January 2006
Volume 47, Issue 1
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Retinal Cell Biology  |   January 2006
Kinase-Dependent Differentiation of a Retinal Ganglion Cell Precursor
Author Affiliations
  • Laura J. Frassetto
    From the Departments of Ophthalmology and Visual Sciences and
  • Christopher R. Schlieve
    From the Departments of Ophthalmology and Visual Sciences and
  • Christopher J. Lieven
    From the Departments of Ophthalmology and Visual Sciences and
  • Amy A. Utter
    Physiology, University of Wisconsin Medical School, Madison, Wisconsin; and the
  • Mathew V. Jones
    Physiology, University of Wisconsin Medical School, Madison, Wisconsin; and the
  • Neeraj Agarwal
    Department of Cell Biology and Genetics, North Texas Health Science Center, Fort Worth, Texas.
  • Leonard A. Levin
    From the Departments of Ophthalmology and Visual Sciences and
Investigative Ophthalmology & Visual Science January 2006, Vol.47, 427-438. doi:10.1167/iovs.05-0340
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      Laura J. Frassetto, Christopher R. Schlieve, Christopher J. Lieven, Amy A. Utter, Mathew V. Jones, Neeraj Agarwal, Leonard A. Levin; Kinase-Dependent Differentiation of a Retinal Ganglion Cell Precursor. Invest. Ophthalmol. Vis. Sci. 2006;47(1):427-438. doi: 10.1167/iovs.05-0340.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. Cell lines are frequently used to elucidate mechanisms of disease pathophysiology. Yet extrapolation of results with cell lines to neurodegenerative disorders is difficult because they are mitotic and usually have other non-neuronal properties. The RGC-5 cell line has many features of retinal ganglion cells (RGCs). Despite its expression of Thy-1 and NMDA receptors, as found in primary RGCs, this line’s ability to proliferate and non-neuronal appearance differentiate it from other central neurons, complicating its use for the study of neuronal survival, electrophysiology, or neurite extension.

methods. A method was identified for differentiating RGC-5 cells using the nonspecific protein kinase inhibitor staurosporine. Cultures were treated with 100 nM to 3.16 μM staurosporine and assessed for a variety of differentiation markers.

results. Differentiated RGC-5 cells expressed numerous neuronal properties, including arrest of proliferation without inducing apoptosis, induction of a neuronal morphology, upregulation of neuronal markers, and establishment of outward rectifying channels. Differentiation was not dependent on a single kinase-dependent pathway, based on profiling multiple kinase phosphorylation targets and attempts to replicate differentiation with multiple specific kinase inhibitors.

conclusions. This method for producing an RGC-like cell from a proliferating cell line facilitates the following previously impractical techniques: high-throughput screening for agents that are neuroprotective or affect ionic channels; straightforward transduction of gene expression in central neurons by nonviral transfection techniques, including production of stable transfectants; biochemical and other assays of pure RGC-like cells without purification on the basis of cell-surface antigens or anatomic location.

