Neurodegeneration with Brain Iron Accumulation, Type 1 Is Characterized by α-, β-, and γ-Synuclein Neuropathology
2000; Elsevier BV; Volume: 157; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)64548-8
ISSN1525-2191
AutoresJames E. Galvin, Benoit I. Giasson, Howard I. Hurtig, Virginia M.‐Y. Lee, John Q. Trojanowski,
Tópico(s)RNA regulation and disease
ResumoNeurodegeneration with brain iron accumulation, type 1 (NBIA 1), or Hallervorden-Spatz syndrome, is a rare neurodegenerative disorder characterized clinically by Parkinsonism, cognitive impairment, pseudobulbar features, as well as cerebellar ataxia, and neuropathologically by neuronal loss, gliosis, and iron deposition in the globus pallidus, red nucleus, and substantia nigra. The hallmark pathological lesions of NBIA 1 are axonal spheroids, but Lewy body (LB)-like intraneuronal inclusions, glial inclusions, and rare neurofibrillary tangles also occur. Here we show that there is an accumulation of α-synuclein (αS) in LB-like inclusions, glial inclusions, and spheroids in the brains of three NBIA 1 patients. Further, β-synuclein (βS) and γ-synuclein (γS) immunoreactivity was detected in spheroids but not in LB-like or glial inclusions. Western blot analysis demonstrated high-molecular weight αS aggregates in the high-salt-soluble and Triton X-100-insoluble/sodium dodecyl sulfate-soluble fraction of the NBIA 1 brain. Significantly, the levels of αS were markedly reduced in the Triton X-100-soluble fractions compared to control brain, and unlike other synucleinopathies, insoluble αS did not accumulate in the formic acid-soluble fraction. These findings expand the concept of neurodegenerative synucleinopathies by implicating αS, βS, and γS in the pathogenesis of NBIA 1. Neurodegeneration with brain iron accumulation, type 1 (NBIA 1), or Hallervorden-Spatz syndrome, is a rare neurodegenerative disorder characterized clinically by Parkinsonism, cognitive impairment, pseudobulbar features, as well as cerebellar ataxia, and neuropathologically by neuronal loss, gliosis, and iron deposition in the globus pallidus, red nucleus, and substantia nigra. The hallmark pathological lesions of NBIA 1 are axonal spheroids, but Lewy body (LB)-like intraneuronal inclusions, glial inclusions, and rare neurofibrillary tangles also occur. Here we show that there is an accumulation of α-synuclein (αS) in LB-like inclusions, glial inclusions, and spheroids in the brains of three NBIA 1 patients. Further, β-synuclein (βS) and γ-synuclein (γS) immunoreactivity was detected in spheroids but not in LB-like or glial inclusions. Western blot analysis demonstrated high-molecular weight αS aggregates in the high-salt-soluble and Triton X-100-insoluble/sodium dodecyl sulfate-soluble fraction of the NBIA 1 brain. Significantly, the levels of αS were markedly reduced in the Triton X-100-soluble fractions compared to control brain, and unlike other synucleinopathies, insoluble αS did not accumulate in the formic acid-soluble fraction. These findings expand the concept of neurodegenerative synucleinopathies by implicating αS, βS, and γS in the pathogenesis of NBIA 1. Neurodegeneration with brain iron accumulation (NBIA 1), also known as Hallervorden-Spatz syndrome or adult neuroaxonal dystrophy, is a rare familial and sporadic neurodegenerative disorder.1Halliday W The nosology of Hallervorden-Spatz disease.J Neurol Sci. 1995; 134: 84-91Abstract Full Text PDF PubMed Scopus (63) Google Scholar, 2Jellinger K Neuroaxonal dystrophy: its natural history and related disorders.in: Zimmerman HM Progress in Neuropathology. vol 2. Grune and Stratton, Inc., New York1973: 129-180Google Scholar, 3Dooling EC Schoene WC Richardson EP Hallervorden-Spatz syndrome.Arch Neurol. 