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Alteration in Brain Presenilin 1 mRNA Expression in Early Onset Familial Alzheimer's Disease
NEURODEGENERATION, Vol. 5, pp 213–218 (1996)
Alteration in Brain Presenilin 1 mRNA Expression in Early Onset Familial Alzheimer’s Disease Amanda J.L. Barton,1 Barry W. Crook,1 Eric H. Karran,1 Frank Brown,1 Deborah Dewar,2 David M.A. Mann,3 R. Carl A. Pearson,4 David I. Graham,5 John Hardy,6 Mike Hutton,6 Karen Duff,6 Alison M. Goate,7 Robert F. Clark7 and Gareth W. Roberts1 1
Department of Molecular Neuropathology Research, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW; 2Wellcome Surgical Institute and Hugh Fraser Neuroscience Laboratories, University of Glasgow, Garscube Estate, Bearsden Road, Glasgow G61 1QH; 3Department of Pathological Sciences, Stopford Building, The University of Manchester, Oxford Road, Manchester M13 9PT; 4Department of Biomedical Science, University of Sheffield, Sheffield S10 2NT; 5Institute of Neurological Sciences , Southern General Hospital NHS Trust, Glasgow, G51 4TF; 6Suncoast Alzheimer’s Disease Laboratory, University of South Florida, Tampa FL33613, USA; 7Department of Psychiatry, Washington University School of Medicine, St Louis, MO 63110, USA The expression of the presenilin 1 (PS-1) gene has been investigated by in situ hybridization in early onset familial Alzheimer’s disease (FAD), late onset Alzheimer’s disease (AD) and normal control brain. Mutations in this gene are responsible for chromosome 14-linked FAD. We have found that presenilin 1 mRNA is present throughout the human brain with a distribution consistent with both a glial and neuronal localization. The in situ hybridization pattern was similar for the controls, the early onset FAD cases and the late onset AD cases. However, one of the two forms of the mRNA for PS-1, the long form (which contains a sequence encoding a four amino acid (VRSQ) insert at its 59 end) was significantly reduced in early onset FAD brain compared with late onset AD. We suggest that this long transcript may alter the normal pathway for processing of amyloid precursor protein, the protein which appears to be central in the pathogenesis of AD. © 1996 Academic Press Limited
Key words: familial Alzheimer’s disease, human brain; in situ hybridization, presenilin 1 mRNA
A NOVEL GENE on chromosome 14, termed presenilin 1 (PS-1) or S182, has recently been identified by molecular genetic studies as being responsible for chromosome 14-linked familial Alzheimer’s disease (FAD) (Rogaev et al., 1995; Sherrington et al., 1995). To date, over 26 FAD associated mutations have been found in this gene (Campion et al., 1995; Clark et al., 1995; Cruts et al., 1995; Sherrington et al., 1995; Sorbi et al., 1995; Tanahashi et al., 1995; Van Broeckhoven, 1995; Wasco
et al., 1995). More recently association has been observed between homozygosity for allele 1 of an intronic polymorphism in the PS-1 gene and increased risk for late onset Alzheimer’s disease (AD) (Wragg et al., 1996). The normal function of PS-1, as well as its role in the pathogenesis of AD, are not known. Sherrington et al. (1995) have demonstrated a wide distribution of PS-1 mRNA expression throughout the normal human brain as well as in other tissues. Analysis of PS-1 mRNA expression in the brains of individuals affected by AD has not been described previously. Recent work has identified two different transcripts of PS-1 (Clark et al., 1995; Cruts et al., 1995). One of these corresponds to the full exon sequence while the
Correspondence to: Dr Amanda J.L. Barton; email: [email protected] Received 22 May 1996; accepted for publication 5 June 1996 © 1996 Academic Press Limited 1055-8330/96/030213 1 6 $18.00/0
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other lacks a sequence encoding the four amino acids valine (V), arginine (R), serine (S) and glutamine (Q): these have been designated PS-1long and PS-1short respectively. In this study we have examined the expression of these two PS-1 mRNA transcripts in the brains of patients dying with early onset FAD. In situ hybridization (ISH) was used to determine the qualitative and quantitative pattern of expression of PS-1 mRNA in the brains of three early onset (presumptive chromosome 14-linked) FAD cases; comparisons with brains from patients with late onset AD and from normal individuals were made.
