Effects of malonate C60 derivatives on activated microglia

Brain Research 940 (2002) 61–68 www.elsevier.com / locate / bres

Research report

Effects of malonate C 60 derivatives on activated microglia Shun-Fen Tzeng a , *, Jia-Ling Lee b , Jon-Sun Kuo b , Chung-Shi Yang c , Periyagamy Murugan d , Lin Ai Tai d , Kuo Chu Hwang d b

a Department of Biology, National Cheng Kung University, Tainan City, Taiwan Department of Research and Education, Taichung Veterans General Hospital, Taichung, Taiwan c Department of Applied Chemistry, National Chi-Nan University, Nantou, Taiwan d Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan

Accepted 21 February 2002

Abstract Activated microglia in acute and chronic neurodegenerative disease of the central nervous system (CNS) can produce large amounts of free radicals, such as reactive oxygen species (ROS), which subsequently contribute to neuropathogenesis. Thus, it is believed that the induction of microglial deactivation can reduce neuronal injury. Buckminsterfullerene (C 60 ) derivatives that possess free radical scavenging properties have been demonstrated to prevent neuronal cell death caused by excitotoxic insult. In this study, we investigated the biological role of two malonic acid C 60 derivatives referred as trans-2 and trans-3 on microglia in the presence of the endotoxin lipopolysaccharide (LPS). Treatment of LPS-activated microglia with trans-2 and trans-3 induced a significant degree of transformation of amoeboid microglia to the ramified phenotype. To understand the mechanism underlying this C 60 mediated microglial morphological transformation, we examined the production of proinflammatory cytokines, interleukin-1b (IL-1b) and tumor necrosis factor-a (TNF-a), as well as the final NO products (nitrate and nitrite) in the microglial culture supernatant. Although inducible nitric oxide (iNOS) mRNA and protein expression in LPS-activated microglia were slightly decreased by trans-2 and trans-3, levels of nitrate and nitrite were unaffected. Paradoxically, trans-2 and trans-3 were found to increase the release of IL-1b in the activated microglial culture. However, trans-2 and trans-3 improved the activity of the antioxidant enzyme, superoxide dismutase (SOD) in LPS-treated microglia. Therefore, our results suggest that the C 60 derivatives might increase microglial SOD enzymatic activity which causes microglial morphological transformation from the activated amoeboid phenotype to the resting ramified form.  2002 Elsevier Science B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Neuroglia and myelin Keywords: Micoglia; Lipopolysaccharide; Free radical; Reactive oxygen species; Interleukin-1b; Tumor necrosis factor-a

1. Introduction Microglia, CNS resident macrophages [30], are normally quiescent in the CNS, and are reactivated by cytokines produced by infiltrating immune effectors [13,21,29]. The reactivated microglia with an amoeboid morphology yield proinflammatory cytokines including IL-1b and tumor necrosis factor-a (TNF-a), and generate reactive oxygen species (ROS) such as superoxide anion (?O 2 2 ) and hydrogen peroxide [6,7,14,31]. Additionally, the reactivated *Corresponding author. Department of Biology, National Cheng Kung University, [1 Ta-Hsueh Rd., Tainan City, Taiwan 70101. E-mail address: [email protected] (S.-F. Tzeng).

microglia produce nitric oxide (NO) that can rapidly react with ?O 2 2 to produce peroxynitrite anions [1–3]. These microglial products are cytotoxic to neurons and glia in neurological disorders such as cerebral ischemia, Alzheimer’s disease and multiple sclerosis, [1,10,11,27–29]. Thus, the reduction in microglial generated ROS and proinflammatory cytokines after CNS injury is believed to be able to suppress neuronal cell death after CNS injury. Antioxidants, vitamin E and vitamin C, have been indicated to induce microglial deactivation [15], suggesting that regulating the oxidative mechanism in microglia may mediate microglial transformation from the activated stage to the resting stage, or vice verse. C 60 molecules, pure carbon spheres, are characterized as a ‘radical sponge’ on

