Summary
The accumulation of amyloid protein Aβ in senile plaques is a key driver and hallmark of Alzheimer disease (AD), a major cause of death and dementia in the elderly. The strongest genetic risk factor in sporadic AD is the ε4 allele of Apolipoprotein E (ApoE4), which potentiates pre-symptomatic endosomal dysfunction and defective clearance of Aβ, although how these two pathways are linked has been unclear. Here, we show that aberrant accumulation of endosomal protons in ApoE4 astrocytes traps the LRP1 receptor in non-productive intracellular compartments, leading to loss of surface expression and Aβ clearance. Hyperacidification of endosomal pH is caused by selective down regulation of the Na+/H+ exchanger NHE6, which functions as a critical proton leak pathway, in ApoE4 brain and astrocytes. In vivo, the NHE6KO mouse model shows elevated Aβ in the brain. Epigenetic restoration of NHE6 expression with histone deacetylase inhibitors normalized ApoE4-specific defects in endosomal pH, LRP1 trafficking and amyloid clearance. Thus, NHE6 is a prominent effector of ApoE4 and emerges as a promising therapeutic target in Alzheimer disease.
Introduction
Alzheimer disease (AD) is a degenerative brain disorder and a leading cause of dementia that affects 47 million people worldwide 1. AD is caused by pathological increase of amyloid β (Aβ) in the brain, resulting from an imbalance between its production and clearance. Recent studies suggest that accumulation of Aβ in the brain begins at least 20 years before symptoms appear 2. 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, neurofibrillary tangles and neuronal death are detected, it is unlikely that disease progression can be halted and reversed 3. Identifying and targeting pre-clinical pathologies may be critical for an effective cure.
In this context, endosomal aberrations constitute the earliest detectable brain cytopathology, emerging before cognitive dysfunction is apparent in neurodegenerative disorders, including Alzheimer disease, Niemann-Pick Type C and Down syndrome 4, 5, 6, 7. Consistent with this finding, genes associated with endosomal trafficking have been implicated as major risk factors in AD 8. Importantly, prominent pre-symptomatic endosomopathy of the brain, evidenced by enlarged and more numerous endosomes, has been observed in the ApoE4 genotype 9, 10, 11, the strongest known genetic risk factor influencing susceptibility to sporadic, late-onset AD (LOAD) 12, 13. The pathological E4 variant of Apolipoprotein E is present in ~50% of patients with AD, and the presence of two copies of the E4 allele increases risk of LOAD by ~12-times as compared to E3 isoform 14. Yet, despite strong evidence implicating endosomal uptake and clearance of Aβ in mediating AD risk in the ApoE4 genotype 14, 15, 16, the underlying mechanism is unknown.
Here we show that a profound dysregulation of endosomal pH in humanized mouse ApoE4 astrocytes leads to intracellular sequestration and cell surface loss of the Aβ receptor, LRP1. Selective down regulation of the endosomal Na+/H+ exchanger NHE6, which functions as a safety valve against excessive acidification by exchanging lumenal protons with cations, is mediated by increased nuclear translocation of the histone deacetylase HDAC4 in ApoE4 astrocytes. HDAC inhibitors that restore NHE6 expression also restore surface expression of LRP1 and effectively correct defective amyloid clearance in ApoE4 astrocytes to non-pathological ApoE3 levels. Consistent with a role in amyloid pathology, in vivo Aβ levels were found to be significantly higher in the brains of NHE6KO mice. These findings could have implications for Christianson syndrome patients who have loss of function mutations in NHE6 and exhibit age-dependent hallmarks of neurodegeneration17, 18. In summary, we identify NHE6 as a novel ApoE effector and suggest potential therapeutic options in the treatment of amyloid disorders.
