Journal of Neuropathology and Experimental Neurology Copyright q 2004 by the American Association of Neuropathologists

Vol. 63, No. 4 April, 2004 pp. 363 380

Intracellular Ferritin Accumulation in Neural and Extraneural Tissue Characterizes a Neurodegenerative Disease Associated with a Mutation in the Ferritin Light Polypeptide Gene R. VIDAL, PHD,* B. GHETTI, MD,* M. TAKAO, MD, C. BREFEL-COURBON, MD, E. URO-COSTE, MD, PHD, B. S. GLAZIER, V. SIANI, MD, M. D. BENSON, MD, P. CALVAS, MD, PHD, L. MIRAVALLE, PHD, O. RASCOL, MD, PHD, AND M. B. DELISLE, MD

Key Words:

Ataxia; Dementia; Extrapyramidal signs; Ferritin; Iron; Neurodegeneration; Tremor.

INTRODUCTION Ferritin, an iron storage protein, is composed of 24 polypeptides assembled into a hollow shell. Each of these polypeptides may be either ferritin light polypeptide (FTL) or ferritin heavy polypeptide (FTH1) (1). The genes encoding FTL and FTH1 have been mapped to chromosomes 11q12-q13 and 19q13.3-q13.4, respectively. FTL and FTH1 are structurally equivalent and have considerable amino acid sequence homology, with over 50% identity (2). The FTL subunit does not have catalytic activity, offering acidic residues on the surface cavity that facilitate iron nucleation. The FTH1 subunit is the main regulator of ferritin activity and contains the ferroxidase center. The ferroxidase activity is essential for iron incorporation in ferritin, but it is also potentially important in the regulation of the redox status of the cells, by removing the potentially more toxic Fe(II) (1). The expression of the 2 ferritin polypeptides is regulated by the iron regulatory proteins ACO1 (or IRP1) and IREB2 (or IRP2). Both proteins are RNA-binding proteins that affect the translation and stabilization of the From Department of Pathology and Laboratory Medicine (RV, BG, MT, BSG, MDB, LM), Indiana University School of Medicine, Indianapolis, Indiana; University Hospital (CBC, EUC, OR, MBD), Purpan Hospital (PC), Toulouse, France; Tarbes Hospital (VS), Tarbes, France. *Both authors contributed equally. Correspondence to: Ruben Vidal, PhD ([email protected]) and Bernardino Ghetti, MD ([email protected]), Indiana University School of Medicine, Department of Pathology & Laboratory Medicine, 635 Barnhill Drive, MS B37A, Indianapolis, IN 46202. This study was supported in part by PHS P30 AG10133, U01AG16976, the Alzheimer’s Association and the American Federation for Aging Research.

FTL and FTH1 mRNAs by binding to stem-loop structures known as iron responsive elements (IREs) found in the 59-untranslated region of FTL and FTH1 mRNAs (1). Mutations in the IRE of FTL are associated with an autosomal dominant hyperferritinemia-cataract syndrome (3). A mutation in the IRE sequence of the FTH1 gene is associated with an autosomal dominant disorder presenting with decreased levels of FTH1 and iron overload (4). These mutations lead to an alteration in the production of the ferritin polypeptides without changing the primary sequence of FTL or FTH1. In addition, neurological symptoms and signs have not been reported in association with any of the IRE mutations. In contrast, a mutation in the coding region of the FTL gene is associated with an autosomal dominant neurodegenerative disease called neuroferritinopathy. This disorder is characterized by extrapyramidal symptoms and abnormal deposition of iron and ferritin in the basal ganglia as well as low serum ferritin levels (5). This paper reports results from a comprehensive analysis of a hereditary neurodegenerative disease clinically characterized by tremor, cerebellar ataxia, parkinsonism and pyramidal signs, behavioral disturbances, and cognitive dysfunction. This symptomatology may appear sequentially over a period of 4 decades. Using a multidisciplinary approach, we have found that this disorder is characterized by intranuclear and intracytoplasmic deposition of ferritin in glia and subsets of neurons in the central nervous system as well as in parenchymal cells of other organ systems. The molecular genetic basis of the disorder is a novel mutation in the coding region of the FTL gene. Preliminary data obtained in the course of these studies were reported in abstract form (6–8).

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Abstract. Abnormal accumulation of ferritin was found to be associated with an autosomal dominant slowly progressing neurodegenerative disease clinically characterized by tremor, cerebellar ataxia, parkinsonism and pyramidal signs, behavioral disturbances, and cognitive decline. These symptoms may appear sequentially over a period of 4 decades. Pathologically, intranuclear and intracytoplasmic bodies were found in glia and subsets of neurons in the central nervous system as well as in extraneural tissue. Biochemical analyses of these bodies isolated from the striatum and cerebellar cortex revealed that ferritin light polypeptide (FTL) and ferritin heavy polypeptide (FTH1) were the main constituents. Molecular genetic studies revealed a 2-bp insertion mutation in exon 4 of the FTL gene. The resulting mutant polypeptide is predicted to have a carboxy terminus that is altered in amino-acid sequence and length. In tissue sections, the bodies were immunolabeled by anti-ferritin and anti-ubiquitin antibodies and were stained by Perls’ method for ferric iron. Synthetic peptides homologous to the altered and wild-type carboxy termini were used to raise polyclonal antibodies. These novel antibodies as well as an antibody recognizing FTH1 immunolabeled the bodies. This study of this disorder has provided additional knowledge and insights in the growing area of ferritin-related neurodegeneration.

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Fig. 1. Pedigree of ‘‘family L.’’ The symbols ● and n represent affected individuals. The symbol # represents unaffected individuals. The arrow indicates the proband. The symbol / indicates deceased individuals.

MATERIALS AND METHODS The family of the proband, identified here as ‘‘family L,’’ has been in France for several generations. We were able to construct a pedigree (Fig. 1) consisting of 56 members over 5 generations.

