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Amyloid Protein and Neurofibrillary Tangles Coexist in the Same Neuron in Alzheimer Disease

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Amyloid Protein and Neurofibrillary Tangles Coexist in the Same Neuron in Alzheimer Disease Proc. Natl. Acad. Sci. USA Vol. 86, pp. 2853-2857, April 1989 Medical Sciences Amyloid protein and neurofibrillary tangles coexist in the same neuron in Alzheimer disease (amyloid 13 peptide/A4 peptide/intraneuronal protein/paired helical rdaments/senile plaques) INGE GRUNDKE-IQBAL*, KHALID IQBAL, LALU GEORGE, YUNN-CHYN TUNG, KWANG Soo KIM, AND HENRY M. WISNIEWSKI New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314 Communicated by Philip Siekevitz, January 3, 1989 (receivedfor review July 27, 1988) ABSTRACT In Alzheimer disease, paired helical filaments tants of mAb to amyloid at dilutions of 1:2-1:20 were used accumulate in the neuron, and amyloid fibers are found in the (IgG subclass in parentheses): 4G5 (IgGi), 2B8 (IgGl), 2F9 extracellular space in the neuropil and brain vessels. Amyloid (IgGl), 4E11 (IgG2a), 4G8 (IgG2b). mAb tau-1 to tau (IgG2a, and paired helical filaments are morphologically distinct. ascites, 1:50,000) was the generous gift of L. I. Binder Although messenger RNA that encodes the amyloid has also (University of Alabama, Birmingham). Mouse ascites fluid been shown in several tissues, including brain, the intracellular from plasmacytoma line MOPC21 was purchased from Litton expression of the protein has not been observed. By using Bionetics. Reagents for the avidin-biotin complex technique monoclonal antibodies to a synthetic amyloid (3 peptide, the were purchased from Vector Laboratories, and reagents for present study demonstrates that amyloid reactivity is present in the peroxidase-anti-peroxidase technique were from Stern- both Alzheimer patients and normal individuals in different bergerMeyer Immunocytochemicals (Jarrettsville, MD). Al- types ofneurons, including the neurons with the neurofibrillary kaline phosphatase-conjugated antibody to mouse IgG and tangles, but not in the tangle itself. the alkaline phosphatase color development reagents 5- bromo4-chloro-3-indolyl phosphate (p-toluidine salt) and Alzheimer nitroblue tetrazolium chloride were from Bio-Rad. Alzheimer disease/senile dementia of the type Immunoblots. Electrophoresis on NaDodSO4/polyacryl- (AD/SDAT) are characterized neuropathologically by the amide gels (80 x 60 x 75 mm slab) was performed according accumulation of two types of aberrant fibrils, the paired to Laemmli (23). Proteins were transferred to Immobilon helical filaments (PHF) and the amyloid. PHF are intraneu- (Millipore) in 25 mM Tris/0.7 M glycine/20% (vol/vol) ronal and accumulate in bundles of parallel fibrils, called methanol (24, 25) at 105 V for 1 hr at room temperature. tangles, in the neuronal cell bodies, their dendrites, and the Residual protein binding sites were blocked with 5% (wt/vol) dystrophic neurites of the neuritic (senile) plaques. In con- defatted dry milk in 10 mM phosphate buffer (pH 7.4) trast, amyloid is not found within the neurons and their containing 0.15 M NaCl for at least 1.5 hr at 22TC. The blots processes but occurs extracellularly in association with the were then incubated for 18 hr at 220C with antibodies diluted plaques either as wisps in the neuropil among the plaque in the blocking solution. Bound IgG was detected by using neurites or as a concentrated mass in the center forming the either alkaline phosphatase-conjugated antibody to mouse plaque core (1). Frequently accumulations of amyloid are IgG (1:2000) or the peroxidase-anti-peroxidase technique also found in the neuropil around the vessels and within the (26) with 5-bromo-4-chloro-indolyl phosphate/nitroblue tet- vessel walls. Amyloid is morphologically readily distinguish- razolium chloride or diaminobenzidine substrates, respec- able from the PHF by the smaller dimensions ofits flibrils and tively. their organization in irregular clusters. Plaque and cere- Immunocytochemistry. Blocks of hippocampus, cerebrum, brovascular amyloid are primarily made up of a 40- to cerebellum, and spinal cord from five patients with histo- 42-amino acid residue peptide, called the P3 peptide or A4 pathologically confirmed AD/SDAT (56-84 years of age, peptide (2-4), whereas mainly ubiquitin (5, 6) and microtu- autopsy at 2-24 hr postmortem) and from eight non- bule-associated protein tau (7-15) have been shown in PHF. Alzheimer individuals (ages 38-81 years, autopsy at 4-14 hr Tau, a family of closely related polypeptides with an average postmortem) were fixed in neutral formaldehyde for an molecular mass of 58 kDa, is abnormally phosphorylated in average of 5 days. Alternatively, some tissue was fixed in PHF and probably also contains other alterations (16-18). Perfix for 2 days. Perfix is a commercial fixative (Fisher) Sequences of amyloid P peptide have also been reported in containing 16.8% isopropyl alcohol, 2.0% (wt/vol) trichloro- preparations enriched in PHF (19-21). However, it has not acetic acid, 4.1% (wt/vol) paraformaldehyde, and 2.0% been resolved as yet whether the