0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )02592-1

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the basis of their potent reactivity with free radicals [20]. Because native C 60 is rarely soluble in water, by adding carboxyl groups to enhance their water solubility, C 60 derivatives have been found to act as a protective agent for cultured cortical neurons against excitotoxic insult [8,9,18,23]. Moreover, in vivo studies have shown that C 60 derivatives can impede cell death and functional degeneration in transgenic mice containing mutant human SOD genes [10]. SOD is an antioxidant enzyme that converts superoxide radicals to H 2 O 2 and oxygen [24]. These findings indicate that the antioxidant properties of C 60 derivatives contribute to neuroprotection. We are particularly interested in understanding the antioxidant effect of C 60 molecules on microglial deactivation. We used malonic acid C 60 derivatives, trans-2 and trans-3, to investigate their effects on the morphological and biochemical changes in activated microglia stimulated with LPS [32]. We found that the C 60 derivatives were indeed able to induce microglial ramification. We also examined whether or not the morphological transformation was related to microglial biochemical changes, such as NO levels, proinflammatory cytokine release, or SOD enzymatic activity.

small amounts of tris malonate C 60 derivatives (,5%). The unreacted C 60 and malonate C 60 derivatives were separated via silica gel column chromatography with toluene / hexane mixture of solvent. Unreacted C 60 was washed from the column using a 10% toluene in hexane solvent. Mono malonate C 60 was then obtained by increasing the toluene percentage to 20%. Regioisomers of bis malonate C 60 were then separated and washed from the column by increasing the percentage of toluene (to 30| 50%). Finally, all C 60 malonate derivatives were separated via normal phase high-pressure liquid chromatography (HPLC) silica gel column using pure toluene as solvent. Saponification of the C 60 malonate derivatives was achieved by refluxing the corresponding malonates (in toluene) under nitrogen in the presence of a 20-fold molar excess of NaH for 5 h. The reaction mixture was then quenched by MeOH at RT, which affords the corresponding malonic acid C 60 derivatives, trans-2 and trans-3. The residual solvent was removed in vacuum to generate a dry powder of trans-2 and trans-3 (Fig. 1). The purity of trans-2 and trans-3 was confirmed by various spectroscopic measurements including HPLC elution sequence, UV, and desorption chemical ionization (DIC)-mass spectrum.

2. Materials and methods

2.2. Primary rat microglia culture

2.1. Synthesis of C60 derivatives

The culture medium constituents, N1 serum supplement and antibiotics were obtained from Gibco (Life Technologies, Grand Island, NY). Fetal bovine serum (FBS) was purchased from HyClone (Logan, Utah). Primary mixed glial cell cultures were prepared as previously described [5] with a slight modification. In brief, cerebral cortices from neonatal Sprague–Dawley rat brains (P1) were removed and carefully dissected. The tissue was dissociated in papsin and passed through a 70 mm-pore nylon mesh. After centrifugation, the cell pellet was resuspended in Dulbecco’s modified Eagle medium (DMEM) / F-12 (1:1) containing 10% heat inactivated fetal bovine serum (FBS), 50 U / ml penicillin and 50 mg / ml

All chemicals used for the synthesis of C 60 derivatives were purchased from Sigma (St. Louis, MO). Malonic acid derivatives of C 60 , trans-2 and trans-3, were synthesized as previously described [16,17,22]. In brief, C 60 in toluene was reacted with 1.5 equivalents of diethyl bromo malonate at 60 8C in the presence of 10 equivalents of sodium hydride (NaH) for 5 h. The unreacted NaH was quenched by methanol at room temperature (RT). The toluene layer was then collected. The resulting deep brown toluene solution contains unreacted C 60 , mono malonate C 60 derivatives, and bis malonate C 60 derivatives along with

Fig. 1. Structures of trans-2 and trans-3 showing dicarboxy groups on the C 60 sphere.

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streptomycin. The cells at a density of 10 7 cells / ml were then plated onto poly-D-lysine (Sigma, St. Louis, MO) coated 75-cm culture flasks (Corning). The culture medium was renewed every 2 days. Eight days later, microglia were collected using the shake-off method. The microglial cells collected were replated either onto 96-well plates at a density of 2310 4 cells / well or onto 35 mm petri dish at a density of 5310 5 cells / well. Subsequently, 18–24 h later, the cells were treated with LPS (Sigma, St. Louis, MO) in DMEM / F-12 medium containing N1 serum supplement (N1 medium). Then, 95% microglia with B4 isolectin (Sigma) positive staining were found in the microglial culture used in this study, while less 5% of cells in the culture were GFAP immunostained astrocytes.

