GM1 ganglioside attenuates convulsions and thiobarbituric acid reactive substances production induced by the intrastriatal injection of methylmalonic acid

The International Journal of Biochemistry & Cell Biology 35 (2003) 465–473

GM1 ganglioside attenuates convulsions and thiobarbituric acid reactive substances production induced by the intrastriatal injection of methylmalonic acid Michele Rechia Fighera a , Juliana Sartori Bonini b , Telma Grendene de Oliveira a , Roberto Frussa-Filho b , João Batista Teixeira Rocha a , Carlos Severo Dutra-Filho c , Maribel Antonello Rubin a , Carlos Fernando Mello a,∗ a

Departamento de Qu´ımica, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil Departamento de Farmacologia, Universidade Federal de São Paulo, 04023-900 São Paulo, SP, Brazil Departamento de Bioqu´ımica, Universidade Federal do Rio Grande do Sul, 90035-003 Porto Alegre, RS, Brazil b

c

Received 13 June 2002; received in revised form 2 October 2002; accepted 2 October 2002

Abstract The effects of the administration of monosialoganglioside (GM1) on methylmalonic acid (MMA)-induced convulsions, production of thiobarbituric acid reactive substances (TBARS) and on the striatal content of ascorbic acid and total non-protein thiol (SH) groups were evaluated in adult male rats. Animals received two intraperitoneal injections of GM1 (50 mg/kg) or saline (0.85% NaCl) spaced 24 h apart. Thirty minutes after the second GM1 or saline injection, l-MMA (6 ␮mol) or NaCl (9 ␮mol) was injected into the right striatum and the animals were observed for the appearance of convulsions for 15 min. The animals were sacrificed and their striatal content of ascorbic acid, SH groups and TBARS was measured. The effect of GM1 on MMA-induced TBARS production in striatal homogenates was also evaluated in vitro. MMA injection caused convulsions (Sal–MMA: 9.8 ± 1.4 episodes, which lasted 271 ± 48 s) and increased the striatal content of TBARS (Sal–MMA: 149.0 ± 11.5 nmol MDA/g tissue), but did not alter total striatal SH or ascorbic acid contents. GM1 pretreatment decreased MMA-induced convulsions (GM1–MMA: 6.3 ± 2.0 episodes, which lasted 115.1 ± 42.2 s) and TBARS increase (GM1–MMA: 102.4 ± 19.5 nmol MDA/g tissue). GM1 pretreatment increased ascorbic acid content of the striata (saline-pretreated: 1514 ± 75.9; GM1-pretreated: 1878.6 ± 102.8 ␮g ascorbic acid/mg tissue). MMA increased TBARS production in vitro, and GM1 had no effect on such MMA-induced effect. This study provides evidence that GM1 increases striatal ascorbic acid content and decreases MMA-induced neurotoxicity assessed by behavioral and neurochemical parameters. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: l-Methylmalonic acid; Convulsion; Monosialoganglioside; Thiobarbituric acid reactive substances; Ascorbic acid

1. Introduction Abbreviations: TBARS, thiobarbituric acid reactive substances; GM1, monosialoganglioside; NMDA, N-methyl-d-aspartate; MDA, malondialdehyde; CNS, central nervous system ∗ Corresponding author. Fax: +55-55-2208031. E-mail address: [email protected] (C.F. Mello).

Methylmalonic acidemias are inherited metabolic disorders caused by a severe deficiency of methylmalonyl-CoA mutase (EC 5.4.99.2) activity, which are clinically characterized by neurological dysfunc-

1357-2725/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 2 ) 0 0 2 7 5 - 3

