Scholarly article on topic 'Defective Transcytosis of APP and Lipoproteins in Human iPSC-Derived Neurons with Familial Alzheimer’s Disease Mutations'

Defective Transcytosis of APP and Lipoproteins in Human iPSC-Derived Neurons with Familial Alzheimer’s Disease Mutations Academic research paper on "Biological sciences"

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{"Alzheimer’s disease" / iPSC / FAD / APP / PS1 / transcytosis / endocytosis}

Abstract of research paper on Biological sciences, author of scientific article — Grace Woodruff, Sol M. Reyna, Mariah Dunlap, Rik Van Der Kant, Julia A. Callender, et al.

Summary We investigated early phenotypes caused by familial Alzheimer’s disease (fAD) mutations in isogenic human iPSC-derived neurons. Analysis of neurons carrying fAD PS1 or APP mutations introduced using genome editing technology at the endogenous loci revealed that fAD mutant neurons had previously unreported defects in the recycling state of endocytosis and soma-to-axon transcytosis of APP and lipoproteins. The endocytosis reduction could be rescued through treatment with a β-secretase inhibitor. Our data suggest that accumulation of β-CTFs of APP, but not Aβ, slow vesicle formation from an endocytic recycling compartment marked by the transcytotic GTPase Rab11. We confirm previous results that endocytosis is affected in AD and extend these to uncover a neuron-specific defect. Decreased lipoprotein endocytosis and transcytosis to the axon suggest that a neuron-specific impairment in endocytic axonal delivery of lipoproteins and other key materials might compromise synaptic maintenance in fAD.

Academic research paper on topic "Defective Transcytosis of APP and Lipoproteins in Human iPSC-Derived Neurons with Familial Alzheimer’s Disease Mutations"

Cell Reports


Defective Transcytosis of APP and Lipoproteins in Human iPSC-Derived Neurons with Familial Alzheimer's Disease Mutations

Graphical Abstract


Grace Woodruff, Sol M. Reyna,

Mariah Dunlap.....Jessica E. Young,

Elizabeth A. Roberts, Lawrence S.B. Goldstein


In Brief

Woodruff et al. find that FAD mutant neurons display abnormal endocytosis and transcytosis of APP and lipoproteins that are mediated by Rab11. Defective endocytosis and transcytosis of lipoproteins are rescued by b-secretase inhibition.


• FAD mutations impair endocytosis and transcytosis of APP and lipoproteins

• Reduced lipoprotein endocytosis and transcytosis are rescued by b-secretase inhibition

Woodruff et al., 2016, Cell Reports 17, 759-773 ciossMark October 11, 2016 © 2016 The Authors.

http://dx.d0i.0rg/l 0.1016/j.celrep.2016.09.034


Cell Reports


Defective Transcytosis of APP and Lipoproteins

in Human iPSC-Derived Neurons

with Familial Alzheimer's Disease Mutations

Grace Woodruff,14 Sol M. Reyna,14 Mariah Dunlap,1 Rik Van Der Kant,1 Julia A. Callender,1 Jessica E. Young,1 Elizabeth A. Roberts,1 and Lawrence S.B. Goldstein1 2 3 5 *

department of Cellular and Molecular Medicine

2Department of Neurosciences

3Sanford Consortium for Regenerative Medicine

University of California, San Diego, La Jolla, CA 92093, USA

4Co-first author

5Lead Contact



We investigated early phenotypes caused by familial Alzheimer's disease (fAD) mutations in isogenic human iPSC-derived neurons. Analysis of neurons carrying fAD PS1 or APP mutations introduced using genome editing technology at the endogenous loci revealed that fAD mutant neurons had previously unreported defects in the recycling state of endocytosis and soma-to-axon transcytosis of APP and lipoproteins. The endocytosis reduction could be rescued through treatment with a b-secretase inhibitor. Our data suggest that accumulation of b-CTFs of APP, but not Ab, slow vesicle formation from an endocytic recycling compartment marked by the transcytotic GTPase Rab11. We confirm previous results that endocytosis is affected in AD and extend these to uncover a neuron-specific defect. Decreased lipo-protein endocytosis and transcytosis to the axon suggest that a neuron-specific impairment in endo-cytic axonal delivery of lipoproteins and other key materials might compromise synaptic maintenance in fAD.


Alzheimer's disease (AD) is a progressive and devastating neurodegenerative disorder that affects more than 30 million people worldwide, including 11% of those over 65 years of age and 32% of those over 85 (Alzheimer's Association, 2014). AD is characterized by progressive cerebral dysfunction, memory loss, synapse loss, and neuronal impairment leading to cell death. To date, there are no disease-modifying treatments that can cure or reduce the progression of AD. Genetically, AD is segmented into two populations: sporadic AD (sAD), where the underlying primary cause is not known, and rare autosomal-dominant mutations causing familial AD (fAD) (Gatz et al.,

2006). The common pathological features of sAD and fAD patients are the accumulation of senile plaques, composed of aggregated amyloid-p (Ap), and neurofibrillary tangles (NFTs), composed of hyper-phosphorylated tau (Spires-Jones and Hyman, 2014). Importantly, many experimental findings regarding AD have come from overexpression studies or studies of non-neuronal cells that lack the unique polarization and compartmentalization of neurons.

The amyloid cascade hypothesis of AD posits that extracellular Ap fragments of proteolytically processed amyloid precursor protein (APP) and intraneuronal tau accumulate abnormally in sAD and fAD. These accumulations are proposed to drive cellular stress, neurotoxicity, and, ultimately, synapse loss and neurodegeneration (Toyn and Ahlijanian, 2014). Previous work also reported apparent defects in early endocytosis in post-mortem brains of AD patients (Cataldo et al., 2001; Ginsberg et al., 2010). Complementary investigations in overexpression and non-neuronal cellular models also pointed to defects in early endocytosis and endocytic dysfunction driven not by Ap but, instead, by p C-terminal fragments (p-CTFs) of APP (von Bartheld, 2004; Cataldo et al., 2000; Ginsberg et al., 2010; Karch and Goate, 2015; Lee et al., 2010; Maxfield, 2014). In addition, unbiased screens in non-neuronal cells consistently identified regulators of endocytic trafficking as key to normal levels of APP processing (Cataldo et al., 2000; Ginsberg et al., 2010; Treusch et al., 2011). However, many of the key investigations did not utilize normal expression levels of AD-related proteins or did not fully examine sorting pathways in highly polarized neurons. Thus, although several important insights into the role of endocytosis in regulating APP processing and sorting have been obtained from various cell models, little is known about endocytosis-dependent trafficking in polarized human neurons with mutations expressed at normal levels.

