RNAs (shRNAs) from a U6 RNA polymerase III promoter and to allow selection of stably transfected cells with G418 (6). The vector (pG418-shRNA) was analyzed using extensive restriction enzyme digestion and partial sequencing. A human syt I plasmid was purchased from American Type Culture Collection (GenBank accession no. BC058917).

Design of shRNA insert. Target sites for silencing syt I were selected using Target Finder (Ambion, Austin, TX). Two complementary DNA oligonucleotides incorporating the chosen target site AGACTTAGGGAAGACCATG (bp 846–864 of GenBank accession no. X52772), a loop sequence, and the reverse complement of the target site were designed. A nine-nucleotide loop sequence was used, TTCAAGAGA (4). The pair of 56-bp DNA oligonucleotides was purchased from Integrated DNA Technologies (Coralville, IA), 5' phosphorylated, and PAGE purified. The pair of oligonucleotides was annealed and ligated into pG418-shRNA digested with XbaI and BpiI. The resulting plasmid, termed shRNA-Syt I, was sequenced to ensure that the insert was present and correct.

Cell culture. An early passage of rat PC12 cells was maintained in culture according to standard methods (21, 22). Cells were grown in medium that consisted of RPMI 1640, 10% heat-inactivated horse serum, 5% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin in a 37°C, 5% CO₂ incubator. The cells were passaged every 7 days, and after 10 passages, a new vial of cells was thawed from stocks frozen in liquid nitrogen to help ensure minimal line drift. The cells were plated for either further passages or experimentation. Cells were treated with 200 ng/ml nerve growth factor (NGF; 2.5S grade II from mouse submaxillary gland; Roche, Indianapolis, IN) for 5–12 days until use.

Transfection. Cells were transfected with 4 µg of transfection grade shRNA-syt I plasmid DNA or shRNA plasmid that did not contain an insert (referred to as control transfection, or CT) per 35-mm tissue culture plate of cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) as described previously (23). Cells were replated 24 h later. Selection media that contained 100 µg/ml G418 (Sigma, St. Louis, MO) was added to the cells 24 h after replating. This concentration of G418 was found to be optimal to kill untransfected cells. Cells grew in selection medium up to 28 days, and single colonies were picked from the plates and expanded for freezing and testing for stable incorporation of the shRNAs.

PCR. Drug-resistant cell lines were screened for insertion of shRNA plasmids into the genome by PCR with genomic DNA, plasmid-specific primers, and MasterTaq PCR reagents (Eppendorf, Westbury, NY). PCR reactions were performed with 35 cycles of 95°C for denaturing (30 s), 54°C for annealing (1 min), and 72°C for elongating (1 min). A final elongation step was run for 10 min at 72°C. The plasmid-specific primers were designed to match sequences on either side of the multiple cloning site to determine whether the plasmid had incorporated into the genome. The forward primer matched the plasmid sequence 100-bp 5' from the KpnI site in the multiple cloning site (GGCGATTAAGTTGGGTAACG) and was used with the reverse primer that was situated about 100 bp into the PGK polyA tail and 3' to the multiple cloning site (GCCTGAAGAACGAGATCAGC). This set of primers resulted in a PCR product of 570 bp for the empty plasmid (shRNA) and 600 bp for the plasmid that contained the insert (shRNA-syt I). Because PC12 cells can develop spontaneous resistance to G418, we determined that cell lines that tested positive with the primers described above also tested positive for the neomycin cassette. A second set of primers was designed to match sequences within the neomycin cassette; the forward primer was GACAATCGGCTGCTCTGATG, and the reverse primer was ACCATGATATTCGGCAAGCA. Incorporation of the neomycin resistance cassette resulted in a PCR product of 512 bp with this set of primers.

Immunoblot analysis. Cells were grown in culture to confluency, removed from the plates, washed, and lysed by resuspension in buffer containing 20 mM Tris, pH 7.5, 1% Triton X-100, 10% glycerol, 2 mM EDTA, and 0.1% protease inhibitor cocktail (Sigma). After 30 min on ice, the cells were sonicated for three short bursts and centrifuged for 2 min at 16,000 g at 4°C. Portions of the supernatant were used to determine protein concentration (see below) or mixed with lithium dodecyl sulfate (LDS) loading buffer (Invitrogen). Crude membranes were prepared from rat cerebellum by homogenization for 10 s on ice in 20 mM Tris, pH 7.5, 2 mM EDTA, and 0.25 M sucrose using a Polytron. After incubation for 1 min on ice, the homogenization was repeated three times. The tissue was further disrupted in a Dounce homogenizer with 15 strokes and centrifuged for 10 min at 1,000 g at 4°C. The supernatant was collected and centrifuged for 30 min at 16,000 g at 4°C. The supernatant was removed, and the pellet was resuspended in homogenization buffer without sucrose. Protein concentration was determined with Coomassie Plus protein assay reagent (Pierce, Rockford, IL) with BSA as a standard. The protein was mixed with LDS loading buffer and heated to 70°C for 10 min.

