Is blunt use among adolescents associated with progressing to current cigar use and more frequent cigar use compared with adolescents who have never used blunts?In this cohort study of 1825 US adolescents, ever vs never blunt use at age 14 years was significantly associated with progression to current cigar use across the following 24 months. Blunt use was not significantly associated with the number of days of cigar use across follow-up.These findings highlight the risk that blunt use may pose for subsequent cigar use among adolescents. This cohort study examines the association of blunt use with cigar use among US adolescents. Almost two-thirds of adolescents who smoke blunts also report using cigars. Although adolescent co-use of blunts and cigars is prevalent, whether adolescent blunt use is associated with progression to current cigar use (past 30-day use) and increased frequency of use is unknown.To determine whether adolescent use of blunts is associated with progression to cigar use (past 30-day use) and increased number of days used (in the past 30 days) across the subsequent 24 months compared with adolescents who have never used blunts.In this prospective cohort study of adolescents from 4 public high schools outside of Philadelphia, Pennsylvania, in-classroom surveys were administered at baseline  and at 6-month intervals for the following 24 months . Data analyses were conducted in September and October 2019.Self-report of lifetime (ever) blunt use at baseline (age 14 years).Progression to current cigar use (use in the past 30 days) and escalation in the frequency of use (number of days used in the past 30 days) across the subsequent 24 months.Of 1825 participants, 907 (49.7%) were female, 1330 (72.9%) were white, and 376 (20.6%) were Hispanic; the mean (SD) age at baseline was 14.38 (0.55) years. In all, 257 participants (14.0%) reported ever blunt use. Mixed-effects models revealed that ever blunt use at baseline vs never blunt use was associated with progression to current cigar use  but not escalation in the number of days used across the following 24 months .These findings highlight the risk that blunt use may pose for subsequent cigar use among adolescents. Policies and public health campaigns addressing marijuana as well as cigars will be important to reduce adolescent blunt use and cigar use. This prospective cohort survey of adolescents in high school sought to determine whether adolescents who ever used blunts, compared with those who did not, were more likely to become current (past 30-day) cigar users and to progress to more frequent use across the following 24 months. Evidence documenting that the use of blunts is associated with cigar use would help guide marijuana- and cigar-related policies to protect adolescents.Cross-sectional research has documented the co-use of blunts and cigars among adolescentsParticipants were adolescents in the ninth grade taking part in a longitudinal cohort study of the associations among combustible cigarette smoking, electronic cigarette use, and other tobacco use. Participants were enrolled in 1 of 4 public high schools in suburban Philadelphia, Pennsylvania. The schools were selected such that our sample would be demographically representative of adolescents nationwide in terms of sex, race, ethnicity, and annual household income. The cohort participants were drawn from 2198 students identified through class rosters at the start of ninth grade. Adolescents were ineligible to participate if they had a severe learning disability or if they did not speak fluent English. Based on the selection criteria, a total of 2017 of the 2198 students (92%) were eligible to participate.Parents were mailed a study information letter (active information) with a telephone number to call to obtain answers to any questions and to decline consent for their adolescent to participate (passive consent). Of the 2017 eligible adolescents, 17 (1%) had a parent who actively declined their adolescent’s participation. Adolescents with parental consent were approached to provide their written assent for study participation. The 124 adolescents (6%) who were absent on the assent/baseline survey days and the 41 adolescents (2%) who did not provide assent owing to lack of interest were not enrolled in the cohort. Thus, 1835 of the 2000 adolescents with consent (92%) provided their assent to participate and completed a 40-minute paper-and-pencil survey. This baseline, or wave 1, survey was completed on site during compulsory classes in the fall of 2016.STROBE) reporting guideline. Data analyses were conducted in September and October 2019.Adolescents completed 4 paper-and-pencil follow-up surveys at 6-month intervals, with 1687 (92%) completing a survey at wave 2 (spring 2017), 1658 (90%) completing a survey at wave 3 , 1643 (89%) completing a survey at wave 4 (spring 2018), and 