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Human Molecular Genetics Advance Access originally published online on October 20, 2005
Human Molecular Genetics 2005 14(23):3673-3684; doi:10.1093/hmg/ddi395
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Induction of HSP70 expression and recruitment of HSC70 and HSP70 in the nucleus reduce aggregation of a polyalanine expansion mutant of PABPN1 in HeLa cells

Qishan Wang, Dick D. Mosser and Jnanankur Bag*

Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ont., N1G 2W1, Canada

* To whom correspondence should be addressed. Tel: +1 519824412 ext 53390; Fax: +1 5198372075; Email: jbag{at}uoguelph.ca

Received August 12, 2005; Accepted October 13, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear inclusions formed by the aggregation of a polyalanine expansion mutant of the nuclear poly(A)-binding protein (PABPN1) is a hallmark of oculopharyngeal muscular dystrophy (OPMD). OPMD is a dominant autosomal disease in which patients exhibit progressive difficulty of swallowing and eyelid elevation, starting around the age of 50. At present, there is no specific treatment to reduce the aggregate burden in patients. However, in cell culture models of OPMD, reduction of protein aggregation can be achieved by ectopic expression of HSP70. As gene transfer may not be the most effective means to elevate HSP70 levels, we tested four pharmacological agents for their ability to induce HSP70, recruit both HSP70 and HSC70 into the cell nucleus and reduce mutant PABPN1 aggregation in a HeLa cell culture model. We show here that exposure to moderate levels of ZnSO4, 8-hydroxyquinoline, ibuprofen and indomethacin produced a robust stress response resulting in the induction of HSP70 in HeLa cells expressing the mutant PABPN1 as a green fluorescent protein (GFP) fusion protein. Both HSP70 and the constitutive chaperone HSC70 localized in the nucleus of cells treated with any one of the four agents. This stress response was similar to what was observed following hyperthermia. All four agents also caused a significant reduction in the cellular burden of protein aggregates, as was judged by confocal microscopy and solubility changes of the aggregates. A concomitant reduction of cell death in drug-treated mutant PABPN1 expressing cells was also observed.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Oculopharyngeal muscular dystrophy (OPMD) is an autosomal-dominant, slowly progressive disease. The symptoms in OPMD patients usually appear at around the age of 50, which include difficulty in swallowing and eyelid elevation (1Go). This hereditary disease has a worldwide distribution with high prevalence among the French Canadian population in Quebec (2Go). OPMD patients have a short expansion of a (GCG)6 trinucleotide repeat in the nuclear poly(A)-binding protein gene (PABPN1). The normal PABPN1 gene has six GCG repeats at the 5' end of exon 1, but in OPMD patients, this GCG repeat is expanded to GCG8–13 (1Go). The six GCG repeats encode the first six alanines in a homopolymeric stretch of 10 alanines located after the first methionine at the N-terminus of the wild-type PABPN1. Therefore, in OPMD patients, there is an expansion of the polyalanine tract to 12–17 alanines (1Go). It is believed that misfolding of mutant PABPN1 due to expansion of the polyalanine tract results in protein aggregation in the nucleus forming intranuclear inclusions (INIs) (3Go). There is a good correlation between protein aggregation and cellular toxicity resulting in cell death. The mutant PABPN1 aggregates appear to be degraded via the ubiquitin-proteasome pathway of protein degradation, as inhibition of this pathway by lactacystin increases formation of the mutant PABPN1 aggregates in COS-7 cells (4Go). It is believed that the ability to clear the protein aggregates through this pathway may be decreased with aging, which could explain the late onset of the disease (4Go).

The pathogenic process resulting from the aggregation of PABPN1 is unclear. PABPN1 serves an essential role in the poly(A) tail elongation process of eukaryotic mRNA (5Go–7Go). Therefore, it is not clear why nuclear inclusions are seen predominantly in muscle cells. In recent years, cell culture models have been developed to study the molecular basis of pathogenesis in OPMD. Ectopic expression of the mutant PABPN1 (8Go) or a reporter green fluorescent protein (GFP) with 19 or 37 alanine repeats (9Go) showed extensive protein aggregates in the nucleus of transfected cells. It has previously been shown that both polyalanine and polyglutamine tracts under different protein contexts cause polypeptides to misfold and aggregate (9Go,10Go). Accumulation of these protein aggregates is believed to induce apoptosis (11Go). The polyalanine expansion induces misfolding of proteins in a way similar to that of polyglutamine expansion-containing proteins (12Go). Several neurodegenerative diseases including Huntington's disease and spinocerebellar ataxia are caused by polyglutamine expansion (13Go,14Go). For both polyglutamine and polyalanine expansions, the protein aggregates produce toxic effects in cells, but the signaling mechanism of cell death is not clear. Recent studies suggest that the PABPN1 aggregates may act by sequestering and trapping poly(A)-containing RNAs (3Go) and various transcription factors (15Go), which could impair a wide range of cellular processes, leading to cell death. The oligomerization domain of PABPN1 is necessary for aggregate formation, and deletion of this domain resulted in a reduced level of cell death in culture (8Go). Transgenic mouse models expressing the mutant PABPN1 have been recently developed. These mice develop nuclear protein aggregates in muscles and neurons, and cell death occurred through both apoptosis and necrosis in muscle tissues (16Go,17Go). The myopathic changes were more prominent in the pharyngeal and eyelid muscles of the transgenic mouse model (16Go). As such, the transgenic mouse model is a valuable tool to study the progression of the disease, and will assist in the development of new approaches to treat OPMD in humans. However, compared with the transgenic mouse, a cell culture model expressing the mutant PABPN1 is inexpensive and easily amenable to experimental manipulations, which allows rapid screening of various potential therapeutic agents for evaluating their effectiveness in dissociating protein aggregates.

Although a direct connection between protein aggregation and OPMD is still disputed (11Go,18Go,19Go), strategies that reduce protein misfolding also reduced aggregate formation and cell death. Expression of the molecular chaperones HSP40 and HSP70 in cells transfected with the mutant PABPN1 reduced aggregate formation (4Go,20Go,21Go). Also the anti-amyloid compounds Congo red and doxycycline reduced PABPN1 aggregate formation and cell death in a cell culture model (21Go).