We tested whether isogenic induced pluripotent stem cell (iPSC)-derived human neurons with fAD PS1 and APP mutations induced by genome editing mutagenesis (transcription activator-like effector nuclease [TALEN] and clustered regularly interspaced short palindromic repeats [CRISPR]/Cas9) at the endogenous loci display amyloid-independent cellular trafficking


Cell Reports 17, 759-773, October 11, 2016 © 2016 The Authors. 759 This is an open access article under the CC BY license (

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Figure 1. PS1DE9 iPSC-Derived Neurons Exhibit Altered Sub-cellular Distribution of APP

(A) PS1DE9 neurons have a gene dose-dependent increase in soma APP intensity. Data are represented as mean ± SEM of all cell values with 10 PS1WT/WT, 6 PS1WT/DE9, and 10 PS1DE9/DE9 biological replicates. (PS1WT/DE9, p < 0.05; ps1iE9/iE9, p < 0.0001). Scale bar, 20 mm.

(B) The surface intensity of APP is increased in PS1WT/DE9 and ps1iE9/iE9 (p < 0.01) neurons compared with PS1WT/WT. Scale bar, 5 mm.

(C) PS1WT/WT neurons exhibited minimal APP CTF staining in unpermeabilized cells and dramatically increased N-terminal fragment (NTF) staining when per-meabilized. Data represent the mean ± SEM from two immunofluorescence experiments with two biological replicates per control (p = 0.0262). Scale bar, 5 mm.

(D) Average density of APP (ps1wt/de9, p < 0.05; PS1DE9/DE9, p < 0.0001) and individual punctum intensities (ps1wt/de9, p < 0.0001; ps1iE9/iE9, p < 0.0001) were decreased in a gene dose-dependent manner in PS1DE9 axons. Data represent the pooled median values with Tukey whiskers from three immunofluorescence experiments representing seven PS1WT/WT, four PS1WT/DE9, and six PS1DE9/DE9 biological replicates. Scale bar, 5 mm.

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and transport phenotypes, beyond the known effects on APP processing, that could account for the development of fAD (von Bartheld, 2004; Cataldo et al., 2000; Ginsberg et al., 2010; Karch and Goate, 2015; Lee et al., 2010; Maxfield, 2014). Human neurons expressing fAD mutant PS1 and APP at normal expression levels from the endogenous loci alter subcellular distribution and trafficking of APP and internalized lipoprotein, leading to elevated levels of APP in the soma and reduced levels in the axons. The redistribution of APP is accompanied by the concurrent accumulation of Rab11 endosomal vesicles in the neuronal soma and reduced Rab11 axonal staining, suggesting that the reduction in axonal APP and lipoproteins can be explained by reduced Rab11-dependent soma-to-axon transcytosis and defects in the recycling endosome (Ascano et al., 2009; von Bartheld, 2004; Buggia-Prévot et al., 2014). Knockdown of Rab11 also leads to reductions in APP axonal density and lipoprotein endocytosis and transcytosis. Our study reveals that a common early defect among fAD PS1 and APP mutations is APP ß-CTF accumulation-induced impairment of a key neuron-specific traffic pathway, soma-to-axon transcytosis, caused by defects in the recycling endosome.


The PS1DE9 Mutation Increases APP in the Soma of Human Neurons and Decreases APP in Axons

PS1 has been reported to have a role in APP trafficking in primary neurons and in non-neuronal cell types (Burns et al., 2003; Cai et al., 2003; Gandy et al., 2007; Zhang et al., 2006). PS1 knockout has been reported to increase cell surface APP (Leem et al., 2002), whereas PS1 fAD mutations have been shown to delay APP arrival at the cell surface (Cai et al., 2003; Gandy et al., 2007). To determine whether the PS1DE9 mutation affects APP localization in human neurons, we generated purified neurons from isogenic iPSC lines from wild-type (PS1WT/WT) and lines heterozygous (PS1WT/DE9) and homozygous (PS1AE9/AE9) for the PS1DE9 mutation (Woodruff et al., 2013). We stained with an APP antibody that has minimal staining in cells from APP knockout mice (Guo et al., 2012; Weissmiller et al., 2015). We found that ps1wt/de9 and PS1AE9/AE9 neurons exhibited a modest but significant increase in intracellular APP staining in the cell body (Figure 1A). To determine whether APP was also increased on the surface of PS1DE9 neurons, we stained unpermeabilized purified neurons with an N-terminal APP antibody that recognizes the extracellular portion of APP (22C11) (Figure 1B). We observed increased APP on the soma surface of PS1DE9 neurons (Figure 1B). As a control, we also stained unpermeabilized neurons with a C-ter-minal antibody and observed minimal staining, as would be expected because the C terminus of APP would not be present on the cell surface (Figure 1C).

We previously published that PS1DE9 human neurons do not exhibit changes in total levels of full-length APP, although there

are increases in the APP CTFs (Woodruff et al., 2013). The increase in soma APP suggests that APP CTFs are accumulating in the soma of purified neurons and/or that APP is missorted, possibly at the expense of axonal APP. We therefore assessed APP staining in the axons of PS1AE9 neurons. To ensure that we were quantifying staining in axons and not dendritic processes, we made use of microfluidic compartments, which separate axons from the bulk somatodendritic population (Figure 1E; Niederst et al., 2015; Selfridge et al., 2015; Taylor et al., 2005). Neurons grown in microfluidic devices extend long processes into the axonal space that do not stain for the somatodendritic marker Map2 and that do stain for the axonal marker neurofilament-H (SMI31) (Selfridge et al., 2015; Figure 1E; Figure S1A). We observed that PS1AE9 axons grown in microfluidic devices have decreased axonal APP puncta and APP punctum intensity (Figure 1D); this decrease is sensitive to PS1AE9 gene dose.

