Although great efforts toward AD drug discovery have been made in recent years, there is currently an impasse with only one new AD therapeutic approved since 2004 (Cummings, Morstorf, & Zhong, 2014 ). While most AD drugs are the result of target‐based screens, a lack of credible drug targets apart from amyloid‐β and tau has limited the search. Phenotypic screens provide an alternative drug discovery strategy that does not require a priori knowledge of a target. Because age is the greatest risk factor for AD, we developed a unique phenotypic screening paradigm specifically designed to recapitulate several of the most common age‐associated central nervous system (CNS) toxicities using cell culture models (Prior et al., 2014 ). We identified a synthetic compound called J147 that is neuroprotective in all of these assays (Chen et al., 2011 ) and promotes the division of neuronal precursor cells in vivo and in vitro (Prior et al., 2016 ). Behaviorally, it enhances memory and restores cognition in APPswe/PS1ΔE9 and the rapidly aging senescence‐accelerated mouse prone (SAMP8) dementia mouse models (Currais et al., 2015 ; Prior, Dargusch, Ehren, Chiruta, & Schubert, 2013 ; Morley, Armbrecht, Farr, & Kumar, 2015). Here, we identify the mitochondrial α‐F1 subunit of ATP synthase (ATP5A) as a high affinity molecular target of J147, a protein previously studied in the context of aging (Chin et al., 2014 ). Therefore, our analysis not only identifies a new AD drug target but also causally connects metabolic regulation, aging, and dementia through a single molecular drug target.

2 RESULTS

2.1 Target identification Three independent approaches were used to initially identify and then confirm the molecular target of J147. First, we used an unbiased small molecule target identification approach called drug affinity responsive target stability (DARTS) (Lomenick, Jung, Wohlschlegel, & Huang, 2011) to detect putative binding partners. Lysates from the HT22 hippocampal nerve cell line were incubated with vehicle, 10 or 50 μm J147 for 15 min before treating with pronase to degrade unbound protein complexes. A band containing proteins preserved by J147 treatment following protein electrophoresis was identified, and mass spectrometry (MS) analysis determined that ATP5A was the most enriched putative target relative to controls (Figure 1a, red arrow, Figure S1). ATP5A is a catalytic subunit of the mitochondrial ATP synthase complex responsible for the synthesis/hydrolysis of ATP. Figure 1 Open in figure viewer PowerPoint Target identification by DARTS and affinity precipitation pull‐downs. (a) DARTS revealed ATP5A as a putative direct J147 target. HT22 cells were treated with 10 and 50 μm J147 for 15 min after which cells were lysed using M‐PER and digested with Pronase. The only band visually preserved among J147‐treated samples (10 and 50 μ m ) (red arrow) was excised and analyzed by LC/MS/MS along with control lanes. ATP5A was the most enriched protein indicating direct target engagement. (b) Affinity precipitation with a biotinylated derivative of J147 (BJ147) pulled down an enriched fraction of mitochondrial‐associated proteins. (c) Affinity precipitation using subventricular zone (SVZ) lysates from adult mice demonstrates BJ147 binding to ATP5A. Unlabeled J147 (100 μ m ) outcompeted ATP5A binding to BJ147 To confirm ATP5A as the molecular target of J147, we performed several additional experiments. First, we incubated HT22 cells and mouse subventricular zone (SVZ) tissue lysates with a biotinylated derivative of J147, BJ147, and used LC/MS/MS to identify coprecipitating proteins. Consistent with the DARTS experiment, a strong enrichment of mitochondria‐associated proteins was present in the streptavidin pull‐down fraction from the BJ47‐incubated samples. The only protein that was reproducibly identified in both the pull‐down and DARTS experiments was ATP5A, while other mitochondrial proteins involved in ion flux and transport such as inositol 1,4,5‐triphosphate receptor 3 (IP3R3), members of the solute carrier family 25 (SLC25a3‐5), and voltage‐dependent anion channel (VDAC) were only present in the pull‐down (Figure 1b). Importantly, the amount of ATP5A was greatly reduced in BJ147‐precipitated lysates incubated with excess unlabeled J147 as a binding competitor (Figure 1c). The most highly enriched protein in both the DARTS and affinity precipitation experiments was ATP5A. Therefore, we asked whether the activity of the ATP synthase complex