A conserved mechanism for regulation of endo-lysosomal pH by histone deacetylases

The pH of the endo-lysosomal system is tightly regulated by a balance of proton pump and leak mechanisms that are critical for storage, recycling, turnover and signaling functions in the cell. Dysregulation of endo-lysosomal pH has been linked to aging, amyloidogenesis, synaptic dysfunction, and various neurodegenerative disorders including Alzheimer’s disease. Therefore, understanding mechanisms that regulate luminal pH may be key to identifying new targets for treatment of these disorders. Meta-analysis of yeast microarray databases revealed that nutrient limiting conditions upregulated transcription of the endosomal Na+/H+ exchanger Nhx1 by inhibition of the histone deacetylase (HDAC) Rpd3, resulting in vacuolar alkalinization. Consistent with these findings, Rpd3 inhibition by the HDAC inhibitor and antifungal drug trichostatin A induced Nhx1 expression and vacuolar alkalinization. Bioinformatics analysis of Drosophila and mouse databases revealed that caloric control of Nhx1 orthologs DmNHE3 and NHE6 respectively, was also mediated by histone deacetylases. We show that NHE6 is a target of cAMP-response element-binding (CREB) protein, providing a molecular mechanism for nutrient and HDAC dependent regulation of endosomal pH. Control of NHE6 expression by pharmacological targeting of the CREB pathway can be used to regulate endosomal pH and restore defective amyloid Aβ clearance in an ApoE4 astrocyte model of Alzheimer’s disease. These observations from yeast, fly, mouse and cell culture models reveal an evolutionarily conserved mechanism for regulation of endosomal NHE expression by histone deacetylases and offer new therapeutic strategies for modulation of endo-lysosomal pH in fungal infection and human disease.


ABSTRACT
The pH of the endo-lysosomal system is tightly regulated by a balance of proton pump and leak mechanisms that are critical for storage, recycling, turnover and signaling functions in the cell. Dysregulation of endo-lysosomal pH has been linked to aging, amyloidogenesis, synaptic dysfunction, and various neurodegenerative disorders including Alzheimer's disease. Therefore, understanding mechanisms that regulate luminal pH may be key to identifying new targets for treatment of these disorders. Metaanalysis of yeast microarray databases revealed that nutrient limiting conditions upregulated transcription of the endosomal Na + /H + exchanger Nhx1 by inhibition of the histone deacetylase (HDAC) Rpd3, resulting in vacuolar alkalinization. Consistent with these findings, Rpd3 inhibition by the HDAC inhibitor and antifungal drug trichostatin A induced Nhx1 expression and vacuolar alkalinization. Bioinformatics analysis of Drosophila and mouse databases revealed that caloric control of Nhx1 orthologs DmNHE3 and NHE6 respectively, was also mediated by histone deacetylases. We show that NHE6 is a target of cAMP-response elementbinding (CREB) protein, providing a molecular mechanism for nutrient and HDAC dependent regulation of endosomal pH. Control of NHE6 expression by pharmacological targeting of the CREB pathway can be used to regulate endosomal pH and restore defective amyloid Aβ clearance in an ApoE4 astrocyte model of Alzheimer's disease. These observations from yeast, fly, mouse and cell culture models reveal an evolutionarily conserved mechanism for regulation of endosomal NHE expression by histone deacetylases and offer new therapeutic strategies for modulation of endolysosomal pH in fungal infection and human disease.
The endosome is a busy way station that handles cargo traffic at the crossroads of degradation and recycling. An important hallmark of the endo-lysosomal system is a pH gradient that is increasingly acidic through the transition from early, recycling and late endosomes to the lysosome (1). Precise tempo-spatial pH lumen regulation within each of these specialized compartments is critical for a range of functions, including controlling cargo sorting, vesicular budding and formation of intraluminal vesicles, dissociation of internalized ligands, receptor recycling and turnover, modulating enzyme activity, antigen processing, and cellular signaling (2).
Endosomes are acidified by the electrogenic pumping of protons by the V-type H + -ATPase in conjunction with vesicular chloride transporters of the CLC family that shunt the membrane potential, effectively allowing a buildup of luminal protons (3,4). Proton leak mechanisms consisting of proton conduction channels or proton-coupled antiporters limit the acidification, and the balance of pump and leak pathways defines the pH set point. Although the molecular identity of vesicular proton channels remains poorly defined, more recently, endosomal Na + /H + exchangers (eNHE) have emerged as the dominant leak pathway for luminal protons. Because exchanger mechanisms have high transport rates of ∼1,500 ions/s (5), they overwhelm the capacity of the proton pump so that even small changes in eNHE activity or expression result in significant pH changes within the endolysosomal system.
The fundamental importance of endosomal pH is highlighted by the fact that a growing number of human diseases are associated with mutations in V-ATPase subunits (e.g., osteopetrosis, renal tubular acidosis, cutis laxa), chloride transporters (e.g., Dent's disease) and eNHE (Christianson syndrome, autism and attention deficit hyperactivity disorder) (3,4,6). Furthermore, defective endo-lysosomal pH regulation is being increasingly linked to cellular aging, amyloidogenesis, synaptic dysfunction and neurodegenerative disorders including Alzheimer's disease (7,8). Therefore, the discovery of novel regulators of endo-lysosomal pH may be crucial to identifying new diagnostic and therapeutic targets for these disorders.
As a starting point, we turned to the yeast S. cerevisiae, which has powerfully informed our understanding of fundamental mechanisms of endocytosis and vesicular traffic that are functionally conserved with higher organisms (9). By leveraging data from yeast to fly and mammalian models, we discovered an evolutionarily conserved mechanism for epigenetic control of eNHE by histone deacetylases (HDAC) in response to nutrient availability.
In mammalian cells, HDACmediated control of endo-lysosomal pH occurs by transcriptional regulation of the endosomal Na + /H + exchanger NHE6 (SLC9A6) via the cAMPresponse element binding protein (CREB) pathway. We show that elevation of cAMP levels by the FDA-approved drug Rolipram results in CREB activation, increased NHE6 expression and restoration of defective Aβ clearance in the ApoE4 astrocyte model of late onset Alzheimer's disease.
Histone acetylation has been widely recognized as a key modulator of global chromatin structure that dynamically couples extracellular signals to gene transcription. Whereas lysine acetylation on histone tails interferes with generation of higher order chromatin structures and promotes access and binding of transcription factors, histone deacetylation favors chromatin packing and represses gene transcription (10)(11)(12). Histone deacetylase (HDAC) inhibitors have been approved for the treatment of hematologic cancers and are being considered as promising therapy to reverse disease-associated epigenetic states in a range of cardiovascular, neurodegenerative and inflammatory diseases (11)(12)(13)(14). Given their broad application, our findings offer potential therapeutic options to treat endo-lysosomal pH dysfunction in autism, AD, and other neurodegenerative disorders.