2.3. MTT colorimetric assay MTT reacts with mitochondrial enzymes in living cells to generate a formazan product. Thus, measurement of the level of formazan product by a colorimetric method is referenced to the relative number of cells. To study the growth effect of trans-2 and trans-3 on microglia, we replated microglia onto a 96-well microtiter plate at a density of 2310 4 cells / well. Then, 18–24 h after treatments, cultures were subjected to MTT assay. MTT solution (5 mg / ml; Sigma) was added to each well. A total of 4 h later, SDS (10% in 0.1 N HCl) was added to each well, and the culture was then incubated overnight at 37 8C. MTT absorbance was measured using an ELISA reader at 595 nm.

2.4. Nitrite /nitrate, TNF-a and IL-1b assay Microglia at a density of 2310 4 cells / well were replated onto 96-well plates. Then, 18 h after treatment with LPS plus trans-2 and trans-3, culture media were collected for NO production assay and cytokine ELISA assay. The production of NO was assessed as the accumulation of nitrate and nitrite in the culture medium using a colorimetric reaction kit from R&D (Minneapolis, MN). Fold production represent the ratio of the production of nitrate and nitrite in the culture medium to that in LPS-treated culture medium. TNF-a and IL-1b were measured using enzyme immunoassay kit from R&D, following the procedure provided by the vendor.

2.5. Reverse-transcription PCR ( RT-PCR) The expression of iNOS mRNA was analyzed by RTPCR assay. Microglia at a density of 1310 6 cells / dish were replated onto 60 mm culture petri dishes. Then, 18 h after treatment with LPS plus trans-2 and trans-3. Total RNA was extracted from the microglial culture according to the method of Chomczynski and Sacchi [4]. A sample of

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2 mg of total RNA were used in the RT-PCR reaction and was mixed with SuperScript one step RT-PCR system components (Gibco, Life Technologies, Grand Island, NY) with the following cycle parameters: 45 8C, 30 min, 94 8C, 2 min; 30 cycles, 94 8C, 15 s; 55 8C, 30 min, 72 8C, 1 min; 72 8C, 7 min, 4 8C, `. Reaction products were then separated on a 1.8% agarose gel, stained with ethidiumbromide, and photographed. Measurements were normalized to GAPDH mRNA levels. Primers for RT-PCR used for gene amplification were described as follows. iNOS (181 bp) sense 59-ctccatagttttcagaagcag-39, antisense 59agttcaatatctcctggtgga-39; GAPDH (671 bp) sense 59-ttcaccaccatggagaaggc-39, antisense 59-accaccctgttgctgtagcc.

2.6. Western blot analysis Microglia at a density of 5310 5 cells / dish were replated onto 35 mm tissue petri dishes. Protein samples were extracted from microglial cultures 18–24 h after treatment with LPS plus trans-2 and trans-3. Cells were harvested and gently homogenized on ice using PBS containing 1% SDS, 1 mM phenylmethyl-sulfonylfluoride (PMSF), 1 mM EDTA, 1.5 mM pepstatin, 2 mM leupeptin, and 0.7 mM aprotinin. Protein concentration was determined using BioRad DC kit. Subsequently, 10–20 mg of total protein was loaded onto 7.5–12.5% SDS–PAGE, and then transferred to the nitrocellulose membrane. The protein was identified by incubating the membrane with anti-iNOS (Calbiochem, Cambridge, MA), anti-MnSOD (StreeGen Biotechnologies Corp., Victoria, Canada), or anti-Cu / Zn SOD (StressGen Biotechnologies Corp., Victoria, Canada) antibodies overnight at 4 8C, followed by horseradish peroxidase conjugated secondary antibodies and ECL solution (NEN Life Science, Boston, MA). Densitometric analysis was performed using Gel Image Digital System equipped with IS-1000 (Version 2.0) Digital Imaging Software (Alpha Innotech Ins.).

