Molecular Psychiatry (1997) 2, 341–342  1997 Stockton Press All rights reserved 1359–4184/97 $12.00

ORIGINAL RESEARCH ARTICLE Table 1 Allele frequency of ApoE

Group n Allele frequency (%) Apolipoprotein E in (Chromosome) progressive supranuclear ⑀2 ⑀3 ⑀4 palsy in Japan PSP 56 10.7* 75.0 14.2 Controls 416 3.6 88.0 8.4 A Sawa1, N Amano1, N Yamada2, H Kajio2, S Yagishita3, T Takahashi4, M Oda5, N Arai6, *P Ͻ 0.05 (by ␹2 test). K Ikeda7, M Tadokoro8 and M Matsushita1

1Department of Neuropsychiatry; 23rd Department of Table 2 Age of onset and duration in the brain stems of the Internal Medicine, Faculty of Medicine, University of Tokyo, PSP patients with differing ApoE alleles Tokyo 113, Japan; 3Department of Pathology, The Kanagawa Rehabilitation Center; 4Department of Age of Neurology, Yokohama City University School of Medicine; 5Department of Pathology and Laboratory Medicine, Tokyo Onset Duration Metropolitan Neurological Hospital; 6Department of Clinical (year) (year) Neuropathology, Tokyo Metropolitan Institute for ⑀ Neuroscience; 7Department of Neuropathology, Tokyo 2 Bearing 55.3 8.7 ⑀ Institute of Psychiatry; 8Department of 1st Pathology, St 4 Bearing 60.3 7.8 Only ⑀3 59.0 8.4 Marianna University School of Medicine, Japan

Keywords: Apolipoprotein E; progressive supranuclear palsy (PSP); Alzheimer ⑀4/⑀4 patient in our study. No significant increase in ⑀4 allele frequency was found in the present series of Progressive supranuclear palsy (PSP) is a rare neuro- PSP cases. degenerative disease which shows several psychiatric We next examined the age of onset and duration in and neurologic symptoms: pseudobulbar palsy, supra- ⑀ + nuclear ocular palsy, extrapyramidal rigidity, gait ataxia, each group. They were almost the same between 4 ⑀ − and dementia.1 Almost all cases seem to be sporadic; and 4 patients (Table 2). Finally we examined NFT therefore, the elucidation of risk factors is important to accumulations in several regions of the brain stem in clarify the pathological mechanism. Apolipoprotein E4 PSP cases. We evaluated the degree of accumulation as (ApoE4) is now well established as a risk factor of Alz- 0, 1, 2, or 3. The value 1 means that the number of heimer’s disease (AD). Here we report the ApoE allele NFT-bearing neurons is less than 5 in one field at 100× frequency in PSP, which shares pathological findings magnification. The value 2 represents the number from such as neurofibrillary tangle (NFT) with AD. NFT is an 5 to 10, and the value 3 is used for more than 10 NFT- important sign for the derangement of normal cytoskele- bearing neurons. The value 0 means no NFT. There tons in degenerating neurons. Although there was no were no differences betwen ⑀4+ and ⑀4− patients in significant increase in ⑀4 allele frequency in the present NFT accumulations in substantia nigra, subthalamic series of PSP cases compared with that in the Japanese ⑀ nucleus, inferior olivary nucleus, and dentate nucleus controls, there was a significant increase in the 2 allele ⑀ frequency in PSP compared to controls. (Table 3). These three findings indicate that 4 allele is Twenty-eight cases of PSP were analyzed. The diag- not a risk factor for PSP and may suggest that ApoE4 nosis of PSP was made neuropathologically based on is not involved in NFT formation. ApoE4 has already the characteristic distribution of NFTs in the brain stem and basal ganglia. By immunohistochemistry, all the brains were found to contain few senile plaques Table 3 NFT accumulation in the brain stems in PSP patients with differing ApoE alleles or Lewy bodies. Our criteria basically accord with the NINDS standard.2 There have been two ApoE genotyp- Accumulation of NFT ing studies on PSP.3,4 They found no increase in the ⑀4 allele frequency,3 or only a slight increase.4 In these substantia subthalamic