Materials and Methods In Situ hybridization PS1 mRNA expression was examined in 4 neurologically normal control cases, 6 late onset AD cases and 3 early onset FAD cases. However, not every brain region was available for each case. The late onset cases were thought to be of a sporadic nature as the mean age at death was 81.2 years (range: 79–84 years); they had a mean post-mortem delay of 8.3 h. The early onset FAD cases were presumed to be linked to chromosome 14 as they all had onset ages, previous family history, clinical presentations and histopathology typical of chromosome 14-linked FAD (Haltia et al., 1994; Lampe et al., 1994). For these the mean age at onset was 38 years (range: 35–41 years), mean age at death was 45 years (range: 44–46 years) and the mean post-mortem delay was 41.7 h. All AD cases had been diagnosed according to standard pathological criteria (Khatchaturian, 1985). The controls had a mean age at death of 68.8 years (range: 57–85 years) and a mean post-mortem delay of 11.8 h. The brain regions examined were the hippocampus, temporal cortex and frontal cortex (regions severely affected by AD pathology), the visual cortex (an area relatively unaffected, but which at the time of death may be in the early stages of the disease process) and the cerebellum (an area not affected by the classic pathology associated with AD and with no clinical involvement). Three different oligoprobes were chosen and synthesized (Table 1): one to detect PS-1long, one to PS-1short and one that recognizes both transcripts, PS-1both. These probes are not
predicted to detect the transcripts of presenilin-2, a closely related gene on chromosome 1 (Levy-Lahad et al., 1995; Rogaev et al., 1995). The ISH methodology is standard and has been described in detail elsewhere (Najlerahim et al., 1990). Ten µm cryostat tissue sections were used. Probes were labelled at their 39 end with 35S-dATP using the NEN DuPont 39 end labelling system. Hybridization and wash temperatures for the various probes are given in Table 1. Hybridized sections were apposed to tritium-sensitive film for the generation of autoradiographs. Hybridization with the PS-1 probes in the sense orientation on adjacent sections was used to control for non-specific background. The signal on autoradiographic film was quantified (blind to diagnosis) using an image analyser (Seescan). A representative area over most of a tissue section was measured: for example, in the hippocampus the different subfields were not separately quantified. The background signal (sense strand hybridization) was subtracted from the antisense signal. Statistical analysis of the data was performed using a two-tailed Student’s t-test.
Northern analysis Northern analysis was carried out with each of the three oligoprobes on Northern blots (Clontech catalogue numbers: 7750-1 and 7755-1) containing polyA1 mRNA from a number of different human brain regions. The same two blots were used and reprobed with each oligo. The probes were 39 end labelled with 32P-dATP using terminal transferase and hybridized under standard conditions (Clontech data sheet).
Results In situ hybridization using all three probes revealed that PS-1 mRNA was present in all of the brain regions examined (Fig. 1). Hybridization with a sense strand control probe gave a very low background signal, as expected (Fig. 1). In the cerebral cortex (three regions) a signal was detected in both the grey and white matter, often with a similar intensity. A diffuse rather than laminar pattern was observed in grey matter and in the hippocampus the different subfields were not
Table 1. PS-1 Oligonucleotide probes Probe PS-1both PS-1long PS-1short
Sense sequence 59-GCACTCAATTCTGAATGCTGCCATCATGAT-39 59-AGCAATACTGTACGTAGCCAGAATGACAAT-39 59-CACCTGAGCAATACT/AATGACAATAGAGAA-39
Bases* 638–667 315–344 309–323 and 336–350
Ti °C
Tw °C
24 23 22
50 49 47
*Refers to EMBL and GenBank entry HUMS182R (accession number: L42110) (Sherrington et al., 1995). Ti represents the hybridization temperature (incubation) and Tw represents the wash temperature for the ISH experiments only (for Northern analysis the temperatures were determined from the Clontech protocol). The underlined bases code for the amino acids V, R, S and Q.
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Figure 1. Distribution of PS-1 mRNA in human brain by ISH. Autoradiograms are of cerebellum (1 and 3), hippocampus (3 and 4) and visual cortex (5 and 6). Pictures 1, 3 and 5 are of antisense hybridization with all three oligoprobes and 2, 4 and 6 are of sense hybridization.