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tion (Fenton, Gravel, & Rosenblatt, 2001). It has previously reported that l-methylmalonic acid (MMA) inhibits succinate dehydrogenase (SDH) and ␤-hydroxybutyrate dehydrogenase activity in vitro (Dutra et al., 1993), and that the intrastriatal administration of MMA induces convulsions through glutamatergic mechanisms (de Mello et al., 1996). In fact, a substantial body of evidence has linked MMA accumulation to ATP depletion, neuronal depolarization and activation of NMDA receptors, which seem to play a critical role in the genesis of the convulsions observed after intrastriatal MMA administration (de Mello et al., 1996; Fighera et al., 1999). The depolarizing action of MMA was confirmed in vitro, reinforcing our behavioral data (McLaughlin, Nelson, Silver, Erecinska, & Chesselet, 1998). A significant amount of work has suggested that reactive oxygen species generation may underlie the neurotoxic effects of succinate dehydrogenase inhibitors in vivo and in vitro (Greenamyre, Garcia-Osuna, & Greene, 1994; Schulz et al., 1996). Malonate- and 3-nitropropionate-induced neurotoxicity is diminished by coenzyme Q administration (Beal et al., 1994) and attenuated in transgenic mice over expressing copper/zinc superoxide dismutase (SOD) activity (Beal et al., 1995), respectively. Malonate increases reactive oxygen species generation and decreases reduced cellular glutathione levels in mesencephalic cultures, reducing cellular viability (Zeevalk, Bernard, & Nicklas, 2000). In fact, cyclic nitrone spin trapping agents that trap reactive oxygen species (Schulz et al., 1995) protect mesencephalic neurons from malonate toxicity, further supporting the involvement of reactive species in the neurotoxic action of complex II inhibitors (McLaughlin et al., 1998). It has also been suggested that reactive oxygen species are involved in the genesis of convulsions (Fighera et al., 1999). We have recently shown that the systemic administration of ascorbic acid or ␣-tocopherol attenuates convulsions induced by the intrastriatal administration of MMA in rats, suggesting that reactive oxygen species may be involved in the convulsive phenomena elicited by MMA (Fighera et al., 1999). Our data agree with the study of Fontella et al., who have demonstrated that MMA stimulates lipid peroxidation and reduces total antioxidant potential in cerebral tissue in vitro, further supporting a possible role of reactive oxygen

species in the pathophysiology of these disorders (Fontella et al., 2000). Gangliosides are sialic acid-containing glycosphingolipids that are synthesized in the Golgi-apparatus and then transported and incorporated into the plasma membrane (Leeden, 1983; Tettamantti, Ghindoni, & Trinchera, 1987). Moreover, exogenous GM1 added to living cells both in vivo and in vitro is taken up by the cells and mixed with the endogenous pool (Moss, Fishman, & Manganiello, 1976). In fact, gangliosides have attracted much attention in the literature due to the various reports of neuroprotection of these compounds against various excitotoxic agents or conditions, such as excitatory amino acid exposure and ischemia (Carolei, Fieschi, Bruno, & Toffano, 1991; Tajrine, Garofalo, Cuello, & Ribeiro-da-Silva, 1997) accompanied by an apparent absence of side effects. In addition, numerous investigations have indicated the involvement of these compounds in different behavioral events associated with adaptive functions (Bellot et al., 1996; Bellot, Vital, Palermo-Neto, & Frussa-Filho, 1997; Vital, Frussa-Filho, & Palermo-Neto, 1995, 1997), neuronal plasticity (Palermo-Neto, Frussa-Filho, & Vital, 1999; Vital et al., 1998) and memory formation (Silva, Bellot, Vital, & Frussa-Filho, 1997; Silva, Felicio, Nasello, Vital, & Frussa-Filho, 1996). In the present study, we investigate whether systemic GM1 administration protects against the convulsant effects of MMA. Moreover, since MMA-induced neurotoxicity seems to involve reactive oxygen species generation (Fighera et al., 1999), the striatal content of TBARS, ascorbic acid and total non-protein thiol group levels were also evaluated.

2. Materials and methods 2.1. Animals and reagents Adult male Wistar rats (270–300 g) maintained on a 12:12 h light:dark cycle, with free access to tap water and standard lab chow (Guabi, Santa Maria, RS, Brazil) were used, and each animal was used only once. All reagents were purchased from Sigma, except GM1 ganglioside, which was kindly donated by Fidia Research Laboratories.

