Evidence for disrupted copper availability in human spinal cord supports CuII(atsm) as a treatment option for sporadic cases of ALS.

The copper compound CuII(atsm) has progressed to phase 2/3 testing for treatment of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). CuII(atsm) is neuroprotective in mutant SOD1 mouse models of ALS where its activity is ascribed in part to improving availability of essential copper. However, SOD1 mutations cause only ~ 2% of ALS cases and therapeutic relevance of copper availability in sporadic ALS is unresolved. Herein we assessed spinal cord tissue from human cases of sporadic ALS for copper-related changes. We found that when compared to control cases the natural distribution of spinal cord copper was disrupted in sporadic ALS. A standout feature was decreased copper levels in the ventral grey matter, the primary anatomical site of neuronal loss in ALS. Altered expression of genes involved in copper handling indicated disrupted copper availability, and this was evident in decreased copper-dependent ferroxidase activity despite increased abundance of the ferroxidases ceruloplasmin and hephaestin. Mice expressing mutant SOD1 recapitulate salient features of ALS and the unsatiated requirement for copper in these mice is a biochemical target for CuII(atsm). Our results from human spinal cord indicate a therapeutic mechanism of action for CuII(atsm) involving copper availability may also be pertinent to sporadic cases of ALS.

Microglial ferroptotic stress causes non-cell autonomous neuronal death.

BACKGROUND: Ferroptosis is a form of regulated cell death characterised by lipid peroxidation as the terminal endpoint and a requirement for iron. Although it protects against cancer and infection, ferroptosis is also implicated in causing neuronal death in degenerative diseases of the central nervous system (CNS). The precise role for ferroptosis in causing neuronal death is yet to be fully resolved. METHODS: To elucidate the role of ferroptosis in neuronal death we utilised co-culture and conditioned medium transfer experiments involving microglia, astrocytes and neurones. We ratified clinical significance of our cell culture findings via assessment of human CNS tissue from cases of the fatal, paralysing neurodegenerative condition of amyotrophic lateral sclerosis (ALS). We utilised the SOD1G37R mouse model of ALS and a CNS-permeant ferroptosis inhibitor to verify pharmacological significance in vivo. RESULTS: We found that sublethal ferroptotic stress selectively affecting microglia triggers an inflammatory cascade that results in non-cell autonomous neuronal death. Central to this cascade is the conversion of astrocytes to a neurotoxic state. We show that spinal cord tissue from human cases of ALS exhibits a signature of ferroptosis that encompasses atomic, molecular and biochemical features. Further, we show the molecular correlation between ferroptosis and neurotoxic astrocytes evident in human ALS-affected spinal cord is recapitulated in the SOD1G37R mouse model where treatment with a CNS-permeant ferroptosis inhibitor, CuII(atsm), ameliorated these markers and was neuroprotective. CONCLUSIONS: By showing that microglia responding to sublethal ferroptotic stress culminates in non-cell autonomous neuronal death, our results implicate microglial ferroptotic stress as a rectifiable cause of neuronal death in neurodegenerative disease. As ferroptosis is currently primarily regarded as an intrinsic cell death phenomenon, these results introduce an entirely new pathophysiological role for ferroptosis in disease.

Evidence for decreased copper associated with demyelination in the corpus callosum of cuprizone-treated mice.

Demyelination within the central nervous system (CNS) is a significant feature of debilitating neurological diseases such as multiple sclerosis and administering the copper-selective chelatorcuprizone to mice is widely used to model demyelination in vivo. Conspicuous demyelination within the corpus callosum is generally attributed to cuprizone's ability to restrict copper availability in this vulnerable brain region. However, the small number of studies that have assessed copper in brain tissue from cuprizone-treated mice have produced seemingly conflicting outcomes, leaving the role of CNS copper availability in demyelination unresolved. Herein we describe our assessment of copper concentrations in brain samples from mice treated with cuprizone for 40 d. Importantly, we applied an inductively coupled plasma mass spectrometry methodology that enabled assessment of copper partitioned into soluble and insoluble fractions within distinct brain regions, including the corpus callosum. Our results show that cuprizone-induced demyelination in the corpus callosum was associated with decreased soluble copper in this brain region. Insoluble copper in the corpus callosum was unaffected, as were pools of soluble and insoluble copper in other brain regions. Treatment with the blood-brain barrier permeant copper compound CuII(atsm) increased brain copper levels and this was most pronounced in the soluble fraction of the corpus callosum. This effect was associated with significant mitigation of cuprizone-induced demyelination. These results provide support for the involvement of decreased CNS copper availability in demyelination in the cuprizone model. Relevance to human demyelinating disease is discussed.

CuII(atsm) improves the neurological phenotype and survival of SOD1G93A mice and selectively increases enzymatically active SOD1 in the spinal cord.