readily delineated (although the dentate gyrus was sometimes visible). In the cerebellum, the granule cell layer contained the most labelling. These data are consistent with PS-1 mRNA expression in both neurons and glia. Northern analysis confirmed that all three PS-1 oligoprobes detected a major transcript in human brain of the correct size for PS-1 mRNA (Sherrington et al., 1995). A major band of approximately 3.4 kb was detected in all brain regions examined with all three probes (Fig. 2, only the data for PS-1short probe shown), indicating a wide distribution in brain for PS-1 mRNA. A very similar pattern of expression was observed for all three probes (data not shown). The observation of PS-1 mRNA in corpus callosum (Fig. 2) is consistent with the interpretation from our ISH data that PS-1 is expressed in glia. Sherrington et al. (1995) also detected a strong signal for PS-1 mRNA in corpus callosum. Kovacs et al. (1996) have reported a diffuse localization in human grey matter similar to that described here but interpret this as a predominantly neuronal distribution. However, Suzuki et al. (1996)
have recently shown both a neuronal and glial localization. A similar anatomical pattern was seen by ISH, in each region, for both PS-1long and PS-1short transcripts. Nevertheless there appeared to be differences between the transcripts in their levels of expression according to brain region; for example PS-1short was relatively less abundant in the cerebellum (Table 2). The hybridization pattern was similar for the controls, sporadic AD and FAD cases. Quantification of the autoradiographic film revealed a statistically significant reduction in the amount of PS-1long mRNA in FAD hippocampus and frontal cortex compared with the sporadic AD cases (Table 2; P 5 0.003 and P 5 0.014 respectively). In the cerebellum there was no significant difference between the controls, sporadic AD and FAD cases. The reduction in hippocampus and frontal cortex may not be due solely to the disease but could also be age related since the FAD cases are younger than the control and sporadic AD cases. However, the reduction in PS-1long appears to be transcript specific because there was no change in the
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Figure 2. Northern blots for PS-1 mRNA in human brain. The blots contain poly A1 RNA and were hybridized with the PS-1short oligo probe. Autoradiographic exposure time was for 18 h. The major band is approximately 3.4 kb and is reported to represent the active gene product; the minor band of approximately 7.2 kb is thought to represent either a rare alternatively spliced transcript or a polyadenylated isoform of the major transcript (Sherrington et al., 1995).
Table 2. Quantification of the ISH signal for PS-1long and PS-1short mRNAs in human brain Brain region
Case
Hippocampus
Control AD FAD AD FAD Control AD FAD FAD FAD
Frontal cortex Cerebellum Temporal cortex Visual cortex
PS-1long (n)
PS-1short (n)
0.025 6 0.014 (2) 0.035 6 0.007 (3) 0.008 6 0.001 (3)* 0.024 6 0.005 (3) 0.012 6 0.0 (3)** 0.036 (1) 0.024 6 0.007 (3) 0.012 6 0.002 (2) 0.014 6 0.009 (3) 0.016 6 0.007 (3)
0.023 (1) 0.026 6 0.01 (3) 0.030 6 0.004 (3) 0.042 6 0.014 (3) 0.022 6 0.011 (3) 0.013 (1) 0.019 6 0.006 (3) 0.014 6 0.005 (2) 0.015 6 0.01 (3) 0.032 6 0.001 (3)
n equals the number of different brains used. Values represent means 6 SD; units are arbitrary (machinery grey levels). *FAD vs AD P 5 0.003; **FAD vs AD P 5 0.014; Student’s t-test.
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level of expression of PS-1short mRNA in any brain region investigated between the three different groups (Table 2); the latter finding indicates an acceptable data reliability between our cases despite variability in post-mortem delay and age at death.
Discussion It is now well established that mutations in the PS-1 gene lead to chromosome 14-associated FAD, though the aetiopathological mechanism involved has not been elucidated. The normal function of PS-1 is unknown but there has been speculation that the gene product is involved in receptor localization or intracellular membrane trafficking by analogy with C. elegans SEL-12 and SPE-4 respectively (L’Hernault & Arduengo, 1992; Levitan & Greenwald, 1995). It is likely that membrane trafficking is part of the normal pathway of synthesis and processing of amyloid precursor protein (APP), the protein which appears to be central in the pathogenesis of AD. We have found that in presumptive chromosome 14 FAD there is a reduction in the expression of the long (VRSQ coding) transcript of PS-1 in anatomical regions of the brain where the pathology of AD is most pronounced. If the normal pathways involved in APP processing depend on PS-1long, then a reduction in its expression (and hence cellular content) might lead to a diversion of APP along alternative processing pathways favouring the generation of beta amyloid peptide (βAP). A study of fibroblasts isolated from chromosome 14 FAD patients and control individuals has provided evidence that PS-1 mutations may affect the normal processing pathway for APP leading to an increase in production of the longer, and more highly aggregable, form of βAP, i.e. βAP1–42 (Scheuner et al., 1995). Furthermore, post-mortem analysis of brains from patients with chromosome 14 FAD show increased deposition of βAP, especially βAP1–42 (Mann et al., 1996). The affected form of PS-1 is the long form containing the VRSQ coding sequence. This sequence has been predicted to be present in the cytoplasmic portion of PS-1 (thought to be a membrane protein) and to represent a potential phosphorylation site for protein kinase C and other kinases; it may make this longer isoform of PS-1 responsive to changes in cytoplasmic calcium concentration or other second messages. Our data suggest that PS-1long expression is altered in specific areas of the brain in FAD.