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2.2. Behavioral evaluation Animals were anesthetized with pentobarbital (40 mg/kg, i.p.) and placed in a rodent stereotaxic apparatus. Under stereotaxic guidance, a cannula was inserted unilaterally into the striatum (coordinates relative to bregma: AP 0 mm, ML 3.0 mm, V 3.0 mm from the dura) (Paxinos & Watson, 1986). Chloramphenicol (200 mg/kg, i.p.) was administered immediately before the surgical procedure. Three days after surgery, the animals received two injections of GM1 (50 mg/kg, i.p.) or saline (NaCl 0.85%, i.p.) spaced 24 h apart. Thirty minutes after the second GM1 or saline injection, buffered MMA (6 ␮mol/2 ␮l) or NaCl (9 ␮mol/2 ␮l) was injected into the right striatum. MMA solutions were neutralized with NaOH to pH 7.4 and injections were performed over a 2 min interval. Immediately after the injections the animals were transferred to a round open field (54.7 cm diameter) with a floor divided into 11 equal areas. The open field session lasted 15 min, and during this time the animals were observed for the appearance of clonic or tonic–clonic convulsions involving forelimbs and/or hindlimbs and the number of convulsive episodes during which the animals presented full lateralization and loss of righting reflex. The number and duration of convulsive episodes and the number of rotational responses contralateral or ipsilateral to the injected hemisphere were recorded (de Mello et al., 1996). 2.3. TBARS, ascorbic acid and total non-protein thiol determination Immediately after the behavioral evaluation, the animals were sacrificed by decapitation and had their brain exposed by the removal of the parietal bone. A punch of the injected striatum was rapidly obtained using a stainless steel puncher 5 mm i.d. around the site of cannula placing. Tissue was homogenized in 1.15% (w/v) KCl with a glass homogenizer and its TBARS content was determined as described by Ohkawa, Ohishi, and Yagi (1979). Ascorbic acid and total non-protein thiol groups were measured in tissue homogenates according to Benderitter et al. (1998) and Ellman (1959), respectively, as modified by Jacques-Silva, Nogueira, Broch, Flores, and Rocha (2001) and Reiter (1995).

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Due to the previously reported antioxidant action of GM1 ganglioside (Maulik, Das, & Gogineni, 1993), we decided to test whether this compound could alter the MMA-induced TBARS generation in rat striatum homogenates in vitro. Striatum from non-cannuled rats was homogenized as described above. An aliquot of 200 ␮l was added to a medium containing MMA (0–20 mM) and 0 or 200 ␮M monosialoganglioside. Incubation was carried out at 37 ◦ C and terminated 1 h later by the addition of acetic acid and SDS. All experiments were conducted according to the ethical rules for animal research at our University. 2.4. Statistical analysis Behavioral data were analyzed by a 2 (saline or GM1) × 2 (NaCl or MMA) factorial ANOVA. Data were log transformed before analysis in order to meet assumptions for ANOVA. Post hoc analysis was carried out by the Student–Newman–Keuls test, when appropriate. In vitro experiment data were analyzed by a 2 (presence or absence of GM1) × 5 (0, 0.66, 2.0, 6.6, 20 mM MMA) factorial ANOVA. Partitioning the total sum of squares into trend components assessed the dose–effect relationship. Post hoc analysis was carried out by the Student–Newman–Keuls test.

3. Results Fig. 1 shows the effect of GM1 administration on the number and duration of convulsive episodes elicited by MMA. Statistical analysis of the number and duration of convulsive episodes revealed that GM1 administration reduced the convulsive behavior elicited by MMA [significant GM1 or saline × MMA or NaCl interaction for number (F(1, 43) = 5.79; P < 0.05) and duration of convulsive episodes (F(1, 43) = 5.84; P < 0.05)]. Fig. 2 shows the effect of GM1 on the striatal TBARS content. Statistical analysis (two-way ANOVA) revealed that MMA increased striatal TBARS content, and that preadministration of GM1 attenuated the MMA-induced increase of striatal TBARS content [significant pretreatment (saline

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Fig. 1. GM1 ganglioside preadministration attenuates the number (A) and duration (B) of convulsive episodes elicited by intrastriatal injection of 6 ␮mol MMA (hatched columns). The control group received 9 ␮mol NaCl (open columns). Data are mean+S.E.M. for n = 9–10 per group. ∗ P < 0.05 compared with Sal–MMA group (Student–Newman–Keuls test). Sal, saline; GM1, monosialoganglioside.

