In this study, we generated hESC lines that stably overexpress wild-type or mutant APP to provide a novel human cell-based approach to examine the molecular and cellular pathways that underlie the development of AD. Interestingly, during the characterization of these APP-expressing lines, we observed that these cells rapidly, robustly, and spontaneously differentiate toward a neural phenotype. Importantly, this phenomenon was not cell line-specific, as we observed the same response in APP-expressing HUES7 clones (, E–H). To determine the underlying mechanism by which APP drives neural differentiation, we examined APP proteolysis and the roles of various APP-derived fragments in neural differentiation. Our results demonstrate that two secreted APP-derived soluble peptides (sAPPα and sAPPβ) drive neural differentiation of hESCs in a concentration-dependent manner.
Previous studies showed that sAPPα can promote neuronal survival and neurite outgrowth (28
). This trophic-like activity has also more recently been shown to promote the proliferation of adult rodent subventricular zone progenitors (31
). However, the potential influence of APP and sAPP fragments on human ES cells has thus far remained largely unexplored. Intriguingly, although sAPPα has been shown by others to mediate much of the neurotrophic properties of sAPPs, here we report the novel findings that sAPPβ is more potent at inducing hESC neural differentiation (). Importantly, our studies have also uncovered a straightforward and reproducible method to generate large numbers of neural cells from hESCs within just 5–6 days. Current protocols, in contrast, typically require 21 or more days to produce an equivalent yield of neural cells (32
To understand why sAPPβ more readily drives differentiation of hESCs than sAPPα, the downstream targets of sAPP signaling will need to be identified. Toward that end, a recent study found that sAPPβ can regulate the transcription of transthyretin and Klotho genes in the absence of full-length APP or APLP1 expression (33
). The mechanism by which sAPPβ modulates gene transcription remains unknown. However, the study by Li et al.
provides some of the first evidence that sAPPs can differentially modulate specific gene targets (33
). Future studies will aim to unravel the specific transcriptional targets of sAPPs that mediate our observed effects on hESC differentiation.
Although our data clearly demonstrate that overexpression of APP can drive neural differentiation of hESCs, a recent examination of mouse ESCs suggests that deletion of APP and its two homologues (APLP1 and APLP2) does not alter differentiation (34
). Two possible explanations may account for this apparent difference. First, our observed neural induction occurs spontaneously in ES media and in the absence of additional factors that can promote neural differentiation. In contrast, Bergmans et al.
) employed a standard neural differentiation protocol, generating embryoid bodies supplemented with retinoic acid and later adding N2 and B27 supplements to further drive differentiation. It is likely that more subtle physiological effects of APP deletion on neural differentiation can be readily overcome by the use of strong neural inducing agents such as retinoic acid. Second, although our experiments utilized hESCs, Bergmans et al.
) studied mouse knockout ESCs. It is possible that in man, modulation and proteolysis of APP plays a more significant role in the development and differentiation of neurons than in mice. In support of this notion, functional differences exist between several human and mouse proteins implicated in AD, including APP, tau, and ApoE (35
). Clearly, to more fully address the physiological function of APP in human cells, knockdown and knockout studies in hESCs will be needed.
In our cell model, we identified an increase in soluble secreted fragments of APP as the major mechanism underlying spontaneous neural differentiation of APP-ESC clones. Intriguingly, although the majority of studies have focused on the trophic activity of sAPPα in neuronal growth, we found instead that sAPPβ is the more potent of the two at driving neural differentiation. This is a significant finding because much effort has been expended in the development of drugs aimed at reducing or abolishing β-secretase activity (39
). Although inhibition of β-secretase would reduce levels of Aβ, our data indicate that β-secretase activity may be involved in neural induction. Whether or not sAPPβ can also modulate adult neurogenesis in a parallel manner to sAPPα remains to be determined.
Interestingly, a recent study showed that soluble and fibrillar Aβ could promote hESC proliferation, whereas treatment of hESCs with a β-secretase inhibitor decreased proliferation and increased nestin expression (5
). We argued that a shift in APP processing from amyloidogenic (β-secretase-mediated) to non-amyloidogenic (α-secretase-mediated) may drive differentiation. Our experiments would argue against this conclusion, as we find that sAPPβ drives robust neural differentiation (). We also observed no differences in the degree of neural induction between wild-type and Swedish mutant APP clones despite the fact that the Swedish mutation increases BACE1 cleavage and decreases α cleavage of APP. One possible explanation for this difference is that BACE1 inhibition would lead to altered processing of many other developmentally relevant BACE1 substrates. For example, a recent study identified several proteins critically involved in neurodevelopment as being substrates for BACE1 cleavage (41
). Most notable among these was the notch ligand Jagged-1, which has itself been shown to drive neural differentiation of ESCs (42
). Thus, the effects of BACE inhibition on neural differentiation likely occur independent of altered APP processing.
A further significant finding of our study was that proteins that comprise the γ-secretase complex are present at very low levels in hESCs. This finding suggests that γ-secretase-mediated cleavage of APP may not be necessary for neural induction. Consistent with this result, we were unable to detect either Aβ40 or 42 productions by hESCs despite the use of a highly sensitive ELISA (data not shown). Interestingly, another group recently showed that AICD interacts with TAG1 and negatively modulates neurogenesis through FE65 in NPCs (11
). This report supports our finding that subsequent interactions and pathways involving γ-secretase-mediated APP cleavage products are not the predominant driver of neural induction.
Studies of patients and transgenic AD models support the notion that altered APP expression or processing may modulate adult neurogenesis. However, the data remain conflicting. Although some groups have reported impaired neurogenesis in animal models of AD (43
), a converse phenotype has been seen by others. For example, studies of AD patients and mouse models that overexpress the human APP gene have revealed an increase in adult neurogenesis (45
). These latter findings clearly fit with our current study and suggest that in patients altered APP processing may well lead to increased neurogenesis, although this change is clearly unable to compensate for the widespread neuronal dysfunction that occurs with the disease.
By generating and examining hESC lines overexpressing either wild-type or mutant forms of APP, we identified a previously unknown role for APP and sAPPs in the neuronal differentiation of hESCs. We also show that the proneural activity of sAPPβ can be readily exploited to generate large numbers of neural cells from hESCs within just 5–6 days as compared with the 21+ days required by current protocols (32
). These APP-overexpressing lines will likely provide a valuable platform for the further dissection of both AD pathogenesis and APP cell biology in a human context. Future studies will be needed to expand our understanding how sAPPβ promotes neural differentiation. For example, it will be critical to identify the downstream targets and signaling pathways activated by sAPPβ in hESCs. By enhancing our understanding of the physiological functions of APP and its role in stem cell differentiation, these studies may also help to guide the development of novel approaches to treat AD.