The accumulated evidence suggests that expression of HSPs in affected cells has a potential therapeutic value in OPMD patients. However, it is a difficult task to express HSPs in OPMD patients by gene therapy or heat-shock treatment. Therefore, we tested whether drug therapy could elevate HSP70 expression and reduce the formation of mutant PABPN1 aggregates in a cell culture model. We show here that ZnSO4 and 8-hydroxyquinoline induced HSP70 expression, and nuclear localization of both HSP70 and the constitutive chaperone HSC70, in mutant PABPN1 expressing HeLa cells. So, this resulted in reduced formation of mutant PABPN1 aggregates and cell death. We also tested whether two well-known pharmacological agents, ibuprofen and indomethacin, which are known to reduce the time and temperature threshold for induction of HSP70 (22Go,23Go), and mobilize HSC70 in the cell nucleus (22Go), could also reduce aggregation of the mutant PABPN1. We show here that these two drugs were as effective as hyperthermia in reducing protein aggregation and cell death.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Formation of Triton X-100 insoluble aggregates of PABPN1-A17–GFP in transfected cells
It is known that overexpression of the mutant PABPN1–GFP fusion protein with a 17 alanine stretch (PABPN1-A17–GFP) in HeLa and COS-7 cells produces bright aggregates similar to the INIs seen in OPMD muscles (4Go,8Go,20Go). Previous studies have shown that at a high expression level, the wild-type PABPN1 also forms aggregates (8Go,20Go,24Go). Therefore, we first examined the optimum transfection conditions for differentiating the propensity of aggregate formation between the wild-type and the mutant proteins. Since the polyalanine stretch is at the N-terminal end of PABPN1, in our studies, we fused the GFP at the C-terminal end of the PABPN1-(A10 and A17) so that it is distal to the region involved in protein aggregation. HeLa cells were transfected with 0.25, 0.5, 1 and 2 µg of plasmid DNA and the distribution of the fusion protein in live cells was examined by confocal microscopy. We examined cells at 24 h intervals after transfection for up to 5 days in culture and found that the best signal was produced 48 h after transfection. The transfection efficiency was only 20–30% with 0.25 and 0.5 µg DNA, whereas 70–80% of the cells expressed GFP when 1 or 2 µg of DNA was used for transfection. Loss of PABPN1-A17–GFP-transfected cells (with 2 µg DNA) was noticeable after 3–4 days in culture, and by day 5 approximately 60–70% of the cells died. In contrast, more than 90% of the cells transfected with PABPN1-A10–GFP remained viable after 5 days in culture. However, the intensity of the GFP signal was reduced after 3 days in culture for both constructs.

Cells transfected with 1–2 µg of the plasmid DNA exhibited three to four different nuclear distribution patterns of the fusion protein. As expected, both mutant and wild-type fusion proteins were localized exclusively in the nuclei and were excluded from the nucleolar regions. When cells were transfected with 2 µg of DNA, PABPN1-A10–GFP appeared diffusely expressed throughout the nucleus in approximately 70–80% of the transfected cells. However, a small number of bright speckles were evident within the diffuse signal (Fig. 1A). Also at this high level of expression of PABPN1-A10–GFP, approximately 15–20% of the cells showed a small number of aggregates, and approximately 5% of the cells showed several large aggregates similar to those observed in PABPN1-A17–GFP-transfected cells (data not shown). Although a subpopulation of cells expressing either the wild-type or the mutant fusion protein had similar protein aggregates, the vast majority of the cells showed distinct distribution patterns for the PABPN1-A10–GFP and PABPN1-A17–GFP fusion proteins. In contrast to the diffuse and amorphic appearance of PABPN1-A10–GFP, distinct aggregates (5Go–10Go) were present in nearly 70% of the PABPN1-A17–GFP expressing cells (Fig. 1B). In approximately 10% of the cells, several (>20) small aggregates were seen, and another 20% of the cells had one to five large aggregates (data not shown) similar to those observed in INIs reported earlier (4Go,8Go,20Go). These large aggregates were rarely (less than 1%) seen in PABPN1-A10–GFP-transfected cells.



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  		Figure 1. Nuclear localization of PABPN1–GFP fusion proteins. HeLa cells grown on coverslips in 35 mm dishes were transfected with 2 µg of either PABPN1-A10–GFP or the PABPN1-A17–GFP expression vectors and intracellular localization of the green fluorescence signal was examined with a confocal microscope following 48 h of transfection. Transfected cells were viewed in 10 viewing fields in self-blinded tests. All experiments were repeated at least three times. (A, B) Nuclear localization of PABPN1-A10–GFP and PABPN1-A17–GFP, respectively. (C, D) Distribution of PABPN1-A10–GFP and PABPN1-A17–GFP, respectively, following treatment with 0.5% Triton X-100 containing buffer.

 
It is known that PABPN-A17–GFP aggregates are resistant to extraction from cells by treatment with a high salt buffer before fixation (8Go). We also noticed similar behavior of mutant PABPN1 aggregates. However, this method of differentiating between the mutant and the wild-type proteins was somewhat inconsistent between experiments. Therefore, we developed an alternative approach to examine the solubility of the fusion protein. It was previously shown that treatment of cells with a mild non-ionic detergent in the presence of sucrose as an osmotic stabilizer could extract the soluble proteins without dissociating the cytoskeletal structures (25Go). In this article, we show that similar treatment of transfected cells with a 0.5% Triton X-100 containing buffer extracted most of the PABPN1-A10–GFP from 90% of the cells (Fig. 1C). The same treatment applied to PABPN1-A17–GFP expressing cells did not remove an appreciable amount of the fluorescence signal. Distinct aggregates or speckles of the mutant protein were seen in 80–90% of the cells (Fig. 1D). These results, therefore, suggest that PABPN1-A17–GFP forms a significant level of aggregates that are biochemically distinct from those formed by PABPN1-A10–GFP.