Neuropathology An autopsy was carried out on the brain of the proband (III12). The fresh brain was hemisected along the midsagittal plane; the right half of the brain was fixed in 10% formalin and the left half was sliced and stored at 2708C. Neurohistology: Following a 72-hour fixation, the left half of the brain was sliced. Tissue samples were processed, cut, and stained according to previously published protocols (9). In addition, the Perls’ method for ferric iron was used. Antibody Production: Following previously published methods (10), polyclonal antibodies (Abs) were raised in rabbits by using a synthetic peptide homologous to residues 166–175 (CLFERLTLKHD) of the wild-type FTL protein [Ab 1277] and a synthetic peptide homologous to residues 166–191 (CLSSKGSLSSTTKSLLSPATSEGPLAK) of the mutated FTL protein [Ab 1283]. Immunohistochemistry: Polyclonal Abs raised against human neuroserpin (1:2,000) (11), glial fibrillary acidic protein (GFAP) (Dako, Carpinteria, CA) (1:100), a synthetic peptide corresponding to residues 119–137 of human a-synuclein (1: 300), ubiquitin (Dako) (1:1,000), ferritin (Dako and Biodesign, Saco, ME) (1:500), ferritin heavy chain (Y-16) (Santa Cruz Biotechnology, Santa Cruz, CA) (1:50), polyglutamine (Chemicon, Temecula, CA) (1:500), and anti-ferritin Abs 1277 and 1283 (1: 1,000) were used, as were monoclonal Abs against the amyloid b protein (Ab) (10D5) (Elan Corporation, San Francisco, CA) (1:100), calcium binding protein (calbindin) (1:500) (12) and microtubule associated protein tau (AT8) (Pierce Biotechnology, Rockford, IL) (1:400). Immunohistochemical labeling was carried out following published protocols (9). Double immunohistochemical studies were performed using the Dako EnVision Doublestain System (Dako) following the manufacturer’s instructions. We used anti-ferritin Abs as primary antibodies and anti-GFAP or anti-calbindin as secondary antibodies.

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Biochemistry To isolate and characterize the intranuclear deposits observed histologically, fresh frozen samples of the putamen and cerebellum of the proband were dissected, minced into 1- to 3-mm pieces and placed in Dulbecco’s-PBS (D-PBS, Sigma, St. Louis, MO) with a cocktail of protease inhibitors (Complete, 1 mM Pepstatin, 100 mM TLCK-HCl, 200 mM TPCK, and 1 mM Leupeptin, all from Roche Molecular Biochemical, Indianapolis, IN) on ice. As a control, cerebellar tissue from an unaffected individual was used. Tissue was then washed by resuspension in D-PBS with protease inhibitors and centrifuged at 5,000 3 g for 10 min at 48C; the procedure was repeated 5 times. The insoluble material was resuspended in 20 volumes of collagenase buffer and digested with collagenase (Sigma) and DNase I (Sigma) as previously described (10). After digestion, the suspension was centrifuged at 5,000 3 g for 30 min at 48C, washed 3 times with 0.1 M Tris-HCl, pH 7.4 and the insoluble-undigested material was subjected to 3 cycles of detergent washes in 1% n-lauryl sarcosine (Sigma). A final wash in the homogenization medium was done. The insoluble material was collected by centrifugation and analyzed by light and electron microscopy. The insoluble material was solubilized in either 7% SDS, 300 mM Tris-HCl, pH 6.8, 36% glycerol (33 solubilization buffer) or 99% formic acid (Sigma), centrifuged at 10,000 3 g for 5 min and the supernatant dried under N2. After

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Pedigree

Electron Microscopy: Tissue obtained from the superior frontal gyrus, the putamen and the cerebellum were fixed with 4% formaldehyde and post-fixed with Dalton’s chrome osmium, dehydrated in graded ethanols, cleared in propylene oxide, and embedded in Epon. One-mm-thick sections were stained with toluidine blue. Ultra-thin sections were contrasted with uranyl acetate and lead citrate. Photographs were obtained using a Philips EM 300 electron microscope. Biopsy Studies: At separate times during the course of the disease, tissue biopsies (liver and skin) were obtained from the proband. Tissue biopsies were also obtained on cases III-11 (muscle and nerve), III-17 (kidney and skin) and IV-6 (skin). Tissue was processed for histological and electron microscopy studies according to established protocols. Eight-mm-thick sections were stained with H&E and immunolabeled with antiferritin Abs (Dako and Biodesign) (1:500). Electron microscopy studies of the biopsy tissue were carried out as described above.

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Genetic Analysis Genomic DNA was extracted from frozen brain tissue of the proband (III-12) and venous blood lymphocytes from 11 members of the family that included affected and nonaffected individuals (II-3, II-5, III-11, III-22 to III-27, IV-6, and IV-11). From the proband’s DNA, the entire coding region and exonintron boundaries of the ACO1 gene and the IREB2 gene were sequenced. The coding region and exon-intron boundaries of the FTL and FTH1 genes were also completely sequenced on the proband. The genomic sequence of the 4 genes was obtained by nucleotide searching (17). The following are the oligonucleotide primers used for the amplification of the 2 ferritin genes and the product lengths are noted in parentheses. FTL exons 1– 2/F, 59-ACG TCC CCT CGC AGT TCG GCG G-39 and 1–2/ R, 59-TGT AGT CCA TTA CCC ACA C-39 (646 bp); exon 3/

F, 59-TGT AGG TTT AGT TCT ATG TG-39 and 3/R, 59- TGT GAA TGA GGC TCT GAA GG-39 (275 bp); exon 4/F, 59-CTG TCA CAT TTT AAT CTG CC-39 and 4/R, 59-AAG CCC TAT TAC TTT GCA AG-39 (293 bp); FTH1 exon 1/F, 59-GGC TAT AAG AGA CCA CAA GCG-39 and 1/R, 59-GCC ACC GCT TAG GCC CGC CC-39 (381 bp); exon 2/F, 59-TAG TAT AAA CAC TTC AGT GTT C-39 and 2/R 59-CCA GTG TAT CAT CAC TTT AT-39 (283 bp); exons 3–4/F 59-AAA CGT GGT GGT TAG AGA TG-39 and 3–4/R 59-ACT TAT AGA AAA GGT AAA GG-39 (559 bp). Polymerase chain reaction (PCR) amplification was performed for 30 cycles of 948C 1 min, 458C 1 min and, 728C 2 min. PCR products were separated on 1.5% agarose gels in TBE and visualized by ethidium bromide staining. PCR fragments were gel purified (Qiagen, Valencia, CA) and sequenced in both directions by direct DNA sequencing on a 377XL Applied Biosystem DNA sequencer (Applied Biosystem) and on a CEQ 2000XL DNA analysis system (Beckman Coulter, Fullerton, CA). PCR products of exon 4 of the FTL gene were subcloned into pCR2.1 vector (TA cloning kit) (Invitrogen, Carlsbad, CA). Recombinant plasmid DNA was isolated from 8 to 10 clones of different PCR reactions and sequenced in both directions.

