Synaptogenic effect of APP-Swedish mutation in familial Alzheimer’s disease

Bo Zhou; Jacqueline G Lu; Alberto Siddu; Marius Wernig; Thomas C Südhof

doi:10.1126/scitranslmed.abn9380

. Author manuscript; available in PMC: 2023 Feb 2.

Published in final edited form as: Sci Transl Med. 2022 Oct 19;14(667):eabn9380. doi: 10.1126/scitranslmed.abn9380

Synaptogenic effect of APP-Swedish mutation in familial Alzheimer’s disease

Bo Zhou ^1,^2,³, Jacqueline G Lu ^1,^2,³, Alberto Siddu ^1,³, Marius Wernig ^2,^3,^*, Thomas C Südhof ^1,^3,^4,^*

PMCID: PMC9894682 NIHMSID: NIHMS1863569 PMID: 36260691

Abstract

Mutations in β-amyloid (Aβ) precursor protein (APP) cause familial Alzheimer’s disease (AD) probably by enhancing Aβ peptides production from APP. An antibody targeting Aβ (aducanumab) was approved as an AD treatment; however, some Aβ antibodies have been reported to accelerate, instead of ameliorating, cognitive decline in individuals with AD. Using conditional APP mutations in human neurons for perfect isogenic controls and translational relevance, we found that the APP-Swedish mutation in familial AD increased synapse numbers and synaptic transmission, whereas the APP deletion decreased synapse numbers and synaptic transmission. Inhibition of BACE1, the protease that initiates Aβ production from APP, lowered synapse numbers, suppressed synaptic transmission in wild-type neurons, and occluded the phenotype of APP-Swedish–mutant neurons. Modest elevations of Aβ, conversely, elevated synapse numbers and synaptic transmission. Thus, the familial AD-linked APP-Swedish mutation under physiologically relevant conditions increased synaptic connectivity in human neurons via a modestly enhanced production of Aβ. These data are consistent with the relative inefficacy of BACE1 and anti-Aβ treatments in AD and the chronic nature of AD pathogenesis, suggesting that AD pathogenesis is not simply caused by overproduction of toxic Aβ but rather by a long-term effect of elevated Aβ concentrations.

INTRODUCTION

Alzheimer’s disease (AD) is the major cause of dementia worldwide, affecting millions of people. A key feature of AD is the formation of plaques containing β-amyloid (Aβ) peptides (1–4). Aβ is produced by site-specific cleavage of amyloid precursor protein (APP) mediated by two proteases, β-secretase 1 (BACE1) and γ-secretase (5–6). Mutations in APP and in γ-secretase alter production of Aβ and cause familial AD, suggesting a role for Aβ in AD pathogenesis (4). These findings prompted the development of antibodies to Aβ as a therapeutic approach, culminating in the recent approval by the U.S. Food and Drug Administration of an Aβ antibody (aducanumab) as a treatment for AD. However, the approval of aducanumab raised controversies due to its unclear therapeutic efficacy (7), especially because an antibody to soluble Aβ worsened the cognitive decline in individuals with AD in other clinical trials (8). Similarly, verubecestat and atabecestat, highly potent small-molecule inhibitors of BACE1, failed to ameliorate AD progression but caused a faster clinical decline (5, 9–10). These clinical data suggest that APP cleavage and Aβ production may have important physiological functions. However, these functions are poorly understood; even the physiological role of the precursor protein APP is largely unknown. The lack of insight into the underlying biology of key genes such as APP has hindered progress in understanding AD (11). For example, to this date, it is uncertain whether presenilin mutations causing familial AD are pathogenic because the mutated catalytic subunits of γ-secretase increase the production of Aβ or change the type of generated Aβ species or because presenilin mutations induce a partial loss of function of γ-secretase (12).

APP, Aβ, and presenilins have been extensively studied in mouse models, providing convincing evidence that high Aβ concentrations are toxic to synapses. For example, transgenic mice overexpressing human APP carrying the so called “Swedish” mutation (K595N/M596L) that increases Aβ and causes early-onset AD in patients (13) exhibit synapse loss and a decrease in synaptic transmission (14–16). Moreover, addition of Aβ to murine cultured neurons or brain slices is toxic to synapses (17–21). Several studies showed that exogenous Aβ promotes glutamate receptor endocytosis and blocks long-term potentiation (LTP) and memory formation (22–27). Other studies, however, reported positive roles of Aβ at synapses. For example, Aβ increases Ca²⁺ influx in cultured neurons and activates synaptic transmission and presynaptic facilitation in mouse hippocampal cultures and slices (28–31), and Aβ is required in young rats for memory stabilization (32). Moreover, studies of neurons generated from patient-derived induced pluripotent stem (iPS) cells with an APP-Swedish mutation revealed increases in the amplitudes and frequency of spontaneous miniature excitatory postsynaptic potentials (mEPSCs) (33). Furthermore, deletion of APP in mice may decrease dendritic spine numbers and reduce LTP (34). The diverse outcomes of these studies on Aβ could be due to the use of different concentrations, forms, and sources of Aβ. Alternatively, rodents may not be an appropriate model for studying the role of Aβ in human health, given the exceptional vulnerability of humans to AD (35) and relatively poor evolutionary conservation of the Aβ sequence. Because of these constraints, extensive studies on human iPS cells and neurons were performed, with hundreds of papers published at present (36–37). However, most of these papers focus on creating AD disease models, and few examined the biological roles and pathological dysfunction of APP or Aβ. Human neurons carrying familial AD-associated APP mutations exhibited elevated Aβ and a modest increase in the size of Rab5-positive endosomes (38), but other features of these neurons, such as their overall development or synaptic connections, were not examined in depth. To address these gaps in our knowledge, we here examined the role of APP and Aβ in human neurons using a tightly controlled genetic approach coupled with a functional electrophysiological analysis.

