Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue Box 290, New York, NY 10021
Received on August 29, 2003; revised on September 30, 2003; accepted on October 1, 2003
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Abstract |
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Key words: plasticity / polysialic acid / polysialyltransferase / PST / STX
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Introduction |
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The primary goal of the present study was to demonstrate the ability to misexpress PSA in vivo by viral delivery of cDNAs encoding either the ST8Sia IV (PST) or ST8Sia II (STX) polysialyltransferase and to determine if PSA misexpression is sufficient to alter the structure of a central nervous system tissue. PST and STX have previously been shown in vitro to synthesize PSA chains specifically on the core carbohydrate of NCAM (Eckhardt et al., 1995; Nakayama et al., 1995
, 1998
; Angata and Fukuda, 2003
). A further motivation for these studies is the possibility that PSA expression might provide a means to promote a permissive environment for repair mechanisms in adult tissues that no longer express PSA (Kiss et al., 2001
).
The embryonic retina was chosen as a test system because it has a highly stereotyped program of morphogenesis and loses its PSA at an early stage in its development (Schlosshauer et al., 1984). Because PSA acts as a permissive factor for tissue remodeling, its action requires a context in which other factors provide the driving force for change. We reasoned that the continuing process of development in the retina would serve this purpose, so that the introduction of ectopic PSA would distort the behavior of the cells, resulting in observable changes in retinal structure. Using retroviral delivery of PST, it has been possible to misexpress PSA and dramatically modify the fundamental structure of the retinal epithelia. The nature of these defects suggests that the primary effect of ectopic PSA is to alter interactions among cells within and at the edges of epithelial margins, consistent with a loss of tissue stability.
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Results |
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Induced expression of PSA-NCAM in CHO cells transfected with CMV-GFP-PST or CMV-GFP-STX
The next step in the study was to evaluate the nature of the PSA produced by transfection with PST and STX. Again, it was important to assess not only the extent of NCAM polysialylation but also the nature of the chains produced, because excessively long chains could produce artifacts unrelated to PSA's physiological function. Furthermore, it was necessary to compare PSA produced by PST and STX, because these enzymes exhibit differences in their ability to polysialylate NCAM (Angata et al., 1998, 2002
; Angata and Fukuda, 2003
). Chinese hamster ovary (CHO) cells were transiently transfected with NCAM180 cDNA and plasmids encoding GFP-PST or GFP-STX (Figure 2). Green fluorescent protein (GFP) fluorescence indicating expression of GFP-PST or GFP-STX is shown in Figures 2C and 2D, and these cells were stained using anti-NCAM (Figures 2A and 2B) and anti-PSA antibodies (Figures 2E and 2F). GFP-STX and GFP-PST were detected predominantly in the juxtanuclear Golgi region as well as at the cell surface, in agreement with previous reports using epitope-tagged STX and PST (Close and Colley, 1998
). As expected, PSA staining was only detected in GFP-positive cells that are stained with anti-NCAM. Expression of GFP-PST resulted in higher levels of staining for PSA than expression of GFP-STX (compare Figures 2E and 2F). Treatment of the transfected cells with endo N prior to addition of antibodies abolished all PSA staining (unpublished data). Similar results were also obtained using COS cells.
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To confirm that these defects specifically reflect cell surface PSA misexpression, endo N was injected along with the RCAS-GFP-PST, so that any PSA expression (ectopic or endogenous) is suppressed. The action of endo N under these conditions is complete and long lasting (Landmesser et al., 1990; Tang et al., 1992
; Yin et al., 1995
), so that no cell surface PSA can be detected throughout the period of the experiment (Figure 4O). Though removal of endogenous PSA by endo N did not by itself alter retinal development, in infected regions it did prevent defects caused by misexpression of PST (Figures 4M, N). This rescue by endo N is scored quantitatively in Table I, showing that expression of GFP-PST in the presence of endo N resulted in mild defects in only 3/120 GFP-positive regions, whereas expression of GFP-PST without endo N resulted in severe or mild defects in 117/120 GFP-positive regions.