Nutrient control of endo-lysosomal pH in yeast
Yeast microarray datasets are a valuable resource for discovery-driven mining efforts. A meta-analysis of 45 microarray experiments that included 937 samples revealed increasing expression of Nhx1 with growth phase stationary>mid-log>early log (Fig. 1A), which we independently confirmed: Nhx1 transcript increased by 3.2-fold in stationary phase cells, compared to mid-log phase (Fig. 1B). Stationary phase growth in yeast is associated with depletion of nutrients. To determine if growth phase regulation of Nhx1 was linked to glucose availability, we tested the effect of glucose removal from mid-log phase yeast cultures. Nhx1 mRNA was increased by nearly 2-fold within 60 min of glucose removal (p<0.001; Fig. 1C). In contrast, addition of glucose (2%) to stationary phase cultures elicited significant decrease in Nhx1 mRNA within 30 min (p<0.001; Fig. 1D).
Nhx1 functions as a leak pathway for protons within the endosome/prevacuolar compartment, in opposition to the proton pump activity of the V-ATPase (15) (Fig. 1E). The balance of proton pump and leak sets the pH within the endo-lysosomal compartments. Therefore, we also analyzed expression of V-ATPase subunits under the same experimental conditions. Although we did not observe a growth phase-dependent transcriptional change in the V-ATPase catalytic subunit Vma1 (Fig. 1B), V-ATPase subunits were significantly downregulated with acute glucose depletion (p<0.001, Vma1; p<0.001, Vma7; Figure 1C) and upregulated upon glucose addition (p<0.05, Vma1; p<0.001, Vma7; Figure 1D) in contrast to Nhx1. Previous studies have shown that yeast V-ATPase subunit Stv1 is selectively targeted to the Golgi/secretory pathway and is not required for vacuolar acidification and function (16,17). In contrast to Vma1 and Vma7, Stv1 expression showed modest or no changes in response to glucose ( Figure 1C-D), suggesting that the effect of glucose regulation on the V-ATPase is largely localized to late endosomal/vacuolar compartments.
We predicted that reciprocal transcriptional changes in Nhx1 and V-ATPase subunits would alter the pH of the yeast vacuole. Vacuolar pH can be probed in situ with the pHsensitive fluorescent dye quinacrine, which accumulates in acidic environments (18). Following glucose depletion, yeast vacuoles showed little or no quinacrine staining, similar to V-ATPase deficient vma7Δ mutants ( Fig. 1F-G; p<0.0001, n=100). Consistent with the potential role for Nhx1 in regulating nutrient control of endo-lysosomal pH in yeast, nhx1Δ mutants showed severely reduced survival during the stationary phase growth (19). Post-translational regulation of V-ATPase disassembly and reassembly in response to glucose concentration is well established (20,21). Here, we conclude that glucose also elicits reciprocal transcriptional changes in proton pump and leak mechanisms to regulate the pH gradient across yeast vacuoles.

