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,
* Kinsmen Laboratory of Neurological Research,
Department of Pathology and Laboratory Medicine and
Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada;
¶ Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania, USA; and
Kinexus Bioinformatics Corporation, Vancouver, British Columbia, Canada
1Correspondence: Kinsmen Laboratory of Neurological Research, University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC V6T 1Z3, Canada. E-mail: aklegeri{at}interchange.ubc.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-synuclein gene as well as by the A30P, E46K, and A53T mutations. The mechanisms are unknown. Reactive astrocytes in the substantia nigra of PD and MPTP-treated monkeys display high levels of the inflammatory mediator intercellular adhesion molecule-1 (ICAM-1), indicating that chronic inflammation contributes to the degeneration. Here we report that -synuclein strongly stimulates human astrocytes as well as human U-373 MG astrocytoma cells to up-regulate both interleukin (IL)-6 and ICAM-1 (ED50=5 µg ml–1). The mutated forms are more potent stimulators than wild-type (WT) -synuclein in these assays. We demonstrate by immunoblotting analysis that this up-regulation is associated with activation of the major mitogen-activated protein kinase (MAPK) pathways. It is also attenuated by PD 98059, an inhibitor of the MAPK/extracellular-regulated kinase kinase MEK1/2, SP 600125, an inhibitor of c-Jun N-terminal kinase (JNK), and SB 202190, an inhibitor of p38 MAPK. The inhibitory effects on human astrocytes have IC50 values of 2, 5, and 1.5 µM respectively. We hypothesize that the neuroinflammation stimulated by release of an excess of normal -synuclein or by release of its mutated forms can be involved in the pathobiology of PD.—Klegeris, A., Giasson, B. I., Zhang, H., Maguire, J., Pelech, S., McGeer, P. L. Alpha-synuclein and its disease-causing mutants induce ICAM-1 and IL-6 in human astrocytes and astrocytoma cells.
Key Words: c-Jun N-terminal kinase • MAPK/ERK kinase • neuroinflammation • p38 mitogen-activated protein kinase • Parkinson disease
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-SYN) IS a 14 kDa acidic protein concentrated in presynaptic neuronal terminals (1
, 2)
. Its hypothesized physiological functions include synaptic vesicle turnover, synaptic plasticity, ubiquitin-proteasome processing, and molecular chaperone (1
2
3
4
5
6)
. Much attention has focused on its involvement in neurodegeneration since its mutations are one cause of autosomal dominant Parkinson disease (PD). Currently there are three known point mutations that result in PD: A30P, E46K, and A53T. Duplication and triplication of a short segment of chromosome 4 encompassing the -syn gene, which leads to three and four functional copies of WT -syn, also cause PD, with the number of copies correlating with the age of onset (7
, 8)
.
Aggregated -syn is a major component of Lewy bodies, which are hallmarks of PD and Lewy body dementia (1
, 2)
. Aggregates of -syn are also found in oligodendrocytes in multiple system atrophy. These disorders, along with other less common diseases, are now collectively known as -synucleinopathies (1
, 2
, 9)
. However, the cellular and molecular mechanisms underlying the pathological action of -syn are not completely understood. While most of the studies have concentrated on the effects of -syn on neuronal cells, including their survival (6
, 10
, 11)
, recent observations have pointed toward an interaction between -syn and glial cells. Immunohistochemical studies have demonstrated that microglia interact with -syn-positive extracellular Lewy bodies (12)
and with oligodendroglial -syn (13
, 14)
. Croisier et al. (15)
reported a positive correlation between -syn deposition in PD substantia nigra (SN) and the intensity of major histocompatibility complex (MHC) class II antigen expression by microglia. In vitro studies have shown that -syn induces a proinflammatory phenotype in rodent microglia (16
, 17)
.
-Syn-containing inclusions have been observed in astrocytes from individuals with PD (18)
and in amyotrophic lateral sclerosis (5)
. Cultured human astrocytes and U251 astrocytoma cell line express -syn mRNA and protein (19)
. Togo et al. (20)
showed that glial fibrillary acidic protein (GFAP)-positive astrocytes attach to -syn-positive extracellular Lewy bodies. It is not known what the consequences are of such an interaction.
