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Originally published In Press as doi:10.1074/jbc.M411439200 on November 4, 2004

J. Biol. Chem., Vol. 280, Issue 2, 1264-1271, January 14, 2005
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Altered Interaction and Expression of Proteins Involved in Neurosecretion in Scrapie-infected GT1-1 Cells*

Malin K. Sandberg{ddagger} and Peter Löw

From the Department of Neuroscience, Karolinska Institutet, Retzius väg 8 B2: 5, Stockholm, S-171 77, Sweden

Received for publication, October 7, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prions cause transmissible and fatal diseases that are associated with spongiform degeneration, astrogliosis, and loss of axon terminals in the brains. To determine the expression of proteins involved in neurosecretion and synaptic functions after prion infection, gonadotropin-releasing hormone neuronal cell line subclone (GT1-1) was infected with the RML scrapie strain and analyzed by Western blotting, real time PCR, and immunohistochemistry. As revealed by Western blotting of lysates exposed to different temperatures, the levels of complexed SNAP-25, syntaxin 1A, and synaptophysin were decreased in scrapie-infected GT1-1 cells (ScGT1-1), whereas the level of monomeric forms of these proteins was increased and correlated to the level of scrapie prion protein (PrPSc). However, when complex formation was prevented by prolonged heating of samples in SDS, the levels of monomeric SNAP-25, syntaxin 1A and synaptophysin in ScGT1-1 cells were decreased in comparison to GT1-1 cells. The reduced level of SNAP-25 was observed as early as 32 days postinfection. Increased mRNA levels of both splice variants SNAP-25a and -b in ScGT1-1 cells were seen. No difference in the morphology, neuritic outgrowth or distribution of SNAP-25, syntaxin 1A, or synaptophysin could be observed in ScGT1-1 cells. Treatment with quinacrine or pentosan polysulfate cleared the PrPSc from the ScGT1-1 cell cultures, and the increase in levels of monomeric SNAP-25 and synaptophysin was reversible. These results indicate that a scrapie infection can cause changes in the expression of proteins involved in neuronal secretion, which may be of pathogenetic relevance for the axon terminal changes seen in prion-infected brains.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prion diseases are neurodegenerative diseases that can be transmissible, inherited, or of sporadic occurrence. They are neuropathologically characterized by a marked astrogliosis, spongiform degeneration, and neuronal loss in the brain (for review see Ref. 1). The vacuoles that give the spongiform character of the degeneration are mainly located in dendrites, which also show varicosities and loss of spines as revealed in Golgi-impregnated specimen (2). In addition to these dendritic changes, reduced expression of presynaptic marker proteins, such as synaptophysin, synaptic-associated protein of molecular weight 25,000 (SNAP-25),1 syntaxin 1, synapsin 1, and {alpha}- and {beta}-synuclein, has been reported in both clinical materials (3–6) and animal experimental models (7, 8) indicating that presynaptic axon terminals are also affected. The mechanisms behind the presynaptic changes and their potential pathogenetic role in the disease are not known. In order to clarify this, a cell culture system would be advantageous.

The GT1-1 cell line is an immortalized mouse hypothalamic gonadotropin-releasing neuronal cell line (9) and represents one of the few cell lines that can be successfully infected by prions (10). These GT1-1 cells express not only key proteins involved in the regulation of secretory exocytosis such as synaptotagmin, synaptobrevin, and SNAP-25, but also synaptophysin that is localized in the membrane of small synaptic vesicles (11). Using this cell system, we have recently reported an impaired function of voltage-gated N-type calcium channels in prion-infected cells (12). Several studies have shown that synaptic vesicle release proteins, i.e. syntaxin 1A and SNAP-25, can interact with presynaptic calcium channels (13, 14) and it has also been reported that these synaptic proteins can modulate the function of presynaptic channels (15–18). In the present study we asked whether alterations in the expression of proteins involved in neurosecretion or synaptic vesicle release could be observed in prion-infected GT1-1 cultures. We here report that the levels of complexed SNAP-25, syntaxin 1A, and synaptophysin were decreased in scrapie-infected GT1-1 cells (ScGT1-1), whereas the levels of monomeric forms of these proteins were increased. When complex formation was prevented by prolonged heating in SDS, the expression of SNAP-25, syntaxin 1A, and synaptophysin in ScGT1-1 cells were decreased in comparison to those in GT1-1 cells. After treatment with quinacrine or pentosan polysulfate, the scrapie prion protein (PrPSc) was cleared from the ScGT1-1 cell cultures, and the increase in levels of monomeric SNAP-25 and synaptophysin were reversible