is modulated by J147. First, we tested J147's effect on ATP synthase enzyme kinetics in isolated bovine heart mitochondria. A dose–response curve is shown after 1 hr of J147 incubation (Figure 2a) that indicates saturating partial inhibition (23.6 ± 3.4%) of ATP synthase activity by J147 with an EC 50 of 20 nm. Importantly, this inhibition is only partial, even at saturating concentrations of J147. These results demonstrate that J147 binds to and partially inhibits the activity of the mitochondrial ATP synthase. Figure 2 Open in figure viewer PowerPoint J147 targets mitochondrial bioenergetics. (a) J147 inhibition of ATP synthase activity from isolated bovine heart mitochondria (F(9,20) = 40.72, ***p = .0004, ****p < .0001, ANOVA). Oligomycin, an inhibitor of both forward (ATP synthesis) and reverse (ATP hydrolysis) ATP synthase activity and an inactive derivative of J147, CAD120, served as positive and negative controls, respectively (not shown) (CAD120: 97.1%; oligomycin: 14 ± 2.4%). (b) BJ147 (green) localizes to mitochondria (red) in HT22 cells within 10 min of addition to tissue culture media. DAPI (cyan), nuclei, BJ147 (green), COXIV (red). Scale bar = 10 μm. (c) Dose‐dependent increase in mitochondrial membrane potential (Δψ m ) in HT22 cells following J147 treatment. Statistics reported for J147 treatments compared to vehicle (*p = .0483, ****p < .0001, ANOVA). (d) ATP5A knockdown in HT22 neuronal cells. (e) ATP5A knockdown phenocopies the effect of J147 on Δψ m (**p = .001, t test, t = 3.385, df = 94). JC1 fluorescence (aggregate:monomer) normalized to oligomycin (10 μ m ). (f) J147 dose‐dependently increases mitochondrial superoxide production in HT22 neuronal cells. (g) siRNA knockdown of ATP5A revealed a similar increase in mitochondrial superoxide production (**p = .001, t test, control siRNA compared to ATP5A siRNA). (h) J147 increases whole cell ATP levels in HT22 cells. FCCP served as a negative control (*p = .0374, ANOVA). (i) ATP5A siRNA‐targeted knockdown increased whole cell ATP levels (*p = .0326) We next determined whether the intracellular localization of J147 in HT22 cells was consistent with a mitochondrial target by confocal fluorescent microscopy using BJ147. An imaging time course demonstrated that J147 colocalized with the mitochondrial marker cytochrome C oxidase IV (COXIV) (Figure 2b, Table S1). Localization of J147 was rapid, occurring within 10 min. Thus, both biochemical and localization experiments support ATP5A as a target of J147.

2.2 J147 targets mitochondrial bioenergetics ATP synthase couples the production or hydrolysis of ATP to the transport of H+ ions across the inner mitochondrial membrane, making it a direct regulator of mitochondrial polarity (Δψ m ). We tested J147's effect on Δψ m using JC1, a ratiometric cationic dye. A significant dose‐dependent increase in mitochondrial membrane potential is observed within 1 hr of J147 treatment (Figure 2c), an effect consistent with the regulation of ATP synthase activity (Perry, Norman, Barbieri, Brown, & Gelbard, 2011). To independently confirm that targeting the α‐subunit of ATP synthase affects its activity and thus Δψ m , we performed siRNA‐targeted knockdown of ATP5A (Figure 2d) and found a similar increase in Δψ m when taken as percent oligomycin‐induced hyperpolarization (Figure 2e). ATP5A siRNA‐treated cells displayed reduced capacity for hyperpolarization after oligomycin addition. Changes in ATP synthase activity occur concomitantly with the production of reactive oxygen species (ROS) (Laura Formentini, Sánchez‐Aragó, Sánchez‐Cenizo, & Cuezva, 2012). Although traditionally thought of as being detrimental, new evidence suggests that inhibition of ATP synthase can elicit a retrograde, ROS‐mediated prosurvival response (Laura Formentini et al., 2012). Both J147 treatment and ATP5A knockdown caused a significant increase in superoxide levels within the mitochondria (Figure 2f,g). In the elderly and in patients with AD, mitochondrial dysfunction leads to reduced levels of ATP which may contribute to disease progression (Reddy et al., 2012; Zhang, Rissman, & Fend, 2015). J147 increased ATP levels in HT22 cells within 4‐6 hr of treatment (Figure 2h) without affecting the rate of glycolysis (Figure S2). Furthermore, ATP5A siRNA‐targeted knockdown similarly increased whole cell ATP levels in these cells (Figure 2i) without affecting protein levels or composition of other oxidative phosphorylation (oxphos) components (Figures S3 andS4).