To further characterize APP fragments that are present in axons, we also measured soluble APP (sAPP) fragments, sAPPa and sAPPp. To quantify secreted sAPP, we plated neurons in compartments and allowed them to extend their axons into the axonal space. We then performed a full medium change, kept the axons in fluidic isolation, and harvested media from the soma and axon sides. When we measured the sAPPa/sAPPp ratio from the soma side of the cultures, we observed no significant differences between ps1wt/wt and PS1AE9/AE9 genotypes (Figure 1F). Additionally, there were no significant differences in the total levels of sAPPa or sAPPp from the soma side (Figure S1B). However, when we quantified the sAPPa/sAPPp ratio from axons only, we observed a significant increase from the ps1ae9/ae9 axons compared with PS1WT/WT (Figure 1F). Upon quantification of the total levels of sAPPa and sAPPp, we determined that the ratio was increased in ps1ae9/ae9 axons primarily because of a reduction in sAPPp (Figure 1F). To test whether the pheno-typic differences we observed in PS1AE9 neurons might be due to differences in neuronal subtypes in our cultures, we stained neurons of each genotype with the neuronal subtype markers gamma-aminobutyric acid (GABA), GAD65/67, and vesicular glutamate transporter (vGlut) (Figure S1C). We found no significant differences in the proportion of cells that stained positive for GABA, GAD65/67, or vGlut between different genotypes (Figure S1C).

Rab11 Distribution Is Altered in PS1DE9 Neurons

There are at least two pathways by which APP can be delivered to the axon. The first is by direct delivery from the frans-Golgi network (TGN), and the second is by an indirect pathway where APP first arrives at the cell surface of the so-matodendritic compartment and then undergoes endocytosis and sorting to the axon. The indirect pathway is a process known as transcytosis, and multiple cargos, including TrkA (Ascano et al., 2009) and L1/NgCAM (Anderson et al., 2005;

(F) sAPPa/p ratios in soma were not statistically different (p = 0.9614), but the ratio of axonally secreted sAPPa/p was Increased in PS1AE9/AE9 axons (p = 0.0199). Shown are relative amounts of secreted sAPPa and sAPPp in PS1AE9/AE9 axons compared with PS1WT/WT. sAPPp levels are reduced (p = 0.0056), but not sAPPa levels (p = 0.0540), in PS1AE9/AE9 axons. Data represent four PS1WT/WT and four PS1AE9/AE9 biological replicates. See also Figure S1.


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Figure 2. ps1DE9/DE9 Neurons Exhibit Altered Rab11 Distribution

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Yap et al., 2008), have been demonstrated to be sorted along this pathway in neurons.

An endocytic regulator that functions at the intersection of the TGN and transcytotic pathways is the Rab GTPase Rab11 (Welz et al., 2014). Rab11 has a well-established role in mediating the recycling of many receptors, including the transferrin receptor (Ullrich et al., 1996), and low-density lipoprotein (LDL) receptors (Sakane et al., 2010; Takahashi et al., 2007). In addition to its role in recycling, Rab11 has also been shown to mediate transcytosis in epithelial cells and neurons. Specifically in neurons, TrkA receptors undergo Rab11-dependent transcytosis to the axon (Ascano et al., 2009; Lazo et al., 2013). Rab11 may also be involved in trafficking of BACE1 to the axon (Buggia-Prevot et al., 2014), colocalizes with some APP in axons (Niederst et al., 2015), and was recently identified in an unbiased Rab GTPase screen in non-neuronal cells as a regulator of Aß and sAPPß production (Udayar et al., 2013). Intriguingly, presenilins have also been reported to interact directly with Rab11 through their hydrophilic loop (Dumanchin et al., 1999).

To determine whether Rab11 could be playing a role in the reduction of axonal APP in PS1DE9 cells, we stained neurons with a Rab11 antibody and measured Rab11 in the somatodendritic and axonal compartments. The Rab11 staining was reminiscent of the altered APP distribution such that PS1AE9/AE9 neurons exhibited increased soma Rab11 intensity, Rab11 puncta, and Rab11 punctum area (Figure 2A). In axons, both the PS1WT/DE9 and PS1AE9/AE9 genotypes had decreased Rab11 punctum density and punctum intensity (Figure 2B). In support of a role of Rab11-dependent trafficking of APP to the axon, short hairpin RNA (shRNA)-mediated knockdown of Rab11 on the soma side of neurons grown in microfluidic devices resulted in an ~40% reduction in APP axonal density (Figure 2C). In keeping with a transcytotic route of APP to the axon, somatodendritic inhibition of endocytosis with Dynasore led to reduced APP (~25% less) and Rab11 (~40% less) density in axons (Figure 2D).

Previous publications have implicated alterations in early en-dosomes and lysosomes in PS1 knockout cells (Coen et al., 2012; Lee et al., 2010; Neely et al., 2011). We therefore looked at the early endosome effector EEA1 and the lysosomal marker Lamp2 even though they are not thought to traffic substantially to axons (Wilson et al., 2000). Despite evidence in the literature that early endosomes and lysosomes are dramatically affected in fAD and sAD, EEA1 and Lamp2 staining in PS1DE9 neurons was not obviously different, suggesting that they are not playing an active role in sorting APP to the axon (Figures S2A and S2B).

