Developing effective therapeutics for complex diseases such as late-onset, sporadic Alzheimer’s disease (SAD) is difficult due to genetic and environmental heterogeneity in the human population and the limitations of existing animal models. Here, we used hiPSC-derived neurons to test a compound that stabilizes the retromer, a highly conserved multiprotein assembly that plays a pivotal role in trafficking molecules through the endosomal network. Using this human-specific system, we have confirmed previous data generated in murine models and show that retromer stabilization has a potentially beneficial effect on amyloid beta generation from human stem cell-derived neurons. We further demonstrate that manipulation of retromer complex levels within neurons affects pathogenic TAU phosphorylation in an amyloid-independent manner. Taken together, our work demonstrates that retromer stabilization is a promising candidate for therapeutic development in AD and highlights the advantages of testing novel compounds in a human-specific, neuronal system.

Recently, novel pharmacological chaperones were developed that stabilize the trimeric core complex of VPS35, VPS29, and VPS26. These molecules, R33 and R55, reduced Aβ in mouse hippocampal neurons in a retromer-dependent manner (). Similarly, the retromer stabilization molecule R55 reduced Aβ in in a murine neuronal cell line harboring the APP Swedish mutation (). These studies support the idea that increasing the levels and/or functions of endocytic trafficking components may be viable options for therapeutic development in AD. However, to move forward in investigation of therapeutic strategies for humans, it is critical to test candidates in a human-specific system. In addition, while Aβ is a critical pathogenic readout for AD, how retromer stabilization affects other components of the disease process, such as TAU hyperphosphorylation, and if these two events are interdependent, is unknown. To address these questions, we used hiPSC-derived neurons from SAD and FAD patients and from genome-edited hiPSC lines. This allowed us to modulate the amounts of Aβ generation and APP expression in neurons and test how retromer stabilization by the small molecule chaperone R33 affects endogenous levels of Aβ and phospho-TAU (pTAU) in a human-specific system.

Retromer is a multiprotein assembly with a primary role in the sorting and trafficking of plasma membrane proteins from the endosomes, to the trans-Golgi network, or directly back to the cell surface (). APP is trafficked through the endocytic network via retromer through its interaction with SORLA, a member of the vacuolar protein-sorting VPS10 family of sorting receptors and a well-defined AD risk gene (). The retromer assembly has two major components, including the cargo-recognition complex (CRC), a trimeric core composed of VPS35, VPS29, and VPS26 (). The second component of retromer comprises the membrane targeting specific nexins, chiefly SNX1, SNX3, SNX5, SNX6, and SNX27 (). Retromer deficiency, primarily loss of VPS35 and VPS26, has been described in SAD patients and increases Aβ peptides in cell culture and cognitive decline in the mouse brain ().

We and others have demonstrated a role for endocytic genes in AD risk (), and elegant microscopic analysis has documented enlarged endosomes and suggested endocytic dysfunction as an early phenotype in AD pathogenesis (). This work has been recapitulated in stem cell-derived neurons (); however, to date, no AD therapy targets the endosomal network, likely due to the complexity of its structure and regulation.

Alzheimer’s disease (AD) is a devastating disorder of the brain and a rapidly growing public health problem. Patients with rare mutations in the amyloid precursor protein (APP) and presenilin 1 and 2 genes (PSEN 1 and PSEN 2) make up about 5% of familial AD cases (FAD) while the vast majority of AD is late-onset, sporadic AD (SAD) (). SAD is a complex and heterogeneous disorder with a clear heritable component that defines genetic risk () but with unknown contributions from the environment. The lack of effective therapies for AD is, in large part, due to our lack of understanding of the cellular and molecular mechanisms that lead to the neuropathological outcomes of the disease. Reasons for this are several-fold. In particular, AD takes decades to manifest clinically, and irreversible cellular damage likely occurs before the overt clinical symptoms are detected. In addition, the human brain is an inaccessible organ, making it difficult to sample tissue from living patients. Human induced pluripotent stem cells (hiPSCs) have facilitated the development of human neuronal models for AD, as they can be differentiated into disease-appropriate cell types and maintain the unique patient genetic background. To date, AD has been successfully modeled using patient and isogenic cell lines, and disease-relevant phenotypes have been well-documented from stem cell-derived neurons for both FAD and SAD ().