1974; 30: 70-83Crossref PubMed Scopus (244) Google Scholar Symptoms usually present in late adolescence or early adult life2Jellinger K Neuroaxonal dystrophy: its natural history and related disorders.in: Zimmerman HM Progress in Neuropathology. vol 2. 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NBIA 1 is characterized clinically by rigidity, dystonia, dyskinesia, and choreoathetosis,5Taylor TD Litt M Kramer P Pandolfo M Angelini L Nardocci N Davis S Pineda M Hattori H Flett PJ Cilio MR Bertini E Hayflick SJ Homozygosity mapping of Hallervorden-Spatz syndrome to chromosome 20p12.3-p13.Nat Genet. 1996; 14: 479-481Crossref PubMed Scopus (140) Google Scholar, 6Maladrini A Cesaretti S Mulinari M Palmeri S Fabrizi GM Villanova M Parrotta E Matagnani A Montagnani M Anichini M Guazzi GC Acanthosytosis, retinitis pigmentosa, pallidal degeneration. Report of two cases without serum lipid abnormalities.J Neurol Sci. 1996; 140: 129-131Abstract Full Text PDF PubMed Scopus (27) Google Scholar, 7Sugiyama H Hainfellner JA Schid-Siegal B Budka H Neuroaxonal dystrophy combined with diffuse Lewy body disease in a young adult.Clin Neuropathol. 1993; 12: 147-153PubMed Google Scholar, 8Swaiman KF Hallervorden-Spatz syndrome and brain iron metabolism.Arch Neurol. 1991; 48: 1285-1293Crossref PubMed Scopus (194) Google Scholar as well as dysarthria, dysphagia, ataxia, and dementia.2Jellinger K Neuroaxonal dystrophy: its natural history and related disorders.in: Zimmerman HM Progress in Neuropathology. vol 2. Grune and Stratton, Inc., New York1973: 129-180Google Scholar, 3Dooling EC Schoene WC Richardson EP Hallervorden-Spatz syndrome.Arch Neurol. 1974; 30: 70-83Crossref PubMed Scopus (244) Google Scholar, 4Jankovic J Kirkpatrick JB Blomquist KA Langlias PJ Bird ED Late onset Hallervorden-Spatz disease presenting as familial parkinsonism.Neurology. 1985; 35: 227-234Crossref PubMed Google Scholar, 8Swaiman KF Hallervorden-Spatz syndrome and brain iron metabolism.Arch Neurol. 1991; 48: 1285-1293Crossref PubMed Scopus (194) Google Scholar NBIA 1 also may be associated with other clinical manifestations such as spasticity and extensor plantar responses,1Halliday W The nosology of Hallervorden-Spatz disease.J Neurol Sci. 1995; 134: 84-91Abstract Full Text PDF PubMed Scopus (63) Google Scholar, 2Jellinger K Neuroaxonal dystrophy: its natural history and related disorders.in: Zimmerman HM Progress in Neuropathology. vol 2. 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25: 363-368Crossref PubMed Scopus (67) Google Scholar whereas in late onset NBIA 1, tau pathology has been demonstrated14Eidelberg D Sotrel A Joachim C Selkoe D Forman A Pendlebury WW Perl DP Adult onset Hallervorden-Spatz disease with neurofibrillary pathology.Brain. 1987; 110: 993-1013Crossref PubMed Scopus (65) Google Scholar, 22Wakabayashi K Fukushima T Koide R Horikawa Y Hasegawa M Watanabe Y Noda T Eguchi I Morita T Yoshimoto M Iwatsubo T Takahasi H Juvenile-onset generalized neuroaxonal dystrophy (Hallervorden-Spatz disease) with diffuse neurofibrillary and Lewy body pathology.Acta Neuropathol. 2000; 99: 331-336Crossref PubMed Scopus (51) Google Scholar, 23Wisniewski K Jervis GA Moretz RC Wisniewski HM Alzheimer neurofibrillary tangles in diseases other than senile and presenile dementia.Ann Neurol. 