References Campion D, Flaman J-M, Brice A, Hannequin D, Dubois B, Martin C, Moreau V, Charbonnier F, Didierjean O, Tardieu S, Penet C, Puel M, Pasquier F, Le Doze F, Bellis G, Calenda A, Heilig R, Martinez M, Mallet J, Bellis M, Clerget-Darpoux F, Agid Y, Frebourg T (1995) Mutations of the presenilin 1 gene in families with early-onset Alzheimer’s disease. Hum Mol Genet 4:2373–2377 Clark RF, Hutton M, Fuldner RA, Froelich S, Karran E, Talbot C, Crook R, Lendon C, Prihar G, He C, Korenblat K, Martinez A, Wragg M, Busfield F, Behrens MI, Myers A, Norton J, Morris J, Mehta N, Pearson C, Lincoln S, Baker M, Duff K, Zehr C, PerezTur J, Houlden H, Ruiz A, Ossa J, Lopera F, Arcos M, Madrigal L, Collinge J, Humphreys C, Ashworth A, Sarner S, Fox N, Harvey R, Kennedy A, Roques P, Cline RT, Philips CA, Venter JC, Forsell L, Axelman K, Lilius L, Johnston J, Cowburn R, Viitanen M, Winblad B, Kosik K, Haltia M, Poyhonen M, Dickson D, Mann D, Neary D, Snowden J, Lantos P, Lannfelt L, Rossor M, Roberts GW, Adams MD, Hardy J, Goate A (1995) The structure of the presenilin 1 (S182) gene and identification of six novel mutations in early onset AD families. Nature Genet 11:219–222 Cruts M, Backhovens H, Wang S-Y, Van Gassen G, Theuns J, De Jonghe C, Wehnert A, De Voecht J, De Winter G, Cras P, Bruyland M, Datson N, Weissenbach J, T.den Dunnen J, Martin J-J, Hendriks L, Van Broeckhoven C (1995) Molecular genetic analysis of familial early-onset Alzheimer’s disease linked to chromosome 14q24.3. Hum Mol Genet 4:2363–2371 Haltia M, Viitanen M, Sulkava R, Ala-Hurula V, Poyhonen M, Goldfarb L, Brown P, Levy E, Houlden H, Crook R, Goate A, Clark, R, Korenblat K, Pandit S, Keller HD, Lilius L, Liu L, Axelman K, Forsell L, Winblad B, Lannfelt L, Hardy J (1994) Chromosme 14-encoded Alzheimer’s disease: Genetic and clinicopathological description. Ann Neurol 36:362–367 Khatchaturian ZS (1985) Diagnosis of Alzheimer’s Disease. Arch Neurol 42:1097–1105 Kovacs DM, Fausett HJ, Page KJ, Kim T-W, Moir RD, Merriam DE, Hollister RD, Hallmark OG, Mancini R, Felsenstein KM, Hyman BT, Tanzi RE, Wasco W (1996) Alzheimer-associated presenilins 1 and 2: Neuronal expression in brain and localization to intracellular membranes in mammalian cells. Nature Med 2:224–229 Lampe TH, Bird TD, Nochlin D, Nemens E, Risse SC, Sumi M, Koerker R, Leaird B, Wier M, Raskind MA (1994) Phenotype of chromosome 14-linked familial Alzheimer’s disease in a large kindred. Ann Neurol 36:368–378 Levitan D, Greenwald I (1995) Facilitation of lin-12-mediated signalling by sel-12, a caenorhabditis elegans S182 Alzheimer’s disease gene. Nature 377:351–354 Levy-Lahad E, Wasco W, Poorkaj P, Romano DM, Oshima J, Pettingell WH, Yu C-E, Jondro PD, Schmidt SD, Wang, K, Crowley AC, Fu Y-H, Guenette SY, Galas D, Nemens E, Wijsman EM, Bird TD, Schellenberg GD, Tanzi RE (1995) Candidate gene for the chromosome 1 familial Alzheimer’s disease locus. Science 269:973–977 L’Hernault SW, Arduengo PM (1992) Mutation of a