1601 (87%) completing a survey at wave 5 . Adolescents were assigned a unique identifier at the baseline survey. A cover page with the adolescent’s name connected to their identifier on the follow-up survey was removed at the time of survey receipt. The participants included in this study are 1825 adolescents who had complete data on the study variables at baseline. Missingness at subsequent waves was largely due to loss to follow-up . The only variable associated with loss to follow-up was non-Hispanic ethnicity . The institutional review board of the University of Pennsylvania and the administration of each of the 4 high schools approved the study. Reporting of the results follows the Strengthening the Reporting of Observational Studies in Epidemiology  at baseline was defined as an increase in the number of days in the past 30 days in which a cigar was used compared with baseline. Cigar use was measured at all 5 waves.In a series of frequently used survey questions, adolescents were asked whether they had ever used large cigars, little cigars, or cigarillos and whether they had used one of these types of cigars in the past 30 days.17 To aid accurate reporting, blunts were defined as marijuana rolled in cigar or tobacco wraps.5 We included sensation seeking and combustible cigarette smoking in the model to control for its potentially overlapping risk for cigar and blunt use.21 Sensation seeking was measured with the 8-item Brief Sensation Seeking Scale .22 Lifetime combustible cigarette smoking was assessed by asking adolescents whether they had ever tried smoking a cigarette (even a few puffs).16Demographic characteristics such as sex, race, and ethnicity were assessed using self-report items. These demographic variables were included in the model to characterize the sample. Similar to the assessment of cigar use, adolescents were asked whether they had ever used a blunt, whether they had used a blunt in the past 30 days, and on how many days in the past 30 days blunts were used.Mixed-effects models were used to evaluate the association between ever blunt use at baseline and (1) progression to current (past 30-day) cigar use and (2) escalation in the number of days cigars were used in the past 30 days across the subsequent 24 months. A logistic regression model was used to assess the association of ever blunt use at baseline with repeated measures of past 30-day cigar use (yes vs no) across waves 2 through 5. A binary mixed-effects model with a logit link and random intercept was fit to the data.General linear mixed modeling was used to assess escalation in the number of days of cigar use in the past 30 days among adolescents who reported cigar use in the past 30 days. For this model, we fit a log-normal mixed-effects model including a random intercept. Unadjusted models were followed by models including the covariates. Cigar use was included in the models at wave 1 to control for any previous use and to assess the prospective association between blunt use and the progression to current (past 30-day) cigar use and escalation in the number of days cigars were used in the past 30 days across waves 2 through 5.P < .05. Both models were run using the GLIMMIX procedure in SAS software version 9.4 (SAS Institute). Supplementary analyses were conducted to examine an alternative association between blunt use and cigar use, that is, whether cigar use was associated with the uptake of blunt use among adolescents.Means and standard deviations were used to describe continuous variables. Frequency distributions and proportions were used to describe categorical variables. Statistical significance was set at 2-tailed Of 1825 participants, 907 (49.7%) were female, 1330 (72.9%) were white, and 376 (20.6%) were Hispanic; the mean (SD) age at baseline (wave 1) was 14.38 (0.55) years. The sample characteristics are presented in Ever blunt use was not associated with an escalation in the frequency of cigar use (number of days that cigars were used in the past 30 days) across the following 24 months  . Only se24 Current blunt use was defined as using blunts on at least 1 day in the past 30 days.17 Escalation in the frequency of blunt use among those adolescents who reported current use (past 30 days) at baseline was defined as an increase in the number of days in the past 30 days in which a blunt was used. Blunt use was included in the models at wave 1 to control for any previous use and to assess the prospective association between cigar use and the progression to current (past 30-day) blunt use as well as escalation in the number of days blunts were used in the past 30 days across waves 2 to 5.In a similar analytic fashion, we evaluated the association between ever cigar use at baseline, progression to current blunt use, and escalation in the number of days blunts were used in the past 30 days across the subsequent 24 