The study of optic neuropathies (e.g., glaucoma, and the neuron primarily involved, the retinal ganglion cell [RGC]), has been limited to primary cell culture and in vivo models. 1 There are difficulties in using cell culture to study RGC pathophysiology. Harvesting retinas and identifying RGCs by retrograde labeling or immunocytochemistry 2 is labor intensive and time-consuming. RGC purification using antibodies to cell-surface antigens 3 or anatomic location 4 is a lengthy and frequently low-yield process that does not result in a homogeneous cell population. Differentiation of neuronal cell lines (e.g., pheochromocytoma-derived PC-12 cells, 5 teratoma-derived NT2-N cells, 6 or neuroblastoma-derived SY5Y cells 7 ) results in cells, that are not retina derived and thus do not share the phenotypic properties of RGCs. 
A transformed retinal ganglion cell line, RGC-5, was derived by transforming postnatal day 1 rat retinal cells with ψ2 E1A virus. This cell line expresses neuronal markers characteristic of RGCs (e.g., Thy-1, Brn-3c, neuritin, and the N-methyl d-aspartate [NMDA]-R1 and γ-aminobutyric acid [GABA]-B receptors), but does not express the astrocyte marker glial fibrillary acidic protein (GFAP). 8 Despite the presence of shared antigens, the RGC-5 line has features significantly different from RGCs, the most significant being that the former is mitotically active. In addition, RGC-5 cells are morphologically more similar to glial cells in culture than to primary RGCs and do not express the repertoire of ion channels characteristic of RGCs. 9  
We sought a method for terminally differentiating RGC-5 cells that would allow pharmacological, biochemical, and electrophysiological studies relevant to primary cultured RGCs. We found that treatment of proliferating RGC-5 cells with the broad-spectrum protein kinase inhibitor staurosporine (SS) for as little as 60 seconds resulted in nondividing cells with multiple branched neurites characteristic of a neuronal morphology, without inducing apoptosis. We also defined several features of these cells and explored the profile of protein kinase inhibition resulting in the differentiated phenotype. 
Methods
Materials
Minocycline was purchased from Fisher Scientific (Fair Lawn, NJ); ZVAD-fmk from Calbiochem (San Diego, CA); thapsigargin, ionomycin, rotenone, 3-(4,5-dimethyl sulfoxide (DMSO) from Sigma-Aldrich (St. Louis, MO); roscovitine, PD98059, LY294002, tyrphostin AG490, tyrphostin AG1295, tyrphostin AG1478, H-89, HA-1077, and bisindolylmaleimide IX methylsulfate from LC laboratories (Woburn, MA); and SS (from Streptomyces staurosporeus; ≥98% purity; catalog number 380-014) from Alexis Biochemicals (San Diego, CA). Cell culture reagents, unless noted, were obtained from BioWhittaker (Rockland, ME). Fluorescent secondary antibodies and fluorescent dyes were obtained from Molecular Probes (Eugene, OR). 
Cell Culture
RGC-5 cells were cultured in Dulbecco’s modified Eagle’s medium (Mediatech, Inc., Herndon, CA) containing 1 g/L glucose with l-glutamine, supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were incubated at 37°C in humidified 5% CO2
Pharmacological agents were used to induce or inhibit various signaling pathways within RGC-5 cells. Cells were replated on 12-mm round coverslips in 24-well plates 24 hours before pharmacological treatment. Drugs were added for various lengths of time and then processed for immunocytochemistry. 
Immunocytochemistry
RGC-5 cells plated on coverslips were fixed with 4% paraformaldehyde (Fisher) in PBS (pH 7.2) for 20 minutes at room temperature, rapidly rinsed with Tris-buffered saline (TBS; 100 mM Tris [pH 7.6], 0.9% NaCl) three times for a few seconds each and two times for 5 minutes each, and blocked with 5% normal goat serum (BioWhittaker) in TBS for 30 minutes at room temperature. The NMDA R1 receptor was dictated by incubating with purified mouse monoclonal anti-NMDA-R1 antibody (BD Biosciences; San Jose, CA; clone 54.1) at 2.5 μg/mL overnight at room temperature, followed by Alexa Fluor 594 (Molecular Probes, Eugene, OR) goat anti-mouse IgG at 10 μg/mL in blocking buffer at room temperature for 90 minutes. Alexa Fluor 594 fluorescence was detected with a Texas red filter set (excitation 560 ± 20 nm, dichroic 595 nm long-pass, emission 630 ± 30 nm). Thy-1 was visualized after fixing and blocking by incubating with rabbit anti-Thy-1 polyclonal IgG (Santa Cruz Biotechnology, Santa Cruz, CA) at 20 μg/mL overnight at 4°C, followed by Alexa Fluor 488 goat anti-rabbit IgG at 4 μg/mL at room temperature for 60 minutes. Alexa Fluor 488 fluorescence was detected with a FITC filter set (excitation 470 ± 20 nm, dichroic 505 nm long-pass, emission 540 ± 20 nm). Nuclear condensation changes that are characteristic of apoptosis were assessed by adding Hoechst 33258 dye at 10 μg/mL for the final 30 minutes of pharmacological treatment before cell fixation and viewed with a 4′,6′-diamino-2-phenylindole (DAPI) filter set (excitation 365 ± 12.5 nm, dichroic 395 nm, emission 420 long pass). Coverslips were then transferred and mounted on microscope slides (Gel/Mount; Biomeda Corp., Foster City, CA). Slides were viewed with an upright microscope (Axiophot; Carl Zeiss Meditec, Dublin CA) with Nomarski optics and epifluorescence, and images acquired (Axiovision software; Carl Zeiss Meditec) at 1300 × 1030 resolution. 
Immunoblot Analysis
Rat RGC-5 cells were grown to approximately 70% confluence on 100-mm tissue culture plates (BD Biosciences, Bedford, MA) and either treated with SS to a final concentration of 316 nM for 3 days or harvested without treatment. Cells were lysed in PBS containing 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (Fisher Scientific), 0.10 mg/mL phenylmethylsulfonyl fluoride (Sigma-Aldrich), and a protease inhibitor (Complete Mini Protease Inhibitor Cocktail Tablet; 5 mg/mL; Roche Diagnostics, Mannheim, Germany) for 15 minutes at 4°C, scraped off the plate, and collected. Cell lysates from two tissue culture plates were pooled for each sample, and incubated on ice for 60 minutes. After centrifugation at 10,000g for 10 minutes at 4°C, the pellets were sonicated on ice for two 15-second bursts and centrifuged again at 10,000g for 10 minutes. The protein concentrations of the supernatants were determined by Bradford assay, and 1 mg of total protein from differentiated and undifferentiated cells was boiled in the presence of 4× lithium dodecyl sulfate (LDS) sample buffer (Invitrogen, Carlsbad, CA) plus 5% β-mercaptoethanol, resolved on a Bis-Tris 4% to 12% polyacrylamide gel (NuPAGE; Invitrogen), and transferred overnight at 50 mA to nitrocellulose membrane in a transfer apparatus (Mini Protean II; Bio-Rad Laboratories, Hercules, CA). 
After transfer, the membrane was blocked with 5% nonfat milk in TBS (pH 8.0) for 30 to 60 minutes and then probed with primary antibodies to RGC marker proteins in blocking buffer. Antibodies used included purified rabbit polyclonal anti-microtubule-associated protein 2 (1:2000; Chemicon, Temecula, CA), polyclonal rabbit anti-Thy-1 (1:100; Santa Cruz Biotechnology), and polyclonal rabbit anti-actin (1:1000; Sigma-Aldrich). Blots were rinsed three times with TBS containing 0.05% Tween-20 (Fisher Scientific), then washed 5 times for 10 minutes each at room temperature on an orbital shaker. Secondary antibodies used were purified horse radish peroxidase (HRP)-conjugated goat anti-rabbit IgG and purified HRP-conjugated goat anti-mouse IgG (1:5000; Jackson ImmunoResearch Laboratories, West Grove, PA) and were incubated for 1 hour at room temperature, followed by 3 rinses and five 10-minute washes with TBS containing Tween-20 at room temperature on an orbital shaker. Blots were treated with freshly prepared ECL solution containing 100 mM Tris-HCl [pH 8.5], 1.25 mM luminol, 225 μM p-coumaric acid (Sigma-Aldrich), and 1 mM H2O2 (Fisher Scientific) for 1 minute, and excess solution was allowed to drip off. The blots were then exposed to film (BioMax XAR; Eastman Kodak Company, Rochester, NY) and developed. The films were scanned at 1600 dpi and band density was determined by comparing total intensity in an area containing the band of interest to the intensity of an equal size area of background using NIH ImageJ software. Band density readings are presented with respect to the density of the band from the control, untreated cell condition. 
Cell Morphology
Photomicrographs were taken at 400× total magnification with Nomarski optics, digitized as above, and stored as JPEG images. The pictures were then batch analyzed off-line, to assess the development of neurites. A total of 50 cells from each condition were analyzed. The 50 cells assessed for neurite outgrowth in each condition were selected to include all cells in each photomicrograph for which the neurite tree was visible, and continuing with a new photomicrograph until 50 cells were analyzed. Projections from the cell were classified as neurites if they were equal to or greater in length than the cell soma. Only branches arising from the soma were counted. The neurite counts were expressed as the mean ± SEM. Neurite length and branching characteristics were assessed using NeuronJ. 10 Individual neurite length was determined by adding the length of all segments from the soma to the end of each branch. 
Cell Proliferation
Cells were incubated with 100 μM bromodeoxyuridine (BrdU; Sigma-Aldrich) at 37°C for 2 hours. The medium was aspirated and the cells immediately fixed with ice-cold glycine-ethanol (150 mM glycine, 70% EtOH [pH 2.0]) for 30 minutes at −20°C. The wells were washed with TBS, incubated with blocking buffer (0.3% Triton X-100, 5% normal goat serum in TBS) for 30 minutes at room temperature, and incubated overnight at 4°C with monoclonal mouse anti-BrdU (Sigma-Aldrich; clone Bu 33) at 4 μg/mL in blocking buffer. After washing, the cells were incubated for 1 hour at room temperature with AlexaFluor 594 (Molecular Probes) goat anti-mouse IgG (2 μg/mL) in TBS. Proliferating cells were identified by superimposing BrdU fluorescence and Nomarski microscopy images of the same fields. 
Cell Number
Cells were placed in 96-well plates, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added immediately to a final concentration of 500 μg/mL. The plates were incubated for 5 hours at 37°C. The medium was then replaced with 200 μL of DMSO, which was pipetted up and down to dissolve the formazan crystals, and the plate was incubated at 37°C for 5 minutes. Absorbance was measured at 550 nm on a microplate reader (ThermoMax; Molecular Devices, Sunnyvale, CA). All readings were normalized to a standard curve derived from known numbers of cells. 
Electrophysiology
Whole-cell voltage-clamp recordings were made at room temperature using borosilicate glass pipettes (3–6 MΩ resistance) filled with (in mM): 140 KCl, 10 EGTA, 2 MgATP, 20 phosphocreatine, and 10 HEPES [pH 7.3], 315 mOsM. The extracellular solution contained (in mM) 145 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 4 glucose (pH 7.4), 325 mOsM. Series resistance was monitored during recordings (5.4 ± 0.65 MΩ, mean ± SEM, n = 17 acceptable recordings). No series resistance compensation was used. Currents were low-pass filtered at 1 to 5 kHz with a four-pole Bessel filter and digitized at a rate no less than twice the filter frequency. Data were collected using an amplifier (200B; Axopatch) and digitizer (1320A Digidata, controlled by AxoGraph software; Axon Instruments Inc., Foster City, CA, running on a Macintosh G4; Apple Computer Inc., Cupertino, CA). Analysis and curve fitting was also performed on computer (AxoGraph; Axon Instruments, Inc.). 
Voltage-gated currents were studied by applying 10-ms voltage steps from −100 to +90 mV in 10-mV increments, from a holding potential of −60 mV. This protocol was run two to six times on each cell, and the currents evoked at each potential were averaged. Passive leak and capacitive currents were removed by scaling the average current evoked at −70 mV, and subtracting the result from the average current obtained at each voltage. Current amplitudes were measured at steady state (i.e., near the end of the voltage step), and the slope conductance was computed as the difference between successive amplitudes, divided by the 10-mV step increment. 
Analysis of Kinase Phosphorylation Targets
Total cell lysates were prepared as previously described. 11 Cells were washed with ice-cold PBS, scraped, and centrifuged at 3200g for 5 minutes. The pellets were resuspended in lysis buffer (Kinexus Bioinformatics, Vancouver, BC, Canada) supplemented with 5 mM pepstatin A (Roche), and protease inhibitor cocktail (Mini Complete tablet; Roche). The suspensions were sonicated on ice for two 15-second bursts, followed by centrifugation at 100,000g for 30 minutes at 4°C. The protein concentrations of the supernatants were determined by Bradford assay, 12 and 500 μg protein from undifferentiated and differentiated cells resolved on 13% single lane SDS polyacrylamide gels. These were then transferred to nitrocellulose membranes. Using a 20-lane multiblotter (Bio-Rad), the membranes were incubated with different mixtures of up to three antibodies per lane that react with a distinct subset of at least 37 known phosphorylation sites of cell signaling proteins of distinct molecular masses (protocol KPSS 2.1; Kinexus Bioinformatics). After further incubation with a mixture of relevant HRP-conjugated secondary antibodies (Santa Cruz Biotechnology), the blots were developed with enhanced chemiluminescence (ECL Plus; GE Healthcare, Piscataway, NJ) and the signals quantified on computer (Quantity One software; Bio-Rad). (Detailed information and protocols of the Kinetworks analysis can be found at the Kinexus Bioinformatics Corporation Web site; www.kinexus.ca.) 
Statistical Analysis
Comparisons between two groups were by Student’s unpaired t-test. Comparisons between more than two groups were by ANOVA and the Dunnett post hoc test. Significant differences required P < 0.05. 
Results
Effect of SS on Morphologic Differentiation of RGC-5 Cells
Treatment with SS from 316 nM to 1.78 μM for 24 hours induced a significant dose-dependent formation of neurites and changed the soma from flat and polygonal to rounded and moderately elevated (Fig. 1A) , consistent with a neuronal morphology. The change in morphology was quantified by counting neurites, defined as cell projections greater than the length of the soma. There was a dramatic increase in neurite counts with SS treatment. Cells treated with vehicle control had 0.16 ± 0.05 neurites per cell, whereas cells treated with 1 μM SS had 2.94 ± 0.11 neurites per cell (Fig. 1D) . SS concentrations higher than 1.78 μM were frequently toxic, as evidenced by a necrotic appearance of the cell body and blebbing and disruption of the neurite tree. Serum-deprivation of RGC-5 cells did not induce neurite expression (0.22 ± 0.06 vs. 0.16 ± 0.05 in control; P = 0.45). 
Our previous experiments demonstrated that RGC-5 cells had differentiated 24 hours after exposure to SS. We tested the length of SS exposure necessary to induce differentiation of the RGC-5 cells after SS was removed from the cell culture medium. Cells were treated with 1 μM SS. At 0, 10, and 30 seconds and 1, 5, 10, 30, and 60 minutes, the medium was aspirated, the wells rinsed once with fresh prewarmed medium, and incubated in fresh medium for 24 hours from the time of treatment. Cells treated for as little as 60 seconds had definite morphologic signs of differentiation (Fig. 1B) , comparable to the results shown in Figure 1A . We studied the progression of neurite development over time by stopping SS treatment after various lengths of exposure (Fig. 1C) . Change in RGC-5 morphology was first observed at 1 hour, and an obvious neuronal morphology was seen as early as 12 hours. 
