Defects in the mammalian Menkes and Wilson copper transporting P-type ATPases cause severe copper homeostasis disease phenotypes in humans. Here, we find that DmATP7, the sole Drosophila orthologue of the Menkes and Wilson genes, is vital for uptake of copper in vivo. Analysis of a DmATP7 loss-of-function allele shows that DmATP7 is essential in embryogenesis, early larval development, and adult pigmentation and is probably required for copper uptake from the diet. These phenotypes are analogous to those caused by mutation in the mouse and human Menkes genes, suggesting that like Menkes, DmATP7 plays at least two roles at the cellular level: delivering copper to cuproenzymes required for pigmentation and neuronal function and removing excess cellular copper via facilitated efflux. DmATP7 displays a dynamic and unexpected expression pattern in the developing embryo, implying novel functions for this copper pump and the lethality observed in DmATP7 mutant flies is the earliest seen for any copper homeostasis gene.

Copper homoeostasis was investigated in the Drosophila melanogaster S2 cell line to develop an insect model for the study of copper regulation. Real-time PCR studies have demonstrated expression in S2 cells of putative orthologues of human Cu regulatory genes involved in the uptake, transport, sequestration and efflux of Cu. Drosophila orthologues of the mammalian Cu chaperones, ATOX1 (a human orthologue of yeast ATX1), CCS (copper chaperone for superoxide dismutase), COX17 (a human orthologue of yeast COX17), and SCO1 and SCO2, did not significantly respond transcriptionally to increased Cu levels, whereas MtnA, MtnB and MtnD (Drosophila orthologues of human metallothioneins) were up-regulated by Cu in a time- and dose-dependent manner. To examine the effect on Cu homoeostasis, expression of several key copper homoeostasis genes was suppressed using double-stranded RNA interference. Suppression of the MTF-1 (metal-regulatory transcription factor 1), reduced both basal and Cu-induced gene expressions of MtnA, MtnB and MtnD, significantly reducing the tolerance of these cells to increased Cu. Suppression of either Ctr1A (a Drosophila orthologue of yeast CTR1) or Ctr1B significantly reduced Cu uptake from media, demonstrating that both these proteins function to transport Cu into S2 cells. Significantly, Cu induced Ctr1B gene expression, and this could be prevented by suppressing MTF-1, suggesting that Ctr1B might be involved in Cu detoxification. Suppression of DmATP7, the putative homologue of human Cu transporter genes ATP7A and ATP7B, significantly increased Cu accumulation, demonstrating that DmATP7 is essential for efflux of excess Cu. This work is consistent with previous studies in mammalian cells, validating S2 cells as a model system for studying Cu transport and identifying novel Cu regulatory mechanisms.

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Background and Purpose: Diacetyl-bis(4-methyl-3-thiosemicarbazonato)copper II (Cu II (atsm)) ameliorates neurodegeneration and delays disease progression in mouse models of amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD), yet the mechanism of action remains uncertain. Promising results were recently reported for separate Phase 1 studies in ALS patients and PD patients. Affected tissue in these disorders shares features of elevated Fe, low glutathione and increased lipid peroxidation consistent with ferroptosis, a novel form of regulated cell death. We therefore evaluated the ability of Cu II (atsm) to inhibit ferroptosis.Experimental Approach: Ferroptosis was induced in neuronal cell models by inhibition of glutathione peroxidase-4 activity with RSL3 or by blocking cystine uptake with erastin. Cell viability and lipid peroxidation were assessed and the efficacy of Cu II (atsm) was compared to the known antiferroptotic compound liproxstatin-1.Key Results: Cu II (atsm) protected against lipid peroxidation and ferroptotic lethality in primary and immortalised neuronal cell models (EC 50 : ≈130 nM, within an order of magnitude of liproxstatin-1). Ni II (atsm) also prevented ferroptosis with similar potency, whereas ionic Cu II did not. In cell-free systems, Cu II (atsm) and Ni II (atsm) inhibited Fe II -induced lipid peroxidation, consistent with these compounds quenching lipid radicals.Conclusions and Implications: The antiferroptotic activity of Cu II (atsm) could therefore be the disease-modifying mechanism being tested in ALS and PD trials. With potency in vitro approaching that of liproxstatin-1, Cu II (atsm) possesses favourable properties such as oral bioavailability and entry into the brain that make it an attractive investigational product for clinical trials of ferroptosis-related diseases.

For more than 150 years, it is known that occupational overexposure of manganese (Mn) causes movement disorders resembling Parkinson's disease (PD) and PD-like syndromes. However, the mechanisms of Mn toxicity are still poorly understood. Here, we demonstrate that Mn dose- and time-dependently blocks the protein translation of amyloid precursor protein (APP) and heavy-chain Ferritin (H-Ferritin), both iron homeostatic proteins with neuroprotective features. APP and H-Ferritin are post-transcriptionally regulated by iron responsive proteins, which bind to homologous iron responsive elements (IREs) located in the 5'-untranslated regions (5'-UTRs) within their mRNA transcripts. Using reporter assays, we demonstrate that Mn exposure repressed the 5'-UTR-activity of APP and H-Ferritin, presumably via increased iron responsive proteins-iron responsive elements binding, ultimately blocking their protein translation. Using two specific Fe -specific probes (RhoNox-1 and IP-1) and ion chromatography inductively coupled plasma mass spectrometry (IC-ICP-MS), we show that loss of the protective axis of APP and H-Ferritin resulted in unchecked accumulation of redox-active ferrous iron (Fe ) fueling neurotoxic oxidative stress. Enforced APP expression partially attenuated Mn-induced generation of cellular and lipid reactive oxygen species and neurotoxicity. Lastly, we could validate the Mn-mediated suppression of APP and H-Ferritin in two rodent in vivo models (C57BL6/N mice and RjHan:SD rats) mimicking acute and chronic Mn exposure. Together, these results suggest that Mn-induced neurotoxicity is partly attributable to the translational inhibition of APP and H-Ferritin resulting in impaired iron metabolism and exacerbated neurotoxic oxidative stress. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/.

SUMMARY) was sensitive to excess Cu and female Mvl 97f flies were also sensitive to Cu limitation. An MtnA-EYFP reporter was used as a proxy measure of Cu distribution within Mvl 97f/+ larvae. Under basal conditions Cu levels were reduced in the anterior midgut and proventriculus relative to control larvae. These results demonstrate Mvl is a functional Cu transporter and that despite partial functional redundancy with the Ctr1 proteins, Cu uptake through this pathway is necessary for optimal viability at the cellular and organismal levels.

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