Amyloid Clearance Defect in Apoe4 Astrocytes Is Reversed by Epigenetic Correction of Endosomal Ph

Amyloid clearance defect in ApoE4 astrocytes is reversed by epigenetic correction of endosomal pH

Hari Prasada and Rajini Raoa,1

aDepartment of Physiology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205

Edited by Reinhard Jahn, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany, and approved June 6, 2018 (received for review January 28, 2018) Endosomes have emerged as a central hub and pathogenic driver ability (7–9). Patients who have CS also show striking age- of Alzheimer’s disease (AD). The earliest brain cytopathology in dependent neurodegeneration, with prominent glial pathology neurodegeneration, occurring decades before amyloid plaques and phosphorylated tau deposits (7). Female carriers have and cognitive decline, is an expansion in the size and number of learning difficulties and behavioral issues, and some present with endosomal compartments. The strongest genetic risk factor for low Mini-Mental State Examination scores suggestive of early sporadic AD is the e4 allele of Apolipoprotein E (ApoE4). Previous cognitive decline (10). Interestingly, NHE6 was among the most studies have shown that ApoE4 potentiates presymptomatic endo- highly down-regulated genes (up to sixfold) in the elderly (70 y) somal dysfunction and defective endocytic clearance of amyloid brain, compared with the adult (40 y) brain (11). These obser- beta (Aβ), although how these two pathways are linked at a cel- vations led us to consider a broader role for NHE6 in neuro- lular and mechanistic level has been unclear. Here, we show that degenerative disorders, including Alzheimer’s disease (AD), a aberrant endosomal acidification in ApoE4 astrocytes traps the major cause of dementia in the elderly. Consistent with this hy- low-density lipoprotein receptor-related protein (LRP1) within in- pothesis, a coexpression analysis of quantitative trait loci in AD tracellular compartments, leading to loss of surface expression and brains revealed NHE6 as a top hub transcript, with 202 network Aβ clearance. Pathological endosome acidification is caused by + + connections and a plethora of potential downstream effects (12). A e4 risk allele-selective down-regulation of the Na /H exchanger knowledge-based approach for predicting gene–disease associa- isoform NHE6, which functions as a critical leak pathway for endo-

tions also identified a link between NHE6 and early-stage AD (13). PHYSIOLOGY KO somal protons. In vivo, the NHE6 knockout (NHE6 ) mouse model Stronger evidence emerged from a recent analysis of the meta- showed elevated Aβ in the brain, consistent with a causal effect. stable aggregation-prone proteome in AD brains that identified Increased nuclear translocation of histone deacetylase 4 (HDAC4) NHE6 as a key component of the proteostasis machinery associ- in ApoE4 astrocytes, compared with the nonpathogenic ApoE3 ated with amyloid plaques and neurofibrillary tangles containing allele, suggested a mechanistic basis for transcriptional down- amyloid beta (Aβ) peptide and tau protein, respectively (14). regulation of NHE6. HDAC inhibitors that restored NHE6 expression Petsko and coworkers (15) recently proposed that endosomal normalized ApoE4-specific defects in endosomal pH, LRP1 trafficking, “traffic jams” are the unifying mediators of downstream pathology and amyloid clearance. Thus, NHE6 is a downstream effector of in AD and interventions designed to “unjam” the endosome have ApoE4 and emerges as a promising therapeutic target in AD. These high therapeutic promise. Endosomal aberrations, evidenced by observations have prognostic implications for patients who have enlarged and more numerous endosomes, are the earliest de- Christianson syndrome with loss of function mutations in NHE6 tectable brain cytopathology, emerging several decades before and exhibit prominent glial pathology and progressive hallmarks cognitive dysfunction is apparent in a subset of neurodegenerative of neurodegeneration. disorders, including AD, Niemann–Pick type C, and Down syndrome (16–19). Genes associated with endosomal trafficking have trichostatin A | amyloid beta | ApoE4 | Na+/H+ exchanger | histone deacetylase Significance he endosome is a central hub for incoming and outgoing traffic Alzheimer’s disease is the most common cause of dementia in and a key recycling/degradation sorting station. Transit through T the elderly. Most cases occur sporadically, with 40–65% of pa- the endolysosomal system is accompanied by an increasingly acidic tients carrying at least one copy of the E4 allele of Apolipopro- pH gradient that controls receptor–ligand uncoupling, vesicle tein E. Because no drug exists that can halt disease progress, budding, exosome formation, membrane turnover, enzyme activa- there is strong interest in understanding the presymptomatic tion, nutrient uptake, and cellular signaling (1). As a defining role of endosomes. We show that excessive endosomal acidifi- feature of compartmental identity and function, the pH of the cation in ApoE4 astrocytes is caused by downregulation of the endolysosomal lumen is precisely set by a balance between proton + + + + Na /H exchanger NHE6 and results in defective clearance of pump and leak pathways (2). The discovery of endosomal Na /H amyloid beta (Aβ) peptide by intracellular sequestration of the exchangers (eNHEs) first in yeast and soon after in plants and LRP1 receptor. Epigenetic modifiers restore NHE6 expression to metazoans, including mammals, established their evolutionarily alkalinize endosomal pH, increase surface expression of LRP1, conserved role as a leak pathway for protons in compartmental pH β + + and correct A clearance in astrocytes. Thus, endosomal pH homeostasis (2–4). Na /H exchangers are estimated to have ex- emerges as a target for the correction of amyloid disorders. ceptionally high transport rates of ∼1,500 ions per second (5), so that even small perturbations in expression or activity result in Author contributions: H.P. and R.R. designed research; H.P. performed research; H.P. and dramatic changes in the ionic milieu within the limited confines of R.R. analyzed data; and H.P. and R.R. wrote the paper. the endosomal lumen. The authors declare no conflict of interest. As a testament to the central role of the endosome at the This article is a PNAS Direct Submission. crossroads of cellular traffic, mutations in eNHEs have been Published under the PNAS license. linked to a host of neurodevelopmental and neurodegenerative 1To whom correspondence should be addressed. Email: [email protected]. + + disorders (2, 6). Loss-of-function mutations in the Na /H ex- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. changer NHE6 (SLC9A6) cause Christianson syndrome (CS), an 1073/pnas.1801612115/-/DCSupplemental. X-linked disorder characterized by autism and intellectual dis-