Reverse Transcription-PCR (RT-PCR) Total cellular RNAs were isolated from fresh frozen brain tissue of the proband by the guanidine isothiocyanate method using Trizol LS (Invitrogen) reagent. Reverse transcription of RNA (1 mg) was performed with the Advantage RT-PCR kit (Clontech, Palo Alto, CA) according to the manufacturer’s instructions. PCR amplification of the first strand cDNA produced by reverse transcription was performed using oligonucleotides E1–2 forward and E4 reverse for the FTL gene and oligonucleotides E1 forward and E4 reversed for the FTH1 gene. As a positive control for the RT-PCR experiment, 0.45 kb of the Glyceraldehyde-3-Phosphate-Dehydrogenase (GAPD) gene was amplified using a human GAPD control oligonucleotide set (Clontech). As negative controls, RNA samples were pretreated with RNase A (Clontech) for 30 min at 378C and subjected to RT-PCR. Each PCR cycle consisting of a denaturation step (948C, 1min), an annealing step (408C, 2 min), and an elongation step (728C, 3 min) was repeated 30 times. RT-PCR products were separated on 1% agarose gels and visualized by ethidium bromide staining. RT-PCR products of the FTL gene were subcloned into pCR2.1 vector and sequenced in both directions.

RESULTS Family History The pedigree (Fig. 1) spans 5 generations. Individuals affected with the autosomal dominant neurological disorder have been identified in 4 generations. The proband’s (III-12) father (II-4), paternal aunt (II-7), brother (III-17), sister (III-11), and nephew (IV-6) were studied clinically and all presented neurological signs. Clinical History of the Proband The proband presented the first neurological signs at age 20. Neurological signs continued for the next 39 years and were characterized by tremor progressively J Neuropathol Exp Neurol, Vol 63, April, 2004

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solubilization, the supernatant fraction (SDS or formic acid) was separated on a 16% Tris-Tricine SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes (Immobilon-P) (Millipore, Bedford, MA) as described (10). Proteins were subjected to N-terminal sequence analysis on a 473A protein sequencer (Applied Biosystems, Foster City, CA). Mass Spectrometry: The insoluble material was solubilized in water:isopropanol:formic acid (4:4:1) and subjected to matrix-assisted laser desorption ionization time-of-flight (MALDITOF) mass spectrometry analysis. For liquid chromatography interfaced with an ion trap (LCQ) mass spectrometry, the SDSsupernatant fraction was separated on a 16% Tris-Tricine SDSPAGE and protein bands were excised and digested in-gel with high performance liquid chromatography (HPLC)-grade trypsin (Sigma). The tryptic peptides were purified on a Perkin Elmer Applied Biosystems capillary HPLC unit and subjected to LCQ quadruple ion trap mass spectrometry analysis using a Finnigan (Thermoquest, San Jose, CA) LCQ mass spectrometer. The peptide masses obtained were used to search the protein sequence database at the National Center for Biotechnology Information (NCBI) using ProFound (13). Peptides corresponding to the obtained mass peaks were also identified by using the Protein Analysis WorkSheet (PAWS) freeware edition (14). Secondary structure prediction analysis was done at Jpred (15) and analysis for alpha helix context was done at ProtScale (16). Immunoblot: The insoluble material was solubilized in solubilization buffer, subjected to 16% acrylamide Tris-Tricine-SDS PAGE, and electrophoretically transferred to Immobilon-P membranes. Recombinant human FTL and FTH1 (Calbiochem, San Diego, CA) were run as controls. The membranes were blocked with 5% nonfat dry milk in 10 mM phosphate buffer, 137 mM NaCl, 2.7 mM KCl (PBS) (Sigma) pH 7.4 with 0.1% Tween-20 (PBS-T) overnight and then incubated for 2 hours at room temperature with the primary Ab. As primary Abs we used anti-ferritin antibody (Dako and Biodesign), Ab 1277 and Ab 1283. All Abs were diluted (1:500) in PBS-T. Horseradish peroxidase-conjugated goat anti-rabbit (Amersham, Piscataway, NJ) was used as the second antibody at a dilution of 1:5,000 in PBS-T. The Biodesign sheep-anti ferritin antibody was previously incubated with a polyclonal rabbit anti-sheep antibody (Chemicon) at a dilution of 1:500 in PBS-T. Immunoblots were visualized by chemiluminescence (Amersham) according to the manufacturer’s specifications.