putative sperm membrane protein in caenorhabditis elegans prevents sperm differentiation but not associated meiotic divisions. J Cell Biol 119:55–68 Mann DMA, Iwatsubo T, Cairns NJ, Lantos PL, Nochlin D, Sumi SM, Bird TD, Poorkaj P, Hardy J, Hutton M, Prihar G, Crook R, Rossor MN, Haltia M (1996) Amyloid (Aβ) deposition in chromosome 14-linked Alzheimer’s disease: Predominance of Aβ42(43). Ann Neurol, in press Najlerahim A, Harrison PJ, Barton AJL, Heffernan J, Pearson RCA (1990) Distribution of messenger RNAs encoding the enzymes glutaminase, aspartate aminotransferase and glutamic acid decarboxylase in rat brain. Mol Brain Res 7:317–333 Rogaev EI, Sherrington R, Rogaeva EA, Levesque G, Ikeda M, Llang Y, Chi H, Lin C, Holman K, Tsuda T, Mar L, Sorbi S, Nacmias B,
218 Piacentini S, Amaducci L, Chumakov I, Cohen D, Lannfelt L, Fraser PE, Rommens JM, St George-Hyslop PH (1995) Familial Alzheimer’s Disease in kindreds with missense mutations in a gene on chromosome 1 related to the Alzheimer’s disease type 3 gene. Nature 376:775–778 Scheuner D, Bird T, Citron M, Lannfelt L, Schellenberg G, Selkoe D, Viitanen M, Younkin SG (1995) Fibroblasts from carriers of familial AD linked to chromosome 14 show increased Aβ production. Soc Neurosci Abstr 21:1500 Sherrington R, Rogaev EI, Liang Y, Rogaeva EA, Levesque G, Ikeda M, Chi H, Lin C, Li G, Holman K, Tsuda T, Mar L, Foncin J-F, Bruni AC, Montesi MP, Sorbi S, Rainero I, Pinessi L, Nee L, Chumakov I, Pollen D, Brookes A, Sanseau P, Polinsky RJ, Wasco W, Da Silva HAR, Haines JL, Pericak-Vance MA, Tanzi RE, Roses AD, Fraser PE, Rommens JM, St George-Hyslop PH (1995) Cloning of a gene bearing a missense mutation in early-onset familial Alzheimer’s disease. Nature 375:754–760 Sorbi S, Nacmias B, Forleo P, Piacentini S, Sherrington R, Rogaev E, St George-Hyslop P, Amaducci L (1995) Missense mutation of S182 gene in Italian families with early-onset Alzheimer’s disease. Lancet 346:439–440
A.J.L. Barton et al. Suzuki T, Nishiyama K, Murayama S, Yamamoto A, Sato S, Kanazawa I, Sakaki Y (1996) Regional and cellular presenilin 1 gene expression in human and rat tissue. Biochem Biophys Res Comm 219:708–713 Tanahashi H, Mitsunaga Y, Takahashi K, Tasaki Hirokazu T, Watanabe S, Tabira T (1995) Missense mutation of S182 gene in Japanese familial Alzheimer’s disease. Lancet 346:440 Van Broeckhoven C (1995) Presenilins and Alzheimer disease. Nature Genet 11:230–232 Wasco W, Pettingell WP, Jondro PD, Schmidt SD, Gurubhagavatula S, Rodes L, Diblasi T, Romano DM, Guenette SY, Kovacs DM, Growdon JH, Tanzi RE (1995) Familial Alzheimer’s chromosome 14 mutations. Nature Med 1:848 Wragg M, Hutton M, Talbot C, Busfield F, Han SW, Lendon, C, Clark RF, Morris JC, Edwards D, Goate A, Pfeiffer E, Crook R, Prihar G, Phillips H, Baker M, Hardy J, Rossor M, Houlden H, Karran E, Roberts G, Craddock N (1996) Genetic association between intronic polymorphism in presenilin-1 gene and late-onset Alzheimer’s disease. Lancet 347:509–512.