Total lysates were electrophoresed on a 10% NuPAGE Bis-Tris gel (Invitrogen), transferred to polyvinylidene difluoride membrane (Millipore, Billerica, MA), and blocked overnight at 4°C in 5% nonfat dried milk in Tris-buffered saline with 0.1% Tween 20. The membrane was incubated with primary antibody (all 1:1,000 dilutions except synaptophysin, used at 1:5,000 dilution), followed by a horseradish peroxidase-conjugated secondary antibody used at dilutions that ranged from 1:30,000 to 1:50,000 (Santa Cruz Biotechnology). Immunoreactive bands were detected and visualized using ECL Advance reagent (GE Healthcare, Piscataway, NJ), exposed to X-ray film, and developed. A positive control tissue or cellular lysate that is known to express the particular syt isoform protein of interest was included on each immunoblot. Rat cerebellar tissue was used for syt I, II, and IX, and the RINm5F pancreatic -cell line was used for syt III and VII (generously provided by Dr. John Corbett, St. Louis University, St. Louis, MO). All immunoblots are representative of three or more independent experiments. Quantitative analysis of the immunoblots was performed with ImageJ (http://rsb.info.nih.gov/ij/) and normalized to arbitrary densitometry units of -actin.

Immunocytochemistry. Cells were replated to collagen-treated Permonox chamber slides and fixed 1 (PC12 cells) or 2 days later (NGF-PC12 cells) with 2% paraformaldehyde. The cells were permeabilized with 0.1% Triton X-100, blocked with 2 mg/ml BSA, and incubated with anti-syt I mouse monoclonal antibody (1:5 dilution) or anti-syt IX mouse antibody (1:200 dilution), blocked, and incubated with a secondary goat-anti-mouse FITC-conjugated antibody (1:200; Jackson ImmunoResearch Laboratories). The cells were washed, mounted with Vectashield (Vector Laboratories, Burlingame, CA), and sealed. Images were acquired using a scanning confocal microscope (Bio-Rad MRC 1024; Hercules, CA) with a 4x zoom on a 60x oil-immersion lens (NA 1.4). For the human syt I rescue experiments, cells were permeabilized with 0.1% Triton X-100, blocked with 10% normal goat serum in PBS, and incubated with anti-syt I mouse monoclonal antibody (1:5 dilution), blocked, and incubated with a secondary rhodamine red-X-conjugated AffiniPure donkey anti-rabbit IgG (H+L) (1:2,000; Jackson ImmunoResearch Laboratories), sealed, and mounted as described above. Images were acquired using a 3x zoom on a 60x oil-immersion lens (NA 1.4). All immunocytochemistry results are representative of three or more independent experiments.

Ca²⁺ imaging. Intracellular Ca²⁺ concentrations ([Ca²⁺]_i) were measured using ratiometric fluorescence imaging with fura-2. Cells were replated 4–8 h before experimentation, loaded with fura-2, and prepared for recording as described previously (23). For each experiment, 20–40 cells were selected and individually imaged using a commercial imaging system (InCytIM2; Intracellular Imaging, Cincinnati, OH). Image pairs were obtained every 10 s at 340- and 380-nm wavelengths. Backgrounds were subtracted from each wavelength, and the 340-nm image was divided by the 380-nm image to provide a ratiometric image. Ratios were converted to free [Ca²⁺]_i by comparing data with fura-2 calibration curves made in vitro by adding fura-2 (50 µM free acid) to solutions that contained known Ca²⁺ concentrations (0 to 602 nM; Molecular Probes-Invitrogen). Cells were exposed to a high-K⁺ solution that contained (in mM) 50 KCl, 87 NaCl, 1 MgCl₂, 5 CaCl₂, 10 D-glucose, and 12 HEPES (pH 7.3). Resting Ca²⁺ level was determined from the average of measurements from all cells in four independent experiments during the 60 s before stimulation. The change in Ca²⁺ was calculated from the five points during the peak change in Ca²⁺ levels that occurred in response to stimulation.