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Neuropsychology A neuropsychological examination at age 54 indicated that the patient had signs of mild cognitive impairment as shown by the following results: Mini Mental State Examination (MMSE) 26/30, memory quotient (MQ) 85/

130 and dementia rating scale (DRS) 124/144. Neuropsychological examination at age 57 showed the presence of a frontal and subcortical dementia. Electrophysiology An electromyogram at age 47 showed postural tremor in all 4 limbs, with predominance in the left upper limb. The frequency of the tremor was 6 cycles/sec. At age 57, eye movement recordings using electrooculography showed normal horizontal and vertical saccades, but saccadic smooth pursuit (reduced gain), and abnormal vestibular ocular reflex cancellation by fixation. There were no square wave jerks. Neuroradiology Several computed tomography studies during the course of the disease revealed the progression of cerebellar atrophy from moderate atrophy of the cerebellar vermis at age 47 to diffuse cerebellar atrophy at age 55. These studies also showed atrophy of the cerebral cortex at age 54. Magnetic resonance imaging (MRI) studies at age 54 and 56 confirmed the cerebral and cerebellar atrophy and detected abnormal T2 hypointense and T1 hyperintense signals in the basal ganglia (Fig. 2A, B). Pontine atrophy was also detected in the second MRI. Laboratory Examination At age 41, liver transaminases and gamma glutamyl transferase were increased and remained abnormal throughout the course of the disease. At age 54, copper metabolism was found to be normal. At age 55, a liver biopsy showed signs of chronic active hepatitis and fibrosis, but no cirrhosis. Intranuclear bodies were seen in fibroblasts within the portal spaces and intracytoplasmic deposits were seen within hepatocytes at the periphery of the lobules. Viral and toxic etiologies were ruled out. Neuropathology Macroscopic Neuropathology: The fresh brain of the proband weighed 1,120 g. There was no atherosclerosis in the major cerebral arteries; however, there was a whitish thickening of the intima. The cerebral hemispheres showed mild to moderate diffuse atrophy; most evident at the level of the frontal lobes. The lateral ventricles

→ Fig. 2. T1 and T2-weighted MRIs of the proband show mild cortical atrophy (A, B). The T1-weighted image shows bilateral hyperintensity associated with central hypointensity in the putamen and caudate nucleus (A). The T2-weighted image shows hypointensity associated with central hyperintensity in the putamen (B). Macroscopic view of the caudate nucleus, putamen, and globus pallidus (C). Note the grayish discoloration and microcavitation in the putamen (arrow). Microscopic section of the putamen and globus pallidus stained with Perls’ method (D). Note the accumulation of numerous iron-containing bodies and the cavitation.

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complicated by cerebellar signs, cognitive dysfunction, dyskinesias, rigidity, and pyramidal signs. Tremors: Tremors were the first to appear. They were predominantly postural and also affected the patient’s handwriting. The tremors worsened; by age 41 they had become postural and action tremors, and by age 49 they had become so severe that they interfered with activities of daily living. Cerebellar Signs: The cerebellar signs were noticed at age 47, and by age 49 she presented dysarthria, dysmetria, and ataxia; however, no nystagmus was noted. At age 56, the ataxic gait was so severe that she had difficulty in maintaining her balance. Cognitive Impairment: Clear signs of frontal and subcortical cognitive impairment were noted at age 54. These signs included disinhibition, verbal fluency, attention deficits, and difficulties in memory and mental calculation. Dyskinesia: Involuntary movement of the face, resembling tardive dyskinesia, hypertony, and cogwheel rigidity were noticed at age 55. At age 58, buccolingual dyskinesia and dystonic posture of the hands and feet were noted. Pyramidal Signs: Brisk tendon reflexes and Babinski signs were seen at age 58. Just prior to the patient’s death, the clinical status deteriorated rapidly with many of the signs and symptoms rapidly progressing. The dyskinesia and dystonia of the extremities became so severe that she was wheelchair bound and unable to feed herself. She became severely dysarthric and dysphagic. The patient had insomnia and anxiety. She was incontinent for urine and developed a urinary tract infection. Treatment with haloperidol to control the involuntary movements led to a severe rigidity, global akinesia, hypertonia, and severe resting tremor. This treatment was interrupted resulting in an improvement in the parkinsonian syndrome and a worsening of the abnormal movements. Within a short period of time, the patient became comatose and died.

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space, mononuclear cells were seen containing intranuclear bodies. The intranuclear bodies were prominent in cells of the choroidal epithelium (Fig. 6G, H). Ependymal cells appeared to be free of intranuclear bodies. Antibodies against ferritin strongly labeled intranuclear bodies of all sizes in glial cells and in nerve cells (Figs. 3C, F, 5H, 6C). Ferritin-immunopositivity was seen not only at the level of the intranuclear bodies, but also in the cytoplasm of glial cells (Fig. 3C, F). Cytoplasmic immunopositivity was diffuse in glia, but well demarcated in Purkinje cells (Fig. 5H). The ferritin-immunopositivity in nuclei and cytoplasms was present throughout the brain. In the neocortex, intranuclear and intracytoplasmic ferritin immunopositivity was present throughout the cortical layers, with the exception of layer I and II. Ferritin-immunopositive bodies were most abundant in the putamen. Occasionally, ferritin immunopositivity was seen in the leptomeningeal and parenchymal vessel walls as well as in leptomeningeal cells. Using double immunohistochemistry for ferritin and GFAP, ferritin-immunopositive bodies of various sizes were seen in the nuclei of astrocytes, whereas ferritin and GFAP immunopositivity were seen in astrocytic cytoplasms (Fig. 4F). Double immunohistochemistry for calbindin and ferritin demonstrated calbindin-immunopositive cytoplasm of Purkinje cells and ferritin-immunopositive bodies in nuclei and cytoplasm of Purkinje cells (Fig. 5I). In Purkinje cell dendrites, the ferritin immunopositive bodies were frequently seen at some distance from the perikaryon. Intranuclear and intracytoplasmic bodies were also strongly immunopositive using an antibody raised against ubiquitin. The bodies did not immunoreact with antibodies against GFAP, polyglutamine, neuroserpin, tubulin, a-synuclein, amyloid b protein, and tau. Electron Microscopy: In toluidine blue-stained sections, the bodies appeared homogeneously blue (Figs. 3E, 4B, C, 5A–C, 6B, D–F, I). In most cells, the bodies occupied a large portion of the nucleoplasm (Figs. 4D–E, 5E). In glial cells, bodies were occasionally seen in both the nucleus and cytoplasm (Fig. 5F). In the neocortex, the material composing the bodies appeared to be less densely packed. The chromatin appeared to be centrifugally displaced to varying degrees, often forming a thin layer adjacent to the nuclear membrane (Fig. 4D, E). Occasionally, a cluster of paracrystalline structures was seen within the intranuclear bodies. The size and electron density of intranuclear bodies in cerebellar granule cells varied (Fig. 5D). In electron micrographs, the bodies appear to be composed of granular electron-dense particles (Fig. 4E). The size of each particle was approximately 8.0 nm. The morphological features of the granular material were very similar to that previously reported for ferritin (18). Dermatopathology, Renal Pathology, and Muscle Pathology Dermatopathology: Epidermal cells did not contain intranuclear bodies; however, numerous fibroblasts in the