www.pnas.org/cgi/doi/10.1073/pnas.1801612115 PNAS Latest Articles | 1of10 Downloaded by guest on October 1, 2021 also been implicated as major risk factors in AD (20). Indeed, the Appendix, Fig. S3 A and B). Meta-analysis of nine independent strongest genetic risk factor in sporadic AD is the e4 allele of gene expression studies from anatomically and functionally dis- Apolipoprotein E (ApoE4), which potentiates both presymptom- tinct brain regions, comprising a total of 103 AD and 87 control atic endosomopathy and defective clearance of Aβ (21–25), al- postmortem brains, also showed no significant changes in LRP1 though how these two pathways are linked at a cellular level has gene expression in AD (SI Appendix, Fig. S3 C and D). Consis- been unclear. tent with these findings, we observed no differences in LRP1 Here, we show that a pathological acidification of endosomal pH transcript (SI Appendix, Fig. S3E) and total LRP1 protein ex- in humanized mouse ApoE4 astrocytes is caused by the selective pression between ApoE3 and ApoE4 astrocytes (SI Appendix, down-regulation of NHE6. This leads to endosomal sequestration Fig. S3 F and G). and cell surface loss of the Aβ receptor, low-density lipoprotein LRP1 undergoes constitutive endocytosis from the membrane receptor-related protein (LRP1). Increased nuclear translocation and recycling back to the cell surface (37). Therefore, we con- of the histone deacetylase 4 (HDAC4) in ApoE4 astrocytes can be sidered the possibility that alterations in LRP1 receptor recycling abrogated by HDAC inhibitors that restore NHE6 expression, could result in differences in plasma membrane expression. ApoE reroute LRP1 to the cell surface, and effectively restore defective isotype-specific surface expression of LRP1 was evaluated using amyloid clearance to nonpathological ApoE3 levels. Consistent four independent approaches (Fig. 2A). First, surface biotinylation with a proposed role in amyloid pathology, we found that in vivo revealed that plasma membrane expression of LRP1 receptor in Aβ levels were significantly higher in the brains of NHE6 knockout ApoE4 astrocytes was lower by ∼50% (Fig. 2B). Second, an an- (NHE6KO) mice. Our findings could have prognostic implications tibody directed against an external epitope of LRP1 to quantify for patients with CS and suggest therapeutic strategies for the surface expression in live cells by flow cytometry analysis showed a treatmentofamyloiddisorders(7,26). reduction of LRP1-positive cells by ∼55% in ApoE4 astrocytes (Fig. 2C)andby∼51% in patient-derived ApoE4/4 fibroblasts (SI Results Appendix,Fig.S2D). Third, this was confirmed by confocal mi- ApoE4 Astrocytes Have Cargo-Specific Defects in Endocytosis. Studies croscopy showing ∼49% lower LRP1 surface labeling by antibody in humans, mouse models, and cell cultures have revealed the in ApoE4 (Fig. 2D). In a fourth approach, surface-bound ligand importance of ApoE isotype-specific differences in Aβ uptake (fluorescent Aβ) measured by confocal microscopy was 66% lower and clearance in AD pathogenesis, although the underlying in ApoE4 astrocytes (Fig. 2E). Notably, the greater attenuation in mechanism remains to be determined (22, 23, 27, 28). The Aβ binding compared with the ∼50% reduction in surface LRP1 ApoE3 variant (Cys112) predominates in the human population at levels suggests additional isotype-specific mechanisms that con- a frequency of 77.9%, whereas the relatively rare ApoE4 variant tribute to Aβ clearance, such as reduced ligand-receptor affinity in (Arg112) is dramatically increased from 13.7 to ∼40% in patients ApoE4 cells or reductions in other Aβ receptors. Confocal mi- with AD (29). The therapeutic focus is on defective Aβ clearance croscopy revealed a striking loss of LRP1 from astrocyte processes in ApoE4 relative to ApoE3 because ApoE knockout (ApoEKO) and increased perinuclear accumulation in ApoE4 cells, rela- mice clear Aβ faster than controls (30), and a rare case of human tive to ApoE3 (Fig. 2F). The rate of LRP1 endocytosis (at 37 °C ApoE knockout showed no evidence of neurodegenerative dis- for 10 min) in ApoE3 and ApoE4 astrocytes was similar (SI ease (31). To this end, we developed a sensitive and quantitative Appendix,Fig.S4), suggesting that LRP1 delivery by exocytosis fluorescent-based assay to monitor cell-associated Aβ peptide or recycling may contribute to the differences in surface ex- (Fig. 1A) in astrocytes from ApoEKO mice with isogenic knock-in pression. Consistent with this possibility, we find significant of human ApoE3 and ApoE4 variants (23). Consistent with evi- colocalization of LRP1 with TFN in ApoE4 astrocytes (Fig. 2G). dence in the literature (32–34), internalized Aβ is sorted to the Thus, ApoE isotype-specific alterations in receptor recycling lysosomal degradation pathway, as shown by high colocalization determine LRP1 surface expression and cellular Aβ uptake, re- with late endosomal-lysosomal markers and low colocalization vealing a pharmacological target for amyloid clearance defects in with the recycling compartment marker transferrin (TFN) (SI the pathological ApoE4 genotype. Appendix, Fig. S1 A–D). Strikingly, cell-associated Aβ was reduced by 78% in ApoE4 astrocytes, relative to ApoE3 (Fig. 1B). To Endolysosomal pH Is Defective in ApoE4 Astrocytes. The pH within distinguish between Aβ uptake and turnover, we monitored the the endolysosomal system plays a critical role in receptor-mediated time course of Aβ internalization by flow cytometry analysis (Fig. endocytosis and recycling (1). We used compartment-specific, pH- 1C) and confocal microscopy (SI Appendix,Fig.S1E). Consistent sensitive fluorescence reporters to probe ApoE isotype-dependent with defective uptake, there was significantly lower cell-associated differences in endosomal, lysosomal, and cytoplasmic pH (Fig. 3A). Aβ in ApoE4 cells relative to ApoE3 at all time points, including Endosomal pH in ApoE4 astrocytes was strongly reduced by very early time points between 1 and 30 min and as long as 12 h ∼0.84 pH unit, relative to ApoE3 (Fig. 3B). A similar acidification (Fig. 1C and SI Appendix, Fig. S1 F and G). In contrast, cell- was observed in endosomes of ApoE4/4 patient fibroblasts, com- associated TFN was 1.5–twofold higher in ApoE4 cells relative paredwithanApoE3/3control(SI Appendix,Fig.S2E). In contrast, to ApoE3 as measured by flow cytometry (Fig. 1D) and confocal we observed >1 pH unit elevation of lysosomal pH in ApoE4 as- microscopy (Fig. 1E). Uptake of dextran (10 kDa) by fluid-phase trocytes (Fig. 3C). Previously, elevated lysosomal pH was observed in endocytosis was not different between ApoE genotypes (Fig. 1F). presenilin 1 (PS1)-deficient cell culture models and neurons, another Similar findings were observed in AD patient-derived fibroblasts genetic model of AD (38). CytoplasmicpHshowednosignificant with the ApoE4/4 genotype compared with an age-matched differences between the two ApoE isotypes (Fig. 3D). ApoE3/3 control (SI Appendix,Fig.S2A–C). These observations To determine if there was a causal link between endolysoso- reveal cargo-selective effects of ApoE isotype in astrocytes and mal pH and defective Aβ clearance in ApoE4 astrocytes, we treated ApoE4 cells with the ionophore monensin, which mediates point to alterations in specific receptor pathways. + + Na /H exchange across acidic compartments (39). Thus, mon- Surface Expression of LRP1 Receptor Is Severely Reduced in ApoE4 ensin treatment (50 μM for 1 h) elevated endosomal pH in Astrocytes. Transcriptional down-regulation of the LRP1 re- ApoE4 knock-in astrocytes from 5.38 ± 0.01–5.74 ± 0.03, relative ceptor has been suggested as an underlying mechanism for de- to the vehicle-treated control (Fig. 3E), without altering cell via- fective Aβ clearance in patients with AD (35). However, we bility (SI Appendix,Fig.S5A). Concomitantly, monensin treatment found no difference in brain LRP1 gene expression at different restored Aβ clearance in ApoE4 astrocytes to ApoE3 levels, as stages of AD (incipient, moderate, and severe), compared with shown by flow cytometry analysis (Fig. 3F). Lower concentrations normal controls, in publicly available microarray data (36) (SI (1 μM) of monensin and shorter Aβ uptake times (1 and 5 min)