The PS1DE9 Mutation Decreases Endocytosis and Transcytosis of APP and LDL

Because we observed alterations in the subcellular distribution of both APP and Rab11 in PS1DE9 neurons, we investigated whether endocytosis and/or transcytosis could account for the APP localization changes. To assess endocytosis of APP, we treated purified neurons with an N-terminal APP antibody (22C11) and allowed cells to internalize the antibody for 30 min, 2 hr, or 4 hr and fixed cells at each of those time points. We then stained with a secondary antibody and quantified the amount of APP endocytosis at each time point (Figure 3A). We observed a decrease in APP puncta in the PS1AE9/AE9 genotype starting at 30 min compared with PS1WT/WT, and this decrease was more prominent at both 2 and 4 hr (Figure 3A). The PS1WT/DE9 genotype also exhibited decreased APP puncta compared with PS1WT/WT at the 2- and 4-hr time points. As a control to confirm whether the APP antibody was being endocy-tosed uniquely via antigen binding, we performed an endocytosis assay with a non-specific antibody and found minimal uptake (Figure S3A). To assess whether this endocytosis defect was specific to APP or common to other cargo, we also measured the uptake of fluorescently labeled LDL at 30 min, 1 hr, 2 hr, and 4 hr (Figure 3B). Similar to what was observed with APP, LDL punctum intensity and LDL punctum density were reduced in ps1wt/de9 and ps1ae9/ae9 neurons at 2 hr and 4 hr (Figure 3B). To test whether the differences observed were due to a non-specific defect in endocytosis, we also quantified the uptake of fluorescently labeled dextran as a marker of bulk endocytosis. We did not observe any significant differences in dextran endocytosis at any time point (Figure 3C). Additionally, we quantified endocytosis of fluorescently labeled transferrin, and, similar to dextran, we did not observe any significant differences (Figures S3B and S3C).

We measured transcytosis of APP and LDL by growing neurons in microfluidic compartments. The axon side of the compartment was kept in fluidic isolation from the soma side, and APP antibody or labeled LDL was added only to the soma side. Transcytotic delivery of cargo from the soma to the axon is a relatively slow process because internalized cargo has to travel long distances (on the order of millimeters in cultured neurons). After 4 hr of endocytosis, cells were fixed, and we quantified the amount of anterogradely transcytosed APP by using an anti-mouse secondary antibody; fluorescent LDL was measured directly (Figures 3D and 3E). We observed that both the PS1WT/DE9 and PS1DE9/DE9 genotypes exhibited decreased APP axonal density and APP intensity after 4 hr of transcytosis (Figure 3D). Similarly, LDL axonal density and intensity were also decreased in PS1WT/DE9 and PS1AE9/AE9 axons after 4 hr

(B) Quantification of Rab11 in axons. Rab11 density (PS1WT/DE9, p = 0.0003; ps1iE9/iE9, p < 0.0001) and punctum intensity (PS1WT/DE9, p < 0.0001; ps1iE9/iE9, p < 0.0001) were reduced in a dose-dependent manner in PS1DE9 neurons. Data represent the mean ± SEM or median with Tukey whiskers of six PS1WT/WT, four PS1WT/DE9, and five ps1iE9/iE9 biological replicates. Scale bar, 5 mm.

(C) Knockdown of Rab11a diminished APP to ~60% of PS1WT/WT levels. Data represent the average of four untreated PS1WT/WT and three Rab11 shRNA-treated PS1WT/WT biological replicates (Rab11 density, p < 0.0001; Rab11 intensity, p < 0.0001; APP density, p < 0.0001; APP intensity, p < 0.0001).

(D) Inhibition of soma endocytosis resulted in an ~50% decrease in Rab11 density (first inset) and approximately a 20% decrease in APP density (second inset). Data represent the mean ± SEM or median with Tukey whiskers from axons of four untreated PS1WT/WT and three Rab11 shRNA-treated PS1WT/WT biological replicates (Rab11 density, p < 0.0001; Rab11 intensity, p < 0.0001; APP density, p = 0.0018; APP intensity, p < 0.0001).

See also Figure S2.


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Figure 3. PS1 Neurons Exhibit Reduced Endocytosis and Transcytosis of APP and LDL

(A) Example images of the internalization of APP 22C11 antibody at 30 min in PS1WT/WTand PS1DE9/DE9 neurons. The bar graphs depict the average APP punctum count and average intensity normalized to cell area at 30 min (PS1WT/DE9, p = 0.1341; PS1iE9/iE9, p < 0.0001), 120 min (PS1WT/DE9, p < 0.0001; PS1iE9/iE9, p < 0.0001), and 240 min (PS1WT/DE9, p = 0.0024; PS1AE9/AE9, p < 0.0001). Data represent the mean ± SEM of a minimum of 50 neurons per time point from four PS1WT/WT, four PS1WT/DE9, and four PS1AE9/AE9 biological replicates. Scale bar, 10 mm.

normalized to cell area. As shown, PS1WT/AE9 and ps1AE9/AE9 neurons have reduced APP and LDL soma endocytosis, with the LDL defect appearing most prominently at 240 min. Punctum data represent the pooled mean ± SEM or median with Tukey whiskers of over 140 cells/time point and 4-6 biological

(B) Example images of LDL-BODIPY labeling in PS1WT/WT and PS1AE9/AE9 neurons. The graphs depict quantification of the LDL punctum count and intensity

Figure 4. Rab11 Mediates Endocytosis and Transcytosis of LDL

(A) psi™™7 neurons were transduced with a lentivirus containing control (CTRL) or Rab11 shRNA(Rab11 knockdown [KD]) and then treated with LDL for 4 hr. Rab11 KD data represent mean ± SEM of soma obtained from three biological replicates of psiWT/WT (p < 0.0001). Scale bar, 10 mm.

(B) Western blot of cells transduced with control or one of three different Rabll shRNAs.

(C) psiWT/WT axons and psiWT/WT axons with Rabll knockdown and co-stains. Scale bar, 5 mm.

(D) Vesicle densities of Rabll and LDL under control and Rabll knockdown conditions. For the LDL density graph, Rabll knockdown (KD) resulted in non-detectable (N.D.) levels of axonal LDL. The punctaseen in Rabll KD were the result of background autofluorescence. LDL transcytosis data represent the mean ± SEM of three biological replicates.

see also Figure s4.

(Figure 3E). These results suggest that the reduction in basal axonal APP in PS1DE9 neurons is due to a decrease in a constitutive transcytosis pathway.