months.As shown in 16 the present findings highlight the risk that marijuana use via blunts poses for cigar uptake.The current study offers new evidence for a prospective association between adolescent blunt use and the use of cigars. Adolescents who had ever used a blunt at age 14 years had more than a 20-fold increase in the odds of becoming a current cigar user across the 24-month follow-up compared with adolescents who had never used a blunt. Whereas previous research has documented an increased vulnerability to marijuana use associated with newer forms of tobacco  among adolescents,25 observed that approximately half of young people who reported having smoked a blunt reported no prior cigar use, while the other half reported that cigar use preceded the initiation of blunt use.Supplementary analyses revealed that adolescents who had ever used a cigar at age 14 years had a 14-fold increase in the odds of progressing to 30-day blunt use, and a 38% increase in the frequency of blunt use across the subsequent 24 months. Coupled with our findings that blunt use at age 14 years was associated with cigar uptake, we can speculate that youth who initiate blunt use may subsequently begin using cigars as intended and that cigar users may subsequently begin to modify their cigars for blunt use. Indeed, using data from the US National Surveys on Drug Use and Health, a recent study26 Adolescents perceive blunt use as more socially acceptable, less harmful to their health, and less addictive than cigarettes.27 Lower risk perceptions associated with marijuana use are accompanied by more perceived benefits.28 Nationwide, the percentage of adolescents who perceive that marijuana use is associated with significant risks has decreased from 30.9% to 21.4% among 10th graders from 2010 to 2018.29 These decreases may reflect the evolving regulatory landscape—as of 2018, 33 states and the District of Columbia had legalized marijuana in some form.30 Educating adolescents about the health consequences associated with blunt smoking, cigar smoking, and the risk for becoming a regular cigar and blunt user seems warranted.Blunt use and cigar use among adolescents are likely fostered through misperceptions regarding the health risks associated with use. Adolescents perceive cigars as less harmful than cigarettes, despite similar health consequences.31 In addition, cigarillos are often sold in a foil pouch, which is popular for storing marijuana.31 Whereas these marketing practices are directed toward those who use cigars for blunt making, they may ultimately benefit cigar manufacturers as adolescents become nicotine dependent and begin using cigars as intended, not just for blunting.Furthermore, some cigars, especially cigarillos, are specifically marketed to promote their use in blunt making. These include features that facilitate blunt making, such as perforations in the tobacco wrap to increase the ease with which the tobacco can be removed and marijuana can be added.33 it is not surprising that coadministering nicotine and cannabis through blunts has been implicated in persistent use of both substances.34 Flavored little cigar and cigarillo use has been tied to blunt smoking,35 as young adults report that flavoring enhances the taste of the marijuana.36 As such, flavoring may further add to the abuse liability and likelihood of persistent use. In November 2018, the US Food and Drug Administration Center for Tobacco Products announced plans to restrict the sale of flavored cigars that were on the market on August 8, 2016. Although this constituted a positive step to reducing exposure to some flavored cigars, those on the market prior to February 15, 2007, are to be “grandfathered” by the Food and Drug Administration and will still be available for purchase. Consequently, this regulatory policy, along with the spread of marijuana legalization in the United States, may inadvertently contribute to continued cigar use and cigar-blunt use among youth.Given that nicotine prolongs and heightens the reinforcing effects of marijuana,As the first study, to our knowledge, to examine the longitudinal association between adolescent blunt use and adolescent cigar use, this study has strengths as well as limitations. The sample was demographically diverse and adolescents were measured during a developmentally vulnerable period for tobacco use. We also used repeated measures of cigar use and blunt use, analyzing the data in a longitudinal fashion across 5 points spanning 2 years. In addition, we controlled for several variables that could account for an association between adolescent cigar use and blunt use. Even after controlling for these variables, the model showed an association between adolescent use of cigars and blunts. However, it is important to note that the present study did not find an association between blunt use and escalation