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were mildly enlarged. The caudate nucleus and cerebellum were mildly atrophic. The caudate nucleus and putamen had a grayish discoloration; small cavities could be seen in the putamen (Fig. 2C). The substantia nigra was hypopigmented. Neurohistology and Immunohistochemistry: Nerve cell loss and gliosis were mild to moderate throughout the cerebral cortex, amygdala, thalamus, substantia nigra, and locus coeruleus; they were most severe in the caudate nucleus, putamen, and globus pallidus. In the putamen, the small cavities measure up to 1.5 mm in diameter (Fig. 2D). In the cerebellum, loss of Purkinje cells was conspicuous. Moderate gliosis was present in the cerebral and cerebellar white matter. With the Heidenhain-Woelcke method for myelin, pallor of the cerebral white matter was demonstrated. The most striking pathologic alteration was the presence of intranuclear and intracytoplasmic bodies in glial cells and in some subsets of neurons (Figs. 3–6, 9). Intranuclear bodies were eosinophilic, homogenous, and could be stained using Perls’ method for iron (Figs. 2D, 3A, D, 4A, 5G, 6A, H). In Bodian preparations, the bodies were generally not argentophilic (Fig. 3B). The bodies were not stained by PAS, Alcian blue, Heidenhain-Woelcke, and they were not fluorescent in thioflavin S preparations. The bodies measured 2 mm to 35 mm in diameter. In many instances, the body occupied almost completely the nucleus and as a result displaced the chromatin up against the nuclear membrane (Figs. 3–5). Many of the nuclei containing these bodies appeared larger than normal (Fig. 3B, E). In the cerebral cortex, intranuclear bodies were seen in perineuronal satellite cells (Fig. 3A, B) and in perivascular glia (Fig. 3D). The caudate nucleus, putamen, and globus pallidus contained the largest numbers of bodies (Figs. 2D, 4). In these areas, numerous bodies were extracellular; they appeared to be larger than intracellular bodies (Fig. 4A, C) and seem to result from the coalescence of multiple bodies (Fig. 4C). Occasionally, intranuclear bodies were seen in neurons of the putamen, globus pallidus, and thalamus. In the cerebellum, intranuclear bodies were seen in glia as well as neurons (Fig. 5). Numerous Purkinje cells and granule cells contained intranuclear bodies (Fig. 5A–D, G). In Purkinje cells, the bodies could also be found in the cytoplasm of the perikaryon and dendrites (Fig. 5G). Among glial cells, the Golgi epithelial cells were most affected (Fig. 5B). In the white matter, numerous glial cells were affected; however, it was not always possible to determine if they were astrocytes or oligodendroglial cells (Fig. 6A–C). Intranuclear bodies were present in the nuclei of endothelial cells of arteries and veins (Fig. 6D–F) as well as in cells of the adventitia (Fig. 6E). Often, in the perivascular

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Fig. 3. Sections of neocortex show numerous intranuclear bodies and intracytoplasmic deposits (arrows) in glial cells (A–F). H&E (A), Bodian (B), Perls’ method (D), toluidine blue (E), and immunohistochemistry using an antibody against ferritin (C, F).

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Fig. 4. Sections of putamen show numerous bodies of various sizes (A–F). H&E (A), toluidine blue (B, C), electron micrographs (D, E) and double immunohistochemistry using antibodies against ferritin (red) and GFAP (brown) (F). Bodies may be intranuclear (arrowhead) and/or intracytoplasmic (arrow) (C). Note that the chromatin (arrow) accumulates toward the nuclear membrane (E). Note the GFAP immunoreactivity in the cytoplasm (arrowhead) and the ferritin immunoreactivity in the nucleus (arrow) (F). Ferritin immunoreactivity is also seen in the cytoplasm. J Neuropathol Exp Neurol, Vol 63, April, 2004

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least 2 polypeptides were the main constituents of the purified bodies (Fig. 7D). A peptide with 19,933.7 units of mass (peak 1) matched the estimated mass of a peptide encoded by the FTL gene. This peptide had an N-terminus that was post-translationally modified, started at position 2 (Ser) and had an N-terminal acetyl group. These results are in agreement with the data obtained by LCQ mass spectrometry. The second peptide appeared as a broader peak (peak 2) and had a molecular mass of 21,221.6 that was consistent with the predicted mass of the full-length FTH1 (21,225.6).

Biochemistry

Genetic Analysis

After purification, the final insoluble fraction consisted of a high quantity of bodies that had morphology comparable to that seen in tissue sections (Fig. 7A). In electron micrographs, the bodies appeared to be composed of granular electron-dense particles measuring approximately 8.0 nm (Fig. 7B). After solubilization of the bodies in solubilization buffer, Tris-Tricine SDS-PAGE analysis revealed a major protein with a molecular weight of ;22 kDa (Fig. 7C, lanes 3 and 4). In addition, a second band corresponding to ;44 kDa was observed. SDS-PAGE analysis of the material solubilized in formic acid showed the presence of mainly the ;22 kDa protein band (not shown). Protein extracts from a control case did not show the presence of protein bands corresponding to ;22 kDa or ;44 kDa (Fig. 7C, lane 2). Our attempts to obtain direct N-terminal amino acid sequence of the ;22 kDa peptide failed, suggesting the presence of a blocked N-terminus. Thus, the ;22 kDa protein was digested with trypsin and purified by HPLC. The mass of the tryptic peptides as determined by LCQ-mass spectrometry is shown in the Table. A peptide search of the nr protein sequence database at the National Center for Biotechnology Information retrieved positive matches for ferritin light and heavy polypeptides (Table). Two tryptic peptides were of special interest. The first was a tryptic peptide with a mass of 631.3, which matched the mass (631.7) of the amino terminal peptide of FTL (SSQIR) with an acetyl group. The acetylation of the N-terminus of the protein precluded direct sequencing by Edman degradation. The second was a tryptic peptide with a mass of 725.4, which matched the mass (725.4) of the C-terminal tryptic peptide corresponding to positions 170–175 (LTLKHD) of FTL. Western blot analyses showed that polyclonal Abs raised against ferritin specifically immunolabeled 2 proteins at ;22 and ;44 kDa (Fig. 7C, lane 5); the molecular weight of these proteins corresponded to the molecular weight of the proteins seen in Coomassie blue stain (Fig. 7C, lanes 3 and 4). The ;22 and ;44 kDa proteins represent the monomeric and dimeric forms of ferritin, respectively. The Abs did not react with the preparation from the control (not shown). MALDI-TOF mass spectrometry analysis showed that at