Amperometry. Carbon fibers (6-µm diameter; Goodfellow, Huntingdon, UK) were threaded into electrodes, pulled with a vertical puller, sealed with epoxy, painted with Sylgard (Dow-Corning, Midland, MI), and stored at 95°C until use. A newly cut surface of a carbon fiber electrode (5) was gently pressed against the side of a cell, because the collection efficiency of detectable events is thought to provide the highest yield in this recording configuration (5, 39). The electrode was backfilled with 3 M KCl and clamped at +700 mV versus a Ag-AgCl ground with an NPI amplifier (ALA Scientific, Westbury, NY) to detect the oxidized catecholamine transmitter. The signal was low-pass filtered at 2 kHz (8-pole Bessel; Warner Instruments, Hamden, CT). A 16-bit analog-to-digital converter (National Instruments, Austin, TX) was interfaced with custom-written software (graciously provided by Dr. Aaron P. Fox, University of Chicago, Chicago, IL), acquired at 10 kHz, and stored on a personal computer. Root mean square (RMS) noise was typically <1 pA. Amperometric spike features such as amplitude, quantal charge, and kinetic parameters were analyzed using custom-written analysis software (graciously provided by Dr. Eugene Mosharov, Columbia University, New York, NY). The detection threshold for an event was set for five times the baseline RMS noise level, and no trace was analyzed with RMS noise >2.5 pA. Overlapping events, those whose spike had not returned to baseline before the next event, were not considered in the analysis even though they were uncommon events. Rise time was measured over 10–90% of the spike's maximal amplitude. The area under individual amperometric spikes is equal to the charge (Q, measured in pC) per release event.

Recording. Cells were treated with NGF for 5–10 days, replated to glass coverslips, and cultured overnight. Before recordings, cells were preloaded with 200 nM norepinephrine (Sigma) for 2–5 h at 37°C. Recordings were conducted on adherent cells exposed to constant and uniform perfusion (1 ml/min) in a recording chamber with an approximate volume of 200 µl. Cells were perfused for 5–10 min with Hanks' buffered salt solution (HBSS), and recording was begun during the HBSS perfusion for 20 s during recording for basal release before changing to a high (50 mM)-K⁺ solution for stimulation of transmitter release for the remainder of the 4-min recording period. All experiments were performed at ambient temperature, 22–24°C.

Catecholamine measurements. Catecholamines were identified and quantified using high-performance liquid chromatography (HPLC)-electrochemical detection, as previously described (7, 11), from cells treated with NGF for 10–12 days. Release was stimulated with a 50 mM K⁺ solution. The system consists of a Varian Pro-Star solvent delivery system and a model 9090 autosampler (Varian) coupled to a C18 column and an ESA Coulochem II detector. Separations were performed isocratically with the use of a filtered and degassed mobile phase consisting of 12% methanol, 0.1 M sodium phosphate, 0.2 mM sodium octyl sulfate, and 0.1 mM EDTA, adjusted to pH 2.7 with phosphoric acid. A computer was used to collect and store the chromatograms that were analyzed using Varian Star software.

Syt I rescue. We confirmed by sequencing that the target sequence of the shRNA for syt I differed by four base pairs from the human syt I plasmid. Because of this four-nucleotide difference, human syt I should not be silenced by the shRNA. Syt I knockdown cells were treated with NGF and transiently transfected for 2 days with the human syt I plasmid before experimentation.

Statistical analysis. All data are displayed as means ± SE, and Student's t-test was used to compare significance.

	RESULTS

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION GRANTS REFERENCES

 
Stable cell lines generated to express shRNAs against syt I. We utilized the plasmid-based approach to RNAi because it allows the generation of stable cell lines. Such stably transfected cell lines continuously transcribe shRNAs from an RNA polymerase III type promoter (e.g., U6 or H1 promoters). These shRNAs are effective in silencing genes (4, 31, 58). This approach has the advantage that it does not require expensive oligonucleotides or RNase-free conditions, and it allows for generation of stably transfected cell lines in which all cells express homogeneous levels of a particular protein. This was an important aspect of our experiments, because we knew from preliminary experiments (not shown) that only 15% or less of our particular line of PC12 cells routinely are transiently transfected, an efficiency that is fairly low for RNAi purposes. To detect a significant reduction in protein expression and to confirm gene silencing by immunoblot analysis, a relatively high transfection efficiency rate is required, 85% or greater. Therefore, we stably transfected cells with a plasmid designed to make hairpin RNAs and generated a homogeneous population of cells that express shRNAs.

PC12 cells were stably transfected with two different plasmids, the shRNA-syt I plasmid and a plasmid that lacked an insert that was used as a positive control for the transfection (referred to as control transfection, or CT). Colonies were expanded, and genomic DNA was prepared from each of the stable cell lines. PCR was used to test for incorporation of the shRNA plasmid into genomic DNA with specific primers that recognized a sequence of the plasmid flanking the insert (and that does not recognize any known genomic sequence), or within the neomycin resistance cassette of the plasmid.