Effect of chemical inducers of the stress response on protein aggregation and cell death
Previously it was shown that co-expression of HSP70 and PABPN1-A17–GFP markedly reduced the formation of nuclear aggregates and cell death (20Go). We tested two relatively non-toxic chemicals for their ability to mimic a stress response in cultured cells and to reduce aggregates of PABPN1-A17–GFP. A search of the literature revealed that ZnSO4 and 8-hydroxyquinoline can induce HSP70 expression in HeLa cells (26Go). We, therefore, chose these two agents for our studies. In addition, we used ethionine, an analogue of methionine, which did not induce HSP70 synthesis in HeLa cells (26Go), as a negative control. Cells were transfected with either the PABPN1-A17–GFP or the PABPN1-A10–GFP plasmids and 48 h after transfection, different concentration of ZnSO4, 8-hydroxyquinoline and ethionine were added to the culture medium and incubated for 6 h. Following this treatment, the cells were allowed to recover for 24 h in fresh medium, free from these chemicals. The percentage of transfected cells showing protein aggregates and the proportion of cells with apoptotic nuclear morphology as a measure of cell death were scored. The results are shown as odds ratios as described in Materials and Methods. We observed that both ZnSO4 and 8-hydroxyquinoline reduced aggregation of PABPN1-A17–GFP. The optimum concentrations of ZnSO4 and 8-hydroxyquinoline were found to be 25 µM. There were fewer cells with protein aggregates in the treated cultures than the untreated control (Fig. 2). In contrast, ethionine treatment showed no significant effect on the extent of aggregation of the mutant fusion protein (Fig. 2). We also observed an improvement in the viability of cells following ZnSO4 and 8-hydroxyquinoline treatments, but not after ethionine treatment. In both ZnSO4- and hydroxyquinoline-treated cultures expressing PABPN1-A17–GFP, fewer cells showed apoptotic nuclei than what was observed in untreated controls. ZnSO4 and 8-hydroxyquinoline reduced cell death optimally at 25 µM, while higher concentrations of these agents produced a small increase in cell death.



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  		Figure 2. Effect of different agents on aggregation of PABPN1-A17–GFP and cell death. HeLa cells grown on coverslips in 35 mm dishes were transfected with PABPN1-A17–GFP vector and 48 h after transfection cells were incubated with the desired agent for 6 h at 37°C or exposed to 42°C for 2 h. Treated cells were allowed to recover for 24 h and fixed and stained with DAPI or directly examined for fluorescence signal by confocal microscopy. The percentage of cells with aggregates as shown in Figure 1 and DAPI stained apoptotic fragmented or condensed nuclei were determined. The results are presented as odds ratios. The error bars were calculated from four independent experiments. HQ, 8-hydroxyquinoline.

 
Several non-steroidal anti-inflammatory drugs (NSAIDs) including ibuprofen and indomethacin are known to decrease the threshold thermal dose (time and temperature combination) necessary to induce a stress response (22Go,23Go). In these studies, we also examined the effect of ibuprofen and indomethacin on PABPN1-A17–GFP aggregation and cell death. Forty-eight hours after transfection, we treated HeLa cells with these drugs at a concentration that is either five times (5x) in excess or equivalent (1x) to what is necessary to inhibit cyclooxygenase activity (22Go) for 1 and 6 h, respectively. In both cases, the treated cells were maintained in fresh drug-free culture medium for 24 h before being examined for protein aggregation and cell death. The results (Fig. 2) show that the 6 h treatment at 1x concentration was slightly more effective than the 1 h treatment at 5x concentration of ibuprofen and indomethacin in reducing protein aggregation and cell death. After the drug treatment, fewer transfected cells showed PABPN1-A17–GFP aggregates than the untreated control cells expressing the mutant protein. Concomitant reduction in the number of apoptotic cells was also observed following the ibuprofen and indomethacin treatments. These two drugs reproducibly showed approximately the same effect on removing protein aggregates and preventing cell death as what was observed with ZnSO4 and 8-hydroxyquinoline. We also used hyperthermia at 42°C for 2 h as a positive control to compare the effectiveness of all four chemicals tested here. We found that the reduction of protein aggregation and cell death achieved by each of the four chemicals was similar to that of the heat-shock treatment (Fig. 2). Ethionine-treated cells showed very little change in aggregate formation and apoptosis and served as a negative control.

Changes in the solubility of PABPN1-A17–GFP following treatment with various chemicals
Representative images illustrating the nuclear distribution of the PABPN1 fusion proteins before and after the chemical treatments are shown in Figure 3. As discussed earlier (Fig. 1), the majority of PABPN1-A17–GFP expressing cells contained protein aggregates (Fig. 3A) and these aggregates were predominantly insoluble in 0.5% Triton X-100-containing buffer (Fig. 3C). After treatment with ZnSO4, 8-hydroxyquinoline, ibuprofen and indomethacin (Fig. 3A), aggregates were absent in the majority of the PABPN1-A17–GFP expressing cells. Only a small percentage (20–30%) of the cells expressing the mutant fusion protein retained some protein aggregates, after the drug treatments. The nuclear distribution of PABPN1-A17–GFP in the majority of the cells treated with the above four chemicals was diffuse and appeared amorphic, similar to that observed for the wild-type fusion protein (Fig. 1A). After treatment, the PABPN1-A17–GFP was readily extractable with 0.5% Triton X-100 (Fig. 3C). In contrast to what was observed with ZnSO4-, 8-hydroxyquinoline-, ibuprofen- and indomethacin-treated cells, the ethionine-treated cells showed no change in the appearance of the protein aggregates and their Triton X-100 solubility (Fig. 3A and C). Similar treatments of PABPN1-A10–GFP-transfected cells with the agents discussed above did not produce any detectable difference in the amorphic distribution, and the Triton X-100 solubility, of the fusion protein (data not shown).



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  		Figure 3. Effect of different agents on nuclear distribution pattern and detergent solubility of PABPN1-A17–GFP. HeLa cells grown on coverslips were transfected with the PABPN1-A17–GFP expression vector as described in Figure 1. Forty-eight hours after transfection, cells were treated with the indicated agents for 6 h and following a 24 h recovery period cells were examined for green fluorescence by confocal microscopy (A). Another batch of similarly treated cells were incubated with a 0.5% Triton X-100 containing buffer on ice for 5 min, washed gently with chilled PBS before being fixed with paraformaldehyde. The Triton X-100-treated cells were also examined by confocal microscopy (C). (D) The Nomarski pictures of cells used in (C). The fluorescence signal of cells shown in (A) by white lines was subjected to line scans (B) using the Leica software. (E) Cells were treated with 10 µM lactacystin as previously described (4Go). All results were reproducible in four independent experiments.

