Effect of the branched-chain α-keto acids accumulating in maple syrup urine disease on S100B release from glial cells

Abstract

Accumulation of the branched-chain α-keto acids (BCKA), α-ketoisocaproic acid (KIC), α-keto-β-methylvaleric acid (KMV) and α-ketoisovaleric acid (KIV) and their respective branched-chain α-amino acids (BCAA) occurs in tissues and biological fluids of patients affected by the neurometabolic disorder maple syrup urine disease (MSUD). The objective of this study was to verify the effect of the BCKA on S100B release from C6 glioma cells. The cells were exposed to 1, 5 or 10 mM BCKA for different periods and the S100B release was measured afterwards. The results indicated that KIC and KIV, but not KMV, significantly enhanced S100B liberation after 6 h of exposure. Furthermore, the stimulatory effect of the BCKA on S100B release was prevented by coincubation with the energetic substrate creatine and with the N-nitro-l-arginine methyl ester (l-NAME), a nitric oxide synthase inhibitor, indicating that energy deficit and nitric oxide (NO) were probably involved in this effect. Furthermore, the increase of S100B release was prevented by preincubation with the protein kinase inhibitors KN-93 and H-89, indicating that KIC and KIV altered Ca²⁺/calmodulin (PKCaMII)- and cAMP (PKA)-dependent protein kinases activities, respectively. In contrast, other antioxidants such as glutathione (GSH) and trolox (soluble vitamin E) were not able to prevent KIC- and KIV-induced increase of S100B liberation, suggesting that the alteration of S100B release caused by the BCKA is not mediated by oxidation of sulfydryl or other essential groups of the enzyme as well as by lipid peroxyl radicals. Considering the importance of S100B for brain regulation, it is conceivable that enhanced liberation of this protein by increased levels of BCKA may contribute to the neurodegeneration characteristic of MSUD patients.

Introduction

Mammalian mitochondrial branched-chain α-keto acid dehydrogenase complex (BCKD) catalyzes the oxidative decarboxylation of the branched-chain α-keto acids (BCKA), α-ketoisocaproic acid (KIC), α-keto-β-methylvaleric acid (KMV), and α-ketoisovaleric acid (KIV), which originate their corresponding branched-chain amino acids (BCAA) leucine, isoleucine and valine, respectively. In patients with maple syrup urine disease (MSUD), or branched-chain keto aciduria, the activity of the BCKD is severely deficient. The metabolic blockage at this step results in the accumulation of BCKA and BCAA in tissues of the affected individuals [1], [2].

The symptomatology of MSUD includes ketoacidosis, failure to thrive, poor feeding, apnea, ataxia, seizures, coma, psychomotor delay and mental retardation [3], [1]. Neuropathologic findings characteristic of the disease are cerebral edema, atrophy of the cerebral hemispheres, spongy degeneration of the white matter and delayed myelination [4], [1]. MSUD usually presents as a heterogeneous clinical phenotype, ranging from the severe classical form to mild variants, possibly due to distinct residual enzyme activity [5]. Although the mechanisms of brain damage in MSUD are still unclear, it appears that leucine and KIC are considered to be the main neurotoxic metabolites since increased plasma concentrations of these metabolites are associated with the appearance of neurological symptoms [6], [1]. In this context, it has been demonstrated that the BCKA may affect energy metabolism in rat brain [7], [8], [9], [10], [11], [12], [13], [14], [15]. Furthermore, it has been postulated that competition of KIV, KIC, and their hydroxyderivatives with l-glutamate for decarboxylation and the consequent reduction of γ-aminobutyric acid (GABA) concentration [16], impairment of myelin development [17], [18], [19], [4], low plasma and brain levels of essential amino acids [20], [21], oxidative stress [22], [23], [24], [25] and reduced brain uptake of essential amino acids leading to decreased neurotransmitter synthesis also contribute to brain injury [26].

Glial cells play a vital role in the homeostatic regulation of the central nervous system (CNS). Astrocytes, the most abundant glial cell type in the brain, are involved in neurotransmitter uptake, neuronal metabolic support, pH regulation, and protection against toxic episodes [27], [28]. In addition, glial cells preserve neuronal survival through inactivation of reactive oxygen species (ROS) [29]. Because of its similarities with primary glial cells in culture, the C6 rat glioma cell line has provided a useful model to study glial cell properties, glial growth factors and sensitivity of glial cells to various substances and conditions [30], [31].

S100B is a calcium-binding protein of 21 kDa expressed primarily in astrocytes and secreted by these cells [32]. Many studies have suggested an intracellular role for this protein particularly in the regulation of cytoskeleton and cell cycle such as modulation of CapZ (an actin-binding protein) [33], microtubules [34], [35], [36], and glial fibrillary acidic protein (GFAP) [37], [35]. Moreover, extracellular S100B stimulates glial proliferation, survival of neurons, and extension of neurites in cell culture [32]. On the other hand, abnormal expression of S100B has supported its role in several neurodegenerative disorders, including Alzheimer's disease [38].

Although several studies demonstrate a relationship between synaptic plasticity and S100B, there are few reports on S100B and glutamatergic transmission. It has been shown that toxic levels of glutamate reduce S100B secretion in hippocampal astrocytes and brain slices [39], [40]. More recently, we have shown that S100B secretion is increased when glutamate uptake is reduced [41]. Interestingly, a decrease of brain glutamate has been observed in MSUD patients during a period of metabolic decompensation [42]. In addition, we found that the BCKA impairs glutamate uptake [43], favoring brain damage in excitotoxic conditions.

Therefore, considering that excitotoxicity, energy depletion and oxidative stress are involved in the neuropathological features observed in patients affected by MSUD in this study we investigated the effect of BCKA on S100B release from C6 glioma cells. We also tested the influence of the energetic substrate creatine, various antioxidants [glutathione (GSH), α-tocopherol (trolox) and N^ω-nitro-l-arginine methyl ester (l-NAME)] and kinase inhibitors (H-89 and KN-93) on the effects elicited by the BCKA.