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Discussion |
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NCAM polysialylation by PST and STX in vitro and in vivo
The first issue raised by these findings is whether the observed biological defects might be an artifact of a grossly nonphysiological level or state of the PSA polymer. That is, the massive overexpression (amount or length) of any such charged polymer might nonspecifically disrupt a cell's ability to behave normally or even survive within a tissue. In vitro experiments using PST and STX have established that either enzyme is capable of polysialylating NCAM (Eckhardt et al., 1995; Kojima et al., 1995
; Nakayama et al., 1995; Scheidegger et al., 1995
; Nakayama and Fukuda, 1996
), and in our study both GFP-STX and GFP-PST produced cell surface PSA-NCAM in CHO, COS, and Neuro2a cells (Figure 2 and unpublished data). Neither the amount of PSA nor the length of the polymers induced appeared to lie outside the observed physiological range, in that all of the NCAM in normal embryonic brain appears to carry PSA and the natural polymer length (as judged by electrophoretic mobility) was not exceeded in the transfection studies. Furthermore, the presence of the GFP moiety did not appear to affect the enzymatic capacity of PST or STX as the GFP-tagged enzymes produced PSA of similar quality as untagged PST. Although it is possible that evaluation of these parameters in cell lines is not entirely representative of polysialylation in intact retina (see discussion of STX), it is also reassuring that the level of PST-induced ectopic PSA in the retina as judged by immunofluorescence was similar to that for endogenous PSA in retina and less than that found naturally in the optic tectum.
The present studies confirm earlier reports that PSA-NCAM produced in vitro by PST contains more sialic acid residues than those obtained with STX (Angata et al., 1998, 2002
; Kitazume-Kawaguchi et al., 2001
). An even more striking difference was seen in vivo, where retroviral expression of PST produced ectopic PSA but STX expression had no detectable effect on PSA levels. This difference may be related to the polysialylation status of NCAM in developing retina, where NCAM is relatively PSA-deficient both in terms of the presence of nonpolysialylated NCAM and the length of PSA polymers. That poorly polysialylated NCAM may be a better substrate for PST than STX is consistent with reports that PST can both increase the length of PSA chains synthesized by STX and add PSA to nonpolysialylated antennas on NCAM N-glycans (Angata et al., 2002
; Angata and Fukuda, 2003
). The expression of PSA by GFP-STX in CHO cells together with the inability of GFP-STX to produce ectopic PSA in retina may suggest that more stringent regulatory mechanisms operate to control NCAM polysialylation in vivo than those operating in vitro.
Effects of ectopic PSA on retinal development
One goal of this study was to establish that PSA is not only necessary for promoting a variety of developmental changes in tissue structure (see introduction) but also sufficient to promote such changes. The PSA gain-of-function approach used here supports this capability in the context of the developing retina. However, it should be noted that if PSA creates permissive conditions for change, then its effects should be detected only when the cells in a tissue are actively attempting to alter their position or shape. In fact, it has been shown that the removal of naturally expressed PSA does not have a detectable effect during periods when a tissue is not undergoing intrinsic reorganization of its architecture (Cremer et al., 1994; Yin et al., 1995
; El Maarouf and Rutishauser, 2003
).
The mechanism whereby PSA misexpression perturbs radial cell morphology and retinal histogenesis likely involves a loss of normal cellcell or cellmatrix interactions between neuroepithelial cell endfeet and the retinal or pial basal lamina. The radial processes of neuroepithelial cells serve as guideposts for the normal migratory pathways of postmitotic retinal cell types (Rakic and Caviness, 1995) and are critical in both establishing retinal polarity and determining the pattern of retinal lamination and the formation of the optic nerve (Halfter, 1980; Mey and Thanos, 2000
; Willbold et al., 2000
; Halfter et al., 2001
). In embryonic chick retina, where the basal lamina is transiently removed during development, the endfeet of neuroepithelial cells become irreversibly retracted, and the orderly migration of cell types, the development of retinal layers, and the growth of RGC axons are perturbed (Halfter, 1998
; Halfter et al., 2001
). Thus, ectopic PSA in radial cells may trigger altered cellcell or cellmatrix interactions that severely disrupts radial cell morphology leading to the observed effects on retinal histogenesis. Ectopic PSA may also reduce the ability of postmitotic cells to interact with and migrate along processes of neuroepithelial cells.
In considering possible molecular targets for PSA in this system, it is interesting to note the role of cadherins in forming and maintaining epithelial structure (Gumbiner, 1996). Masai et al. (2003)
demonstrate that zebrafish with mutations in N-cadherin have impaired organization and maintenance of adherens junctions in retinal neuroepithelial cells and are unable to organize their retinal neurons into correct layers. Such an association of cadherin function with the effects induced by PSA is also consistent with the fact that PSA has been shown to be a potent negative regulator of cadherin adhesive function (Fujimoto et al., 2001
).
Just as up-regulation of PSA is valuable for promoting morphogenesis, so also is its down-regulation important for maintaining the structures that have been assembled. Expression of the polysialylated form of NCAM during an active phase of tissue development is typically followed by a progressive loss of PSA as more stable mature adult tissues are formed. For example, in the chick tectum, PSA-NCAM expression is down-regulated immediately following the innervation of retinal axons (Yamagata et al., 1995). Similarly, PSA expression is tightly regulated during synaptogenesis, being highly expressed on the surface of ciliary neurons prior to synapse formation and then progressively lost from points of synaptic contact (Bruses et al., 1995
, 2002
). Another example is the transient expression of PSA as myotubes separate from myotube clusters to form individual muscle fibers (Fredette et al., 1993
). In the present study, the importance of this down-regulation to tissue stability is illustrated functionally by the fact that an abnormal persistence of PSA severely degrades the ability of the retina to maintain its overall epithelial integrity and thus the formation of organized layers.