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture, Infection, and Procedures to Induce Differentiation— The neuronal cell line GT1-1 (kindly provided by Dr. P. Mellon, The Salk Institute, La Jolla, CA) was grown in 25-cm2 or 75-cm2 culture flasks (Corning Inc., Corning, NY). Briefly, the cells were cultivated in Dulbecco's MEM containing Glutamax-I, supplemented with 5% horse serum, 5% fetal calf serum, and 1% penicillin/streptomycin (supplemented DMEM; all obtained from Invitrogen Life Technologies, Inc., Paisley, UK), and maintained at 37 °C in a humidified 5% CO2 atmosphere. The medium was changed every 2nd to 3rd day, and the cells were dissociated with trypsin (Invitrogen Life Technologies, Inc.) and subcultured every 5th day. For infection with scrapie, the cells were dissociated and seeded into 24-well plates (Corning Inc.), 4 x 103 cells/well, and grown in supplemented DMEM. After reaching a 70–90% confluence, the cells were incubated with a brain homogenate from mice infected with the RML (Rocky Mountain Laboratory) strain of scrapie (obtained from Prof. S. B. Prusiner, Department of Biochemistry, UCSF, San Francisco, CA). The homogenate was diluted 1:10 in supplemented DMEM and added to the cells for 72 h at 32 °C. The medium was then removed, the cells cultivated in supplemented DMEM at 37 °C and subcultured five times before they were analyzed for the presence of PrPSc by Western blotting. Uninfected GT1-1 cells were cultivated simultaneously under the same conditions as the ScGT1-1 cells. To induce differentiation, GT1-1 cells and ScGT1-1 cells were plated in poly-L-lysine hydrobromide-coated (0.1 mg/ml; Sigma) 35 mm x 10 mm culture dishes (Corning Inc.), 5 x 105 cells/dish, and grown in supplemented DMEM at 37 °C for 2 days before 1 mM dibutyryl cyclic AMP (db-cAMP; Sigma) and 200 µM 3-isobutyl-1-methylxanthine (IBMX, Sigma) were added to the culture medium for 3 days.

Quinacrine and Pentosan Polysulfate Treatment—For clearance of PrPSc, ScGT1-1 cells were grown in 25-cm2 culture flasks (Corning Inc.) in supplemented DMEM, together with 0.5 µM quinacrine (Sigma) in 37 °C in a humidified 5% CO2 atmosphere. Fresh culture medium and quinacrine were added every second day, and the cells were subcultured every 5th day for 2–3 weeks. Pentosan polysulfate (5 µg/ml; Sigma) was also used to clear PrPSc from the cultures. Fresh culture medium and pentosan polysulfate were added every 3rd day, and the cells were subcultured every 5th day for 2 weeks. Untreated ScGT1-1 cells and uninfected GT1-1 cells were cultivated simultaneously under the same conditions as the quinacrine or pentosan polysulfate-treated cells.

Western Blot Analyses—For detection of the normal (PrPC), and the abnormal isoform of the prion protein (PrPSc), syntaxin 1A, synaptophysin, SNAP-25, and GAPDH, Western blot analyses were performed. GT1-1 cells and ScGT1-1 cells were plated in poly-L-lysine hydrobromide-coated (0.1 mg/ml; Sigma) 35 mm x 10 mm culture dishes (Corning Inc.), 5 x 105 cells/dish, and grown in supplemented DMEM at 37 °C for 5 days. The cells were lysed in 100 µl of lysis buffer containing 0.5% deoxycholic acid sodium salt, 0.5% Triton X-100, 150 mM NaCl, and 10 mM Tris-HCl, (pH 8.0; all obtained from Sigma) and a protease inhibitor mixture (Complete, Mini, EDTA-free from Roche Diagnostics GmbH, Mannheim, Germany). The lysates were centrifuged at 13,000 rpm for 1 min and the pellets discarded. Protein concentrations were determined by a Bio-Rad protein assay. For analysis of PrPSc, the supernatant was split in two aliquots. One aliquot was treated with proteinase K (PK, Roche Applied Science), 10 µg/ml per 1 mg of protein, for 30 min at 37 °C. The proteinase K treatment exceeded the effects of the protease inhibitor mixture and degraded the proteinase K-sensitive scrapie prion protein. The PK degradation was stopped by incubation with 5 mM phenylmethylsulfonyl fluoride (Sigma). Thereafter, the PK-treated and the other untreated aliquots were boiled in 4x SDS sample buffer (1x; 62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 0.003% bromphenol blue, 1% {beta}-mercaptoethanol) for 10 min if no other information is stated. The samples were loaded on NuPAGE 12% Bis-Tris gels (Invitrogen, Groningen, The Netherlands) and electrophoresed in Nu-PAGE MOPS SDS running buffer (Sigma) according to the manufacturer's instructions. The gels were blotted to an Immobilon-PSQ polyvinylidene difluoride sequencing membrane (Millipore Corporation, Bedford, MA). Primary antibodies used were D13 raised against PrPC (HuM-Fab antibody, 1:700; InPro Biotechnology, Inc., San Fransisco, CA), anti-syntaxin 1A (mouse, purified IgG; 1:5000; Synaptic Systems, Göttingen, Germany), anti-synaptophysin 1 (rabbit serum, 1:1000; Synaptic Systems), anti-SNAP-25 (rabbit serum, 1:2500) (19), and antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH; mouse-purified IgG, 0.25 µg/ml, Ambion, Austin, Tex). The antibodies were diluted in 0.3% (w/v) Tween-20/phosphate-buffered saline solution (PBS-T; Sigma) and 5% (w/v) bovine serum albumin (BSA; Sigma). Secondary antibodies used were horseradish peroxidase-labeled goat anti-human IgG F(ab')2 (Pierce), goat anti-mouse IgG (DAKO Danmark A/S, Glostrup, Denmark) and swine anti-rabbit (DAKO), all diluted 1:5000 in PBS-T and 5% (w/v) nonfat dry milk. The blots were developed using the ECL+ detection system and Hyperfilm ECL (Amersham Biosciences). Optical density was determined using Gel Doc 2000 with the software Quantity one 4.2.2 (Bio-Rad).