two reports, cases showing high densities of senile nigra nucleus plaques were included, suggesting that some may be complicated with AD. Thus, the sample size of auth- ⑀2 Bearing 1.6 ± 0.6 1.8 ±0.5 entic PSP in their studies is only 10. Our larger sample ⑀4 Bearing 1.3 ± 1.0 1.5 ± 0.5 size is better suited for this type of study. Allele fre- Only ⑀3 1.2 ± 0.6 1.5 ± 0.7 quencies of ⑀2, ⑀3, and ⑀4 were 10.7%, 75.0%, and 14.2% in PSP, and 3.6%, 88.0%, and 8.4% in age- The extent of NFT accumulation is quantitated as: 1, less matched Japanese controls5 (Table 1). Every ⑀4-bearing abundant; 2, abundant; 3, fully abundant. The means with individual was ⑀3/⑀4. There was no ⑀2/⑀4, ⑀2/⑀2, or standard deviations are shown in this table. Apolipoprotein E in PSP in Japan A Sawa et al 342 been established as a risk factor of AD.6,7 PSP and AD The genotyping in the 208 age-matched Japanese con- share the same neuropathological characteristics, cyto- trols was performed in our previous study by this iso- skeletal derangement which leads to tau dissociation electric focusing method.5 Differences in the frequency from microtubules and the formation of NFT.8,9 Our of ApoE genotype between the groups were evaluated results suggest that ⑀4 may not affect their cytoskeletal by the ␹2 test. The individual alleles were tested separ- derangement. The accumulation of senile plaques is ately. unique in AD. It is more probable to attribute ⑀4 effect to modulation of this plaque accumulation. In fact, the References aggregation of ␤ protein, the main component of senile plaques, is affected by ApoE4 in vitro.10 1 Steele JC, Richardson JC, Olszewski J. Progressive supranuclear On the other hand, there was a significant increase palsy. A heterogeneous degeneration involving the brain stem, ⑀ basal ganglia and cerebellum with vertical gaze and pseudobulbar in the 2 allele frequency in PSP compared to the con- palsy, nuchal dystonia and dementia. Arch Neurol 1964; 10: 4,11 trols in this and another study (Table 1). The age of 333–359. onset was slightly earlier in ⑀2+ patients compared 2 Hauw J-J, Daniel SE, Dickson D, Horoupian DS, Jellinger K, Lantos with ⑀2− patients, although the duration of the illness PL, McKee A, Tabaton M, Litvan I. Preliminary NINDS neuro- pathologic criteria for Steel–Richardson–Olszewski syndrome was similar (Table 2). To the extent of our sample size, (progressive supranuclear palsy). Neurology 1994; 44: 2015–2019. no statistically significant difference was found in age 3 Tabaton M, Rolleri M, Masturzo P, Cammarata S, Angelini G, of onset, duration, and number of NFTs between ⑀2+ Hansen LA, Saito T, Petersen RB, Perry G, Richey P, Gambetti P, and ⑀2− patients. We also examined the allele fre- Bertolini S. Apolipoprotein E ⑀4 allele frequency is not increased quency in eight living PSP patients, and two of them in progressive supranuclear palsy. Neurology 1995; 45: 1764–1765. ⑀ ⑀ 4 Schneider JA, Gearing M, Robbins RS, de l’Aune W, Mirra SS. Apo- were found to have 2 allele. The role of 2 is still con- lipoprotein E genotype in diverse neurodegenerative disorder. Ann troversial. van Duijn et al suggested that ⑀2 may be a Neurol 1995; 38: 131–135. risk factor for AD,12 while others have reported the pro- 5 Noguchi S, Murakami K, Yamada N. Apolipoprotein E genotype tective effect of ⑀2.13 Recently a remarkable elevation and Alzheimer’s disease. Lancet 1993; 342: 737–738. ⑀ 6 Namba Y, Tomonaga M, Kawasaki H, Otomo E, Ikeda K. Apolipo- of 2 allele was found in a subset of very elderly protein E immunoreactivity in cerebral amyloid deposits and neur- patients with senile dementia, in whom abundant ofibrillary tangles in Alzheimer’s disease and kuru plaque amyloid NFTs are present without an increase of senile pla- in Creutzfeldt–Jakob disease. Brain Res 1991; 541: 