RESULTS

Generation of human neurons with a conditional Swedish mutation in the APP gene

Among APP mutations associated with familial AD, the Swedish mutation (K595N/M596L; located in exon 16 of APP) has been studied most intensely because it promotes β-secretase over α-secretase cleavage of APP and thereby increasing Aβ production (13, 39–40). Although many studies were performed on neurons derived from patients’ iPS cells with familial AD (41–44), such experiments are difficult to perform under truly isogenic conditions. Introduction or reversal of disease mutations into iPS cells by genetic engineering produces genetically similar cell lines (38, 44), but these procedures require clonal selection and passaging of cells that induce genetic changes. Therefore, to investigate the effect of the APP-Swedish mutation under truly isogenic conditions, we introduced the Swedish mutation conditionally in human embryonic stem (hES) and iPS cells, thereby avoiding clonal variation (Fig. 1A). Using homologous recombination, we inserted into the APP gene tandem copies of exon 16 that were either wild type (WT) or carried the Swedish mutation flanked by loxP and FRT sites (Fig. 1A and fig. S1; see Materials and Methods for details). As a result, Flp recombination excises the mutant but not WT exon 16, whereas Cre recombination excises the WT but not mutant exon 16; without Flp or Cre recombination, the engineered APP allele represents a loss-of-function state (Fig. 1A). Using this strategy, we generated four correctly targeted hES and iPS cell lines. We then derived excitatory neurons from these clones using forced expression of neurogenin 2 (Ngn2) (see fig. S2 for characterization) (45) and infected the developing postmitotic neurons with viruses expressing Flp or Cre recombinase. Quantifications confirmed a nearly complete infection rate (fig. S3, B and C). In this manner, we produced human neurons that are either homozygous for the WT APP gene or heterozygous for the Swedish-mutant and the WT APP gene but contain an otherwise identical genetic background.

Swedish-mutant human neurons exhibited no survival phenotype (fig. S3, A to C) and expressed the same amount of APP as WT neurons (Fig. 1B and fig. S3, D and E) but produced more Aβ₄₀ and Aβ₄₂ than WT neurons (Fig. 1C). Swedish-mutant neurons exhibited a small increase in phosphorylated Tau protein in human neurons (fig. S3, F to I) but no major changes in dendritic arborization or soma size (Fig. 2, A and B, and fig. S3, J to L). Imaging of Rab5-positive endosomes, however, detected a modest but consistent increase (15 to 20%) in the size of Rab5-positive endosomes in both hES and iPS cell–derived neurons (Fig. 2, C and D, and fig. S3M), confirming previous observations with APP-Swedish–mutant human neurons generated with a different protocol (38).

The Swedish mutation of APP increases functional synapse numbers

We next imaged synapses in WT and Swedish-mutant human neurons by immunocytochemistry for synapsin (Fig. 2E and fig. S4A). We observed an increased (~20%) synapse density in human neurons carrying the Swedish mutation. This increase was detected in neurons derived from all conditionally mutant hES and iPS cells (Fig. 2F and fig. S4B) and was confirmed by staining neurons for PSD95 as a postsynaptic marker (Fig. 2G and fig. S4C). Moreover, analyses of the protein composition of Swedish-mutant human neurons revealed a relatively large elevation (20 to 80% depending on the protein) in the expression of synaptic marker proteins (Fig. 2H and fig. S4D), consistent with an increase in synapse numbers.

The increase in synapses induced by the APP-Swedish mutation is unexpected because the Swedish mutation leads to a two- to three-fold elevation in Aβ production (Fig. 1C), which is thought to be neuro- and synaptotoxic. Thus, we expected a decrease instead of an increase in synapses (46–48). Although the increase in synapses was found using two independent approaches (imaging and protein expression measurements), we sought to further validate this result using electrophysiological recordings. Measurements of the frequency and amplitude of spontaneous mEPSCs in isogenic WT and Swedish-mutant neurons derived from independent stem cell clones demonstrated a robust increase (~30%) in mEPSC frequency without a change in mEPSC amplitude, consistent with the observed elevation of synapse numbers (Fig. 3, A to F). In addition, recordings of EPSCs evoked by action potentials documented a similar increase in synaptic strength, monitored as the EPSC amplitude, without a significant change in the coefficient of variation as a measure of the release probability (p > 0.05; Fig. 3, G to J). Viewed together, these data establish with multiple independent approaches that under isogenic conditions, the Swedish mutation in APP induces an increase in the number of functional synapses in human neurons.