Rab11 Mediates Transcytosis of APP and LDL

To further investigate the role of Rab11 in endocytosis of LDL and transcytosis of APP and LDL, we used shRNA-medi-ated knockdown of Rab11 in wild-type neurons (Figure 4B). Knockdown of Rab11 in PS1WT/WT neurons almost completely abolished LDL endocytosis in the somatodendritic region

(Figure 4A), and transcytosed LDL was undetectable under Rab11 knockdown conditions (Figures 4C and 4D). Further support for a role of Rab11-mediated transcytosis of APP comes from co-staining experiments where axonal transcytosed APP (22C11) was stained with Rab11. As seen in Figure S4, 35% of Rab11 overlapped with transcytosed APP in PS1WT/WT axons, and about 25% of transcytosed APP overlapped with Rab11 in ps1wt/wt axons. The percentage of Rab11 with transcytosed 22C11 was reduced in PS1AE9/AE9 axons (as expected given the reduced density of transcytosed

replicates/genotype/time point at 30 min (PS1WT/DE9, p = 0.0671; ps1iE9/iE9, p = 0.1637), 60 min (PS1WT/DE9, p = 0.0013; ps1iE9/iE9, p < 0.0001), 120 min (PS1WT/DE9, p < 0.0001; PS1DE9/DE9, p < 0.0001), and 240 min (PS1WT/DE9, p < 0.0001; PS1iE9/iE9, p < 0.0001). Scale bar, 20 mm,

(C) Example images and a graph of intensity quantification are depicted for endocytosis of the fluid-phase fixable marker Dextran-tetramethylrhodamine. Data represent the mean ± SEM of soma intensities across 23-28 images/time point representing eight PS1WT/WT, eight PS1WT/DE9, and eight ps1iE9/iE9 biological replicates at 30 min (PS1WT/DE9, p = 0.1683; PS1iE9/iE9, p = 0.6532), 60 min (PS1WT/DE9, p = 0.6265; PS1iE9/iE9, p = 0.8126), 120 min (PS1WT/DE9, p = 0.9117; PS1DE9/DE9, p = 0.5285), and 240 min (PS1WT/DE9, p = 0.1595; ps1iE9/iE9, p = 0.6465). Scale bar, 20 mm.

(D and E) Neurons were grown in microfluidic devices and then allowed to internalized either APP antibody (D) (densities: PS1WT/DE9, p< 0.0001; ps1iE9/iE9, p = 0.0011) or LDL-BODIPY (E) (densities: PS1WT/DE9, p < 0.0001; PS1DE9/DE9, p = 0.0024) on the soma side with the axons in fluidic isolation. All intensity comparisons were p < 0.0001. Axons that passed through the channels were imaged, and punctum densities and intensities were evaluated. Data represent the mean ± SEM of four PS1WT/WT, three PS1WT/DE9, and four PS1DE9/DE9 biological replicates per stain. Scale bar, 5 mm. See also Figure S3.

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22C11), but the percent of 22C11 with Rab11 remained steady (Figure S4A).

PS1DE9 Impairs Recycling of LRP1

The absence of a lipoprotein endocytic defect at early time points in PS1 DE9 neurons suggested that transcription, levels, degradation, or recycling of the LDL receptor may be driving the reduction in transcytosis. Although there are many potential LDL receptors, LRP1 was an attractive candidate because of its high expression in brain and neuronal samples (Zhang et al., 2014). To determine whether transcription or degradation of LRP1 was affected in PS1DE9 neurons, we treated purified human neurons with unlabeled LDL and harvested neurons for mRNA and protein. We did not observe differences in LRP1 mRNA or protein levels (Figure 5A) at baseline or after LDL treatment, suggesting that transcription, total levels, and degradation of LRP1 after LDL treatment are not playing a role in the reduced endocytosis of LDL. Although

PS1 has been hypothesized to drive reduced degradation of proteins because of alterations in lysosomal pH (McBrayer and Nixon, 2013; Nixon and Yang, 2011), we did not observe changes in lysosomal pH in the PS1 DE9 neurons, as assessed by the two ratiometric pH probes LysoSensor yellow/blue dextran and fluorescein-Dextran-tetramethylrhodamine (Figure S5A). In addition, degradation of full-length APP is not different in ps1ae9/ae9 neurons treated with cycloheximide (Figure S5B) or when lysosome degradation is blocked with chloroquine.

To determine whether the amount of lipoprotein receptors at the somatodendritic surface are driving endocytosis defects at later time points, we measured the LDL receptor amount at baseline and after 4 hr of LDL treatment using two different methods. Purified neurons were incubated at 4°C with labeled LDL for 30 min and then fixed (0-h time point); this gives a measure of total LDL receptors at the cell surface (Figure 5B). Neurons were also incubated for 4 hr with unlabeled LDL and then



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brought to 4°C, unlabeled LDL was washed off, and then cells were incubated with labeled LDL followed by fixation. This gives a measure of LDL receptors at the surface after 4 hr of endocy-tosis (Figure 5B). Under these conditions, there were no differences in surface LDL punctum count in PS1AE9/AE9 neurons compared with PS1WT/WT neurons (Figure 5B). Because LDL could be binding non-specifically to surface proteins, we evaluated specific receptor populations by biotinylating the surface of neurons with a cleavable biotin and used streptavidin beads to pull down surface proteins before and after LDL treatment. Probing for LRP1 demonstrated that the surface levels of LRP1 at baseline were not significantly different between PS1WT/WT and PS1AE9/AE9. However, after 4 hr of LDL treatment, LRP1 receptor levels trend to decrease in PS1AE9/AE9 (p = 0.1293) (Figure 5C). Thus, the decrease in LDL endocytosis we observed in PS1AE9/AE9 neurons (Figure 3B) can be accounted for in part by the modest (~50%) LRP1 reduction at the neuronal surface after 4 hr of LDL treatment.