 
To test whether the effect of the four chemical agents tested here on PABPN1-A17–GFP aggregation were similar to that observed with hyperthermia, we performed similar analyses after subjecting the transfected cells to heat shock at 42°C for 2 h, followed by a 24 h recovery period. The results show that induction of HSP70 by heat shock achieved a similar level of reduction of PABPN1-A17–GFP aggregates and also increased the Triton X-100 solubility of the mutant protein (Fig. 3A and C).

We analyzed the distribution profiles of the fusion proteins in individual cells by line scans of the cells indicated in Figure 3. The line scan (Fig. 3B) of PABPN1-A17–GFP expression in the untreated cell shows the presence of high levels of GFP as distinct peaks, whereas those of the treated cells, with the exception of ethionine, appeared more diffuse and evenly distributed throughout the nucleus. These results confirm our visual observation of confocal images that ZnSO4, 8-hydroxyquinoline, ibuprofen and indomethacin treatments resulted in reduced aggregation of PABPN1-A17–GFP in HeLa cells.

To obtain a better quantitative estimate of the changes in PABPN1-A17–GFP protein aggregates following the drug treatment, we determined the distribution of the fusion protein between Triton X-100 soluble and insoluble fractions by immunoblotting. Cell equivalent amounts of both Triton X-100 soluble and insoluble fractions were analyzed for the presence of PABN1-A17 (or A10)–GFP. The results show that although 80–90% of the wild-type fusion protein was extracted by the detergent, the majority (70–80%) of the mutant fusion protein was retained in the detergent-treated cells (Fig. 4). This distribution pattern changed markedly following ZnSO4, 8-hydroxyguinoline, ibuprofen and indomethacin treatments. Almost 65–75% of the PABPN1-A17–GFP was readily extracted by the detergent from the drug-treated cells. Similar change in the detergent solubility of this protein was observed in heat-shock-treated cells. There was also no significant difference in the cellular levels of PABPN1-A17–GFP between the treated and untreated transfected cells, suggesting that the reduction in the presence of insoluble mutant PABPN1 aggregates following treatments with ZnSO4, hydroxyquinoline, ibuprofen, indomethacin and hyperthermia was not due to an increase in the degradation of the aggregates.



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  		Figure 4. Detergent solubility of PABPN1-A17–GFP aggregates following different drug treatment. HeLa cells grown on 35 mm dishes were transfected with 2 µg of PABPN1-A17–GFP expression vector and treated with various agents as described in Materials and Methods. The cells on the dish were incubated with 0.7 ml of 0.5% Triton X-100 containing buffer for 5 min on ice. The detergent containing buffer (soluble fraction) was then removed from the dish and cells were lysed in 400 µl of SDS containing SDS–PAGE gel-loading buffer (pellet fraction). The detergent solubilized fraction was centrifuged at 12 000g for 1 min to remove any contaminating cell and cell debris. Cell equivalent amounts of soluble and insoluble fractions were analyzed for the presence of PABPN1-A17–GFP and ß-actin (loading control) by western blotting. These experiments were repeated three times using extracts from separate transfection studies.

 
We also analyzed the distribution of ß-actin between the Triton X-100 soluble and insoluble fractions. In all cases, approximately 50% of ß-actin was present in the detergent insoluble fraction (Fig. 4). As ß-actin is a major component of the cytoskeleton, a significant portion of the cellular ß-actin is expected to be retained in the detergent-treated cells. These results, therefore, suggest that the change in the solubility of PABPN1-A17–GFP following the drug treatment was not a non-specific effect of the drugs used in our studies. The change in the solubility of the mutant fusion protein following drug treatments was also confirmed by testing its solubility in chilled sodium dodecylsulfate (SDS) as previously described (4Go).

Inhibition of proteasome activity
It was previously shown that inhibition of proteasome activity by lactacystin increases aggregation of the mutant PABPN1, but does not change aggregation of the wild-type protein (4Go), suggesting that the proteasome pathway is involved in degrading the misfolded mutant PABPN1. We, therefore, tested whether the ability of the four chemicals used in our studies to clear the aggregates were due to an increase in proteasome activity. PABPN1-A17–GFP-transfected cells were treated with various agents for 24 h in the presence of 10 µM of the proteasome inhibitor lactacystin, and PABPN1 aggregates were viewed by confocal microscopy as described earlier. Results show (Fig. 3E) that lactacystin did not affect the ability of ZnSO4, hydroxyquinoline, ibuprofen, indomethacin and heat-shock treatments to reduce the aggregate burden in PABPN1-A17–GFP expressing cells. However, there were small increases in the presence of PABPN1 aggregates in the untreated and ethionine-treated control cells by lactacystin. As such, these results suggest that the four chemicals and heat-shock treatment reduced aggregate burden without increasing degradation of the mutant PABPN1 aggregates via proteasomes.