Fig. 3. — (A to F) Assessment of the frequency and amplitude of spontaneous miniature excitatory postsynaptic currents (mEPSCs), monitored in the presence of tetrodotoxin in *APP*^+/+ and *APP*^Swe/+ human neurons derived from H1.2 (A to C) and 5d1.1 clones (D to F) [(A and D) representative traces; (B and E) cumulative probability plots of the interevent times and summary graph of the mEPSC frequency (inset); (C and F) cumulative probability plots and summary graphs (inset) of mEPSC amplitudes]. (G to J) Assessment of the amplitude and coefficient of variation of the evoked EPSCs measured on isogenic *APP*^+/+ and *APP*^Swe/+ human neurons derived from H1.2 (G and I) and 5d1.1 clones (H and J) [(G and H) representative traces; (I and J) summary graphs of EPSC amplitudes (left) and coefficients of variation (c.v.) (right)]. All neurons were analyzed 5 weeks after neuronal induction; additional data are shown in fig. S4. All numerical data are means ± SEM; numbers of cells/experiments analyzed are indicated in bars. Statistical significance was assessed by Kolmogorov-Smirnov (KS) test (all cumulative probability plots) or Student’s t test (all bar graphs), with*P < 0.05 and ****P < 0.0001. Nonsignificant comparisons are not indicated.

BACE1 inhibition blocks the effect of the Swedish mutation of APP in human neurons

The key consequence of the Swedish mutation is thought to be an enhancement of APP cleavage by BACE1, leading to increased Aβ production (39–40, 49), as also observed in our experiments using isogenic human neurons (Fig. 1C). The unexpected effect of the Swedish mutation on synapse numbers suggests that, contrary to its supposedly synaptotoxic effect, Aβ may support synapses. If correct, then this hypothesis would imply a central role for BACE1 in regulating synapses. To test this hypothesis, we asked whether pharmacological inhibition of BACE1 alters synaptic function and whether a potential effect of BACE1 inhibition is altered by the Swedish mutation in APP.

Chronic treatment of WT human neurons with the BACE1 inhibitor LY2886721 (50–51) suppressed production of Aβ and of the secreted extracellular domains of APP that contain a BACE1-generated C-terminal sequence (sAPPβ) (fig. S5, A to C). Chronic BACE1 inhibition did not lower the secreted extracellular domains of APP that contain an α-secretase–generated C-terminal sequence (sAPPα and had no effect on APP expression, demonstrating its selectivity (fig. S5, C and D). Chronic BACE1 inhibition robustly decreased synapse numbers (~30%) in WT neurons, as measured by synapsin immunocytochemistry, without affecting synapse size (Fig. 4, A to C). Moreover, chronic BACE1 inhibition completely blocked the increase in synapse numbers induced by the conditional heterozygous APP-Swedish mutation, again without affecting the synapse size (Fig. 4, A to C).

Fig. 4. — (A to C) Quantification of synapse density in WT *APP*^+/+ and Swedish-mutant *APP*^Swe/+ neurons with or without BACE1 inhibition measured by immunocytochemistry for synapsin in human neurons derived from two independent stem cell clones with conditional Swedish mutations [(A) representative images of *APP*^+/+ and *APP*^Swe/+ human neurons with or without BACE1 inhibitor (BACEi) treatment; neurons were stained for synapsin (SYN; green) and MAP2 (red; bottom: high magnification of dendrites); (B and C) summary graphs of the density (left) and size (right) of synapsin-positive synaptic puncta in neurons derived from H1.2 (B) or 5d1.1 cells (C)]. Neurons were treated with the BACE1 inhibitor LY2886721 at 2 μM starting 1 week after neuronal induction and analyzed 4 weeks later. (D to I), The frequency and amplitude of mEPSCs in isogenic *APP*^+/+ and *APP*^Swe/+ neurons with or without BACE1 inhibition monitored by whole-cell patch-clamp recordings in human neurons derived from two independent stem cell clones [H1.2 (D to F) and 5d1.1 (G to I)] with conditional *APP*-Swedish mutations [(D and G) representative mEPSC traces; (E and H) cumulative probability plots of the mEPSC interevent interval and summary graph of the mEPSC frequency (inset); (F and I) cumulative probability plot and summary graph (inset) of the mEPSC amplitude]. All numerical data are means ± SEM (cells/experiments are indicated in all bars); statistical significance was assessed by two-way ANOVA with post hoc corrections (all bar graphs) or KS test (all cumulative probability plots), with*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Nonsignificant comparisons are not indicated.

To independently confirm these results suggesting that the synaptic effects of the Swedish mutation in APP require BACE1 cleavage, we measured spontaneous synaptic activity using electrophysiological recordings (Fig. 4, D to I). The conclusions were the same as for morphological synapse measurements. Specifically, chronic BACE1 inhibition decreased (~20%) the frequency of mEPSCs in WT neurons, whereas, as described above, the Swedish mutation in APP increased (30 to 40%) the mEPSC frequency. BACE1 inhibition of Swedish-mutant neurons markedly suppressed the mEPSC frequency (40 to 60% decrease), such that it was similar to the mEPSC frequency observed in BACE1-inhibited WT neurons (Fig. 4, D to I). None of these manipulations altered the mEPSC amplitude. Together, these results indicate that BACE1 inhibition reduces the synapse density in a manner dependent on APP that occludes the effect of the APP-Swedish mutation.