Fig. 2. GM1 ganglioside preadministration attenuates MMAinduced (6 ␮mol) increase of striatal TBARS content. The control group was injected with 9 ␮mol NaCl. Data are presented as nmol MDA/g tissue (mean+S.E.M.) for n = 9–10 per group. ∗ P < 0.05 compared with Sal–MMA group (Student–Newman–Keuls test).

or GM1) × treatment (NaCl or MMA) interaction (F(1, 37) = 4.93; P < 0.05)]. Table 1 shows the effect of GM1 on the striatal content of ascorbic acid in the injected and in the control striatum. Statistical analysis (three-way ANOVA with the striata treated as within subject factor) revealed only a significant effect of GM1 pretreatment (F(1, 36) = 9.51; P < 0.005), indicating that GM1 administration increased total striatal ascorbic acid levels, regardless of the injection of MMA into the striatum. Since MMA had no effect on striatal ascorbic levels, for the statistical purposes and for the sake of clarity, pooled means are presented. Total striatal non-protein thiol content was not altered by GM1 pretreatment (Table 2).

Table 3 shows the effect of MMA and GM1 on TBARS production in striatal homogenates. Statistical analysis (two-way ANOVA) revealed that MMA increases TBARS production in striatal homogenates regardless of the presence of GM1 (200 ␮M) [significant main effect of MMA (F(4, 30) = 47.95; P < 0.001)]. Partitioning of the sum of squares into trend components revealed that TBARS production increased linearly with the amount of MMA added to the reaction medium [significant linear trend: in the absence of GM1 (F(1, 15) = 85.58; P < 0.001); in the presence of GM1 (F(1, 15) = 97.64; P < 0.001)]. These data suggest that GM1 ganglioside had no intrinsic antioxidant activity in this assay.

Table 1 Effect of GM1 ganglioside (50 mg/kg, i.p.) and of MMA (6 ␮mol) on the striatal content of ascorbic acid (␮mol ascorbic acid/g tissue)

Saline GM1

NaCl (9 ␮mol per striatum)

MMA (6 ␮mol per striatum)

Right (injected)

Left (control)

Right (injected)

Left (control)

1417.6 ± 90.9 1815.3 ± 143.7

1507.3 ± 105.6 1631.0 ± 136.4

1467.6 ± 107.9 1984.1 ± 183.5

1748.2 ± 136.9 2051.9 ± 163.6

Pooled mean

1514.7 ± 75.9 1878.6 ± 102.8∗

Data are mean ± S.E.M. for n = 9–14 per group. Statistical analysis (three-way ANOVA with the striata treated as within subject factor) revealed only a significant effect GM1 pretreatment (F(1, 36) = 9.51). MMA, l-methylmalonic acid; GM1, monosialoganglioside. ∗ P < 0.005.

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Table 2 Absence of effect of GM1 ganglioside (50 mg/kg, i.p.) and of MMA (6 ␮mol) on the total striatal non-protein thiol content (␮g SH/g tissue) NaCl (9 ␮mol per striatum)

Saline GM1

MMA (6 ␮mol per striatum)

Right (injected)

Left (control)

Right (injected)

Left (control)

1.22 ± 0.11 1.32 ± 0.11

1.32 ± 0.10 1.28 ± 0.10

1.45 ± 0.20 1.29 ± 0.11

1.40 ± 0.11 1.26 ± 0.15

Data are mean ± S.E.M. for n = 9–14 per group. Statistical analysis (three-way ANOVA with the striata treated as within subject factor) revealed no significant effect. Table 3 GM1 ganglioside (200 ␮M) does not alter MMA-induced TBARS generation in vitro Saline Control MMA (0.66 mM) MMA (2.00 mM) MMA (6.66 mM) MMA (20.0 mM)

170.6 196.9 236.1 333.3 487.0

GM1 ± ± ± ± ±

21.6 21.7 23.8 30.8∗ 28.6∗

183.4 202.7 245.5 391.5 518.8

± ± ± ± ±

18.8 23.1 42.8 37.1∗ 17.1∗

Data are nmol of MDA per hour per gram of striatum of four independent experiments (mean ± S.E.M.). MMA, l-methylmalonic acid; GM1, monosialoganglioside. ∗ P < 0.05 compared with respective control (Student– Newman–Keuls test).