Regulation of endo-lysosomal pH in yeast is mediated by a histone deacetylase
We extended our unbiased bioinformatics approach to analyze data from 284 microarray studies that comprised a wide range of experimental conditions including gene deletion, overexpression and mutations, drug/toxin treatment, nutrient limitation or excess, metabolic/environmental stress and more. We identified experimental conditions eliciting a minimum of ±2-fold change in NHX1 gene expression ( Fig. 2A, Fig. S1A). The highest up regulation of Nhx1 was observed under conditions of (i) genetic deletion of Rpd3, (ii) glucose depletion, and (iii) nitrogen depletion. Pathway analysis revealed that loss or inhibition of histone deacetylase (HDAC) activity of Rpd3 was common to all three of these conditions (22,23) (Fig. 2B). Thus, nutrient (glucose or nitrogen) depletion elicits histone acetylation by replacement and inactivation of the HDAC/Rpd3 complex from the promoters of specific genes (22). Consistent with this finding, Rpd3 deletion phenocopies calorie restriction by enhancing lifespan of yeast cells (24,25). Conversely, all five conditions leading to maximal down regulation of Nhx1 were linked to depletion of Abf1, a transcription factor that is negatively regulated by Rpd3 (26-28) ( Fig. 2A, and S1A). Recent evidence suggests that Abf1 is activated during nutritional stress (29). Consistent with this finding, Abf1 is predicted to bind to the NHX1 promoter ( Fig.  S1B). For comparison, depletion of another general regulatory transcription factor Rap1 had no effect on Nhx1 expression ( Fig. 2A and S1A). Thus, all top hits in Nhx1 gene expression (both up-and down regulation) funneled into a single pathway: the Rpd3-Abf1-Nhx1 axis (Fig. 2C).
To confirm the microarray data, we independently demonstrated that Nhx1 transcript was elevated by 1.9-fold in the rpd3Δ mutant, relative to isogenic wild type yeast (Fig. 3A). Other histone deacetylase gene deletions conferred more modest induction (1.35-fold in hda1Δ; Fig.  3A), or repression (0.6-fold in sir2Δ; Fig. 3A). All three yeast deletion strains showed similar growth (Fig. S2A). Restoration of Rpd3 by ectopic expression under a galactose-inducible promoter in rpd3Δ not only abolished Nhx1 upregulation in stationary phase, but also caused further repression (68% lower) in mid-logarithmic cells and inhibited yeast growth ( Fig. S2B-C).
Yeast Rpd3 is a target of the histone deacetylase inhibitor and fungal antibiotic trichostatin A (TSA), and the transcriptional profile of TSA-treated wild-type yeast is similar to that of rpd3Δ yeast (23). We show that that TSA treatment of wild-type yeast elicited significant 2.9-fold up regulation of Nhx1 transcript (Fig. 3B) mimicking the effect of Rpd3 deletion. In contrast, we observed significant down regulation of V-ATPase subunits Vma1 (∼30% lower; Fig. 3C) and Vma7 (∼37% lower; Fig. 3D) in TSA-treated yeast, compared to vehicle treatment. In contrast to Vma1 and Vma7, the levels of Stv1 did not change significantly with TSA treatment (Fig.  S2D), suggesting that the effect of TSA is specific to V-ATPase localized to the vacuolar compartment.
The transcriptional upregulation of Nhx1 and concomitant downregulation of vacuolar V-ATPase subunits by TSA is predicted to alkalinize the yeast vacuole (3). Compared to robust quinacrine fluorescence in wild-type yeast vacuoles (18), TSA treatment drastically reduced vacuolar accumulation of quinacrine, similar to the effect of the well-characterized V-ATPase inhibitor concanamycin A, pointing to increased vacuolar pH (Fig. 3E-F). Alkaline pH sensitivity is a defining phenotype of V-ATPase mutants, indicative of the reduced ability to acidify vacuoles (30,31). We found that TSA treated cells show dose-dependent reduction of growth at alkaline media pH (Fig. 3G). Hypersensitivity to TSA in the vma7Δ yeast, lacking V-ATPase activity, is evidenced by synthetic lethality (Fig.  3H). TSA synergizes with the antifungal action of an azole drug fluconazole that we previously showed to act via inhibition of V-ATPase activity (30) (Fig. S2E-F). Together, these data provide strong evidence for regulation of yeast vacuolar pH by HDAC inhibition.