Conditional deletion of APP impairs synaptic function

The results of the experiments with Swedish-mutant neurons and BACE1 inhibition suggest that APP cleavage serves to promote synapses. If so, then deletion of APP should decrease synaptic function. To test this prediction, we introduced a conditional loss-of-function mutation into the APP gene in hES cells (Fig. 5A). Different from the conditionally APP-Swedish mutation (Fig. 1A), the strategy for generating conditional deletions of APP is much simpler. We introduced loxP sites that flank exon 3 of the APP gene, which lead to a deletion of APP expression upon Cre-mediated excision (Fig. 5A and fig. S6, A to C). However, in contrast to engineering of the heterozygous Swedish mutation, we had to target both alleles of the APP gene to create a complete loss of APP function.

Conditional deletion of APP in human neurons greatly lowered (>80%) APP expression (Fig. 5B and fig. S6, D and E). Because Cre virus infection rate of neurons was close to 100%, the remaining APP protein is likely derived from cocultured nonmutant glia. As expected, the APP deletion also severely suppressed Aβ secretion (>90%) (Fig. 5C). The APP deletion had no effect on neuronal survival or dendritic arborization (Fig. 5, D and E, and fig. S7, A to E). However, the APP deletion caused a decrease (10 to 25%) in the size of Rab5-positive endosomes (Fig. 5, F and G, and fig. S7F). Thus, there is a matching increase in the size of Rab5-positive endosomes in APP-Swedish–mutant human neurons (Fig. 2, C and D) but a decrease in APP-deficient neurons (Fig. 5, F and G).

The APP deletion caused a robust decrease (20 to 35%) in synapse density, as measured by immunocytochemistry for either synapsin or PSD95 (Fig. 6, A to C, and fig. S7, G to I). This decrease was detected in two independent clones on an isogenic background. Electrophysiological recordings, analyzing neurons derived from two independent clones, demonstrated that the APP deletion caused a decline in mEPSC frequency (20 to 30%) without affecting the mEPSC amplitude or intrinsic electrical properties (Fig. 6, D to I, and fig. S7J). Last, the APP deletion suppressed the amplitude of evoked EPSCs (30 to 40% decrease), which was at least in part likely due to a decrease in release probability as judged by the increase (10 to 25%) in the coefficient of variation (Fig. 6, J to M). Thus, the deletion and Swedish mutation of APP induce mirror-image phenotypes: The APP deletion suppresses synapse numbers and synaptic function, but the APP-Swedish mutation enhances them. In the set of experiments described in the following, we explored how these phenotypes are produced.

Exogenous Aβ promotes synapse formation in human neurons

Our data indicate that a BACE1-dependent cleavage products of APP regulate synapses. Three APP cleavage products are increased by the APP-Swedish mutation and suppressed by the APP deletion: the secreted extracellular APPs fragment after BACE1 cleavage (sAPPβ), the remaining, transient C-terminal APP fragment after BACE1 cleavage (C99, also referred to as CTFβ), and the secreted Aβ peptide released from the C99 fragment by γ-secretases. To identify which of these products mediates the synaptic effects of APP cleavage by BACE1, we transfected human embryonic kidney (HEK) 293T cells with full-length APP (positive control), sAPPβ, and C99, using enhanced green fluorescent protein (EGFP)–only transfections as a negative control (Fig. 7A). The supernatants from HEK293T cells expressing full-length APP or C99, but not from HEK293T cells expressing sAPPβ or EGFP only, contained elevated Aβ as expected (Fig. 7B), whereas the supernatants from HEK293T cells expressing full-length APP or sAPPβ, but not from HEK293T cells expressing C99 or EGFP only, contained higher amounts of sAPPβ (fig. S8A). We added the supernatants from the transfected HEK293T cells to matching WT and APP-deleted human neurons and analyzed these neurons using morphological and electrophysiological assays (Fig. 7A). The amount of added supernatant produced a final Aβ concentration in the medium of the neurons (~0.4 to 0.5 ng/ml) comparable to that observed in the medium of neurons carrying the APP-Swedish mutation. Immunoblotting demonstrated that Aβ in fresh HEK293T cell supernatants, as added to the neuronal culture medium, was largely monomeric, whereas upon storage, the Aβ became oligomeric (fig. S8C).