Alteration in Brain Presenilin 1 mRNA Expression in Early Onset Familial Alzheimer’s Disease Amanda J.L. Barton,1 Barry W. Crook,1 Eric H. Karran,1 Frank Brown,1 Deborah Dewar,2 David M.A. Mann,3 R. Carl A. Pearson,4 David I. Graham,5 John Hardy,6 Mike Hutton,6 Karen Duff,6 Alison M. Goate,7 Robert F. Clark7 and Gareth W. Roberts1 1
Department of Molecular Neuropathology Research, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW; 2Wellcome Surgical Institute and Hugh Fraser Neuroscience Laboratories, University of Glasgow, Garscube Estate, Bearsden Road, Glasgow G61 1QH; 3Department of Pathological Sciences, Stopford Building, The University of Manchester, Oxford Road, Manchester M13 9PT; 4Department of Biomedical Science, University of Sheffield, Sheffield S10 2NT; 5Institute of Neurological Sciences , Southern General Hospital NHS Trust, Glasgow, G51 4TF; 6Suncoast Alzheimer’s Disease Laboratory, University of South Florida, Tampa FL33613, USA; 7Department of Psychiatry, Washington University School of Medicine, St Louis, MO 63110, USA The expression of the presenilin 1 (PS-1) gene has been investigated by in situ hybridization in early onset familial Alzheimer’s disease (FAD), late onset Alzheimer’s disease (AD) and normal control brain. Mutations in this gene are responsible for chromosome 14-linked FAD. We have found that presenilin 1 mRNA is present throughout the human brain with a distribution consistent with both a glial and neuronal localization. The in situ hybridization pattern was similar for the controls, the early onset FAD cases and the late onset AD cases. However, one of the two forms of the mRNA for PS-1, the long form (which contains a sequence encoding a four amino acid (VRSQ) insert at its 59 end) was significantly reduced in early onset FAD brain compared with late onset AD. We suggest that this long transcript may alter the normal pathway for processing of amyloid precursor protein, the protein which appears to be central in the pathogenesis of AD. © 1996 Academic Press Limited
Key words: familial Alzheimer’s disease, human brain; in situ hybridization, presenilin 1 mRNA
A NOVEL GENE on chromosome 14, termed presenilin 1 (PS-1) or S182, has recently been identified by molecular genetic studies as being responsible for chromosome 14-linked familial Alzheimer’s disease (FAD) (Rogaev et al., 1995; Sherrington et al., 1995). To date, over 26 FAD associated mutations have been found in this gene (Campion et al., 1995; Clark et al., 1995; Cruts et al., 1995; Sherrington et al., 1995; Sorbi et al., 1995; Tanahashi et al., 1995; Van Broeckhoven, 1995; Wasco
et al., 1995). More recently association has been observed between homozygosity for allele 1 of an intronic polymorphism in the PS-1 gene and increased risk for late onset Alzheimer’s disease (AD) (Wragg et al., 1996). The normal function of PS-1, as well as its role in the pathogenesis of AD, are not known. Sherrington et al. (1995) have demonstrated a wide distribution of PS-1 mRNA expression throughout the normal human brain as well as in other tissues. Analysis of PS-1 mRNA expression in the brains of individuals affected by AD has not been described previously. Recent work has identified two different transcripts of PS-1 (Clark et al., 1995; Cruts et al., 1995). One of these corresponds to the full exon sequence while the
Correspondence to: Dr Amanda J.L. Barton; email: [email protected] Received 22 May 1996; accepted for publication 5 June 1996 © 1996 Academic Press Limited 1055-8330/96/030213 1 6 $18.00/0
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other lacks a sequence encoding the four amino acids valine (V), arginine (R), serine (S) and glutamine (Q): these have been designated PS-1long and PS-1short respectively. In this study we have examined the expression of these two PS-1 mRNA transcripts in the brains of patients dying with early onset FAD. In situ hybridization (ISH) was used to determine the qualitative and quantitative pattern of expression of PS-1 mRNA in the brains of three early onset (presumptive chromosome 14-linked) FAD cases; comparisons with brains from patients with late onset AD and from normal individuals were made.