1979; 5: 288-294Crossref PubMed Scopus (228) Google Scholar consisting of both paired helical filaments and straight filaments without amyloid β-protein (Aβ) deposition. In this study, we examined three cases of NBIA 1 immunohistochemically with antibodies to NFs, purified LBs, tau, Aβ, APP, αS, as well as β-synuclein (βS) and γ-synuclein (γS). Moreover, we also mapped topographically-distinct epitopes extending throughout αS in lesions that were immunostained by anti-αS antibodies, and we demonstrated species of normal and abnormal αS in the NBIA 1 brain by Western blot analysis. Three autopsy proven cases (one infantile, one adult, one late-onset) of NBIA 1 were obtained from the brain banks at the Center for Neurodegenerative Disease Research, Case Western Reserve University, and the Universities of Miami and Maryland (see Table 1 for clinical demographics). Tissue was fixed in either 10% neutral buffered formalin or 70% ethanol/150 mmol/L NaCl and paraffin-embedded. The following brain regions were examined: motor cortex, sensory cortex, midfrontal lobe, orbitofrontal lobe, cingulate gyrus, superior temporal gyrus, amygdala, hippocampus (anterior and posterior), visual cortex, insula, caudate, putamen, globus pallidus, nucleus accumbens, basal forebrain, thalamus, hypothalamus, midbrain/substantia nigra, pons/locus ceruleus, medulla, cervical spinal cord, olfactory bulb, and trigeminal ganglion when available. Serial sections of 6 μm thickness were analyzed.Table 1Clinical Demographics and Neuropathology of NBIA 1 CasesCaseAge at onsetAge at death (years)Cause of deathFamily historyPresenting symptomOther featuresPMI (hrs)Brain wt (grams)Macroscopic and microscopic neuropathologyUniv. of Miami14 months8Aspiration pneumoniaNOLoss of developmental milestonesFlaccidity, hearing loss, muscle cramps, dementia, dysphagia, seizures51024Severe atrophy, mild ventricular enlargement, severe neuronal loss in spinal cord, iron deposition, spheroids, NCIs, GCIsCNDR18 years29InanitionNOTremorParkinsonism, optic atrophy, dementia, dysphagia, dysarthria, spasticity, pes cavus61080Severe atrophy, mild ventricular enlargement, depigmentation of locus ceruleus, iron deposition, spheroids, NCIs, GCIs, glial tanglesCase Western Reserve75 years77Aspiration pneumoniaNOConfusionDementia, behavioral abnormalities601530No atrophy, severe ventricular enlargement, small cortical infarct, iron deposition, spheroids, NCIs, GCIsPMI, postmortem interval; NCI, neuronal cytoplasmic inclusions; GCI, glial cytoplasmic inclusion; CNDR, Center for Neurodegenerative Disease Research. Open table in a new tab PMI, postmortem interval; NCI, neuronal cytoplasmic inclusions; GCI, glial cytoplasmic inclusion; CNDR, Center for Neurodegenerative Disease Research. Histochemical studies were performed using hematoxylin and eosin (H&E) and silver stains whereas immunohistochemistry was performed using well-characterized antibodies as described.24Galvin JE Lee VMY Baba M Mann DMA Dickson DW Yamaguchi H Schmidt ML Iwatsubo T Trojanowski JQ Monoclonal antibodies to purified cortical Lewy bodies recognize the mid-size neurofilament subunit.Ann Neurol. 1997; 42: 595-603Crossref PubMed Scopus (45) Google Scholar, 25Galvin JE Uryu K Lee VMY Trojanowski JQ Axon pathology in Parkinson's disease and Lewy body dementia hippocampus contains α-, β- and γ-synuclein.Proc Natl Acad Sci USA. 1999; 96: 13450-13455Crossref PubMed Scopus (376) Google Scholar The antibodies used here included those specific for NF subunits (RMdO20, NFL),26Schmidt ML Murray J Lee VMY Hill WD Werkin A Trojanowski JQ Epitope map of neurofilament protein domains in cortical and peripheral nervous system Lewy bodies.Am J Pathol. 1991; 139: 53-65PubMed Google Scholar ubiquitin (mAb 1510, Chemicon, Temecula, CA),27Shaw G Chau V Ubiquitin and microtubule-associated protein tau immunoreactivity each define distinct structures with differing distributions and solubility properties in Alzheimer brain.Proc Natl Acad Sci USA. 1988; 85: 2854-2858Crossref PubMed Scopus (56) Google Scholar purified cortical LBs (LB48),24Galvin JE Lee VMY Baba M Mann DMA Dickson DW Yamaguchi H Schmidt ML Iwatsubo T Trojanowski JQ Monoclonal antibodies to purified cortical Lewy bodies recognize the mid-size neurofilament subunit.Ann Neurol. 