Fig. 7. — (A) Experimental strategy for (B) to (F). The supernatant of HEK293T cells expressing EGFP (control), full-length APP (flAPP), or the C99 and sAPPβ fragments of APP was added to human *APP*^+/+ and *APP*^−/− neurons 7 days after neuronal Ngn2 induction; neurons were analyzed 4 weeks afterwards. (B) Total Aβ peptides measured by ELISA in supernatants of HEK293T cells expressing EGFP, full-length APP, C99, and sAPPβ. (C) Quantification of synapse density measured by synapsin immunocytochemistry on human *APP*^+/+ and *APP*^−/− neurons treated with indicated supernatants from (A). For representative images, see fig. S8F. (D to F) Assessment of the mEPSC frequency in human *APP*^+/+ and *APP*^−/− neurons treated with indicated supernatants from (A). [(D) Representative mEPSC traces; (E and F) cumulative probability plots of mEPSC interevent intervals (insets, summary graphs of the mEPSC frequency) from *APP*^+/+ (E) and *APP*^−/− (F) neurons]. (G) Experimental strategy for (H) to (K). Human *APP*^−/− neurons were infected with lentiviruses encoding mOrange (control), full-length APP (flAPP), or the C99 and sAPPβ fragments of APP 4 days after neuronal Ngn2 induction; neurons were analyzed 5 weeks after neuronal induction. Uninfected *APP*^+/+ and *APP*^−/− neurons were used as further controls in (H) to (K). (H) Total Aβ peptides in supernatants of human *APP*^−/− neurons expressing EGFP, full-length APP, C99, and sAPPβ. Values were measured by ELISA at 17 days after neuronal Ngn2 induction. (I) Quantification of synapse density by synapsin immunocytochemistry on human *APP*^−/− neurons expressing EGFP, full-length APP, C99, and sAPPβ. For representative images, see fig. S9F. (J and K) Assessment of the mEPSC frequency on human *APP*^−/− neurons expressing EGFP, full-length APP, C99, and sAPPβ. [(J) Representative mEPSC traces; (K) cumulative probability plot of mEPSC interevent intervals and summary graph of the mEPSC frequency(inset)]. Additional data are shown in figs. S8 and S9. All summary graphs show means ± SEM [number of experiments (B and H) or of images or cells/experiments (all other graphs) are indicated in bars]; statistical significance was assessed by one-way ANOVA (B, H, I, and K) or two-way ANOVA (C, E, and F) with post hoc corrections, or by KS test (cumulative probability plots), with *P < 0.05, **P < 0.01, and ***P < 0.001. Nonsignificant comparisons are not indicated.

Supernatants from HEK293T cells did not change the size of Rab5-positive endosomes compared to the supernatant from EGFP control in both WT and APP-deficient human neurons, although there was a trend for an increase in size in neurons treated with supernatants from HEK293T cells expressing full-length APP or C99 (fig. S8, D and E). However, supernatants from HEK293T cells expressing full-length APP or C99, but not the other supernatants, increased (~25%) the density of synapsin-positive puncta in WT human neurons without changing their size (Fig. 7C and fig. S8, F and G). As before, APP-deficient neurons exhibited a decreased synapse density (~20%), but, again, the full-length APP and C99 supernatants increased the synapse density (~25%). An almost identical pattern of responses was detected when we recorded mEPSCs, with the full-length APP and C99 supernatants robustly increasing the mEPSC frequency (~30%) in both WT and APP-deficient human neurons without changing the mEPSC amplitude (Fig. 7, D to F, and fig. S8, H and I). Together, these results can be explained by an effect of Aβ because Aβ is very likely the only product that is present in the full-length APP and C99 supernatants but lacking in the sAPPβ and control supernatants.

A potential role of Aβ in promoting synaptic function is unexpected given the vast literature suggesting the opposite, albeit on the basis of experiments applying rather high Aβ concentrations to rodent neurons (52–54). We also find that direct applications of Aβ to human neurons are highly toxic. To definitively establish a role for Aβ in synapse formation, we sought to test its role using a second, complementary approach. We generated matching WT and APP-deficient human neurons and used lentiviruses to express in the APP-deficient neurons either mOrange as a negative control or full-length APP, sAPPβ, or C99 (Fig. 7G). Measurements of Aβ and sAPPβ secreted into the medium demonstrated that only expression of full-length APP and C99 (but not of mOrange or sAPPβ) resulted in physiological Aβ production, whereas conversely expression of full-length APP and sAPPβ (but not of mOrange and C99) induced secretion of physiological sAPPβ (Fig. 7H and fig. S9, A to C).

In contrast to the supernatant experiments, the decreased size of Rab5-positive endosomes (~25%) in APP-deleted neurons was fully reversed by expression of full-length APP or C99 but not by expression of mOrange or sAPPβ (fig. S9, D and E). Expression of full-length APP and C99, but not of mOrange or sAPPβ, fully restored synapse numbers that were suppressed (~25%) by the APP deletion (Fig. 7I and fig. S9, F and G). Furthermore, recordings of mEPSCs demonstrated that expression of full-length APP and C99, but not of mOrange or sAPPβ, rescued most of the decrease in mEPSC frequency induced by the deletion of APP, although the rescue achieved statistical significance only in the cumulative distributions (p < 0.0001) and not in the means (Fig. 7, J and K, and fig. S9H). Thus, Aβ at physiological amount reverses the synaptic phenotypes of APP-deficient human neurons.