Results
ApoE4 astrocytes have cargo specific defects in endocytosis
Studies in human, mouse models and cell-culture have revealed the importance of ApoE-isotype-specific differences in Aβ uptake and clearance in AD pathogenesis, although the underlying mechanism remains to be determined 14, 16, 19, 20. To this end, we developed a sensitive and quantitative fluorescent-based assay to monitor cell-associated Aβ peptide (Fig. 1A) in astrocytes from ApoEKO mice with isogenic knock-in of human ApoE3 and ApoE4 variants 14. Internalized Aβ is sorted to the lysosomal degradation pathway as evidenced by high colocalization with late endosomal-lysosomal markers and low colocalization with the recycling compartment marker transferrin (TFN) (Fig. S1A-D). Strikingly, cell-associated Aβ was reduced by 78% in ApoE4 astrocytes, relative to ApoE3 (Fig. 1B). To distinguish between Aβ uptake and turnover, we monitored the time course of Aβ internalization by flow cytometry analysis (Fig. 1C) and confocal microscopy (Fig. S1E). Consistent with defective uptake, there was significantly lower cell-associated Aβ in ApoE4 cells relative to ApoE3 at all time points (Fig. 1C, Fig. S1F). In contrast, cell-associated TFN was 1.5-2 fold higher in ApoE4 cells relative to ApoE3 as measured by flow cytometry (Fig. 1D) and confocal microscopy (Fig. 1E). Uptake of dextran by fluid-phase endocytosis was not different between ApoE genotypes (Fig. 1F). These observations reveal cargo selective effects of ApoE isotype in astrocytes and point to alterations in specific receptor pathways.
Surface expression of LRP1 receptor is severely reduced in ApoE4 astrocytes
Transcriptional down regulation of the LRP1 receptor has been suggested as an underlying mechanism for defective Aβ clearance in AD patients21. However, we found no difference in brain LRP1 gene expression at different stages of AD (incipient, moderate, and severe), as compared with normal controls, in publicly available microarray data 22 (Fig. S2A-B). Meta-analysis of nine independent gene expression studies from anatomically and functionally distinct brain regions, comprising a total of 103 AD and 87 control post-mortem brains also showed no significant changes in LRP1 gene expression in AD (Fig. S2C-D). Consistent with these findings, we observed no differences in LRP1 transcript and total protein expression between ApoE3 and ApoE4 astrocytes (Fig. S2E-G).
LRP1 undergoes constitutive endocytosis from the membrane and recycling back to the cell surface 23. Therefore we considered the possibility that alterations in LRP1 receptor recycling could result in differences in plasma membrane expression. ApoE isotype-specific surface expression of LRP1 was evaluated using four independent approaches (Fig. 2A). First, surface biotinylation revealed that plasma membrane expression of LRP1 receptor in ApoE4 astrocytes was lower by ~50% (Fig. 1B). Second, an antibody directed against an external epitope of LRP1 to quantify surface expression in live cells by flow cytometry analysis showed a reduction of LRP1-positive cells by ~43% (Fig. 1C). Third, this was confirmed by confocal microscopy showing ~49% lower LRP1 surface labeling by antibody in ApoE4 (Fig. 1D). In a fourth approach, surface-bound ligand (fluorescent Aβ) measured by confocal microscopy was 66% lower in ApoE4 astrocytes (Fig. 1E). Notably, the greater attenuation in Aβ binding when compared to the ~50% reduction in surface LRP1 levels suggests additional isotype-specific mechanisms that contribute to Aβ clearance, such as reduced ligand-receptor affinity in ApoE4 cells or reductions in other Aβ receptors. Thus, ApoE isotype-specific alterations in receptor recycling determine LRP1 surface expression and cellular Aβ uptake, revealing a new pharmacological target for amyloid clearance defects in the pathological ApoE4 genotype.
Endo-lysosomal pH is defective in ApoE4 astrocytes
The pH within the endo-lysosomal system plays a critical role in receptor-mediated endocytosis and recycling 24. We used compartment-specific, pH-sensitive fluorescence reporters to probe ApoE-isotype dependent differences in endosomal, lysosomal and cytoplasmic pH (Fig. 3A). Endosomal pH in ApoE4 astrocytes was strongly reduced by ~0.84 pH unit, relative ApoE3 (Fig. 3B). In contrast, we observed >1 pH unit elevation of lysosomal pH in ApoE4 astrocytes (Fig. 3C). Previously, elevated lysosomal pH was observed in presenilin 1 (PS1)-deficient cell culture models and neurons, another genetic model of AD 25. Cytoplasmic pH showed no significant differences between the two ApoE isotypes (Fig. 3D).
