A mutant light-chain ferritin that causes neurodegeneration has enhanced propensity toward oxidative damage.

Intracellular inclusion bodies (IBs) containing ferritin and iron are hallmarks of hereditary ferritinopathy (HF). This neurodegenerative disease is caused by mutations in the coding sequence of the ferritin light chain (FTL) gene that generate FTL polypeptides with a C-terminus that is altered in amino acid sequence and length. Previous studies of ferritin formed with p.Phe167SerfsX26 mutant FTL (Mt-FTL) subunits found disordered 4-fold pores, iron mishandling, and proaggregative behavior, as well as a general increase in cellular oxidative stress when expressed in vivo. Herein, we demonstrate that Mt-FTL is also a target of iron-catalyzed oxidative damage in vitro and in vivo. Incubation of recombinant Mt-FTL ferritin with physiological concentrations of iron and ascorbate resulted in shell structural disruption and polypeptide cleavage not seen with the wild type, as well as a 2.5-fold increase in carbonyl group formation. However, Mt-FTL shell disruption and polypeptide cleavage were completely inhibited by the addition of the radical trap 5,5-dimethyl-1-pyrroline N-oxide. These results indicate an enhanced propensity of Mt-FTL toward free radical-induced oxidative damage in vitro. We also found evidence of extensive carbonylation in IBs from a patient with HF together with isolation of a C-terminal Mt-FTL fragment, which are both indicative of oxidative ferritin damage in vivo. Our data demonstrate an enhanced propensity of mutant ferritin to undergo iron-catalyzed oxidative damage and support this as a mechanism causing disruption of ferritin structure and iron mishandling that contribute to the pathology of HF.

Abnormal iron metabolism in fibroblasts from a patient with the neurodegenerative disease hereditary ferritinopathy.

BACKGROUND: Nucleotide duplications in exon 4 of the ferritin light polypeptide (FTL) gene cause the autosomal dominant neurodegenerative disease neuroferritinopathy or hereditary ferritinopathy (HF). Pathologic examination of patients with HF has shown abnormal ferritin and iron accumulation in neurons and glia in the central nervous system (CNS) as well as in cells of other organ systems, including skin fibroblasts. To gain some understanding on the molecular basis of HF, we characterized iron metabolism in primary cultures of human skin fibroblasts from an individual with the FTL c.497_498dupTC mutation. RESULTS: Compared to normal controls, HF fibroblasts showed abnormal iron metabolism consisting of increased levels of ferritin polypeptides, divalent metal transporter 1, basal iron content and reactive oxygen species, and decreased levels of transferrin receptor-1 and IRE-IRP binding activity. CONCLUSIONS: Our data indicates that HF fibroblasts replicate the abnormal iron metabolism observed in the CNS of patients with HF. We propose that HF fibroblasts are a unique cellular model in which to study the role of abnormal iron metabolism in the pathogenesis of HF without artifacts derived from over-expression or lack of endogenous translational regulatory elements.

Motor neuron-immune interactions: the vicious circle of ALS.

Because microglial cells, the resident macrophages of the CNS, react to any lesion of the nervous system, they have for long been regarded as potential players in the pathogenesis of several neurodegenerative disorders including amyotrophic lateral sclerosis, the most common motor neuron disease in the adult. In recent years, this microglial reaction to motor neuron injury, in particular, and the innate immune response, in general, has been implicated in the progression of the disease, in mouse models of ALS. The mechanisms by which microglial cells influence motor neuron death in ALS are still largely unknown. Microglial activation increases over the course of the disease and is associated with an alteration in the production of toxic factors and also neurotrophic factors. Adding to the microglial/macrophage response to motor neuron degeneration, the adaptive immune system can likewise influence the disease process. Exploring these motor neuron-immune interactions could lead to a better understanding in the physiopathology of ALS to find new pathways to slow down motor neuron degeneration.

Lead exposure stimulates VEGF expression in the spinal cord and extends survival in a mouse model of ALS.

Exposure to environmental lead (Pb) is a mild risk factor for amyotrophic lateral sclerosis (ALS), a paralytic disease characterized by progressive degeneration of motor neurons. However, recent evidence has paradoxically linked higher Pb levels in ALS patients with longer survival. We investigated the effects of low-level Pb exposure on survival of mice expressing the ALS-linked superoxide dismutase-1 G93A mutation (SOD1(G93A)). SOD1(G93A) mice exposed to Pb showed longer survival and increased expression of VEGF in the ventral horn associated with reduced astrocytosis. Pretreatment of cultured SOD1(G93A) astrocytes with low, non toxic Pb concentrations upregulated VEGF expression and significantly abrogated motor neuron loss in coculture, an effect prevented by neutralizing antibodies to VEGF. The actions of Pb on astrocytes might explain its paradoxical slowing of disease progression in SOD1(G93A) mice and the improved survival of ALS patients. Understanding how Pb stimulates astrocytic VEGF production and reduces neuroinflammation may yield a new therapeutic approach for treating ALS.

