Differential regulation of amyloid-beta-protein mRNA expression ...

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D. CARLETON GAJDUSEK§, WARREN G. YOUNGt, JOHN H. MORRISONt, AND MICHAEL C. WILSON*¶. Departments of *Molecular Biology and tBasic and ...
Proc. Natl. Acad. Sci. USA Vol. 85, pp. 1297-1301, February 1988 Neurobiology

Differential regulation of amyloid-,B-protein mRNA expression within hippocampal neuronal subpopulations in Alzheimer disease (quantitative in situ hybridization/neuritic plaques/neurofibrillary tangles/neurobiology of aging)

GERALD A. HIGGINS*t, DAVID A. LEWISt, SINA BAHMANYAR§, DMITRY GOLDGABER§, D. CARLETON GAJDUSEK§, WARREN G. YOUNGt, JOHN H. MORRISONt, AND MICHAEL C. WILSON*¶ Departments of *Molecular Biology and tBasic and Clinical Research, Research Institute of Scripps Clinic, La Jolla, CA 92037; and §Laboratory of Central Nervous System Studies, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, MD 20892

Contributed by D. Carleton Gajdusek, October 12, 1987

differences in the amount of amyloid-,B-protein mRNA between hippocampal neuronal subpopulations and to study the regulation of the mRNA during the disease process. Our results show that, although the amyloid-f3-protein gene is expressed widely in neurons of the hippocampal formation, differences are found in mRNA abundance between specific neuronal subpopulations in normal aged and AD hippocampus. This suggests that neurons of the subicular complex and entorhinal cortex, which are sensitive to the pathological consequences of AD, may express the amyloid-p-protein gene at elevated levels in the disease.

We have mapped the neuroanatomical distriABSTRACT bution of amyloid-fi-protein mRNA within neuronal subpopulations of the hippocampal formation in the cynomolgus monkey (Macaca fascicularis), normal aged human, and patients with Alzheimer disease. Amyloid-8-protein mRNA appears to be expressed in all hippocampal neurons, but at different levels of abundance. In the central nervous system of monkey and normal aged human, image analysis shows that neurons of the dentate gyrus and cornu Ammonis fields contain a 2.5-times-greater hybridization signal than is present in neurons of the subiculum and entorhinal cortex. In contrast, in the Alzheimer disease hippocampal formation, the levels of amyloid-fi-protein mRNA in the cornu Ammonis field 3 and parasubiculum are equivalent. These rmdings suggest that within certain neuronal subpopulations cell type-specific regulation of amyloid-,B-protein gene expression may be altered in Alzheimer disease.

EXPERIMENTAL PROCEDURES Sources and Preparation of Central Nervous System Tissues. Brains from three neurologically normal patients (aged 66, 78, and 79 years) were obtained between 2 and 5 hr postmortem. Brains from five patients (aged 68, 71, 82, 86, and 87 years) with a clinical diagnosis of AD were obtained within 1-7 hr after death. The clinical diagnosis in the AD patients was confirmed by neuropathological analysis that showed generalized atrophy and the presence of both NP and NFT in multiple regions of the cerebral cortex and in the hippocampal formation. Immediately after autopsy, coronal tissue slabs were cut and placed in either 4% paraformaldehyde or 0.215% periodate/0.685% lysine/2% paraformaldehyde for at least 48 hr and then washed in a series of graded sucrose solutions. Twenty-micron-thick coronal sections were collected on a cryostat and mounted on gelatin-coated slides. Adjacent sections were stained with cresyl violet for localization of neuroanatomical structures or with 1% aqueous thioflavin S for visualization of extracellular amyloid and NFT. Tissue from four young adult male cynomolgus monkeys (Macacafascicularis) was also used for in situ hybridization. Animals were deeply anesthetized with ketamine hydrochloride (25 mg/kg intramuscularly) and sodium pentobarbital (10 mg/kg intraperitoneally). Animals were then perfused transcardially with cold 1% paraformaldehyde in 0.15 M phosphate buffer at pH 7.4 (PB) for 30-60 sec followed by perfusion with cold 4% paraformaldehyde in PB. The latter solution was perfused for 8-10 min at a flow rate of 250-500 ml/min, depending upon the size of the animal. Immediately following the perfusion, the brain was removed and sliced into blocks (3-5 mm thick), which were placed in cold fixative for an additional 6 hr. Tissue blocks were then washed in a series of cold, graded sucrose solutions and