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1801612115 Prasad and Rao Downloaded by guest on October 1, 2021 ABSchematic Aβ endocytosis- Confocal Microscopy ApoE3 Labelled Aβ DAPI ApoE4 cargo **** 200

Flow

cytometry ApoE3 150

Confocal 50 Astrocyte culture Endocytosis Wash microscopy ApoE4 ApoE4/4 Normalised Aβ clearance 0

Aβ endocytosis- Flow Cytometry C ApoE3 16 hours 1 hour 4 hours ApoE4 **** ApoE3 ApoE3 ApoE3 15000

10000 102 103 104 105 102 103 104 105 102 103 104 105

ApoE4 ApoE4 ApoE4 5000 **** ****

0 PHYSIOLOGY 102 103 104 5 102 103 104 105 102 103 104 105 Aβ clearance (Mean fluorescence) E3 E4 E3 E4 E3 E4

Count 10 Aβ clearance (Log scale) 1 hour 4 hours 16 hours

Transferrin endocytosis- Flow Cytometry Transferrin endocytosis- Dextran endocytosis- D EFConfocal Microscopy Flow Cytometry ApoE3 ApoE3 ApoE3

1 hour 4 ApoE4 TFN DAPI ApoE4 ApoE4 400 150 ApoE3 **** 3X10

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1X10 100 ApoE4 ApoE4/4 Normalised TFN uptake Normalised dextran uptake tn 0 0 100 0 0 TFN uptake (Mean fluorescence) uoC 103 104 105 TFN uptake (Log scale)

Fig. 1. ApoE isotype-specific differences in Aβ clearance and specific receptor pathways. (A) Fluorescent-based assay to monitor clearance of Aβ peptides by astrocytes. (B) Representative micrographs (Left) and quantification (Right) of ApoE3 and ApoE4 astrocytes subjected to 24 h of Aβ uptake. The fluorescence intensity and exposure settings were kept constant. Following background subtraction, fluorescence signal from cell-associated Aβ was reduced by 78% in ApoE4 astrocytes, relative to ApoE3 (****P = 9.6 × 10−71, Student’s t test; n = 100 per condition). (Scale bar, 50 μm.) (C, Left) Representative fluorescence- activated cell sorting (FACS) histograms demonstrating Aβ internalization by ApoE3 (Top, orange) and ApoE4 astrocytes (Bottom, gray) at 1 h, 4 h, and 16 h (n = 10,000 cells per experimental condition). The x axis depicts Aβ clearance in logarithmic scale, and the vertical dashed line represents median fluorescence intensity. (C, Right) Quantification of biological triplicate measurements of Aβ clearance from FACS analysis of ApoE3 and ApoE4 cells. Note the significantly lower cell-associated Aβ in ApoE4 relative to ApoE3 at all time points (53% lower at 1 h, 59% lower at 4 h, and 65% lower at 16 h; ****P < 0.0001, Student’s t test; n = 3). (D) Representative FACS histograms (Left) and quantification of mean fluorescence intensity of biological triplicates (Right) demonstrating TFN − uptake following 60 min of endocytosis by ApoE3 (green) and ApoE4 (gray) astrocytes (∼1.5-fold higher; ****P = 6.8 × 10 5, Student’s t test; n = 3). The x axis of the FACS histograms depicts TFN uptake in logarithmic scale, and the vertical dashed line represents median fluorescence intensity. (E) ApoE3 and ApoE4 astrocytes were incubated with fluorescent TFN for 1 h to compare steady-state TFN uptake by confocal microscopy. Fluorescence intensity and exposure settings were kept constant. Representative images are shown (Left), and mean fluorescence ± SE was plotted (Right). Following background sub- − traction, fluorescence signal was increased by approximately twofold in ApoE4 astrocytes, relative to ApoE3 (****P = 3.5 × 10 32, Student’s t test; n = 100 per condition). (Scale bar, 10 μm.) (F) Quantification of mean fluorescence intensity of biological triplicates demonstrating dextran uptake by ApoE3 and ApoE4 astrocytes (P = 0.870, Student’s t test; n = 3). NS, not significant. (SI Appendix, Figs. S1 and S2).