RNA Extraction, cDNA Synthesis, and Real Time PCR—Cells were plated in poly-L-lysine hydrobromide-coated (0.1 mg/ml; Sigma) 35 x 10 mm culture dishes (Corning Inc.), 5.0 x 105 cells/dish, and grown in supplemented DMEM at 37 °C for 5 days before experiments were performed. From these cultures, total RNA was extracted, using the RNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The amount and purity of the RNA was assessed by spectrophotometry (Ultrospec Plus, Amersham Biosciences). 500 ng of total RNA was subsequently treated with 1 unit of amplification grade DNase I (Invitrogen) for 15 min at room temperature and inactivated by the addition of 2.5 mM EDTA followed by incubation at 65 °C for 10 min. The DNase-treated RNA was reverse-transcribed in a 20-µl reaction containing the following reagents from Invitrogen: 150 ng of random hexamer primers, 1x RT buffer, 10 mM dithiothreitol, 500 µM each of dNTPs, and 50 units of MoMLV reverse transcriptase (Superscript II). cDNA synthesis was allowed to proceed for1 h at 42 °C before inactivation at 70 °C for 15 min. For quantification of the relative mRNA levels of the SNAP-25a and -b isoforms, primers corresponding to SNAP-25 (AB003991 [GenBank] ) exon 2, F (5' to 3' AGG ACG CAG ACA TGC GTA ATG AAC TGG AGG) together with SNAP-25a (AB003991 [GenBank] ) exon 5a, R (5' to 3' TTG GTT GAT ATG GTT CAT GCC TTC TTC GAC ACG A) and SNAP-25b (AB003992 [GenBank] ) exon 5b, R (5' to 3' CTT ATT GAT TTG GTC CAT CCC TTC CTC AAT GCG) were used (20). mRNA encoding cyclophilin was amplified using the following primers: F (5' to 3' GCT TTT CGC CGC TTG CT and R (5' to 3' CTC GTC ATC GGC CGT GAT) (X52803 [GenBank] ), designed in Primer express. 1 µl of cDNA template was amplified in triplicate 25-µl reactions containing the following reagents; Platinum® SYBR® Green qPCR Supermix UDG, and 250 nM of each primer (all from Invitrogen). An ABI Prism® 7000 sequence detection system (Applied Biosystems) under the following cycling conditions: 95 °C for 15 s and 60 °C for 1 min, for 45 cycles. Real-time PCR data was analyzed using the ABI Prism 7000 software (Applied Biosystems). Data analysis was done using the 2-{Delta}{Delta}CT method for relative quantification (21), and all samples were normalized to cyclophilin, which was used as an endogenous control.

Immunohistochemistry—The cell cultures were fixed in 4% paraformaldehyde (PFA, Merck KGaA, Darmstadt, Germany) for 10 min and thereafter quenched in 0.1 M NH4Cl (Sigma) for 5 min. The cultures were preincubated with 0.5% Triton X-100, 5% BSA, and PBS for 10 min in room temperature. For determination of PrPSc, the cell cultures were fixed in 4% PFA for 30 min, washed in 1% (w/v) NH4Cl/PBS, and permeabilized with 0.1% Triton X-100 for 5 min. The cultures were treated with 3 M guanidinium thiocyanate (Merck-Schuchardt, Hohenbrunn, Germany) diluted in PBS for 5 min, and thereafter preincubated with 5% (w/v) BSA diluted in PBS for 30 min. The cultures were then incubated with the rabbit polyclonal antibodies against synaptophysin 1 (1:50), or SNAP-25 (1:1000), or the mouse monoclonal antibody against syntaxin 1A (1:1000) diluted in 0.2% Triton X-100, 2.5% (w/v) BSA and PBS at 4 °C overnight. For labeling against PrPSc, D13 raised against PrPC (HuM-Fab antibody, 1:200; 4 °C overnight; InPro Biotechnology, Inc., San Francisco, CA), diluted in 5% (w/v) BSA/PBS was used. After washing, the cells were incubated with Rhodamin RedTM-X-conjugated AffiniPure donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and Cy2TM-conjugated AffiniPure donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.), diluted 1:500 and 1:250 in 0.2% Triton X-100, 2.5% (w/v) BSA and PBS, for 1 h at room temperature. PrPSc was visualized with Cy3TM-conjugated AffiniPure donkey anti-human IgG (Jackson ImmunoResearch Laboratories, Inc.), diluted 1:200 in 2.5% (w/v) BSA/PBS for 40 min. The cultures were mounted in glycerol containing 2.5% 1,4-diazabicyclo-(2, 2, 2)-octane (Sigma). Images of the immunolabeled neurons were obtained with a CCD camera (AxioCam, Carl Zeiss Vision GmbH, Hallbergmoos, Germany) connected to a fluorescence microscope (Microphot FX, Nikon Corp., Tokyo, Japan) using AxioVision software (Carl Zeiss Vision GmbH), and prepared for illustration with Adobe software (Adobe Systems Inc., San Jose, CA).