We previously reported that intercellular adhesion molecule-1 (ICAM-1) is highly expressed in reactive astrocytes in PD SN along with its ligand lymphocyte function-associated antigen 1 (LFA-1) in activated microglia. This same combination is expressed in the SN of monkeys that had been treated with MPTP years previously (21)
. A possible interpretation is that the ICAM-1-LFA-1 system is responsible for sustaining inflammation in the SN and that this inflammation, in turn, is responsible for autodestruction of SN dopaminergic neurons.
We report here on the interaction of -syn and its mutated forms with human astrocytes and human astrocytoma U-373 MG cells. We demonstrate up-regulation of ICAM-1 expression by these cells where mutated forms are more potent than WT -syn on ICAM-1 expression by U-373 MG cells.
We also show that -syn is able to induce secretion of IL-6, which is blocked by specific inhibitors of the p38, extracellular-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinase pathways. Taken together, these data indicate that release of -syn may contribute to PD by virtue of its ability to stimulate inflammation in glial cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-syn and its mutated forms A30P, E46K, and A53T were prepared as described previously (22
, 23)
. They were used without preaggregation. All -syn samples tested negatively for the presence of endotoxin by E-TOXATE kit (from Sigma, St. Louis, MO, USA). The detection limit of the kit was 0.06 endotoxin units (U) ml–1.
Human recombinant IFN-
(IFN-) and IL-6 were purchased from PeproTech Canada (Ottawa, ON, Canada). PD 98059, a selective cell-permeable inhibitor of MAP kinase (MAPK) kinase (also known as MAPK/ERK kinase or MEK1/2), SB 202190, a highly selective, cell-permeable inhibitor of p38 MAPK, and SP 600125, a selective inhibitor of c-Jun NH2-terminal kinase (JNK), were purchased from Sigma. Bacterial lipopolysaccharide (LPS, from E. coli 055:B5), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), and phosphatase substrate Sigma 104 were also obtained from Sigma. Antibodies purchased for enzyme-linked immunoabsorbent assay were as follows: for IL-6 capture, a rat monoclonal (1:500 dilution, PharMingen, San Diego, CA, USA); for IL-6 detection, a biotinylated rabbit polyclonal (1:250, PeproTech Canada). For Western blot, the following rabbit polyclonal antibodies (1:750 dilution) were used: pan-specific ERK1/2 and p38 MAPK (StressGen Biotechnologies, Victoria, Canada); pan-specific JNK (R&D Systems, Minneapolis, MN, USA); and phospho-specific T202+Y204 ERK1/2, T180+Y182 p38 MAPK, and T183+Y185 JNK (Biosource International, Camarillo, CA, USA).
ICAM-1 (CD54) detection was performed using mouse monoclonal antibodies (1:1000) supplied by either Biogenex (MU326-UC, 1H4, San Ramon, CA, USA) or DAKO (clone 6.5B5; Carpinteria, CA, USA). The rabbit polyclonal anti-GFAP antibody (Ab) from DAKO was used at 1:20,000 dilution. The alkaline phosphatase-labeled anti-mouse Ab (1:3000) was supplied by Invitrogen Canada (Burlington, ON, Canada), whereas ExtrAvidin-alkaline phosphatase (1:20,000) was from Sigma. Antibodies used for immunocytochemistry included rabbit polyclonal anti-GFAP used at 1:20,000 dilution and mouse monoclonal anti-CD68 used at 1:400 (both from DAKO).
Cell culture
The human astrocytic U-373 MG cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were grown in Dulbecco’s modified Eagle medium (DMEM) nutrient mixture F12 Ham (DMEM-F12) supplemented with 10% fetal bovine serum (FBS) (FBS, Invitrogen) and gentamicin (50 µg ml–1), and were used without initial differentiation.