gave similar results (SI Appendix,Fig.S5B). These observations NHE6 Restores Defective Aβ Clearance in ApoE4 Astrocytes. Luminal were independently confirmed by confocal microscopy (Fig. 3G), pH in the endolysosomal network is set by the precise balance of + suggesting that defective pH regulation could underlie the ob- proton pump and leak pathways, mediated by V-type H -ATPase served Aβ clearance defects. (V-ATPase) and endosomal NHE6, respectively (Fig. 4A)(2,6,8,

Prasad and Rao PNAS Latest Articles | 3of10 Downloaded by guest on October 1, 2021 A Schematic B Surface biotinylation C Flow cytometry

Surface biotinylation (B) ApoE3 ApoE3 Flow cytometry (C) ApoE4 ApoE4 kDa 1.5 150 Surface immunofluorescence (D) ~200 Ligand binding (E) ** ****

Surface LRP1 expression 1.0 100

A (ligand) LRP1 80 LRP1 (receptor) 0.5 50 (Normalised)

Na+/K+- (Normalised intensity) Surface LRP1 expression 100 Surface LRP1 positivity ATPase 0.0 0 123

D E Surface immunofluorescence Ligand binding ApoE3 ApoE3 LRP1 DAPI ApoE4 Aβ DAPI ApoE4 300 300 **** **** ApoE3

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LRP1 DAPI DIC ApoE3 LRP1 TFN ApoE4/4 ApoE4/4 Z ApoE4 MERGE

Fig. 2. Reduced surface expression of LRP1 receptor in ApoE4 astrocytes. (A) Four independent approaches to evaluate ApoE isotype-specific surface expression of LRP1. (B) Surface biotinylation (Left) and quantification (Right) of biological triplicates showing that plasma membrane levels of LRP1 are de- + + pressed by ∼50% in ApoE4 astrocytes, relative to ApoE3 (**P = 0.0022, Student’s t test; n = 3). Plasma membrane protein Na /K ATPase is used as a loading control. (C) Fraction of LRP1-positive cells quantified following surface antibody labeling and fluorescence-activated cell sorting (FACS) analysis of 10,000 live, nonpermeabilized cells in biological triplicates. Unstained cells were used as a control. Note the ∼55% lower surface LRP1 positivity in ApoE4 relative to −− ApoE3 (****P = 4.8 × 10 5, Student’s t test; n = 3). (D) Representative surface immunofluorescence micrographs (Left) and quantification (Right) showing prominent LRP1 staining on the cell surface and processes and faint, ∼49% lower, labeling on ApoE4 cells (****P = 2.4 × 10−16, Student’s t test; n = 75 per condition). (E) Plasma membrane level of LRP1 receptor was monitored by a ligand (fluorescent Aβ)-binding assay performed on ice that only allows Aβ to bind surface receptors. Representative images are shown (Left), and mean fluorescence ± SD was plotted (Right). Surface-bound Aβ was 66% lower in − ApoE4 astrocytes, relative to ApoE3 (****P = 2.2 × 10 24, Student’s t test; n = 75 per condition). (F, Left) Confocal micrographs revealing a striking loss of LRP1 from astrocyte processes (white arrow) and increased perinuclear accumulation in ApoE4 cells, relative to ApoE3. (F, Right) Zoomed-in images of the boxed regions. Increased perinuclear accumulation of LRP1 in ApoE4 cells is also evident in orthogonal slices (Z) (black arrows). (G) Confocal micrographs revealing prominent colocalization of LRP1 (green) with TFN (red) in DAPI (blue)-stained ApoE4 astrocytes. Colocalization is evident in the merge and orthogonal slices (Z) as yellow puncta (SI Appendix, Figs. S2–S4). DIC, differential interference contrast. (Scale bars, 10 μm.)

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1801612115 Prasad and Rao Downloaded by guest on October 1, 2021 A Schematic B Endosomal pH C Lysosomal pH D Cytoplasmic pH TFN- FITC TFN- 633 ApoE3 ApoE3 ApoE3 DXN- pHrodo green ApoE4 ApoE4 ApoE4 7 6 8 DXN- 647 ** *** NS BCECF 6.5 5.5 7.5

6 6.21 5 5.20 7 7.19 Cytoplasm 7.01 4.5 pH 5.5 pH 6.5 pH pH Endosome 5.37 5 4 4.08 6 Lysosome 4.5 3.5 5.5

3 4 12 12 5 12 Axis Title Axis Title Axis Title

E Endosomal pH FGAβ clearance – Aβ clearance – confocal microscopy flow cytometry ApoE3 ApoE4 Aβ DAPI ApoE4 ApoE4 ApoE4+Monensin ApoE4+Monensin 6 ApoE4+Monensin 500 **** *** **** 100 400 5.74 **** 5.5 Vehicle clearance clearance 300 5.38 pH 50 200 5 100 ApoE4/4 Normalised A β Normalised A β Monensin

4.5 0 0 z

Fig. 3. Endolysosomal pH is defective in ApoE4 astrocytes. (A) Compartment-specific, ratiometric, pH-sensitive fluorescence reporters used to probe ApoE PHYSIOLOGY isotype-dependent differences in endosomal, lysosomal, and cytoplasmic pH. Endosomal pH was measured by incubations with pH-sensitive TFN-FITC, together with pH-nonsensitive Alexa Fluor 633-TFN (TFN-633). Lysosomal pH was measured by incubations with pH-sensitive pHrodo-green-Dextran (DXN- pHrodo green), together with pH-nonsensitive Alexa Fluor 647-Dextran (DXN-647). Cytoplasmic pH was measured ratiometrically using pH-sensitive green and pH-nonsensitive red fluorescence of 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) dye. (B) Endosomal pH in ApoE4 astrocytes was strongly reduced by ∼0.84 pH unit, relative to ApoE3 (**P = 0.0037, Student’s t test; n = 3). (C) Lysosomal pH was elevated by >1 pH unit in ApoE4 astrocytes (***P = 0.0009, Student’s t test; n = 3). (D) Cytoplasmic pH showed no significant (NS) differences between ApoE3 and ApoE4 astrocytes (P = 0.2904, Student’s t test; n = 3). (E) Monensin treatment (50 μM for 1 h) corrected hyperacidic endosomal pH in ApoE4 astrocytes, relative to vehicle treatment (***P = 0.0005, Student’s t test; n = 3). (F) Quantitation of Aβ clearance from fluorescence-activated cell sorting (FACS) analysis of 10,000 cells in biological triplicates confirmed restoration of Aβ clearance (1 h) in ApoE4 astrocytes to ApoE3 levels with monensin treatment (****P = 6.5 × 10−7, Student’s t test; n = 3). (G) Representative micrographs (Left) and quantification (Right) showing an ∼2.9-fold increase in cell-associated Aβ following 1 h of uptake in ApoE4 astrocytes with monensin treatment (****P = 2.4 × 10−20, Student’s t test; n = 50) (SI Appendix, Figs. S2 and S5). (Scale bar, 10 μm.)