Statistics—Optical density and real time PCR data were analyzed using Student's t test together with Welch's correction. All optical density data were converted to logarithmic values to get a Gaussian distribution. Differences were considered statistically significant if p < 0.05, and bars represent mean values. All statistical analyses were made using Graph Pad Prism 3.0 (Graph Nad Software Inc., San Diego).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ScGT1-1 Showed a Decreased Level of Complexed SNAP-25, Syntaxin 1A, and Synaptophysin Compared with Uninfected GT1-1 Cells—Using Western blotting, we could confirm previous observations on the occurrence of SNAP-25 (25 kDa), syntaxin 1A (33 kDa), and synaptophysin (38 kDa) in the GT1-1 cells (Fig. 1) (9, 11, 22). To investigate the interaction between different synaptic proteins we compared ScGT1-1 (90 days postinfection; p.i.) and corresponding uninfected GT1-1 samples incubated at 4 and 100 °C in SDS over 20 min. When ScGT1-1 cell lysates, incubated at 100 °C, were compared with lysates incubated at 4 °C, no difference in the level of monomeric SNAP-25 could be observed by Western blotting (Fig. 1). However, a major increase in the level of monomeric SNAP-25 could be seen in GT1-1 cell lysates incubated at 100 °C compared with lysates incubated at 4 °C, indicating a complex formation between SNAP-25 and other proteins. In addition, a reduced level of monomeric SNAP-25 could be observed in ScGT1-1 cell lysates incubated at 100 °C compared with GT1-1 cell lysates incubated at 100 °C, showing that the SNAP-25 expression is decreased in ScGT1-1 cells (Fig. 1). When syntaxin 1A was investigated by Western blotting, a band of ~90 kDa could be observed in uninfected GT1-1 cell lysates incubated at 4 °C that was absent in ScGT1-1 cell lysates incubated at 4 °C. This band of complexed syntaxin 1A could not be observed in GT1-1 cell lysates incubated at 100 °C. In addition, no difference in the level of monomeric syntaxin 1A could be seen in ScGT1-1 cell lysates incubated at 4 or 100 °C where the GT1-1 cell lysates incubated at 100 °C showed a higher level of monomeric syntaxin 1A compared with GT1-1 cell lysates incubated at 4 °C. When ScGT1-1 and uninfected GT1-1 cell lysates incubated at 100 °C were compared, a reduced level of monomeric syntaxin 1A was seen in lysates from ScGT1-1 (Fig. 1). When the protein expression of synaptophysin was determined in ScGT1-1 and uninfected GT1-1 cell lysates incubated at 4 and 100 °C, a similar trend as with SNAP-25 and syntaxin 1A could be observed. No marked difference in the level of GAPDH, a protein used as a loading control, could be demonstrated.



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  		FIG. 1.
The expression of complexed and monomeric neurosecretory proteins in GT1-1 cells infected with the RML scrapie strain (ScGT1-1) and uninfected GT1-1 cells. Western blots showing the level of monomeric SNAP-25, syntaxin 1A, and synaptophysin in addition to complexed syntaxin 1A, in ScGT1-1 cell and corresponding GT1-1 cell lysates (Sc batch A; 90 days p.i.), exposed to different temperatures. No difference in the level of monomeric SNAP-25 or synaptophysin could be observed in ScGT1-1 cell lysates incubated at 100 °C and ScGT1-1 cell lysates incubated at 4 °C, whereas an increased level of monomeric SNAP-25 and synaptophysin could be seen in GT1-1 cell lysates incubated at 100 °C compared with the levels in lysates incubated at 4 °C. The SNAP-25 antibody used did not recognize complexed forms of SNAP-25. A complexed form of syntaxin 1A (~90 kDa) could be observed in GT1-1 cell lysates incubated at 4 °C that could not be seen in ScGT1-1 cell lysates incubated at 4 °C. These results indicate a disturbed complex formation in ScGT1-1 cells. GAPDH was used as a loading control.