Neurite branching patterns of differentiated RGC-5 cells were examined and the number of branches counted. Neurite branching, average neurite length, and total neurite length were negligible in untreated cells. Differentiation induced by exposure to 316 nM SS led to minimally branched neurites, whereas higher concentrations induced more branching (Fig. 1E) . In addition to increased branching, a moderate (1 μM) concentration of SS promoted longer average (Fig. 1F)and total (Fig. 1G)neurite outgrowth. At high (3.16 μM) SS concentrations, the neurite branching was greater, but the average and total process length decreased somewhat. All comparisons between differentiated and undifferentiated RGC-5 cells of neurite number, branches, average neurite length, and total length were significant at P < 0.05, for each concentration of SS. 
Effect of SS on RGC-5 Postmitotic Cell Viability
RGC-5 cells are mitotically active. To test whether SS induces terminal differentiation, we measured incorporation of the thymidine analogue BrdU during the S phase in SS-treated and control cells. We found that SS (1 μM for 24 hours) causes RGC-5 cells to become BrdU (i.e., postmitotic; Fig. 2 ). BrdU incorporation was seen in 69.5% of untreated control cells, compared with 2.2% of SS-treated cells, consistent with a switch to a nonproliferating state. 
SS is commonly used to initiate apoptosis, and the lack of BrdU incorporation could be due to cell death and not the transition to a postmitotic phenotype. To explore this possibility, we used the MTT assay in differentiated and undifferentiated RGC-5 cells. The MTT assay is a quantitative colorimetric method for measuring cell proliferation and viability. The number of cells after SS treatment increased slightly in number over a 24-hour period (7,133 ± 266 cells at 1 hour after culture versus 10,094 ± 1,023 cells at 24 hours; P = 0.012), whereas untreated cells predictably increased dramatically (7,666 ± 471 cells at 1 hour vs. 16,579 ± 653 at 24 hours; P < 0.000001). Given the lack of BrdU incorporation in SS-treated cells, the small increase in MTT metabolisms indicates that differentiated cells have moderately increased redox activity. 13  
Expression of Retinal Ganglion Cell Markers in Differentiated RGC-5 Cells
RGC-5 cells express NMDAR1 and Thy-1, which are both seen in mature RGCs. 8 We hypothesized that differentiated RGC-5 cells would retain their expression of Thy-1 and NMDAR1. NMDAR1 and Thy-1 labeling was seen in both undifferentiated and differentiated RGC-5 cells, with visibly more intense staining in the latter (Figs. 3A 3B)
To quantify the comparative levels of ganglion cell marker expression, protein samples prepared from RGC-5 cells differentiated for 3 days in the presence of 316 nM SS were analyzed by immunoblot. Western blot analysis revealed increased levels of Thy-1 and microtubule-associated protein (MAP)-2, a dendritic marker, after differentiation (Figs. 3C 3D)
Expression of Voltage-Gated Ion Channels after SS Treatment
To determine whether the differentiating effects of SS treatment also involves changes in the electrophysiological properties of RGC-5 cells, we examined membrane currents using whole-cell voltage-clamp recording. Untreated cells had an input resistance of 55 ± 10 MΩ (mean ± SEM), an apparent capacitance of 28 ± 2 pF, and a resting potential of −34 ± 0.5 mV (n = 4). After subtracting passive components, a small residual outward current was present at depolarized potentials, yielding a maximum conductance of 3.1 ± 0.5 nS at +90 mV (Fig. 4A) . Three SS treatment protocols were tested (given as hours in SS with duration of the following incubation in medium alone in parentheses): 1 hour (24 hours), 1 hour (48 hours), and 22 hours (2 hours). SS induced some changes in passive properties, as judged by ANOVA and Dunnett’s post hoc test. After treatment, input resistances were 397 ± 171 MΩ (1 hour [24 hours], n = 5, not significant, NS), 322 ± 89 MΩ (1 hour [48 hours], n = 4, NS), and 843 ± 201 (22 hours [2 hours]; n = 3, P < 0.05), apparent capacitances were 31 ± 5 pF (1 hour [24 hours], NS), 44 ± 3 pF (1 hour [48 hours], P < 0.05), and 38 ± 3 (22 hours [2 hours]; NS), and resting potentials were −27 ± 9 mV (1 hour [24 hours], n = 5, NS), −9 ± 6 mV (1 hour [48 hours], NS), and −11 ± 3 (22 hours [2 hours]; NS). The larger capacitance in some cells is consistent with the elaboration of neuronlike processes. In addition, although not significant by ANOVA, SS induced increases in voltage-gated conductance in all groups of treated cells. After leak subtraction, outward currents had conductances at +90 mV of 14 ± 5 nS (1 hour [24 hours], NS), 7 ± 3 nS (1 hour [48 hours], NS), and 5 ± 3 nS (22 hours [2 hours], NS). Inspection of the spread in the data (Fig. 4B)reveals that some treated cells had small conductance similar to untreated cells, whereas others had much larger conductance. This bimodal distribution probably accounts for the inability to detect significant differences. We therefore further examined cells with conductances greater than the mean of the untreated group plus twice its SD. The probability of finding such values should be extremely low if there were truly no differences between groups. However, half of the treated cells had conductances in this range, suggesting that SS induces the expression of voltage-gated current in a large fraction of cells. In these cells, the current increased monotonically as a function of voltage (Fig. 4C)and could be fit with a Boltzmann function in the most robust treatment group (i.e., 1 hour [24 hours]), yielding a maximum conductance of 26 nS and an apparent half-activation voltage of +48 mV. Because treated cells displayed neuronlike processes, we cannot be assured of there being an adequate space clamp. Thus, the strongly positive activation range and shallow slope factor we observed may not accurately reflect the biophysical characteristics of the expressed channels. Nonetheless, it seems clear that SS altered the electrical properties of a subset of treated cells. 
SS Differentiation and Induction of Apoptosis
SS can induce apoptosis, and its differentiating effect on RGC-5 cells could be a side effect of apoptotic cell death. We tested this possibility in three ways: exposing cells to SS while inhibiting apoptosis, attempting to differentiate cells with other agents that induce apoptosis, and looking for nuclear changes of apoptosis in SS-treated cells. 
We differentiated RGC-5 cells with SS in the presence of two apoptosis inhibitors with different mechanisms of action, minocycline (which inhibits cytochrome c release) and ZVAD-fmk (a broad-spectrum caspase inhibitor). After 30-minute pretreatment with 10 μM minocycline or 20 μM ZVAD-fmk, SS was added to RGC-5 cells at a final concentration of 1 μM and incubated for 24 hours. Cells treated with minocycline or ZVAD-fmk and subsequently exposed to SS underwent morphologic differentiation, similar to cells that were not pretreated with apoptosis inhibitors (Fig. 5) . Undifferentiated cells treated with minocycline or ZVAD-fmk alone did not differentiate. 
We then induced apoptosis in RGC-5 cells with three different drugs and measured whether there were any differentiating effects. Rotenone (10 μM) was used to inhibit complex I of the mitochondrial electron transport chain, ionomycin (20 μM), to elevate intracellular calcium by acting as an ionophore, and thapsigargin (10 μM) to elevate intracellular calcium by inhibiting endoplasmic reticulum Ca2+-ATPase. None resulted in morphologic evidence of differentiation, although ionomycin and thapsigargin caused the cells to become more spindle-shaped (Fig. 6 , left). 
Finally, we assessed SS-treated RGC-5 cells for nuclear morphologic changes of apoptosis. After a 24-hour incubation with SS, Hoechst 33258 (10 μg/mL) was added for 30 minutes and the cells examined. SS-treated cells were the same as the control, with finely granular nuclei which were not compacted. As expected, ionomycin, rotenone, and thapsigargin induced nuclear condensation (Fig. 6 , right). 
Reproduction of Differentiation with SS by More Kinase-Specific Inhibitors
SS is a broad-spectrum kinase inhibitor. To determine which specific kinase(s) could be responsible for differentiation, we attempted to differentiate RGC-5 cells with 12 different relatively specific kinase inhibitors, alone or in combination, at a wide range of concentrations. Inhibitors were chosen based on published data 14 demonstrating that SS inhibited the respective kinase (Table 1) . None of the kinase inhibitors alone or in combination differentiated RGC-5 cells to the same degree as SS, although two produced observable changes in morphology. H-1152, which primarily inhibits Rho-kinase (K i = 1.6 nM) and H-89, which primarily inhibits protein kinase A (K i = 50 nM), induced mild elevation in neurite counts, but did not produce rounding of the cell soma (Fig. 7) . SS-treated (3.16 μM) cells expressed 3.04 ± 0.16 neurites, whereas H-1152-treated (1 μM) and H-89-treated (56.2 μM) RGC-5 cells expressed 1.06 ± 0.15 and 1.80 ± 0.16 neurites, respectively. Despite mildly increased neurite expression with some kinase inhibitors, none reproduced the morphologic appearance of RGCs (Fig. 8)
Effect of SS on Changes in Phosphorylation Targets of Several Protein Kinases
Because of the difficulty in replicating the differentiating effects of SS with a broad array of relatively specific protein kinase inhibitors, we used a kinomic approach, studying the change in phosphorylation status of multiple kinase targets with immunoblot analysis. 15 Because we wanted to determine the earliest changes associated with signaling differentiation and not those associated with its execution, we analyzed cells that had been exposed to SS for 5 minutes, a period that we had previously established committed the cells to differentiation without altering morphology. 
Compared with sham-treated cells, there was a 90% increase in the S722 target of focal adhesion kinase (FAK). Other phosphorylation targets with notable differences from control were c-KitY703 (43% increase), ERK1T202/Y204 (not present in control), ERK2T185/Y187 (59% decrease), CDK1T14/Y15 (64% increase), MEK1S298 (100% decrease), and PKRT451 (100% decrease; Fig. 9 ). 
Discussion
These results demonstrate that a short exposure to the broad-spectrum kinase inhibitor SS differentiates the RGC precursor cell line RGC-5 into cells that have many of the morphologic, postmitotic, electrophysiologic, and antigenic properties of mature RGCs (Table 2) . Differentiated cells have multiple long neurites that branch and contact other cells. Unlike RGC-5 cells, differentiated cells do not divide, nor does the exposure to SS induce nuclear changes of apoptosis or decrease cell number. 
The electrophysiological changes seen with SS are in the same direction as mature RGCs, because both the latter and SS-differentiated RGC-5s have large voltage-gated conductances, whereas undifferentiated RGC-5 cells do not. However, SS treatment did not lead to the establishment of a more negative resting potential, even though K+ conductance was increased. SS treatment also caused expression of only a very narrow set of new channels (i.e., one or two types of high-voltage activated K+, or possibly Cl, channels), but not nearly the full range of Na+, Ca2+, and K+ channels reported in RGCs. We also saw no evidence of functional synapses. Together, these electrophysiological findings represent differentiation toward an RGC-like phenotype, but are still distinct from the electrophysiology of a mature RGC. 
There are other examples of differentiation of cell lines with protein kinase inhibition, although there are significant differences between the mechanism in RGC-5 cells and other cell types. For example, the human prostatic cancer cell line TSU-Pr1 is differentiated by SS, with most cells assuming a neuronal morphology after 3 days. 16 The mechanism is inhibition of CDK2 and consequent arrest in G1. 17 Our studies demonstrated that RGC-5 differentiation did not occur with kenpaullone or roscovitine, inhibitors of CDK2, even at 13 to 200 times the IC50. 14 SS also potentiates differentiation of human promyelocytic leukemic HL-60 cells, but requires the presence of another differentiating agent (e.g., retinoic acid). 18  
SS is a differentiating agent in retinal cells and induces rhodopsin expression and decreases green cone opsin expression in chick embryonic retinal cells. 19 This differs from the better characterized CNTF-mediated differentiation pathway, in that the latter increases green cone opsin expression. 19 The mechanism by which SS differentiates chick photoreceptors has not yet been elucidated. SS induces neurite extension in the PC12 pheochromocytoma cell line at high concentrations, 20 although at lower concentrations it inhibits neurite induction by nerve growth factor. 21 The differentiating effect involves upregulation of epidermal growth factor (EGF) receptor expression, along with dephosphorylation of EGF receptors. 22 In contrast, we did not find evidence of EGF receptor phosphorylation in either undifferentiated or differentiated RGC-5 cells. Another difference between SS differentiation of PC12 cells and RGC-5 cells is decreased ion channel expression in the former 23 and upregulation of at least two different ion channels in the latter. 
SS differentiates the neuroblastoma-derived SH-SY5Y cell line, causing neurite extension and expression of voltage-gated calcium channels. 24 Unlike RGC-5 cells, many (but not all 25 ) of the SH-SY5Y–differentiated phenotype can be reproduced with protein kinase C inhibition. 26 Also, unlike RGC-5 cells, these cells undergo transient apoptosis after treatment. 27  
Thus, the mechanism by which SS induces RGC-5 cell differentiation is different from that underlying SS differentiation of other cell types. It is unlikely to be a result of apoptosisbecause SS, a known apoptosis inducer, does not activate the apoptotic cascade in RGC-5 cells at concentrations that were used to induce differentiation. This is an important distinction, because differentiation resulting in apoptosis would not be useful for studying RGC pathophysiology. Instead, it likely reflects the inhibition of an as yet unidentified kinase not normally known to be inhibited by SS. 
Differentiating RGC-5 cells has significant advantages for the study of RGC pathophysiology and elucidating diseases of the optic nerve. The undifferentiated RGC-5 cell line, which has some RGC characteristics, is significantly different from RGCs with respect to proliferation, morphology, and electrophysiology. Differentiation of RGC-5 cells into a RGC phenotype could make the following methods possible: high-throughput screens of neuroprotective and other agents, nonviral gene transduction and production of stable lines, reduced volume of experimental animals needed for primary cultures, production of ρ0 cells for studying mitochondrial (mt)DNA and making cybrids, large-scale biochemical and other assays without need for purification of RGCs, and possible extrapolation to understanding the differentiation of stem cells or retinal neuronal precursors. 
In summary, we showed that SS induces RGC-5 cells to differentiate, express neurites, become postmitotic and nonapoptotic, and alter their kinase phosphorylation patterns. A subset of differentiated cells also display a much larger voltage-gated conductance than undifferentiated cells, with the outward rectification typical of most voltage-gated potassium channels and some voltage-gated chloride channels. The mechanism for differentiation is not the result of inhibition of kinases normally inhibited by SS, nor by induction of apoptosis, and presumably results from other effects of SS. Differentiated RGC-5 cells are potentially useful targets for studying neuronal pathophysiology and screening new therapies for nervous system and visual system disease. 
 