40). Changes in expression and activity of the pump and leak could account for the observed ApoE isotype-specific shifts in pathways could lead to significant dysregulation of endosomal pH endolysosomal pH. in AD brains. Consistent with this possibility, analysis of a publicly Taken together, these data suggest an important, hitherto un- available microarray dataset (GSE5281) comprising a total of derappreciated role of proton transport and endosomal pH reg- 15 sporadic, late-onset AD and 12 matched control postmortem ulation in AD. We hypothesized that NHE6 is a potential ApoE brains from the middle temporal gyrus (41) revealed that genes effector, and that down-regulation of NHE6 in disease-associated involved in hydrogen ion transmembrane transport, including the ApoE4 variants is causal to a subset of AD phenotypes. We show endosomal NHE6 and V-ATPase subunits, comprised 10% of the that amyloid Aβ levels are elevated in brains from NHE6KO mice top 100 down-regulated genes, exhibiting the highest enrichment (Fig. 4D), together with diminished brain weight (SI Appendix, Fig. scores (>15-fold; SI Appendix,Fig.S5C and D). In patients who S5G), suggesting an underlying neurodegenerative pathology have AD with the ApoE4/4 genotype, NHE6 was among the consistent with microcephaly and extensive gliosis in patients with transcripts differentially down-regulated in the hippocampus, com- CS (2). Furthermore, NHE6KO brains show ∼22% lower levels of pared with ApoE3/3 (42). We validated these findings using an neurofilament light chain (SI Appendix,Fig.S5H), characteristic of independent, large human brain transcriptome dataset (n = 363; AD brains (43) (SI Appendix,Fig.S5D). In contrast, mRNA levels GSE15222) to show ApoE4 isotype-specific differential gene of the neuronal protein TUBB3 remained unchanged in NHE6KO expression of NHE6 in the aging brain (12) (Fig. 4B). Although brains (SI Appendix,Fig.S5H). the NHE6 transcript was similar in ApoEKO mouse astrocytes Next, we tested if ectopic expression of GFP-tagged NHE6 (SI and ApoEKO astrocytes with knock-in of human ApoE3, it was Appendix, Fig. S6 A and B) could correct defective Aβ uptake in ∼56% reduced in ApoE4 knock-in cells (SI Appendix, Fig. S5E). ApoE4 astrocytes. For comparison, we also evaluated the NHE6 NHE6 protein was concomitantly decreased by ∼45% in ApoE4 variants L188P and G383D found in patients with CS (2, 44, 45), astrocytes, compared with ApoE3 (Fig. 4C). There was also an which localize to highly conserved sequences predicted to be within ApoE4-specific reduction in the transcript for the related endo- the membrane-embedded transporter domain (SI Appendix,Fig.S6 somal isoform NHE9 (∼70% lower) and the lysosomal V- C–E). Wild-type NHE6 and CS variants were expressed at similar ATPase V0a1 subunit (∼67%), but not for the plasma mem- levels and localized to TFN-positive endosomes (SI Appendix,Fig. brane NHE1 isoform (SI Appendix, Fig. S5F). Similar changes in S6 F and G). Like monensin, NHE6 alkalinized the endosomal gene expression were observed in AD patient-derived ApoE4/4 lumen in ApoE4 astrocytes, but the CS patient mutations did not fibroblasts compared with ApoE3/3 fibroblasts from age-matched (SI Appendix,Fig.S6H). Ectopic NHE6 expression does not alter control (SI Appendix,Fig.S2F–H). These large expression changes lysosomal pH, suggesting that compartmental pH regulation is

Prasad and Rao PNAS Latest Articles | 5of10 Downloaded by guest on October 1, 2021 ADSchematic B Human brain C Astrocytes Mouse brain ApoE4- ApoE3 ApoE4 WT KO ApoE4+ kDa NHE6 Pump ****p=1.2X10-10 14 **** 150 V-ATPase NHE6 ** 10

a.u.) 12

2 76 10 β-actin 52 9 38 8 + H 6 + 1.5 ApoE3 H 8 *** concentration ApoE4 4

1.0 A β

Leak pmol/gram of protein) ( 2 NHE6 0.5

Endosomal pH NHE6 expression (Log 7 0.0 0 22 23 33 24 34 44 NHE6 protein ApoE genotype

A clearance Surface LRP1 expression EFApoE3 ApoE4 ApoE4 ApoE4+NHE6 DIC sLRP1 ApoE4+NHE6 105 150 500

)ecnecsero **** **** 400 **** cnaraelc 100 ApoE4 clearance clearance 300 ulf 104 elacsgoL( 200

Ae 50 Normalized A 100 Normalised A β Surface LRP1 expression (Normalised fluorescence) 103 0 ApoE4+NHE6 0

