In humans, approximately 20% of heritable, familial amyotrophic lateral sclerosis (ALS) cases have been linked to mutations in the copper, zinc superoxide dismutase 1 gene (SOD1; Cu, Zn SOD), and mice that over-express the mutant SODG93A protein are one of the most widely used animal model for ALS research. In this model, overexpression of the mutant SOD results in a pathological gain-of-function that leads to the deterioration and death of motor neurons in the spinal cord. For SOD protein to function copper must be delivered to it by the Copper-Chaperone-for-SOD (CCS) protein. Interestingly, the co-expression of human CCS with the human SODG93A protein in mice results in rapid ALS-like neurodegeneration. This accelerated disease onset results from a copper deficiency that affects mitochondria during development, and is consistent with copper deficiency seen in the spinal cord in ALS patients. In a January 2016 a collaborative team led by Dr. Joseph Beckman of the College of Science at Oregon State University capitalized on the accelerated disease progression in SODG93A mice over-expressing human CSS to evaluate a novel and effective copper-based ALS therapy. (Williams et al. 2016).
The Oregon State team created SODG93A x CSS double transgenic mice by breeding mice expressing high SODG93A protein levels (B6SJL-Tg(SOD1*G93A)1Gur/J (002726) to mice overexpressing human CCS (B6;SJl-Tg(Prnp-CCS)17Jlel/J(018917)), and treated both strains with CuATSM, a PET-imaging agent that releases copper in tissues with damaged mitochondria. CuATSM has low toxicity, penetrates the blood-brain barrier of ALS patients within minutes, and was previously shown to moderately prolong survival by 15% in SODG93A low-expressing mice (B6SJL-Tg(SOD1*G93A)1Gur/J (002300)). CuATSM dissolved in DMSO can be delivered safely and effectively transdermally by applying it directly to an animal’s skin.
Three treatment groups were compared to evaluate the effectiveness of CuATSM application on survival rate: continuous treatment from embryonic development through adulthood, treatment through postnatal day 21, and treatment following symptom development. High-expressing SODG93A x CSS pups whose mothers’ received CuATSM treatment during pregnancy and that received continuous treatment post-delivery remained active and healthy for over a year; neurodegenerative signs typically did not appear until the mice were~600 days old. When CuATSM treatment was stopped at 21 days, the mice started to show motor impairment by 70-90 days of age, with mortality occurring by ~125 days. Interestingly, if treatment was stopped at 21 days, but then re-started when motor impairment emerged, the mice gradually recovered and remained healthy until progressive ALS symptoms re-emerged at ~340 days. Low-expressing SODG93A x CSS double transgenic mice that received treatment beginning at 24 days – roughly the time when motor deficits are observed – all recovered, gained weight, and remain symptom-free at 480 days. (Without treatment, the high SODG93A x CSS and low SODG93A x CSS double transgenic mice typically died at between 8-13 and 22-50 days, respectively.)
Understanding the processes behind the accelerated ALS disease progression in SODG93A x CSS mice was a main focus of Dr. Beckman's team and directed their therapeutic development. In SODG93A mice, misfolded human SOD is over-expressed and accumulates in a zinc-bound, but copper-deficient state in the spinal cord. The zinc stabilizes the unfolded SOD protein as it waits for CCS to recruit copper and to complete its functional maturation. Copper is primarily bound by cytochrome c oxidase and Cu, Zn SOD. Overexpression of human CCS in the SODG93A x CSS mouse model recruits the available intracellular copper preferentially to SOD, exacerbating the copper deficiency in these mice. Copper-dependent cytochrome c oxidase activity was detected at similar levels in wild-type, SODG93A, and SODG93A x CSS mice that received continuous CuATSM treatment. When CuATSM treatment was stopped in SODG93A x CSS mice, however, cytochrome c oxidase levels in the spinal cord were reduced by 75%, supporting the role of human CCS in the depleting copper from mitochondrial cytochrome c oxidase and highlighting copper’s importance in perinatal development.