Figure 1.
 
SS treatment induced RGC-5 differentiation. (A) Twenty-four hour exposure to increasing concentrations of SS causes increasing degrees of neurite expression. Concentrations beyond those depicted resulted in cell necrosis after 24 hours of incubation. (B) Short-term exposure to SS causes differentiation 24 hours after removal of the chemical from the culture medium. Morphologic changes were observed after as little as 30 seconds of exposure. (C) The cells were exposed to SS for various durations and immediately examined. They had increasingly well-developed branching projections with increasing duration of exposure to SS. (D) RGC-5 cells were treated with various concentrations of SS. The number of neurites in each condition is expressed as average ± SEM from 50 cells at SS concentrations ranging from 100 nM to 3.16 μM. (E) The degree of neurite branching was quantified after 24 hours of exposure to SS. The number of neurite branches per cell is expressed as average ± SEM from 10 cells at SS concentrations from 0 to 3.16 μM. (F) Lengths of cellular projections were measured after SS-induced differentiation. Using NeuronJ, we measured the length of each projection from the soma to its end. Neurite length is expressed as average ± SEM from 10 cells. (G) The total length of all projections from each cell was measured. Total neurite length is expressed as the average ± SEM of the projections of 10 cells. All comparisons between differentiated and undifferentiated RGC-5 cells of neurite number, branches, average neurite length, and total length were significant at P < 0.05, at each concentration of SS.
Figure 1.
 
