Expression of a Mutant Form of the Ferritin Light Chain Gene Induces Neurodegeneration and Iron Overload in Transgenic Mice

Abstract

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.

Iron-mediated Aggregation and a Localized Structural Change Characterize Ferritin from a Mutant Light Chain Polypeptide That Causes Neurodegeneration

“…We observed that ferritin precipitates obtained in vitro were composed of fully assembled 24-mers, similar to what has been reported in inclusions in patients with HF (9, 12) and in transgenic mice expressing the MT-FTL polypeptide (29). At iron: ferritin ratios above 2000:1, under the same experimental conditions in which we analyzed WT-and MT-FTL, the majority of FTH1 but not WT-FTL ferritins precipitate (16).…”

“…In this mouse model, the human MT-FTL polypeptide forms heteropolymers with the endogenous murine wild type Ftl and Fth1 polypeptides, which are seen to aggregate in neurons and glia throughout the life span of the mice (29). After treatment of the cells with FAC to increase their intracellular iron stores, we observed a switch of ferritin from the detergentsoluble fraction to the detergent-insoluble fraction, suggesting a change in the solubility (Fig.…”

“…Pups were obtained from transgenic dams homozygous for the FTL498 -499InsTC mutation in C57BL/6J genetic background (29). Briefly, cerebral cortices were removed, and the tissue was minced and dissociated in 0.25% trypsin (Invitrogen) for 15 min at 37°C.…”

Mutant Ferritin L-chains That Cause Neurodegeneration Act in a Dominant-negative Manner to Reduce Ferritin Iron Incorporation

“…The common property of the two mutants is a major conformational alteration of the C terminus, which was exposed to solvent and accessible to proteolytic enzymes. Moreover, the expression of human L167fs in transgenic mice was shown to cause a phenotype with cytosolic and intranuclear ferritin aggregates analogous to those found in the patients (27), and to cause a deregulation of brain iron with up-regulation of the endogenous ferritin, down-regulation of transferrin receptor, and reduced RNA binding activity of IRP1 (26).…”

“…In contrast, the mutant L167fs was shown to be as soluble as L-wild type (Lwt), to be less thermostable, and to form aggregates when incubated with an excess of Fe(II) in aerobic conditions (22,24,25). It was proposed that these iron-rich ferritin aggregates contribute to the formation of ferritin bodies in vivo and alter ferritin functionality (26,27). Studies on cellular models showed that the expression of L154fs and L167fs caused remarkably similar phenotypes, with the accumulation of endogenous ferritins, an increase of labile iron pool and oxidative damage, joined to a reduction of ferritin half-life and proteasome activity (22).…”