RNAi stably knocks down syt I expression. We tested whether syt I expression was specifically "silenced" in cells stably transfected with shRNA-syt I using immunoblot analysis. Lysates were made from cells expressing the shRNA designed to recognize and reduce the expression of syt I (shRNA-syt I) and from cells stably transfected with the empty vector (CT). Expression of proteins from these cells was compared with expression of syt I in untransfected PC12 cells and rat cerebellum as controls. Blots were probed with an anti-syt I antibody to detect a band of 65 kDa, the expected apparent molecular weight. Six of the seven shRNA empty plasmid cell lines (CT) tested positive for the plasmid with PCR and did not exhibit a reduction in expressed syt I as expected. In contrast, the four shRNA-syt I cell lines that tested positive for the shRNA insert by PCR exhibited varying degrees of reduction in expressed syt I protein. We continued experimentation with one of each of the stable cell lines, and all experiments described were performed with lines termed CT and shRNA-syt I. In addition, both of these lines tested positive for incorporation of neomycin resistance cassette by PCR. Figure 1A, top, shows a blot probed with the anti-syt I antibody. Both the untransfected PC12 cells (control) and the empty vector stable cells (CT) have a detectable band of protein at 65 kDa compared with cells expressing the shRNA designed specifically to knockdown syt I (shRNA-Syt I). Syt I levels in the knockdown cells were 0.02% of those in control cells (Fig. 1A, bottom), which was not different from zero.

View larger version (53K): [in this window] [in a new window]   Fig. 1. RNA interference (RNAi) is specific for targeting and knocking down expression of synaptotagmin I (syt I). A, top: syt I expression is knocked down in stably transfected cell lines. Immunoblot analysis shows that syt I is reduced to undetectable levels in the cells stably transfected with the short hairpin RNA (shRNA)-syt I plasmid (lane 2) compared with either control untransfected PC12 cells (lane 1) or cells transfected with a plasmid that does not contain an shRNA insert (CT; lane 3). All lanes were loaded at 2 µg/lane. Expression of syt IX, the most abundant isoform, is unaffected in the cells expressing the shRNA-syt I plasmid (0.1 µg/lane). Expression of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins involved in vesicle release or expression of a structural protein, -actin, also was unaffected (1 µg/lane). A, bottom: syt I expression was significantly reduced compared with control PC12 cells and normalized to arbitrary densitometry units of -actin (n = 5, P < 0.001). This is the only significant difference between any expressed proteins on the blots. B: increasing amounts of loaded lysate (shown above each lane) for the syt I knockdown cells show that there was no detectable syt I expression for loaded lysate to 20 µg/lane. C: knocking down expression of syt I did not cause an increase of expression of syt II, III, or VII. Lane 1 shows lysates made from control tissues: cerebellum for syt II and a pancreatic cell line, RINm5F cells, for syt III and VII. Lane 2 shows lysates from control untransfected PC12 cells; lane 3, from syt I knockdown cells; and lane 4, from CT cells (syt II and III, 2 µg/lane, and syt VII, 3 µg/lane).

To determine whether suppression of protein expression is specific for syt I, we performed immunoblot analysis to test whether the shRNA-syt I and CT cell lines exhibited a reduction in protein expression for syt IX, the most abundantly expressed syt isoform and one that is similar in sequence to Syt I. We also tested whether additional proteins involved in regulated secretory vesicle release were altered in their expression, such as SNAP-25, VAMP-2 (or synaptobrevin), syntaxin I, and a structural protein, -actin. Figure 1A shows that cells stably transfected with shRNA-syt I specifically suppressed the expression of syt I. The shRNA-syt I cells were tested for low abundance expression by using increasing concentrations of loaded protein amounts. Even with 20 µg of lysate protein, the shRNA-syt I cells did not express detectable syt I protein (Fig. 1B). Syt IX was not affected by the shRNA-syt I, because the shRNA-syt I cells express syt IX at levels equivalent to that of the CT cells. Furthermore, none of the tested proteins exhibited any significant changes in expressed protein levels in the syt I knockdown cells compared with the CT cells. Thus RNAi targeted against syt I effectively and specifically knocked down expression of syt I in stably transfected cells.

In addition to specificity of knockdown, a primary concern with RNAi is whether suppression of one gene product results in compensatory upregulation of closely related gene products. We used immunoblot analysis to determine whether syt II, III, or VII was upregulated. Figure 1C shows that neither syt III nor syt VII exhibit an upregulation of protein expression in the shRNA-syt I cells compared with the CT cells. Each of the isoforms is shown compared with control tissue, cerebellum (syt II) or RINm5F pancreatic cells (syt III and VII). Syt II was not detectable as expected.