Because hiPSCs are amenable to genome engineering, we generated an APP knockout (APP KO) hiPSC line from our parental APP duplication hiPSC line (APP1) using CRISPR/Cas9 genome editing. APP KO hiPSCs were generated from FAD APPparental line by excision of one copy of APP and the introduction of two premature stop codons via non-homologous end-joining in the remaining copies (R. Van der Kant et al., personal communication; Figures S5 A and S5B). APP KO hiPSCs have no detectable APP protein by western blot analysis ( Figure S5 C). We differentiated and purified neurons from APP KO hiPSCs and confirmed that these neurons do not have detectable levels of secreted Aβ peptides by ELISA ( Figure 6 A). We next treated APP KO neurons with R33 and observed decreased pTAU from APP KO neurons ( Figure 6 B) with no change in tTAU ( Figure 6 C), again leading to a significant reduction in the pTAU/tTAU ratio in APP KO neurons ( Figure 6 D). When APP KO neurons were transduced with shVPS35-c, we documented a significant increase in the amount of pTAU ( Figure 6 E), and this effect was confirmed with a second shRNA, shVPS35-d ( Figure S6 A). Finally, we tested whether VPS35 knockdown would change the effect of R33 on pTAU levels in APP KO neurons. Interestingly, knockdown of VPS35 did not affect the R33-mediated reduction in pTAU in these cells ( Figure S6 B). However, this result is not overly surprising, as we have an incomplete loss of VPS35 (∼50%) and the R33 chaperone is still stabilizing the remaining retromer. Indeed, previous work demonstrates that retromer stabilization chaperones continued to stabilize two of the three cargo-recognition core proteins (VPS29 and VPS35) under single knockdown conditions (shVPS26) (). Taken together, these experiments suggest that retromer stability influences TAU phosphorylation and that this is mediated through an APP-independent mechanism. Furthermore, these data imply that retromer enhancement may be beneficial even under less than optimal retromer conditions, such as loss of VPS35 expression, a condition documented in AD patient tissue ().

We next asked if modulation of Aβ levels affected the decrease in pTAU by R33. We measured pTAU from isogenic neurons harboring the APP Swedish mutation, which leads to a 2- to 3-fold increase in endogenous Aβ in heterozygous and homozygous carriers, respectively ( Figure 5 C). Interestingly, we did not observe increased levels of pTAU/tTAU ratio in cells harboring these mutations alone. Previous work using isogenic cell lines of another penetrant AD mutation, PSEN1ΔE9, also failed to show increased pTAU/tTAU ratios at baseline conditions (). However, treatment with R33 reduced the pTAU/tTAU ratio in the isogenic neurons to the same extent across this allelic series, with no significant difference in R33-mediated TAU ratio reduction between the wild-type cells and the Swedish genotypes ( Figure 5 D), suggesting that retromer stabilization reduces TAU phosphorylation regardless of genetic condition. To test whether modulating level of the APP β-CTF in our cell cultures affected the R33-mediated reduction of the TAU ratio, we treated wild-type purified neurons with the gamma-secretase inhibitor compound E, which inhibits the generation of Aβ but increases the amount of the β-CTF of APP. Interestingly, R33 treatment further reduced the amount of Aβ remaining after compound E treatment ( Figure 5 E). The presence of compound E had no effect on the decrease pTAU/tTAU ratio after treatment with R33 ( Figure 5 F). Taken together, these data suggest that while the pTAU/tTAU reduction by R33 is highly correlative with Aβ peptide levels, modulation of APP processing, either up or down, does not change the R33-mediated reduction of pTAU.

Because the magnitude of decrease in pTAU after R33 treatment was similar to the decreases observed in APP processing, we performed a linear regression analysis on absolute levels of pTAU and absolute levels of Aβ in the cultures across all 13 patient cell lines and found a positive correlation between the reduction of Aβ and the reduction in pTAU ( Figure 5 A). Previous work in hiPSC-derived neurons has suggested that beta cleavage of APP, rather than Aβ, is more highly associated with TAU phosphorylation (), so we also examined the levels of sAPPβ compared with pTAU and observed a similar, but less robust, correlation between the reduction in sAPPβ with pTAU ( Figure 5 B). Taken together, however, these data suggest that the reduction in pTAU by R33 is highly correlative with a reduction in amyloidogenic processing of APP.