in the frequency of cigar use. Progression to significantly more frequent use may unfold across a longer period of time.A potential limitation of this study is that we did not measure how blunts were made. While blunt use and cigar use were assessed as 2 separate behaviors, it is unclear whether blunts were made with little cigars, large cigars, cigarillos, or tobacco leaf wraps. Relatedly, we did not measure preference for flavored cigars and its association with blunt use. To offer increasing clarity into the association between adolescent blunt use and subsequent cigar use, future research will need to delineate specific features of blunt making and cigar use among adolescents. In addition, given that our overall marijuana use item assessed use from all sources, including blunts, we were not able to include it in the model and control for other sources of marijuana use.37 Policies and public health campaigns addressing marijuana as well as cigars will be important to reduce adolescent blunt use and cigar use.The findings of this cohort study highlight the risk that blunt use may pose for subsequent cigar use among adolescents. In addition, the findings point to the risk that cigar use may pose for subsequent and escalating blunt use among adolescents. This is concerning, as co-use of tobacco and marijuana through blunting may present an additive risk for toxicant and carcinogen exposure.


Because old age is the greatest risk factor for dementia, a successful therapy will require an understanding of the physiological changes that occur in the brain with aging. Here, two structurally distinct Alzheimer's disease (AD) drug candidates, CMS121 and J147, were used to identify a unique molecular pathway that is shared between the aging brain and AD. CMS121 and J147 reduced cognitive decline as well as metabolic and transcriptional markers of aging in the brain when administered to rapidly aging SAMP8 mice. Both compounds preserved mitochondrial homeostasis by regulating acetyl-coenzyme A (acetyl-CoA) metabolism. CMS121 and J147 increased the levels of acetyl-CoA in cell culture and mice via the inhibition of acetyl-CoA carboxylase 1 (ACC1), resulting in neuroprotection and increased acetylation of histone H3K9 in SAMP8 mice, a site linked to memory enhancement. These data show that targeting specific metabolic aspects of the aging brain could result in treatments for dementia. Age is the greatest risk factor for Alzheimer's disease (AD) and related dementias. While genetic risk factors have been the major focus of AD drug discovery, it is important to consider the progressively detrimental metabolic processes that take place with normal aging as possible therapeutic targets. Clinical evidence shows that a decline in cerebral glucose metabolism (hypometabolism) precedes the pathology and symptoms of AD and is more severe than that observed in normal aging . Given tMost of the energy derived from glucose oxidation is produced in mitochondria. Not surprisingly, a number of mitochondrial-dependent functions are found to be impaired during aging and AD . In factC. elegans , at least in part, by the inhibition of acetyl-CoA carboxylase 1 (ACC1). Importantly, the compounds increase histone acetylation in cultured neurons and SAMP8 mice at a site on histone H3 that is required for memory formation . TogetheTo identify age-dependent changes in brain metabolism that are causally associated with dementia, we tested CMS121 and J147 in SAMP8 mice. The SAMP8 mice are a model of accelerated aging that develop a progressive, age-associated decline in brain function as well as a number of brain pathologies similar to human dementia and AD patients . Some ofWe asked whether J147 and CMS121 could prevent the development of the aging phenotype in SAMP8 mice when administered in the last quadrant of their lifespan, reflecting the therapeutic context for human AD patients. Treatment with the diets containing the compounds started at 9 months of age and continued for four months (13 months of age); the median lifespan of these mice is 15 months.We began our analyses with an unbiased approach by studying the entire transcriptome of brain hippocampal tissue. Principle component analysis (PCA) showed a clear distinction in the variation of the transcriptome between the 9 and 13 months old SAMP8 groups, indicating that aging has a strong effect on gene expression . ImportaTo support the PCA and drift analysis, we examined the effects of the compounds on the expression of individual genes. 485 differently expressed (DE) genes were found between the 9 and 13 months old SAMP8 mice . 