Neither syt I protein expression nor knockdown of syt I decreases with time in culture. Because PC12 cells are maintained in culture for multiple passages, it was important to verify that endogenous syt I protein levels do not decrease with time in culture and therefore do not provide a false interpretation of RNAi in syt I knockdown stable cells. Immunoblot analysis was used to evaluate the expression levels of syt I with time in passage for PC12 cells and stably transfected shRNA-syt I cells. The expression levels of syt I did not decrease with time in culture for at least seven passages in control cells (data not shown). Furthermore, syt I in the stably transfected cell line remained stably knocked down for at least 9 wk when the cells were cultured in the presence of selection media.

To determine whether the cellular expression patterns of syt I and IX were affected in the syt I knockdown cells, we performed immunocytochemistry on both undifferentiated and NGF-treated cells to detect syt I and syt IX in untransfected control cells, shRNA-syt I cells, and CT cells. Figure 2 shows confocal images of single cells expressing syt I (A) and syt IX (B). The syt I knockdown cells did not express detectable syt I, whereas control cells and cells stably transfected with the shRNA plasmid did express syt I. syt IX expression was not affected in the syt I knockdown cells. From the images, regardless of NGF treatment, both syt I and IX appear to be expressed in the cytoplasm of the cell and along the surface of the cellular membrane, but are largely excluded from the nucleus. These data are similar to those reported for syt I and IX proteins localized to vesicle membrane (16, 17, 29). This experiment confirms at the cellular level that RNAi targeted against syt I completely and specifically knocked down expression of syt I in individual PC12 cells without affecting the other abundantly expressed syt IX protein.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Expression of syt I is abolished in single cells. PC12 (–NGF) or NGF-treated PC12 cells (+NGF) were prepared for immunocytochemistry with a primary anti-syt I mouse antibody (A) or a primary anti-syt IX mouse antibody (B). Representative photos are shown for control untransfected cells (control) and stable cells transfected with either shRNA-Syt I or empty vector (CT). Scale bar, 10 µm.

Stable syt I knockdown cells have normal resting Ca²⁺ levels and respond to high-K⁺ stimulation. To determine whether cells stably transfected with shRNA-syt I have altered resting Ca²⁺ levels or altered Ca²⁺ influx in response to stimulation, we performed Ca²⁺ imaging experiments on individual cells with the Ca²⁺ indicator fura-2. Figure 3 shows averaged fura-2 imaging experiments. Each panel shows a representative group of cells imaged from one dish of cells. Average Ca²⁺ concentration is plotted as a function of time for control PC12 cells (Fig. 3A), shRNA-syt I cells (Fig. 3B), and CT cells (Fig. 3C). For each group of cells, data were averaged at each time point for all of the cells imaged. The cells were stimulated with 50 mM K⁺ solution (solid bar above each graph). Average values for resting Ca²⁺ and the change in [Ca²⁺]_i are shown in the summary graph (Fig. 3D). Syt I knockdown cells did not exhibit any differences in resting Ca²⁺ compared with control untransfected or CT cells. When the cells were stimulated, syt I knockdown cells responded with a somewhat greater change in Ca²⁺ influx (P < 0.01) that is most likely due to the cells lacking Ca²⁺ binding sites, because each syt I molecule can coordinate 5 Ca²⁺ ions (14, 38).

View larger version (24K): [in this window] [in a new window]   Fig. 3. Resting and stimulated Ca2+ levels are similar in syt I knockdown cells compared with control cells. Ca2+ imaging was performed with fura-2 to measure resting [Ca2+]i level and changes in Ca2+ in response to stimulation with 50 mM K+ (solid bars). A representative experiment for each group of cells is plotted as the average Ca2+ level (±SE) vs. time for untransfected control cells (A), cells stably expressing shRNA-syt I plasmid (B), or CT cells (C). The summary graph shows the average resting Ca2+ and change in Ca2+ during stimulation (D). The data represent an average of 183 control cells, 199 shRNA-Syt I cells, and 152 shRNA cells. *P < 0.01.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Knockdown of syt I causes a reduction in secretion that is rescued with human syt I. A: representative amperometric traces are shown for control (untransfected) and syt I knockdown cells (shRNA-syt I), each with multiple events. Arrows depict the time that the cell was switched from a basal solution to a stimulating high (50 mM)-K+ solution. The total number of events for each 4-min recording is indicated below each representative trace. B: four individual events are shown on an expanded time scale for the control (left) and shRNA-syt I cells (right). C: the averaged number of events per cell is shown in the bar graph for control untransfected cells (n = 7), syt I knockdown cells (n = 7), CT cells (n = 9), and the syt I knockdown cells transiently transfected with human syt I (rescue; n = 7). Although the number of events was reduced dramatically for the syt I knockdown cells, the reduction was not statistically significant (P = 0.1). D: human syt I plasmid is expressed in the syt I knockdown cells (rescue). NGF-treated cells were prepared for immunocytochemistry with a primary anti-syt I mouse antibody. Scale bar, 10 µm. E: norepinephrine (left) and dopamine (right) release were reduced in the syt I knockdown cells compared with control cells. Release of stimulated catecholamine is expressed as a percentage of total content of norepinephrine or dopamine content of the cells (n = 6 for each set of cells).