Human astrocytes were isolated from surgically resected temporal lobe tissues. Protocols established by De Groot et al. (24)
were modified as described previously (25)
. Briefly, tissues were washed with HBSS and chopped into small (<2 mm3) pieces using a sterile scalpel. The fragments were incubated in 10 ml of 0.25% trypsin solution at 37°C for 20 min. Subsequently DNase I (from bovine pancreas, Pharmacia Biotech, Baie d’Urfé, PQ, Canada) was added to reach a final concentration of 50 µg ml–1. Tissues were incubated for an additional 10 min at 37°C. The cell suspension was diluted with 10 ml of DMEM-F12 with 10% FBS and gently triturated by using a 10 ml pipette with a wide mouth. After centrifugation at 275 g for 10 min, the cell pellet was resuspended in the serum-containing medium, triturated several times, and passed through a 100 µm nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ, USA). The cell suspension was then centrifuged once more (275 g for 10 min), resuspended into 10 ml of DMEM-F12 with 10% FBS containing gentamicin (50 µg ml–1), and plated onto uncoated 10 cm tissue culture plates (Becton Dickinson). Plates were placed in a humidified 5% CO2, 95% air atmosphere at 37°C for 2 h in order to achieve adherence of microglial cells. Nonadherent cells with myelin debris were removed and transferred into 10 cm tissue culture plates. These plates were incubated for 48 h, after which nonadherent cells and myelin debris were discarded. Astrocyte cultures were allowed to expand by replacing the medium once a week. New passages of cells were generated by harvesting confluent cultures of astrocytes by using trypsin-EDTA cell culture medium (0.25% trypsin with EDTA from Invitrogen). Passages up to the third passage of human astrocytes from four surgical cases were used in this study.
Immunostaining with antibodies against CD68, which stains microglia as well as macrophages, and GFAP, a marker of astrocytes, was performed as described earlier (26)
. It confirmed the observations that unstimulated human microglial cells, unlike astrocytes, do not significantly divide in vitro (24
, 27)
, and therefore human astrocyte cultures that had been expanded to reached confluency contained <0.5% microglial cells. Microglial contamination was even lower, or in some cases undetectable, after second and third passages.
For IL-6 and ICAM-1 measurements, astrocytes were detached by trypsin-EDTA treatment and seeded onto 96-well culture plates by adding to each well 5 x 104 U-373 MG cells or 1 x 104 human astrocytes in 100 µl of DMEM-F12 medium containing 5% FBS. Cells were allowed to adhere for 24 h, after which the cell medium was replaced and various stimuli were added in a final volume of 150 µl. Effects of protein kinase inhibitors were studied by adding drugs 15 min before stimulation. Duplicate or triplicate wells were used for each experiment with U-373 MG cells, while single wells were analyzed in the case of human astrocytes. After a 48 h incubation period, 100 µl of cell supernatants was removed for IL-6 analysis. The rest of the supernatant was discarded and the astrocytes were fixed by air-drying.
Measurement of IL-6
The concentrations of IL-6 in cell-free supernatants were measured by an enzyme-linked immunoabsorbent assay (ELISA) (28)
. Briefly, cytokine capture antibodies were diluted in 0.1 M bicarbonate coating buffer, pH 8.2. Aliquots (50 µl) were added to each well of 96-well plates and incubated overnight at 4°C. Nonspecific binding sites were blocked by incubation of wells with 200 µl of 3% BSA in PBS for 2 h at room temperature. Samples and recombinant IL-6 standards were added at 100 µl per well and plates were incubated for 3 h at room temperature. Biotinylated IL-6 detection antibodies were diluted in PBS/3% BSA and added at 100 µl to each well. Plates were incubated for 1 h at room temperature. ExtrAvidin-alkaline phosphatase conjugate was added at 1:20,000 dilution in PBS/3% BSA at 100 µl per well, followed by a 40 min incubation at room temperature. After each of the above experimental steps, plates were washed two to six times with 0.5% Tween in PBS, pH 7.0. Optical density at 405 nm was read after incubation of wells with substrate buffer containing 1 mg ml–1 Sigma 104 phosphate substrate in 0.1 M diethanolamine buffer, pH 9.8, for 3 h at room temperature. Concentrations of IL-6 in the experimental samples were calculated according to the optical densities obtained from wells containing standards of recombinant cytokine.
Measurement of ICAM-1 (CD54) expression
Measurement of ICAM-1 expression was performed as described for other cell surface receptors (29)
. Cells were fixed first by air-drying, followed by incubation in ice-cold 4% paraformaldehyde in PBS for 30 min. After several washes with PBS, the plates were blocked at room temperature with 3% BSA in PBS for 2 h, then incubated with one of the two monoclonal anti-ICAM-1 antibodies diluted 1:1000 in 3% BSA for 1 h. They were washed four times in PBS, followed by incubation for 1 h with goat anti-mouse IgG alkaline phosphatase conjugate diluted 1:3000 in blocking solution. After washing six more times with PBS, the presence of surface receptors in each well was estimated using a microplate reader. Optical densities using a 405 nm filter were measured after incubating the wells for 2 h with substrate buffer containing 1 mg ml–1 Sigma 104 phosphate substrate in 0.1 M diethanolamine buffer, pH 9.8.