(A and B) A linear regression analysis was performed between Aβ and pTAU (A) and sAPPβ and pTAU (B). R 2 values reported on graphs. (C) For each comparison, a one-way ANOVA with a Tukey multiple comparisons posttest was performed. (D and F) For each comparison, a two-tailed t test was performed. n = 3 independent experiments per treatment/condition.

(E) Compound E treatment substantially decreases Aβ peptides in hiPSC-derived neurons and treatment with R33 further decreases Aβ peptides in this condition, ∗∗∗ represents statistical difference from vehicle; ∗ represents statistical difference between compound E alone and compound E + R33.

(C) hiPSC neurons with the APP Swedish (Swe) mutation introduced by genome editing show substantial increases in Aβ peptides in either heterozygous (Swe/wild-type [WT]) or homozygous (Swe/Swe) genotypes compared with the WT isogenic controls.

(B) Absolute levels of sAPPβ peptides from 13 patient cell lines were graphed against absolute values of pTAU from hiPSC-derived neurons and show a positive correlation between decreases in sAPPβ and decreases in pTAU.

(A) Absolute levels of Aβ peptides from 13 patient cell lines were graphed against absolute values of pTAU from hiPSC-derived neurons and show a positive correlation between decreases in Aβ and decreases in pTAU.

The Decrease of Phosphorylated TAU Mediated by Retromer Stabilization Is Correlative with, but Not Dependent on, Decreases in Aβ and sAPPβ

Figure 5 The Decrease of Phosphorylated TAU Mediated by Retromer Stabilization Is Correlative with, but Not Dependent on, Decreases in Aβ and sAPPβ

We next tested whether destabilization of the retromer complex by knockdown of VPS35 had the opposite effect of R33 treatment in hiPSC-derived neurons. Using lentiviral transduction, we expressed two independent short hairpin RNAs (shRNAs) against VPS35 mRNA (determined from a pool of four shRNAs, Figure S4 A) in purified neurons and documented significant increases in Aβ peptides similar to what has been previously reported in mice ( Figures 4 A and 4B ) (). We next measured phosphorylated TAU protein (Thr 231) from purified neurons transduced with two VPS35 shRNA and observed a small but significant increase in pTAU at Thr 231 ( Figure 4 C). The magnitude of the increase in pTAU after VPS35 knockdown is similar to that of the decrease in pTAU we observe after R33 treatment. Because shRNA VPS35-c gave the strongest effect, we confirmed the knockdown of VPS35 protein and VPS35 mRNA using that shRNA in multiple experiments ( Figures 4 D and 4E). We consistently observed an approximately 50% knockdown of VPS35 protein in our neuronal cultures ( Figure 4 D). Although we are only able to reduce the levels of VPS35 protein by half, it should be noted that germline deletion of VPS35 is results in embryonic lethality in mice and haploinsufficiency of VPS35 is sufficient to increase AD neuropathology in a transgenic mouse model (). These data suggest that even modest changes in retromer subunit levels may have a large impact on cellular phenotypes. In terms of AD, we note that duplication of APP resulting in one extra copy of the gene and 50% more APP expression is sufficient to cause severe and early-onset AD (). Thus, for factors that may increase or decrease risk but not the deterministic probability of AD, changes on the order of 20% are likely relevant in human disease.

(A–C) For each comparison, a one-way ANOVA with a Tukey multiple comparisons posttest was performed. (D and E) For each comparison, a two-tailed t test was performed.p < 0.05,p < 0.01,p<0.001. Error bars represent SD. See also Figure S4

(C) pTAU levels increase in hiPSC-derived neurons when VPS35 levels are reduced by VPS35 shRNA (two shRNA, c and d) compared with scrambled shRNA (SCR shRNA).

(A and B) Aβ peptides (A, Aβ 1-40 ; B, Aβ 1-42 ) measured from the cell culture media of hiPSC-derived neurons are increased when VPS35 levels are reduced by VPS35 shRNA (two shRNA, VPS35-c and VPS35-d) compared with a scrambled shRNA (SCR shRNA).