 
An Increased Level of Monomeric SNAP-25, Syntaxin 1A, and Synaptophysin Was Correlated to the PrPSc—When two ScGT1-1 cell batches infected at different time points were analyzed for the occurrence of PrPSc, the first ScGT1-1 cell batch (Sc batch A) showed a higher level of PrPSc than the other (Sc batch B) (Fig. 2A). There was no apparent difference in the morphology of GT1-1 and ScGT1-1 cells (Sc batch A) (Fig. 2B). SNAP-25, syntaxin 1A, and synaptophysin were observed in the GT1-1 cells by Western blotting (Fig. 2, C–H). The Sc batch A of ScGT1-1 cells showed an increase in the monomeric forms of SNAP-25 (27%), synaptophysin (16%), and syntaxin 1A (18%), 60–85 days p.i., compared with control cells (Fig. 2, C–H), while the increase in monomeric SNAP-25 in Sc batch B, which contained lower levels of PrPSc, was more modest, 60–70 days p.i. (Fig. 2, C and D). There was no difference in the expression of GAPDH that was used as a loading control. When the presence of PrPSc in Sc batch A and Sc batch B cultures were determined by immunohistochemistry using the antibody D13 in combination with guanidinium thiocyanate treatment, ~85% of the Sc batch A cells showed a visible staining for PrPSc in comparison to ~35% of the Sc batch B cells (Fig. 3).



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  		FIG. 2.
The expression of monomeric neurosecretory proteins in GT1-1 cells infected with the RML scrapie strain (ScGT1-1) at two different time points (Sc batch A and B). The lysates were incubated at 100 °C for 10 min. A, Western blot showing bands characteristic for PrPSc after PK treatment. B, no difference in morphology is seen between ScGT1-1 cells (Sc batch A) and uninfected GT1-1 cells. Phase contrast microscopy, bar 20 µm. C, E, and G, Western blots showing SNAP-25, synaptophysin and syntaxin 1A expression in Sc batch A and B cells and corresponding uninfected GT1-1 cells. (D, F, and H) Scatter diagrams showing the expression of SNAP-25, synaptophysin, and syntaxin 1A distribution (logarithmic, optical density values) in Sc batch A cells, 60–85 days p.i; Sc batch B cells 65–70 days p.i., and the corresponding uninfected GT1-1 cells. A significantly increased expression of SNAP-25 (D), synaptophysin (F), and syntaxin 1A (H) was observed in the Sc batch A cells containing the higher level of PrPSc, whereas a more modest, but significant, increase in expression of SNAP-25 could be seen in the Sc batch B cells (D). No increased expression of synaptophysin or syntaxin 1A could be seen in the Sc batch B cells (F and H). GAPDH was used as a loading control. Bar, mean values; Student's t test together with Welch's corrections: ***, p < 0.001; **, p < 0.01; *, p < 0.05.

 



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  		FIG. 3.
The level of PrPSc in cell cultures derived from Sc batch A and B, infected at different time points with the RML scrapie strain. ~85% of the cells in cultures from Sc batch A exhibited a visible PrPSc staining (100 days p.i.) in comparison to ~35% of the cells in cultures from Sc batch B (70 days p.i.), as shown by immunohistochemistry using the D13 HuM-Fab antibody, in combination with guanidinium thiocyanate treatment; bar, 25 µm.

 
ScGT1-1 Cells Showed a Decreased Expression of SNAP-25, Syntaxin 1A, and Synaptophysin Compared with Uninfected GT1-1 Cells, When Complex Formation Was Eliminated— When ScGT1-1 cell lysates, incubated at 100 °C for 20 min to avoid complex formation, were compared with uninfected GT1-1 cell lysates incubated at 100 °C, a reduced level of SNAP-25, syntaxin 1A and synaptophysin could be observed (Fig. 1). When a new batch of GT1-1 cells was infected, the levels of monomeric SNAP-25, syntaxin 1A, and synaptophysin were determined in ScGT1-1 and GT1-1 cell lysates incubated at 100 °C for 20 min, at different time points after infection (Fig. 4). A reduced expression of SNAP-25 could be observed 32 days p.i., and the difference became more pronounced 45 days p.i. A similar trend could be demonstrated when the expression of synaptophysin was investigated. However, no marked difference in the expression of syntaxin 1A could be seen after 45 days p.i., and there was no difference in the GAPDH loading controls (Fig. 4).



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  		FIG. 4.
The expression of SNAP-25, syntaxin 1A, and synaptophysin in GT1-1 cells infected with the RML scrapie strain (ScGT1-1) and uninfected GT1-1 cells at different time points after infection. Western blots showing the difference in SNAP-25, syntaxin 1A, and synaptophysin expression at 15, 32, and 45 days p.i, when complex formation was prevented by incubation of ScGT1-1 and GT1-1 lysates in 100 °C for 20 min. A decreased expression of SNAP-25 could be observed in ScGT1-1 cells 32 days p.i. compared with corresponding uninfected GT1-1 cells, and this difference became more pronounced 45 days p.i. A decreased synaptophysin expression in ScGT1-1 cells could be observed 45 days p.i., whereas the expression of syntaxin 1A was not markedly changed at 45 days p.i. GAPDH was used as a loading control.