Induction of HSP70 and nuclear localization of HSP70 and HSC70
The precise biochemical nature of the changes in PABPN1-A17–GFP aggregates following treatment of transfected cells with ZnSO4, 8-hydroxyquinoline, ibuprofen and indomethacin is not clear, but may involve the correction of protein misfolding by heat-shock proteins. In our studies, both hyperthermia and treatment with the pharmacological agents resulted in comparable reduction in protein aggregates. Therefore, whether these agents can produce a robust heat-shock response was examined. We found that (Fig. 5) compared with the untreated and ethionine-treated PABPN1-A17–GFP-transfected cells, there were significantly higher levels of HSP70 in cells treated with ZnSO4, 8-hydroxyquinoline, ibuprofen and indomethacin. This increased HSP70 level was comparable to that observed following heat-shock treatment. This effect of increasing HSP70 level was not limited to the PABPN1-A17–GFP expressing cells; untreated HeLa cells and mock- and PABPN1-A10–GFP-transfected cells also showed an increase of HSP70 level following treatment with ZnSO4, 8-hydroxyquinoline, ibuprofen and indomethacin; however, this increase was not as robust as what was observed following heat-shock treatment. We also observed that the HSP70 level in untreated and ethionine-treated PABPN1-A17–GFP-transfected cells was routinely higher than what was observed in mock- and PABPN1-A10–GFP-transfected cells. These results suggest that expression of PABPN1-A17–GFP, which forms nuclear aggregates, produced a stress response in cells and this was further enhanced by exposure to the four chemical agents. We also performed similar analyses of the constitutive chaperone HSC70 and found that its level did not change significantly following treatment with any of the five chemicals or heat-shock treatment (Fig. 5). Both non-transfected and transfected cells behaved similarly with respect to HSC70 levels. We used ß-actin level as loading controls, which demonstrated that equivalent amounts of cellular proteins were loaded in all wells of the gel.



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  		Figure 5. Expression of HSP70 and HSC70 in PABPN1-A17–GFP-transfected cells. HeLa cells were either mock-transfected or transfected with PABPN1-A10 (or A17)–GFP expression vectors and treated with the indicated chemicals as described earlier. Total cell lysate was subjected to SDS–PAGE and cellular levels of HSP70, HSC70 and ß-actin (loading control) were examined by western blotting. As controls, exponentially growing Hela cells were also treated with the indicated chemicals and HSP70, HSC70 and ß-actin levels were analyzed by western blotting. Similar results were obtained in three independent experiments.

 
Redistribution of HSC70 from the cytoplasm to the nucleus has been observed in HeLa and NIH3T3 cells following treatment with ibuprofen and indomethacin (22Go). However, whether ZnSO4 and 8-hydroxyquinoline induce a similar redistribution was not known. Results of our analyses of the subcellular distribution of HSC70 show that it was localized predominantly in the nucleus of ZnSO4-, 8-hydroxyquinoline-, ibuprofen- and indomethacin-treated transfected cells (Fig. 6). The nuclear distribution of HSC70 in these treated cells was similar to that of the heat-shock-treated cells. In contrast, HSC70 was present, predominantly in the cytoplasmic fraction of mock-transfected cells, and in the ethionine-treated and -untreated PABPN1-A17–GFP-transfected cells. Similar analyses of HSP70 distribution (Fig. 6) also revealed predominantly nuclear localization of HSP70 in ZnSO4-, 8-hydroxyquinoline-, ibuprofen- and indomethacin-treated cells. Again, the nuclear localization of HSP70 in drug-treated cells was similar to that of the heat-shock-treated cells. Although a low level of HSP70 was easily detected in the total cell extract of PABPN1-A17–GFP expressing cells, we could not detect the same after subcellular fractionation, unless we used more cell extract. Most likely, this was caused by the distribution of HSP70 between the nuclear and soluble fractions. However, the cellular HSP70 in untreated PABPN1-A17–GFP-transfected cells was detectable only in the nuclear fraction (Fig. 6).



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  		Figure 6. Nuclear distribution of HSP70 and HSC70. Nuclear and cytoplasmic extracts from PABPN1-A17–GFP-transfected cells and mock-transfected HeLa cells were analyzed for HSP70, HSC70 and ß-actin levels by western blotting. For detecting HSP70 in untreated PABPN1-A17–GFP-transfected cells, the amount of protein used was twice of what was employed to examine HSP70 in drug-treated cells. Results were reproducible in three separate experiments.

 
Immunofluorescence studies of nuclear localization of HSP70 and HSC70
To analyze how HSP70 and HSC70 were distributed in individual transfected cells, we examined cells by confocal microscopy following immunostaining of specimens. The cells were examined for localization of both GFP and HSP70 or the constitutively expressed chaperone HSC70. The results (Fig. 7A) show that HSP70 was present in the nucleus of almost all transfected cells following ZnSO4, 8-hydroxyquinoline, ibuprofen, indomethacin and heat-shock treatments. The nuclear distribution pattern of HSP70 in these cells was similar to what was observed in heat-shock-treated cells. As in the drug-treated cells, the PABPN1-A17–GFP distribution was diffuse with small speckles or aggregates, nuclear distribution of HSP70 was also diffuse, but some of the HSP70 co-localized with the speckles or remaining aggregates. In the majority of untreated cells expressing PABPN1-A10 or (A17)–GFP, HSP70 was not detectable in the nucleus, only in a small percentage of cells a weak HSP70 signal was noticed. However, in approximately 20% of PABPN1-A17–GFP-transfected cells that had large distinct aggregates confined near the nuclear periphery, a significant level of HSP70 was present in the nucleus and it was co-localized with PABPN1-A17–GFP aggregates. Also, the signal for the very low level of HSP70 detected in some cells expressing PABPN1-A10 or (A17)–GFP appeared to co-localize with PABPN1 aggregates. These observations suggest that in a subpopulation of cells PABPN1-A17–GFP aggregates produced a robust stress response and co-localized with the aggregates. Why the same level of HSP70 was not detectable in most of the transfected cells is not known. However, it is likely that induction of HSP70 and its co-localization with the protein aggregates is a progressive event and the stress response increases with the aggregate burden. Future studies on the dynamics of the co-localization of HSP70 and the mutant PABPN1 aggregates may provide a better understanding of this process.