2.7. Measurement of SOD activity Microglia at a density of 5310 5 cells / dish were replated onto 35 mm tissue culture dishes. The cells were harvested 18 h after treatment with LPS plus trans-2 and trans-3, and homogenized in 5% monophosphoric acid. After centrifugation at 3000 rev. / min for 2 min at 4 8C, the supernatant was analyzed for total SOD activity (Cu / Zn-SOD and Mn-SOD) using the BIOXYTECH SOD-525 kit from R&D (Minneapolis, MN). The method was based on the SOD-mediated increase in the rate of autoxidation of 5,6,6a,11b-tetrahydro-3,9,10-trihydroxybenzo[c]fluorine in an aqueous alkaline solution that yields a chromophore with maximum absorbance at 525 nm [26]. Total protein was measured using Bio-Rad DC kit. The specific activity was expressed as units per milligram of protein.

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Fig. 2. Trans-2 and trans-3 induce a morphological change of LPS-activated microglia. The malonic acid C 60 derivatives, trans-2 (C,D) and trans-3 (E,F) at 10 mM concentration, was added to the microglia culture in N1 medium with (B,D,F) or without (A,C,E) 10 ng / ml LPS. Then, 18 h after treatment, the culture was harvested. The amoeboid morphology of LPS-activated microglia (arrowheads, B) was observed under phase-contrast microscopy. LPS-stimulated microglia treated with trans-2 (D) and trans-3 (F) had ramified processes (arrows). Similarly, trans-2 and trans-3 at 30 mM also induced the ramification of LPS-activated microglia. Bar in A–F, 100 mM.

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3. Results

3.1. C60 derivatives induce a change in microglial morphology In general, microglia in the resting stage exhibit a ramified morphology and become amoeboid in response to various stimuli, such as endotoxin LPS. As shown in Fig. 2, microglia with amoeboid morphology were observed in the culture stimulated with 10 ng / ml LPS. However, similar to the resting culture, greater than 95% microglia showed the process-bearing morphology when treated with LPS in combination with trans-2 or trans-3 at 10 mM concentration. In the absence of LPS, trans-2 and trans-3 at 10 mM concentration can cause approximately a 50% increase in MTT metabolism (Fig. 3). However, in the presence of LPS, MTT metabolism in microglia was unchanged by trans-2 and trans-3 at 10 mM concentration, indicating that trans-2 and trans-3 had no influence on activated microglial cell viability. Similar results were observed when microglia were treated with trans-2 or trans-3 at 30 mM concentration either in the absence or presence of LPS. Additionally, the combination of trans-2 and trans-3 did not enhance microglial ramification and MTT metabolism when compared to that in either trans-2 or trans-3 treatment separate treatments (data not shown).

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tion. In LPS-treated microglia, iNOS mRNA and protein were highly expressed while trans-2 and trans-3 slightly reduced the transcriptional level of iNOS (Fig. 4A). Moreover, the addition of trans-2 or trans-3 caused moderate inhibition in microglial iNOS production in the presence of LPS at 10 and 50 ng / ml (Fig. 4B). However, the

3.2. Effect of the C60 derivatives on NO production The effect of C 60 derivatives on microglial NO production was studied by examining iNOS expression and the level of nitrate and nitrite in the culture medium. In the absence of LPS, no expression of iNOS mRNA or protein was observed in mircoglia with or without trasn-2 and trans-3. The small amount of nitrate and nitrite was found in the culture medium of microglia without LPS stimula-

Fig. 3. Effect of trans-2 and trans-3 on microglial cell viability. Microglial cells activated with or without LPS at 50 ng / ml were treated with trans-2 and trans-3 at 10 mM in N1 medium. Then, 18 h after treatment, the culture was subjected to MTT assay for the examination of microglial viability. Without LPS stimulation, trans-2 and trans-3 caused a 50% increase in MTT metabolism when compared to that observed in control. However, no significant increase in MTT metabolism was found when microglia were treated with LPS plus trans-2 and trans-3. Data are mean6S.E.M. values (n58). *** P,0.005; ** P,0.01 (Student’s t-test).