Finally, we tested whether other molecules hypothesized to be neurotrophic or protective against AD phenotypes had a similar effect to R33 on the pTAU/tTAU ratio. We have previously shown that the neurotrophin brain-derived neurotrophic factor (BDNF) reduced Aβ peptides in stem cell-derived neurons and that this reduction was correlated with protective variants in the SORL1 gene, whose protein product, SORLA, is a receptor of the retromer complex (). BDNF has been shown to reduce TAU phosphorylation, although at a different epitope than is analyzed in this study, in retinoic acid-differentiated SH-SY5Y cells (). We treated hiPSC-derived neurons from three cell lines harboring SORL1 protective variants with BDNF and measured the pTAU/tTAU ratio. Under these conditions, however, we did not observe a significant change in the TAU ratio ( Figure S3 C). Taken together, these data suggest that the decrease in the pTAU/tTAU ratio by R33 has a more specific effect on the stabilization of the trimeric core cargo-recognition complex than on the various retromer receptor or interacting proteins.

We probed a second pTAU epitope, paired helical fragment (PHF) TAU, and observed that in the presence of compound E, a gamma-secretase inhibitor, levels of PHF TAU increased, but when neurons were concomitantly treated with both compound E and R33, the PHF signal decreased ( Figure S3 A). We tested whether R33 affected the activation of the TAU kinase GSK3β ( Figure S3 B); however, we did not observe a significant effect of R33 treatment on the activity of GSK3β, suggesting that the decrease in pTAU by R33 is mediated through a different mechanism.

In addition to amyloid plaques, another neuropathological hallmark of AD is the presence of neurofibrillary tangles comprising hyperphosphorylated TAU protein. The connection between Aβ and TAU is complex, with the dominant theory that Aβ pathology precedes and drives TAU pathology (), while other evidence suggests that these pathways can occur independently (). Neurons purified from hiPSCs have detectable levels of phosphorylated TAU on Thr231 and this correlates with increases in Aβ from APPpatients (). We tested the effect of retromer stabilization by R33 on the pTAU/total TAU (tTAU) ratio on our cohort of patient neurons and found that R33 treatment significantly decreased the pTAU/tTAU ratio in all cell lines, whether they were derived from NDC or SAD individuals ( Figures 3 A and 3B ). Interestingly, this decrease in the ratio was due to an effect on pTAU ( Figure 3 C), while the levels of tTAU in the cultures remained unchanged ( Figure 3 D). R33 treatment had a similar effect on neurons derived from an APPpatient, with a reduction of pTAU ( Figure 3 E), but not tTAU ( Figure 3 F), leading to a lowered pTAU/tTAU ratio in these neurons ( Figure 3 G).

Non-normally distributed data (A–D) were analyzed by Wilcoxon test.p < 0.05,p < 0.001. NS, nonsignificant. Error bars represent SD. (E–G) For APP duplication: n = 1 FAD individual, three independent experiments/treatment. For each comparison, a two-tailed t test was performed.p < 0.01. NS, nonsignificant. Error bars represent SD. See also Figure S3

(D) Levels of tTAU in all samples (NDC and SAD neurons) are unaffected by R33 treatment. n = 13 individuals (SAD + NDC) represented by dots/squares; two to four independent experiments/treatment.

(C) R33 reduces the TAU ratio by lowering phosphorylated TAU on Thr 231 in all samples (NDC and SAD neurons). n = 13 individuals (SAD + NDC) represented by dots/squares; two to four independent experiments/treatment.

(B) pTAU/tTAU ratio in purified neurons derived from NDC (SAD) individuals treated with vehicle (dots) or R33 (squares) for 72 hr. n = 7 SAD individuals (represented by dots/squares); two to four independent experiments/treatment.

(A) pTAU/tTAU ratio in purified neurons derived from NDC individuals treated with vehicle (dots) or R33 (squares) for 72 hr. n = 6 NDC individuals (represented by dots/squares); two to four independent experiments/individual/treatment.

