Hereditary ferritinopathy Ruben Vidal a, * , Marie Bernadette Delisle b , Olivier Rascol b , Bernardino Ghetti a,1 a Department of Pathology and Laboratory Medicine, Division of Neuropathology, Indiana University School of Medicine, 635 Barnhill Drive MS A142, Indianapolis, IN 46202, USA b University Hospital, Toulouse, 31059 France Non-heme iron deposits in the brain occur in several neurodegenerative diseases. They are the Hallervorden-Spatz syndrome (pantothenate kinase associated neurodegenera- tion [PKAN]; Hayflick in this symposium), aceruloplasmi- nemia (Harris in this symposium), and a recently described autosomal dominant extrapyramidal disease caused by a mutation in the ferritin light-chain gene [1]. For the last, Curtis et al. [1] proposed the name ‘‘neuroferritinopathy’’. The hallmark of this condition is the increase of iron and ferritin in the extracellular spaces and cytoplasm of cells in the basal ganglia of affected individuals associated with low serum ferritin levels. As a catalyst of lipid peroxidation, iron may also contribute to the pathogenesis of other degenerative brain disorders, including Parkinson’s disease, Huntington’s chorea, and Friedreich’s ataxia [2]. We studied a novel autosomal dominant neurodegener- ative disease characterized by intracytoplasmic and intra- nuclear accumulation of ferritin in the central nervous system (CNS). Inclusions were not limited to the CNS as they were also observed in skin, liver, muscle, nerve, and kidney biopsy samples of affected family members. Clinically, the disease is characterized by the appearance, early in the third decade of life, of tremor of the upper extremities followed later by cerebellar ataxia, dysarthria, extrapyramidal signs, and cog- nitive dysfunction. Ceruloplasmin, ferritin, copper and iron concentrations in serum were within the normal range. Neuropathologically, this condition is characterized by the presence of nuclear and cytoplasmic, spherical, eosino- philic inclusions. At the ultrastructural level, they are composed of amorphous, granular, osmiophilic material in neurons and glia throughout the central nervous system. Inclusions are present in large numbers in the neurons of striatum and cerebellum as well as in glial cells throughout the brain. When a major portion of the nucleus is occupied by an inclusion, the nuclear membrane is thin, with inclu- sions having a diameter that ranges from 2 to 35 Am. In addition to the intranuclear inclusions, numerous large, strongly eosinophilic and argyrophilic deposits are seen in the basal ganglia. They may be located in the cytoplasm or outside of nerve cells. Occasionally, these deposits appear fused with each other. In the cerebellar cortex, the inclusions are located in the nuclei of Purkinje cells and granule cells. In addition, inclusions are also seen in the somata and dendrites of Purkinje cells. Occasionally, both intranuclear and intracytoplasmic deposits are seen in the same Purkinje cell. The inclusions are immunolabeled by antibodies raised against ferritin and ubiquitin, and are positive with Perls’s stain for ferric iron. They are periodic acid-Schiff negative and are not stained with Alcian Blue. Amyloid and neuro- fibrillary pathology are absent after staining with thioflavin S or Congo Red. Immunostaining with antibodies against Ah (10D5) and tau protein (AT8) are negative. To determine the nature and composition of the inclu- sions, we isolated them from frozen cerebellum and puta- men of the proband. The technique included repeated cycles of homogenization and centrifugation in Dulbecco’s phos- phate-buffered saline, followed by detergent washes. The quality and purity of the preparation were assessed by staining the fraction with hematoxylin and eosin. Electron microscopy of the final fraction strongly resembled the cerebral inclusion bodies. Protein analysis of the purified inclusions by Tris – tricine sodium dodecylsulfate-polyacry- lamide gel electrophoresis (SDS-PAGE) revealed a major protein band with an apparent molecular weight (MW) of f 22 kDa. In addition, a second band with a MW of f 44 kDa was observed. Additional SDS-PAGE of the material solubilized by formic acid showed mainly the f 22 kDa protein band. Protein extracts prepared in the same way from cerebellum of a control case did not show the presence of the 22 kDa band. Our attempts to obtain direct N-terminal amino acid sequences of the f 22 kDa peptide failed, suggesting a blocked N-terminus. The 22 kDa protein was digested with trypsin, and the digest was purified by high- performance liquid chromatography. The mass of the pep- tides was determined using a Finnigan LCQ ion trap mass spectrometer. A search of the database of the National 0022-510X/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. PII:S0022-510X(02)00435-5 * Corresponding author. Tel.: +1-317-274-1729; fax: +1-317-274- 4882. E-mail addresses: rvidal@iupui.edu (R. Vidal), bghetti@iupui.edu (B. Ghetti). 1 Tel.: +1-317-274-7818; fax: +1-317-274-4882. www.elsevier.com/locate/jns Journal of the Neurological Sciences 207 (2003) 110– 111