amyloid peptide is actually (wt/vol) zinc chloride. In the developing nervous system, it a component of the PHF or a contaminant. has been shown to preserve the antigenic activity of glial fibrillary protein better than formaldehyde- or glutaralde- MATERIALS AND METHODS hyde-containing fixatives (27). The tissue was embedded in Paraplast, and 6-,um serial sections were prepared. Neuronal Antibodies and Immunological Reagents. Monoclonal anti- cell bodies were isolated from AD/SDAT frontal cortex as bodies (mAbs) to a synthetic peptide corresponding to the described previously (28), and cell smears were dried onto first 24 amino acids of the ,B peptide were generated as gelatin-coated slides and either fixed with 10% (vol/vol) previously described (22). All antibodies reacted with the 17- neutral formalin or with 10o neutral formalin containing 5% 24 amino acid sequence fragment of 83 peptide in ELISA and (vol/vol) dimethyl sulfoxide for 10 min at room temperature with plaque and vascular amyloid by immunocytochemistry (29) or fixed with -20°C acetone for 3 min. Some sections (22). For the studies in the present paper, culture superna- Abbreviations: AD/SDAT, Alzheimer disease/senile dementia of The publication costs of this article were defrayed in part by page charge the Alzheimer type; PHF, paired helical filaments; mAb, monoclonal payment. This article must therefore be hereby marked "advertisement" antibody. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 2853 Downloaded by guest on September 29, 2021 2854 Medical Sciences: Grundke-Iqbal et al. Proc. Natl. Acad Sci. USA 86 (1989) were treated with 86% (vol/vol) formic aciid for 5-60 min of about 93, 55 (a doublet), 40, and 31 kDa. In calf brain, the prior to immunostaining according to the met! hod of Kitamoto 93-kDa band instead of the 125-kDa doublet was most et al. (30). Immunocytochemistry was perft )rmed on tissue prominently labeled. The major bands (i.e., the 125-kDa sections or cell smears by using the avidin--biotin complex doublet in human brain and the 93-kDa band in calf brain) technique (29). In some cases, immunostainitng was followed were easily visible when as little as 10 ug of protein was by staining with 1% aqueous solution of tLhioflavine S to applied (Fig. 1). In some other cases, an additional doublet of visualize immunonegative tangles and amylloid. The slides about 220 kDa was stained. No labeling was seen with as were photographed immediately after the Ithioflavine step much as 50 ttg of homogenate per lane when mouse IgG from because thioflavine-positive fluorescence raptidly faded when myeloma MOPC21 at a concentration similar to that of the used together with diaminobenzidine immunoostaining. mAb to amyloid was used as a control antibody. In sections of Alzheimer brain, the mAb reacted strongly with plaque and vascular amyloid (Fig. 2). The latter was RESULTS found as heavy deposits within the walls of the cortical and On Western blots (Fig. 1) all amyloid mAbs Ireacted with the meningeal vessels and in addition as loose fibrous accumu- synthetic 1-24 residue 13 peptide. No cros;sreactivity was lations either perivascular or scattered in the neuropil of the observed with ubiquitin, tau, or the polypelptides in micro- most affected areas. Similarly, in the brains of non-demented tubule and neurofilament preparations. Hoowever, in total aged individuals, the sparsely occurring plaque and vascular brain homogenate, several polypeptides w,ere labeled. In amyloid was also stained strongly. On treatment of the homogenates from an Alzheimer and an ageed control indi- sections with formic acid prior to application of the antibod- vidual, the mAb prominently labeled a polypeptide doublet of ies, a dramatic increase in the number and intensity of 125 kDa and, in addition, weakly labeled polypeptidebands immunostained plaque, vascular, and amorphous amyloid deposits in the neuropil was observed (data not shown). 1 2 3 4 5 6 7 In addition to the extraneuronal amyloid, dark labeling of accumulations of intracellular granules in various types of 8 9 neurons in cerebral and cerebellar cortices and spinal cord was observed (Figs. 3 and 4). Dilutions of the culture supernatants for optimal staining ranged from 1:10 to 1:50 depending upon the myeloma line and was the same in each case for both intracellular and extracellular amyloid staining. The immunostaining was not affected when the salt concen- tration was increased from 0.15 M to 0.3 M and 0.05% Tween-20 was added to the antibodies. Like the extraneu- _ ronal amyloid, the immunoreactivity of this intraneuronal -ir~.materialX increased in the formic acid-treated tissue sections (data not shown). The intraneuronal amyloid immunoreac- tivity appeared to be expressed in cerebellum and cerebrum _ s of AD/SDAT as well as of the non-Alzheimer control group - irrespective of age or postmortem time investigated. In the cerebellum, the labeling was most conspicuous in the cytoplasm ofthe Purkinje cells, which were almost all stained in the cases studied ranging from a 38-year-old control individual to an 81-year-old Alzheimer patient (Fig.
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