163–166. ques.14 This type of dementia and PSP in our report 7 Strittmatter WJ, Saunders AM, Schmechel D, Pericak-Vance M, share an important characteristic, cytoskeletal derange- Enghild J, Salvesen GS, Roses AD. Apolipoprotein E: high-avidity binding to ␤-amyloid and increased frequency of type 4 allele in ment which leads to NFT without the remarkable sen- late-onset familiar Alzheimer disease. Proc Natl Acad Sci USA ile plaque accumulation. ⑀2 allele frequency is slightly 1993; 90: 1977–1981. lower in Japanese than in Caucasians. Even if we take 8 Joachim CL, Morris JH, Kosik KS, Selkoe DJ. Tau antisera recognize account of this, the significant elevations of ⑀2 allele neurofibrillary tangles in a range of neurodegenerative disorders. ⑀ Ann Neurol 1987; 22: 514–520. frequency in these two dementias suggest that 2 may 9 Iwatsubo T, Hasegawa M, Ihara Y. Neuronal and glial tau-positive be implicated in neurodegeneration. inclusions in diverse neurologic disease share common phos- In summary, we found no association of PSP with phorylation characteristic. Acta Neuropathol 1994; 88: 129–136. ⑀4, but significant association with ⑀2. 10 Ma J, Yee A, Brwer Jr HB, Das S, Potter H. Amyloid-associated proteins a1-antichymotypsin and apolipoprotein E promote assembly of Alzheimer ␤-protein into filaments. Nature 1994; 372: 92–94. Methods 11 Muramatsu T, Higuchi S, Arai H, Sasaki H, Yamada K, Hayashida M, Trojanowski JQ. Apolipoprotein E ⑀4 allele distribution in The neuropathological diagnosis and assessment of 28 alcoholic dementia and in Alzheimer’s disease in Japan. Ann Neu- cases of PSP were performed by one neuropathologist rol 1994; 36: 797–799. (NA). Genomic DNAs were extracted from formalin- 12 van Duijn CM, Knijff P, Wehnert A, Voecht JD, Bronzova JB, Havekes LM, Hofman A, Broeckhoven CV. The apolipoprotein E fixed paraffin-embedded sections of the PSP brains, ⑀ 15 2 allele is associated with an increased risk of early-onset Alzhei- and amplified by polymerase chain reaction. The pri- mer’s disease and a reduced survival. Ann Neurol 1995; 37: 605– mers used in our study were 5′-TAAGCTTGGCTGGGC 610. GCGGACATG-3′ (corresponding to bases 422–438), 5′- 13 Benjamin R, Leake A, McArthur FK, Ince PG, Candy JM, Edward- ACAGAATTCGCCGCGGTACTGCAC-3′ (corresponding son JA, Morris CM, Bjertness E. Protective effect of apoE epsilon 2 to bases 476–460) for discrimination of ⑀4, and 5′-ATT in Alzheimer’s disease. Lancet 1994; 344: 473. ′ 14 Ikeda K, Akiyama H, Arai T, Sahara N, Mori H, Usami M, Sakata CGAACGTAAGCGGCTCCTCCGC-3 (corresponding to M, Mizutani T, Wakabayashi K, Takahashi H. A subset of senile bases 547–564), 5′-TGTCTTAAGGCCCCGGCCTGGTA dementia with high incidence of the ApoE ⑀2 allele. Ann Neurol CAC-3′ (corresponding to bases 611–595) for discrimi- (in press). nation of ⑀2. These primers are designed as comp- 15 Wright DK, Manos MM. Sample preparation from paraffin-embed- ded tissues. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds). lementary nucleotides of corresponding ApoE PCR Protocols: a Guide to Methods and Applications. Academic sequence with several additional nucleotides at their Press: San Diego, 1990, pp 153–158. 5′ ends to make the reaction easier. Amplified frag- 16 Hixson JE, Vernier DT. Restriction isotyping of human apolipo- ments were digested by HhaI, followed by gel electro- protein E by gene amplification and cleavage with Hha I. J Lipid phoretic analysis.16 The serum of those who were clini- Res 1990; 31: 545–548. cally diagnosed as PSP was examined by 1-D, flat-gel, Correspondence: M Matsushita MD, 7-3-1 Hongo Bunkyo-ku, Tokyo iso-electric focusing using a commercial kit 113, Japan (Phenotyping ApoE IEF System, Joko, Tokyo, Japan). Received 26 March 1997; revised and accepted 8 April 1997 Apolipoprotein E in PSP in Japan A Sawa et al 343