LDL Endocytosis Defects in PS1DE9 Neurons Are Rescued by p-Secretase Inhibition

We demonstrated previously that the PS1AE9 mutation impairs g-secretase activity and that APP CTFs accumulate in human PS1AE9 neurons (Woodruff et al., 2013). A previous study also demonstrated that g-secretase inhibition in mouse embryonic fibroblasts (MEFs) reduces LDL endocytosis (Tamboli et al., 2008). To determine whether g-secretase activity or the p-CTF fragment might be responsible for the impaired LDL endocytosis in human neurons, we treated PS1WT/WT neurons with p- and g-secretase inhibitors and measured LDL endocytosis (Figures 6A and 6B). Inhibition of g-secretase severely decreased LDL endocytosis at 4 hr, whereas p-secretase inhibition had no significant effect (Figures 6A and 6B). Treatment with both p-and g-secretase inhibitors caused a marked accumulation of a-CTFs while ablating p-CTFs (Figure 6C) but had no effect on LDL endocytosis at 4 hr (Figure 6B). These results suggest that the p-CTF, not a-CTF, of APP is responsible for impairing LDL endocytosis in AE9 neurons. To further test this possibility, we treated PS1 AE9 neurons with a p-secretase inhibitor and measured LDL endocytosis (Figures 6A and 6D). We observed that, upon treatment with a p-secretase inhibitor, PS1AE9/AE9 neurons were rescued in the ability to endocytose LDL (Figure 6D). Treatment of PS1AE9/AE9 neurons with both p- and g-secretase inhibitors, which abolishes p-CTF and increases a-CTF, also rescued the LDL endocytosis defect (Figure 6D), which suggests that accumulation of only the p-CTF is respon-

sible for impaired LDL endocytosis. To test whether p-secretase inhibition could rescue the transcytosis defect we observed, we treated PS1AE9/AE9 neurons grown in microfluidic compartments with a p-secretase inhibitor and then measured LDL transcytosis (Figures 6E and 6F). Similar to the endocytosis result, we also observed that p-secretase inhibition significantly increased the amount of transcytosed LDL in PS1AE9/AE9 neurons (Figure 6E).

LDL Endocytosis Defects Are Common to fAD APP Mutations

Accumulation of APP p-CTFs is a phenotype shared by many APP and PS1 fAD mutations (Chang and Suh, 2005; Sinha and Lieberburg, 1999). To assess whether other fAD mutations might share a common phenotype of impaired LDL endocytosis, we generated additional isogenic cell lines harboring either the APP V717F (APPV717F) or APP Swedish (APPswe) mutations (Sherrington et al., 1995). In neurons homozygous for the AppV717F mutation, there was significantly reduced LDL endocytosis after 4 hr, and this defect was rescued by p-secretase inhibition (Figures 7A and 7B). Similar to both the PS1AE9 and AppV717F mutations, neurons homozygous for the APPswe mutation also exhibited decreased LDL endocytosis (Figures 7A and 7B). In contrast to the other fAD mutations, p-secretase inhibition in the APPswe neurons did not rescue the LDL endocytosis defect (Figures 7A and 7B). However, mutant APP Swedish protein has been reported previously to decrease the potency of p-secretase inhibitors (Yamakawa et al., 2010), which could explain why, in our study, p-secretase inhibition did not rescue the APPswe neurons. These findings demonstrate that the reduction in LDL endocytosis is not specific to the PS1AE9 mutation but is a phenotype common to fAD APP mutations. Similar to PS1AE9 axons, we also observed reduced basal levels of axonal APP and Rab11 densities and reduced endocytosis of 22C11 at 4 hr in APPV717F neurons (Figure S6). Thus, endocytosis defects, and perhaps transcytosis defects, are common among at least one fAD PS1 mutation and two fAD APP mutations.


Here we demonstrate that, in human neurons with endogenous expression of fAD mutations induced with genome editing technology, increased p-CTF of APP alters the subcellular localization of APP and the distribution of Rab11 and decreases endocytosis and soma-to-axon transcytosis of LDL. LDL endocytosis and transcytosis defects are rescued by p-secretase inhibition in at least some of the fAD mutations. Our results show

Figure 6. LDL Endocytosis Defects in PS1DE9 Neurons Are Rescued by p-Secretase Inhibition

(A) Example images from drug-treated PS1WT/WT, ps1WT/AE9, and PS1AE9/AE9 neurons. Scale bar, 20 mm.

(B) Quantification of LDL punctum count per soma in 4-hr LDL-treated PS1WT/WT neurons. Treatment with a g-secretase inhibitor (GSI) resulted in a decrease in LDL endocytosis in PS1WT/WT neurons, whereas treatment with a p-secretase inhibitor (BSI) had no effect. Data represent the mean ± SEM of 12 PS1WT/WT, 6 GSI 100 nM (p < 0.0001), 6 GSI 1mM (p < 0.0001), 12 BSI 4 mM (not significant [n.s.]), and 4 GSI 1 mM + BSI 4 mM (n.s) biological replicates.

(C) Example western blot of neurons treated with p- and g-secretase inhibitors. a-CTFs and p-CTFs are indicated by arrows.

(D) Quantification of LDL endocytosis in PS1AE9/AE9 neuronstreated with inhibitors. n = 10 PS1AE9/AE9 forvehicle (VEH), 12 PS1 AE9/AE9for BSI, and 4 PS1AE9/AE9 for 4 mM BSI + 1 mM GSI biological replicates (***p < 0.0001 compared with vehicle).

(E) Quantification of LDL transcytosis in PS1AE9/AE9 under control and p-secretase inhibition. Treatment with 4 mM of BSI increased the levels of transcytosed, axonal LDL compared with untreated ps1AE9/AE9 from an average density of ~0.33 count/mm to ~0.43 count/mm (p = 0.0044).

(F) Example images of ps1AE9/AE9 axons with and without p-secretase inhibition after 4 hr of LDL transcytosis. Axons were co-stained with the axonal marker SMI31 for neurofilament-H.



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Figure 7. LDL Endocytosis Defects Are Common in Other fAD Mutations

(A) Representative images of LDL uptake in APPv717f and APPswe neurons co-stained with the somatodendritic marker Map2. Scale bar, 20 mm.



p = 0.5545) but not APPswe. Data represent the mean ± SEM of six APPWT/WT, three appV717F/V717F, and three APPs'

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(C) Model of APP and lipoprotein transcytosis in neurons.