To determine if there was a causal link between endo-lysosomal pH and defective Aβ clearance in ApoE4 astrocytes, we treated ApoE4 cells with the ionophore monensin that mediates Na+/H+ exchange across acidic compartments26. Thus, monensin treatment (50μM for 1 h) elevated endosomal pH in ApoE4 knock-in astrocytes from 5.38±0.01 to 5.74±0.03, relative to the vehicle treated control (Fig. 3E). Concomitantly, monensin treatment restored Aβ clearance in ApoE4 astrocytes to ApoE3 levels, as shown by flow cytometry analysis (Fig. 3F). This was independently confirmed by confocal microscopy (Fig. 3G), suggesting that defective pH regulation could underlie the observed Aβ clearance defects.
NHE6 restores defective Aβ clearance in ApoE4 astrocytes
Luminal pH in the endo-lysosomal network is set by the precise balance of proton pump and leak pathways, mediated by V-type H+-ATPase and endosomal Na+/H+ exchangers (NHE6), respectively (Fig. 4A)27, 28, 29, 30. Changes in expression and activity of the pump and leak pathways could lead to significant dysregulation of endosomal pH in Alzheimer brains. Consistent with this possibility, analysis of a publicly available microarray dataset (GSE5281) comprising a total of 15 sporadic, late-onset AD (LOAD) and 12 matched control post-mortem brains 31 revealed that genes involved in hydrogen ion transmembrane transport, including the endosomal NHE6 and V-ATPase subunits, comprised 10% of the top 100 down regulated genes, exhibiting highest enrichment scores (>15-fold; Fig. S3A). In AD patients with ApoE4/4 genotype, NHE6 was among the transcripts differentially down regulated in hippocampus, by up to ~4-fold compared to ApoE3/3 32, 33. We validated these findings using an independent, large human brain transcriptome dataset (n=363, GSE15222) to show ApoE4 isotype-specific differential gene expression of NHE6 in aging brain 34 (Fig. 4B). Although NHE6 transcript was similar in ApoEKO mouse astrocytes and ApoEKO astrocytes with knock-in of human ApoE3, it was ~56% reduced in ApoE4 knock-in cells (Fig. 4C). There was also ApoE4 specific reduction in transcript for the related endosomal isoform NHE9 (~70% lower) and lysosomal V-ATPase V0a1 subunit (~67%), but not for the plasma membrane NHE1 isoform (Fig. S3B). These large transcript differences could account for the observed ApoE-isotype specific shifts in endo-lysosomal pH.
Taken together, these data suggest an important, hitherto underappreciated role of proton transport and endosomal pH regulation in AD. We hypothesized that NHE6 is a potential ApoE effector, and that down regulation of NHE6 in disease-associated ApoE4 variants is causal to a subset of AD phenotypes. Consistent with this hypothesis, amyloid Aβ levels were found to be elevated in mouse brains from NHE6KO (Fig. 4C), together with diminished brain weight (Fig. S3C), suggesting an underlying neurodegenerative pathology.
Similar to monensin treatment, lentiviral vector mediated expression of NHE6 alkalinized the endosomal lumen in ApoE4 astrocytes (Fig. S3D). Therefore, we tested if ectopic expression of GFP-tagged eNHE isoforms could correct defective Aβ uptake in ApoE4 astrocytes. Remarkably, Aβ clearance was restored to ApoE3 levels in ApoE4 astrocytes transfected with NHE6 (Fig. 4E), but not with NHE9 (Fig. S3E-F), pointing to an isoform-specific role for NHE6 in Aβ clearance.