Ubiquitous expression of mutant Cu/Zn-superoxide dismutase (SOD1) selectively affects motor neurons in the central nervous system (CNS), causing the adult-onset degenerative disease amyotrophic lateral sclerosis (ALS). The CNS-specific impact of ubiquitous mutant SOD1 expression is recapitulated in transgenic mouse models of the disease. Here we present outcomes for the metallo-complex CuII(atsm) tested for therapeutic efficacy in mice expressing SOD1G93A on a mixed genetic background. Oral administration of CuII(atsm) delayed the onset of neurological symptoms, improved locomotive capacity and extended overall survival. Although the ALS-like phenotype of SOD1G93A mice is instigated by expression of the mutant SOD1, we show the improved phenotype of the CuII(atsm)-treated animals involves an increase in mature mutant SOD1 protein in the disease-affected spinal cord, where concomitant increases in copper and SOD1 activity are also evident. In contrast to these effects in the spinal cord, treating with CuII(atsm) had no effect in liver on either mutant SOD1 protein levels or its activity, indicating a CNS-selective SOD1 response to the drug. These data provide support for CuII(atsm) as a treatment option for ALS as well as insight to the CNS-selective effects of mutant SOD1.

In Vivo Modeling of the Pathogenic Effect of Copper Transporter Mutations That Cause Menkes and Wilson Diseases, Motor Neuropathy, and Susceptibility to Alzheimer's Disease.

Copper is an essential biometal, and several inherited diseases are directly associated with a disruption to normal copper homeostasis. The best characterized are the copper deficiency and toxicity disorders Menkes and Wilson diseases caused by mutations in the p-type Cu-ATPase genes ATP7A and ATP7B, respectively. Missense mutations in the C-terminal portion of ATP7A have also been shown to cause distal motor neuropathy, whereas polymorphisms in ATP7B are associated with increased risk of Alzheimer's disease. We have generated a single, in vivo model for studying multiple pathogenic mutations in ATP7 proteins using Drosophila melanogaster, which has a single orthologue of ATP7A and ATP7B. Four pathogenic ATP7A mutations and two ATP7B mutations were introduced into a genomic ATP7 rescue construct containing an in-frame C-terminal GFP tag. Analysis of the wild type ATP7-GFP transgene confirmed that ATP7 is expressed at the basolateral membrane of larval midgut copper cells and that the transgene can rescue a normally early lethal ATP7 deletion allele to adulthood. Analysis of the gATP7-GFP transgenes containing pathogenic mutations showed that the function of ATP7 was affected, to varying degrees, by all six of the mutations investigated in this study. Of particular interest, the ATP7BK832R Alzheimer's disease susceptibility allele was found, for the first time, to be a loss of function allele. This in vivo system allows us to assess the severity of individual ATP7A/B mutations in an invariant genetic background and has the potential to be used to screen for therapeutic compounds able to restore function to faulty copper transport proteins.

Evidence for a role for the putative Drosophila hGRX1 orthologue in copper homeostasis.

Glutaredoxins are a family of small molecular weight proteins that have a central role in cellular redox regulation. Human GRX1 (hGRX1) has also been shown to play an integral role in copper homeostasis by regulating the redox activity of the metalated sites of copper chaperones such as ATOX1 and SOD1, and the copper efflux proteins ATP7A and ATP7B. To further elucidate the role of hGRX1 in copper homeostasis, we examined the impact of RNA interference-mediated knockdown of CG6852, a putative Drosophila orthologue of hGRX1. CG6852 shares ~41 % amino acid identity with hGRX1 and key functional domains including the metal-binding CXXC motif are conserved between the two proteins. Knockdown of CG6852 in the adult midline caused a thoracic cleft and reduced scutellum, phenotypes that were exacerbated by additional knockdown of copper uptake transporters Ctr1A and Ctr1B. Knockdown of CG6852 in the adult eye enhanced a copper-deficiency phenotype caused by Ctr1A knockdown while ubiquitous knockdown of CG6852 resulted a mild systemic copper deficiency. Therefore we conclude that CG6852 is a putative orthologue of hGRX1 and may play an important role in Drosophila copper homeostasis.

Reduced glutathione biosynthesis in Drosophila melanogaster causes neuronal defects linked to copper deficiency.

Glutathione (GSH) is a tripeptide often considered to be the master antioxidant in cells. GSH plays an integral role in cellular redox regulation and is also known to have a role in mammalian copper homeostasis. In vitro evidence suggests that GSH is involved in copper uptake, sequestration and efflux. This study was undertaken to further investigate the roles that GSH plays in neuronal copper homeostasis in vivo, using the model organism Drosophila melanogaster. RNA interference-mediated knockdown of the Glutamate-cysteine ligase catalytic subunit gene (Gclc) that encodes the rate-limiting enzyme in GSH biosynthesis was utilised to genetically deplete GSH levels. When Gclc was knocked down in all neurons, this caused lethality, which was partially rescued by copper supplementation and was exacerbated by additional knockdown of the copper uptake transporter Ctr1A, or over-expression of the copper efflux transporter ATP7. Furthermore, when Gclc was knocked down in a subset of neuropeptide-producing cells, this resulted in adult progeny with unexpanded wings, a phenotype previously associated with copper dyshomeostasis. In these cells, Gclc suppression caused a decrease in axon branching, a phenotype further enhanced by ATP7 over-expression. Therefore, we conclude that GSH may play an important role in regulating neuronal copper levels and that reduction in GSH may lead to functional copper deficiency in neurons in vivo. We provide genetic evidence that glutathione (GSH) levels influence Cu content or distribution in vivo, in Drosophila neurons. GSH could be required for binding Cu imported by Ctr1A and distributing it to chaperones, such as Mtn, CCS and Atox1. Alternatively, GSH could modify the copper-binding and transport activities of Atox1 and the ATP7 efflux protein via glutathionylation of copper-binding cysteines.