Expression of human syt I can rescue catecholamine secretion. To determine whether the reduced secretion in shRNA-syt I knockdown cells is actually due to lack of syt I, we attempted to rescue secretion by transiently transfecting the shRNA-Syt I knockdown cells with a plasmid designed to express human syt I. Human syt I differs from rat syt I in 4 of the 19 nucleotides in the shRNA target region. Therefore, human syt I mRNA should not be subject to RNAi elicited by the shRNA used in this study, which targets rat syt I. Using immunocytochemistry, we established that human syt I is indeed expressed in syt I knockdown cells transiently transfected with the human syt I expression plasmid (Fig. 4D). To determine whether the exogenously expressed human syt I protein functions similarly to endogenous rat syt I, we measured release events using amperometry from syt I cells transiently cotransfected with plasmids expressing human syt I and green fluorescent protein. Expression of green fluorescent protein was used to identify transfected cells. The average number of release events per cell measured in the syt I knockdown cells expressing human syt I (114 events ± 10, n = 7 cells, 798 total events) was not significantly different from the number of release events measured in either the control untransfected or CT cells (Fig. 4C). Because expression of human syt I rescued the secretion deficit observed in syt I knockdown cells, the decrease in secretion most likely is due to lack of syt I and is unlikely to be due simply to clonal variability between cell lines.

Catecholamine release is reduced determined by HPLC. To confirm that the reduction in release events measured in single cells with amperometry is reflective of a reduction of release from the cell lines, we used HPLC to quantify stimulated release of catecholamine from fields of cells. NGF-treated cells were stimulated with high-K⁺ solution for 5 min to evoke Ca²⁺-dependent vesicle release of the catecholamines norepinephrine and dopamine. Figure 4E shows the stimulated fractional release of norepinephrine and dopamine expressed as a percentage of total norepinephrine or dopamine content of the cells, respectively. Average stimulated fractional release of norepinephrine was reduced by 58% and that of dopamine by 51% in syt I knockdown cells compared with control cells (P < 0.001, n = 6 for each set of cells). Average basal fractional release was <0.85% (data not shown) for the same sets of experiments.

Quantal charge and kinetic characteristics are reduced in syt I knockdown cells. Quantification of the individual events shows that peak amplitude was reduced in shRNA-syt I cells. Peak amplitude of the averaged events was 15.6 ± 0.9 pA for control cells (n = 1,119) and 7.2 ± 0.6 pA for syt I knockdown cells (n = 417; P < 0.01) (Fig. 5A). Similarly, the kinetic rate of rise was significantly reduced from 27.3 pA/ms (±2.3 pA/ms) in control cells to 13.4 pA/ms (±1.9 pA/ms; P < 0.01) in syt I knockdown cells (Fig. 5B). The rising phase was reduced from 1.35 ms (±0.05 ms) in control cells to 1.11 ms (±0.08 ms) in syt I knockdown cells (P < 0.01). The bar histograms shown in Fig. 5, A and B, for peak amplitude and rate of rise kinetic parameters reveal that the distribution of events is different in the syt I knockdown cells compared with control cells.

View larger version (20K): [in this window] [in a new window]   Fig. 5. Peak amplitude and rate of rise time of all individual events are reduced in syt I knockdown cells. Averaged data are shown in the bar graph at left for all events from control and syt I knockdown cells for amplitude (A) and kinetic rate of rise time (B). The amplitude distribution of the total individual events is shown in the graphs at right. The reduction of peak amplitude and rate of rise was significantly reduced in the syt I knockdown cells compared with control cells. *P < 0.01.

View larger version (30K): [in this window] [in a new window]   Fig. 6. Quantitative analysis reveals that the amplitude, quantal charge, and kinetics are reduced in the syt I knockdown cells. A: two cells were chosen from each of the control and shRNA-syt I knockdown cells that had a similar number of events (170). Spike amplitude (Amp), rising phase (RP), falling phase (FP), time to half-width (T1/2), and quantal charge (Q) were measured as shown by arrows for the averaged spike event from each cell. Quantal charge was calculated by integrating the area under the curve. The kinetic parameters of quantal charge (B), half-width (C), and falling phase (D) were significantly reduced in the syt I knockdown cells compared with control cells. *P < 0.01.