Colocalization of NHE6 with EEA1 and LRP1 (Fig. S3G-H) suggested a potential role for NHE6 in endosomal recycling of LRP1 receptors. Compared to the weak surface LRP1 staining in vector-transfected ApoE4 astrocytes, we observed prominent, ~2.5-fold higher LRP1 staining in ApoE4 cells expressing ectopic NHE6 (Fig. 4F). Similar results were obtained in surface biotinylation experiments that showed robust ~5.7-fold higher surface LRP1 levels in ApoE4 cells with restored NHE6 expression, compared to transfection with empty vector (Fig. S3I). We confirmed that there were no concomitant changes in LRP1 transcript (Fig. S3J) or total protein expression levels (Fig. S3I), suggesting that increased surface LRP1 was due to posttranslational redistribution of the existing cellular LRP1 pool. Taken together, our data point to diminished NHE6 expression as a major underlying cause for defective Aβ clearance in ApoE4 astrocytes. Furthermore, since LRP1 is a receptor for multiple ligands, loss of NHE6 may contribute to other ApoE4 defects, including defective synaptosome uptake and synapse pruning 12, 23.
HDAC inhibitors rescue NHE6-mediated Aβ clearance deficits
Reports of increased nuclear translocation of multiple histone deacetylases (HDACs) in ApoE4 isotype, relative to ApoE3 (Fig. 5A), in post-mortem brains and neurons suggested a mechanistic basis for our observations 35. Fractional colocalization of HDAC4 with DAPI revealed prominent overlap, consistent with increased nuclear translocation in ApoE4 astrocytes (Fig. 5B). This was independently verified in Western blots of nuclear fractions, which showed higher HDAC4 in ApoE4 astrocytes relative to ApoE3 (Fig. S4A).
To translate these observations, we screened a panel of nine HDAC inhibitors comprising several different chemical classes for their potential to augment the expression of NHE6 in ApoE4 astrocytes. Whereas inhibitors of class I (CI994) or class II (MC1568) HDACs resulted in minimal changes in NHE6 expression, broad-spectrum drugs inhibiting both classes, including sodium butyrate, sodium valproate, LBH589, TSA, and SAHA (vorinostat), resulted in significant restoration of NHE6 expression levels in ApoE4 astrocytes to levels comparable to ApoE3 astrocytes (Fig. 5C). Other narrow spectrum HDAC inhibitors studied here (tubacin and clioquinol) had no significant effect. Both TSA and SAHA elicited dose dependent NHE6 increases with half-maximal response (EC50) of 6.50±0.36μM and 6.81±0.53μM (Fig. S4B-C), respectively, comparable to their therapeutic plasma concentrations 36. Neither TSA nor SAHA significantly altered NHE9 levels (Fig. S4D). We confirmed that both TSA and SAHA stimulated acetylation of histone H3 and H4 in ApoE4 astrocytes following 60 minute of treatment (Fig. S4E-F). Next, we sought to determine if enhanced NHE6 expression resulting from inhibition of histone deacetylases was physiologically effective in correcting hyperacidic endosomal pH in ApoE4 astrocytes. TSA treatment (5μM for 12h) exhibited a compartment-specific effect of significantly elevating endosomal pH (Fig. 5D) without effect on lysosomal pH (Fig. 5E). Of note, TSA or SAHA treatment in ApoE4 astrocytes did not significantly affect cell viability measured using trypan blue exclusion (Fig. S4G).
Key to the potential efficacy of HDAC inhibitors in AD therapy is their ability to rescue Aβ clearance deficits in ApoE4 astrocytes. 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 and tubacin) conferred minimal changes in Aβ clearance. Notably, broad-spectrum HDAC inhibitors (e.g. TSA and SAHA) that significantly restored NHE6 expression also elicited proportionally complete correction of defective Aβ clearance in ApoE4 astrocytes to levels similar (up to 92.4%) to ApoE3 cells (Fig. 5F and S4H). ApoE4 cells treated with SAHA showed prominent, vesicular Aβ staining relative to vehicle control (Fig. 5G). Taken together, these findings reveal differential effects of ApoE3 and ApoE4 genotypes on nucleo-cytoplasmic shuttling of the HDACs leading to a novel molecular mechanism, with clinical implication, for ApoE4 associated down regulation of NHE6 in post-mortem brain and astrocyte models.
