Figure 7. DCA reduces motor neuron loss and astrocyte reactivity in the spinal cord of SOD1G93A mice. Representative Toluidine blue stain (left column) and GFAP immunofluorescence (red, right column) in anterior horn spinal cord sections from non Tg (top), SOD1G93A control (middle) or DCA-treated SOD1G93A (bottom) mice. Dotted lines in right column panels indicate the limit between grey and white matter. The graphs indicate the number of neuronal somas located in Rexed lamina IX (left) and the percentage of GFAP immunoreactive area in the ventral horn (right) in the indicated groups of animals. The corresponding measurement areas are drawn in the top. Data are mean 6 SEM from at least three animals per group as indicated in Methods. *p,0.05, significantly different from non Tg control, **p,0.05, significantly different from SOD1G93A control. Scale bars: 50 mm. doi:10.1371/journal.pone.0034776.g007 DCA Effects in ALS Models preclinical data that systemic DCA administration decreases astrocytosis, motor neuron death and prolongs motor performance and survival of SOD1G93A ALS mice. Since DCA has been used in humans for decades in the treatment of lactic acidosis and inherited mitochondrial diseases [18], these results suggest that DCA may be employed in clinical trials for ALS. How DCA attenuates the damage induced by the expression of mutated SOD1 in astrocytes still needs to be clarified. Mitochon- dria represent a specific site of mutated SOD1 accumulation. In particular, it accumulates in the outer membrane and the intermembrane space, where it induces mitochondrial damage and metabolic dysfunction [3,5]. Oxidative stress to astrocyte mitochondria may underlie the transformation to a neurotoxic phenotype [5,24]. Reactive oxygen species (ROS) produced in mitochondria may affect signal transduction pathways through oxidation/reduction of cysteine residues in kinases, phosphatases, and other regulatory factors [25] leading to different, even opposite cellular responses. By stimulating pyruvate consumption, DCA improves the redox balance of mitochondria, which may result in normalization of altered mitochondria-regulated signaling [26]. Furthermore, DCA applied to mice at doses similar to the ones we have used but for a longer period of time significantly induced antioxidant enzyme activities, including superoxide dismutase, catalase and glutathione peroxidase [27], suggesting an indirect antioxidant effect of DCA. Interestingly, DCA beneficial effects on survival and motor performance were more pronounced in males than in females. Similar results have been described in previous studies where different approaches resulted in beneficial effects only in males [28]. Men have higher risk of suffering ALS than women [29], and sex differences have also been reported in SOD1G93A mutant rats [30] and mice [31–34] particularly when large numbers of animals are compared [35]. These observations suggest that gender differences may be partially a result of sex hormone action and in fact estrogen modulates disease progression in SOD1G93A mice [36,37]. Among the various neuroprotective mechanisms proposed for estrogen actions, the ability to modulate mitochondrial function [38] and oxidative stress is particularly interesting in this context. Furthermore, the fact that increased ROS levels were observed in SOD1G93A males but not in females [28] suggests that in the latter the redox balance between pro- and anti-oxidant mechanisms is already shifted toward neuroprotection, masking DCA beneficial effects. Previous works have suggested an alteration of mitochondrial redox metabolism in ALS. Indeed, recent metabolomic analysis of cerebrospinal fluid [39] or serum [40] by (1)H NMR spectroscopy in ALS patients, revealed abnormal metabolite patterns that may indicate perturbation of glucose metabolism. Among the ap- proaches that aim to restore mitochondrial function and energy production, pyruvate administration to ALS mice has been assessed [41,42]. However, results were contradictory probably due to differences in doses and animal strains. Nonetheless, pharmacological administration of DCA did improve mitochon- drial function underscoring the potential of metabolic modulation to neutralize the progression of the disease. In addition to mitochondrial dysfunction, SOD1G93A astrocytes also exhibited an increased proliferation rate. Although these two events might seem unconnected, evidence obtained in cancer cells indicates that in fact they are mutually related. Otto Warburg in the 1920s described that increased proliferation rate in cancer cells is associated to glycolytic metabolism rather than to mitochondrial oxidation of pyruvate [43]. In accordance, DCA reverses the glycolytic metabolism in several cancer cell lines along with reduced proliferation [44]. Our data showing that DCA treatment of SOD1G93A astrocytes improved mitochondrial function and PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e34776