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  		Figure 7. Subcellular localization of HSP70 and HSC70 by immunofluorescence. HeLa cells grown on coverslips were transfected with PABPN-A17–GFP and treated with the indicated chemicals. The cells were then fixed in paraformaldehyde and processed for immunostaining with HSP70- (A) and HSC70 (B)-specific antibodies and counterstained with Texas red-conjugated anti-rabbit antibody. The stained specimens were examined with a confocal microscope. Approximately 25 transfected cells were examined for each of three separate experiments. (A) Cells stained with HSP70 antibody. Very little HSP70 is detectable in the majority of untreated PABPN1-A17–GFP- or PABPN1-A10–GFP-transfected cells. Transfected cells with large INIs showed a significant level of HSP70 in the nucleus, co-localized with PABPN1-A17–GFP aggregates. A significant increase of HSP70 and its nuclear localization was observed in ZnSO4-, 8-hydroxyquinoline-, ibuprofen- and indomethacin-treated cells. (B) Cells stained with HSC70 antibody. HSC70 is predominantly in the cytoplasm of the majority of mock- and PABPN1-A10 (or A17)–GFP-transfected cells. HSC70 is present in the nucleus and co-localized with the PABPN1-A17–GFP aggregates in a small population of INI-containing cells. Following treatments of transfected cells with ZnSO4, 8-hydroxyquinoline, ibuprofen and indomethacin, the HSC70 signal is predominantly nuclear and shows a diffuse distribution with co-localized PABPN1-A17–GFP.

 
Analysis of the nuclear distribution of the constitutively expressed chaperone HSC70 by immunostaining showed that after ZnSO4, 8-hydroxyquinoline, ibuprofen and indomethacin treatments, the HSC70 appeared almost exclusively in the cell nuclei of most of the cells (Fig. 7B). Similar to HSP70, approximately 20% of untreated PABPN1-A17–GFP-transfected cells with large aggregate burdens, HSC70 appeared in the nuclei and co-localized with the aggregates. Also in drug-treated transfected cells, the HSC70 co-localized with the PABPN1-A17–GFP and appeared diffuse. In non-transfected and PABPN1-A10–GFP-transfected cells, there was almost no detectable HSC70 in the nuclei. Most of the HSC70 in these cells was present around the perinuclear cytoplasm.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Protein aggregation and cellular toxicity
Aggregation of misfolded proteins generally proceeds through multiple stages, starting with the oligomerization of proteins, which undergoes further polymerization to form fibrillar structures, that is seen as nuclear inclusions by microscopy (27Go,28Go). In polyglutamine and ß-amyloid disease models, there is considerable agreement that the oligomers are more toxic to the cell than the large aggregates (29Go). Recent studies showed that in fact formation of the protein inclusions is helpful for the survival of cells (29Go–32Go), as it probably sequesters the toxic aggregates in the nucleus. In OPMD, as few as expansion of two alanines in the 10 alanine long stretch of the wild-type PABPN1 results in increased aggregation of PABPN1. However, the presence of stretches of 12–17 alanines per se does not lead to protein aggregation. The proteins HOXA13, FOXL2, CBFA1 and ZIC2 all contain 14–17 alanines, but are not known to misfold to form aggregates (15Go). It appears that the wild-type PABPN1 itself is susceptible to aggregation due to the presence of two overlapping oligomerization domains. These domains are necessary for the formation of oligomers of both wild-type and mutant PABPN1 (8Go). There are considerable agreements between ours and other studies (4Go,20Go) that both mutant and the wild-type PABPN1 could form oligomeric and polymeric aggregates, but the alanine-expanded mutant protein forms these aggregates considerably more frequently. As such, in cells expressing the mutant protein the aggregate burden increases with time causing cell death. Therefore, agents that could enhance proteosomal clearance of the aggregates and/or prevent oligomerization of the mutant PABPN1 could have therapeutic potential.

We tested four different chemicals for their ability to reduce aggregation of PABPN1-A17–GFP. Four of these agents, ZnSO4, 8-hydroxyquinoline, ibuprofen and indomethacin, are capable of mimicking some aspects of the stress response in cultured cells. The effect of stress-inducing agents on the presence of aggregates in cells was measured by two different methods including confocal microscopy and solubility in a non-ionic detergent. The confocal microscopic analysis showed a change from bright aggregates of PABPN1-A17–GFP to more amorphic forms in more than 70–80% of treated cells. The remaining approximately 20% of treated cells still showed detectable levels of speckles and aggregates. Results of our studies also showed that ZnSO4, 8-hydroxyquinoline, ibuprofen and indomethacin were equally effective as the heat-shock treatment in preventing aggregation of mutant PABPN1. This conclusion is also supported by the increase in the Triton X-100 solubility of the mutant PABPN1–GFP fusion protein, following treatments with the chemicals mentioned earlier. We also observed a good correlation between the effectiveness of various agents in reducing protein aggregates and protection from cell death.

Stress response and PABPN1-A17–GFP aggregation
Induction of heat-shock proteins by prior exposure to non-toxic stress conditions is known to protect cells against a variety of toxic conditions including hyperthermia and heavy metal exposure (33Go). Although hyperthermia, arsenite and other heavy metals including cadmium and copper and the amino acid analogue canavanine are well-known inducers of the heat-shock response (33Go,34Go), we were interested in determining whether non-toxic treatment of cells with ZnSO4 or 8-hydroxyquinoline could evoke a stress response similar to that observed by hyperthermia. We used two criteria to compare the stress response induced by ZnSO4 or 8-hydroxyquinoline treatment to that of hyperthermia. We first showed that similar levels of HSP70 induction were achieved by ZnSO4, 8-hydroxyguinoline and heat-shock treatments. Also, all three treatments stimulated nuclear localization of both HSP70 and HSC70.

As the level of HSP70 and HSC70 was low in mutant PABPN1–GFP expressing cells, we could not detect nuclear HSP70 or HSC70 in the majority of these cells. However, both proteins were detected in the nucleus of approximately 20% of the cells containing large nuclear inclusions as shown in Figure 7. Although, in these cells both HSP70 and HSC70 were present in the nucleus, and co-localized with the PABPN1-A17–GFP aggregates, it is likely that the nuclear level of chaperone proteins was not sufficient to renature the misfolded PABPN1-A17–GFP aggregates. Further increase in the cellular level of these chaperones by hyperthermia or drug treatments was required to reduce the aggregate burden. We also observed that the stress induced by hyperthermia and drugs also reduced the presence of small aggregates and speckles normally seen in the PABPN1-A10–GFP expressing cells (data not shown). These speckles are probably the oligomeric form of PABPN1. Therefore, it is likely that the presence of HSP70 and HSC70 in the cell nucleus interfere with the oligomerization process of PABPN1. However, in the absence of a clear understanding of the underlying molecular process of protein aggregation, we cannot determine how the chaperone proteins reduced PABPN1-A17–GFP aggregate formation.