 
ScGT1-1 Cells Exhibited an Increased Level of Both the SNAP-25a and -b Isoform mRNA Compared with Uninfected GT1-1 Cells—To investigate whether the decreased SNAP-25 expression detected in ScGT1-1 cells was reflected at the mRNA level, real time PCR was used. This method also made it possible to distinguish between the two different SNAP-25 isoforms (SNAP-25a and -b). mRNA corresponding to SNAP-25a and -b confirmed previous results where GT1-1 cells have been found to express mRNA for SNAP-25 (9). The expression of the genes encoding SNAP-25a and -b in ScGT1-1 cells, 100 days p.i., were elevated by 77 and 78% respectively, compared with uninfected GT1-1 cells (Fig. 5).



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  		FIG. 5.
Relative mRNA levels of the SNAP-25a and -b isoforms in GT1-1 cells infected with the RML scrapie strain (ScGT1-1; Sc batch A; 100 days p.i.) compared with mRNA levels in corresponding, uninfected GT1-1 cells using real time PCR. The mRNA level of cyclophilin is used as an endogenous control. Both SNAP-25a and -b isoform transcripts are significantly increased in ScGT1-1 cells. Bar, mean values; Student's t test: ***, p < 0.001; **, p < 0.01; *, p < 0.05.

 
db-cAMP/IBMX-treated ScGT1-1 and GT1-1 Cells Showed a Comparable Increase in SNAP-25 and Synaptophysin Expression and Were Morphologically Similar—Using immunohistochemistry, the SNAP-25 labeling was mainly localized to the plasma membrane of the soma in a diffuse and non-clustered way, both in ScGT1-1 cells and GT1-1 cells (Fig. 6, A and B). Also, a few neurites could be seen diffusely immunolabeled for SNAP-25 in both GT1-1 and ScGT1-1 cells. Synaptophysin labeling occurred as small puncta localized throughout the soma of the ScGT1-1 and GT1-1 cells. The neurites observed were also immunolabeled (Fig. 6, C and D).



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  		FIG. 6.
GT1-1 cells infected with the RML scrapie strain (ScGT1-1; Sc batch A; 90 days p.i.) and uninfected GT1-1 cells, treated or not treated with 1 mM db-cAMP and 200 µM IBMX. Cultures were treated for 3 days and thereafter fixed and immunolabeled with SNAP-25 or synaptophysin antibodies. SNAP-25 is concentrated to the plasma membrane both in GT1-1 cells (A) and ScGT1-1 cells (B). The synaptophysin distribution shows a fine punctate pattern over the cell soma in uninfected GT1-1 cells (C) and in ScGT1-1 cells (D). Treatment with db-cAMP and IBMX induced neuritic outgrowth in both GT1-1 (E) and ScGT1-1 (F) cells, and the SNAP-25 localization was similar in these cells; bar, 25 µm. Exposure to db-cAMP and IBMX induced an increased expression of SNAP-25 and synaptophysin in both GT1-1 and ScGT1-1 cells (G) as determined by Western blotting. GAPDH was used as a loading control.

 
An increased expression of SNAP-25 and neuritic outgrowth have been observed upon exposure of PC-12 cells to db-cAMP (23). To investigate whether ScGT1-1 cells were different from GT1-1 cells in this respect, we compared the effects of combined treatment with db-cAMP and IBMX in these cell populations. When ScGT1-1 cells and GT1-1 cells were treated with db-cAMP/IBMX for 3 days, outgrowth of neurites (Fig. 6, E and F) as well as an increased expression of SNAP-25 and synaptophysin (Fig. 6G), were seen to a similar extent in both cell populations. The cellular localization of SNAP-25 and synaptophysin in the treated ScGT1-1 or GT1-1 cells showed no marked changes compared with the corresponding untreated cells, although the number and length of neurites had increased markedly (Fig. 6, E and F). These results indicate that the increased level of monomeric SNAP-25 or synaptophysin does not reflect a primary change in neuritic outgrowth in the cell cultures.

Quinacrine and Pentosan Polysulfate Cleared PrPSc from the ScGT1-1 Cell Cultures, and the Increase in SNAP-25 and Synaptophysin Expression Was Reversible—Quinacrine and pentosan polysulfate treatment of prion-infected cell cultures have previously been shown to clear PrPSc from the cultures (24–26). Treatment of ScGT1-1 cells with 0.5 µM quinacrine for 2–3 weeks caused a clearance of PrPSc from the cultures as shown by Western blotting (Fig. 7A). Such quinacrine treatment caused no marked changes in the levels of monomeric SNAP-25, synaptophysin, or syntaxin 1A in uninfected GT1-1 cells and reduced the monomeric SNAP-25 and synaptophysin protein levels in the ScGT1-1 cells (85 days p.i., Fig. 7B). However, the increased level of monomeric syntaxin 1A in ScGT1-1 cells remained after quinacrine treatment (Fig. 7B). ScGT1-1 cells treated with pentosan polysulfate for 2 weeks showed no presence of PrPSc (data not shown). Also, in these cultures, the changes in expression of SNAP-25 had become less evident 110 days p.i. (data not shown).