To determine the genetic defect associated with the disorder, we sequenced the entire coding regions and surrounding intronic sequences of the ACO1, IREB2, FTH1, and FTL genes in the proband. The ACO1 gene, located on chromosome 9, is organized into 20 exons (19). IREB2, located on chromosome 15, is organized into 23 exons (20). The coding regions and adjacent intronic sequences of the ACO1 and IREB2 genes were found to be normal. FTL and FTH1 Genes: The IRE sequences of the FTL and FTH1 genes were found to be normal. The sequence of exons 1 to 4 of the FTH1 gene, exons 1 to 3 of the FTL gene and adjacent intronic sequences of both genes were found to be normal. In exon 4 of the FTL gene, a 2-bp insertion mutation was found (Fig. 8A). The mutation consists in a thymine (T) and a cytosine (C) insertion (TC sequence) between bases 494 and 499 (498– 499InsTC). PCR products containing the mutation were cloned and sequenced in both directions, verifying the TC insertion (Fig. 8B). The same mutation was found in individuals III-11, III-12, III-24, III-25, III-27, and IV-6. The mutation was not found in individuals II-3, II-5, III22, III-23, III-26, and IV-11 as well as in 20 unrelated normal controls and after BLASTN searching of Expressed Sequence Tags (EST) databases (17). Sequencing of cloned RT-PCR products revealed that the wild-type and mutant FTL genes (Fig. 8D) and the FTH1 gene were expressed in the brain of the proband. The open reading frame (ORF) of the mutant FTL gene is predicted to be translated into a protein of 191 amino acids in length. Compared to the wild-type FTL protein of 175 amino acids, the mutant FTL protein has different amino acids at residues 167 to 175 and an additional 16 amino acids in positions 176 to 191 (Fig. 8C). Analyses of the secondary structure and the a-helical context of the mutant protein predict the loss of the C-terminal secondary structure (a-helix). Analysis of the Distribution of FTH1 and Wild-Type and Mutant FTL Polypeptides In Western blot analysis, Ab 1277 and Ab 1283 did not recognize recombinant FTH1 (Fig. 8E, lanes 1 and J Neuropathol Exp Neurol, Vol 63, April, 2004

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papillary dermis showed intranuclear bodies similar to those seen in the brain (Fig. 6I, J). Using antibodies against ferritin, the intranuclear bodies were strongly labeled (Fig. 6K). Renal Pathology: A small tissue sample containing only renal tubules, but not glomeruli, showed the presence of ferritin-immunoreactive intranuclear bodies in the tubular epithelium (Fig. 6L). Muscle Pathology: Intranuclear bodies appeared to be present in endothelial cells of muscle capillaries, but not nuclei of muscle cells.

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4), and only Ab 1277 recognized recombinant FTL (Fig. 8E, lanes 2 and 5). Both antibodies recognized the 22 kDa and 44 kDa proteins present in the isolated bodies (Fig. 8E, lanes 3 and 6). Antibodies 1277 and 1283 were also used for immunohistochemistry and gave comparable patterns of labeling (Fig. 9A, B, D, E). The pattern of labeling of intranuclear and intracytoplasmic ferritin deposits was indistinguishable from that using the anti-ferritin Abs from Dako and Biodesign. Immunohistochemistry using Ab Y-16 showed that FTH1 was also present in the deposits (Fig. 8C, F). DISCUSSION

167 to 175 and 16 additional amino acids at residues 176 to 191. As a consequence, the mutant FTL polypeptide is predicted not to have the last a-helical domain (E helix). This conformational change may have a significant impact on the structure of the FTL and the stability of the ferritin molecules. The E helix appears to stabilize the subunit conformation of ferritin by making several hydrophobic contacts with apolar side chains near the start of B helix and the end of D helix as well as by being linked via hydrogen bonds to the N-terminal ends of B and C helices (1, 22, 23). The consequences of the absence of the E helix in FTL have not been studied. Point mutations in the E helix domain of FTH1 lead to the aggregation of ferritin by either preventing its full assembly or by causing the precipitation of entirely assembled molecules (23, 24). The biochemical analysis of ferritin bodies showed that they contain mutant FTL, wild-type FTL, and FTH1 polypeptides. Electron microscopic studies of tissue sections and isolated bodies from the proband showed the presence of ;8-nm granular electron-dense particles resembling ferritin. Thus, it is possible that the mutant FTL polypeptide is a constituent of the assembled ferritin molecules. Further studies are needed to clarify this issue. Ferritin can be seen by electron microscopy as dense particles dispersed in the cytosol of the cells (18). Data regarding the presence of ferritin in the cell nucleus are limited. Ferritin has been shown to be present in the nucleus of human astrocytoma cells (25), avian corneal epithelial cells (26, 27), and cells in animal models of iron overload (28–34). Since ferritin does not have any known nuclear localization signals (NLS) and is not affected by inhibitors of the translocation of NLS-containing proteins (25), the mechanism of transport of ferritin to the nucleus and its specific role in the cell nucleus remains unknown (25, 26, 35). Using FTH1-myc-tagged constructs having C-terminal deletions and therefore no E helix, Cai and Linsenmayer (27) showed nuclear transport without supramolecular assembly of ferritin into the nucleus of corneal epithelial cells. This suggests that supramolecular assembly is not necessary for nuclear transport to occur and that transport does not need the presence of the Ehelix domain. Recently, a novel protein, ferritoid, has been found to be involved in the nuclear transport of ferritin in chicken corneal epithelial cells (36). Whether a mammalian protein homologous of ferritoid exists remains to be determined.

