Introduction
Approximately 20% of familial human Amyotrophic Lateral Sclerosis (fALS) cases are attributed to mutations in the gene encoding Cu, Zn Superoxide Dismutase (SOD) (Rosen et al., 1993). Mice highly over-expressing mutant human SOD recapitulate the human disease closely, presenting with upper and lower motor neuron loss, progressive paralysis, and death in ~150 days (Gurney et al., 1994).
[we still don’t know how SOD causes disease, but here are some hypotheses. immature SOD is significantly destabilized and aggregation prone. some evidence suggests immature SOD in a soluble form may have toxic effects, too]
SOD normally undergoes a series of posttranslational modifications that activate and stabilize the protein: forming an intramolecular disulfide bond, binding one copper atom and one zinc atom per monomer, and dimerizing. However, a large fraction of SOD accumulates as zinc-containing, copper-deficient protein in the spinal cords of
mice overexpressing mutant SODG37R or SODG93A[JM1] (Roberts et al., 2014; Williams et al., 2016). The human Copper Chaperone for SOD (CCS) catalyzes SOD copper binding and formation of the SOD intramolecular disulfide via a process requiring oxygen (Banci et al., 2012; Brown et al., 2004; Furukawa et al., 2004; Lamb et al., 2000). Co-expression of human CCS with human SOD in mice instigates a systemic copper deficiency early in development that is linked to rapid development of cellular and behavioral deficits. Mice
co-expressing CCS[JM2] with “low” levels of SODG93A exhibit diminished copper-dependent cytochrome
c oxidase (COX) activity compared to nontransgenic mice, rapidly develop [movement impairment] within 2 weeks and die within 20-50 days (Son et al., 2007; Williams et al., 2016). Mice expressing “high” levels of SODG93A with CCS die even sooner, within 3 weeks (Williams et al., 2016).
Treatment with the copper delivery agent CuATSM (diacetyl-bis(N4-methylthiosemicarbazone) rescues this early developmental crisis, restores COX activity, restores additional copper to SOD, and massively extends survival of high-expressing SODG93AxCCS mice from ~15 days to over 600 days, which is significantly longer than the SODG93A lifespan of ~150 days (Williams et al., 2016). CuATSM treatment also extends survival of SODG93A or SODG37R mice not co-expressing CCS (Hilton et al., 2017; McAllum et al., 2013; Roberts et al., 2014; Williams et al., 2016). Chemical reduction of the ligated CuATSM copper (II) atom is proposed to trigger copper release in vivo, resulting in Cu delivery to the CNS (Donnelly et al., 2012).
We hypothesized that chemical derivatives of CuATSM that are measurably easier to reduce will deliver more copper to the CNS and more robustly improve the SOD and COX measures of copper deficiency seen in SODxCCS mice. Here we test this reductive release model by administering CuATSM derivatives to mice co-expressing SODWT with CCS, a model which we show experiences lethal non-ALS developmental copper deficiency without intervention. [this is important because…]
Summary of Results
Here we report that human SODWT, when co-expressed with human CCS in mice, causes lethal copper deficiency that is rescue-able by treatment with CuATSM, but not by more-easily-reducible CuATSM chemical derivatives. [This suggests that there is more to the mechanism of CuATSM than getting lots of copper to the CNS.]
SODWTxCCS mice experience an early development crisis similar to G93AxCCS. They exhibit motor deficits, diminished CNS Cu-dependent COX activity, accumulated CNS Cu-deficient SOD, and death within 3 weeks. We designed a number of CuATSM derivatives based on the reductive release hypothesis. All of the derivatives delivered copper to the CNS, as measured by increased COX and Cu-replete SOD, but not all treatments rescued the early development crisis.
[JM1]what about in human patients?
[JM2]should talk about how CNS is only over-expressed in CNS, how Cu-deficiency is probably only in CNS