Rosaria Ingrassia

Ferritins: A family of molecules for iron storage, antioxidation and more

Ferritins are characterized by highly conserved three-dimensional structures similar to spherical shells, designed to accommodate large amounts of iron in a safe, soluble and bioavailable form. They can have different architectures with 12 or 24 equivalent or non-equivalent subunits, all surrounding a large cavity. All ferritins readily interact with Fe(II) to induce its oxidation and deposition in the cavity in a mineral form, in a reaction that is catalyzed by a ferroxidase center. This is an anti-oxidant activity that consumes Fe(II) and peroxides, the reagents that produce toxic free radicals in the Fenton reaction. The mechanism of ferritin iron incorporation has been characterized in detail, while that of iron release and recycling has been less thoroughly studied. Generally ferritin expression is regulated by iron and by oxidative damage, and in vertebrates it has a central role in the control of cellular iron homeostasis. Ferritin is mostly cytosolic but is found also in mammalian mitochondria and nuclei, in plant plastids and is secreted in insects. In vertebrates the cytosolic ferritins are composed of H and L subunit types and their assembly in a tissues specific ratio that permits flexibility to adapt to cell needs. The H-ferritin can translocate to the nuclei in some cell types to protect DNA from iron toxicity, or can be actively secreted, accomplishing various functions. The mitochondrial ferritin is found in mammals, it has a restricted tissue distribution and it seems to protect the mitochondria from iron toxicity and oxidative damage. The various functions attributed to the cytosolic, nuclear, secretory and mitochondrial ferritins are discussed.

Late-onset Parkinsonism in NFκB/c-Rel-deficient mice

Activation of the nuclear factor κB/c-Rel can increase neuronal resilience to pathological noxae by regulating the expression of pro-survival manganese superoxide dismutase (MnSOD, now known as SOD2) and Bcl-xL genes. We show here that c-Rel-deficient (c-rel−/−) mice developed a Parkinson’s disease-like neuropathology with ageing. At 18 months of age, c-rel−/− mice exhibited a significant loss of dopaminergic neurons in the substantia nigra pars compacta, as assessed by tyrosine hydroxylase-immunoreactivity and Nissl staining. Nigral degeneration was accompanied by a significant loss of dopaminergic terminals and a significant reduction of dopamine and homovanillic acid levels in the striatum. Mice deficient of the c-Rel factor exhibited a marked immunoreactivity for fibrillary α-synuclein in the substantia nigra pars compacta as well as increased expression of divalent metal transporter 1 (DMT1) and iron staining in both the substantia nigra pars compacta and striatum. Aged c-rel−/− mouse brain were characterized by increased microglial reactivity in the basal ganglia, but no astrocytic reaction. In addition, c-rel−/− mice showed age-dependent deficits in locomotor and total activity and various gait-related deficits during a catwalk analysis that were reminiscent of bradykinesia and muscle rigidity. Both locomotor and gait-related deficits recovered in c-rel−/− mice treated with l-3,4-dihydroxyphenylalanine. These data suggest that c-Rel may act as a regulator of the substantia nigra pars compacta resilience to ageing and that aged c-rel−/− mice may be a suitable model of Parkinson’s disease.

1B/(-)IRE DMT1 expression during brain ischemia contributes to cell death mediated by NF-κB/RelA acetylation at Lys310.

The molecular mechanisms responsible for increasing iron and neurodegeneration in brain ischemia are an interesting area of research which could open new therapeutic approaches. Previous evidence has shown that activation of nuclear factor kappa B (NF-κB) through RelA acetylation on Lys310 is the prerequisite for p50/RelA-mediated apoptosis in cellular and animal models of brain ischemia. We hypothesized that the increase of iron through a NF-κB-regulated 1B isoform of the divalent metal transporter-1 (1B/DMT1) might contribute to post-ischemic neuronal damage. Both in mice subjected to transient middle cerebral artery occlusion (MCAO) and in neuronally differentiated SK-N-SH cells exposed to oxygen-glucose-deprivation (OGD), 1A/DMT1 was only barely expressed while the 1B/DMT1 without iron-response-element (−IRE) protein and mRNA were early up-regulated. Either OGD or over-expression of 1B/(−)IRE DMT1 isoform significantly increased iron uptake, as detected by total reflection X-ray fluorescence, and iron-dependent cell death. Iron chelation by deferoxamine treatment or (−)IRE DMT1 RNA silencing displayed significant neuroprotection against OGD which concomitantly decreased intracellular iron levels. We found evidence that 1B/(−)IRE DMT1 was a target gene for RelA activation and acetylation on Lys310 residue during ischemia. Chromatin immunoprecipitation analysis of the 1B/DMT1 promoter showed there was increased interaction with RelA and acetylation of H3 histone during OGD exposure of cortical neurons. Over-expression of wild-type RelA increased 1B/DMT1 promoter-luciferase activity, the (−)IRE DMT1 protein, as well as neuronal death. Expression of the acetylation-resistant RelA-K310R construct, which carried a mutation from lysine 310 to arginine, but not the acetyl-mimic mutant RelA-K310Q, down-regulated the 1B/DMT1 promoter, consequently offering neuroprotection. Our data showed that 1B/(−)IRE DMT1 expression and intracellular iron influx are early downstream responses to NF-κB/RelA activation and acetylation during brain ischemia and contribute to the pathogenesis of stroke-induced neuronal damage.