Evolutionary conservation of eNHE regulation by nutrient availability and histone deacetylases
To investigate if the mechanism of upregulation of eNHE in response to nutrient limitation and HDAC activity is evolutionarily conserved, we evaluated publicly available expression data in the fruit fly Drosophila melanogaster. Fruit fly has one eNHE isoform DmNHE3, which, like its mammalian cousin NHE6, is highly expressed in the brain (32) (Fig.  4A). Analysis of a microarray dataset studying the effects of starvation in Drosophila larvae (GSE14531) revealed significant downregulation of class I HDAC DmRpd3 (-2.14-fold; Fig. 4B) and reciprocal upregulation of class III HDAC DmSir2 (1.66-fold; Fig. 4C) in fly larvae starved for 24 h (33). Importantly, consistent with findings from yeast, there was significant upregulation (2.12-fold; Fig. 4D) of DmNHE3 expression with starvation (33). No changes in expression with starvation were seen for other NHE isoforms, DmNHE1 and DmNHE2 ( Fig. S3A-B). We also evaluated the effect of nutrient depletion on transcript levels of Vha68 and Vha14 that are orthologs of Vma1 and Vma7, respectively. Unlike our observations in yeast, no change in expression of these V-ATPase subunits was noted in Drosophila, suggesting that the transcriptional effect of starvation might be limited to endosomal NHE in higher eukaryotes.
Chromatin-modifying complexes are conserved between yeast and mammals. Eighteen mammalian HDACs have been described, grouped into four functional classes based on similarity to yeast orthologs (10). Independent studies have shown that insulin, the principal hormone orchestrating response to glucose availability, alters HDAC nuclear translocation in mammalian cells (34). Calorie restriction has been shown to improve insulin sensitivity and mimic the effects caused by HDAC inhibitors, including on histone acetylation and activation of cAMP-response element-binding (CREB) protein (35-38). Intriguingly, the mammalian eNHE ortholog NHE6 is a reported insulin responsive protein and its subcellular trafficking is regulated by insulin (39). Furthermore, in neurons, loss of NHE6 has been reported to attenuate brain-derived neurotrophic factor (BDNF) signaling that is downstream of insulin and CREB cascade (40,41). Therefore, to test if calorie restriction or HDAC inhibitors enhance mammalian eNHE expression, we first analyzed publicly available expression data on caloric restriction (approximately 10% lower than ad lib intake) in the mouse (Fig. 5A). These data revealed significant upregulation of NHE6 in the neocortex (Fig. 5B), but not of the closely related endosomal NHE9 or V-ATPase subunit Vma1/ATP6V1A ( Fig. S4A-B) (42). Next, we analyzed two independent microarray experiments studying the effect of TSA in cell culture at shorter (2 h; GSE8488) and longer (18 h; GSE9247) time points (43,44). Expression data from both these experiments revealed significant up regulation of NHE6 with TSA treatment ( . Activation of transcription factor CREB is a common response to both calorie restriction and pharmacological HDAC inhibition (12,38). Thus, for comparison, we evaluated a prototypic CREB target BDNF and documented significant upregulation with calorie restriction in mice (Fig.  S4C) and HDAC inhibition in cell culture ( Fig.  S5C-F), as predicted. These observations from yeast, fly, mouse and cell culture models support our hypothesis of an evolutionarily conserved mechanism for regulation of eNHE expression by histone deacetylases.

NHE6 is a transcriptional target of CREB
Next, we attempted to identify the HDACregulated transcription factor equivalent to yeast Abf1 in higher eukaryotes. In the absence of direct Abf1 orthologs in the Drosophila or human genome, we looked for parallel starvation response pathways between human and yeast. Various studies suggest that CREB transcription factor regulates response to nutrient deprivation and glucose availability in mammalian cells, analogous to the Abf1 transcription factor in yeast (38,45). Both class I (HDAC1) and class II (HDAC4) HDACs directly interact with CREB and negatively regulate its function (46,47). Furthermore, similar to the Abf1 transcription factor in yeast, the phosphorylation state of CREB is tightly regulated in response to nutrient and glucose availability (38,48). Based on all these observations, we hypothesized NHE6 as a potential CREB target (Fig. 6A).
Analysis of a publicly available dataset of CREB target genes, from chromatin immunoprecipitation with DNA microarray (ChIPon-chip)(49), using the recommended cutoff for ChIP positive genes (binding ratio ≥1.5 and Log 10 p-value ≥3) revealed NHE6 as a potential CREB target. The ChIP binding data for NHE6 was similar to that of DUSP1, a known CREB target, whereas no CREB occupancy was detected for the related NHE7 isoform ( Fig. 6B-C). Consistent with our hypothesis in Fig. 6A, TSA treatment resulted in ∼5.2-fold increase in NHE6 expression in HEK293 cells (Fig. 6D), whereas, modest increases were observed in NHE7 (∼1.3-fold; Fig   S6A) and NHE9 (∼1.4-fold; Fig. 6D), and no changes in expression of V-ATPase subunits V0a1 (lysosomal) and V0a2 (putative endosomal) were observed ( Fig. S6B-C). We used the firefly and Renilla luciferase reporter gene system to demonstrate significant activation of cyclic AMPresponse element (CRE) with TSA treatment (10µM, 12 h), relative to vehicle control (p=0.0064; Fig. 6D-E). To establish a causal link between CREB activation and NHE6 expression, we treated HEK293 cells with forskolin, an adenylyl cyclase activator, and confirmed significant increase in NHE6 transcript levels that was blocked by expression of a constitutively active/nuclear HDAC4 mutant (S246/467/632A) (Fig. 6G).
To directly test if NHE6 is a CREB target, we expressed Flag-tagged CREB1 plasmid in HEK293 cells and assessed (i) functional expression of NHE6 and (ii) binding of CREB to NHE6 promoter by ChIP-qPCR (Fig. 7A). Expression of CREB1 in HEK293 cells significantly elevated NHE6 transcript (Fig. 7B), resulting in alkalization of endosomal pH from 5.9±0.03 to 6.12±0.05 (Fig. 7C). In contrast, no changes in NHE6 transcript or endosomal pH were observed with the non-phosphorylatable mutant of CREB1 (S133A; Fig. 7B-C). Chromatin immunoprecipitation was performed with a ChIP grade anti-Flag mouse antibody (Fig. 7D). Using real time qPCR we amplified three predicted CREB binding sites in the promoter of NHE6 (SLC9A6) at positions -12Kb, +01Kb, and +02Kb relative to transcription start site (TSS) (Fig. 7D  and S6D). For comparison, we used primers targeted to NHE9 (SLC9A9) promoter at position -01Kb relative to TSS (Fig. 7E). We confirmed CREB binding to NHE6 promoter at all three predicted sites (Fig. 7F-H). In contrast, we observed no significant binding of CREB to NHE9 promoter (Fig. 7I). Specific pull-down of CREB1 was confirmed by probing the ChIP samples with an antibody against CREB raised in rabbit (Fig.  7J). Consistent with our ChIP-qPCR findings, we note that the pattern of NHE6 expression closely parallels that of CREB1 through normal human brain development ( Fig. S7K; Pearson correlation: 0.62) and in all areas of the brain (Fig. 7L). In contrast, NHE9 expression showed no correlation with CREB ( Fig. 7M; Pearson correlation: -0.018).
Taken together, our findings reveal a novel mechanism of CRE mediated transcriptional regulation of NHE6 and provides a molecular mechanism for nutrient and HDAC dependent regulation of endosomal pH.

