also decreased their proliferation rate suggest common activation of transduction pathways between cancer cells and SOD1G93A astrocytes. Moreover, we have found evidence of unregulated proliferation and lack of replicative senescence in a population of phenotypically aberrant astrocytes isolated from SOD1G93A rats [12]. Thus, the possibility exists that chronic mitochondrial dysfunction in neonatal astrocytes promotes long term changes in astrocyte phenotype and their neurotoxic potential. Although not analyzed in our study, several cellular pathways might mediate the increased proliferation rate of SOD1G93A astrocytes. Among them we can include up-regulation of the isoform A of the lactate dehydrogenase (LDH-A) [45], activation of E3 ubiquitin ligase APC/C-Cdh1 [46], or modulation of transduction pathways by depolarized mitochondria [47]. Systemic DCA likely improves mitochondrial status in several other cell types relevant to ALS, including neurons and skeletal muscle. In the central nervous system, astrocytes seem to preferentially respond to DCA due to having a greater fraction of total PDH in the inactive phosphorylated form than neurons [15]. DCA might also exert a direct effect on skeletal muscle. It is interesting to note that DCA-treated animals remained with increased grip strength until death compared to untreated animals. Noticeably, the EDL muscle fibers of DCA-treated mice displayed neuromuscular junctions with normal size and shape, suggesting DCA could delay the progressive neuromuscular junction destruc- tion characteristic of animals with ALS [48]. Furthermore, DCA has been effective in recovering the function of ischemic muscle [49], suggesting an effect stimulating muscle trophism or decreasing deleterious inflammation. The present study supports the potential use of DCA in multi- drug approaches to treat ALS. DCA has been also shown to be protective in models of Huntington’s disease, which also involves non-cell-autonomous mechanisms [19], suggesting a more general effect in neurodegenerative diseases. Taken together these data raise the possibility that DCA might have therapeutic benefits in ALS patients. Materials and Methods Materials Culture media and serum were obtained from Invitrogen (Carlsbad, CA). All other reagents were from Sigma Chemical Co (Saint Louis, MO) unless otherwise specified. Ethics Statement Procedures using laboratory animals were in accordance with international guidelines and were approved by the Institutional Animal Committee (Comisio´n honoraria de experimentacio´ n animal de la Universidad de la Repu´ blica http://www.chea.csic. edu.uy; CHEA). Animals Transgenic ALS mice carrying the G93A mutation for human SOD1, strain B6SJL-TgN(SOD1-G93A)1Gur [50], were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and genotyped as previously described [51]. Mice were housed under controlled conditions with free access to food and water. Sprague-Dawley NTac:SD-TgN(SOD1G93A)L26H rats were obtained from Ta- conic (Hudson, NY; [52]) and were bred locally by crossing with wild-type Sprague-Dawley female rats. Dichloroacetate treatment trial Male and female transgenic mice and non-transgenic littermates were divided randomly into the following groups (n = 9 per group): DCA Effects in ALS Models A) transgenic control and B) non-transgenic control groups which received regular drinking water; C) transgenic DCA treatment and D) non-transgenic DCA treatment groups, administered with dichloroacetate (DCA; Sigma, St. Louis, MO). DCA was added to tap water in a 500 mg/L concentration and placed into water bottles. A daily dose of 100 mg/kg was used based on a daily water intake of 5 ml. The DCA solution was made fresh twice a week, with the total consumed volume measured in order to ensure a constant dose. The treatment was performed from presymptomatic stage (70 days old) to death. Animals were observed weekly for onset of disease symptoms, as well as progression to death. Onset of disease was scored as the first observation of abnormal gait or overt hind limb weakness. End-stage of the disease was scored as complete paralysis of both hind limbs and the inability of the animals to right themselves after being placed on their side. Cell cultures Astrocyte cultures: Primary rat spinal cord astrocyte cultures were prepared from transgenic SOD1G93A and non-transgenic 1- day-old pups, genotyped by PCR, as previously described [4,53]. Briefly, cells were plated at a density of 26104 cells/cm2 in 35 mm Petri dishes or 24-well plates (Nunc, Naperville, IL, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), HEPES (3.6 g/ L), penicillin (100 IU/mL) and streptomycin (100 mg/mL). Astrocyte monolayers were .98% pure as determined by GFAP immunoreactivity. Astrocyte-motor neuron cocultures: motor neuron preparations were obtained from embryonic day 15 (E15) rat spinal cord by a combination of optiprep (1:10 in L15 medium, SIGMA St. Louis, MO) gradient centrifugation and immunopanning with the monoclonal antibody IgG192 against p75 neurotrophin receptor as previously described [53], then plated on rat astrocyte monolayers at a density of 300 cells/cm2 and maintained for 48 h in L15 supplemented medium as described [53]. Treatment of cultures and motor neuron counting Confluent astrocyte monolayers were changed to L15 supple- mented media prior treatment. Stock solution of DCA (SIGMA St. Louis, MO) was prepared in distilled water and directly applied to astrocyte monolayers at the indicated concentrations. For co- culture experiments, astrocyte monolayers were treated with DCA for 24 h and motor neurons were plated in fresh L15 supplemented media, after washing twice with Dulbecco’s phosphate buffered saline (DPBS). Motor neuron survival was assessed after 48 h by directly counting all p75 immunoreactive cells displaying neurites longer than 4 cell bodies in diameter [53]. Proliferation assessment Confluent astrocyte cultures were incubated with 10 mg/ml bromo deoxyuridine (BrdU) and 5 mM DCA or vehicle in DMEM 2% FBS for 24 h. Cells were fixed in 4% paraformal- dehyde, incubated in 1 N HCl for 1 h and immunofluorescence to detect BrdU using 1:300 diluted mouse monoclonal antibody (Dako) was performed. Alexa Fluor488 conjugated goat anti-mouse (Invitrogen) was used as secondary antibody, and propidium iodide to counterstain total nuclei. At least 600 nuclei were counted for each point. Proliferation is expressed as the percentage of BrdU immunoreactive nuclei with respect to total propidium iodide stained nuclei. PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e34776