SS treatment induced RGC-5 differentiation. (A) Twenty-four hour exposure to increasing concentrations of SS causes increasing degrees of neurite expression. Concentrations beyond those depicted resulted in cell necrosis after 24 hours of incubation. (B) Short-term exposure to SS causes differentiation 24 hours after removal of the chemical from the culture medium. Morphologic changes were observed after as little as 30 seconds of exposure. (C) The cells were exposed to SS for various durations and immediately examined. They had increasingly well-developed branching projections with increasing duration of exposure to SS. (D) RGC-5 cells were treated with various concentrations of SS. The number of neurites in each condition is expressed as average ± SEM from 50 cells at SS concentrations ranging from 100 nM to 3.16 μM. (E) The degree of neurite branching was quantified after 24 hours of exposure to SS. The number of neurite branches per cell is expressed as average ± SEM from 10 cells at SS concentrations from 0 to 3.16 μM. (F) Lengths of cellular projections were measured after SS-induced differentiation. Using NeuronJ, we measured the length of each projection from the soma to its end. Neurite length is expressed as average ± SEM from 10 cells. (G) The total length of all projections from each cell was measured. Total neurite length is expressed as the average ± SEM of the projections of 10 cells. All comparisons between differentiated and undifferentiated RGC-5 cells of neurite number, branches, average neurite length, and total length were significant at P < 0.05, at each concentration of SS.
Figure 2.
 
SS differentiated RGC-5 cells became postmitotic[b]. RGC-5 cells were incubated with 100 μM BrdU for the final 2 hours of a 24-hour incubation with1 μM SS, followed by anti-BrdU immunofluorescence staining (red fluorescence overlapped with Nomarski images). Only 2.2% of SS-differentiated cells were positive for BrdU incorporation, compared to 69.5% of control cells.
Figure 2.
 
SS differentiated RGC-5 cells became postmitotic[b]. RGC-5 cells were incubated with 100 μM BrdU for the final 2 hours of a 24-hour incubation with1 μM SS, followed by anti-BrdU immunofluorescence staining (red fluorescence overlapped with Nomarski images). Only 2.2% of SS-differentiated cells were positive for BrdU incorporation, compared to 69.5% of control cells.
Figure 3.
 
RGC-5 cells increased the expression of RGC markers after differentiation. (A) Untreated (left) or SS-differentiated (right) RGC-5 cells incubated for 24 hours stained with mouse anti-glutamate receptor (anti-NMDAR1; clone 54.1) followed by goat anti-mouse IgG. (B) Untreated (left) or SS-differentiated (right) RGC-5 cells incubated for 24 hours with rabbit anti-Thy-1 polyclonal IgG stain followed by goat anti-rabbit IgG. (C) Protein levels of Thy-1 and MAP-2 measured by immunoblot analysis was increased after SS differentiation. Actin was used as the loading control. (D) Changes in Thy-1 and MAP-2 expression were quantified by densitometry. Thy-1 and MAP-2 protein levels were increased after 3 days of differentiation with 316 nM SS. RGC-5 actin content was unchanged.
Figure 3.
 
RGC-5 cells increased the expression of RGC markers after differentiation. (A) Untreated (left) or SS-differentiated (right) RGC-5 cells incubated for 24 hours stained with mouse anti-glutamate receptor (anti-NMDAR1; clone 54.1) followed by goat anti-mouse IgG. (B) Untreated (left) or SS-differentiated (right) RGC-5 cells incubated for 24 hours with rabbit anti-Thy-1 polyclonal IgG stain followed by goat anti-rabbit IgG. (C) Protein levels of Thy-1 and MAP-2 measured by immunoblot analysis was increased after SS differentiation. Actin was used as the loading control. (D) Changes in Thy-1 and MAP-2 expression were quantified by densitometry. Thy-1 and MAP-2 protein levels were increased after 3 days of differentiation with 316 nM SS. RGC-5 actin content was unchanged.
Figure 4.
 
SS treatment transiently induced voltage-gated outward currents in RGC-5 cells. (A) Example of voltage-clamp recordings from two RGC-5 cells. Top traces: an untreated cell displaying little or no voltage-gated currents. Bottom traces: cell treated with SS, revealing large voltage-gated outward currents that activated rapidly and showed no inactivation during the 10-ms step. The cell was exposed to SS (1 μM) for 1 hour, followed by a 24-hour wash period before recording. (B) A summary of voltage-gated conductance under different treatment conditions. SS treatment increased the size of voltage-gated outward current in a subset of cells. The duration of treatment is given below the data points, with the following wash period in parentheses. (• and error bars) Mean ± SEM of conductance in five untreated cells. Dashed line: twice the SD above the mean, used as a cutoff criterion for classifying cells as having significantly larger outward current than the control (♦ and ▴). (C) Conductance versus voltage curves for the six cells in which SS treatment was effective. Filled symbols: from the 1-hour (24 hours) condition in (B). Solid line: a fit to their mean, using the equation G = G max/{1+ exp[(V 1/2V)/slope]}, and yielding a maximum conductance (G max) of 26 nS, a half-maximum voltage (V 1/2) of +48 mV, and a slope factor of 31 mV. The parameters from individual fits were (mean ± SEM): G max = 29 ± 5 nS, V 1/2 = +55 ± 12 mV, and slope = 33 ± 6 mV. Open symbols: from the 1-hour (48 hours) and 22-hour (2 hours) treatment conditions. These cells did not approach their maximum conductance even at +90 mV. Thus, they could not be fit, but appeared to display a different voltage dependence than that in the 1-hour [24-hour] condition.
Figure 4.
 