Fig. 4. NHE6 restores defective Aβ clearance in ApoE4 astrocytes. (A) Endosomal pH is precisely tuned by a balance of proton pumping (acidification) through the V-ATPase and proton leak (alkalization) via NHE6. (B) Box plots of NHE6 transcript levels in postmortem brains extracted from a large microarray dataset (GSE15222; n = 363) showing significant down-regulation of NHE6 expression in ApoE4+ AD brains (****P = 1.2 × 10−10, Student’s t test). (C, Top) Western blot analysis of NHE6 protein revealed that it was ∼45% lower in ApoE4 astrocytes, relative to ApoE3. (C, Bottom) Densitometric quantification of the monomeric and dimeric forms of NHE6 relative to β-actin (***P = 0.0009, Student’s t test; n = 4). (D)Aβ levels were significantly higher in the brains of the NHE6-null mouse model (NHE6KO), relative to WT (**P = 0.0013, Student’s t test; n = 5 per condition), consistent with our hypothesis. Aβ was measured using ELISA and normalized to total protein concentration in the brain homogenate. (E) Fluorescence-activated cell sorting (FACS) scatter plots (Left) and quantification (Right) demonstrating Aβ internalization by an empty vector expressing ApoE3 (orange) and ApoE4 (gray) astrocytes and ApoE4 astrocytes with restored NHE6 expression (green). Note the remarkable correction of Aβ clearance in ApoE4 astrocytes with NHE6 expression to ApoE3 levels (****P = 4.9 × 10−6, Student’s t test; n = 3). (F) Representative surface immunofluorescence images (Left) and quantification (Right) of nonpermeabilized cells showing an ∼2.5- − fold increase in plasma membrane LRP1 expression in ApoE4 cells expressing exogenous NHE6 (****P = 6.02 × 10 12, Student’s t test; n = 40 per condition) (SI Appendix, Figs. S2 and S5–S7). DIC, differential interference contrast. (Scale bars, 2.5 μm.)

specific and localized (SI Appendix,Fig.S6I). Remarkably, Aβ NHE6 may contribute to other ApoE4 defects, including de- clearance was restored to ApoE3 levels in ApoE4 astrocytes fective synaptosome uptake and synapse pruning (37, 46). transfected with NHE6 (Fig. 4E). However, CS variants failed to correct Aβ clearance deficits in ApoE4 astrocytes, consistent with HDAC Inhibitors Rescue NHE6-Mediated Aβ Clearance Deficits. Re- loss of function (SI Appendix,Fig.S6J). Intriguingly, ectopic de- ports of increased nuclear translocation of multiple HDACs in livery of NHE9 resulted in robust expression in TFN-positive the ApoE4 isotype, relative to ApoE3 (Fig. 5A), in postmortem endosomal compartments but failed to restore defective Aβ brains and neurons suggested a mechanistic basis for our ob- clearance in ApoE4 astrocytes (SI Appendix,Fig.S6K and L), servations (47). Fractional colocalization of HDAC4 with DAPI pointing to an isoform-specific role for NHE6 in Aβ clearance. revealed prominent overlap, consistent with increased nuclear Colocalization of NHE6 with EEA1 and LRP1 (SI Appendix, translocation in ApoE4 astrocytes (Fig. 5B). This was indepen- Fig. S7 A and B) suggested a potential role for NHE6 in endo- dently verified in Western blots of nuclear fractions, which showed somal recycling of LRP1 receptors. Compared with the weak higher nuclear HDAC4 in ApoE4 astrocytes relative to ApoE3 (SI surface LRP1 staining in vector-transfected ApoE4 astrocytes, Appendix,Fig.S8A). We recently discovered an evolutionarily we observed prominent, ∼2.5-fold higher LRP1 staining in conserved mechanism for nutrient and HDAC-dependent regulation ApoE4 cells expressing ectopic NHE6 (Fig. 4F). Similar results of NHE6 gene expression (48). HDAC inhibitors could therefore were obtained in surface biotinylation experiments that showed potentially correct human pathologies resulting from NHE6 down- robust, ∼5.7-fold higher surface LRP1 levels in ApoE4 cells with regulation and aberrant endosomal hyperacidification. To extend restored NHE6 expression, compared with transfection with and translate these observations, we screened a panel of nine HDAC empty vector (SI Appendix, Fig. S7C). We confirmed that there inhibitors comprising several different chemical classes for their were no concomitant changes in LRP1 transcript (SI Appendix, potential to augment the expression of NHE6 in ApoE4 astro- Fig. S7D) or total protein expression (SI Appendix, Fig. S7C) cytes. Whereas inhibitors of class I (CI994) or class II (MC1568) levels, suggesting that increased surface LRP1 was due to post- HDACs resulted in minimal changes in NHE6 expression, struc- translational redistribution of the existing cellular LRP1 pool. turally distinct, broad-spectrum drugs inhibiting both classes, in- Consistent with these findings, confocal microscopy revealed a cluding sodium butyrate, sodium valproate, LBH589, trichostatin reduction in perinuclear accumulation of LRP1 and partial res- A (TSA), and suberoylanilide hydroxamic acid (SAHA) (vorino- toration of staining on astrocyte processes in ApoE4 cells with stat), resulted in significant restoration of NHE6 expression levels NHE6 expression (SI Appendix, Fig. S7E). Taken together, our in ApoE4 astrocytes to levels comparable to ApoE3 astrocytes data point to diminished NHE6 expression as a major underlying (Fig. 5C). Other narrow-spectrum HDAC inhibitors studied here cause for defective Aβ clearance in ApoE4 astrocytes. Further- (tubacin and clioquinol) had no significant effect. We confirmed a more, since LRP1 is a receptor for multiple ligands, loss of robust increase in NHE6 protein in ApoE4 astrocytes following

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1801612115 Prasad and Rao Downloaded by guest on October 1, 2021 HDAC localization A Schematic B ApoE3 HDAC4 HDAC4 DAPI Z ApoE4 ApoE3 1.0 **** TF 0.8

ApoE3 0.6 HAT HDAC HDAC inhibitors 0.4 Nuclear HDAC4 0.2

ApoE4 ApoE4

0.0

C HDAC inhibition kD DEEndosomal pH Surface LRP1 NHE6− −76 ApoE3 DMSO DMSO Actin− ApoE4 −38 TSA TSA 250 ApoE4+HDACi panel ** ** 2.5 7 200

noisserpxe6EHN 2.0 )egnahcdloF( 6 150 1.5 pH 100 1.0 5

0.5 50 Normalised LRP1 levels 4 0.0 0

Narrow spectrum Broad spectrum

F Aβ clearance – flow cytometry G Aβ clearance – confocal microscopy DMSO PHYSIOLOGY R2=0.7884 Aβ DAPI SAHA 160 800 SAHA DMSO 300 **** LBH589 ecnaraelc SVAL 200 CI994 600 140 TSA 100 DMSO

SBUT 0 clearance 102 103 104

β 400 AdesilamroN 120 300 SAHA CQ 200 200 SAHA Normalised A β MC1568 100 ApoE4/4 100 DMSO TUB 0 0 2 3 4