1997; 42: 595-603Crossref PubMed Scopus (45) Google Scholar αS (LB509, Syn204, Syn208, SNL1),28Jakes R Crowther A Lee VMY Trojanowski JQ Iwatsubo T Goedert M Epitope mapping of LB509, a monoclonal antibody directed against human α-synuclein.Neurosci Lett. 1999; 269: 13-16Crossref PubMed Scopus (91) Google Scholar, 29Giasson BI Ross J Goedert M Duda JE Leight S Trojanowski JQ Lee VMY A panel of epitope-specific antibodies detect protein domains distributed throughout human α-synuclein in Lewy bodies of Parkinson's disease.J Neurosci Res. 2000; 59: 528-533Crossref PubMed Scopus (194) Google Scholar αS/βS (Syn202, Syn205, Syn214, SNL4),29Giasson BI Ross J Goedert M Duda JE Leight S Trojanowski JQ Lee VMY A panel of epitope-specific antibodies detect protein domains distributed throughout human α-synuclein in Lewy bodies of Parkinson's disease.J Neurosci Res. 2000; 59: 528-533Crossref PubMed Scopus (194) Google Scholar βS (Syn207),18Tu PH Galvin JE Baba M Giasson B Tomita T Leight S Nakajo S Iwatsubo T Trojanowski JQ Lee VM-Y Glial cytoplasmic inclusions in white matter oligodendrocytes of multiple systems atrophy brains contain insoluble α-synuclein.Ann Neurol. 1998; 44: 415-422Crossref PubMed Scopus (603) Google Scholar γS (antisera20),18Tu PH Galvin JE Baba M Giasson B Tomita T Leight S Nakajo S Iwatsubo T Trojanowski JQ Lee VM-Y Glial cytoplasmic inclusions in white matter oligodendrocytes of multiple systems atrophy brains contain insoluble α-synuclein.Ann Neurol. 1998; 44: 415-422Crossref PubMed Scopus (603) Google Scholar tau (17026),30Hong M Zhukareva V Vogelsberg-Ragaglia V Wszolek Z Reed L Miller BI Geschwind DH Bird TD McKeel D Goate A Morris JC Wilhelmsen KC Schellenberg GD Trojanowski JQ Lee VMY Mutation-specific functional impairments in distinct tau isoforms of hereditary FTDP-17.Science. 1998; 82: 1914-1917Crossref Scopus (836) Google Scholar APP (LN39),31Arai H Lee VMY Messinger ML Greenberg BD Lowery DE Trojanowski JQ Expression patterns of β-amyloid precursor protein (β-APP) in neural and non-neural human tissues from Alzheimer's disease and control subjects.Ann Neurol. 1991; 30: 686-693Crossref PubMed Scopus (140) Google Scholar and Aβ (2332).32Schmidt ML DiDario AG Otvos L Hoshi N Kant A Lee VMY Trojanowski JQ Plaque associated neuronal proteins: a recurrent motif in neuritic amyloid deposits throughout diverse cortical areas of the Alzheimer's disease brain.Exp Neurol. 1994; 130: 311-322Crossref PubMed Scopus (30) Google Scholar Sections were probed by immunohistochemistry, developed using the avidin-biotin-peroxidase complex method (Vector Labs, Burlingame, CA) and then lightly counterstained with hematoxylin. Sections were examined and graded semiquantitatively using the following system: 0 = no pathology, 1 = rare pathology, 2 = mild pathology, 3 = moderate pathology, and 4 = severe pathology. Each section was analyzed for synuclein IR spheroids, LBs, NCIs, GCIs, and DNs as well as for tau and amyloid pathologies. Western blotting and biochemical fractionation were performed as described previously.29Giasson BI Ross J Goedert M Duda JE Leight S Trojanowski JQ Lee VMY A panel of epitope-specific antibodies detect protein domains distributed throughout human α-synuclein in Lewy bodies of Parkinson's disease.J Neurosci Res. 2000; 59: 528-533Crossref PubMed Scopus (194) Google Scholar, 33Lippa CF Fujiwara H Mann DMA Giasson B Baba M Schmidt ML Nee LE O'Connell B Pollen DA George-Hyslop P Ghetti B Nochlin D Bird TD Cairns NJ Lee VMY Iwatsubo T Trojanowski JQ Lewy bodies contain altered α-synuclein in brains of many familial Alzheimer's disease patients with mutations in presenilin and amyloid precursor protein genes.Am J Pathol. 