SS treatment transiently induced voltage-gated outward currents in RGC-5 cells. (A) Example of voltage-clamp recordings from two RGC-5 cells. Top traces: an untreated cell displaying little or no voltage-gated currents. Bottom traces: cell treated with SS, revealing large voltage-gated outward currents that activated rapidly and showed no inactivation during the 10-ms step. The cell was exposed to SS (1 μM) for 1 hour, followed by a 24-hour wash period before recording. (B) A summary of voltage-gated conductance under different treatment conditions. SS treatment increased the size of voltage-gated outward current in a subset of cells. The duration of treatment is given below the data points, with the following wash period in parentheses. (• and error bars) Mean ± SEM of conductance in five untreated cells. Dashed line: twice the SD above the mean, used as a cutoff criterion for classifying cells as having significantly larger outward current than the control (♦ and ▴). (C) Conductance versus voltage curves for the six cells in which SS treatment was effective. Filled symbols: from the 1-hour (24 hours) condition in (B). Solid line: a fit to their mean, using the equation G = G max/{1+ exp[(V 1/2V)/slope]}, and yielding a maximum conductance (G max) of 26 nS, a half-maximum voltage (V 1/2) of +48 mV, and a slope factor of 31 mV. The parameters from individual fits were (mean ± SEM): G max = 29 ± 5 nS, V 1/2 = +55 ± 12 mV, and slope = 33 ± 6 mV. Open symbols: from the 1-hour (48 hours) and 22-hour (2 hours) treatment conditions. These cells did not approach their maximum conductance even at +90 mV. Thus, they could not be fit, but appeared to display a different voltage dependence than that in the 1-hour [24-hour] condition.
Figure 5.
 
RGC-5 cells underwent differentiation independent of cytochrome c release or caspase activation. RGC-5 cells were pretreated for 30 minutes with 10 μM minocycline, which inhibits cytochrome c release, or 20 μM ZVAD-fmk, a broad-spectrum caspase inhibitor. Cells were subsequently exposed to 1 μM SS in the presence of minocycline or SS for 24 hours. Minocycline or ZVAD-fmk did not inhibit differentiation with SS.
Figure 5.
 
RGC-5 cells underwent differentiation independent of cytochrome c release or caspase activation. RGC-5 cells were pretreated for 30 minutes with 10 μM minocycline, which inhibits cytochrome c release, or 20 μM ZVAD-fmk, a broad-spectrum caspase inhibitor. Cells were subsequently exposed to 1 μM SS in the presence of minocycline or SS for 24 hours. Minocycline or ZVAD-fmk did not inhibit differentiation with SS.
Figure 6.
 
Apoptosis inducers other than SS did not differentiate RGC-5 cells. RGC-5 cells were treated for 24 hours with SS, the calcium ionophore ionomycin, the complex I inhibitor rotenone, or thapsigargin, which causes calcium release from the endoplasmic reticulum. Differentiation, assessed by morphology (left) was at its maxim with SS. Some induction of a spindle-shaped morphology occurred with ionomycin and thapsigargin. Apoptosis, assessed by Hoechst 33258 staining of nuclear condensation (right), was seen with ionomycin, rotenone, and thapsigargin, but not SS.
Figure 6.
 
Apoptosis inducers other than SS did not differentiate RGC-5 cells. RGC-5 cells were treated for 24 hours with SS, the calcium ionophore ionomycin, the complex I inhibitor rotenone, or thapsigargin, which causes calcium release from the endoplasmic reticulum. Differentiation, assessed by morphology (left) was at its maxim with SS. Some induction of a spindle-shaped morphology occurred with ionomycin and thapsigargin. Apoptosis, assessed by Hoechst 33258 staining of nuclear condensation (right), was seen with ionomycin, rotenone, and thapsigargin, but not SS.
Table 1.
 
Profile of the Effects of Kinase Inhibitors on Various Kinases at the Concentrations Used for RGC-5 Cells
Table 1.
 
Profile of the Effects of Kinase Inhibitors on Various Kinases at the Concentrations Used for RGC-5 Cells
Kinase Roscovitine Tyrphostin AG490 Tyrphostin AG1295 Tyrphostin AG1478 H-89 HA-1077 Bisindolyl-Maleimide IX Kenpaullone H-1152 IBMX PD98059 LY290042 SS
CDK1/cyclin B X X X
CDK2/cyclin A X X X
CDK5 X X X
JAK-2 X X
EGFR X X X
PDGFR X X
PKA X X
MLCK X X
CAM X X
PKC X X
Rho-Kinase X X
cAMP PDE X (via PKC)
cGMP PDE X (via PKC)
MEK X X
Akt X X
Figure 7.
 
Inhibition of several protein kinases did not reproduce the differentiating effect of SS. Cells were treated for 24 hours with specific kinase inhibitors at concentrations chosen to result in complete inhibition of the respective kinase(s). No kinase inhibitor, alone or in combination, led to morphology similar to that observed after SS treatment.
Figure 7.
 
Inhibition of several protein kinases did not reproduce the differentiating effect of SS. Cells were treated for 24 hours with specific kinase inhibitors at concentrations chosen to result in complete inhibition of the respective kinase(s). No kinase inhibitor, alone or in combination, led to morphology similar to that observed after SS treatment.
Figure 8.
 
The effect of relatively specific kinase inhibitors, alone or in combination, on RGC-5 differentiation. Although certain kinase inhibitors caused some morphologic changes, none reproduced the neuronal appearance observed when RGC-5 cells were incubated with SS.
Figure 8.
 
The effect of relatively specific kinase inhibitors, alone or in combination, on RGC-5 differentiation. Although certain kinase inhibitors caused some morphologic changes, none reproduced the neuronal appearance observed when RGC-5 cells were incubated with SS.
Figure 9.
 
SS treatment rapidly changes kinase phosphorylation patterns in RGC-5 cells. (A) RGC-5 cells were treated with 1 μM SS for 5 minutes. Western blot analysis was used to examine kinase phosphorylation at 39 known sites. Bands of interest are numbered as follows: (1) c-Kit; (2) extracellular signal-regulated kinase 1 (ERK 1); (3) extracellular signal-regulated kinase 2 (ERK 2); (4) cyclin-dependent kinase 1 (CDK 1); (5) focal adhesion kinase (FAK); (6) MAP kinase kinase 1 (MAP 1); and (7) Double-stranded RNA-dependent protein kinase (PKR). (B) Normalized differences between SS treatment and the control revealed several proteins with marked changes in phosphorylation levels. Incr, present only in SS; Decr, present only in the control.
Figure 9.
 
SS treatment rapidly changes kinase phosphorylation patterns in RGC-5 cells. (A) RGC-5 cells were treated with 1 μM SS for 5 minutes. Western blot analysis was used to examine kinase phosphorylation at 39 known sites. Bands of interest are numbered as follows: (1) c-Kit; (2) extracellular signal-regulated kinase 1 (ERK 1); (3) extracellular signal-regulated kinase 2 (ERK 2); (4) cyclin-dependent kinase 1 (CDK 1); (5) focal adhesion kinase (FAK); (6) MAP kinase kinase 1 (MAP 1); and (7) Double-stranded RNA-dependent protein kinase (PKR). (B) Normalized differences between SS treatment and the control revealed several proteins with marked changes in phosphorylation levels. Incr, present only in SS; Decr, present only in the control.
Table 2.
 
Similarities and Differences among RGC-5 Cells, Differentiated RGC-5 Cells, and Primary RGCs
Table 2.
 