DISCUSSION

Our results suggest that contrary to currently prevailing concepts, Aβ at physiological concentrations is not neurotoxic and synaptotoxic but supports synapse function in human neurons. We used a rigorous genetic approach with isogenic controls and four independent approaches to arrive at this conclusion: analysis of human neurons carrying the APP-Swedish mutation that causes familial AD, demonstration of the role of BACE1 using a well-validated BACE1 inhibitor in WT and APP-mutant human neurons, analysis of human neurons carrying a deletion of APP, and measurements of the effects of increasing Aβ directly. These experiments suggest that, physiologically, APP promotes synapse formation in human neurons at least in part via release of Aβ peptides.

Why did previous studies arrive at a different conclusion? Studies in human and mouse neurons often involved high concentrations of Aβ and were performed under conditions that made control for genetic background and for clonal variation difficult (41–43). It is possible that oligomeric Aβ is neurotoxic but not normally produced by neurons (55), whereas monomeric Aβ may be synaptogenic. This would agree with the delayed development of AD in patients, suggesting that secreted endogenous Aβ may only become oligomeric as it accumulates during a person’s lifetime, similar to what is observed in vitro. Moreover, recombinant or overexpressed Aβ is more likely oligomeric than endogenous Aβ, thereby enhancing its toxicity. Our results confirm the previously observed increase in the size of Rab5-positive endosomes in human neurons with APP mutations that are associated with familial AD (38) but reveal that relative to the synaptic phenotype produced by the APP-Swedish mutation, the endosomal change is rather small. Given the increase in endosome size and synapse numbers in APP-Swedish–mutant and APP-deficient neurons, further research is needed to reveal the relation of endosomes to synapse formation in neurons.

Using cultured human neurons, we found that the APP-Swedish mutation increases synaptic function, which is quite counterintuitive. In the same vein, we and others observed an increase in synapse numbers with apolipoprotein E4 (ApoE4) similar to that detected here with Aβ released from APP, which is also puzzling in suggesting that ApoE4, the most important genetic risk factor for sporadic AD, predisposes to AD but enhances instead of suppressing synapse formation (44, 56, 57). ApoE4 also increases the size of endosomes in human neurons (44), similar to endogenous Aβ in our experiments. Neither of these findings was anticipated, nor can we offer a ready explanation for why a genetic predisposition to AD should be enhancing synapse numbers and endosome sizes. A key to reconciling the synaptogenic and neuropathological effects of Aβ and ApoE4 may be the timelines involved. Whereas synapse formation occurs in hours, AD pathogenesis operates over decades. The relation of synapse formation to synaptic degeneration is unclear, especially because synapse formation proceeds throughout life, and many synapses are short lived (58). Although unexpected, our findings offer a potential explanation for the consistent clinical finding that pharmacological suppression of Aβ by BACE1 inhibition leads to cognitive dysfunction (9–10), which could be due to a loss of synapses. We find this hypothesis intriguing because the BACE1 inhibition–mediated cognitive decline in patients with AD is reversible (59). Again, these questions will need to be addressed with new experimental approaches—time will tell.

Our study has important limitations. All of our work was performed with cultured excitatory neurons with a relatively, although not completely, homogeneous composition. In a living brain, many different types of neurons are connected into vast communicating networks that are supported by active types of glia, whereas cultures represent a less complex two-dimensional preparation. However, cultures reduce the environmental complexity and may serve as an appropriate model for the effects of single molecules/genes in human neuronal diseases. It seems likely that the fundamental properties of the basic units of circuits, such as their synapses, will be similarly affected by the APP mutations we analyzed in two- or three-dimensional states, but the effect of these mutations is difficult to analyze in a complex brain context. Different types of neurons, especially excitatory and inhibitory neurons, may be affected differentially by the APP mutations. Moreover, the impact of the mutations we examined on circuits is difficult to predict, not only because we only studied a restricted range of excitatory neurons but also because we did not examine these neurons in a real brain with all of its complexity. However, human neurons and their synapses are not readily amenable for studies in an intact brain. Although organoids have provided enormous progress in this regard, neuronal maturation in organoids is generally more limited than in two-dimensional cultures and little synaptic circuit analyses have been possible.

Mouse models of AD that involve transgenic expression of mutant APP have produced diverse phenotypes, with some results agreeing well with the present findings. Transgenic mice expressing APP with AD-associated mutations exhibit hyperexcitability, consistent with increased synaptic function (60). In young mice, the APP-Swedish mutation increases the spine density as a proxy for synapse density, but in aging mice, it decreases the spine density (61). Similarly, recent studies suggest that elevated Aβ in mice enhance synaptic protein expression (62), whereas the APP knockout may decrease spine and synapse numbers (63). Although more detailed studies on synapses in mutant mice will be required to conclude how general these findings are, they are congruent with the notion proposed here that Aβ is a pro-synaptogenic factor in young neurons at physiological concentrations.

In summary, we show here that in human neurons, the Swedish mutation in APP that enhances Aβ secretion promotes synapse formation, whereas deletion of APP or pharmacological BACE1 inhibition suppresses synapse formation. Taken together, these data provide a direct link of AD pathogenesis to synapse function that may help in designing AD prevention strategies.

