Alzheimer disease A68 proteins injected into rat brain induce codeposits of beta-amyloid, ubiquitin, and alpha 1-antichymotrypsin.

Abstract

Aberrandy phosphorylated tau proteins (i.e., A68 or PHF-tau) and (-amyloid MATERIALS AND METHODS Isolation and Characterization of A68, Dephosphorylated A68 (DEP-A68), and Normal Tau. A68 was purified from the brains of four AD patients and one Down syndrome patient with . To render A68 water soluble for injection, A68 was further purified as follows. After sucrose gradient centrifugation (14,15), the 1.25-2.0 M and 2.25-2.5 M sucrose fractions were extracted in 2 M guanidine isothiocyanate at 37°C for 60 min, and the guanidine-insoluble material was removed by further centrifugation for 30 min at 100,000 x g. The supernatant was exhaustively dialyzed against distilled water, and the water-insoluble material was removed by centrifugation once again. The resulting supernatant was lyophilized and used for injection into rats as well as for the generation of DEP-A68. This guanidine-extracted, water-soluble supernatant was shown by Western blots (see below) to contain purified A68. Although the A68 preparation contained PHFs (14) prior to guanidine extraction, the guanidine-extracted, water-soluble A68 did not reassemble into PHFs or straight filaments in vitro (data not shown), as monitored by negative staining and electron microscopy (14). DEP-A68 was generated from A68 by enzymatic dephosphorylation after overnight incubation in type III-N Escherichia coli alkaline phosphatase (20 units/ml) at 37°C as described (14). Normal adult human tau was prepared exactly as described (14,15). Aliquots of the A68, DEP-A68, and normal adult human tau preparations that were used for injection were analyzed by gel electrophoresis and by Western blots with epitope-specific antibodies to A68 and tau according to described methods (2,3,14,15). The anti-tau and anti-A68 antibodies used in this study included Alz5O to residues 2-10; T60 to residues 119-150; T14 to residues 141-178; T46 to residues 404-441; Taul, which recognizes tau and DEP-A68, but not A68, and binds to a nonphosphorylated epitope within residues 189-207; T3P, which recognizes A68, but not tau or DEP-A68, and binds to an epitope within residues 389-402 that contains a phosphate at Ser-396; and PHF1, which is similar to the T3P antiserum (for further information on these antibodies, see refs. 2, 3, and 14-21 and citations therein; the numbering system for the amino acids in tau referred to here is based on the largest tau isoform, as described in ref. 22

Amyloid β-Protein (Aβ) 1–40 But Not Aβ1–42 Contributes to the Experimental Formation of Alzheimer Disease Amyloid Fibrils in Rat Brain

“…Five microliters of these A␤1-40 (10 g/ l) or A␤1-42 (10 g/ l) were injected into the right side of the hippocampus or deep cerebral cortex of female Sprague Dawley rats (200 -300 gm; n ϭ 50). After different postinjection survival times, the brains removed from the rats were fixed in 70% ethanol /0.15 M NaC l and embedded in paraffin as reported previously (Shin et al, 1993(Shin et al, , 1994. The postinjection survival times included 1 d (n ϭ 5), 1 week (n ϭ 5), 3 weeks (n ϭ 5), 5 weeks (n ϭ 5), and 7 weeks (n ϭ 5) for A␤1-40 as well as for A␤1-42.…”

“…After different postinjection survival times, the rats were perf used with PBS, followed by perf usion with 4% glutaraldehyde/0.1 M cachodylate buffer, pH 7.4, as described (Shin et al, 1993). The postinjection survival times included 1 week (n ϭ 8), 2 weeks (n ϭ 5), and 3 weeks (n ϭ 5).…”

Therapeutic strategies for tau mediated neurodegeneration

“…The concept that AD Aβ pathology can be transmitted from injections of AD brain lysates was first demonstrated in primate by Baker et al ,130 131 but this could not be replicated with injections of purified AD PHF tau proteins into non-Tg rat brains 132. However, more recently it was shown that tau pathology can propagate in a cell to cell manner 32.…”

Transmission of Tau Pathology from Human to Rodent Brain: How to Humanise Animal Models for Alzheimer’s Disease Research

“…An early study on transmissibility of AD was performed on hamsters which were inoculated with a buffy coat preparation from AD patients [68]. These animals developed CJD-like histopathological alterations.…”