Molecular characterization of the new defective Pbrescia alpha1-antitrypsin allele†

Alpha1‐antitrypsin (α1AT) deficiency is a hereditary disorder associated with reduced α1AT serum level, predisposing adults to pulmonary emphysema. Among the known mutations of the α1AT gene (SERPINA1) causing α1AT deficiency, a few alleles, particularly the Z allele, may also predispose adults to liver disease. We have characterized a new defective α1AT allele (c.745G>C) coding for a mutant α1AT (Gly225Arg), named Pbrescia. The Pbrescia α1AT allele was first identified in combination with the rare defective Mwürzburg allele in an 11‐year‐old boy showing significantly reduced serum α1AT level. Subsequently, the Pbrescia allele was found in the heterozygous state with the normal M or the defective Z allele in nine and three adults respectively. In cellular models of the disease, we show that the Pbrescia mutant is retained in the endoplasmic reticulum as ordered polymers and is secreted more slowly than the normal M α1AT. This behaviour recapitulates the abnormal cellular handling and fate of the Z α1AT and suggests that the mutation present in the Pbrescia α1AT causes a conformational change of the protein which, by favouring polymer formation, is etiologic to both severe α1AT deficiency in the plasma and toxic protein‐overload in the liver.

A mutational analysis of the epitopes of recombinant human H-ferritin

Murine monoclonal antibodies were elicited by the recombinant human H-ferritin overexpressed in Escherichia coli. They had a specificity analogous to that of the antibodies elicited by natural human H-chain, and all of them showed low additivity in binding the recombinant ferritin. Four antibodies of each group were challenged with four H-ferritin mutants overexpressed in E. coli, altered in different accessible areas of the molecule. They consisted of deletions of the first 13 and last 22 amino acids, a duplication of an 18 amino acid sequence in the loop region, and a substitution of a 5 amino acid stretch in the three-fold symmetry axis region. Double diffusion, immunodot analyses and inhibition plots indicated that: (1) all the mutants were recognized by at least one antibody; (2) the deletion of the N-terminus and the duplication in the loop region had the strongest effect on antibody binding; and (3) epitope boundaries of the various antibodies could not be recognized. The antibodies were tested with H-containing ferritins from rat and hen hearts, and showed low or absent reactivities despite their high structural homology with human ferritin. Comparison of the amino acid sequences of human, mouse, rat and hen H-chains, together with mutational data, suggested that; (i) ferritin epitopes are large, probably encompassing a large portion of the subunit surface and (ii) Thr-5 and Cys-90 have a role in H-ferritin immunogenicity.

Ferrous Iron Up-regulation in Fibroblasts of Patients with Beta Propeller Protein-Associated Neurodegeneration (BPAN)

Mutations in WDR45 gene, coding for a beta-propeller protein, have been found in patients affected by Neurodegeneration with Brain Iron Accumulation, NBIA5 (also known as BPAN). BPAN is a movement disorder with Non Transferrin Bound Iron (NTBI) accumulation in the basal ganglia as common hallmark between NBIA classes (Hayflick et al., 2013). WDR45 has been predicted to have a role in autophagy, while the impairment of iron metabolism in the different NBIA subclasses has not currently been clarified. We found the up-regulation of the ferrous iron transporter (-)IRE/Divalent Metal Transporter1 and down-regulation of Transferrin receptor in the fibroblasts of two BPAN affected patients with splicing mutations 235+1G>A (BPAN1) and 517_519ΔVal 173 (BPAN2). The BPAN patients showed a concomitant increase of intracellular ferrous iron after starvation. An altered pattern of iron transporters with iron overload is highlighted in BPAN human fibroblasts, supporting for a role of DMT1 in NBIA. We here present a novel element, about iron accumulation, to the existing knowledge in field of NBIA. Attention is focused to a starvation-dependent iron overload, possibly accounting for iron accumulation in the basal ganglia. Further investigation could clarify iron regulation in BPAN.