← Fig. 5. Sections of cerebellum show intranuclear bodies (arrows) in neurons (A–D, G–I) and glial cells (A–C, E, F) as well as intracytoplasmic bodies in Purkinje cells (G–I) and glial cells (F). Toluidine blue (A–C), electron micrographs (C–F), H&E (G), immunohistochemistry using an antibody against ferritin (H) and double immunohistochemistry using antibodies against ferritin (red) and calbindin (brown) (I).

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The current study describes a novel autosomal dominant neurodegenerative disorder associated with a mutation in exon 4 of the FTL gene and ferritin deposits in cells of the brain and other tissues. The disease, which is characterized by tremor, ataxia, parkinsonian and pyramidal signs, as well as cognitive decline, progresses gradually, first affecting striatal and cerebellar function then later cortical functions. The brain of the proband showed mild cerebral and cerebellar atrophy as well as cavitation of the putamen. Astrocytes and oligodendroglia containing intranuclear and intracytoplasmic ferritin bodies were found in gray and white matter regions. Ferritin bodies in glia were most abundant in the caudate nucleus, putamen, and globus pallidus. In these areas, nerve cell loss was most severe and led to the formation of extracellular ferritin deposits and loss of neuropil. Intranuclear ferritin bodies were present in neurons, primarily cerebellar granule cells and Purkinje cells, which also had intracytoplasmic ferritin bodies. Ferritin bodies in neurons and glia had a high content of iron. Biochemical and immunohistochemical studies revealed that ferritin bodies contained wild-type and mutant FTL as well as FTH1. In the physiological state, ferritin is composed of FTL and FTH1 (1). At the 3-dimensional level, each ferritin polypeptide (FTL and FTH1) consists of a bundle of 4 long helices, a fifth short helix and a long extended loop (1, 21). The a-helices domains, named A, B, C, D, and E, correspond approximately to residues 12 to 39, 47 to 73, 94 to 121, 126 to 155, and 162 to 175 of the amino acid sequence of FTL. The wild-type FTL polypeptide consists of 175 amino acids; whereas, the mutant FTL polypeptide described in the present report consists of 191 amino acids. As compared to wild-type FTL, the mutant polypeptide has different amino acids at residues

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← Fig. 6. Sections of the proband’s cerebral and cerebellar white matter (A–C), cerebral blood vessels (D–F), and choroid plexuses (G, H). Skin (I–K) and renal (L) biopsies of individuals IV-6 and III-17, respectively. H&E (A), toluidine blue (B, D– F, I), immunohistochemistry using antibodies against ferritin (C, G, K, L), Perls’ method (H) and electron micrograph (J). Intranuclear bodies (arrows) are demonstrated using various methods in glial cells of the white matter (A–C). Intranuclear bodies are also observed in cells of arteries (D, E) and veins (F), choroidal epithelium (G, H), skin (I–K) and kidney (L).

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Fig. 7. Eosin-stained preparation (A) and electron micrograph (B) of the post-detergent-treated pellet. Coomassie blue stained Tris-Tricine SDS-PAGE of inclusions isolated from the cerebellum (line 2) of a control and the cerebellum (line 3) and the putamen (line 4) of the proband (C). Western blot (line 3) using anti-ferritin Abs (Dako) (line 5). Low molecular-mass markers (Amersham) in kDa (line 1). Mass/charge ratio (m/z) in MALDI-MS (D).

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TABLE

FTL

FTH1

Tryptic peptide (amino acids)

Mass (m/z) m/c

2–6* 54–60 54–65 61–76 84–98 99–105 99–106 144–154 145–154 170–175 11–23 65–80 73–77

631.3/631.7 873.4/873.4 1,507.6/1,508.6 1,938.6/1,935.2 1,701.8/1,702.9 732.3/732.9 860.4/860.4 1,320.8/1,320.7 1,192.6/1,192.5 725.4/725.4 1,544.6/1,545.6 1,898.0/1,896.0 658.3/657.7

Corresponding sequence SSQIR ELAEEKR ELAEEKREGYER EGYERLLKMQNQRGGR KPAEDEWGKTPDAMK AAMALEK AAMALEKK KMGDHLTNLHR MGDHLTNLHR LTLKHD QNYHQDSEAAINR EHAEKLMKLQNQRGGR LQNQR

No. of missed cleavages 0 1 2 3 1 0 1 1 0 1 0 3 0

The mutation presented in this study is the second mutation found in the coding sequence of the FTL gene. The first mutation described is an adenine (A) insertion at position 460–461 (FTL460–461InsA) and is associated with an autosomal dominant neurodegenerative disease (5). The disorder is characterized by dystonia, low serum ferritin levels, deposition of ferritin and iron in the brain, and cavitation of the basal ganglia. Both mutations are located in exon 4 of the FTL gene and result in the production of FTL polypeptides that contain a carboxy terminus that is altered in amino-acid sequence and length. The presence of mutant C-terminal FTL polypeptides in ferritin molecules may affect the ability of ferritin to carry out its function as an iron storage protein. This, in turn, may lead to an excess of intracellular iron causing an increase in the translation of the ferritin polypeptides and accumulation of ferritin and iron (37). Based on a comparison of published reports (5, 37, 38) describing the FTL460–461InsA mutation and the data presented in this report on the FTL498–499InsTC mutation, the associated phenotypes appear to have some differences. Individuals with the FTL460–461InsA mutation clinically present, in the fifth or sixth decade of life, severe choreoathetosic, parkinsonian, and pyramidal signs, but not consistently significant cerebellar involvement or cognitive decline. Serum ferritin levels were abnormally low in the presence of normal levels of iron. Liver function was normal (5). This contrasts with the FTL498–499InsTC mutation in that individuals present in the third decade of life clinical signs that include tremor, cerebellar ataxia, and significant cognitive decline. Iron levels in the serum were normal; however, ferritin serum levels were not tested. Liver dysfunction was first J Neuropathol Exp Neurol, Vol 63, April, 2004