	DISCUSSION

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION GRANTS REFERENCES

 
We have generated a PC12 cell line stably transfected with a plasmid expressing an shRNA insert that targets a specific region of syt I mRNA. Expression of this shRNA specifically and completely knocked down the expression of syt I protein without affecting cell survival, the ability of cells to respond to treatment with NGF, or stimulated Ca²⁺ influx. The lack of syt I reduced, but did not abolish, secretion of catecholamines from the cells, suggesting that additional protein(s) can act as a Ca²⁺ sensor for regulated release. A primary advantage of generating stable knockdown cell lines is that electrophysiological measurements can be performed on single cells within a homogenous population that have equivalently reduced levels of one specific protein. Other advantages to using RNAi include lack of developmental problems and lethality often observed in knockout mice, as well as simultaneous or sequential knockdown of multiple proteins. Furthermore, expression of most proteins can be knocked down within a relatively short period of time, with relative ease and at a lower cost to the researcher compared with generating knockout mice. By establishing stable knockdown cell lines, the uncertainty that the targeted protein is knocked down to the same extent in each cell is reduced.

Syt I knockdown was specific in that expression of syt I was abolished in the syt I knockdown cells, whereas other vesicle and structural protein expression levels were unaffected. Syt I knockdown cells did not exhibit either a reduction or compensatory upregulation of other endogenously expressed syt isoforms that bind phospholipids in a Ca²⁺-dependent manner, including the highly abundant syt IX isoform or the much less abundant syt III or VII isoforms. syt II, an isoform that is closely related to syt I in sequence and that has been postulated to be functionally complementary to syt I, is an isoform that is not expressed in the cells as protein even though the transcript is made in these cells. Syt II expression was not upregulated when syt I was abolished in the syt I knockdown cells.

We have begun to characterize the functional effects of removing syt I on catecholamine secretion from the neuroendocrine cells. Syt I knockdown cells exhibited reduced secretion events as measured using amperometry and reduced fractional catecholamine release as measured using HPLC. As a test of specificity of the shRNA, expression of human syt I not targeted by the shRNA reversed the effects of the syt I knockdown by functionally rescuing the secretion events. Quantitative kinetic analysis of the amperometric events shows that amplitude, quantal charge, rate of rise, half-width, and falling phase all were significantly reduced in the syt I knockdown cells compared with control cells. The amplitude of the spike events was reduced to 50% of control, and quantal charge was reduced, which translated to a sixfold decrease in catecholamine molecules released. This result may be due to 1) reduced time or size of pore opening, 2) reduced vesicle size and thus volume of stored transmitter, or 3) reduced numbers of transmitter molecules packaged in vesicles. Although our current results do not allow us to directly distinguish among these three possibilities, the measurements of reduced rate of rise time, reduced half-width, and reduced falling phase of the amperometric spike events in syt I knockdown cells are consistent with the idea that the fusion pore may remain open for a shorter time than in control cells.

The partial reduction of catecholamine secretion seen in our syt I knockdown cells is similar to the 60% reduction in catecholamine release measured using real-time amperometry in cracked PC12 cells after the introduction of recombinant C2A-C2B domain from syt I (51). The C2A-C2B domain presumably competes with endogenous Syt I for binding to Ca²⁺ and the SNARE complex, thus inhibiting the usual function of syt I. However, our data vary from results in chromaffin cells obtained from syt I knockout mice (53). In their studies of secretion from Ca²⁺-dialyzed chromaffin cells in adrenal slices, the authors found that the Ca²⁺ dependence of release was altered, exocytosis of large dense-core vesicles was delayed, and Ca²⁺-dependent fusion rates were slowed as measured using membrane capacitance techniques (53). They did not detect differences in amperometric release events between wild-type and syt I knockout cells. One possibility for the differences between our study and that of Voets et al. (53) may be the stimulation protocols for vesicle release. Because Voets et al. used Ca²⁺ dialysis to stimulate, the release machinery may have become saturated with Ca²⁺ and caused the subsequent shift in Ca²⁺ dependence of vesicle release, whereas our stimulation protocol may not have saturated the release machinery. This possible explanation would fit well with results measured in wild-type chromaffin cells that exhibit a strong Ca²⁺ dependence for release of vesicle content; that is, for small Ca²⁺ influx, only a fraction of the contents of vesicles is released, but with strong stimulation and hence a large Ca²⁺ influx, all of the vesicle content is released (13).