The hippocampal formation is a preferential site for the pathological appearance of neurofibrillary tangles (NFT) as well as amyloid deposition in neuritic plaques (NP) and cerebrovascular amyloidosis of Alzheimer disease (AD) (1-5). It now appears that these neuropathological markers all contain the same 4-kDa P peptide (6-9). Recently, the amino acid sequence of the amyloid-P peptide has been used to identify cDNA clones of a 3.5-kilobase mRNA (10-13). The open reading frame of this mRNA predicts a polypeptide of 695 amino acids, which resembles a cell surface receptor (12) and contains the 4-kDa P peptide as part of a putative hydrophobic transmembrane domain (10-13). The expression of amyloid-,B-protein mRNA, in both normal and AD brain (10-14) as well as in nonneuronal tissues (11), suggests that the amyloid-,3-peptide precursor is a normal cellular protein, whose 4-kDa proteolytic cleavage product may accumulate during the neurodegenerative process of AD. The involvement of the amyloid-,/-protein gene in AD is suggested by genetic linkage analysis, mapping of the amyloid-13-protein gene on chromosome 21 at or near the locus responsible for familial AD (15), and the reported duplication of the gene in some cases of sporadic AD as well as karyotypically normal Down syndrome (16). In order to define precisely those cell types that specifically contain the amyloid-p-protein mRNA and thus may be involved in the aberrant deposition of the 4-kDa P peptide in AD, we have mapped the neuroanatomical distribution of the mRNA by in situ hybridization within neuronal subpopulations of the hippocampal formation in monkeys, normal aged humans, and patients with AD. Quantitation of the hybridization signal has allowed us to determine relative

Abbreviations: AD, Alzheimer disease; NFT, neurofibrillary tangles; NP, neuritic plaques; CA, cornu Ammonis. tPresent address: Department of Neurobiology and Anatomy, University of Rochester School of Medicine, Rochester, NY 14642. $To whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge

payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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were sectioned coronally in a cryostat at 20 ,um. Tissue sections were mounted on gelatin-coated slides, air-dried, and stored at either 4°C for 1-3 days or at - 70°C for up to 4 weeks. In Situ Hybridization. In situ hybridization was performed as described previously (17) with 35S-labeled RNA transcripts generated from a pGEM3 plasmid vector (Promega Biotec, Madison, WI) containing the 1.1-kilobase EcoRI amyloid-/-protein cDNA fragment isolated by Goldgaber et al. (10). To ensure that the signal obtained with antisenseprobe hybridization reflected the presence of genuine amyloid-(-protein mRNA and was not due to nonspecific effects of RNA or cellular density (17), control hybridizations were also done with a sense-strand probe not complementary to the mRNA. T7 RNA polymerase was used for generation of antisense transcripts and SP6 RNA polymerase was used for synthesis of the sense-strand probe. The transcription was performed in 40 mM Tris HCl, pH 7.5/6 mM MgCl2/2 mM dithiothreitol/5 units of RNasin (Promega Biotec)/400 ,uM ATP/400 ,uM CTP/25 ,uM each guanosine 5'-[a-35S]thiotriphosphate and uridine 5'-[a-35S]thiotriphosphate (800-1000 Ci/mmol; 1 Ci = 37 GBq; New England Nuclear)/1-2 ,ag of linearized DNA template containing 5-10 units of SP6 RNA polymerase (Boehringer Mannheim) or T7 RNA polymerase (Stratagene Cloning Systems, La Jolla, CA). Additionally, for quantitative determination of optical density in comparisons of normal versus AD hybridization, control hybridizations were performed using an antisense RNA probe homologous to proteolipid protein mRNA (18). For in situ hybridization, slide-mounted sections were postfixed in 4% paraformaldehyde (in PB) for 5 min at room temperature and then were rinsed twice in PB. Slides were immersed in proteinase K (Boehringer Mannheim) at 50 ,tg/ml in 5 x TE (1 x TE = 10 mM Tris/1 mM EDTA, pH 8.0) for 7.5 min at room temperature, rinsed in PB, placed in 0.05 M HCO for 7.5 min, then rinsed and dehydrated in graded alcohols containing 0.33 M ammonium acetate, and air-dried at room temperature. Following application of the prehybridization buffer containing 50% formamide, 0.75 M NaCl, 25 mM Pipes, 25 mM EDTA, S x Denhardt's (1 x Denhardt's = 0.02% bovine serum albumin/0.02% Ficoll/ 0.02% polyvinylpyrrolidone), 250 mM dithiothreitol, 0.2% NaDodSO4, 10% dextran sulfate, and denatured yeast RNA and salmon sperm DNA at 500 ,g/ml, 35S-labeled RNA probe was added for hybridization overnight at 52°C. Following hybridization, coverslips were removed in 4 x SSC (1 x SSC = 0.15 M NaCl/0.015 M sodium citrate, pH 7) containing 300 mM 2-mercaptoethanol, and the slidemounted sections were digested with pancreatic RNase (50 ,ug/ml) in 0.5 M NaCl/1 x TE for 30 min at 37°C and rinsed with 0.5 x SSC at 420C. The slides were air-dried and exposed to x-ray film (DuPont Cronex) for 24-48 hr at room temperature. The slides were then dipped in emulsion (Kodak NTB-2), exposed for 5-10 days, and developed for

Proc. Natl. Acad. Sci. USA 85 (1988)

tive quantitative determinations were limited to comparison of cell populations within a section. In the present study, we have used the term hippocampal formation to include the dentate gyrus, the hippocampus proper (Ammon's horn), the subicular complex, and the entorhinal cortex. Within the hippocampal formation, we have used nomenclature that we believe is most appropriate for studying AD pathology within the cytoarchitectonic context of the primate hippocampal formation. Thus, although the designation of H subdivisions of the hippocampus has been favored by pathologists and regio superior and regio inferior of Ramon y Cajal may be more appropriate for the primate, we have chosen to use the nomenclature of Lorente de No (20). We have included CA4 as the hilar region of the dentate gyrus as has recently been suggested (21).

RESULTS In situ hybridization to amyloid-f-protein mRNA in the human brain and the cynomolgus monkey brain shows that it is expressed in neurons in telencephalic structures such as the hippocampal formation and neocortex (14) and at lower abundance in the cerebellum (Fig. 1), brainstem, and spinal cord. The specificity of the hybridization to amyloid-,3 protein mRNA is also demonstrated in Fig. 1. Virtually no signal was obtained in the hippocampal formation of normal human central nervous system following sense-strand hybridization, as compared to the intense hybridization obtained with the antisense strand probe complementary to amyloid-f3-protein mRNA. Within the hippocampal formation, the amyloid-,-protein gene appears to be widely expressed by neurons although the level of the mRNA varies between neurons of different fields. This is illustrated at low magnification in Fig. 2, which shows the distribution of amyloid-,-protein mRNA hybridization within the hippocampal formation of cynomolgus monkey (Fig. 2A), normal aged human (Fig. 2B), and a patient with AD (Fig. 2C). In the dentate gyrus, both granule and hilar neurons contain amyloid-p-protein mRNA. Within the hippocampus proper of normal primate brain, it appears that most, if not all, of the pyramidal neurons in the CA1 and CA3 fields exhibit intense hybridization (Fig. 2), indicating high abundance of the mRNA. In contrast, there was less amyloid-f3protein mRNA hybridization in neurons of the

ronal subpopulations was determined on a video-based image analysis system (19). For any given dark-field microscopic image of the emulsion autoradiograph, optical density measurements were made of individual "grain clusters," which we assume represent individual cells. The mean optical density per cell was measured for a given x 200 magnification field, and cells in the cornu Ammonis (CA) field 3 (CA3) were compared to adjacent parasubiculum and to subiculum, presubiculum, and entorhinal cortex. Because of the possibility of variability between in situ hybridization experiments and between slides within an experiment, rela-