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  		FIG. 7.
GT1-1 cells infected with the RML scrapie strain (ScGT1-1) and uninfected GT1-1 cells, treated with 0.5 µM quinacrine for 2–3 weeks and analyzed for PrPSc and proteins involved in neurosecretion by Western blotting. A, no bands characteristic for PrPSc are detected in the quinacrine-treated (+Q) ScGT1-1 cells (Sc batch A; 85 days p.i). B, the level of monomeric SNAP-25 and synaptophysin was reduced in the quinacrine-treated ScGT1-1 cells compared with the level in non-treated (–Q) ScGT1-1 cells. The level of monomeric SNAP-25 and synaptophysin was comparable in the treated and non-treated uninfected GT1-1 cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, marked alterations in the interaction and expression of proteins involved in neurosecretion in scrapie-infected GT1-1 cells were found. The GT1-1 cell clone, which is derived from hypothalamic neurons immortalized by genetically targeted tumorigenesis using the promotor region of the gonadotropin-releasing hormone (GnRH) gene, expresses endogenous GnRH mRNA as well as neuronal cell markers like neuron-specific enolase (NSE) (9) and neuron-specific tubulin (TUJ1) (12). These cells contain two types of secretory vesicles involved in regulated secretion, namely large dense core vesicles (LDCVs) and small synaptic-like microvesicles (SLMVs) (11). While the LDCVs are involved in the release of GnRH, the SLMVs have been shown to release GABA upon depolarization with extracellular K+ (11). Synaptophysin, which is associated with SLMVs, shows a punctate pattern in the GT1-1 cells when immunolabeled. SNAP-25, which is involved in both types of regulated secretion, is associated with the plasma membrane (11). In the present study, we observed a distribution of both SNAP-25 and synaptophysin similar to that previously described. The pattern of distribution of these two proteins and syntaxin 1A was comparable in the uninfected GT1-1 and ScGT1-1 cells. However, the levels of complexed SNAP-25, syntaxin 1A, and synaptophysin were clearly reduced in ScGT1-1 cells while a corresponding increase of monomeric forms of these proteins could be seen and correlated to the level of PrPSc in the cultures. The change in the level of monomeric SNAP-25 was most likely a result of the presence of PrPSc in the cells, since the alterations were, at least partly, reversible upon treatment with quinacrine and pentosan polysulfate, which both abolished the PrPSc. The increased level of monomeric synaptophysin showed a similar trend upon quinacrine treatment.

The protein SNAP-25 exists as two isoforms (27). SNAP-25a has been implicated in neuritic outgrowth and fusion of vesicles delivering components to the plasma membrane throughout the neurite and in the growth cones (28, 29). SNAP-25b on the other hand, is important for neuronal synaptic vesicle release and neuropeptide secretion in neurons and neuroendocrine cells (28, 30). However, it has been shown that SNAP-25a also can be involved in secretory vesicle release (31, 32). In addition, recent observations concerning neuroexocytosis in chromaffin cells indicate that SNAP-25b is more potent in its ability to stabilize vesicles in the primed state compared with SNAP-25a (32).

In the present study we observed an up-regulation of mRNA for both isoforms during the infection, but no differences in growth or extension of neurites between uninfected and infected cells could be seen. Both GT1-1 and ScGT1-1 cells extended neurites and showed a similar cellular distribution of SNAP-25 and synaptophysin upon treatment with db-cAMP/IBMX. In addition, a comparable increase in SNAP-25 and synaptophysin level was seen in db-cAMP/IBMX-treated GT1-1 and ScGT1-1 cells using Western blotting. Thus, there was no evidence that the observed changes in the level of monomeric SNAP-25 were a result of any change in neuritic outgrowth caused by the infection. It is therefore more likely that the increased mRNA expression reflects another type of reaction to the infection. Such effects of the infection could either be directly caused by the pathological isoform PrPSc or reflect a cellular response to the infection.

The scrapie infection caused a disturbance in the SNAP-25 and syntaxin 1A complex formation and this may create a non-functional SNARE complex. The expression of SNAREs is highly regulated both during the development of the nervous system and in mature neurons. In cultured cerebellar granulae cells, the expression of syntaxin 1A and SNAP-25 increases with maturation, as well as complex formation between these two proteins (33). In addition, the turnover rate of SNAP-25 decreases in mature neurons (34), indicating that the binary complex formation might have a stabilizing ability on this protein. Therefore, a disturbed interaction between SNAP-25 and syntaxin 1A might induce a degradation of SNAP-25 and syntaxin 1A. In addition, an alteration in the endocytotic machinery caused by PrPSc, which is present in lysosomes (35), could tentatively lead to dysfunctions in the endocytotic recycling pathway. In either way, an increased degradation of SNAREs may result in an increased expression of mRNA for these proteins. On the other hand, an up-regulation of SNAP-25 protein expression has previously been observed in the brain as a response to kainate and colchicine exposure as well as axonal, mechanical lesions (36–40). The up-regulation of SNAP-25 was not related to sprouting or growth in some of these studies, since there was no parallel increase in GAP-43 expression (37, 39, 40). However, no discrimination between complexed and monomeric SNAP-25 was performed in these studies.

