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. 1998 Dec 1;18(23):9629-37.
doi: 10.1523/JNEUROSCI.18-23-09629.1998.

Alzheimer amyloid protein precursor in the rat hippocampus: transport and processing through the perforant path

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Alzheimer amyloid protein precursor in the rat hippocampus: transport and processing through the perforant path

J D Buxbaum et al. J Neurosci. .

Abstract

Amyloid deposition is a neuropathological hallmark of Alzheimer's disease. The principal component of amyloid deposits is beta amyloid peptide (Abeta), a peptide derived by proteolytic processing of the amyloid precursor protein (APP). APP is axonally transported by the fast anterograde component. Several studies have indicated that Abeta deposits occur in proximity to neuritic and synaptic profiles. Taken together, these latter observations have suggested that APP, axonally transported to nerve terminals, may be processed to Abeta at those sites. To examine the fate of APP in the CNS, we injected [35S]methionine into the rat entorhinal cortex and examined the trafficking and processing of de novo synthesized APP in the perforant pathway and at presynaptic sites in the hippocampal formation. We report that both full-length and processed APP accumulate at presynaptic terminals of entorhinal neurons. Finally, we demonstrate that at these synaptic sites, C-terminal fragments of APP containing the entire Abeta domain accumulate, suggesting that these species may represent the penultimate precursors of synaptic Abeta.

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Figures

Fig. 1.
Fig. 1.
Elements of the perforant path. The perforant path originates in the entorhinal cortex, from which it “perforates” the hippocampal fissure before terminating in all parts of the hippocampus. A major target of the pathway is the granule cells of the dentate gyrus that in turn project to region CA3 via the mossy fiber axons.
Fig. 2.
Fig. 2.
APP695 mRNA in enriched in the entorhinal cortex. For analysis of APP mRNA, RNA was reverse transcribed using antisense primer AS1219, and the reverse-transcribed products were incubated in a PCR with 32P-5′ end-labeled sense primer. A, PCR analysis of APP mRNA. Amplified products generated after 16, 18, 20, or 22 cycles of the PCR procedure were fractionated by agarose electrophoresis and visualized by EtBr staining (left) and autoradiography (right). PCR products were generated from transcripts encoding APP695 (350 bp), APP751 (518 bp), and APP770 (575 bp) M, 1 kb ladder (Life Technologies, Rockville, MD). B, Quantification of PCR analysis. Autoradiograms were subjected to quantitative densitometry, and the cycle number was plotted versus the log of the relative density. Regression analysis was performed for each product, and the equations (and regression coefficients) were determined as follows: APP695 y = 0.290x − 0.062 (1.000); APP751y = 0.271 x − 1.156 (1.000); and APP770 y = 0.312x − 2.297 (0.998).
Fig. 3.
Fig. 3.
APP undergoes axonal transport in the rat CNS.A, [35S]Methionine was injected into rat entorhinal cortex (EC), and transport was allowed to proceed for 6 hr. Subsequently, the entorhinal cortex and the hippocampus were dissected and homogenized, and the labeled APP was analyzed by immunoprecipitation with antibodies reactive with the C or N terminal of APP. Full-length APP species that were reactive with anti-C-terminal antibodies and present in the entorhinal cortex but not the hippocampus were identified as incompletely glycosylated (immature) APP. APP species that were reactive with anti-N-terminal antibodies but not anti-C-terminal antibodies were identified as C-terminal-truncated (secreted) APP. Protein molecular weight standards are in kilodaltons. B, Full-length APP polypeptides in detergent lysates of uninjured rat entorhinal cortex and hippocampus were visualized by immunoblotting with anti-C-terminal antibodies. C, Patterns of full-length APP polypeptides in rat EC and hippocampus (H) are compared with human APP-695 and APP-770 transiently expressed in transfected COS-1 cells; 25 μg of detergent-soluble homogenate from EC or Hand 5 μl of detergent-soluble cell lysate from COS-1 cells were fractionated on 7% Tris-glycine gels. The right panelis a longer exposure of the left panel and allows visualization of mature APP-770 (770 Gly)Gly, Mature; Im, immature.
Fig. 4.
Fig. 4.
Potentially amyloidogenic CTFs of APP accumulate in the synaptic and/or axonal compartment in the rat CNS. [35S]Methionine was injected into rat entorhinal cortex, and transport was allowed to proceed for 6 hr. Subsequently, the entorhinal cortex and the hippocampus were dissected and homogenized, and the labeled APP was analyzed by immunoprecipitation with antibodies reactive with the C terminal of APP. The precipitates were fractionated on Tris-tricine gels to resolve the low molecular weight APP-derived species. A, Visualization of APP CTFs. Left, Five C-terminal fragments were identified in the entorhinal cortex and in the hippocampus by immunoprecipitation of35S-labeled protein. Center, Similar bands were also observed in ECL-labeled immunoblots of proteins extracted from crude synaptosomal fractions (P2). Right, Two of the bands observed in brain tissue had an apparent molecular mass that was the same as or higher than that of C100 extracted from CHO cells, stably expressing C100. DG, Dentate gyrus. B, Effects of entorhinal cortex extracts on APP degradation. CHO cells, overexpressing APP770, were labeled with [35S]methionine and lysed. The lysate was divided into two aliquots and incubated in the absence (−EC) or presence (+EC) of extracts of nonlabeled entorhinal cortex. After incubation, the 35S-labeled C-terminal fragments were analyzed from both aliquots on 16% Tris-glycine gels.
Fig. 5.
Fig. 5.
The identity of APP CTFs. A, Detergent lysates were prepared from rat entorhinal cortex and hippocampus. APP CTFs were immunoprecipitated using CT15or 3134N antibodies, treated with protein phosphatase, and analyzed by immunoblotting with CT15. Lanes 1, 2, Immunoblot analysis of total lysates withCT15. Lanes 3–8, Nontreated (−) and λ phosphatase-treated (+) APP immunoprecipitates (CT15 and3134N) or control immunoprecipitates (anti-Myc, raised against an epitope of the protooncogene c-myc) probed with CT15.B, The schematic represents our interpretation of the identity of APP CTFs as deduced from the data in A and in earlier biochemical characterization of CTFs (Seubert et al., 1992;Naslund et al., 1994; Simons et al., 1996; Wang et al., 1996; Xu et al., 1998). Circled P, Phosphate.
Fig. 6.
Fig. 6.
Full-length APP, secreted APP, and C-terminal fragments of APP accumulate in synaptic sites in the dentate gyrus. [35S]Methionine was injected into rat entorhinal cortex, and transport was allowed to proceed for 6 hr. Subsequently, the entorhinal cortex and the hippocampus were dissected. The hippocampus was sectioned, and the dentate gyrus was removed. The entorhinal cortex, the dentate gyrus, and the remainder of the hippocampus (dorsal hippocampus) were homogenized, and the labeled APP was analyzed by immunoprecipitation with antibodies reactive with the C or N terminal of APP. Recovered immune complexes were resolved on Tris-tricine gels, and the dried gels were exposed for 2 months to x-ray film. APP species were identified as described in the legends to Figures 2 and 3.

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