Although the misexpression of PSA has detrimental effects in the developing retina, it could have beneficial effects in promoting tissue plasticity and repair in response to pathological insult. The fact that the brief period of nerve sprouting following nerve injury is functionally correlated with a transient up-regulation of PSA (Kiss et al., 2001), suggests that a more persistent expression might be useful. Moreover, the nature of the tissue perturbations found in the present studies suggest that PSA expression can be effective in disrupting interactions involving nonneuronal cellular elements, without affecting the ability of neurons to extend axons.
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Materials and methods |
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To produce retroviral particles DF-1 cells were transfected with retroviral constructs using LipofectAMINE 2000 (Life Technologies, Carlsbad, CA). Cells were then propagated and expanded over 10 days in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 2% chick serum, by which time 100% of the cells were infected. When cells reached confluency after the last split, a minimal volume of low-serum media was added (DMEM with 2% FCS and 0.2% chick serum). After 24 h, media was removed and filtered through a 0.45-µm cellulose acetate filter. Media was replaced, and a second harvest was carried out 24 h later. Viral conditioned media was centrifuged for 3 h at 4°C in a Beckman SW28 rotor at 21,000 rpm. Viral pellets were resuspended in Optimem on ice (Life Technologies, Cergy Pontoise, France) using 0.05 times the original volume. Retroviral titers were determined according to published protocols by immunocytochemistry with AMV3C2 anti-gag antibody (Morgan and Fekete, 1996; Developmental Studies Hybridoma Bank, Iowa City, IA). Immunocytochemistry for titering was carried out with DF-1 cells in chamber slides as described shortly, and titers for all virus preparations were in the range of 3 x 106 to 5 x 106 infectious units/ml.
Expression of NCAM, STX, and PST in CHO Cells
CHO cells obtained from the American Type Tissue Collection were transiently transfected at 90% confluency with pCMV-GFP-PST and pCMV-NCAM180, pCM-GFP-STX and pCMV-NCAM180, or pCMV-NCAM180 alone using LipofectAMINE2000 according to the manufacturers instructions. Cells were grown in 60 mm dishes or in eight-well chamber slides (Tissue-Tek, Naperville, IL) in DMEM supplemented with 10% FCS. Thirty-six hours after lipofection cells were harvested in extraction buffer containing 20 mM TrisHCl, 150 mM NaCl, 2 mM ethylenediamine tetra-acetic acid, and 0.8% Nonidet P-40 containing a protease inhibitor mixture (Boehringer Mannheim). Suspensions were sonicated on ice three times for 10 s and incubated at 4°C for 1 h. Samples were then centrifuged at 15,000 x g for 10 min, and the protein concentration in the supernatant was determined using the BCA method (Pierce, Rockford, IL).
Immunocytochemistry for PSA and NCAM in CHO Cells
CHO cells were transfected as described in eight-well chamber slides. Thirty-six hours after transfection cells were fixed for 15 min at room temperature in phosphate buffered saline (PBS)/4% paraformaldehyde and rinsed 4 times 10 min in PBS. Blocking was carried out for 60 min at room temperature in PBS-T containing 0.05% Triton X-100 and 1% ultra-pure IgG-free bovine serum albumin (Jackson Immunoresearch, West Grove, PA). Cells were then incubated overnight at 4°C with 1:1500 dilutions of the monoclonal antibodies 5e that recognizes chick NCAM (Watanabe et al., 1986) and 5a5 that recognizes PSA (Acheson et al., 1991
). Cells were washed three times 10 min in PBS-T and incubated for 1 h in blocking buffer with the appropriate Cy5- (1:400) or Cy3-conjugated (1:800) antibodies (Jackson Immunoresearch). After rinsing four times 15 min, slides were coverslipped with mowiol containing 0.01% triethylenediamine (Sigma-Aldrich, Deisenhofen, Germany). Staining for NCAM and PSA was abolished in control experiments where either primary antibody was omitted.