Count 10 10 10 1.0 1.5 2.0 2.5 Aβ clearance (Log scale) NHE6 expression

Fig. 5. HDAC inhibitors rescue NHE6-mediated Aβ clearance deficits. (A) Schematic showing HDAC activation in ApoE4, compared with increased histone acetylase (HAT) activity and transcription factor (TF) binding in ApoE3. HDAC inhibitors could potentially exert therapeutic effects by reducing ApoE4-induced nuclear translocation of HDACs. (B) Representative micrographs (Left) and quantification using the Pearson correlation coefficient (Right) of colocalization of HDAC4 (red) with nuclear DAPI (blue) in ApoE3 and ApoE4 astrocytes. Colocalization is evident in the merge and orthogonal slices (Z) as magenta puncta. Note the prominent overlap between HDAC4 and DAPI, consistent with increased nuclear translocation, in ApoE4 astrocytes (Pearson’s correlation coefficient for ApoE3: 0.17 ± 0.01 vs. ApoE4: 0.51 ± 0.02; ****P = 4.6 × 10−44, Student’s t test; n = 150 per condition). (C) Quantitative PCR analysis to determine the efficacy of HDAC inhibitors to augment the expression of NHE6 in ApoE4 astrocytes following 12 h of treatment. Broad-spectrum drugs, including sodium butyrate, sodium valproate, LBH589, TSA, and SAHA, restored NHE6 expression in ApoE4 astrocytes to levels comparable to ApoE3 astrocytes. (Inset) Robust increase of NHE6 protein with HDAC inhibition by SAHA and TSA treatment. The complete Western blot is shown in SI Appendix, Fig. S8B.(D) HDAC inhibition by TSA treatment (5 μM for 12 h) corrected hyperacidic endosomal pH in ApoE4 astrocytes, relative to DMSO treatment (**P = 0.0014, Student’s t test; n = 3). (E) HDAC inhibition by TSA treatment significantly increased surface levels of LRP1, as determined by surface labeling at 4 °C and flow cytometry, in ApoE4 astrocytes (**P = 0.0037, Student’s t test; n = 3). The fluorescence-activated cell sorting (FACS) histogram is shown in SI Appendix, Fig. S8G.(F, Left) Quantitation of Aβ clearance by FACS analysis (10,000 cells in biological triplicates) to determine the efficacy of HDAC inhibitors to rescue Aβ clearance deficits in ApoE4 astrocytes. Note the prominent linear relationship between Aβ clearance and the fold change in NHE6 expression (R2 = 0.7884, P < 0.0001) elicited by DMSO and nine HDAC inhibitors. CQ, clioquinol; SBUT, sodium butyrate; SVAL, sodium valproate; TUB, tubacin. (Right) Representative FACS histograms demonstrating the increase in Aβ internalization by ApoE4 with SAHA treatment. (G) Representative micrographs (Left) and quantification (Right) dem- − onstrating internalized Aβ following SAHA treatment. Note the prominent, vesicular Aβ staining in SAHA-treated ApoE4 cells (****P = 9.9 × 10 13, Student’s t test; n = 160 per condition) (SI Appendix, Fig. S8). (Scale bars, 10 μm.)

HDAC inhibition (Fig. 5C and SI Appendix, Fig. S8B). Both TSA of significantly elevating endosomal pH (Fig. 5D) without effect and SAHA elicited dose-dependent NHE6 increases with a half- on lysosomal pH (SI Appendix,Fig.S8F). Importantly, we ob- maximal response of 6.50 ± 0.36 μMand6.81± 0.53 μM(SI served prominent, approximately twofold higher surface LRP1 Appendix,Fig.S8C and D), respectively, comparable to their expression in ApoE4 cells with TSA treatment (Fig. 5E and SI therapeutic plasma concentrations (49). Neither TSA nor SAHA Appendix,Fig.S8G). We confirmed that both TSA and SAHA significantly altered NHE9 levels (SI Appendix,Fig.S8E). Next, stimulated acetylation of histones H3 and H4 in ApoE4 astrocytes we sought to determine if enhanced NHE6 expression resulting following 60 min of treatment (SI Appendix,Fig.S8H and I). Of from inhibition of HDACs was physiologically effective in cor- note, TSA or SAHA treatment in ApoE4 astrocytes did not sig- recting hyperacidic endosomal pH in ApoE4 astrocytes. TSA nificantly affect cell viability measured using trypan blue exclusion treatment (5 μM for 12 h) exhibited a compartment-specific effect (SI Appendix, Fig. S8J).