Measurement of nitrite
The concentration of nitrite in tissue culture medium was measured as described before (30)
by mixing equal volumes of medium and Griess reagent (1% sulfanilamide, 0.1% N-1-naphthyl-ethylenediamine dihydrochloride, 2.5% phosphoric acid) and measuring absorbency at 550 nm.
Immunoblot analysis
About 3.5 x 106 U-373 MG cells in 8 ml of DMEM-F12 medium containing 5% FBS were plated onto uncoated 10 cm tissue culture plates. Cells were allowed to adhere for 24 h, after which the cell medium was replaced with fresh and WT -syn was added to reach the final concentration of 10 µg ml–1. After treatments for 0–60 min, cells were washed with ice-cold PBS, scraped into lysis buffer (pH 7.4, 20 mM Tris, 20 mM ß-glycerophosphate, 150 mM NaCl, 3 mM EDTA, 3 mM EGTA, 1 mM Na3VO4, 0.5% Nonidet P-40, and 1 mM dithiothreitol), supplemented with protease inhibitors (1 mM phenylmethanesulfonylfluoride (PMSF), 2 µg/ml leupeptin, 4 µg/ml aprotinin, and 1 µg/ml pepstatin A), and sonicated twice for 15 s. Cell debris was removed by centrifugation at 100,000 rpm for 30 min at 4°C. The concentration of soluble proteins was determined by the Bradford assay (31)
. Approximately 25 µg of total lysate protein per lane was resolved on 13% sodium dodecyl sulfate (SDS) -polyacrylamide gels, then transferred to nitrocellulose membranes for standard Western blot with primary antibodies. After further incubation with relevant horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the blots were developed using enhanced chemiluminescence Plus reagent (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) and signals were quantified using Quantity One software (Bio-Rad, Hercules, CA, USA).
Statistical analysis
Data are presented as means ± SE. The concentration-dependent effects of various inhibitors were evaluated statistically by the randomized blocks design ANOVA. Fisher’s least significant differences (LSD) test was used for multiple comparisons.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-syn on human astrocytes, we applied WT -syn and its mutated forms A30P, E46K, and A53T (0.1 to 10 µg ml–1) alone or in combination with IFN- (150 U ml–1) to cultured human U-373 MG astrocytoma cells (Fig. 1
, Fig. 2
) and astrocytes (Fig. 3
). We measured two inflammatory markers: cell surface ICAM-1 and IL-6 secretion into the cell culture medium. Figure 1A
shows that all WT and mutant forms (A30P, A53T, and E46K) of -syn up-regulated ICAM-1 expression by U-373 MG cells after 48 h incubation. The concentration-dependent effects of all -syn forms were significant according to 1-way randomized blocks ANOVA (F and P values are displayed). The mutated forms of -syn were more potent than the WT protein. The differences between the dose-response curves of various forms of -syn were tested by the 2-way ANOVA method. The A30P mutation was more potent than WT -syn (P=0.002), as were the E46K (P=0.03) and A53T (P=0.008), at stimulating ICAM-1 expression. The A53T form of -syn was not only more potent than the WT -syn in terms of the amplitude of response, but was also effective at lower concentrations. The ED50 value for the WT, A30P, and E46K forms of -syn was 
5 µg ml–1; for A53T, this value was 2.5 µg ml–1. Levels of ICAM-1 expression after 10 µg ml–1 -syn were roughly half as high as those observed after stimulation with 150 U ml–1 of the potent proinflammatory cytokine IFN-.