A

B

C

D

anti -sense

autoradiography. Image Analysis. Measurement of relative differences in

amyloid-3-protein mRNA abundance between different neu-

Cb

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FIG. 1. Control for the specificity of amyloid-3-protein mRNA hybridization in the hippocampal formation of normal brain. Antisense-probe hybridization to hippocampal formation (HF) (A) and cerebellum (Cb) (B). Sense-strand hybridization to adjacent tissue sections of the HF (C) and Cb (D) shows no discernible specific hybridization pattern. X-ray film images of coronal sections hybridized with "S-labeled RNA probe are shown.

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brain. Fig. 2C also shows a marked decrease of amyloid-3protein mRNA hybridization in the subiculum, presumably due to cell death in this AD brain. Of five AD brains, cell loss and concomitant lack of hybridization in this field of the hippocampal formation were consistently observed. In order to estimate the relative difference in the hybridization signal, reflecting differences in mRNA abundance between the subiculum and neurons of the CA3 fields, we compared optical density measurements of hybridization intensity between selected regions. Table 1 shows such comparisons between CA3 pyramidal neurons and neurons in the parasubiculum. In the hippocampal formation of normal aged human brain, the mean optical density of pyramidal neurons was approximately 2.5 (1.7-3.2) times that of the parasubiculum, suggesting that the amyloid-j3 protein mRNA is expressed at higher abundance in the CA3 than in the parasubiculum. Representative dark-field photomicrographs of similar regions from which these measurements were made are shown in Fig. 3 A and B. In contrast to the hybridization pattern in the monkey brain and the normal aged human brain, optical density analysis shows that in the AD brain, surviving neurons of the parasubiculum exhibit approximately the same hybridization intensity as pyramidal neurons of CA3 (Table 1 and Fig. 3 C and D). Thus, in five different tissue sections from three AD patients, the ratio of hybridization intensity between the CA3 and parasubiculum was approximately 1, indicating relatively equivalent mRNA abundance. In one normal human brain and one AD brain, a comparison was made between the intensity of amyloid-3-protein mRNA hybridization relative to the hybridization for proteolipid protein mRNA, expressed by oligodendrocytes. The abundance of proteolipid protein mRNA was not altered in the diseased tissue (data not shown), indicating that the hybridization intensity of amyloid-,B3protein mRNA reflects an increase of expression within subicular neurons in the AD brain rather than a decrease in CA3 neurons.

DISCUSSION The hippocampal formation is particularly vulnerable to the pathological mechanisms that operate in AD, adult Down syndrome, and to a lesser extent in normal human aging. All of the associated neurodegenerative events, including cell death, NP, NFT, cerebrovascular amyloidosis, and granulovacuolar degeneration, are expressed preferentially in the hippocampal formation during the disease process (1, 2), and in some AD patients the only neuropathological findings are cell loss as well as NP and NFT localization to the hippocampal formation (1, 2, 22). NP appear to be found most often in the molecular layer of the dentate gyrus (a terminal zone for afferents from the entorhinal cortex), within the dorsal portion of the subicular pyramidal cell layer, and to a lesser extent within the CA1-CA3 pyramidal cell layers (3, 5, 23). Similarly, NFT preferentially affect certain cell populations within the hippocampal formation, including projection neurons in layer II of entorhinal cortex, and within pyramidal neurons of subiculum and CA1 (24). We have found that apparently all neurons in the hippocampal formation contain amyloid-f-protein mRNA, from which the brain amyloid peptide is derived. However, within the hippocampal formation, large differences in the levels of amyloid-,-protein mRNA exist between different neuronal subpopulations. Thus, although dentate and CA pyramidal neurons express this mRNA at high levels, amyloid-f3protein mRNA appears to be present normally at a relatively lower abundance in neurons within the ventromedial portion of the temporal lobe throughout the entorhinal cortex and especially within the subicular complex throughout the rostrocaudal extent of the hippocampal formation. Quantitation of the hybridization signal shows that neurons in the para-