noted around the midpoint of the clinical course and remained present throughout the course. Pathologically, individuals with the FTL460–461InsA mutation have ironpositive ferritin-immunopositive inclusions present throughout the brain, but most abundant in the globus pallidus (5). These inclusions appear to be mainly extracellular, but are observed colocalizing with microglia and oligodendrocytes throughout the forebrain and cerebellum and appear to colocalize with neurons in the globus pallidus. This differs from the pathology seen in individuals with the FTL498–499InsTC mutation in that ferritin bodies are seen in cells of the brain and extraneural tissues. Within the CNS, both glia and neurons have intranuclear and intracytoplasmic ferritin bodies. The neurons most involved are the granule and Purkinje cells of the cerebellum. While it appears that ferritin is the primary culprit in these individuals, the impact of the abnormal accumulation of iron on neurodegeneration is not entirely clear. There are other genetic diseases in humans and one in a transgenic mouse that have as a pathologic characteristic an abnormal accumulation of iron in the brain. Two of the human diseases share some similar clinical and pathologic characteristics with those associated with mutations in FTL. These diseases are pantothenate kinase-associated neurodegeneration (PKAN), also known as Hallervorden–Spatz disease (39), and aceruloplasminemia (40), and are associated with mutations in the Pantothenate Kinase gene (PANK2) and Ceruloplasmin (CP) genes, respectively. The adult/late clinical onset variant of PKAN has a clinical phenotype that includes cerebellar ataxia, tremor, parkinsonian syndrome, and dementia similar to that seen

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Mass to charge ratio (m/z) and measured/computed masses (m/c) of tryptic peptides obtained after digestion of the purified bodies. * Indicates the presence of 1 acetyl group.

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Fig. 8. Wild-type sequence (top) and mutant sequence (bottom) of the FTL gene showing the 2-bp duplication (underlined) (A). Sequence of the mutant allele with the duplication (underlined) (B). Nucleotide and translated amino acid sequence of the C-terminal portions of wild-type and mutant FTL (C). The 2-bp insertion is in italic and underlined. The new C-terminal amino acid sequence is also underlined. RT-PCR of full-length FTL cDNA (lane 4) and a positive control (GAPD) (lane 2) (D). RNA pretreated with RNase A showed no amplification (lane 3). Markers are 123 molecular mass marker (Gibco) (lane 1) and Hind III digestion of Lambda DNA (Gibco) (lane 6). Western blot analysis using Abs 1277 (lanes 1–3) and 1283 (lanes 4–6) of recombinant FTH1 (lanes 1, 4), recombinant FTL (lanes 2, 5) and inclusions isolated from the cerebellum of the proband (lanes 3, 6) (E).

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in individuals with the FTL498–499InsTC mutation. T2weighted images of the globus pallidus and substantia nigra in PKAN and of the caudate nucleus and putamen of individuals with the FTL498–499InsTC mutation show a decreased intensity with hyperintensive areas in the internal segment. These images are consistent with increased iron and ferritin content surrounding gliosis and vacuolation. In PKAN, the basic histopathological findings are the presence of axonal spheroids containing iron, ferritin in microglial cells, Lewy bodies in neurons, and neurofibrillary tangles (39, 41); whereas, the main histopathological findings associated with the FTL498– 499InsTC mutation are the presence of ferritin and iron in astrocytes, oligodendroglia and some neuronal populations. Aceruloplasminemia is clinically characterized by adult-onset diabetes mellitus, retinal degeneration, and a neurological syndrome characterized by involuntary movements, cerebellar ataxia, and dementia (40). A T2weighted MRI of the brain reveals low signal in the caudate nucleus and putamen as well as thalamus, substantia nigra, red nucleus, and dentate nucleus, suggesting the J Neuropathol Exp Neurol, Vol 63, April, 2004

presence of iron overload (42). Neuropathologically, the caudate nucleus and putamen show severe neuronal loss and iron deposition as well as the presence of globular structures that may be enlarged astrocytic processes containing iron. Iron deposition and iron loss is mild in the neocortex. The knock-out mouse model was generated with a targeted deletion of the gene encoding the mouse iron regulatory protein 2 (Ireb2). These mice develop a movement disorder characterized by ataxia, bradykinesia and tremor (43). Adult Ireb2-deficient mice show a significant cytosolic accumulation of iron and ferritin in neurons and oligodendrocytes. The relationship among these genetic mutations, neurodegeneration, and the accumulation of ferritin and iron remains to be elucidated. Understanding these relationships may help us understand if iron accumulation is responsible for neurodegeneration in each of these diseases. Even if it is determined that iron accumulation is the final event, it would be important to elucidate the pathways that connect the genetic mutation to iron accumulation in each of these diseases.

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Fig. 9. Sections of cerebellar cortex (A–C) and cerebellar white matter (D–F). Immunohistochemistry using Ab 1277 (A, D), Ab 1283 (B, E), and antibody Y-16 (C, F). Intranuclear and intracytoplasmic immunopositivity is seen with all 3 antibodies.

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ACKNOWLEDGMENTS We gratefully acknowledge P. Piccardo, J.J. Liepnieks and M. Yazaki for useful discussions, B. Dupree, R. Richardson, B. Dennis and C. Alyea for technical help, Urs Ku¨derli for photographic assistance. The authors are grateful to Y. Lu and T. Neubert at the New York University Protein Analysis Facility, Skirball Institute, New York University School of Medicine, New York and J. Hawes at the Biochemistry Biotechnology Facility, Indiana University School of Medicine, Indianapolis, Indiana. Genbank accession numbers: FTL, X03742, X03743; FTH1, M14211, M14212; ACO1, NM002197, AF261088, RP11–334P12; IREB2, M58511, AC027228. The mutant FTL cDNA sequence has been deposited in GenBank under accession number AY466472.

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Received September 2, 2003 Revision received January 8, 2004 Accepted January 12, 2004

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