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Figure 1B
shows that ICAM-1 levels expressed by IFN--stimulated U-373 MG cells were enhanced by all forms of -syn. The up-regulation was 2- to 3-fold higher than observed with IFN- alone and was statistically significant for all forms of -syn by the 1-way ANOVA (see Fig. 1B
for F and P values). The mutated forms of -syn, when combined with IFN-, again were more potent than the WT protein. According to 2-way ANOVA, the effects of E46K (P=0.003) and A53T (P=0.004) were significantly higher compared with WT -syn, although the increase for A30P was only of marginal significance (P=0.059). ICAM-1 expression in the presence of 10 µg ml–1 -syn and IFN- was similar to that induced by the potent proinflammatory combination of LPS and IFN- (Fig. 1B
).
Figure 1
shows data obtained by using a mouse monoclonal antibody (mAb) to ICAM-1 supplied by Biogenex. The effects of -syn at 10 µg ml–1 with and without IFN- were also measured by using a different mouse mAb to ICAM-1 supplied by DAKO. Almost identical results were obtained by using these two different antibodies (data not shown). Furthermore, no changes in the expression level of the astrocytic marker GFAP were detected in the samples prepared in the same manner as those represented in Fig. 1
(data not shown).
Figure 2
shows the effects of -syn on IL-6 secretion by U-373 MG cells. As reported previously (28)
, unstimulated U-373 MG cells expressed high levels of IL-6. Therefore, even such potent stimuli as IFN- alone or in combination with LPS (see right bars on Figs. 2A, B
) only modestly increased secretion of IL-6 by this cell line. With the numbers of observations made, only two of the -syn forms (wild-type and E46K) showed a significant concentration-dependent effect while the other two exhibited only trends toward significance. Nevertheless, the concentration of IL-6 in -syn-stimulated samples reached levels comparable to those induced by IFN- and LPS, indicating that the full secretory capacity of these cells is being reached under these conditions (Fig. 2B
). We also measured nitrite levels after 48 h incubation in the U-373 MG cell culture media in the samples described in Fig. 2
. No significant changes were detected (data not shown).
Activation of U-373 MG cells was assessed after 48 h since preliminary experiments indicated that extending incubation times to 72 h and 96 h increased both ICAM-1 expression and IL-6 secretion in -syn stimulated and unstimulated cells. The difference remained approximately the same for ICAM-1 expression (compare Fig. 1A
and Fig. 4
C) and was slightly reduced for IL-6 secretion (data not shown).
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Figure 3
shows the effects of various forms of -syn on human astrocytes. Although human astrocytes were seeded at a 5-fold lower density than U-373 MG cells, their basal expression of ICAM-1 was 6- to 8-fold higher. Unstimulated human astrocytes, unlike U-373 MG cells, secrete very little IL-6. Nevertheless, levels of both ICAM-1 expression and IL-6 secretion in samples that had been maximally stimulated (e.g., -syn+IFN- or LPS+IFN-) were similar in the two types of cultured cells.
At 10 µg ml–1, all forms of -syn, when applied, caused a significant up-regulation of the two activation parameters in human astrocytes. The effects on ICAM-1 expression appeared to be weaker, presumably due to the high basal level of expression of this adhesion molecule. Even LPS plus IFN-, a combination of two potent inducers (32)
, increased expression of this adhesion molecule <2-fold. Similarly, a near maximal increase was observed with IFN- alone (54% up-regulation), which could explain why -syn failed to further enhance ICAM-1 expression by cells already stimulated with IFN- (Fig. 3A
). Figure 3B
shows that all forms of -syn were more potent than IFN- as inducers of IL-6 secretion by astrocytes. Concentrations of IL-6 in samples treated by -syn alone were comparable to those from cells stimulated by a combination of LPS plus IFN-.
Secretion of IL-6 by astrocytes and U-373 MG cells stimulated by -syn was decreased by selective inhibitors of the MAP kinase kinase MEK1/2 (PD 98059), JNK (SP 600125), and p38 MAPK (SB 202190). These inhibitors displayed a significant concentration-dependent effect in cultures of human astrocytes (Fig. 4A
) and U-373 MG cells (Fig. 4B, C
). The inhibitory effects on human astrocytes after 48 h incubation were estimated to have the following IC50 values: 2 µM for PD 98059, 5 µM for SP 600125, and 1.5 µM for SB 202190.