Control of NHE6 expression by pharmacological targeting of CREB pathway
Christianson syndrome (CS) patients, with loss of function mutations in NHE6, clinically present with neurodevelopmental disorders of intellectual disability and severe autism, and show age-dependent hallmarks of neurodegeneration, including loss of cerebellar and cortical mass, prominent glial pathology and hyperphosphorylated tau deposition (3,(50)(51)(52). Recent revelations on the shared pathology of defective Aβ clearance and altered processing of amyloid precursor protein (APP) between autism and Alzheimer disease suggest that endosomal pH regulation may be one critical mechanistic link between neurodevelopmental and neurodegenerative disorders (53). NHE6 gene expression is depressed in both AD and autism (54,55). CRE mediated transcriptional regulation of NHE6 expression offers opportunities to expand the repertoire of drugs that could potentially correct human pathologies resulting from NHE6 down regulation and aberrant endosomal hyperacidification (Fig. 8A). For example, Rolipram, a PDE4 inhibitor that elevates cAMP levels resulting in CREB activation, has shown efficacy as memory enhancer and for amelioration of Aβ related neuropathology in mice models (56).
We used mouse astrocytes with knock-in of human ApoE alleles that alter genetic risk for Alzheimer disease (57). Recently, we showed that NHE6 expression is severely reduced in astrocytes carrying the pathological ApoE4 variant, relative to the isogenic non-risk ApoE3 allele (8). Consistent with this observation, CREB is downregulated in ApoE4 brains (58). First, we confirmed CREB-mediated NHE6 regulation in ApoE3 astrocytes using the CREB inhibitor (KG501): we observed dose-dependent down regulation of NHE6 transcript levels with KG501 treatment (Fig. 8B). Rolipram was effective in dose-dependent activation of CREB targets in ApoE4 astrocytes including the prototypic CREB target BDNF (Fig. S7A), and astrocyte specific CREB targets, ALDH1A1 and FBXO2(59) (Fig.  S7B-C). We also observed significant induction of CREB mRNA by Rolipram as well (Fig. 8C), consistent with similar observations in rat hippocampus (60). Here, we demonstrate dosedependent increase in NHE6 transcript levels with Rolipram treatment (1.25µM to 10µM) in ApoE4 astrocytes (Fig. 8D). To evaluate the efficacy of Rolipram treatment on NHE6 mediated function, we monitored Aβ clearance in cultured ApoE4 astrocytes by flow cytometry and confocal microscopy (Fig. 8E). We show that Rolipram elicits significant increase in Aβ clearance, evidenced by increase in cell-associated fluorescence by flow cytometry of ApoE4 astrocytes (Fig. 8F). Similarly, endocytosed Aβ, monitored by confocal microscopy, exhibited an average of 2.66-fold increase following treatment with Rolipram ( Fig. 8G-H). Taken together, our findings show that CREB activation of NHE6 expression is translatable into correction of Aβ clearance deficits in ApoE4 astrocytes.