Prasad and Rao PNAS Latest Articles | 7of10 Downloaded by guest on October 1, 2021 Key to the potential efficacy of HDAC inhibitors in AD therapy A is their ability to rescue Aβ clearance deficits in ApoE4 astrocytes. LRP1 We observed a prominent linear relationship between Aβ clearance and the fold change in NHE6 expression (R2 = 0.7884; Fig. 5F) elicited by the panel of nine HDAC inhibitors. HDAC inhibitors with lower induction of NHE6 expression (e.g., MC1568, HDACi ApoE3 tubacin) conferred minimal changes in Aβ clearance. Notably, broad-spectrum HDAC inhibitors (e.g., TSA, SAHA) that signif- RE icantly restored NHE6 expression also elicited proportionally NHE6 complete correction of defective Aβ clearance in ApoE4 astro- Na+/K+ cytes to levels similar (up to 92.4%) to ApoE3 cells (Fig. 5F and SI LRP1 Appendix K , Fig. S8 ). ApoE4 cells treated with SAHA showed H+ prominent, vesicular Aβ staining relative to vehicle control (Fig. G ApoE4 5 ). Taken together, we show distinct effects of ApoE3 and EE ApoE4 genotypes on nucleocytoplasmic shuttling of HDAC4. These findings lead to a molecular mechanism, with clinical implications, for ApoE4-associated down-regulation of NHE6 in postmortem brain and astrocyte models. V-ATPase β β Discussion Fig. 6. Proposed role for NHE6 in A clearance in astrocytes. A receptor LRP1 is constitutively recycled to the cell surface through early (EE) and AD is characterized by a pathological increase of amyloid Aβ in recycling (RE) endosomes in ApoE3 astrocytes. Loss of NHE6 expression in the brain, resulting from an imbalance between its production ApoE4 astrocytes hyperacidifies endosomes and impairs trafficking of and clearance. Recent studies suggest that accumulation of Aβ LRP1 receptor, resulting in defective Aβ clearance. HDAC inhibitors (HDACi) in the brain begins at least 20 y before symptoms appear (50). restore expression of NHE6 and Aβ clearance in ApoE4 cells. Although several promising drugs targeting the amyloid cascade have been developed, their astoundingly high failure rates (99.6%) in the clinic suggest that by the time amyloid plaques, Abnormalities in histone acetylation have been linked to neu- neurofibrillary tangles, and neuronal death are detected, it is rodegenerative diseases, including AD, and HDAC inhibitors unlikely that disease progression can be halted and reversed (51). appear to show a neuroprotective effect, improving memory and In this context, understanding and targeting preclinical endo- cognition in mouse models (59, 60). Here, we link increased nu- somal pathologies may be critical for an effective cure. clear translocation of HDAC4 in ApoE4 astrocytes to down- Endosomes take the center stage in an emerging model of AD. regulation of NHE6 expression. We show that broad-spectrum Dysfunction of endosomes is proposed to be a pathogenic hub HDAC inhibitors restore NHE6 expression, normalize endo- β and driver of the disease (15). Previously, we showed that somal pH, and correct A clearance defects in ApoE4 astrocytes. NHE6 limits the acidification of early endosomes to regulate Thus, the amelioration of AD pathogenesis observed in vitro and trafficking and BACE1-mediated processing of the amyloid in vivo by small-molecule inhibitors of HDACs may be mediated, precursor protein (APP), effectively limiting production of Aβ in part, by NHE6. Future work could test the efficacy of these peptide (52). In this study, we demonstrate a critical role for pharmacological agents on amyloid pathology in well-defined NHE6 in the uptake and clearance of Aβ in astrocytes. Aβ animal models. Given the well-known link between NHE6 dys- clearance in cell culture is defined as the endocytic uptake of Aβ function and epilepsy (2, 9), we suggest that increased NHE6 from the extracellular milieu and is quantified by measuring in- expression could potentially contribute to antiepileptic mecha- ternalized Aβ (23, 34, 53, 54). We used mouse astrocytes expressing nisms of the HDAC inhibitor drug sodium valproate. Importantly, human ApoE isoforms that produce, lipidate, package, and se- our data demonstrate a hitherto unrecognized ability of HDAC crete ApoE in a brain-relevant physiological fashion (23). The inhibitors to specifically enhance endosomal pH, which could well-documented pathogenic deficiency of ApoE4 astrocytes to potentially correct human pathologies resulting from aberrant clear Aβ is mediated by decreased expression of NHE6, which endosomal hyperacidification. Our recent observations showing results in endosomal overacidification and reduced surface levels that NHE6 is a target of the transcription factor cAMP-response of the Aβ receptor LRP1. Thus, NHE6 is an important ApoE4 element-binding protein (CREB), known to be negatively regu- effector in astrocytes. The precise pH-sensitive perturbation in lated by HDACs, provide a molecular mechanism for HDAC trafficking remains to be determined. We suggest that hyper- inhibitor-mediated activation of NHE6 expression in ApoE4 as- acidification of early and recycling endosomes blocks receptor trocytes (48). Of note, reduced phosphorylation of CREB has recycling to the plasma membrane, trapping the endosomes in a been described in brains of ApoE4 carriers (58, 61, 62). nonproductive intracellular pool (Fig. 6). Recent observations Dysfunction in endolysosomal pH is an emerging theme in AD also point to a role for ApoE4 in binding and trapping insulin with clear potential for intervention to exploit the disease- receptor within intracellular compartments (55), which could result modifying effects of endosomal pH (19). Amphipathic drugs, from acid pH-mediated ApoE4 aggregation within the endosomes such as bepridil and amiodarone, partition into acidic compart- (56). Taken together, we propose that loss of NHE6 function ments, alkalinize endosomes, and correct Aβ pathology in cell contributes to the endosomal pathology observed in presymp- culture and animal models (63). Our study supports a rational, tomatic AD brains both by accelerating Aβ production and by mechanistic basis for such repurposing of existing US Food and inhibiting Aβ clearance, promoting the development of amyloid Drug Administration-approved drugs with well-established safety plaques and culminating in neurodegeneration. Finally, given and pharmacokinetic profiles, known to have off-label activity of our previous studies suggesting a profound panspecific effect of endosomal alkalization, to target the cellular microenvironment eNHE activity on membrane persistence of multiple cell surface in AD. Similar to our observations in AD, down-regulation of proteins (2, 40, 57), we propose that pathogenic endosomal NHE6 gene expression has been reported in autism brains (64). acidification can occur as an upstream event and impair endo- We suggest that endosomal pH may be a critical mechanistic link cytic recycling of multiple cell surface receptors and transporters, between neurodevelopmental and neurodegenerative disorders. including glutamate and insulin receptors previously reported to Thus, a subset of patients with autism who have dysregulated be reduced in brains of ApoE4 carriers (55, 58). NHE6 activity, either from loss-of-function mutations or by

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1801612115 Prasad and Rao Downloaded by guest on October 1, 2021 down-regulated gene expression, is likely to have a high risk of Aβ Assay on Mouse Brain. A human/rat/mouse β-amyloid ELISA kit from Wako developing neurodegenerative disorders, thereby providing a (no. 294-64701) was used for the estimation of Aβ40 levels in brain homoge- rational basis to stratify patients for targeted therapies. nates, as per the manufacturer’s instructions. Briefly, brains of mice were dis- sected on ice, weighed, and homogenized in ice-cold radioimmunoprecipitation Methods assay buffer (PBS + 1% Triton + 0.1% SDS + 0.5% deoxycholate) containing – – Animals. All procedures were carried out with the approval of the Institutional protease inhibitor (Roche). Lysate was centrifuged for 8 10 min at 6,000 Animal Care and Use Committee of the University of California, San Francisco 7,000 × g, and supernatant was collected and used for ELISA. The bicinchoninic and the Johns Hopkins University School of Medicine, Baltimore. The Slc9a6 acid method was used to measure the total protein concentrations. Aβ40 was knockout mice (no. 005843, strain name B6.129P2-Slc9a6

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