Similarities and Differences among RGC-5 Cells, Differentiated RGC-5 Cells, and Primary RGCs
Feature RGC-5 Differentiated RGC-5 RGC
Morphology Flat, polygonal, few neurites Small soma, several long neurites Small soma, several long neurites
Mitotic state Proliferating Nonproliferating Nonproliferating
Ion channels Small whole-cell conductance Clv, Kv, Kir Large whole-cell conductance, consistent with Kv or Clv Nav1.1, Nav1.2, Nav1.3, and Nav1.6 Cav3.1, Cav3.2, and Cav3.3 Kv Kir1.1, Kir2.1, Kir2.3, Kir3.1, Kir3.2, and Kir3.3 ASIC1a, ASIC2a, ASIC2b, ASIC3, and ASIC4
Neuronal markers Thy-1, NMDAR1, GABA-B receptor, Brn-3C, neuritin, synaptophysin Thy-1, NMDAR1 (others not tested) Thy-1, NMDAR1, Brn-3C, neuritin, TrkA, TrkB, P75, CNTF, BDNF, GDNF, GABA-B, synaptophysin
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Figure 1.
 
SS treatment induced RGC-5 differentiation. (A) Twenty-four hour exposure to increasing concentrations of SS causes increasing degrees of neurite expression. Concentrations beyond those depicted resulted in cell necrosis after 24 hours of incubation. (B) Short-term exposure to SS causes differentiation 24 hours after removal of the chemical from the culture medium. Morphologic changes were observed after as little as 30 seconds of exposure. (C) The cells were exposed to SS for various durations and immediately examined. They had increasingly well-developed branching projections with increasing duration of exposure to SS. (D) RGC-5 cells were treated with various concentrations of SS. The number of neurites in each condition is expressed as average ± SEM from 50 cells at SS concentrations ranging from 100 nM to 3.16 μM. (E) The degree of neurite branching was quantified after 24 hours of exposure to SS. The number of neurite branches per cell is expressed as average ± SEM from 10 cells at SS concentrations from 0 to 3.16 μM. (F) Lengths of cellular projections were measured after SS-induced differentiation. Using NeuronJ, we measured the length of each projection from the soma to its end. Neurite length is expressed as average ± SEM from 10 cells. (G) The total length of all projections from each cell was measured. Total neurite length is expressed as the average ± SEM of the projections of 10 cells. All comparisons between differentiated and undifferentiated RGC-5 cells of neurite number, branches, average neurite length, and total length were significant at P < 0.05, at each concentration of SS.
Figure 1.
 
SS treatment induced RGC-5 differentiation. (A) Twenty-four hour exposure to increasing concentrations of SS causes increasing degrees of neurite expression. Concentrations beyond those depicted resulted in cell necrosis after 24 hours of incubation. (B) Short-term exposure to SS causes differentiation 24 hours after removal of the chemical from the culture medium. Morphologic changes were observed after as little as 30 seconds of exposure. (C) The cells were exposed to SS for various durations and immediately examined. They had increasingly well-developed branching projections with increasing duration of exposure to SS. (D) RGC-5 cells were treated with various concentrations of SS. The number of neurites in each condition is expressed as average ± SEM from 50 cells at SS concentrations ranging from 100 nM to 3.16 μM. (E) The degree of neurite branching was quantified after 24 hours of exposure to SS. The number of neurite branches per cell is expressed as average ± SEM from 10 cells at SS concentrations from 0 to 3.16 μM. (F) Lengths of cellular projections were measured after SS-induced differentiation. Using NeuronJ, we measured the length of each projection from the soma to its end. Neurite length is expressed as average ± SEM from 10 cells. (G) The total length of all projections from each cell was measured. Total neurite length is expressed as the average ± SEM of the projections of 10 cells. All comparisons between differentiated and undifferentiated RGC-5 cells of neurite number, branches, average neurite length, and total length were significant at P < 0.05, at each concentration of SS.
Figure 2.
 
SS differentiated RGC-5 cells became postmitotic[b]. RGC-5 cells were incubated with 100 μM BrdU for the final 2 hours of a 24-hour incubation with1 μM SS, followed by anti-BrdU immunofluorescence staining (red fluorescence overlapped with Nomarski images). Only 2.2% of SS-differentiated cells were positive for BrdU incorporation, compared to 69.5% of control cells.
Figure 2.
 
SS differentiated RGC-5 cells became postmitotic[b]. RGC-5 cells were incubated with 100 μM BrdU for the final 2 hours of a 24-hour incubation with1 μM SS, followed by anti-BrdU immunofluorescence staining (red fluorescence overlapped with Nomarski images). Only 2.2% of SS-differentiated cells were positive for BrdU incorporation, compared to 69.5% of control cells.
Figure 3.
 
RGC-5 cells increased the expression of RGC markers after differentiation. (A) Untreated (left) or SS-differentiated (right) RGC-5 cells incubated for 24 hours stained with mouse anti-glutamate receptor (anti-NMDAR1; clone 54.1) followed by goat anti-mouse IgG. (B) Untreated (left) or SS-differentiated (right) RGC-5 cells incubated for 24 hours with rabbit anti-Thy-1 polyclonal IgG stain followed by goat anti-rabbit IgG. (C) Protein levels of Thy-1 and MAP-2 measured by immunoblot analysis was increased after SS differentiation. Actin was used as the loading control. (D) Changes in Thy-1 and MAP-2 expression were quantified by densitometry. Thy-1 and MAP-2 protein levels were increased after 3 days of differentiation with 316 nM SS. RGC-5 actin content was unchanged.
Figure 3.
 
RGC-5 cells increased the expression of RGC markers after differentiation. (A) Untreated (left) or SS-differentiated (right) RGC-5 cells incubated for 24 hours stained with mouse anti-glutamate receptor (anti-NMDAR1; clone 54.1) followed by goat anti-mouse IgG. (B) Untreated (left) or SS-differentiated (right) RGC-5 cells incubated for 24 hours with rabbit anti-Thy-1 polyclonal IgG stain followed by goat anti-rabbit IgG. (C) Protein levels of Thy-1 and MAP-2 measured by immunoblot analysis was increased after SS differentiation. Actin was used as the loading control. (D) Changes in Thy-1 and MAP-2 expression were quantified by densitometry. Thy-1 and MAP-2 protein levels were increased after 3 days of differentiation with 316 nM SS. RGC-5 actin content was unchanged.
Figure 4.
 
SS treatment transiently induced voltage-gated outward currents in RGC-5 cells. (A) Example of voltage-clamp recordings from two RGC-5 cells. Top traces: an untreated cell displaying little or no voltage-gated currents. Bottom traces: cell treated with SS, revealing large voltage-gated outward currents that activated rapidly and showed no inactivation during the 10-ms step. The cell was exposed to SS (1 μM) for 1 hour, followed by a 24-hour wash period before recording. (B) A summary of voltage-gated conductance under different treatment conditions. SS treatment increased the size of voltage-gated outward current in a subset of cells. The duration of treatment is given below the data points, with the following wash period in parentheses. (• and error bars) Mean ± SEM of conductance in five untreated cells. Dashed line: twice the SD above the mean, used as a cutoff criterion for classifying cells as having significantly larger outward current than the control (♦ and ▴). (C) Conductance versus voltage curves for the six cells in which SS treatment was effective. Filled symbols: from the 1-hour (24 hours) condition in (B). Solid line: a fit to their mean, using the equation G = G max/{1+ exp[(V 1/2V)/slope]}, and yielding a maximum conductance (G max) of 26 nS, a half-maximum voltage (V 1/2) of +48 mV, and a slope factor of 31 mV. The parameters from individual fits were (mean ± SEM): G max = 29 ± 5 nS, V 1/2 = +55 ± 12 mV, and slope = 33 ± 6 mV. Open symbols: from the 1-hour (48 hours) and 22-hour (2 hours) treatment conditions. These cells did not approach their maximum conductance even at +90 mV. Thus, they could not be fit, but appeared to display a different voltage dependence than that in the 1-hour [24-hour] condition.
Figure 4.
 