Nutrient control of endo-lysosomal pH
The discovery of endosomal Na + /H + exchangers (eNHE) in the mid-90's and subsequent work in a range of model organisms and mammalian systems has established the fundamental importance of these ion transporters in human health and disease (3,61). Although the contribution of the V-ATPase in nutrient control of endo-lysosomal pH is well established in yeast (20,21), the role of eNHE in this mechanism was previously unknown. Here, we leveraged public data from yeast, fly and mammalian studies to make an unbiased in silico prediction leading to the discovery of an evolutionarily conserved mechanism for regulation of eNHE expression by nutrients and histone deacetylases (HDACs). Previous studies have established that glucose triggers rapid, post-translational regulation of disassembly and reassembly of V-ATPase to regulate the pH gradient across yeast vacuoles (20,21). In this study, we show that glucose also elicits reciprocal transcriptional changes in Nhx1 and V-ATPase subunits to control vacuolar pH. This finding is in agreement with previous work showing severely reduced survival of Nhx1 mutants during nutrient-depleted stationary phase (19).
The regulation of endo-lysosomal pH by Nhx1 and V-ATPase (or their orthologs in higher eukaryotes) might promote cell survival during nutrient depletion or starvation by at least three potential mechanisms: (i) Vacuole acidity facilitates proton-dependent import of amino acids into the lumen through Avt1, a neutral amino acid transporter important for starvation resistance (18,62). In nutrient depleted cells, decrease in vacuolar acidification would reduce vacuolar import of amino acids and help maintain cytoplasmic amino acid homeostasis (Fig. S8A-B).
(ii) Transcriptional control of V-ATPase subunits could supplement post-translational mechanisms of V-ATPase disassembly as an energy-saving mechanism to conserve ATP under nutrient limiting conditions. (iii) We have previously shown that Nhx1 regulates vesicle trafficking and lysosomal biogenesis to control internalization of plasma membrane proteins and degradation in vacuoles (15,61). Increased Nhx1 expression in glucose-depleted cells might therefore help promote endocytosis and provide resources for survival during glucose starvation (63). Intriguingly, eNHE mutants in C. elegans (Nhx5) and fly (DmNHE3) were found to be resistant to metformin, a biguanide drug commonly used to treat type-2 diabetes, potentially through dysregulation of autophagy (64). Given that metformin is known to induce a caloric restrictionlike state (65), our findings suggest that upregulation of eNHE and alkalinization of endosomal pH may underlie response to metformin therapy.

Antifungal drugs target endo-lysosomal pH
Pathogenic fungi have become a leading cause of morbidity and mortality in the world (66). Fungi need V-ATPase function to infect their hosts, and genetic inactivation of vacuolar specific V o subunit Vph1, but not secretory pathway specific subunit Stv1, abolished virulence and invasive growth of C. albicans, underscoring the importance of endo-lysosomal pH as a drug target (16,17). Consistent with this finding, the antifungal activity of azoles, which inhibit biosynthesis of fungal-specific sterol, ergosterol, is mediated by reduced V-ATPase activity in ergosterol-deficient vacuoles (30). Similarly, decreased V-ATPase activity may underlie the antifungal activity of amphotericin B, which is known to directly bind and sequester ergosterol in yeast cells (67). Furthermore, many naturally occurring antifungal compounds such as terpenoid phenols, function at least in part by disrupting V-ATPase function (68). Recent studies have documented the potential benefits of repurposing FDA approved amphipathic drugs such as amiodarone, already in clinical use as calcium antagonists, that partition into acidic compartments, alkalinize endo-lysosomes and potentiates antifungal effects and reverse resistance to azole drugs (69). Here we show that an antifungal HDAC inhibitor trichostatin A (TSA) increases vacuole pH and enhances the antifungal effects of fluconazole. Intriguingly, both amiodarone and HDAC inhibitors confer significant neuroprotection in cell culture and animal models (11,12,70). Together, these studies and our current findings makes a strong case for (i) repurposing antifungal drugs as potential drugs to correct human pathologies arising from dysregulation of endolysosomal pH and (ii) repurposing FDA approved drugs having offtarget effects on pH homeostasis as candidates for the development of new therapeutic strategies for fungal disease.

Targeting endo-lysosomal pH in human diseases
Dysregulation of endo-lysosomal pH is an emerging theme in pathogenesis of a wide range of human diseases, ranging from neurodegeneration to cancer (7,71). There is clearly potential for intervention to exploit the disease-modifying effects of endosomal pH, and this should be investigated further. From lessons learnt with Baker's yeast, we known that gene disruption of NHX1 leads to cellular phenotypes reminiscent of Alzheimer's disease (AD) that include enlarged endosome/prevacuolar compartment, hyperacidic luminal pH, enhanced proteolysis of the chaperone protein Vps10 (a homolog of the AD susceptibility factor SORL1), mistrafficking of vacuolar hydrolases and profound deficiencies in lysosomal cargo delivery (15,72). Several studies have identified links between eNHE and a host of neurological disorders including autism, intellectual disability, epilepsy, Parkinson's disease, multiple sclerosis and more recently to late-onset AD (LOAD) (3,8,54,73), although mechanisms underlying eNHE regulation have remained obscure. Here we show that expression of the Christianson Syndrome protein NHE6 is under control of CREB transcription factor. A translatable finding from our studies is that pharmacological HDAC inhibition (by TSA) or CREB activation (by Forskolin or Rolipram) elevate endosomal pH and have the potential to mitigate human pathologies such as AD and autism associated with aberrant endosomal hyperacidification. Furthermore, regulation of endo-lysosomal pH may at least partially explain the working mechanism of HDAC inhibitors in cancer therapy. Tumor cells in the periphery have better blood supply, whereas cells inside tumor masses are under starved conditions, since nutrients are not fully supplied to these cells. As we have shown earlier, a subset of brain cancer glioblastoma multiforme show significant upregulation of eNHE isoform NHE9 that might help in survival of tumor cells in nutrient limited conditions (74). Together, these data suggest that, by governing fundamentally critical endosomal pH homeostasis, eNHE plays an important role in mediating nutrient control of endo-lysosomal pH. This importance makes them an attractive target as antifungal drugs and in several human diseases. Exploration of the conserved mechanism for regulation of endo-lysosomal pH by histone deacetylases provides a foundation for understanding the pathways regulating pH homeostasis and a platform for the development of drugs that could potentially correct human pathologies resulting from dysregulated endolysosomal acidification.