(C1) FL-APP is internalized in Rab5+ sorting endosomes that contain LDL associated with LRP1 as well as p-secretase (BACE1) and possibly PS1.

(C2) As vesicles become more acidic along the endocytic pathway, LDL dissociates from LRP1, and FL-APP is cleaved by p-secretase. A proportion of the

cleaved APP population resides in Rab11+ endosomes containing LRP1, p-CTF, and LDL.

(C3) When p-CTF is cleaved by g-secretase, LRP1 can be recycled back to the cell surface, and transcytosis of LDL and FL-APP to the axon can occur. fAD mutations increase p-CTFs either by enhancing p-secretase processing or impairing g-secretase-mediated cleavage of APP. p-CTFs impair recycling of LRP1 and decrease transcytosis of APP and lipoproteins, possibly by directly sequestering LRP1 in a Rab11+ endosome. See also Figure S6.




that impaired LDL endocytosis and transcytosis are present in multiple types of fAD mutations, (Figure 7C) and that together they define an apparent defect in the Rab11 recycling endo-

some. Epidemiologic evidence implicating cholesterol as a major player in AD also dovetails with these molecular and cellular findings (reviewed in Fonseca et al., 2010, and Wolozin, 2004).

We demonstrate that APP p-CTFs may cause impaired LDL uptake by reducing recycling of LRP1 receptors back to the cell surface. One possible mechanism is that p-CTFs bind to LRP1 (Kounnas et al., 1995; Pietrzik et al., 2002; Tamboli et al., 2008), or other LDL receptors and retain LDL receptors in a Rab11-containing compartment until the p-CTF is cleaved by g-secretase. This would possibly explain why g-secretase inhibition impairs LDL uptake and why that defect can be rescued by p-secretase inhibition. The observation that basal Rab11 is reduced in axons of fAD mutant neurons suggests that a common constitutive recycling/transcytotic pathway is impaired and raises the possibility that modulating Rab11 activity could also rescue fAD phenotypes. Although we cannot rule out prote-olysis of the 22C11 or labeled LDL probe following endocytosis as a possible explanation for the reduced transcytosis, our data are most consistent with the possibility reported in previous work that early endocytosis is affected (Cataldo et al., 2000).

The finding that fAD neurons have defects in lipoprotein trans-cytosis is intriguing in light of previous work showing that neurons are dependent on the uptake of extracellular cholesterol from lipoprotein particles to perform functions such as axon elongation and synapse formation and maintenance (Barres and Smith, 2001; Lane-Donovan et al., 2014; Mauch et al., 2001; Nagleretal., 2001; Pfrieger, 2003; Pierrot et al., 2013). In fact, glia-derived cholesterol was reported to enhance synapto-genesis of the adult rat CNS (Mauch et al., 2001; Nagler et al., 2001), suggesting that a defect in endocytosis and transcytosis of extracellularly derived cholesterol could have long-term functional consequences leading to impaired neurotransmitter release and synaptic function. For instance, many studies of APOE function have focused on its potential role in mediating Ap clearance, but APOE has also been identified as the major lipoprotein carrier in the brain, and the e4 allele is less efficient in transporting brain cholesterol (Liu et al., 2013). Interestingly, post-mortem studies comparing sAD patients to age-matched controls found that brain cholesterol levels are reduced in the areas of learning and memory, the hippocampus and cortex (Svennerholm and Gottfries, 1994).

Previous work implicated early endosome dysfunction in sAD and some forms of fAD (Cataldo et al., 2001; Ginsberg et al., 2010). In contrast to previous studies, we report abnormalities in a Rab11-marked recycling endosome in fAD mutations, which is consistent with previous work on transcytosis that identified Rab11 as an endocytic regulator with a unique role in polarized cells, including neurons (Ascano et al., 2009; Buggia-Prevot et al., 2014; Welz et al., 2014). Thus, we describe a defect in a neuron-specific pathway that could contribute to the reported early endosomal defects and could produce AD pathology. In contrast, other recent findings have suggested that lysosomal pathology is the major driver of pathology in PS1 fAD mutations (Lee et al., 2010, 2015). Our data suggest that dysfunction in endosomal sorting could drive changes in lysosomal function rather than an original defect in the lysosome that drives such changes (Peric and Annaert, 2015).

We thus propose that transcytotic trafficking defects could be at the root of many types of fAD and, potentially, sAD. In fact, endocytic trafficking changes have been reported for APOE4 relative to APOE3 and 2 (Chen et al., 2010). These types of defects

could lead to previously reported axonal amyloid-dependent and -independent transport defects (Kim et al., 2015; Stokin et al., 2008; Vossel et al., 2015). Similarly, many fAD mutations thought to act solely by changing Ap production may, in fact, also act by changing sorting and trafficking signals in p-CTF of APP, leading to changes in constituents of axonal vesicles derived by transcytotic trafficking of lipoproteins and other key synaptic constituents. Although it may seem counter-intuitive that cleavage of p-CTF is needed to generate axonal vesicles containing p-CTF or full-length APP, we suggest that p-CTF cleavage happens in the portion of the recycling endosome that remains in the soma and that buds off axonal vesicles with APP, lipoproteins, and other components (Figure 7C). Finally, we note that the defects we report occur in the absence of overexpression of any of the proteins involved and, thus, may accurately reflect the earliest changes from normal behavior generated by fAD.


Statistics were performed using GraphPad Prism. Normality for each dataset was assessed using D'Agostino-Pearson test. When data were normally distributed, a two-way ANOVA with a post hoc Tukey test was used to compare genotypes. For precise p value comparisons, a multiple t test was done after ANOVA calculations. Most immunofluorescence data were non-normally distributed, and a nonparametric Kruskal-Wallis test with Dunn's multiple comparison was used to compare genotype medians. Data are depicted with bar graphs of the mean ± SEM of all values in an experiment or boxplots where the median is depicted with a line, and whiskers indicate the Tukey distribution as determined by GraphPad Prism.