SS treatment transiently induced voltage-gated outward currents in RGC-5 cells. (A) Example of voltage-clamp recordings from two RGC-5 cells. Top traces: an untreated cell displaying little or no voltage-gated currents. Bottom traces: cell treated with SS, revealing large voltage-gated outward currents that activated rapidly and showed no inactivation during the 10-ms step. The cell was exposed to SS (1 μM) for 1 hour, followed by a 24-hour wash period before recording. (B) A summary of voltage-gated conductance under different treatment conditions. SS treatment increased the size of voltage-gated outward current in a subset of cells. The duration of treatment is given below the data points, with the following wash period in parentheses. (• and error bars) Mean ± SEM of conductance in five untreated cells. Dashed line: twice the SD above the mean, used as a cutoff criterion for classifying cells as having significantly larger outward current than the control (♦ and ▴). (C) Conductance versus voltage curves for the six cells in which SS treatment was effective. Filled symbols: from the 1-hour (24 hours) condition in (B). Solid line: a fit to their mean, using the equation G = G max/{1+ exp[(V 1/2V)/slope]}, and yielding a maximum conductance (G max) of 26 nS, a half-maximum voltage (V 1/2) of +48 mV, and a slope factor of 31 mV. The parameters from individual fits were (mean ± SEM): G max = 29 ± 5 nS, V 1/2 = +55 ± 12 mV, and slope = 33 ± 6 mV. Open symbols: from the 1-hour (48 hours) and 22-hour (2 hours) treatment conditions. These cells did not approach their maximum conductance even at +90 mV. Thus, they could not be fit, but appeared to display a different voltage dependence than that in the 1-hour [24-hour] condition.
Figure 5.
 
RGC-5 cells underwent differentiation independent of cytochrome c release or caspase activation. RGC-5 cells were pretreated for 30 minutes with 10 μM minocycline, which inhibits cytochrome c release, or 20 μM ZVAD-fmk, a broad-spectrum caspase inhibitor. Cells were subsequently exposed to 1 μM SS in the presence of minocycline or SS for 24 hours. Minocycline or ZVAD-fmk did not inhibit differentiation with SS.
Figure 5.
 
RGC-5 cells underwent differentiation independent of cytochrome c release or caspase activation. RGC-5 cells were pretreated for 30 minutes with 10 μM minocycline, which inhibits cytochrome c release, or 20 μM ZVAD-fmk, a broad-spectrum caspase inhibitor. Cells were subsequently exposed to 1 μM SS in the presence of minocycline or SS for 24 hours. Minocycline or ZVAD-fmk did not inhibit differentiation with SS.
Figure 6.
 
Apoptosis inducers other than SS did not differentiate RGC-5 cells. RGC-5 cells were treated for 24 hours with SS, the calcium ionophore ionomycin, the complex I inhibitor rotenone, or thapsigargin, which causes calcium release from the endoplasmic reticulum. Differentiation, assessed by morphology (left) was at its maxim with SS. Some induction of a spindle-shaped morphology occurred with ionomycin and thapsigargin. Apoptosis, assessed by Hoechst 33258 staining of nuclear condensation (right), was seen with ionomycin, rotenone, and thapsigargin, but not SS.
Figure 6.
 
Apoptosis inducers other than SS did not differentiate RGC-5 cells. RGC-5 cells were treated for 24 hours with SS, the calcium ionophore ionomycin, the complex I inhibitor rotenone, or thapsigargin, which causes calcium release from the endoplasmic reticulum. Differentiation, assessed by morphology (left) was at its maxim with SS. Some induction of a spindle-shaped morphology occurred with ionomycin and thapsigargin. Apoptosis, assessed by Hoechst 33258 staining of nuclear condensation (right), was seen with ionomycin, rotenone, and thapsigargin, but not SS.
Figure 7.
 
Inhibition of several protein kinases did not reproduce the differentiating effect of SS. Cells were treated for 24 hours with specific kinase inhibitors at concentrations chosen to result in complete inhibition of the respective kinase(s). No kinase inhibitor, alone or in combination, led to morphology similar to that observed after SS treatment.
Figure 7.
 
Inhibition of several protein kinases did not reproduce the differentiating effect of SS. Cells were treated for 24 hours with specific kinase inhibitors at concentrations chosen to result in complete inhibition of the respective kinase(s). No kinase inhibitor, alone or in combination, led to morphology similar to that observed after SS treatment.
Figure 8.
 
The effect of relatively specific kinase inhibitors, alone or in combination, on RGC-5 differentiation. Although certain kinase inhibitors caused some morphologic changes, none reproduced the neuronal appearance observed when RGC-5 cells were incubated with SS.
Figure 8.
 
The effect of relatively specific kinase inhibitors, alone or in combination, on RGC-5 differentiation. Although certain kinase inhibitors caused some morphologic changes, none reproduced the neuronal appearance observed when RGC-5 cells were incubated with SS.
Figure 9.
 
SS treatment rapidly changes kinase phosphorylation patterns in RGC-5 cells. (A) RGC-5 cells were treated with 1 μM SS for 5 minutes. Western blot analysis was used to examine kinase phosphorylation at 39 known sites. Bands of interest are numbered as follows: (1) c-Kit; (2) extracellular signal-regulated kinase 1 (ERK 1); (3) extracellular signal-regulated kinase 2 (ERK 2); (4) cyclin-dependent kinase 1 (CDK 1); (5) focal adhesion kinase (FAK); (6) MAP kinase kinase 1 (MAP 1); and (7) Double-stranded RNA-dependent protein kinase (PKR). (B) Normalized differences between SS treatment and the control revealed several proteins with marked changes in phosphorylation levels. Incr, present only in SS; Decr, present only in the control.
Figure 9.
 
SS treatment rapidly changes kinase phosphorylation patterns in RGC-5 cells. (A) RGC-5 cells were treated with 1 μM SS for 5 minutes. Western blot analysis was used to examine kinase phosphorylation at 39 known sites. Bands of interest are numbered as follows: (1) c-Kit; (2) extracellular signal-regulated kinase 1 (ERK 1); (3) extracellular signal-regulated kinase 2 (ERK 2); (4) cyclin-dependent kinase 1 (CDK 1); (5) focal adhesion kinase (FAK); (6) MAP kinase kinase 1 (MAP 1); and (7) Double-stranded RNA-dependent protein kinase (PKR). (B) Normalized differences between SS treatment and the control revealed several proteins with marked changes in phosphorylation levels. Incr, present only in SS; Decr, present only in the control.
Table 1.
 
Profile of the Effects of Kinase Inhibitors on Various Kinases at the Concentrations Used for RGC-5 Cells
Table 1.
 
Profile of the Effects of Kinase Inhibitors on Various Kinases at the Concentrations Used for RGC-5 Cells
Kinase Roscovitine Tyrphostin AG490 Tyrphostin AG1295 Tyrphostin AG1478 H-89 HA-1077 Bisindolyl-Maleimide IX Kenpaullone H-1152 IBMX PD98059 LY290042 SS
CDK1/cyclin B X X X
CDK2/cyclin A X X X
CDK5 X X X
JAK-2 X X
EGFR X X X
PDGFR X X
PKA X X
MLCK X X
CAM X X
PKC X X
Rho-Kinase X X
cAMP PDE X (via PKC)
cGMP PDE X (via PKC)
MEK X X
Akt X X
Table 2.
 
Similarities and Differences among RGC-5 Cells, Differentiated RGC-5 Cells, and Primary RGCs
Table 2.
 
Similarities and Differences among RGC-5 Cells, Differentiated RGC-5 Cells, and Primary RGCs
Feature RGC-5 Differentiated RGC-5 RGC
Morphology Flat, polygonal, few neurites Small soma, several long neurites Small soma, several long neurites
Mitotic state Proliferating Nonproliferating Nonproliferating
Ion channels Small whole-cell conductance Clv, Kv, Kir Large whole-cell conductance, consistent with Kv or Clv Nav1.1, Nav1.2, Nav1.3, and Nav1.6 Cav3.1, Cav3.2, and Cav3.3 Kv Kir1.1, Kir2.1, Kir2.3, Kir3.1, Kir3.2, and Kir3.3 ASIC1a, ASIC2a, ASIC2b, ASIC3, and ASIC4
Neuronal markers Thy-1, NMDAR1, GABA-B receptor, Brn-3C, neuritin, synaptophysin Thy-1, NMDAR1 (others not tested) Thy-1, NMDAR1, Brn-3C, neuritin, TrkA, TrkB, P75, CNTF, BDNF, GDNF, GABA-B, synaptophysin
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