Materials and Methods In Situ hybridization PS1 mRNA expression was examined in 4 neurologically normal control cases, 6 late onset AD cases and 3 early onset FAD cases. However, not every brain region was available for each case. The late onset cases were thought to be of a sporadic nature as the mean age at death was 81.2 years (range: 79–84 years); they had a mean post-mortem delay of 8.3 h. The early onset FAD cases were presumed to be linked to chromosome 14 as they all had onset ages, previous family history, clinical presentations and histopathology typical of chromosome 14-linked FAD (Haltia et al., 1994; Lampe et al., 1994). For these the mean age at onset was 38 years (range: 35–41 years), mean age at death was 45 years (range: 44–46 years) and the mean post-mortem delay was 41.7 h. All AD cases had been diagnosed according to standard pathological criteria (Khatchaturian, 1985). The controls had a mean age at death of 68.8 years (range: 57–85 years) and a mean post-mortem delay of 11.8 h. The brain regions examined were the hippocampus, temporal cortex and frontal cortex (regions severely affected by AD pathology), the visual cortex (an area relatively unaffected, but which at the time of death may be in the early stages of the disease process) and the cerebellum (an area not affected by the classic pathology associated with AD and with no clinical involvement). Three different oligoprobes were chosen and synthesized (Table 1): one to detect PS-1long, one to PS-1short and one that recognizes both transcripts, PS-1both. These probes are not
predicted to detect the transcripts of presenilin-2, a closely related gene on chromosome 1 (Levy-Lahad et al., 1995; Rogaev et al., 1995). The ISH methodology is standard and has been described in detail elsewhere (Najlerahim et al., 1990). Ten µm cryostat tissue sections were used. Probes were labelled at their 39 end with 35S-dATP using the NEN DuPont 39 end labelling system. Hybridization and wash temperatures for the various probes are given in Table 1. Hybridized sections were apposed to tritium-sensitive film for the generation of autoradiographs. Hybridization with the PS-1 probes in the sense orientation on adjacent sections was used to control for non-specific background. The signal on autoradiographic film was quantified (blind to diagnosis) using an image analyser (Seescan). A representative area over most of a tissue section was measured: for example, in the hippocampus the different subfields were not separately quantified. The background signal (sense strand hybridization) was subtracted from the antisense signal. Statistical analysis of the data was performed using a two-tailed Student’s t-test.
Northern analysis Northern analysis was carried out with each of the three oligoprobes on Northern blots (Clontech catalogue numbers: 7750-1 and 7755-1) containing polyA1 mRNA from a number of different human brain regions. The same two blots were used and reprobed with each oligo. The probes were 39 end labelled with 32P-dATP using terminal transferase and hybridized under standard conditions (Clontech data sheet).
Results In situ hybridization using all three probes revealed that PS-1 mRNA was present in all of the brain regions examined (Fig. 1). Hybridization with a sense strand control probe gave a very low background signal, as expected (Fig. 1). In the cerebral cortex (three regions) a signal was detected in both the grey and white matter, often with a similar intensity. A diffuse rather than laminar pattern was observed in grey matter and in the hippocampus the different subfields were not
Table 1. PS-1 Oligonucleotide probes Probe PS-1both PS-1long PS-1short
Sense sequence 59-GCACTCAATTCTGAATGCTGCCATCATGAT-39 59-AGCAATACTGTACGTAGCCAGAATGACAAT-39 59-CACCTGAGCAATACT/AATGACAATAGAGAA-39
Bases* 638–667 315–344 309–323 and 336–350
Ti °C
Tw °C
24 23 22
50 49 47
*Refers to EMBL and GenBank entry HUMS182R (accession number: L42110) (Sherrington et al., 1995). Ti represents the hybridization temperature (incubation) and Tw represents the wash temperature for the ISH experiments only (for Northern analysis the temperatures were determined from the Clontech protocol). The underlined bases code for the amino acids V, R, S and Q.
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Figure 1. Distribution of PS-1 mRNA in human brain by ISH. Autoradiograms are of cerebellum (1 and 3), hippocampus (3 and 4) and visual cortex (5 and 6). Pictures 1, 3 and 5 are of antisense hybridization with all three oligoprobes and 2, 4 and 6 are of sense hybridization.