We next asked if retromer stabilization by R33 had an effect in genetic backgrounds of penetrant mutations leading to early-onset, FAD, either by increasing the levels of APP (APP duplication, APP) or by enhancing beta cleavage of APP (APP Swedish, APP). In either patient neurons from FAD APP) or in isogenic neurons in which the Swedish mutation was introduced via CRISPR/Cas9 genome editing ( Figure S2 D) (), we observed a similar fold-decrease in Aβ peptides from APPpatients and the APPisogenic neurons as with the NDC and SAD patient neurons ( Figures 2 A and 2B), suggesting that this small molecule chaperone is a potent reducer of Aβ, despite patient or cell line genetic background. Indeed, we observed a significant decrease in all forms of APP processing ( Figure 2 C) and observed a reduction in APP β-CTF in purified neurons treated with R33 ( Figure 2 D). Interestingly, we did not observe a significant increase in FL APP ( Figure S2 C), although this may be due to the variability of APP expression across all our different patient genetic backgrounds and the dynamic process between APP cleavage and recycling, where changes in APP cleavage may affect the overall expression of APP. Future experiments are needed to determine how retromer stabilization affects these processes. Taken together, these data corroborate previous work in the mouse and suggest that retromer stabilization keeps APP out of intracellular compartments that generate β-cleavage of APP and, subsequently, Aβ.

We then tested a cohort of cell lines derived from participants in the University of California, San Diego (UCSD) Alzheimer's Disease Research Center (ADRC), which included six NDCs and seven SAD patients (n = 13 patient cell lines) ( Figure 1 A). We found that in cell lines derived from either NDC or SAD subjects, R33 treatment for 72 hr significantly reduced both Aβ 1–40 and 1–42 peptide species ( Figures 1 B–1E). Because this reduction occurred equally for both Aβ species, we did not observe significant changes in the Aβ 42:40 ratio ( Figures 1 F and 1G). This is similar to what was previously observed in mouse hippocampal neurons, where retromer stabilization decreased Aβ 40 and 42 peptides equally (). We observed variability among neuronal cultures in the amount of increase in VPS35 stability, which could be due to inherent variability between hiPSC lines, individual patient genomes, or the limited amount of material obtained from our purified neuron protocol ( Figure S2 A). However, in representative experiments, we documented detectable increases in VPS35 stability from purified neuronal cultures ( Figure S2 B).

Non-normally distributed data (A–E) were analyzed by Wilcoxon test.p < 0.05. Normally distributed data (F and G) were analyzed by two-tailed t test. NS, nonsignificant. All error bars represent SD. See also Figures S1 and S2

(F and G) Aβ 42:40 ratios in purified neurons derived from NDC (F) or SAD (G) patients treated with vehicle (dots) or R33 (squares) for 72 hr. n = 6 NDC individuals (represented by dots/squares); two to four independent experiments/individual/treatment. n = 7 SAD individuals (represented by dots/squares); two to four independent experiments/treatment.

(B and C) Aβ 1-40 (B) and Aβ 1-42 (C) peptides in purified neurons derived from NDC individuals treated with vehicle (dots) or R33 (squares) for 72 hr.

(A) Schematic diagram of experimental design. hiPSCs were generated from fibroblast biopsies of six NDCs and seven probable SAD patients from the UCSD ADRC. These lines have been previously published and characterized inand. FACS purified neural stem cells and purified neurons were generated following previously published methods ().

The pharmacological chaperone R55 was previously shown to stabilize the retromer core complex, reduce Aβ peptides, and decrease APP in endosomes in mouse primary hippocampal neurons, suggesting that retromer stabilization is beneficial in terms of AD pathogenesis (). We extended these studies to test the effect of retromer stabilization in human neurons using an hiPSC approach. We tested both R55 and its analog R33 in FACS-purified neurons derived from non-demented control (NDC) hiPSCs. Our differentiation protocol has consistently been shown to recapitulate key cellular AD phenotypes in a purified system, allowing us to accurately quantitate analytes coming from a particular cell type (i.e., human neurons) (). We found that, in contrast to mouse neurons, R33 had a greater effect on Aβ peptide reduction than R55 in human neurons differentiated from hiPSCs ( Figure S1 ).

Discussion

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