Fig. 4. Effect of trans-2 and trans-3 on microglial iNOS expression and nitrate / nitrite production. A. RT-PCR assay was performed to examine the expression of microglial iNOS mRNA in the treatment of LPS plus trans-2 and trans-3 for 18 h. Trans-2 and trans-3 in the presence of LPS slightly reduced the expression of iNOS mRNA in microglial activated by 10 ng / ml LPS. The expression of microglial iNOS mRNA was not detected in the absence of LPS. B. Western Blot analysis was performed to examine the levels of iNOS protein in microglia incubated with LPS plus trans-2 and trans-3 for 18 h. Note that iNOS protein levels were not detectable in microglia without LPS treatment. C. The production of nitrate and nitrite in the supernatant of microglia was analyzed using a colorimetric method. The amount of nitrate and nitrite in mircoglia treated with LPS alone was represented as 1. Fold production was defined as the ratio of the amount of nitrate and nitrite in each treatment to LPS alone. These experiments were repeated with similar results.

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amount of nitrate and nitrite in the culture supernatant of LPS-treated microglia was not decreased by the treatment of trans-2 and trans-3 (Fig. 4C).

3.3. Effect of C60 derivatives on microglial TNF-a and IL-b release Activated microglia can secrete proinflammatory cytokines such as TNF-a and IL-1b [12,19]. To determine the effect of trans-2 and trans-3 on the cytokine production of LPS-activated microglia, microglial culture media were collected 18 h after treatment, and then subjected to TNF-a and IL-1b ELISA assay. In the absence of LPS, TNF-a and IL-1b were not detectable in the culture supernatant of microglia with or without C 60 derivative treatment (Fig. 5). High levels of TNF-a and IL-1b were detected in the culture supernatant of microglia stimulated with LPS at 10 ng / ml (data not shown) and 50 ng / ml (Fig. 5). The level of TNF-a was slightly increased in the culture supernatant of microglia with LPS and trans-2 treatments, while trans3 treatment showed no effect on the increase of TNF-a

release in LPS-treated microglia (Fig. 5). Hence, the levels of IL-1b in the culture supernatant of LPS-treated microglia were biostatistically increased by either trans-2 or trans-3 when compared to that in LPS-treated microglial culture without the addition of the C 60 derivatives (P, 0.0001 for trans-2; P,0.0001 for trans-3).

3.4. Effect of the C60 derivatives on SOD enzymatic activity To investigate whether the C 60 derivatives have an antioxidant effect on activated microglia, we analyzed microglial SOD enzymatic activity in the absence or presence of LPS. We examined the expression of Mn-SOD and Cu / Zn-SOD. Western Blot analysis indicated that LPS treatment induced the upregulation of microglial Mn SOD protein expression (Fig. 6). In the absence or presence of LPS, trans-2 and trans-3 caused either no increase or a slight decrease in the levels of microglial Mn SOD protein. A similar expression pattern of microglial Cu / Zn SOD protein levels was also observed (data not shown). Examination of total SOD enzymatic activity indicated that the C 60 derivatives had no effect on altering microglial SOD enzymatic activity in the absence of LPS. However, LPS caused 1- to 3-fold reduction in microglial SOD activity. Interestingly, trans-2 and trans-3 increased microglial SOD activity in the presence of LPS at 10 or 50 ng / ml.

4. Discussion

Fig. 5. Trans-2 and trans-3 increase TNF-a and IL-1b accumulation in LPS-activated microglia. Microglia at the density of 5310 4 cells / well were replated onto 96-well plates. Then, 18–24 h later, the culture was incubated with various treatments as indicated above. Subsequently, 18 h after treatment, the culture supernatant was subjected to TNF-a (A) and IL-1b (B) immunoassay. Data are the mean6S.E.M. (n53–5) of TNF-a and IL-1b protein (pg / ml). The experiment was repeated twice with similar results. *** P,0.005; * P,0.05 (Student’s t-test).