1998; 153: 1365-1370Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar Briefly, gray matter (0.3 gram) from the midfrontal cortex of NBIA 1 (Case Western Reserve) and a normal age-matched control brain was homogenized in 2 ml/gram of tissue of high-salt (HS) buffer (50 mmol/L Tris, pH 7.4, 750 mmol/L NaCl, 10 mmol/L NaF, 5 mmol/L ethylenediaminetetraacetic acid with protease inhibitors) and centrifuged at 100,000 ×g for 30 minutes. The pellets were re-extracted and the supernatants were pooled. The pellets were sequentially extracted twice with 2 ml/gram of HS buffer/1% Triton X-100 (HS/T) and once with 1 ml/gram sodium dodecyl sulfate (SDS) sample buffer (1% SDS, 10% sucrose, 10 mmol/L Tris, pH 6.8, 1 mmol/L ethylenediaminetetraacetic acid, 40 mmol/L dithiothreitol). The pellets were extracted with 0.67 ml/gram 70% formic acid (FA) and disrupted with two sequential 2-second sonication bursts. FA was evaporated in an Automatic Environment SpeedVAc System (Savant Instruments, Holbrook, NY). SDS sample buffer (0.67 ml/gram) was added to the dried pellets, followed by vigorous vortex, and the pH was adjusted with NaOH. SDS sample buffer was added to the HS and HS/Triton fraction and all of the samples were boiled for 5 minutes. The FA samples were centrifuged at 13,000 × g for 5 minutes to remove insoluble debris. Five μl of each fraction was loaded in separate lanes for SDS-polyacrylamide gel electrophoresis followed by Western blot analysis.29Giasson BI Ross J Goedert M Duda JE Leight S Trojanowski JQ Lee VMY A panel of epitope-specific antibodies detect protein domains distributed throughout human α-synuclein in Lewy bodies of Parkinson's disease.J Neurosci Res. 2000; 59: 528-533Crossref PubMed Scopus (194) Google Scholar, 33Lippa CF Fujiwara H Mann DMA Giasson B Baba M Schmidt ML Nee LE O'Connell B Pollen DA George-Hyslop P Ghetti B Nochlin D Bird TD Cairns NJ Lee VMY Iwatsubo T Trojanowski JQ Lewy bodies contain altered α-synuclein in brains of many familial Alzheimer's disease patients with mutations in presenilin and amyloid precursor protein genes.Am J Pathol. 1998; 153: 1365-1370Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar Two of the three brains exhibited severe atrophy with mildly dilated ventricles. The third brain (late onset) had marked ventricular dilatation without significant atrophy (see Table 1). All of the cases examined had iron accumulation in the globus pallidus, substantia nigra, red nucleus, and dentate nucleus whereas microscopic evaluation revealed neuronal loss with gliosis. The infantile onset case also displayed marked loss of anterior horn cells in cervical and lumbar spinal cord. Dystrophic axon spheroids were noted with H&E and silver stains in the basal ganglia and brainstem in all three cases. αS pathology was seen throughout all three NBIA 1 brains (Figure 1). For example, αS IR NCIs and spheroids were abundant in the globus pallidus, putamen, thalamus, midfrontal gyrus, precentral gyrus, postcentral gyrus, amygdala, and entorhinal cortex (Figure 1, A–H). In addition, most cortical regions displayed numerous αS-positive DNs, but they were most prominent in the CA2/3 region of the hippocampus (Figure 1I). Antibodies specific for βS and γS labeled axonal spheroids but not NCIs or DNs. Panels J to M in Figure 1 illustrate representative βS IR spheroids in the cingulate gyrus, inferior olive, cervical spinal cord, and putamen, respectively, whereas panels N to O in Figure 1 show typical γS IR spheroids in the cingulate and midfrontal
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