ApoE4 astrocyte-conditioned media increased APP expression and Aβ 42 secretion in hiPSC-derived neurons
To address the effect of ApoE4 astrocytes on neuronal APP expression and Aβ generation, we utilized an iPSC line derived from healthy individuals carrying the ApoE3 allele and its isogenic line in which ApoE3 is converted to ApoE4 . Both ApoE3 and ApoE4 iPSC lines were differentiated in astrocytes or excitatory neurons as described previously [14, 15] with some modifications (Fig. 1A), and immunostaining confirmed the identity of these cells (Supplementary Fig. 1). In order to investigate whether secretory factors from ApoE4 astrocytes could affect neuronal Aβ production, we generated healthy hiPSC-derived neurons carrying ApoE3 and cultured them in conditioned media from other healthy iPSC-derived astrocytes (ApoE3/E4 heterozygote) for five weeks. The media were then replaced with either ApoE3 or ApoE4 astrocyte conditioned media (ACM) and cultured for four days (Fig. 1B). We found that ApoE4 ACM positively regulated the expression of APP in hiPSC-derived neurons carrying ApoE3 (Fig. 1C, D). Immunoblotting from neuronal lysates revealed that APP levels were significantly increased by ApoE4 ACM (Fig. 1E). We further measured the secreted levels of Aβ40 and Aβ42 and found that Aβ42 secretion was significantly increased by ApoE4 ACM. These data show that secretory factors from ApoE4 astrocytes positively regulate neuronal APP expression and Aβ secretion.
Cholesterol positively regulated the formation of lipid rafts and APP expression in hiPSC-derived neurons
Accumulation of intracellular cholesterol in hiPSC-derived ApoE4 astrocytes compared to isogenic ApoE3 astrocytes has been recently reported [11, 16], and Lin et al. further reported increased cholesterol secretion from ApoE4 astrocytes. Astrocytes supply cholesterol to neurons to support synapse formation and regulate membrane fluidity [9, 17]. Moreover, cholesterol, along with ganglioside and triglyceride, is a critical component of membrane lipid rafts, which provide a suitable platform for various membrane-bound proteins, including glutamate receptors. APP and its processing secretases, β- and γ-secretase, are also known to be located in lipid rafts, while α-secretase is mainly expressed in non-lipid rafts [18, 19].
Previous studies have shown that increasing cholesterol in the membrane induced the formation of lipid rafts and increased Aβ production [20, 21]. Therefore, we hypothesized that increased levels of cholesterol in ApoE4 ACM could be a key factor in upregulating APP and its processing by facilitating the formation of lipid rafts. First, the conditions were optimized to regulate environmental cholesterol levels by treating neurons with cholesterol or methyl β-cyclodextrin (MβCD; to deplete cholesterol) in a rat primary neuron culture system (Supplementary Fig. 2A). We found that treatment with 20 µM of cholesterol for four days was sufficient to increase neuronal cholesterol, as visualized by filipin III staining, which is naturally fluorescent upon cholesterol binding (Supplementary Fig. 2B, C). We then measured the levels of lipid rafts by staining for cholera toxin B (CTX-B), a well-known lipid raft marker, and found a significant increase in lipid rafts following cholesterol treatment (Supplementary Fig. 2D-E). APP expression was also increased in cholesterol-treated rat primary neurons (Supplementary Fig. 2F). There was no reduction in both neuronal cholesterol and lipid rafts following MβCD treatment in rat primary neurons (Supplementary Fig. 2B-E), which could be due to the homeostatic mechanism of neurons to compensate for the loss of intracellular cholesterol. To address whether cholesterol treatment is sufficient to mimic the effect of ApoE4 ACM on neuronal APP expression and Aβ secretion, as shown in Fig. 1C-F, we treated hiPSC-derived neurons with cholesterol or MβCD (Fig. 2A). The upregulation of cholesterol in neurons by exogenous cholesterol treatment was confirmed by filipin III staining (Fig. 2B, C). Consistent with the observation in rat primary neurons, MβCD treatment did not alter the levels of cholesterol in hiPSC-derived neurons (Fig. 2B, C). We then measured levels of lipid rafts in neurons and found that cholesterol treatment increased the area of CTX-B signals without affecting intensity, suggesting the expansion of lipid rafts (Fig. 2D, E). We also measured APP levels in neurons and found significant upregulation of APP expression by cholesterol treatment, due to increased area of APP signals rather than intensity (Fig. 2F), which is consistent with rat primary neurons treated with cholesterol (Supplementary Fig. 2F). We further found that the co-localization of APP and CTX-B was significantly increased by cholesterol treatment in both hiPSC-derived neurons and rat primary neurons (Fig. 2G, Supplementary Fig. 2G). To determine whether APP upregulation is caused simply by the expansion of lipid rafts or if more APP is recruited to the given area of lipid rafts, we measured the intensity of APP in the CTX-B/APP co-localized area. The data showed that there was no alteration in APP intensity in these regions (Fig. 2G, Supplementary Fig. 2G), suggesting that increased APP expression by extracellular cholesterol supply is mainly due to the increased area of lipid rafts.
Cholesterol in ApoE4 ACM increased the formation of lipid rafts in hiPSC-derived neurons
To investigate whether the effects of ApoE4 ACM on neuronal cholesterol levels and lipid raft formation were due to cholesterol oversupply, we measured cholesterol levels in ApoE3 and ApoE4 ACM. Consistent with a previous report , we found higher cholesterol levels in ApoE4 ACM than in ApoE3 ACM (Fig. 3A). We then measured filipin III in hiPSC-derived neurons cultured with conditioned media from either ApoE3 or ApoE4 astrocytes as described in Fig. 1B, and ApoE4 ACM-treated neurons displayed increased filipin III signals (Fig. 3B, C). We also found that the area and total levels of CTX-B were significantly increased compared to those of ApoE3 ACM-treated neurons (Fig. 3D-E). To determine whether the cholesterol in ApoE4 ACM is the major cause of the upregulation of lipid rafts and APP in neurons, we added MβCD to ApoE4 ACM during neuronal culture (Fig. 3F) and found that the upregulation of neuronal cholesterol by ApoE4 ACM was significantly attenuated by MβCD, potentially due to its scavenging effect toward exogenous cholesterol (Fig. 3G, H). The addition of MβCD to ApoE4 ACM abolished the ApoE4 ACM-induced increase in lipid raft expansion (Fig. 3I, J).
Reducing cholesterol attenuated ApoE4 ACM-induced APP upregulation and Aβ 42 secretion in hiPSC-derived neurons
To determine whether cholesterol in ApoE4 ACM was the major cause for the upregulation of APP and its metabolism to produce Aβ in hiPSC-derived neurons, we added MβCD to ApoE4 ACM during neuronal culture (Fig. 4A). As shown in Fig. 1E, neurons cultured with ApoE4 ACM showed increased expression of APP compared to those cultured with ApoE3 ACM. However, in the presence of MβCD, ApoE4 ACM was not able to induce significant upregulation of APP. Increased co-localization of lipid rafts and APP by ApoE4 ACM was also abolished in neurons treated with MβCD (Fig. 4B-E). Furthermore, MβCD treatment inhibited the ApoE4 ACM-induced increase in Aβ42 secretion in hiPSC-derived neurons (Fig. 4F). Taken together, these data suggest that an excess supply of cholesterol is responsible for ApoE4 ACM-mediated neuronal Aβ42 overproduction.