684 andWe next determined if the changes in gene transcription in old SAMP8 mice and the effects by CMS121 and J147 were associated with functional alterations in cognition.Assessment of spontaneous behavior in the open field assay showed only a decline in average velocity between the 9 and 13 months SAMP8 mice . The comThe elevated plus maze examines disinhibition behavior based on the aversion of normal mice to open spaces. Dementia is clinically associated with disinhibition and AD mouse models tend to exhibit increased disinhibition . 13 montThe Barnes maze is used to analyze spatial learning and hippocampal-dependent memory. In this assay, mice use visual cues to locate a hidden box. No changes between the groups were found in the escape latencies during the learning and the retention phases (data not shown). However, when tested during the reversal phase, which is more sensitive to deficits in memory and learning, CMS121 significantly improved the learning of the new location . J147 haWe next asked whether there were specific cellular processes that were targeted by the compounds that could explain their beneficial effects. To address this, we carried out pathway analysis with the 485 DE genes that were altered with aging  and withCMS121 affected a number of different pathways , some ofA similar conclusion was reached when we used an alternative approach by clustering the whole transcriptome according to gene expression patterns. We found one large cluster (cluster 2) that showed increased changes in gene expression with aging that were prevented by the compounds , and whoThe analysis of all genes that encode proteins associated with the mitochondria shows both up- and down-regulated changes in expression during aging . In ordeWe next tested the hypothesis that the coordinated responses at the gene expression level with aging are associated with dysregulation at the metabolic level. To do this, we carried out large-scale, untargeted metabolic profiling of brain cortical tissue from 9 months, 13 months and 13 months+compound-treated SAMP8 mice. This study not only assessed the levels of metabolites that directly participate in brain mitochondrial metabolic pathways, such as the tricarboxylic acid (TCA) cycle and fatty acid synthesis/oxidation, but also metabolites that are involved in many other pathways. A total of 496 small molecules were measured. 205 metabolites were significantly altered between 9 and 13 months in the SAMP8 mice and the alterations in some of these metabolites were prevented by CMS121 and J147 . Global We also measured the levels of metabolites in the plasma of the same animals. Both compounds showed a partial suppression of the changes that took place with aging . AlthougA detailed investigation of individual metabolites measured in the brain that participate in the TCA cycle revealed profound changes with aging that were partially prevented by both CMS121 and J147 . In factPathway enrichment analysis with all of the brain metabolites that were significantly altered with aging confirmed that the TCA cycle was among the top pathways preferentially affected . In addiWe then carried out pathway enrichment analysis with the brain metabolites that were altered by the compounds in relation to the 13 months old mice and found that both compounds shared a common top pathway related to the metabolism of acyl carnitines . The levAlthough no pathway analysis with the metabolic data from the plasma was pursued due to the heterogeneous nature of plasma metabolites derived from diverse tissues and organs, the data demonstrate that the compounds also had a systemic effect on some parameters of aging.Specifically, these data show that the maintenance of mitochondrial gene expression in the brain by CMS121 and J147 is associated with a preservation of key mitochondrial metabolic intermediates. Moreover, the build-up in acyl carnitines with both compounds is suggestive of a mitochondrial target underlying the action of the compounds.To further delineate the overall changes in metabolism shared by both compounds, network analysis was carried out by GAM integrating both the transcriptomic and the metabolomic data. Analysis of the networks generated for each compound showed that acetyl-CoA is a central metabolite shared by CMS121 and J147 . To ruleTo provide further support for the central role of acetyl-CoA in the effects of the compounds, network analysis was performed on transcriptomic data from cultured, differentiated rat cortical neurons treated with CMS121 and J147. This analysis also identified acetyl-CoA as a central metabolite regulated by both compounds .Acetyl-CoA is used by histone acetyltransferases to acetylate histones and thereby regulate memory by restructuring chromatin in the brain . In primc-, leading to depletion of intracellular glutathione (GSH), production of ROS and cell death −2-furoic acid (TOFA) or ACC1 expression was reduced with a specific siRNA. Both of these approaches significantly increased the levels of acetyl-CoA in the HT22 cells . If acetFinally, similar to what we found previously with J147 , the inhTogether, these