In this study, we demonstrate that the C 60 derivatives, trans-2 and trans-3, induce the morphological change of microglia from amoeboid activated shape to ramified resting appearance. The morphological transformation by trans-2 and trans-3 may be related to the increase of the SOD enzymatic activity in LPS-activated microglia. The two C 60 derivatives have no effect on the reduction of NO production in LPS-treated microglia although they downregulate the levels of iNOS mRNA and protein. However, the two C 60 derivatives can increase IL-1b release in LPS-activated microglia. Evidence indicates that antioxidants such as vitamin E and vitamin C can induce ramification of microglia [15]. Similarly, the malonate C 60 derivatives induce the ramification of amoeboid activated microglia. The C 60 induced morphological change can be observed as early as 6 h after treatment (data not shown), suggesting that fast microglial response to the C 60 derivatives may happen in order to induce the transformation of the microglial cytoskeleton. In the absence of LPS, MTT metabolism is upregulated by trans-2 and trans-3. However, treatment of microglia with trans-2 and trans-3 did not cause an increase in the production of microglial NO, IL-1b and TNF-a, indicating that the malonate C 60 derivatives did not act as the trigger

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Fig. 6. Treatment with trans-2 and trans-3 induces an increase in microglial SOD activity. In the presence of LPS at various concentrations as indicated above, microglia were treated with trans-2 and trans-3 at 10 mM for 18 h. Western Blot analysis was performed to examine the level of Mn-SOD (A). Thee similar results were obtained in Cu-SOD protein expression. The SOD activity was performed twice as indicated above. The results were expressed as fold induction that is the ratio of the SOD activity (Unit / mg) in treatment of trans-2 and trans-3 to controls (LPS, 0, 1, 10, 50 ng / ml) as 1.

of microglial activation. In contrast, based on the findings shown in this study, the C 60 derivatives may be capable of mediating microglia deactivation in the presence of stimuli, such as LPS or agents produced after CNS injury. O2 2 can react with NO to generate the potent neuronal toxin, peroxynitrite anion [2,3]. It has been reported that C 60 molecules can effectively eliminate O 2 2 to protect neurons from excitotoxic injury [9]. Accordingly, the C 60 derivatives used in this study may also reduce the level of O 22 in LPS-activated microglia. Although we have no evidence showing the C 60 -induced alternation of ROS levels in LPS-activated microglia, we demonstrate that SOD enzymatic activity in LPS-activated microglia is elevated by trans-2 and trans-3. O 2 2 dismutation by SOD generates hydrogen peroxide and oxygen that can react with NO to produce NO final products, nitrate and nitrite. However, we show here that the C 60 derivatives do not change the levels of nitrate and nitrite in LPS-activated microglia, although the C 60 derivatives reduce the expression of iNOS mRNA and protein in LPS-activated microglia. Yet, the C 60 derivatives may induce their effect on

accelerating O 2 2 dismutation by upregulating the enzymatic activity of SOD, which may subsequently contribute to the ramification of amoeboid microglia. Nevertheless, the precise mechanism by which the C 60 derivatives induce this microglial morphological transformation toward the resting phenotype remains to be elucidated. Microglial activation is always accompanied by high production of proinflammatoy cytokines, such as TNF-a and IL-1b [25,28]. Do the C 60 derivatives mediate antiinflammatory reaction in LPS-activated microglia? The addition of the C 60 derivatives to microglia in the presence of LPS increases the release of TNF-a and IL-1b to a certain extent. The findings seem to be contradictory to our hypothesis that the C 60 derivatives are able to deactivate microglia via their antioxidant properties. Is the increase in the release of TNF-a and IL-1b caused by the process of microglial ramification that was induced by the C 60 derivatives? Certainly, the mechanism for the C 60 -modulated TNF-a and IL-1b release remains to be clarified. In summary, C 60 derivatives induce microglial ramification, indicating their important role in microglial deactiva-

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tion. The action of C 60 derivatives on microglial deactivation may result from the elimination of O 2 2 by increasing microglial SOD enzymatic activity. Together, our results that suggest a potential role of C 60 molecules in microglia deactivation may lead to a possible treatment for oxidative stress-induced neurodegenerative disorders.

Acknowledgements This study was supported by the Taicung Veterans General Hospital of Taiwan (TCVGH-897316D) and the National Health Research Institutes of Taiwan (NHRI-GTEX89B907C).

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