MATERIALS AND METHODS

Study Design

This study was designed to thoroughly characterize APP-Swedish mutant and APP-deficient human neurons, particularly on synapse formation and synapse activity. For this purpose, we made hES/iPS cell lines carrying conditional APP-Swedish mutation or conditional APP-deficient alleles, and generated APP-Swedish mutant and APP-deficient human neurons along with their isogenic controls, with Cre or Flp induced genome recombination. Moreover, BACE1 inhibitor was applied to test whether the phenotype was caused by APP Cleavage in human neurons. In addition, we also elevated different APP cleavage products in human neuron medium to identify the one(s) elevating synapse formation. For all experiments, at least three biological replicates (independent experiments) were performed. All analyses were performed out in a ‘blinded’ fashion whereby the experimenter was unaware of the sample identity.

Statistics

No statistical methods were used to predetermine sample size because effect sizes were unknown before experiments. Statistical significances for comparisons between two conditions were called with two-tailed t-test in Microsoft Excel or GraphPad Prism. For comparisons between more than two conditions, one-way ANOVA was performed with Tukey’s post hoc in GraphPad Prism. Interactions between two factors were tested with two-way ANOVA with Tukey’s post hoc in GraphPad Prism.

The Kolmogorov-Smirnov test was applied for cumulative curves. All data in bar graphs and summary plots are shown as means ± SEM of independent biological replicates in all figures. Numbers in bars represent number of cells/cultures analyzed, with statistical significance (∗ = p < 0.05, ∗∗ = p < 0.01, ∗∗∗ = p < 0.001 and ∗∗∗∗ = p < 0.0001).

See Supplementary Material section for detailed materials and methods.

Supplementary Material

Table S1

Table S1. Individual subject-level data for statistics with n<20. (See supporting files)

NIHMS1863569-supplement-Table_S1.xlsx^{(24.3KB, xlsx)}

supplemental material

Fig. S1. Generation and verification of human ES and iPS cells with heterozygous knockin mutations in the APP gene that produces a conditionally Swedish-mutant APP allele.

Fig. S2. Characterization of induced human neurons derived from the human ES cell line H1.

Fig. S3. Further detailed characterizations of human neurons carrying APP-Swedish mutation, with the analysis data for additional clones that were only partly included in Fig. 1 & 2.

Fig. S4. Additional data on synapse formation and intrinsic electrical properties of APP^+/+ and APP^Swe/+ human neurons.

Fig. S5. Additional data demonstrating the effect of the BACE1 inhibitor LY2886721 (BACEi) on Aβ/sAPP secretion and on the relative concentration of APP mRNAs in human APP^+/+ and APP^Swe/+ neurons.

Fig. S6. Strategy for, and verification of, homozygous conditional KO mutations of APP in multiple engineered ES cell clones.

Fig. S7. Additional experiments characterizing human APP^−/− neurons compared to their genetically precisely matched APP^+/+ controls that were derived from the same conditionally mutant parental ES cells line, including dendrite development, synapse puncta sizes, and intrinsic electrical properties.

Fig. S8. Assessments of the secretion and aggregation of Aβ and sAPPβ that were obtained in the supernatants of transfected HEK293T cells, and measurements of their effects on the size of Rab5-positive endosomes, size of synaptic puncta, and mEPSC amplitudes in isogenic APP^+/+ and APP^−/− human neurons.

Fig. S9. Validation of the lentiviral expression of full-length APP (flAPP) or of the C99 and sAPPβ fragments of APP in HEK293T cells and human neurons, and measurements of the effects of the expression of full-length APP (flAPP) or of the C99 and sAPPβ fragments of APP on the size of Rab5-positive endosomes, size of synaptic puncta, and mEPSC amplitudes in human APP^−/− neurons compared to precisely matched APP^+/+ controls.

NIHMS1863569-supplement-supplemental_material.pdf^{(3MB, pdf)}

Acknowledgments:

We thank Dr. Peter Davies (Feinstein Institute for Medical Research, North Shore-LIJ Health) for Tau antibodies, Dr. Koji Tanabe (Stanford U.) for the 5d1 human iPS cell line, Stanford Neuroscience Microscopy Service (NS069375), and Dr. Samuele Marro for advice. The figure cartoons were adapted from Servier Medical Art (smart.servier.com). This study was supported by grants from National Institutes of Aging grants AG048131 (to M.W. and T.C.S.) and AG070919 (to T.C.S.).

Footnotes

Competing interests: T.C.S. serves on the Board of Directors for Sanofi S.A. and for CytoDel Inc., and is a member of the Scientific Advisory Board of REATA Pharmaceuticals, Simcere America Inc., Alector, Elysium, Jupiter, Danaher, Boost, Recognify, and Neuralight. In addition, T.C.S. has equity in Sanofi S.A., CytoDel, REATA, Alector, Jupiter, Boost, Recognify and NeuraLight. None of the consulting activity is directly related to the studies described in the current manuscript, and no research funding was obtained from any company for the present studies or for other studies in T.C.S.’ laboratory. No patents were filed in connection with the current study.

Data and materials availability:

All data are available in the main text or the supplementary materials.