data show that the preservation of brain mitochondrial gene expression and metabolism in the aging brain by both CMS121 and J147 is mediated by a shared mechanism involving the inhibition of ACC1 and increased levels of the central mitochondrial metabolite acetyl-CoA.Despite the observation that old age is the greatest risk factor for AD, few clinical approaches have focused on treating AD and other dementias by targeting detrimental age-related processes that are relevant to disease onset and development. In part, this is because we still lack a deep understanding of the aging brain. As such, our laboratory has developed a novel drug discovery program based upon cell-based screening assays that mimic multiple aspects of old age-associated neurodegeneration and AD pathology  and has CMS121 and J147 were able to reduce age-related cognitive dysfunction, even when administered to SAMP8 mice at a late stage of their lives. In addition, CMS121 and J147 share a common mechanism of action that is associated with the maintenance of mitochondrial health in terms of transcriptional stability and metabolism. Mitochondrial dysfunction is one of the hallmarks of aging , and an Consistent with these observations, CMS121 and J147 reduced several molecular parameters of brain aging. PCA and drift analysis of the transcriptome showed that the two compounds significantly prevented changes in gene expression associated with aging. The level of suppression by CMS121 was particularly remarkable as evidenced in the correlation plots. Although not as strong, J147 also significantly prevented transcriptomic aging, which is in accordance with what we found in the SAMP8 mice treated from a young age (3 months old) with this drug candidate .The drift suppression was not as strong at the level of the brain metabolites as it was with gene expression. Instead, numerous metabolites that did not change with age were directly altered by the compounds. The effects of CMS121 and J147 on certain metabolic pathways and individual metabolites were very specific, namely the increase in acyl carnitines. Since acyl carnitines are direct substrates for fatty acid oxidation in mitochondria and a source of acetyl-CoA, they further support the conclusion that the compounds have a direct effect on mitochondrial metabolism in the brain. CMS121 and J147 also suppressed the metabolic drift in the plasma, indicating that the compounds may prevent the aging process at the systemic level as well.Pathway analysis of the transcriptome identified genes that code for proteins that are part of the ETC and ATP synthase as being preferentially maintained at the younger level by the compounds. Mitochondria-related genes were also the top pathways affected by aging, indicating that mitochondrial dysfunction is part of the SAMP8 aging process. Because a decrease in mitochondrial DNA copy number was found with aging, the increased expression of gene transcripts associated with the ETC with aging is likely a compensatory response to a lack of adequate mitochondrial metabolism. The conclusion is supported by the observation that deficits in the respiratory chain and mitochondrial bioenergetics increase as a function of age , and by Acetyl-CoA is a key metabolite that bridges glycolysis, fatty acid β-oxidation, fatty acid synthesis and the TCA cycle in the mitochondria. Its metabolism in the TCA cycle leads to the production of metabolites that are used in other metabolic pathways essential for the proper functioning of the cell, including the neurotransmitter acetylcholine which is reduced in forebrain neurons at early stages of the disease . Acetyl-Although acetyl-CoA can be derived from multiple sources, our data argue that the elevation of acetyl-CoA by the compounds is due to the inhibition of ACC1 via its phosphorylation by AMPK. This conclusion is based on the following observations: 1) both compounds cause ACC1 phosphorylation in vivo, primary neurons, HT22 cells and fibroblasts, and increase acetyl-CoA levels in all four models; 2) knockdown of ACC1 increases acetyl-CoA levels and provides neuroprotection; 3) chemical inhibition of ACC1 also increases acetyl-CoA levels and provides neuroprotection; and 4) absence of AMPK prevents the inhibition of ACC1, increases in acetyl-CoA levels and reduces the protection against oxytosis.One of the few clinical approaches targeting mitochondrial metabolism that has been tested in the context of AD is the use of ketogenic diets. In animals, ketone bodies are mainly produced in the liver from fatty acid β-oxidation through acetyl-CoA and are then transported via blood to other tissues, such as the brain, where they are once again metabolized to generate acetyl-CoA. Administration of a variety of ketogenic diets has been shown to have some positive effects in both AD transgenic mice as well as human AD patients , and