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FIG. 2. Distribution of amyloid-,B-protein mRNA within neurons of the hippocampal formation in cynomolgus monkey (M. fascicularis) (A), a normal aged human (B), and a patient with AD (C) as detected by antisense probe hybridization. Notice the lack of neuronal hybridization between arrows, presumably due to cell death, and the elevated abundance of the mRNA in the presubiculum (PrS) of AD hippocampus versus the normal aged human. Dark-field photomicrographs of autoradiographically processed coronal tissue sections from mid-rostrocaudal levels of the hippocampal formation are shown. DG, dentate gyrms; S, subiculum; PaS, parasubiculum; EC, entorhinal cortex; PRC, perirhinal cortex; TF, temporal neocortex area F; ots, occipitotemporal sulcus. (Bars = 1 mm.)

adjacent subiculum, presubiculum, parasubiculum, and entorhinal cortex of both monkey brain and normal aged human

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Table 1. Change in the relative abundance of amyloid-/-protein mRNA in the parasubiculum in AD Optical density per cell, mean ± SD CA3/parasubiculum CA3 Parasubiculum Normal aged 2.63 48.00 ± 11.75 (82) 126.00 ± 18.62 (60) N1 1.72 74.59 ± 10.28 (37) N2 128.29 ± 11.21 (33) 3.28 39.55 ± 7.32 (35) N3 129.73 ± 10.19 (48) AD 1.00 122.77 ± 8.55 (49) 122.74 ± 7.97 (42) AD1 1.06 AD2 102.11 ± 4.78 (39) 96.43 ± 7.36 (17) 0.99 148.71 ± 8.93 (34) 147.90 ± 12.25 (28) AD3 0.70 AD4 101.37 ± 12.86 (20) 144.43 ± 8.73 (38) 0.95 88.13 ± 9.60 (44) 83.62 ± 9.12 (26) AD5 CA3/parasubiculum, ratio of optical density in the CA3 to that in the parasubiculum. Numbers in parentheses indicate the number of cells. Comparison of optical densities shows that in the normal aged human brain, CA3 pyramidal neurons exhibit 1.72-3.28 times the hybridization intensity of parasubicular neurons. Conversely, in five AD tissue sections, the CA3/parasubiculum optical density ratios ranged from 0.70-1.06, showing relatively equal hybridization intensities between these regions in the disease.

subiculum exhibit approximately 2-3 times less optical density than CA3 pyramidal neurons. However, this difference in expression is not apparent when comparing these neuronal populations in AD hippocampus, suggesting the possibility that either mRNA abundance is increased in surviving neurons of the subiculum and entorhinal cortex during the disease process or that levels in the rest of the hippocampus are reduced in AD. Overexpression of the amyloid-f3protein gene in these populations, possibly as a simple consequence of gene duplication (16) or as a result of cell-type differences in the regulation of mRNA expression, may therefore ac-

count for the early and severe pathological changes seen in these regions in AD (25). These findings suggest that subicular and entorhinal neurons may be particularly sensitive to increased levels of amyloid 3 protein, which may cause aberrant deposition of the smaller, 42-amino acid 8 peptide in NP and NFT. However, this correlation of high levels of amyloid-/-protein gene expression with vulnerability does not hold for all cell populations in AD. Granule and hilar neurons of the dentate gyrus and CA3 pyramidal neurons normally express this mRNA at high abundance, yet they appear to be spared the pathological consequences of AD