We observed an increased expression of HSP70 in ibuprofen- and indomethacin-treated cells. Previous studies showed that these NSAIDs can reduce the temperature and the length of exposure necessary for the stress-induced expression of heat-shock proteins in cultured cells. Nuclear localization of HSC70 and activation HSF DNA binding activity following NSAID treatments were shown at a dose approximately 5 times higher than that required to inhibit cyclooxygenase-2 activity (22Go). Here we showed that either a short 1 h treatment at this 5x dose or a longer 6 h treatment at the 1x dose was able to induce HSP70 expression in PABPN1-A17–GFP-transfected cells. We believe that as the mutant protein expression already produced a weak stress response in these cells, ibuprofen and indomethacin were able to enhance this effect, and significantly increased HSP70 production to reduce aggregate burden on cells and improve cell survival. In these studies, we examined the expression and nuclear mobilization of only HSP70 and HSC70, but the role of additional chaperones like HSP40 in clearing protein aggregates cannot be ruled out. Also one should be cautious in implying a direct relationship between nuclear HSP70 and HSC70 levels and reduction of PABPN1-A17–GFP aggregates. Whether the chemicals used in our studies directly or by some other mechanism interfered with the folding of ß-sheets of PABPN1 is not known. We, however, consider this possibility unlikely, as all four agents and hyperthermia behaved similarly in reducing protein aggregates and producing the stress response. Also, as we do not yet know why protein aggregates are toxic to cells, it is difficult to establish clearly a causal relationship between reduced protein aggregation and cell death observed in our studies. Furthermore, it is known that expression of HSP70 can directly protect cells from apoptosis (35Go). In other diseases caused by misfolded proteins, it appears that the oligomeric form of the protein is more toxic than the fibrillar inclusions (30Go–32Go). If the same is true for OPMD, the increased survival of cells could result from preventing the oligomerization of PABPN1-A17–GFP by HSP70 and HSC70. The failure of the inhibitor of proteasome activity to abolish the ability of HSP70 and HSC70 to renature the mutant PABPN1 aggregates (Fig. 3E) supports the above model.

Potential therapeutic agents for OPMD
All four agents shown here to reduce protein aggregates and cell death in our OPMD cell culture model are good candidates for further studies as therapeutic agents for OPMD and possibly other diseases related to protein misfolding. Zinc sulfate is an essential mineral nutrient, and the average Zn2+ concentration in human plasma is approximately 15–20 µM (36Go). This is close to the concentration (25 µM) of ZnSO4 used in our studies in the culture medium to reduce protein aggregation in HeLa cells. 8-Hydroxyquinoline is a certified non-toxic agent used in animal feeds as an anti-microbial anti-parasitic agent and to treat infections in humans (37Go,38Go). Various derivatives of this drug have also shown an effect in reducing ß-amyloid plaques in early clinical trials on patients with Alzheimer's disease (39Go,40Go). Its effect on reducing amyloid plaques is believed to be due to its chelating properties of Zn2+ and Cu2+ which are involved in facilitating the process of pathological protein aggregation (41Go). Therefore, it is not certain whether the observed effect of 8-hydroxyquinoline in our studies on PABPN1-A17–GFP aggregation is due to metal chelation or stress response. The normal therapeutic dose of quinoline derivatives in adult humans is approximately 2 g/day for 20 days (38Go). In our cell culture model, we used ~3.5 mg of 8-hydroxyquinoline per liter of culture medium; therefore, it is effective at a relatively low concentration and has a good therapeutic potential in treating OPMD. The other two agents used in our studies, ibuprofen and indomethacin, are well-known anti-inflammatory drugs already approved for treating humans. We have shown here that at approximately the same concentration that is required to inhibit cyclooxygenase activity, ibuprofen and indomethacin were effective in dissociating PABPN1-A17–GFP aggregates. There are also other compounds including dimethyl sulfoxide, Congo red, doxycycline and benzothiazoles that were previously shown to reduce both polyglutamine and polyalanine aggregates (20Go,28Go,42Go,43Go). To the best of our knowledge, the effectiveness of ZnSO4, 8-hydroxyquinoline and ibuprofen in renaturing polyglutamine or polyalanine aggregates has not been previously reported. As this work was in progress, Ishihara et al. (44Go) reported the effectiveness of indomethacin in inducing a heat-shock response and preventing polyglutamine-induced cytotoxicity; therefore, all four agents tested here may be useful in preventing the pathogenic processes resulting from both polyalanine and polyglutamine aggregates. The effectiveness of these agents may be tested in the future on the available transgenic mouse model of OPMD (16Go,17Go). Furthermore, it will be interesting to examine whether combining two or more of these agents could enhance their effectiveness.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Culture and transfection of cells
HeLa cells were grown as monolayer cultures in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 10 units/ml penicillin and 100 µg/ml streptomycin (Invitrogen, Burlington, Ont., Canada) at 37°C in the presence of 5% CO2. Approximately 3x105 cells grown on a 35 mm dish in DMEM medium with 10% FBS were used for transfection. One to two micrograms of plasmid DNA was incubated with 8 µl of lipofectamine in 200 µl of Opti-MEM (Invitrogen) for 30 min at room temperature before being added to the cells. Cells were incubated for 5 h at 37°C with the DNA/liposome mixture in 1 ml of Opti-MEM. Following the incubation, 1 ml of growth medium containing 20% FBS was added to the culture. After 12 h of incubation, the medium was replaced by fresh, complete DMEM medium containing 10% FBS. The cells were examined with a fluorescence microscope 48 h after transfection.