2.3 ATP synthase inhibition protects from neurotoxic insults We next asked whether inhibiting ATP synthase activity elicits a similar neuroprotective response as seen with J147 in our age‐associated toxicity screens that were the basis for J147 development (Prior et al., 2013). If so, modulating ATP synthase activity either by siRNA‐targeted knockdown of ATP5A or overexpression of its endogenous inhibitor, ATPase inhibitor factor 1 (IF1), should protect in models of amyloid proteotoxicity, glutamate‐induced glutathione depletion (oxytosis), and iodoacetic acid (IAA)‐induced energy depletion. First, we tested protection against amyloid proteotoxicity using human MC65 neuronal cells conditionally expressing the C99 fragment of amyloid precursor protein (APP) under the control of a tetracycline (tet) promoter. Upon induction, C99 is processed to produce Aβ polymers and this leads to cell death, an effect that is blocked by J147 (Chen et al., 2011). Similarly, ATP5A knockdown also prevented intracellular amyloid‐induced cell death in MC65 cells (Figure 3a). Figure 3 Open in figure viewer PowerPoint Knockdown of ATP5A phenocopies the neuroprotective effects of J147. (a) ATP5A knockdown efficiency in MC65 cells (left, Western blot). Both J147 and ATP5A knockdown protect MC65 cells from death in a proteotoxicity model of Aβ (right). (b) ATP5A knockdown protects HT22 cells from cell death in a model of IAA‐induced energy depletion (****p < .0001, t test). (c) ATP5A knockdown protects HT22 cells from cell death in a model of glutamate‐induced oxytosis (p = .0002, ****p < .0001, t test). (d, e) ATP5A knockdown does not provide an additive effect to J147‐induced protection during oxytosis and IAA toxicity. (f, g) Overexpression of IF1 is neuroprotective against glutamate (2 m m ) and IAA‐induced energy depletion (7.5 μ m ) in HT22 cells (*p = .0180, t test) Next, we tested the neuroprotective effects of ATP5A knockdown in toxicity models of oxytosis and energy depletion. Oxytosis occurs when high levels of extracellular glutamate block cystine import resulting in glutathione depletion and cell death (Tan, Schubert, & Maher, 2001), while IAA irreversibly inhibits the glycolytic enzyme glyceraldehyde 3‐phosphate dehydrogenase to induce energy loss (Maher, Salgado, Zivin, & Lapchak, 2007). We have previously shown that J147 protects HT22 cells from both oxytosis and IAA toxicity (Chen et al., 2011). Knockdown of ATP5A phenocopies the protection conferred by J147 in both assays (Figure 3b,c). As expected if the target of J147 is ATP5A, cell viability is not further improved by J147 treatment in ATP5A siRNA‐targeted knockdown cells (Figure 3a,d,e). To corroborate the neuroprotection induced by ATP5A knockdown, we overexpressed a constitutively active, pH‐insensitive mutant (H49K) of the endogenous inhibitor of ATP synthase, IF1 (Figure 3f) in HT22 cells. IF1 binds to the catalytic F1‐portion of ATP synthase and inhibits its activity (García‐bermúdez & Cuezva, 2016). Similar to J147 and ATP5A knockdown, IF1‐overexpression significantly protected HT22 cells from glutamate and IAA‐induced toxicity (Figure 3g). Together, these data demonstrate that modulating ATP synthase activity, whether by siRNA‐mediated knockdown of ATP5A or IF1 overexpression, phenocopies the neuroprotective effects of J147 in aging‐associated and AD‐like toxicities, and further support ATP5A as the molecular target for J147.