FIG. 3. In situ hybridization shows differential regulation of amyloid-f-protein mRNA expression within hippocampal neuronal subpopulations in normal aged human brain versus AD brain. In the normal hippocampal formation, CA3 pyramidal neurons (A) express this mRNA at much higher abundance than do neurons of the parasubiculum (B). In contrast, in AD hippocampus, CA3 pyramidal cells (C) and neurons in the parasubiculum (D) appear to express amyloid-f-protein mRNA at relatively equal abundance. Dark-field photomicrographs of emulsion-dipped, coronal tissue sections. (Bar = 50 ,um.)

Neurobiology: Higgins et al. relative to other neuronal subpopulations in the hippocampal formation (4). Similarly, we have not observed changes or increases in amyloid-,&protein mRNA levels in vulnerable neocortical cell populations in AD (26), suggesting that other factors may be necessary for cellular devastation in the disease and that a complex relationship must exist between amyloid-,f3protein gene expression and pathology in AD. An alternative explanation for the increase in amyloid-,& protein mRNA abundance within subicular and entorhinal neurons in AD is that the protein may be involved in a regenerative response to the primary pathological events of the disease. Lesion-induced loss of entorhinal cells, which project to the dentate gyrus, has been shown in the rat to induce sprouting of remaining afferents to the molecular layer (27, 28). Geddes et al. (29) have shown that loss of these perforant path neurons in AD produces a similar, sprouting-induced expansion of cholinergic inputs to the dentate gyrus and have suggested that the preferential deposition of plaques in afferent terminal zones of the molecular layer of the dentate gyrus may result from or participate in the sprouting process that occurs in AD (30). In this context, it is possible that the deposition of the 4-kDa P peptide in NP represents a reduced capacity of neurons to mount a plastic response to the pathology of AD (31). Finally, in view of recent genetic analyses linking the jprotein gene to the AD locus (15, 16), defects in specific alleles of this gene may predispose the 69-kDa amyloid-fprotein precursor to aberrant proteolytic processing, which may be exacerbated during neuronal regeneration. The widespread expression of amyloid-f3-protein mRNA in hippocampal neurons and its elevated abundance in certain cell populations in AD in which NFT are most often seen suggest that it is the loss of these NFT-containing neurons that leads to the memory disturbances of the disease. Isolation of the hippocampal formation from its connections with the neocortex may result from destruction of subicular and entorhinal cells causing disconnection of its afferent and efferent limbs (4, 5). Such a model would predict that elevated expression of the amyloid-j3protein gene would also occur in these neurons during normal human aging in which declarative memory deficits, cell loss, and the accumulation of NFT mimic, in reduced magnitude, the pathological events that occur in AD (32). It is therefore important to examine the temporal progression of expression of the amyloid-,-protein gene within the subiculum and entorhinal cortex during normal human aging. Note Added in Proof. It has recently been reported that the amyloid(-protein gene is not duplicated in the majority of cases of sporadic (33-35) or familial (33) AD and can be genetically distinguished from the defect in familial AD (36, 37), thus suggesting that altered expression of the gene is consequential to the primary defect. We wish to gratefully acknowledge the provision of human brain tissue by the Institute for Biogerontology Research (Phoenix, AZ) and Dr. Constantine Bouras (Geneva). We also thank Drs. Robert Terry, Michael Campbell, and George Oyler for comments on the manuscript, Dr. David Amaral for his assistance with the lowmagnification photomicrography, and Michelle L. Dietrich for her excellent and patient secretarial assistance. These studies were supported by Hereditary Disease Foundation and National Institutes of Health postdoctoral fellowships (G.A.H.), the Alzheimer's Disease and Related Disorders Association, and National Institutes of Health Grants AG 05131 and NS 22347 (J.H.M.), RSDA MH 00519 (D.A.L.), and NS 23038 and CA 33730 (M.C.W.). This is publication number 4770MB from the Research Institute of Scripps Clinic.

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