Yeast strains and growth
All Saccharomyces cerevisiae strains used were derivatives of BY4741 (gift from Dr Susan Michaelis, Johns Hopkins University or purchased from transOMIC technologies). Yeast cells were grown in YPD (yeast extract, peptone and dextrose) medium at 30°C with shaking at 250 rpm. Acute glucose depletion was performed by growing yeast in in YP (yeast extract and peptone) medium. Yeast growth was determined by optical density of the culture at 600 nm (OD600). Spotting assay on YPD agar plates was performed to detect drug-induced growth inhibition phenotypes.

Alkaline pH sensitivity and quinacrine staining
Increased luminal pH in the yeast vacuole was detected using two defining phenotypes: alkaline pH sensitivity and quenching of quinacrine staining. Sensitivity to alkaline media pH was conducted in YPD medium, which was adjusted to pH 7 or pH 4.3. For quinacrine staining, cells were suspended in YPD medium containing 100 mM HEPES pH 7.6, 200 µM quinacrine and stained for 10 min at 30°C. Stained cells were washed 3X with 100 mM HEPES pH 7.6, 0.2% glucose and examined by fluorescence microscopy immediately. Quantification of images was done using ImageJ software.

Aβ clearance assays
To quantify Aβ clearance, ApoE4 astrocytes were washed with serum-free medium (SFM) followed by incubation with 100 nM fluorescently-labeled HiLyte Fluor 647-Aβ40 (#AS-64161, AnaSpec). Following several washed with PBS cells were 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 FACSCalibur instrument (BD Biosciences). Unstained cells without any exposure to fluorescently-labeled Aβ were used as a control for background fluorescence. Quantification of confocal images was done using ImageJ software.

Endosomal pH measurement
Endosomal pH was measured using flow cytometry, as we previously described (54,73). Briefly, cells were rinsed and incubated in SFM for 30min, to remove residual transferrin and then incubated with pH-sensitive FITC-Transferrin (#T2871, Thermo Fisher Scientific) (75 µg/ml) together with pH non-sensitive Alexafluor 633-Transferrin (#T23362, Thermo Fisher Scientific) (25 µg/ml) at 37°C for 1hr. Cells were placed on ice to stop transferrin uptake and excess transferrin was removed by washing 2X with ice-cold serumfree DMEM and PBS. Bound transferrin was removed by washing with ice-cold pH 5.0 PBS and pH 7.0 PBS. Cells were trypsinized and pH was determined by flow cytometry analysis of ∼10,000 cells in biological triplicates using the FACSCalibur instrument (BD Biosciences). A four-point calibration curve with different pH values (4.5, 5.5, 6.5 and 7.5) was generated using Intracellular pH Calibration Buffer Kit (#P35379, Thermo Fisher Scientific) in the presence of 10µM K + /H + ionophore nigericin and 10µM K + ionophore valinomycin.

Bioinformatics
Unbiased in silico approach was used to identify experimental conditions that significantly altered Nhx1 expression, by analyzing data from 284 Saccharomyces cerevisiae microarray studies, that comprised a wide range of studies including gene deletion, overexpression and mutations, drug/toxin treatment, nutrient limitation or excess, metabolic/environmental stress and many others. Experimental conditions with a minimum gene expression fold change of ±2 were selected for further thorough pathway analysis. A predictive model for regulation of Nhx1 expression was created based on the overlap of pathways from experimental conditions that resulted in highest degree of changes in gene expression (both upand downregulation) and selected for in vitro validation. Meta-analysis of 45 microarray experiments including 937 samples determined effect of growth phase on Nhx1 expression. Normalized yeast expression data was obtained from Genevestigator (Nebion AG). Drosophila microarray experiment GSE14531 was analyzed to study starvation effect on fly larvae. Mammalian gene expression datasets included in the study were GSE11291, GSE8488 and GSE9247. Human brain and Drosophila gene expression data were obtained from the Allen Brain and FlyBase atlas, respectively.

