Identification of a stable fragment of the Alzheimer amyloid precursor containing the beta-protein in brain microvessels.

Abstract

Altered proteolysis of the (3-amyloid precursor protein (PAPP) (36 and 38 yr), and 18 non-AD subjects (fetal-78 yr). Frozen nonneural tissues (renal cortex, heart, spleen, and adrenal) and fresh or frozen animal brains (rat, guinea pig, rabbit, and cow) were also used to prepare microvessels. Microvessels were purified at 40C by a modification of the method of Tsuji et al. (9). Cerebral gray matter (usually 5-10 g of wet weight) was minced into -2 mm3 blocks, suspended in 5-10 vol of Tris-buffered saline (TBS) (50 mM Tris/150 mM sodium chloride/2 mM EDTA/0.1 mM diisopropyl fluorophosphate/ leupeptin at 1 Ag/ml/pepstatin at 0.1 ug/ml/7-amino-1-chloro-3-tosylamide-2-heptanone at 1 tig/ml, pH 7.6), and homogenized with a motor-driven, loosely fitting Teflon pestle (5-10 up-and-down strokes). The homogenate was sieved through 73-,.m nylon mesh. The flow-through fraction was saved as the nonvascular brain fraction, and the mesh was washed in TBS buffer to resuspend the retained material.This suspension was placed on a column of 425-to 600-Iumdiameter glass beads (Sigma) with a sheet of 73-,um nylon mesh at the bottom. The column was washed with 5-10 vol of TBS, and its contents were placed in a beaker of 50-100 ml of TBS. Microvessels were separated from the glass beads by agitation with a metal rod. The beads settled, and the microvessel suspension was decanted and centrifuged at 10,000 x g for 30 min. The resultant pellet was resuspended in 0.25 M sucrose, homogenized (five strokes), and layered on a discontinuous sucrose-density gradient (8 ml each of 1.0 M, 1.3 M, and 1.5 M sucrose in H20). After centrifugation at 58,000 x g in an SW 28 rotor for 120 min, the microvessel fraction was recovered as a pellet from the bottom of the tube and resuspended in 1-2 ml of TBS. Some microvessel fractions were prepared from fresh brain tissue by an alternate but comparable purification method described by Kalaria and Harik (10).To separate large from small microvessels, the suspension retained on the 73-pum mesh was sieved through a 153-,um nylon mesh, and the latter was washed in TBS to resuspend the retained material. This suspension was centrifuged at 10,000 x g for 30 min at 40C, and the resultant pellet was layered on a sucrose gradient as described above. For purification of small microvessels, the flow-through fraction off the 153-,um mesh was successively processed over the glass bead column and the sucrose gradient as described above.Purified microvessel fractions were homogenized (10 strokes) in a Teflon homogenizer, mixed 3:1 with 4x SDS Abbreviations: 8APP, 13-amyloid precursor protein; FL-f3APP, fulllength fAPP; A,8P, amyloid ,1-protein; AD, Alzheimer disease. 1345The 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.

An Etiological Role of Amyloidogenic Carboxyl‐Terminal Fragments of the β‐Amyloid Precursor Protein in Alzheimer's Disease

“…A/I-bearing CT fragments have been detected in leptomeningeal vessels and microvessels and choroid plexus purified from the brain of aged normal individuals and AD patients (Nordstedt et al, 1991;Estus et al, 1992;Ghiso et al, 1992;Tamaoka et al, 1992;Kalaria et al, 1996). The 19-kDa amyloidogenic A/I-bearing CT fragment was also found in gray matter, and the levels of the mature /IAPP isoform (133 kDa) and 19-kDa amyloidogenic CT fragment increased two-to threefold with age (Nordstedt et al, 1991).…”

“…It has been reported that the CT fragment of /I-APP in the medium under the conditions used has not been detected, whereas such fragments have been detected intracellularly in tissues and cultured cells (Nordstedt et al, 1991;Estus et al, 1992; Fukuchi et al, 1992a,b; Haass et al, 1992a,b;Tamaoka et al, 1992).…”

“…The recent concentration on other potentially amyloidogenic products of /IAPP has produced interesting candidates, the most promising of which are the amyloidogenic carboxyl-terminal (CT) fragments of /IAPP. The transgenic mice expressing /IAPP and presenilins were not, however, examined for the presence of CT 100.First, CT peptides have been found not only in various cultured cells (Maruyama et al, 1990;Wolf et al, 1990;Dyrks et al, 1992;Estus et al, 1992; Gandy et al., 1992a,b;Golde et al, 1992;Haass et al, 1992b) but also in paired helical filaments (Caputo et al, 1992), in senile plaques (Selkoe et al, 1988), in microvessels (Tamaoka et al, 1992), and in choroid plexus from human brain in AD, in the white matter of Down's syndrome brains (Tokuda et al, 1995), and in human platelets (Tables 1 and 2). CT fragments with molecular masses of 12-16 kDa have also been found in media and cytosol of lymphoblastoid cells obtained from patients with earlyor late-onset FAD (Matsumoto, 1994) and Down's syndrome (Kametani et al, 1994).…”

“…The A/I is composed of 39-43 amino acids and proteolytically derived from larger /3-amyloid precursor protein (/3APP) isoforms of 695, 714, 751, and 770 amino acids (Kang et al, 1987). Several mutations of /IAPP around the A/I domain have been discovered in certain types of earlyonset familial AD (FAD) (for review, see Tanzi et al, 1993), supporting its causal role in the pathogenesis of AD.To date, at least three processing pathways have been described: the nonamyloidogenic secretory pathway (a-secretase), which releases soluble ectodomain and prevents A/I formation (Esch et al, 1990); the endosomal-lysosomal pathway, in which some cell surface /IAPPs are reinternalized (Haass et al, 1 992a,b) and cleaved around at the N-terminus of the A/I sequence by /3-secretase to produce A/3-bearing amyloidogenic breakdown products of various sizes (14-22 kDa) Golde et al, 1992; Haass et al, 1992a,b;Shoji et al, 1992;Tamaoka et al, 1992) and subsequently possibly cleaved by 'ysecretase to release soluble 4-kDa A/I (Koo and Squazzo, 1994); and the 4-kDa A/I-producing pathway (/3-and y-secretases), involving coated pit-mediated endocytosis, which may be independent of the constitutive secretory pathway (Koo and Squazzo, 1994).Classically, A/I is the marker for AD, and it has been linked to the accompanying neurodegeneration (see, e.g., Sisodia et al, 1990). Several lines of evidence suggest that the overexpression of /3APP and the subsequent production of A/I could be linked to the genesis of AD (for review, see Checler, 1995).…”

“…To date, at least three processing pathways have been described: the nonamyloidogenic secretory pathway (a-secretase), which releases soluble ectodomain and prevents A/I formation (Esch et al, 1990); the endosomal-lysosomal pathway, in which some cell surface /IAPPs are reinternalized (Haass et al, 1 992a,b) and cleaved around at the N-terminus of the A/I sequence by /3-secretase to produce A/3-bearing amyloidogenic breakdown products of various sizes (14-22 kDa) Golde et al, 1992; Haass et al, 1992a,b;Shoji et al, 1992;Tamaoka et al, 1992) and subsequently possibly cleaved by 'ysecretase to release soluble 4-kDa A/I (Koo and Squazzo, 1994); and the 4-kDa A/I-producing pathway (/3-and y-secretases), involving coated pit-mediated endocytosis, which may be independent of the constitutive secretory pathway (Koo and Squazzo, 1994).…”