Western blotting
Protein extracts from chick retina and tectum were prepared by dissecting tissues in cold oxygenated Tyrode's solution (134 mM NaCl, 3 mM KCl, 20.5 mM NaHCO3, 3 mM CaCl2, 1 mM MgCl2, and 12 mM glucose, pH 7.2). Tissues were mechanically dissociated in extraction buffer using a fire-polished Pasteur pipette, then proteins were extracted as described. Equal amounts of protein were divided into aliquots and in some cases endo N, isolated as described (Hallenbeck et al., 1987), was added to a sample for 1 h at 37°C to remove PSA. Samples were heated at 65°C for 20 min and loaded on 6% sodium dodecyl sulfatepolyacrylamide gel electrophoresis gels using a Bio-Rad minigel system (BioRad, Munich, Germany). Proteins were transferred by electrophoresis onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked for 1 h in TBS-T (0.1% Tween 20) containing 5% dry milk (BioRad), then incubated overnight at 4°C in a 1:1500 dilution of anti-NCAM antibody. Blots were incubated with peroxidase-conjugated secondary antibodies (Jackson Immunoresearch) diluted 1:5000 in TBS-T. After incubation for 1 h at room temperature, membranes were washed four times for 5 min with TBS-T. Detection was carried out using the ECL method according to the manufacturers instructions (Amersham Pharmacia Biotech, Little Chalfont, U.K.).
Viral infection and coinjection of virus with endo N
Fertile white leghorn eggs, subtype 0 (Spafus, North Franklin, CT), were incubated at 38°C in a 100% humidified incubator. At 36 h embryos were windowed and staged according to Hamburger and Hamilton (1951). Embryos at Hamburger and Hilton stage 1012 were used for injections. Approximately 100 nl of retroviral concentrates containing 0.02% fast green and 80 µl/ml of polybrene (Sigma-Aldrich) was pressure injected into the right optic vesicle using a picospritzer (Parker Kannafin Corp, Fairfield, NJ) attached to a micromanipulator (Narishige, East Meadow, NY). The solution was injected until it filled the telencephalon and both optic vesicles. Embryos were sealed with parafilm and incubated until the desired stage. In preliminary experiments using RCAS(B)GFP, this method was found to produce a high level of infection in the retina as shown by staining with anti-gag antibody as well as by monitoring GFP expression. Injection at Hamburger and Hilton stage 1012 with RCAS(B)GFP-PST or RCAS(B)GFP-STX typically resulted in infection of 4050% of the retina, and infection with RCAS(B)GFP produced slightly higher rates of infection. In all RCAS injection experiments, representative sections were stained with anti-gag to assess the extent of infection. In additional sets of control experiments, endo N was coinjected together with RCAS(B)GFP-PST. Equal volumes of either PBS or endo N was added to viral concentrates and injected as described. Three embryos injected with RCAS(B)GFP-PST with endo N and three embryos injected with virus alone were analyzed from each of two independent experiments. Representative sections were stained with anti-PSA and anti-GAG antibodies to ensure complete removal of PSA by endo N and to access the extent of infection. The effects of GFP-PST expression in the presence or absence of endo N were then scored as described in Table I.
Immunohistochemistry and confocal microscopy
Chick eyes were dissected from white leghorn embryos (Spafus) of different ages in cold-oxygenated Tyrode's solution and then fixed in 4% paraformaldehyde-PBS overnight at 4°C. Eyes were then rinsed extensively in PBS with six changes over 4 h and placed in 20% sucrose overnight at 4°C for cryoprotection. The tissue was then embedded in optimal cutting temperature compound (Tissue-Tek) and sectioned at 16 µM using a cryostat. Tissue sections were air-dried and stored at -80°C. For immunohistochemistry on retinal sections, slides were incubated in 100 mM glycine in PBS for 10 min followed by 1 h in blocking solution (PBS containing 1% bovine serum albumin and 0.05% Triton X-100). Primary antibodies diluted in blocking buffer were incubated overnight at 4°C. NCAM distribution was visualized using the 5e antibody (1:1500), and PSA was detected with the 5A5 antibody (1:1500). Other antibodies used in this study (obtained from the Developmental Studies Hybridoma Bank) are anti-NF-M to label retinal axons (clone 4H6, 1:50), anti-vimentin to label glial cells (clone H5, 1:100), and islet-1 to label RGCs (clone 39.4D5, 1:100). Sections were then rinsed twice with PBS and incubated for 1 h at room temperature in blocking buffer and the appropriate cy3- or cy5-conjugated secondary antibodies. Single confocal scans were obtained using a Zeiss LSM 510 confocal microscope.
Analysis of cell division and death
Fifty microliters of BrdU solution (50 µg/µl; Amersham, Piscataway, NY) was placed onto E8 embryos that had been injected with RCAS(B)GFP-PST. Embryos were then incubated for 4 h at 38°C. Eyes were dissected and processed for immunohistochemistry as described, and anti-BrdU immunohistochemistry was carried out according to the manufacturer's instructions (Amersham). To visualize cells undergoing cell death in the retina, the in situ Cell Death Detection Kit, TMR Red was used according to the manufacturer's instructions (Roche Applied Science, Indianapolis, IN).
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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