4. Discussion In the present study we show, for the first time, that intrastriatal MMA administration in vivo induces TBARS generation, measured ex vivo. In addition, we demonstrate that the systemic preadministration of GM1 ganglioside attenuates MMA-induced convulsions, prevents MMA-induced TBARS generation and increases the ascorbic acid content in the striatum. Interestingly, GM1 ganglioside had no effect on MMA-induced TBARS generation in vitro, suggesting that the presently reported neuroprotective action of GM1 ganglioside is not related to an intrinsic antioxidant activity of this glycolipid, but may be due to a secondary increase of the ascorbic acid content in the brain. Reactive species can be highly damaging to cells due to the oxidation of essential cellular constituents such as lipids, proteins and DNA, which can be measured by identification of their byproducts, such as malondialdehyde (Reiter, 1995). The brain is particularly susceptible to oxidation by reactive species because of its dependency on aerobic metabolism,

large content of polyunsaturated lipid in the mitochondrial and plasma membranes of brain cells and its low antioxidant defenses (Reiter, 1995). Mitochondrial dysfunction and consequent ATP depletion are a major cause of oxidative stress and calcium homeostasis alterations (Cassarino & Bennett, 1999) in the central nervous system, which ultimately produces loss of cellular integrity and cell death. Indeed, various neurodegenerative disorders, including Parkinson, Huntington and Alzheimer’s diseases have been associated with mitochondrial dysfunction, activation of excitotoxic mechanisms and reactive species generation (Beal, 2000; Cassarino & Bennett, 1999). Various lines of evidence have suggested that some inborn errors of metabolism, particularly those accompanied by mitochondrial dysfunction, may share a similar mechanism of acute neurotoxicity due to the accumulation of metabolic inhibitors and/or defective activity of enzymes involved in energy metabolism. This seems to be the case for MMA, the main metabolite that accumulates in methylmalonic acidemia, which is a competitive inhibitor of succinate dehydrogenase that causes convulsions through glutamatergic mechanisms and reactive species generation in vitro (Fenton et al., 2001; Fighera et al., 1999; Fontella et al., 2000; McLaughlin et al., 1998). In fact, MMA-induced convulsions are attenuated by ascorbic acid and ␣-tocopherol, further supporting the involvement of reactive species in the effects of MMA effects (Fighera et al., 1999). Moreover, it has been recently reported that MMA inhibits ouabain-sensitive Na+ , K+ -ATPase in synaptosomes, but not in synaptic membranes, suggesting that mitochondria, the main site of reactive species production, are necessary for the appearance of the deleterious effects of MMA (Wyse et al., 2000). The MMA-induced increase of striatal TBARS content ex vivo observed here is in agreement with the hypothesis that MMA increases

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reactive species production and lipoperoxidation, initially described as an in vitro phenomenon (Fontella et al., 2000). Various hypotheses have been proposed to explain the neuroprotective role of GM1. It has been reported that gangliosides affect the functional dynamics of the cell membranes in several ways: they contribute to the structural rigidity of membranes, facilitate the action of neurotrophic factors, modulate neuronal development, and differentiation (Hakomori, 1981; Hakomori et al., 1990) and inhibit neurotransmitter release (Schroeder, Schroeder, & Sabel, 1996). Moreover, a direct antioxidant action of GM1 has also been reported (Avrova, Tyurin, Tyurin, & Kagan, 1994; Maulik et al., 1993). In the present study we demonstrated that GM1 protects against the MMA-induced increase of striatal TBARS content ex vivo, but has no effect on MMA-induced lipoperoxidation in vitro. These findings suggest that the protective action of GM1 may require a certain degree of cellular intactness, since it did not occur in striatal homogenates. Moreover, it makes the previously reported intrinsic antioxidant role of GM1 (Maulik et al., 1993) an unlikely explanation for such protection. It is worth pointing out that there is evidence that the neuroprotective effects of GM1 ganglioside are mediated in part by tyrosine kinases associated with growth factor as well as protein kinase C and activation of transcription factors (Ariga, Jarvis, & Yu, 1998). Therefore one might speculate that activation of this cascade is involved in the protective effects of GM1 against MMA-induced convulsions and TBARS generation. Since it is well known that ascorbic acid and glutathione are reactive species scavengers that are present at high concentrations in the CNS (Reiter, 1995), we decided to investigate whether MMA and/or GM1 treatment altered glutathione and ascorbic acid levels in the striatum. Systemic GM1 administration did not alter the striatal content of total non-protein SH groups (Table 2). Such a lack of effect of GM1 on total non protein SH groups agrees with previous findings reported by Bondy and Guo, who have shown that chronic administration of a GM1 derivative does not alter the total glutathione concentration in the brain (Bondy & Guo, 1996). It is important to point out, however, that the biochemical method used to measure glutathione levels in the present study