Chromatin Immunoprecipitation (ChIP)-qPCR
To assess physical binding of the flag tagged CREB to the NHE6 Gene promoter, ChIP-qPCR was carried out. ChIP was performed using the EpiQuik Chromatin Immunoprecipitation (ChIP) Kit (#P-2002, Epigentek), according to the manufacturer's instructions. In brief, cells were treated with 1% formaldehyde for 10 minutes to cross-link proteins to DNA. To generate DNA fragments approximately 200 to 1000 bp long, the cross-linked chromatin was sonicated on ice. ChIP assays were performed using using a ChIP-grade Anti-flag antibody. Specific pull-down of CREB1 was confirmed by probing the ChIP samples with an antibody against CREB raised in rabbit using Western blotting. After reversal of the cross-links, qPCR was performed using the EpiTect ChIP-qPCR primer assay for NHE6/SLC9A6 (GPH1013950(-)12A, −12 kb from the transcription start site; GPH1013950(+)01A, +1 kb from the transcription start site; and GPH1013950(+)02A, +2 kb from the transcription start site). ChIP-qPCR primer assay for NHE9/SLC9A9 promoter (GPH1023313(-)01A, -1 kb from the transcription start site) was used as a control (all Qiagen). The resulting amplification and melt curves were analyzed to ensure specific PCR product. Threshold cycle (Ct) values were used to calculate the fold enhancement.

Luciferase assay
pCRE-Luc (encoding for the cAMP-responsive element CRE and firefly luciferase), and pSV40-RL (encoding for a SV40 promoter and Renilla luciferase) plasmids (gift of Dr Jennifer L. Pluznick, Johns Hopkins University) were transiently transfected into HEK293T cells using lipofectamine 2000 reagent, as per manufacturer's instructions. The ratio of firefly luciferase and Renilla luciferase was measured using the Dual-Luciferase Assay System (#E1910, Promega) with data collected using a FLUOstar Omega automated plate reader (BMG LabTech) to evaluate the CREB activation following treatment with a HDAC inhibitor.

18.
Hughes  A. Waterfall plot depicting fold-change in Nhx1 expression (y-axis) obtained from an unbiased bioinformatics analysis of 284 microarray studies (x-axis), as described in the "Experimental Methods". Note that all five conditions leading to maximal down regulation of Nhx1 were linked to depletion of Abf1 transcription factor, known to be negatively regulated by Rpd3. B. Inhibition of histone deacetylase (HDAC) activity of Rpd3 was common to all three microarray experiments that gave maximal up regulation of Nhx1 expression, namely (i) genetic deletion of Rpd3, (ii) glucose depletion, and (iii) nitrogen depletion. C. HDAC/Rpd3-Abf1-Nhx1 axis for regulation of Nhx1 expression. This model predicts that inhibition of Rpd3 activity releases brake on Abf1 that in turn binds to Abf1 binding site (ABS) on Nhx1 promoter and enhances its expression. See related Figure S1.   Figure S2.   A. Schematic to draw parallels between regulation of eNHE between yeast (top) and mammalian cells (bottom). CREB transcription factor regulates response to nutrient deprivation and glucose availability in mammalian cells, analogous to the Abf1 transcription factor in yeast. Both class I (HDAC1) and class II (HDAC4) HDACs directly interact with CREB and negatively regulate its function. Based on this predictive model, we hypothesized NHE6 as a potential CREB target. (B-C). ChIP-on-chip (chromatin immunoprecipitation with DNA microarray) data analyzed using the recommended cutoff for ChIP positive genes (binding ratio ≥1.5 and Log 10 p-value ≥3) revealed NHE6 as a potential CREB target. Note that the ChIP binding data for NHE6 was similar to that of DUSP1, a known CREB target, whereas no CREB occupancy was detected for the related NHE7 isoform. D. qPCR analysis demonstrating prominent ∼5.2-fold increase in NHE6 expression (****p=9.7X10 -5 ; n=3; Student's t-test), and modest increases in NHE9 (∼1.4-fold; **p=0.0011; n=3; Student's t-test) with TSA treatment of HEK293 cells. (E-F). Luciferase assay to confirm TSA induced activation of CREB using a cyclic AMP-response element (CRE) that drives a firefly luciferase reporter gene and is measured luminometrically (E). Renilla luciferase driven by a constitutively active SV40 promoter is used to normalize for variation of cell number and transfection efficiency. Significant activation of cyclic AMP-response element (CRE) seen with TSA treatment (10µM; 12h; F) in HEK293 cells, relative to vehicle control (**p=0.0064; n=3; Student's t-test). G. qPCR analysis to determine the potential of forskolin to augment the expression of NHE6 in HEK293 cells. Note significant increase in NHE6 transcript levels with forskolin treatment (***p=0.0005; n=3; Student's t-test) that was blocked by expression of a constitutively active/nuclear HDAC4 mutant (S246/467/632A) (p=0.116; n=3; Student's t-test). See related Figure S6.