Menkes disease is a multi-systemic copper metabolism disorder caused by mutations in the X-linked ATP7A gene and characterised by progressive neurodegeneration and severe connective tissue defects. The ATP7A protein is a Copper (Cu)-transporting ATPase expressed in all tissues and plays a critical role in the maintenance of copper homeostasis in cells of the whole body. ATP7A participates in copper absorption in the small intestine and in copper transport to the CNS across the blood-brain-barrier and blood–cerebrospinal fluid- barrier. Cu is essential for synaptogenesis and axonal development. In cells, ATP7A participates in the incorporation of copper into Cu-dependent enzymes during the course of its maturation in the secretory pathway. There is a high degree of homology (>80% ) between the human ATP7A and murine Atp7a genes. Mice with mutations in the Atp7a gene, called mottled mutants, are well-established and excellent models of Menkes disease. Mottled mutants closely recapitulate the Menkes phenotype and are invaluable for studying Cu-metabolism. They provide useful models for exploring and testing new forms of therapy in Menkes disease. Recently, non-mammalian models of Menkes disease, Drosophila melanogaster and Danio rerio mutants were used in experiments which would be technically difficult to carry out in mammals.

Heme oxygenase-1 (HO-1, HMOX1) degrades heme protecting cells from heme-induced oxidative damage. Beyond its well-established cellular functions, heme has emerged as a stabilizer of G-quadruplexes. These secondary DNA structures interfere with DNA replication. We recently revealed that nuclear HO-1 colocalizes with DNA G-quadruplexes and promotes their removal. Here, we investigate whether HO-1 safeguards cells against replication stress.

G-quadruplexes (G4) are stacked nucleic acid structures that are stabilized by heme. In cells, they affect DNA replication and gene transcription. They are unwound by several helicases but the composition of the repair complex and its heme sensitivity are unclear. We found that the accumulation of G-quadruplexes is affected by heme oxygenase-1 (Hmox1) expression, but in a cell-type-specific manner: hematopoietic stem cells (HSCs) from Hmox1^−/− mice have upregulated expressions of G4-unwinding helicases (e.g., Brip1, Pif1) and show weaker staining for G-quadruplexes, whereas Hmox1-deficient murine induced pluripotent stem cells (iPSCs), despite the upregulation of helicases, have more G-quadruplexes, especially after exposure to exogenous heme. Using iPSCs expressing only nuclear or only cytoplasmic forms of Hmox1, we found that nuclear localization promotes G4 removal. We demonstrated that the proximity ligation assay (PLA) can detect cellular co-localization of G-quadruplexes with helicases, as well as with HMOX1, suggesting the potential role of HMOX1 in G4 modifications. However, this colocalization does not mean a direct interaction was detectable using the immunoprecipitation assay. Therefore, we concluded that HMOX1 influences G4 accumulation, but rather as one of the proteins regulating the heme availability, not as a rate-limiting factor. It is noteworthy that cellular G4–protein colocalizations can be quantitatively analyzed using PLA, even in rare cells.

In the visual system of Drosophila melanogaster the retina photoreceptors form tetrad synapses with the first order interneurons, amacrine cells and glial cells in the first optic neuropil (lamina), in order to transmit photic and visual information to the brain. Using the specific antibodies against synaptic proteins; Bruchpilot (BRP), Synapsin (SYN) and Disc Large (DLG), the synapses in the distal lamina were specifically labeled. Then their abundance was measured as immunofluorescence intensity in flies held in light/dark (LD 12:12), constant darkness (DD), and after locomotor and light stimulation. Moreover, the levels of proteins (SYN and DLG), and mRNAs of the brp, syn and dlg genes, were measured in the fly’s head and brain, respectively. In the head we did not detect SYN and DLG oscillations. We found, however, that in the lamina, DLG oscillates in LD 12:12 and DD but SYN cycles only in DD. The abundance of all synaptic proteins was also changed in the lamina after locomotor and light stimulation. One hour locomotor stimulations at different time points in LD 12:12 affected the pattern of the daily rhythm of synaptic proteins. In turn, light stimulations in DD increased the level of all proteins studied. In the case of SYN, however, this effect was observed only after a short light pulse (15 min). In contrast to proteins studied in the lamina, the mRNA of brp, syn and dlg genes in the brain was not cycling in LD 12:12 and DD, except the mRNA of dlg in LD 12:12. Our earlier results and obtained in the present study showed that the abundance of BRP, SYN and DLG in the distal lamina, at the tetrad synapses, is regulated by light and a circadian clock while locomotor stimulation affects their daily pattern of expression. The observed changes in the level of synaptic markers reflect the circadian plasticity of tetrad synapses regulated by the circadian clock and external inputs, both specific and unspecific for the visual system.

Heme oxygenase-1 (HO1, Hmox1 ) degrades excess heme and is considered an anti-oxidative and anti-inflammatory enzyme. Our previous studies in Hmox1 knockout mice revealed induction of interferon-stimulated genes (ISGs) in all cell types analyzed, despite unchanged interferon production. Here, we sought to identify the pathway underlying HO1-dependent ISG regulation and determine how ISG expression changes in cultured cells in response to stressors typical of Hmox1 -deficient mice. Using murine wild-type and Hmox1 -deficient (KO-Hmox1) fibroblasts, we showed that in cells cultured under control conditions, the expression of most of the tested ISGs was independent of cellular HO1 status. We then analyzed the effect of extrinsic stressors: hemolytic, oxidative, genotoxic, and replication stress, proinflammatory TNFα, and endogenous heme overload. TNFα (upregulated in Hmox1 knockout mice) was the sole and universal ISG inducer in both wild-type and KO-Hmox1 fibroblasts. Unexpectedly, the response of KO-Hmox1 cells to exogenous TNFα was weakened, probably due to impaired NF-κB activity and reduced p65 nuclear retention. A similar decrease we observed for STAT1. Additionally, the presence of TREX1 exonuclease in the nucleus indicated impaired nuclear envelope integrity. Noteworthy, HO1 colocalizes with PARP1, a protein regulating cytoplasmic-nuclear transport. Olaparib-mediated PARP1 inhibition abolished TNAα-induced nuclear accumulation of p65 and STAT1 in wild-type cells, but not in KO-Hmox1 counterparts. In summary, the inflammation typical of Hmox1 -deficient mice appears to be a major inducer of ISGs in vivo . Despite this, the inflammatory response to exogenous TNFα is attenuated in KO-Hmox1 cells, likely due to decreased nuclear retention of NF-κB and STAT1.