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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that is characterized by progressive degeneration of motor neurons in the spinal cord, motor cortex, and brainstem. There is currently no effective treatment for ALS. Transgenic mice expressing familiar ALS-linked mutant superoxide dismutase (SOD1) have been commonly used for preclinical trials; however, successful trails in mice mostly fail in subsequent human trials. Most preclinical trials start treatment before the onset of symptoms, while ALS patients in clinical trials have surpassed the onset of disease. In addition, ALS is not only a multi-factorial disease but also a multi-systemic disease that affects several cell types [1]. Therefore, therapies should be aimed at the interception of multiple mechanisms. In the present study, we investigated (1) whether the therapeutic agents, which were previously tested starting before disease onset, still showed protective effects when the treatments were initiated after disease onset and (2) whether combination therapies offered better effects after disease onset. Minocycline, which inhibits microglia activation and apoptotic cascade, was previously found to be effective at ameliorating motor impairment and increasing the lifespan of SOD1G93A mice when administered at 5 weeks of age [2]. However, minocycline fails in human trials [3]. We assessed whether similar beneficial effects could be seen when treatment began at 90 days of age. This time point was chosen because SOD1G93A mice [4] exhibited 10~20% decline in motor function at this age, which may be equivalent to the stage that ALS patients first visit a doctor. The results showed no delay in impaired motor function (Fig. 1A&B, Mino) and no extension in survival (Fig. 1C&D, Mino). This result is consistent with a recent report [5]. Ceftriaxone, which reduces excitotoxicity via activation of glial glutamate transporter EAAT2, was found to slow disease progression in SOD1G93A mice when administered at 12 weeks of age [6]. Ceftriaxone is currently in clinical trials for ALS. We found that when ceftriaxone was administered starting from 90 days of age, treatment no longer slowed disease progression (Fig. 1A&B, Cfx) and survival (Fig. 1C&D, Cfx). We then assessed whether the combined treatments of ceftriaxone and minocycline, starting at 90 days of age, would have protective effects. The results showed no delay in impaired motor function (Fig. 1A&B, Double) but a significant increase in survival by ~12 days (Fig. 1C&D, Double). Figure 1 Effects of single and combination treatments on disease onset and survival of SOD1G93A mice. SOD1G93A mice (B6SJL-Tg(SOD1-G93A)1Gur/J with high copy number of the mutant human SOD1 gene, Jackson Laboratory) were used in this study (the cited previous ... Vitamin E, which reduces oxidative damage, can delay disease onset when administered at 30 days of age, but has no effect in extending the lifespan of SOD1G93A mice [7], only slightly slowing disease progression in human ALS trials [8,9]. We found that when mice were treated with vitamin E starting at 30 days of age and then with ceftriaxone and minocycline starting at 90 days of age, a significant delay in motor dysfunction by ~18 days (Fig. 1A&B, Triple D30) and an extension in survival by ~16 days (Fig. 1C&D, Triple D30) were observed. Furthermore, we assessed the combined three treatments starting at 90 days of age. This triple treatment resulted in greater improvement of motor performance than that seen in the double treatment (ceftriaxone and minocycline) (Fig. 1A&B, Triple D90). The prolonged survival for the triple treatment was similar to that seen in the double treatment (Fig. 1C&D, Triple D90). To confirm that the protective effects of combined treatments were due to the inhibition of targeted mechanisms, we performed immunofluorescent staining on lumbar spinal cord sections prepared from treated SOD1G93A mice and wild-type littermates at 115 days of age. As shown in Fig. 2A, EAAT2 protein levels were restored in treated mice (in the double and triple groups). Oxidative damage as measured RNA oxidation (15A3 staining) was also reduced [7]. Astrogliosis (GFAP staining) and microglial activation (CD11b staining) in SOD1G93A mice were increased compared to that seen in wild-type littermates. There was even greater enhanced glial activation by the cocktail treatments - the double and triple groups, which is consistent with a recent report [5]. Furthermore, to confirm that the delayed disease onset and prolonged survival were associated with reduced neuronal loss, we performed cresyl violet staining on lumbar spinal cord adjacent sections. A significant reduction of motor neuron loss was observed in the triple groups (Fig. 2B). Figure 2 Effects of combination treatments on oxidative damage, EAAT2 expression, glial activation, and motor neuron loss in SOD1G93A mice. Immunofluorescent (A) and Cresyl violet (B) stainings were performed on lumbar spinal cord sections prepared from treated ... Transition from preclinical mouse studies to human clinical trials is difficult for most diseases. As the first step, proper design of preclinical trials is very important. The results of this study suggest that inappropriate pre-symptomatic treatment in mice may partially provide explanations for the failure of some clinical trials in ALS patients. In addition, combination treatments may serve as a therapeutic strategy for human trials. The agents that have been unsuccessful in human trials may prove to be more beneficial when used in combination with one another.