References and Notes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1

Table S1. Individual subject-level data for statistics with n<20. (See supporting files)

NIHMS1863569-supplement-Table_S1.xlsx^{(24.3KB, xlsx)}

supplemental material

Fig. S1. Generation and verification of human ES and iPS cells with heterozygous knockin mutations in the APP gene that produces a conditionally Swedish-mutant APP allele.

Fig. S2. Characterization of induced human neurons derived from the human ES cell line H1.

Fig. S3. Further detailed characterizations of human neurons carrying APP-Swedish mutation, with the analysis data for additional clones that were only partly included in Fig. 1 & 2.

Fig. S4. Additional data on synapse formation and intrinsic electrical properties of APP^+/+ and APP^Swe/+ human neurons.

Fig. S6. Strategy for, and verification of, homozygous conditional KO mutations of APP in multiple engineered ES cell clones.

NIHMS1863569-supplement-supplemental_material.pdf^{(3MB, pdf)}

Data Availability Statement

All data are available in the main text or the supplementary materials.

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Synaptogenic effect of APP-Swedish mutation in familial Alzheimer’s disease

Bo Zhou

Jacqueline G Lu

Alberto Siddu

Marius Wernig

Thomas C Südhof

Abstract

INTRODUCTION

RESULTS

Generation of human neurons with a conditional Swedish mutation in the APP gene

Fig. 1. Generation of conditional Swedish-mutant APP^Swe/+ human neurons with APP expression and Aβ secretion measurements.

Fig. 2. Characterization of dendrite development, endosome sizes, and synapse densities in human conditional Swedish-mutant APP^Swe/+ neurons compared to their perfect isogenic controls.

The Swedish mutation of APP increases functional synapse numbers

Fig. 3. Synaptic transmission in human wild-type APP^+/+ and Swedish-mutant APP^Swe/+ neurons.

BACE1 inhibition blocks the effect of the Swedish mutation of APP in human neurons

Fig. 4. The effect of pharmacological BACE1 inhibition on synapse formation and synaptic transmission in human wild-type APP^+/+ and Swedish-mutant APP^Swe/+ neurons.

Conditional deletion of APP impairs synaptic function

Fig. 5. Generation and characterization of isogenic human APP^+/+ and APP^−/− neurons derived from the same conditional APP knockout stem cell subclones.

Fig. 6. Synapse density and synaptic transmission in human APP^+/+ and APP^−/− neurons.

Exogenous Aβ promotes synapse formation in human neurons

Fig. 7. The effect of modest elevations of Aβ on synapse formation in human neurons.

DISCUSSION

MATERIALS AND METHODS

Study Design

Statistics

Supplementary Material

Acknowledgments:

Footnotes

Data and materials availability:

References and Notes

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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Cite

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PERMALINK

Synaptogenic effect of APP-Swedish mutation in familial Alzheimer’s disease

Bo Zhou

Jacqueline G Lu

Alberto Siddu

Marius Wernig

Thomas C Südhof

Abstract

INTRODUCTION

RESULTS

Generation of human neurons with a conditional Swedish mutation in the APP gene

Fig. 1. Generation of conditional Swedish-mutant APPSwe/+ human neurons with APP expression and Aβ secretion measurements.

Fig. 2. Characterization of dendrite development, endosome sizes, and synapse densities in human conditional Swedish-mutant APPSwe/+ neurons compared to their perfect isogenic controls.

The Swedish mutation of APP increases functional synapse numbers

Fig. 3. Synaptic transmission in human wild-type APP+/+ and Swedish-mutant APPSwe/+ neurons.

BACE1 inhibition blocks the effect of the Swedish mutation of APP in human neurons

Fig. 4. The effect of pharmacological BACE1 inhibition on synapse formation and synaptic transmission in human wild-type APP+/+ and Swedish-mutant APPSwe/+ neurons.

Conditional deletion of APP impairs synaptic function

Fig. 5. Generation and characterization of isogenic human APP+/+ and APP−/− neurons derived from the same conditional APP knockout stem cell subclones.

Fig. 6. Synapse density and synaptic transmission in human APP+/+ and APP−/− neurons.

Exogenous Aβ promotes synapse formation in human neurons

Fig. 7. The effect of modest elevations of Aβ on synapse formation in human neurons.

DISCUSSION

MATERIALS AND METHODS

Study Design

Statistics

Supplementary Material

Acknowledgments:

Footnotes

Data and materials availability:

References and Notes

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Fig. 1. Generation of conditional Swedish-mutant APP^Swe/+ human neurons with APP expression and Aβ secretion measurements.

Fig. 2. Characterization of dendrite development, endosome sizes, and synapse densities in human conditional Swedish-mutant APP^Swe/+ neurons compared to their perfect isogenic controls.

Fig. 3. Synaptic transmission in human wild-type APP^+/+ and Swedish-mutant APP^Swe/+ neurons.

Fig. 4. The effect of pharmacological BACE1 inhibition on synapse formation and synaptic transmission in human wild-type APP^+/+ and Swedish-mutant APP^Swe/+ neurons.

Fig. 5. Generation and characterization of isogenic human APP^+/+ and APP^−/− neurons derived from the same conditional APP knockout stem cell subclones.

Fig. 6. Synapse density and synaptic transmission in human APP^+/+ and APP^−/− neurons.