Recently, it has been shown that SNAP-25 plays an important role in dampening the calcium response evoked by depolarization with KCl in hippocampal glutamatergic neurons. Hippocampal GABAergic neurons lack SNAP-25 expression and upon transfection with this protein, lower calcium responses are seen in these neurons when depolarized with KCl (41). Decreased KCl-evoked N-type calcium channel responses has previously been described using fluorometric calcium measurements in the GT1-1 cell system after scrapie infection (12). The increased monomeric SNAP-25 expression in ScGT1-1 cells might therefore be implicated in the reduced KCl-evoked calcium responses, similar to the situation described in hippocampal neurons.

Syntaxin 1A binding with the synaptic protein interaction (synprint) site in voltage-gated N-type calcium channels can modulate gating and reduce inward currents in expression systems (16, 18, 42, 43) as well as in isolated nerve terminals (15). SNAP-25b interaction with the synprint site also reduces inward currents when co-expressed with N-type calcium channels in tsA-201 cells (18). The importance of a balance between SNARE proteins has been emphasized by the finding that co-expression of SNAP-25b and syntaxin 1A counteracts modulatory effects induced by independently expressed syntaxin 1A (18).

The increased level of monomeric synaptophysin, a protein which is localized to the membrane of small synaptic vesicles in neurons and neuroendocrine cells (44–47), is of interest, since this indicates that the synaptic vesicles containing classical neurotransmitters may be affected in scrapie-infected neurons. The function of synaptophysin is unclear, and no essential changes in the neurotransmitter release could be observed in knockout mice (48). However, both antisense oligonucleotides complementary to the synaptophysin mRNA and microinjection of synaptophysin antibodies have been demonstrated to reduce calcium-dependent neurotransmitter secretion (49, 50). Furthermore, an interaction between synaptophysin and synaptobrevin has been demonstrated indicating implications in the control of exocytosis (51). This interaction has also been suggested to be important for synaptic vesicle maturation (52). In addition, inhibition of SNAP-25 expression using antisense has previously been shown to induce decreased expression of synaptophysin (29) indicating that the expression of these proteins may be related.

When complex formation was prevented by using prolonged heating of samples in SDS, a reduced expression of SNAP-25, syntaxin 1A, and synaptophysin was observed in ScGT1-1 cells. These observations confirm previous studies of synaptic protein expression due to prion infection in vivo. Prion diseases are associated with synaptic disorganization and reduction as seen in brains from both patients with CJD (3, 4) and scrapie-infected mice (7, 53–56). The degeneration and loss of axon terminals occur early during scrapie infections, and in mice it precedes a dendritic spine loss and neuronal cell death (53, 55, 56). The loss of axon terminals is paralleled by decreased levels of several presynaptic proteins in both CJD (5, 6) and in murine scrapie (8). In the latter disease, no marked cell death in the brain could be seen, but an increased level of c-Jun/AP-1, mainly in reactive microglial cells, indicated phagocytosis of exogenous elements including synaptic debris (8). In conclusion, marked changes were observed in the interaction and expression of SNAP-25, synaptophysin, and syntaxin 1A in our cell system following scrapie infection.

These results clearly demonstrate that a scrapie infection can cause changes in the expression of proteins involved in neuronal secretion, which may be implicated in the reduced N-type calcium channel responses described previously in the scrapie-infected GT1-1 cells. In addition, the changes observed may be involved in the pathogenesis of synaptic alterations that are prominent features of prion diseases.


   FOOTNOTES
 
* This study was supported by grants from the Swedish Foundation for Strategic Research (EU QLK2-CT-2002.8162 and FOOD-CT-2004-5065) and the United States Army (DAMD17-03-102288). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Neuroscience, Karolinska Institutet, Retzius väg 8 B2:5, S-171 77 Stockholm, Sweden. Tel.: 46-8-524-878-10; Fax: 46-8-325-325; E-mail: malin.sandberg{at}neuro.ki.se.

1 The abbreviations used are: SNAP-25, soluble NSF attachment protein 25; DMEM, Dulbecco's modified Eagle's medium; MOPS, 4-morpholinepropanesulfonic acid; PBS, phosphate-buffered saline; BSA, bovine serum albumin; GT1-1, gonadotropin-releasing hormone neuronal cell line subclone; ScGT1-1, scrapie-infected GT1-1 cells; p.i., postinfection; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PK proteinase K; db-cAMP, dibutyryl cyclic AMP; IBMX, 3-isobutyl-1-methylxanthine dibutyrl cAMP; PrPSc, scrapie prion protein. Back


   ACKNOWLEDGMENTS
 
We are grateful to Prof. Krister Kristensson for discussions and valuable comments on this manuscript.



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