In a spontaneously occurring mutant line of PC12 cells that lacked the syt I isoform, Ca²⁺-dependent secretion of dopamine as measured by HPLC was normal compared with that in wild-type PC12 cells (41). This observation led to the suggestion that neuroendocrine cells do not require syt I and that an alternative protein may function as the Ca²⁺ sensor for release (41). Our data differ from the mutant PC12 cell data, because the syt I knockdown cells exhibited differences in release of catecholamine compared with the parent cell line. It is not known at this time whether the differences between our results may arise from methodological measurement of secretion, variations between the PC12 cell lines, or the proteins that act to regulate Ca²⁺-dependent release. Yet another experimental difference between our results and the data for mutant PC12 cells that lack syt I is that the mutant cells exhibited an upregulation of syt IX expression compared with control cells, whereas our syt I knockdown cells did not exhibit any upregulation in syt IX. Even so, our results agree with the interpretation that additional proteins may function as regulators of evoked vesicle release.

Previous work has led to the suggestion that a protein other than Syt I has a role as a Ca²⁺ sensor in rapid, regulated release. Syt I deletion studies performed in various organisms such as Drosophila (29), Caenorhabditis elegans (34), and mice (19) revealed that syt I was required for fast synaptic release of neurotransmitter. In syt I knockout mice, fast synchronous release was abolished, but slow asynchronous release was still evident (19) and even increased to match the synchronous release of wild-type neurons, which led to the hypothesis that syt I inhibits slow asynchronous release (32, 33). Furthermore, upon either altering or neutralizing the Ca²⁺-binding pockets of the C2 domains by point mutagenesis, the Ca²⁺ dependency of release was shifted, which suggested that the C2A domain cannot represent the Ca²⁺ sensor in its entirety but, rather, is a necessary but not sufficient component of the sensor required for fast neurotransmission in the central nervous system (42). Additional experiments in a variety of systems have shown that interfering with either the C2A or C2B domain, or with Ca²⁺ ions, SNARE complex proteins, and/or phospholipid binding of syt I, results in altered secretion of vesicles (18, 27, 30, 49, 55).

To account for the remaining Ca²⁺-dependent fusion events, cells would require an additional protein to compensate as a Ca²⁺ sensor. Likely candidates include one of the additional Syt isoforms endogenously expressed by the cells, such as syt IX, or the less abundant syt isoforms, syt III or VII. Our results showing that syt I and IX are the two most abundant isoforms of syt expressed agree well with other reports of syt isoform expression in PC12 cells (16, 51, 59). Low expression levels of syt VII have been reported in PC12 cells (51). There is a discrepancy as to whether syt III is expressed; for example, Tucker et al. (51) did not detect syt III, but Sugita et al. (47) did detect this isoform. Both syt III and VII have been proposed to act as Ca²⁺ sensors (24, 46, 47), because recombinant fragments of the C2 domains of these isoforms inhibited secretion from cracked PC12 cells (51), and syt VII can function as a high-affinity Ca²⁺ sensor for secretion of large dense-core vesicles in PC12 cells (24).

In conclusion, this study has established a neuroendocrine cell line that utilizes the plasmid-based RNAi method to stably, specifically, and completely knock down expression of syt I in a homogenous population of cells. Release studies show that syt I knockdown cells have reduced release events and reduced amounts of transmitter released that may be due to a reduced open time of the fusion pore. These results support a role for syt I as a functional Ca²⁺ sensor, but not an exclusive Ca²⁺ sensor. RNAi-induced effects are unlikely to be due to a lack of Ca²⁺ ions, given that Ca²⁺ influx was similar between knockdown and control cells. Furthermore, it is unlikely that one of the other endogenously expressed syt isoforms or the closely related isoform, syt II, has compensated functionally for the lack of syt I.

	GRANTS

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION GRANTS REFERENCES

 
The research was funded by NIH Grant NS-42681 (to A. B. Harkins) and a Monsanto Fellowship (to J. M. Moore).

	ACKNOWLEDGMENTS

 
We thank Dr. Aaron P. Fox (University of Chicago) for helpful discussion and critical review of the manuscript. We thank Drs. Joseph Baldassare and Alan Stephenson (Saint Louis University) and Kyle McMillan (University of Chicago) for technical assistance. We thank William Roden and Dr. Heather Macarthur (Saint Louis University) for technical assistance with the HPLC experiments. We are grateful to Drs. Aaron P. Fox (University of Chicago) and Eugene Mosharov (Columbia University) for software development and to Dr. Hans Peter Zassenhaus (Saint Louis University) for the generous use of a fluorescence microscope. We thank Dr. Bryan Wolf (University of Pennsylvania) for the generous gift of Syt III antibody. The anti-Syt I monoclonal antibody developed by Dr. Louis Reicherdt and the anti--actin antibody developed by Drs. Jim Jung and Ching Lin were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by The University of Iowa Dept. of Biological Sciences, Iowa City, IA 52242.

	FOOTNOTES

 

Address for reprint requests and other correspondence: A. B. Harkins, Dept. of Pharmacological and Physiological Science, St. Louis Univ. School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104 (e-mail: harkinsa{at}slu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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