2.4 J147 and ATP5A modulate AMPK/mTOR signaling As age is the greatest risk factor for AD, interventions that slow aging or extend health span might serve as potential therapies that delay disease onset (Currais, 2015). Recent studies have highlighted a role for ATP synthase in the regulation of mTOR and lifespan extension in flies and worms (Chin et al., 2014; Sun et al., 2014). Inhibition of mTOR via activation of AMPK is a canonical longevity‐associated pathway (Johnson, Rabinovitch, & Kaeberlein, 2013). Activation of AMPK is achieved through phosphorylation of threonine (Thr) 172 on the α‐subunit, lowering activity of some ATP‐consuming pathways while promoting ATP synthesis through others such as fatty acid oxidation. Therefore, we asked whether J147 modulated AMPK/mTOR signaling via ATP synthase. We monitored AMPK/mTOR activity using site‐specific phosphorylation antibodies against proteins involved in this pathway. In Figure 4a, two different cell types were used to assay the activity of the AMPK/mTOR pathway following treatment with J147. In mouse HT22 (left panel) and human MC65 neuronal cells (right panel), there is a clear, time‐dependent activation of AMPK (pAMPK) by J147. AMPK phosphorylation of raptor at Ser792 is critical for AMPK‐mediated inhibition of mTOR. An increase in raptor Ser792 phosphorylation was observed in both cell types treated with J147 (Figure 4a). Raptor‐mediated inhibition of mTORC1 activity reduces unnecessary ATP expenditure by decreasing S6 kinase activity resulting in reduced protein translation. AMPK‐mediated phosphorylation of acetyl‐CoA carboxylase (ACC1) promotes energy production by reducing fatty acid synthesis and increasing β‐oxidation of fatty acids (Hardie, Ross, & Hawley, 2012). J147 decreases S6 activity and increases ACC1 phosphorylation in both cell types. These data show that the AMPK/mTOR signaling pathway, known to promote aging, is downstream of J147 target engagement. Importantly, siRNA‐mediated knockdown of ATP5A in MC65 cells (Figure 4b) phenocopied the effects of J147 on AMPK/mTOR signaling (Figure 4b‐f). Figure 4 Open in figure viewer PowerPoint J147 and ATP5A modulate AMPK/mTOR signaling. (a) Time course of 100 n m J147 activation of the AMPK/mTOR signaling pathway. Increasing phosphorylation of AMPK (α‐Thr172), raptor (Ser792), ACC1 (Ser79) and decreasing phosphorylation of S6 (Ser235/236) in HT22 and MC65 cells. Corresponding quantification graphs for AMPK/mTOR targets are below their respective Western blots. (b) ATP5A knockdown in MC65 cells phenocopies the J147 effect on the AMPK/mTOR pathway. Corresponding quantifications for each target are shown (c–e). Grey columns represent control siRNA, and all other colors represent ATP5A siRNA. (c) Increases in phosphorylation of AMPK (red) (**p = .0011 (phospho/total); (d) decrease in phosphorylation of S6 (blue) (Ser235/236) (**p = .0017 (phospho/total), ***p = .0002 (tot/GAPDH); (e) increase in phosphorylation of raptor (green) (Ser792) (**p = .0027 (phospho/total), **p = .0073 (tot/GAPDH); (f) increase in phosphorylation of ACC1 (black) (Ser79) (**p = .003 (phosphor/total), ****p < .0001 (tot/GAPDH) J147 caused an increase in AMPK phosphorylation despite modestly increasing ATP levels, suggesting an alternative mode of AMPK activation to that of sensing the AMP:ATP ratio. The only known alternative in the brain is calcium/calmodulin‐dependent protein kinase kinase β (CamKK2) activation of AMPK (Racioppi & Means, 2012), suggesting Ca2+‐mediated activation of AMPK by J147. Therefore, we asked whether J147 might regulate CamKK2 activity by modulating resting Ca2+ homeostasis. Measurement of cytosolic Ca2+ upon J147 treatment in HT22 cells revealed a dose‐dependent increase in cytosolic Ca2+ levels (Figure 5a). To determine whether CamKK2 mediates J147 activation of AMPK, we treated rat cortical neurons with J147 and a potent inhibitor of CamKK2, STO‐609. STO‐609 prevented J147‐mediated activation of AMPK and its downstream signaling effectors, ACC1, S6, and raptor (Figure 5b), thereby identifying CamKK2 as key mediator of the modulation of AMPK/mTOR signaling by J147 (statistics shown for 30 min and 4 hr time points in Figure S5). An AMPK knockout (KO) fibroblast cell line was used to further examine the role of AMPK in J147 signaling. We found a significant decrease in J147‐mediated protection by J147 in the oxytosis assay in the KO cells as compared to WT cells (Figure 5c). Figure 5 Open in figure viewer PowerPoint J147 modulates resting Ca2+ homeostasis to activate the AMPK/mTOR axis. (a) J147 increases the levels of cytosolic Ca2+ in HT22 cells. A23187 was used as a positive control (*p = .0162, ***p = .0002, ***p < .0001, ANOVA). (b) The CamKK2 inhibitor STO‐609 attenuated J147‐induced activation (100 n m ) of AMPK targets in rat primary cortical neurons. Phosphorylation targets include AMPK (α‐Thr172), Raptor (Ser792), ACC1 (Ser79), and S6 (Ser235/236); corresponding quantifications for each are shown below. (c) J147 protection against glutamate (2.5 m m ) is reduced in AMPK KO fibroblasts demonstrating a direct role for AMPK in the protective effects of J147 (****p < . 0001 (10 n m J147),****p < . 0001 (25 n m J147),***p < .0001 (100 n m J147))