Construction and expression of PABPN1–GFP fusion proteins
The cDNA clones containing the entire coding regions of the wild-type with six GCG repeats (GCG6) and a mutant with 13 GCG repeats (GCG13) of human PABPN1 gene were obtained from Dr G.A. Rouleau (1Go). Expression vectors encoding PABPN1–GFP fusion proteins, in which the GFP was present at the C-terminal end of PABPN1, were made by subcloning PABPN1 cDNA clones into the pEFGP-N3 vector (Clontech, MA, USA). The entire coding regions of wild-type (PABPN1-A10) and mutant (PABPN1-A17) proteins from the parent plasmids (1Go) were amplified by PCR using appropriate primers. The sense primer for PCR (5'-CCGGAATTCGGCGATGGCGGCGGCGGC-3') included a start codon and contained an EcoR1 site. The antisense primer (5'-CAGGGTACCGTAAGGGGAATACCATGATGTC-3') contained a Kpn1 site. The appropriate PABPN1 cDNA clones were amplified using these primers and the PCR products were gel purified, digested with EcoR1, and Kpn1 and inserted into the EcoR1/Kpn1 site of the pEGFP-N3 vector.

Confocal microscopy
Cells were grown on a glass coverslip placed in a 35 mm tissue culture dish and transfected as described above. Transfected cells were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 20 min. Confocal microscopy was performed to determine the cellular localization of GFP fusion proteins as previously described (21Go,45Go). The specimen was mounted in PBS containing 70% glycerol and examined with a Leica laser scanning confocal microscope. The GFP fluorescence was excited at 488 nm and emission was examined at 516 nm. Cells in 10 different fields of view were analyzed and the images were recorded as TIF files. Images representative of the GFP localization in the majority of cells were used for presentation.

Measurement of protein levels
Approximately 3x105 cells were transfected with a PABPN1–GFP plasmid as described earlier. Two days after transfection, the cells were washed three times with PBS and lysed by adding 400 µl of gel-loading buffer [50 mM Tris–Cl, pH 6.8, 2% (w/v) SDS, 1% (w/v) bromophenol blue, 10% (v/v) glycerol and 100 mM DTT]. The samples were subjected to SDS–12% polyacrylamide gel electrophoresis (PAGE) (46Go). The separated polypeptides were electrophoretically transferred from the gel to a nitrocellulose membrane for 4 h in a transfer buffer containing 25 mM Tris, 192 mM glycine and 20% methanol, pH 8.3. After treating the transfer membrane with a blocking buffer (2% non-fat dry milk and 0.2% Tween-20 in PBS) for 16 h at 4°C, the membrane was incubated with diluted (1:1000) primary antibody for 2 h at room temperature, washed several times with a washing buffer (0.2% Tween-20 in PBS) and further incubated with an alkaline phosphatase conjugated secondary antibody for another 1 h. The bound antibody was detected with NBT (4-nitroblue tetrazolium chloride) and BCIP (5-bromo-4-chloro-3-phosphate) as previously described (47Go). The PABPN1–GFP and ß-actin (loading control) were detected by a GFP antibody (Living Colours A.V. peptide antibody, Clontech, MA, USA) and the ß-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), respectively. The Hsp70 and Hsc70 were detected by using antibodies specific for Hsp70 and Hsc70, respectively (Santa Cruz Biotechnology).

Induction of Hsp70 in transfected cells
Twenty-four hours after transfection, different chemicals were added to the culture medium as sterile solutions in 70% ethanol. The final concentration of ethanol in the culture medium was between 0.2 and 0.75%. Cells were exposed to the indicated chemicals for 6 h and then allowed to recover for 24 h in fresh growth medium. For heat-shock treatment, the transfected cells were incubated at 42°C for 2 h in a 5% CO2 incubator, followed by a 24 h recovery period at 37°C. Following the appropriate treatment, cells were either processed for microscopic studies or biochemical analyses.

Proteasome inhibition
Twenty-four hours after transfection of HeLa cells with the PABPN1-A17–GFP expression vector, 10 µM of the proteasome inhibitor lactacystin (Calbiochem, La Jolla, CA, USA) was added to the culture medium. Transfected cells were then treated with the chemicals indicated in Figure 3 for an additional 24 h. Transfected control cells were incubated with the carrier solvent DMSO for 24 h.

Extraction of proteins from cells
Transfected cells on a coverslip were washed twice in chilled PBS and then treated with 200 µl of 0.5% Triton X-100 containing buffer (10 mm PIPES, 100 mm NaCl, 2.5 mm magnesium acetate, 0.3 M sucrose, pH 6.8) for 5 min on ice. The cells were briefly rinsed in 100 µl chilled PBS and fixed with 4% paraformaldehyde.

Measurement of nuclear aggregates and cells death
Cells were fixed in 4% paraformaldehyde and visualized by confocal microscopy for the appearance and localization of the GFP-fusion proteins. Approximately 50 cells were examined in 10 different fields of view. Cells were considered to have protein aggregates if the GFP signal appeared extremely bright in several places within the nuclei. More than 80% of untreated mutant PABPN1-transfected cells showed several (>10) bright aggregates. For better quantification of aggregates, the nuclear signal was line scanned using Leica software. To measure cell death, paraformaldehyde-fixed cells were stained with DAPI and the nuclei were viewed with a fluorescence microscope. The cells were considered dead if the nuclei appeared fragmented and pyknotic as previously described (21Go). Approximately 200 cells were examined to determine the percentage of dead cells. The changes in nuclear protein aggregates and cell death following various treatments were calculated as odds ratios in multiple experiments as previously described (21Go). The ratio between the % of treated cells with and without nuclear aggregates was divided by the ratio between the % of untreated cells with and without nuclear aggregates to calculate odds ratio.

Immunostaining of proteins
Cells grown on a coverslip were fixed with methanol at –20°C for 10 min and permeabilized with 0.1% Triton X-100/PBS for 2 min. The fixed cells were treated with 10% normal blocking serum derived from the same species as the primary antibody in PBS for 20 min. The presence of HSP70 and HSC70 was then detected by using specific antibodies. After a 1 h incubation at 20°C with the primary antibody, the cells were washed with PBS and incubated for another 1 h at 20°C with a Texas red conjugated anti-mouse IgG (1:100 dilution, Santa Cruz Biotechnology). Finally, the cells were washed with PBS, mounted in 70% glycerol/PBS (pH 7.5) and observed with a fluorescence microscope.


   ACKNOWLEDGEMENTS
 
PABPN1 clones were generously provided by Dr G.A. Rouleau of Centre for Research in Neuroscience, McGill University. This work was supported by grants from NSERC and CIHR, Canada.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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