The exosome secretory pathway transports amyloid precursor protein carboxyl-terminal fragments from the cell into the brain extracellular space

Abstract

In vitro studies have shown that neuronal cell cultures secrete exosomes containing amyloid-β precursor protein (APP) and the APP-processing products, C-terminal fragments (CTFs) and amyloid-β (Aβ). We investigated the secretion of full-length APP (flAPP) and APP CTFs via the exosome secretory pathway in vivo. To this end, we developed a novel protocol designed to isolate exosomes secreted into mouse brain extracellular space. Exosomes with typical morphology were isolated from freshly removed mouse brains and from frozen mouse and human brain tissues, demonstrating that exosomes can be isolated from post-mortem tissue frozen for long periods of time. flAPP, APP CTFs, and enzymes that cleave both flAPP and APP CTFs were identified in brain exosomes. Although higher levels of both flAPP and APP CTFs were observed in exosomes isolated from the brains of transgenic mice overexpressing human APP (Tg2576) compared with wild-type control mice, there was no difference in the number of secreted brain exosomes. These data indicate that the levels of flAPP and APP CTFs associated with exosomes mirror the cellular levels of flAPP and APP CTFs. Interestingly, exosomes isolated from the brains of both Tg2576 and wild-type mice are enriched with APP CTFs relative to flAPP. Thus, we hypothesize that the exosome secretory pathway plays a pleiotropic role in the brain: exosome secretion is beneficial to the cell, acting as a specific releasing system of neurotoxic APP CTFs and Aβ, but the secretion of exosomes enriched with APP CTFs, neurotoxic proteins that are also a source of secreted Aβ, is harmful to the brain.

FIGURE 1.

Brain exosome isolation experimental flow chart. The steps of the experimental procedure (right) designed to isolate and purify brain exosomes are described along with the associated objectives of each step of the procedure (left).

FIGURE 2.

Characterization of exosomes isolated from murine hemi-brains. A, wide-field EM imaging showed multiple mouse brain exosomes of sizes ranging from 50 to 150 nm and no other structures or cellular debris. Scale bar = 100 nm. B and C, exosomes isolated from the brains of WT and APP transgenic (Tg2576) mice were found in sucrose step gradient fractions b–d. Exosomal protein content (B) and exosomal AChE activity levels (C) standardized to total brain protein content were not different between transgenic and non-transgenic brain exosomes collected from fractions b–d. D, Western blotting demonstrated the presence of the exosomal marker flotillin in fractions b–d. The enzymes α-secretase (ADAM10), β-secretase (BACE1), and γ-secretase (nicastrin) were also detected in exosomal fractions. E, immuno-EM showed that exosomes isolated from mouse brains were identified by antibodies to the exosomal markers flotillin and TSG101 and were immunoreactive to antibodies that react with flAPP and APP β-CTF (C1/6.1) and with flAPP, APP β-CTF, and Aβ (4G8). Scale bar = 100 nm.

FIGURE 3.

Exosomes isolated from frozen human brain are immunoreactive to anti-flotillin and anti-flAPP, APP CTFs and Aβ. A, wide-field EM imaging showed multiple human brain exosomes of sizes ranging from 50 to 150 nm and no other structures or cellular debris. B, immuno-EM showed that exosomes isolated from human brains were identified by antibodies to the exosomal marker flotillin and were immunoreactive to antibodies that react with flAPP and APP metabolites (6E10). Scale bars = 100 nm.

FIGURE 4.

Exosomes isolated from brain tissues of Tg2576 and wild-type control mice contain flAPP, APP CTFs, and Aβ. flAPP and APP CTFs were revealed by Western blot analysis of exosome lysates (A) and brain homogenates (C) of Tg2576 (Tg) and WT mice with antibody C1/6.1. Protein bands were quantified and are presented as the ratio between Tg2576 and wild-type levels of either flAPP or APP CTFs. flAPP in the brains (C′) and brain exosomes (A′) of Tg2576 mice was 5.8 times (p ≤ 0.0001, Student's t test; n = 3) and 4.9 times (p = 0.0177, Student's t test; n = 3) higher, respectively, than in wild-type control mice. APP CTFs levels in the brains (C′) and brain exosomes (A′) of Tg2576 animals were 5.6 times (p = 0.0036, Student's t test; n = 3) and 4.0 times (p = 0.0056, Student's t test; n = 3) higher, respectively, than in wild-type control mice. Western blot analysis with antibody 6E10 showed Aβ associated with exosomes in the brains of Tg2576 mice (B). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 5.

Brain exosomes are enriched with APP CTFs. Shown are the results from Western blot analysis with antibody C1/6.1 of brain homogenates and exosomes isolated from the brains of WT and Tg2576 mice retrieved prior to purification on a sucrose step gradient column (Pre-column) or from fractions b–d collected from the sucrose gradient column. Protein bands were quantified and are presented as the ratio between APP CTFs and flAPP in brain homogenates and exosomes. The APP CTF:flAPP ratio was higher in exosomes compared with brain homogenates in the brains of wild-type control mice (p = 0.025) and Tg2576 mice (p = 0.027) (analysis of variance with Fisher's least significant difference post-hoc test; n = 3). The APP CTF:flAPP ratio in Tg2576 brain exosomes compared with wild-type control brain exosomes was not different (p > 0.05). *, p < 0.05.