Measurements of ICAM-1 expression showed that effects of the above MAPK inhibitors on this parameter of U-373 MG cell activation could be demonstrated after 96 h incubation with -syn (Fig. 4C
). Extension of the incubation time was needed in order to achieve higher levels of ICAM-1 expression (see Fig. 1A
for ICAM-1 levels after 48 h). None of the inhibitors reduced the viability of astrocytic cells as measured by the MTT assay (26)
, indicating they were nontoxic at the concentrations used (data not shown).
Data presented in Fig. 5
and Table 1
demonstrate the activation of all three major MAPK pathways. This was established by immunoblotting with kinase-specific and phosphorylation site-specific antibodies detecting ERK1/2, JNK, and p38 MAPK. Figure 5
illustrates a time course experiment where U-373 MG cells were stimulated with WT -syn for 0, 5, 10, 20, or 60 min. There was a marked increase in the relative phosphorylation of both ERK1/2 (31% increase) and p47 JNK (205% increase) as early as 10 min after the exposure of cells to the -syn. This increase appeared to persist for the 60 min duration of the experiment. Relative phosphorylation of p40 JNK did not change as dramatically, with only 20% and 25% increases being observed after 20 and 60 min incubation, respectively.
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These observations were confirmed by analyzing an additional eight U-373 MG cell cultures, half of which were stimulated for 20 min with WT -syn (Table 1)
. In this series of immunoblots, we estimated the relative phosphorylation of p40 and p47 JNK as well as that of p38 MAPK and were able to distinguish phosphorylation of the two ERK isoforms, ERK1 and ERK2. Phosphorylation of four kinases was significantly up-regulated, the highest relative increase being observed for ERK2 (81%) and ERK1 (66%), followed by p38 (44%) and p47 JNK (35%). The increase in p40 JNK phosphorylation after 20 min incubation was not statistically significant, being only 24%.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-syn can stimulate the expression of ICAM-1 and the secretion of IL-6. Complementary studies were conducted by using the astrocytic cell line U-373 MG. Direct challenge of both types of cultured cells by -syn increased the expression of ICAM-1 (Fig. 1A
, Fig. 3A
) and further enhanced the effect of IFN- in U-373 MG cells. The addition of -syn to primary astrocytes treated with IFN- resulted in only a small increase in the levels of ICAM-1 presumably due to the high basal expression in these cells that can only be slightly increased even with LPS (Fig. 3A
). This indicates that a maximal effect had been reached. Significantly, all the mutant forms of -syn were more potent than the WT protein in increasing ICAM-1 expression in U-373 MG cells.
Exposure to -syn also stimulated the secretion of IL-6 in both types of cultured cells (Fig. 2
, Fig. 3B
). There was only a small effect in U-373 MG cells due to the high basal secretion of IL-6 in these cells, but the effect induced by -syn in primary astrocytes was dramatic. -Syn was a far more potent inducer of IL-6 secretion than IFN- (Fig. 3B
). -Syn also stimulated the secretion of IL-6 in primary astrocytes challenged with IFN- (Fig. 3B
). However, in the presence of IFN-, -syn had no effect on IL-6 secretion by U-373 MG cells, presumably due to maximal activation by this cytokine (Fig. 2B
). It is also important to note that not all cellular functions of astrocytic type cells were affected by -syn. For example, we failed to detect changes in GFAP levels and nitrite secretion by U-373 MG cells. Both of these parameters have been used before to detect the inflammatory status of astrocytes.
Increased phosphorylation and abnormal accumulation of MAPKs have been reported in neurons of postmortem brain tissues affected by several synucleinopathies including PD and dementia with Lewy bodies (33
34
35)
. Furthermore, increased levels of phosphorylated JNK were recently reported in mice overexpressing the A30P form of -syn (36)
.
There are three major MAPK pathways (37
, 38)
—ERK1/2, JNK, and p38—all of which have been associated with the actions of -syn either in vitro or in vivo (16
, 33
, 34
, 36
, 39
, 40)
. Therefore, we tested for the involvement of these pathways by using specific inhibitors and immunoblotting for their protein and phosphorylation levels. Inhibitors of the three MAPK pathways, in a concentration-dependent manner, lowered IL-6 secretion by human astrocytes, as well as IL-6 and ICAM-1 expression by U-373 MG astrocytoma cells. None of the drugs at the concentrations used had any effect on the viability of astrocytes or astrocytoma cells.