Discussion
The discovery of endosomal Na+/H+ exchangers (eNHE) first in yeast, and soon after in plants, metazoans, and mammalian systems, established their evolutionarily conserved role as a leak pathway for protons in compartmental pH homeostasis, critical for cargo trafficking and vesicular transport 27, 37, 38. Na+/H+ exchangers are estimated to have exceptionally high transport rates of ~1,500 ions/s 39, so that even small perturbations in expression result in dramatic changes in ionic milieu within the limited confines of the endosomal lumen. Genetic studies have linked eNHE to a host of neurodevelopmental and neurodegenerative disorders, including Christianson syndrome (CS) with symptoms of autism, intellectual disability and epilepsy, Parkinsons disease, multiple sclerosis and AD, although underlying mechanisms remain to be determined 27.
One clue emerged from network analysis of 1697 genes in a late-onset AD dataset (Cases n=176, Controls=188), in which the endosomal Na+/H+ exchanger NHE6 (SLC9A6) was identified as a top-five hub transcript in AD, with 202 network connections and a plethora of potential downstream effects 34. More recent network analysis of the metastable subproteome associated with AD also converged on NHE6 as a major hub gene regulating protein trafficking and clearance mechanisms 40. In addition to prominent neurodegeneration phenotypes in CS patients, female carriers have learning difficulties and behavioral issues, and some present with low Mini Mental Status Exam (MMSE) scores suggestive of early cognitive decline 41. Interestingly, NHE6 was among the most highly down regulated genes (up to 6-fold) in elderly (70 years) brain, compared to adult (40 years)42. These observations point to a more widespread role for NHE6 in neurodegenerative disorders and unexpectedly common pathological pathways between CS and Alzheimer disease.
Previously, we showed that NHE6 regulates trafficking and BACE1-mediated processing of amyloid precursor protein APP to limit production of amyloidogenic peptides 43. In this study, we demonstrate a critical role for NHE6 in the uptake and clearance of soluble, secreted Aβ in astrocytes. Using ApoE-isoform-expressing isogenic astrocytes that produce, lipidate, package, and secrete ApoE in a brain-relevant physiological fashion, we showed that the well-documented pathogenic deficiency of ApoE4 astrocytes to clear Aβ is mediated by decreased expression of NHE6, which results in endosomal over-acidification and reduced surface levels of the Aβ receptor LRP1. Thus, NHE6 is a novel and important ApoE4 effector in astrocytes (Fig. 6). The precise pH-mediated perturbation in trafficking remains to be determined. It is likely that hyper-acidification of early and recycling endosomes redistributes plasma membrane proteins to the lysosome at the expense of recycling to the cell surface. Taken together, we propose that loss of NHE6 function contributes to the endosomal pathology observed in pre-symptomatic AD brains both by accelerating Aβ production and by inhibiting Aβ clearance, promoting the development of amyloid plaques and culminating in neurodegeneration and dementia.
Abnormalities in histone acetylation have been linked to several neurodegenerative diseases including AD, and HDAC inhibitors appear to show a neuroprotective effect, improving memory and cognition in mouse models 44, 45. Here, we link increased nuclear translocation of HDAC4 in ApoE4 astrocytes to down regulation of NHE6 expression. We show that broad-spectrum HDAC inhibitors restore NHE6 expression, normalize endosomal pH and correct Aβ clearance defects in ApoE4 astrocytes. Thus, the amelioration of AD pathogenesis observed in vitro and in vivo by small molecule inhibitors of HDACs may be mediated, in part, by NHE6. Future work could test the efficacy of these pharmacological agents on amyloid pathology in well-defined animal models. Given the well-known link between NHE6 dysfunction and epilepsy 27, 46, we suggest that increased NHE6 expression could potentially contribute to anti-epileptic mechanisms of HDAC inhibitor drug sodium valproate. Importantly, our data demonstrate a hitherto unrecognized ability of HDAC inhibitors to specifically enhance endosomal pH that could potentially correct human pathologies resulting from aberrant endosomal hyperacidification.