Microfluidic Compartments

Microfluidic compartments were made in-house as described previously (Nie-derst et al., 2015). Briefly, Sylgard 182 (Ellsworth Adhesives) was used to mold devices. When cured, the devices were cut and then washed with isopropanol, water, and 70% ethanol. The devices were plasma-treated and mounted onto glass coverslips. The mounted device was this coated with poly-ornithine/ laminin (PO/L) as described above.

sAPP Measurements

sAPP was measured from conditioned media from microfluidic compartments from the soma side (contained soma, axons, dendrites) and the axon side (axons only) 48 hr after a full medium change in which the axons were kept in fluidic isolation. sAPP was measured using the sAPPa/sAPPp human kit (Meso Scale Discovery). The kit has a sensitivity down to 120 pg/mLfor sAPPa and 52 pg/mL for sAPPp, and all measurements were in the linear range.


Purified neurons were grown in 384-well imaging plates at a density of 25,000 cells/well for 7-9 days after sorting. Neurons were fixed in 4% paraformaldehyde and PBS for 30 min at 37°C, permeabilized with 0.1% Triton X-100, and blocked (10% donkey serum, 3% BSA, and 0.1% Triton X-100 in PBS). For surface labeling experiments, neurons were not permeabilized. For compartment experiments, polydimethylsiloxane (PDMS) microfluidic devices were plasma-bonded directly onto glass coverslips (Niederst et al., 2015). Neurons were seeded at a density of one to three million cells per compartment and grown for 7-10 days (until axons passed through the channels). Compartments were then fixed as above and imaged. The antibodies used for immunofluorescence experiments were anti-Rab11 (1:1,000, Life Technologies, 71-5300), anti-APP Y188 (1:200, Abcam, ab32136), anti-APP 22C11 (1:100, EMD Millipore, MAB348), anti-EEA1 (1:100, BD 610457), anti-NF-H (1:1,000, BioLegend, SMI-31r), and anti-Map2 (1:500, ab5392). Secondary antibodies were Alexa Fluor donkey anti-mouse and anti-rabbit

immunoglobulin G (IgG) (Invltrogen), and Dylight 405 donkey anti-chicken IgY (Jackson ImmunoResearch Laboratories, 703-475-155) was used at 1:200. Images were acquired on a Zeiss confocal microscope.

Endocytosis and Transcytosis

For constitutive uptake endocytosis assays, neurons were incubated with LDL-boron-dipyrromethene (BODIPY) (20 mg/mL, Invitrogen L3483) or Dextran-tetramethylrhodamine (TMR), molecular weight (MW) 10,000 (250 mg/mL, Invitrogen, D1817) at 37°C for the indicated times, fixed, and imaged. For all fixed endocytosis assays, a custom ImageJ program was used to identify Map2-positive soma and automatically generate regions of interest (ROIs) corresponding to soma. Mean intensity and punctum count per soma were determined and averaged across images and experiments. All endocytosis assays were repeated at least three times. In experiments where secretase inhibitors were used, cells were treated with the inhibitor 24 hr prior to the experiment and kept in the presence of the inhibitor for the duration of the experiment. Compound E (GSI, EMD Chemicals) was used at 200 nM in endocytosis assays and at 100 nM and 1 mM for western blotting. ßIV inhibitor (b Inh, Millipore) was used at 4 mM.

For transcytosis experiments, neurons were grown in compartments and treated on the soma side with LDL-BODIPY (20 mg/mL), LDL-DiI (1,1'diocta-decyl-3,3,3',3-Tetramethyllindocarbocycanine Percholorate) (12.5 mg/mL), or mouse anti-22C11 (1:100) for 4 hr with axons in fluidic isolation. Axonal punctum analysis was done as described previously (Szpankowski et al., 2012). Axons were imaged at x100, and a custom Gaussian-fitting colocalization package in MATLAB (MathWorks) was used to calculate axonal density, punctum intensity, and percent colocalization per axon. All iPSCs were generated following informed consent and Institutional Review Board approval.

Surface Biotinylation Assay

Neuronal medium was changed to warm fresh medium or medium supplanted with 12.5 mg/mL of unlabeled LDL for 4 hr. At the end of the incubation, neurons were washed twice with ice-cold PBS and then incubated at 4°C with 2 mM EZ-Link Sulfo-NHS-SS-Biotin (Life Technologies) in PBS for 30 min. Cells were then lysed in equal volumes of radioimmunoprecipatation assay lysis (RIPA) buffer. For pull-down experiments, 200 mg of harvested protein at 0.5 mg/mL was incubated with 50 mL of pre-washed Pierce streptavidin magnetic beads (Life Technologies, 88817) overnight at 4°C. The next day, beads were washed to remove residual, unbound proteins, and biotinylated proteins were released from the streptavidin beads by boiling samples in loading buffer at 100°C. Western blots were run with 5% of input, 5% of supernatant, and 50% of pull-down. Quantification of recycling was determined based on input signal (pull-down/input).


Supplemental Information includes Supplemental Experimental Procedures and seven figures and can be found with this article online at http://dx.doi. org/10.1016/j.celrep.2016.09.034.


S.M.R., G.W., and L.S.B.G. wrote the manuscript and designed the experiments. G.W., J.A.C., J.E.Y., and E.A.R. designed and generated the isogenic iPSC lines. S.M.R., G.W., M.D., and R.V.D.K. performed the experiments.


We thank Angels Almenar for helpful suggestions and scientific advice. G.W. was supported by an institutional training grant (2T32AG000216-21). S.M.R. was supported by NIH Predoctoral Training Grant T32 GM008666, a CIRM predoctoral training grant, and a Tina Nova scholarship. R.V.D.K. is supported by an ERC Marie Curie International Outgoing Fellowship and an Alzheimer Netherlands Fellowship. This work was supported by California Institute of Regenerative Medicine Grant RT2-01927 (to L.S.B.G.) and NIA RF1 AG048083 (to L.S.B.G.).

Received: October 14, 2015 Revised: July 22, 2016 Accepted: September 12, 2016 Published: October 11, 2016


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