phenotype (referred to as ‘‘AbA cells’’) from primary spinal cord cultures of symptomatic SOD1G93A rats with unprecedented proliferative and neurotoxic capacity [12] further supports a role for astrocytes in ALS progression. It remains to be determined whether the neurotoxic phenotype of SOD1G93A-expressing astrocytes may be reverted by the improvement of mitochondrial metabolism and in turn slow disease progression. The organohalide dichloroacetate (DCA) is a well-characterized inhibitor of the protein kinase of the pyruvate dehydrogenase (PDH) [13]. PDH, located in the mitochondrial matrix, in its active unphosphorylated state mediates acetyl coenzyme-A formation from pyruvate, which feeds the electron transport chain responsible for ATP synthesis and oxygen consumption. Phos- phorylation of PDH by PDH kinase (PDK) generates its inactive phosphorylated state. DCA-mediated inhibition of PDK renders most of PDH in the active form and then pyruvate metabolism switches towards glucose oxidation to CO2 in the mitochondria (Fig. 1). Another mechanism by which DCA may favor PDH activity is to decrease degradation of the E1 alpha subunit of the complex. It has been claimed that changes in E1 alpha subunit phosphorylation could affect susceptibility to proteases that may lead to an increase in the amount of the total enzyme [14]. In the central nervous system, DCA enhances glucose and lactate oxidation to CO2 and reduces lactate release mainly in astrocytes compared to having almost no effects on neurons, which supports the compartmentalization of glucose metabolism between astroglia and neurons [15]. The fraction of total PDH in the inactive phosphorylated form is normally greater in astroglia than in neurons, a situation that favors lactate export from astroglia to neurons but it can be modulated by DCA [15]. DCA administration in vivo activates brain PDH activity [16], indicating that it crosses the blood-brain barrier, and DCA is currently used clinically to lower elevated lactate levels in cerebrospinal fluid of patients with mitochondrial disorders [17,18]. However, it is unknown whether DCA may offer benefits in neurological disorders associated to mitochondrial dysfunction. Specifically, it is not known whether DCA may prevent the in vivo and in vitro neurotoxic influence of SOD1G93A astrocytes in ALS models by regulating their mitochondrial respiration. Here we provide evidence that DCA reduced astrocyte neu- rotoxicity to motor neurons in culture and furthermore, applied to Figure 1. Site of action of dichloroacetate. DCA inhibits the mitochondrial enzyme PDH kinase, thereby maintaining the PDH complex in its unphosphorylated catalytically active state and facilitating the aerobic oxidation of glucose. doi:10.1371/journal.pone.0034776.g001 DCA Effects in ALS Models ALS mice, slowed disease progression and enhanced motor strength. Results Effect of DCA on SOD1G93A astrocytes To evaluate the effects of DCA on PDH we exposed cultures of SOD1G93A astrocytes to DCA (5 mM, 24 h) and total and phosphorylated forms of PDH were quantified by western blotting assay using specific antibodies (anti PDH-E1a subunit and anti PDH-E1a-pSer293 respectively). As expected, exposure of both non-transgenic (non Tg) and SOD1G93A astrocytes to DCA reduced phosphorylated PDH relative levels (Fig. 2A–B). Howev- er, this effect was greater in SOD1G93A astrocytes than in non Tg ones. DCA also increased total levels of PDH in non Tg astrocytes according to previous reports [14]. Total PDH levels were basally increased in SOD1G93A untreated astrocytes. DCA treatment improved mitochondrial coupling in SOD1G93A astrocytes as determined by high-resolution respirom- etry expressed by the respiratory control ratio (RCR). The RCR value calculated for untreated SOD1G93A astrocytes was signifi- cantly reduced (by 45%) compared to non Tg astrocytes as described previously [5]. DCA-treated SOD1G93A astrocytes showed a significant increase in the RCR to the level of that shown by non Tg astrocytes (Fig. 2C). We observed that DCA also reduced the proliferation rate in SOD1G93A astrocytes that was otherwise increased by 100% with respect to non Tg ones (Fig. 2D). DCA prevented the toxicity of SOD1G93A astrocytes to motor neurons SOD1G93A astrocytes display neurotoxic influence for motor neurons in culture, which can be reverted by mitochondria- targeted antioxidants [5]. In order to elucidate whether improving mitochondrial function with DCA in SOD1G93A astrocytes was also beneficial for motor neuron survival we plated purified motor neurons on top of DCA-pretreated (0.5–5 mM, 24 h) SOD1G93A astrocyte monolayers. This treatment significantly increased motor neuron survival grown on top of SOD1G93A astrocytes to the level of that shown by non Tg astrocytes (Fig. 3). DCA increased survival of SOD1G93A mice To assess whether DCA could also exert protective effects on the progressive paralysis in SOD1G93A mice, the compound was administered from 70 days of age until death in the drinking water (500 mg/L) as previously described in an animal model of Huntington’s disease [19]. DCA was well tolerated and did not show apparent signs of intoxication, such as weight loss, disease or premature death, when compared to non-treated SOD1G93A and non Tg control mice. Treatment with DCA significantly increased survival both in males and females as compared with control mice treated with water only (males ctrl n = 9: 126.962.6 days, DCA n = 9: 138.062.8 days; females ctrl n = 10: 130.061.87 days DCA n = 9: 138.462.42 days; Fig. 4A, B). Disease onset was not significantly affected by DCA (males ctrl n = 7: 99.463.0 days; DCA n = 6: 106.262.3 days; females ctrl n = 5: 104.2 days65.1; DCA n = 8: 111.366.7 days). DCA improved motor performance and maintained motor unit integrity in SOD1G93A mice ALS is characterized by progressive muscle weakness and paralysis. Therefore, we sought to determine the effect of DCA on motor performance of SOD1G93A mice. Notably, DCA signifi- PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e34776