Dysfunction in endo-lysosomal pH is an emerging theme in AD with clear potential for intervention to exploit the disease-modifying effects of endosomal pH 7. Amphipathic drugs such as bepridil and amiodarone partition into acidic compartments, alkalinize endosomes, and correct Αβ pathology in cell culture and animal models 47. Our study supports a rational, mechanistic basis for such repurposing of existing FDA-approved drugs with well-established safety and pharmacokinetic profiles, known to have off-label activity of endosomal alkalization, to target the cellular microenvironment in AD. Similar to our observations in AD, down regulation of NHE6 gene expression has been reported in autism brains 48. We suggest that endosomal pH may be a critical mechanistic link between neurodevelopmental and neurodegenerative disorders. Thus, a subset of autism patients with dysregulated NHE6 activity, either from loss-of-function mutations or by down-regulated gene expression, are likely to have a high risk of developing neurodegenerative disorders, thereby providing a rational basis to stratify patients for targeted therapies. In conclusion, this work presents, (i) a novel ApoE4 regulated cellular mechanism and druggable target in AD, namely, regulation of amyloid pathology by intra-endosomal pH; (ii) a new focus on endosome trafficking in astrocyte function, a neglected area in neurodegenerative disorders; (iii) a new link between an autism gene and the AD risk allele ApoE4; (iv) a new strategy for mechanism-based therapies for AD and related devastating disorders with important implications for early intervention to limit progressive, severe and debilitating neurodegeneration seen in Christianson syndrome patients.
Author Contributions
H.P. designed, conducted and analyzed experiments and wrote the paper. R.R. designed and interpreted experiments and wrote the paper.
Materials and Methods
Animals
All procedures were carried out in accordance with The Institutional Animal Care and Use Committee of the University of California, San Francisco and the Johns Hopkins University School of Medicine, Baltimore. The Slc9a6 knockout mice (#005843, strain name B6.129P2-Slc9a6<tm1Dgen) were obtained from Jackson Laboratories. The model was engineered by inserting the LacZ reporter gene, which encodes β-galactosidase into the Slc9a6 genomic locus (Deltagen). In all experiments, male Slc9a6−/Y mice were used as mutants and wild-type male Slc9a6+/Y mice as controls. On average, five mice of each genotype were used in each experiment.
Aβ clearance assays
Human ApoE isoform-expressing (ApoE3 and ApoE4) astrocyte cells were plated in six-well plates and were grown to confluence. To measure Aβ uptake, cells were washed with serum-free medium (SFM) followed by incubation with 100 nM fluorescently-labeled HiLyte Fluor 647-Aβ40 (#AS-64161, AnaSpec) for various timepoints. Cells were washed with PBS and fixed for confocal imaging using the LSM 700 Confocal microscope (Zeiss), or trypsinized for flow cytometry analysis of ~10,000 cells in biological triplicates using the FACSAria instrument (BD Biosciences). Unstained cells without any exposure to fluorescently-labeled Aβ were used as a control for background fluorescence.
Aβ assay on mouse brain
The human/rat/mouse β amyloid ELISA kit was from Wako (#294–64701) was used for the estimation of Aβ40 levels in brain homogenates, as per manufacturer’s instructions. Briefly, brains of mice were dissected on ice, weighed and homogenized in ice-old RIPA buffer (PBS+ 1% Triton+ 0.1% SDS+ 0.5% deoxycholate) containing protease inhibitor (Roche). Lysate was centrifuged for 8–10 minutes at 8,000–9,000 rpm and supernatant was collected and used for ELISA. BCA method was used to measure the total protein concentrations. Aβ40 was normalized to total protein concentration in the lysate.
Endosomal, lysosomal and cytoplasmic pH measurement
Detailed protocols are provided in the Extended Experimental Procedures
Statistical Analysis
All data were analyzed statistically by Student’s t test, ANOVA and linear regression test using GraphPad Prism. All data are presented as mean ± SD.
Acknowledgements
We thank Drs. Robert Edwards and Julie Ullman of University of California San Francisco for providing mouse brains and Dr. David M Holtzman, Washington University, St. Louis for the gift of ApoE immortalized astrocytes. We are very grateful to Dr. Seth S. Margolis for helpful discussions, and Richard L. Blosser for assistance with the flow cytometry analysis. This work was made possible by support from the Johns Hopkins Medicine Discovery Fund to R.R. Additional support came from a grant to R.R. from the National Institutes of Health (DK054214). H.P. is Fulbright Fellow supported by the International Fulbright Science and Technology Award.