does not allow assessment of the intracellular reduced glutathione. This is particularly important in view of a recent report by Zeevalk et al., who have described that exposure of mesencephalic cultures to the SDH inhibitor malonate causes an increase in the efflux of reduced glutathione, decreasing its intracellular levels (Zeevalk et al., 2000). Therefore, our results do not rule out an alteration in the intracellular reduced glutathione by MMA or GM1. Interestingly, we found that systemic GM1 administration increased total striatal ascorbic acid levels in the striatum. It has long been known that ascorbic acid is present in the brain tissue at high concentrations compared to other organs (Kaufman, 1966), and that there is a greater than 10-fold gradient between the concentration of ascorbic acid in brain and serum (Kaufman, 1966; Schreiber & Trojan, 1991). Reduced ascorbic acid (the predominant form in serum) seems to be transported into the brain by a family of Na+ -dependent l-ascorbic acid transporters (Dhariwal, Hartzell, & Levine, 1991; Tsukaguchi et al, 1999). On the other hand, the oxidized form of ascorbic acid, dehydroascorbic acid, is transported into the brain through GLUT1 glucose transporters and is trapped in the brain and in cells by reduction to ascorbic acid (Aguset, 1997; Huang, Agus, & Winfree, 2001; Rice, 2000; Vera et al., 1995). Besides playing an important role in the catecholamine biosynthesis as a cofactor of dopamine ␤-hydroxylase, ascorbic acid inhibits peroxidation of membrane phospholipids, and acts as a scavenger of reactive species in the brain (Englard & Seifter, 1986; Padh, 1990). The ascorbic acid content of the CNS varies with gender (Ferris, Kume-Kick, Russo-Menna, & Rice, 1995; Kume-Kick, Ferris, Russo-Menna, & Rice, 1996; Rodriguez, Warkentin, Risberg, & Rosadini, 1988) and can be altered by different treatments, such as ascorbic acid (Jacques-Silva et al., 2001), dehydroascorbic acid (Huang et al., 2001) and estrogen administration (Das, Das, Bagchi, & Dey, 1993; Kume-Kick & Rice, 1998; Kume-Kick et al., 1996), ovariectomy (Padh, 1990; Kume-Kick et al., 1996), ischemia (Das et al., 1993; Khandwekar, Nath, & Nath, 1974; Kume-Kick & Rice, 1998; Kume-Kick et al., 1996), and other forms of oxidative stress. The present report adds GM1 to the list of compounds that alter ascorbic acid content in the central nervous system and, although we lack experimental evidence of the neurochemical

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mechanisms underlying the presently reported effect of GM1 on ascorbic acid levels, these results are important in the sense that they unravel a possible mechanism for the antioxidant and anticonvulsant action of GM1, particularly if one considers the previously reported anticonvulsant action of ascorbic acid against the MMA-induced convulsions (Fighera et al., 1999). It is worth pointing out that other neuroprotective mechanisms than antioxidant activity have been proposed for ascorbate, such as glutamate removal from the synaptic cleft through a glutamate–ascorbate heteroexchange transporter and a direct inhibitory action on NMDA-mediated currents (Grünewald, 1993). In conclusion, the present study reports for the first time that MMA induces lipoperoxidation in vivo, and that GM1 ganglioside attenuates MMA-induced convulsions and lipoperoxidation, an effect that occurs concomitantly with an increase in the ascorbic acid content of the striatum. These results suggest that GM1 decreases convulsions and lipoperoxidation by increasing the cerebral content of ascorbic acid. Importantly, these results also suggest that GM1 ganglioside may be of value to attenuate neurological deficits of methylmalonic acidemic patients and may introduce a new class of drugs in the management of methylmalonic acidemia.

Acknowledgements This work was supported by Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnológico (CNPq) PronEx—MCT: 661061-1997-3 and FAPERGS 99/1367.3. C.F.M., R.F.-F., M.A.R. and J.B.T.R. are recipients of CNPq fellowships (numbers 351127/97-6, 522975/95 and 523761/95-3-RE, respectively). TRB Pharma, the Brazilian representative of Fidia Research Laboratories, kindly donated GM1 ganglioside.

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