readily delineated (although the dentate gyrus was sometimes visible). In the cerebellum, the granule cell layer contained the most labelling. These data are consistent with PS-1 mRNA expression in both neurons and glia. Northern analysis confirmed that all three PS-1 oligoprobes detected a major transcript in human brain of the correct size for PS-1 mRNA (Sherrington et al., 1995). A major band of approximately 3.4 kb was detected in all brain regions examined with all three probes (Fig. 2, only the data for PS-1short probe shown), indicating a wide distribution in brain for PS-1 mRNA. A very similar pattern of expression was observed for all three probes (data not shown). The observation of PS-1 mRNA in corpus callosum (Fig. 2) is consistent with the interpretation from our ISH data that PS-1 is expressed in glia. Sherrington et al. (1995) also detected a strong signal for PS-1 mRNA in corpus callosum. Kovacs et al. (1996) have reported a diffuse localization in human grey matter similar to that described here but interpret this as a predominantly neuronal distribution. However, Suzuki et al. (1996)
have recently shown both a neuronal and glial localization. A similar anatomical pattern was seen by ISH, in each region, for both PS-1long and PS-1short transcripts. Nevertheless there appeared to be differences between the transcripts in their levels of expression according to brain region; for example PS-1short was relatively less abundant in the cerebellum (Table 2). The hybridization pattern was similar for the controls, sporadic AD and FAD cases. Quantification of the autoradiographic film revealed a statistically significant reduction in the amount of PS-1long mRNA in FAD hippocampus and frontal cortex compared with the sporadic AD cases (Table 2; P 5 0.003 and P 5 0.014 respectively). In the cerebellum there was no significant difference between the controls, sporadic AD and FAD cases. The reduction in hippocampus and frontal cortex may not be due solely to the disease but could also be age related since the FAD cases are younger than the control and sporadic AD cases. However, the reduction in PS-1long appears to be transcript specific because there was no change in the
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Figure 2. Northern blots for PS-1 mRNA in human brain. The blots contain poly A1 RNA and were hybridized with the PS-1short oligo probe. Autoradiographic exposure time was for 18 h. The major band is approximately 3.4 kb and is reported to represent the active gene product; the minor band of approximately 7.2 kb is thought to represent either a rare alternatively spliced transcript or a polyadenylated isoform of the major transcript (Sherrington et al., 1995).
Table 2. Quantification of the ISH signal for PS-1long and PS-1short mRNAs in human brain Brain region
Case
Hippocampus
Control AD FAD AD FAD Control AD FAD FAD FAD
Frontal cortex Cerebellum Temporal cortex Visual cortex
PS-1long (n)
PS-1short (n)
0.025 6 0.014 (2) 0.035 6 0.007 (3) 0.008 6 0.001 (3)* 0.024 6 0.005 (3) 0.012 6 0.0 (3)** 0.036 (1) 0.024 6 0.007 (3) 0.012 6 0.002 (2) 0.014 6 0.009 (3) 0.016 6 0.007 (3)
0.023 (1) 0.026 6 0.01 (3) 0.030 6 0.004 (3) 0.042 6 0.014 (3) 0.022 6 0.011 (3) 0.013 (1) 0.019 6 0.006 (3) 0.014 6 0.005 (2) 0.015 6 0.01 (3) 0.032 6 0.001 (3)
n equals the number of different brains used. Values represent means 6 SD; units are arbitrary (machinery grey levels). *FAD vs AD P 5 0.003; **FAD vs AD P 5 0.014; Student’s t-test.
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level of expression of PS-1short mRNA in any brain region investigated between the three different groups (Table 2); the latter finding indicates an acceptable data reliability between our cases despite variability in post-mortem delay and age at death.
Discussion It is now well established that mutations in the PS-1 gene lead to chromosome 14-associated FAD, though the aetiopathological mechanism involved has not been elucidated. The normal function of PS-1 is unknown but there has been speculation that the gene product is involved in receptor localization or intracellular membrane trafficking by analogy with C. elegans SEL-12 and SPE-4 respectively (L’Hernault & Arduengo, 1992; Levitan & Greenwald, 1995). It is likely that membrane trafficking is part of the normal pathway of synthesis and processing of amyloid precursor protein (APP), the protein which appears to be central in the pathogenesis of AD. We have found that in presumptive chromosome 14 FAD there is a reduction in the expression of the long (VRSQ coding) transcript of PS-1 in anatomical regions of the brain where the pathology of AD is most pronounced. If the normal pathways involved in APP processing depend on PS-1long, then a reduction in its expression (and hence cellular content) might lead to a diversion of APP along alternative processing pathways favouring the generation of beta amyloid peptide (βAP). A study of fibroblasts isolated from chromosome 14 FAD patients and control individuals has provided evidence that PS-1 mutations may affect the normal processing pathway for APP leading to an increase in production of the longer, and more highly aggregable, form of βAP, i.e. βAP1–42 (Scheuner et al., 1995). Furthermore, post-mortem analysis of brains from patients with chromosome 14 FAD show increased deposition of βAP, especially βAP1–42 (Mann et al., 1996). The affected form of PS-1 is the long form containing the VRSQ coding sequence. This sequence has been predicted to be present in the cytoplasmic portion of PS-1 (thought to be a membrane protein) and to represent a potential phosphorylation site for protein kinase C and other kinases; it may make this longer isoform of PS-1 responsive to changes in cytoplasmic calcium concentration or other second messages. Our data suggest that PS-1long expression is altered in specific areas of the brain in FAD.
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