Abnormal iron metabolism and oxidative stress in mice expressing a mutant form of the ferritin light polypeptide gene.

Insertional mutations in exon 4 of the ferritin light chain (FTL) gene are associated with hereditary ferritinopathy (HF) or neuroferritinopathy, an autosomal dominant neurodegenerative disease characterized by progressive impairment of motor and cognitive functions. To determine the pathogenic mechanisms by which mutations in FTL lead to neurodegeneration, we investigated iron metabolism and markers of oxidative stress in the brain of transgenic (Tg) mice that express the mutant human FTL498-499InsTC cDNA. Compared with wild-type mice, brain extracts from Tg (FTL-Tg) mice showed an increase in the cytoplasmic levels of both FTL and ferritin heavy chain polypeptides, a decrease in the protein and mRNA levels of transferrin receptor-1, and a significant increase in iron levels. Transgenic mice also showed the presence of markers for lipid peroxidation, protein carbonyls, and nitrone-protein adducts in the brain. However, gene expression analysis of iron management proteins in the liver of Tg mice indicates that the FTL-Tg mouse liver is iron deficient. Our data suggest that disruption of iron metabolism in the brain has a primary role in the process of neurodegeneration in HF and that the pathogenesis of HF is likely to result from a combination of reduction in iron storage function and enhanced toxicity associated with iron-induced ferritin aggregates in the brain.

Iron-mediated aggregation and a localized structural change characterize ferritin from a mutant light chain polypeptide that causes neurodegeneration.

Nucleotide insertions in the ferritin light chain (FTL) polypeptide gene cause hereditary ferritinopathy, a neurodegenerative disease characterized by abnormal accumulation of ferritin and iron in the central nervous system. Here we describe for the first time the protein structure and iron storage function of the FTL mutant p.Phe167SerfsX26 (MT-FTL), which has a C terminus altered in sequence and extended in length. MT-FTL polypeptides assembled spontaneously into soluble, spherical 24-mers that were ultrastructurally indistinguishable from those of the wild type. Far-UV CD showed a decrease in alpha-helical content, and 8-anilino-1-naphthalenesulfonate fluorescence revealed the appearance of hydrophobic binding sites. Near-UV CD and proteolysis studies suggested little or no structural alteration outside of the C-terminal region. In contrast to wild type, MT-FTL homopolymers precipitated at much lower iron loading, had a diminished capacity to incorporate iron, and were less thermostable. However, precipitation was significantly reversed by addition of iron chelators both in vitro and in vivo. Our results reveal substantial protein conformational changes localized at the 4-fold pore of MT-FTL homopolymers and imply that the C terminus of the MT-FTL polypeptide plays an important role in ferritin solubility, stability, and iron management. We propose that the protrusion of some portion of the C terminus above the spherical shell allows it to cross-link with other mutant polypeptides through iron bridging, leading to enhanced mutant precipitation by iron. Our data suggest that hereditary ferritinopathy pathogenesis is likely to result from a combination of reduction in iron storage function and enhanced toxicity associated with iron-induced ferritin aggregates.

Expression of a mutant form of the ferritin light chain gene induces neurodegeneration and iron overload in transgenic mice.

Increased iron levels and iron-mediated oxidative stress play an important role in the pathogenesis of many neurodegenerative diseases. The finding that mutations in the ferritin light polypeptide (FTL) gene cause a neurodegenerative disease known as neuroferritinopathy or hereditary ferritinopathy (HF) provided a direct connection between abnormal brain iron storage and neurodegeneration. HF is characterized by a severe movement disorder and by the presence of nuclear and cytoplasmic ferritin inclusion bodies in glia and neurons throughout the CNS and in tissues of multiple organ systems. Here we report that the expression in transgenic mice of a human FTL cDNA carrying a thymidine and cytidine insertion at position 498 (FTL498-499InsTC) leads to the formation of nuclear and cytoplasmic ferritin inclusion bodies. As in HF, ferritin inclusions are seen in glia and neurons throughout the CNS as well as in cells of other organ systems. Our studies show histological, immunohistochemical, and biochemical similarities between ferritin inclusion bodies found in transgenic mice and in individuals with HF. Expression of the transgene in mice leads to a significant decrease in motor performance and a shorter life span, formation of ferritin inclusion bodies, misregulation of iron metabolism, accumulation of ubiquitinated proteins, and incorporation of elements of the proteasome into inclusions. This new transgenic mouse represents a relevant model of HF in which to study the pathways that lead to neurodegeneration in HF, to evaluate the role of iron mismanagement in neurodegenerative disorders, and to evaluate potential therapies for HF and related neurodegenerative diseases.