Activation of the MAPK pathways by -syn could also be observed by measuring the relative phosphorylation of the proteins by phospho-specific antibodies. These experiments showed that phosphorylation of ERK1, ERK2, p47 JNK, and p38 MAPK were significantly up-regulated by -syn treatment of the cells. This observation, taken together with data obtained by using the specific inhibitors of MAPKs, indicates that the cellular effects of -syn (in this case, secretion of IL-6 and expression ICAM-1) could be mediated through activation of these MAPK pathways. These observations are consistent with data obtained by Seo et al. (16)
, who showed that the effects of extracellularly added -synuclein on rat microglia were blocked by inhibitors of ERK1/2 and p38 MAP kinase signaling.
The fast activation of MAPKs after only 10–20 min (see Fig. 5
and Table 1
) implicates the direct activation of cell surface receptors. Cellular uptake and diffusion throughout the cytoplasm would require a longer delay time than surface activation, but intracellular receptor mechanisms cannot be ruled out. A similarly short time course for ERK1/2 activation, with a maximum between 10 and 30 min, has been reported for two types of cell surface receptors in rat astrocytes: P2 purinergic receptors (41)
and protease-activated receptors (PARs) (42)
. A direct intracellular binding of -syn to ERK2 has been reported (40)
, but this direct interaction resulted in attenuation rather than activation of ERK2. This family of MAPKs was originally shown to be activated by ligation of cell surface receptors. We are currently trying to identify which receptors might be responsible for -syn action. It appears that they are unlikely to be ICAM-1 since the two antibodies used in this study failed to block the cellular effects of -syn on U-373 MG cells (data not shown).
Our data show that unaggregated -syn is an effective stimulator of astrocytes, indicating that any leakage or excretion of this protein from normal or damaged neurons into the extracellular space (10)
could potentially stimulate glial cells into an inflammatory state. Soluble -syn can be detected in extracellular biological fluids, including human cerebrospinal fluid and blood plasma (43
44
45)
. Its overexpression in cultured cells can result in extracellular release (46)
. The mechanism that leads to the presence of extracellular -syn could be membrane permeability resulting from cell death, but recently it was reported that monomeric and aggregated -syn may be secreted by an unconventional endoplasmic reticulum/Golgi-independent exocytosis pathway (45)
. Such a secretion was enhanced by proteasomal and mitochondrial dysfunction, both of which have been implicated in the etiology of PD.
Here we show that -syn can directly stimulate astrocyte production of IL-6. Astrocytes are a major source of this proinflammatory cytokine (47)
. IL-6 has many physiological roles in the central nervous system, but it can also propagate the inflammatory reactions by stimulating other immune cells and, under some circumstances, even cause neurotoxicity (reviewed in ref. 47
48
49
).
ICAM-1 (CD54) and its counter receptors LFA-1 (CD11a) and Mac-1 (CD11b, CR3) also participate in neuroinflammation. We previously reported that overexpression of ICAM-1 occurs on activated astrocytes in PD SN along with activated microglia. Perhaps more significantly, this same combination was observed in monkeys who had been treated with MPTP 5 to 14 yr previously (21)
. These data indicate that sustained neuroinflammation can occur in the SN and that ICAM-1 may be one of the provocative stimulants. In turn, -syn could be the agent that initiates and sustains astrocytic ICAM-1 expression.
Aberrant ICAM-1 expression by astrocytes has already been observed in a number of degenerative disorders, including multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer’s disease, Pick’s disease, and progressive supranuclear palsy (50
, 51)
. Imamura et al. (52)
also described ICAM-1 up-regulation in microglial cells in PD, including cells surrounding Lewy bodies.
Enhanced proinflammatory effects of mutated forms of -syn could explain why they induce autosomal dominant PD. Other mechanisms that have been suggested include greater susceptibility of neurons to various toxic insults (53
54
55)
as well as impairment of synuclein degradation (56)
.
In this study we demonstrate that all three major MAPK pathways are involved in the proinflammatory effects of -syn on astrocytes. These inflammatory effects could contribute to the pathogenesis of PD. Accordingly, inhibitors of these or other pathways stimulated by -syn could be useful as therapeutic agents in the treatment of PD and related neurodegenerative diseases.
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ACKNOWLEDGMENTS |
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Received for publication March 21, 2006. Accepted for publication May 25, 2006.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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