twenty-two and twenty-two 9 months old female SAMP8 mice were fed with CMS121 and J147 diets, respectively . Diet treatment lasted for four months until mice reached 13 months of age. At 9 months of age, SAMP8 mice already present a strong phenotype . The dosThe effect of the compounds was assessed in older SAMP8 mice after the four months of treatment and any age-related changes are defined by comparison to 9 months old SAMP8 mice. All mice were randomly assigned to experimental groups. The number of mice per group was determined based on previous experiments  and was The SAMP8 line, a naturally occurring mouse line that was developed based on its phenotype of accelerated aging, was acquired from Harlan Laboratories (U.K.). Mouse body weights were measured regularly and no significant differences were found between the groups . All expMice were anesthetized and their blood collected by cardiac puncture. After perfusing with PBS, their brains were removed and dissected to collect cortex and hippocampus. Tissue was prepared for Western blotting, RNA extraction and metabolomic analysis.Metabolite measurement and analysis were conducted at Metabolon as previously described .Acetyl-CoA levels were determined in protein-free lysates of primary neurons or HT22 cells treated as indicated in the figure legends using a kit from Sigma (MAK039) according to the manufacturer's instructions. The levels were normalized to the protein in the solubilized pellet using the BCA assay.Mouse hippocampal HT22 cells, generated in our laboratory, were propagated as previously described . AMPK knPrimary cortical neurons were prepared from day 17 rat embryos and used at 7 days in vitro (7 DIV) .Samples from line 85 APPswe/PSEN1dE9 transgenic AD mouse mice and respective wild type (Wt) mice treated with J147 in a reversal experimental paradigm were obtained from our previous study . In anotElevated plus maze - The maze consisted of four arms  30 cm long and 5 cm wide in the shape of a plus. A video-tracking system (Noldus EthoVision) was used to automatically collect behavioral data. The software was installed on a PC computer with a digital video camera mounted overhead on the ceiling, which automatically detected and recorded when mice entered the open or closed arms of the maze and the time spent in each. Mice were habituated to the room 24 hr before testing and habituated to the maze for 1 min before testing by placing them in the center of the maze and blocking entry to the arms. Mice were then tested for a 5 min period and their behavior recorded. Disinhibition was measured by comparing the time spent on the open arms to time spent on the closed arms.Barnes maze - The maze consisted of a flat circular surface (36’ diameter) with 20 equally spaced holes (2’ diameter) along the outer edge. One of the holes led to a dark hide box while the other 19 led to false boxes that were too small to be entered. The latency to enter the hide box was recorded. The test was conducted in three phases. Phase 1 (Training): A hide box was placed under one of the holes. Animals were placed into an opaque cylinder in the center of the maze for 30 s to promote spatial disorientation at the start of the test. After 30 s, the cylinder was removed and the animal explored the maze until it found and entered the hide box. The number of incorrect entries was scored. If the mouse failed to enter the box within 3 min, it was gently led into the box. The animal remained in the box for an additional 20 s before it was removed from the boxed and gently placed into the home cage. Training is repeated three times a day for four days. The location of the hide box remained the same during every trial but it was shifted between subjects to reduce the potential for unintended intra-maze cues. Phase 2 (Retention): This phase measures retention of spatial memory following a delay. After a two day break from training, each animal was re-tested for a one day, three-trial session using the same hide box location as before. Phase 3 : This phase examines memory reversal. On the day following the retention phase, a new hide box location was established 180 degrees to the original location. The same method as before was used and trials were repeated three times a day over two consecutive days.Western blots were carried out as described previously . The prihttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE101112).RNA was isolated from the hippocampus of SAMP8 mice or primary neurons using the RNeasy Plus Universal mini kit (Qiagen). RNA-Seq libraries were prepared using the Illumina TruSeq Stranded mRNA Sample Prep Kit according to the manufacturers instructions. Briefly, poly-A RNA was selected using poly dT-beads. mRNA was then fragmented and reverse transcribed. cDNA was end-repaired, adenylated and ligated with Illumina adapters with indexes. Adapter-ligated cDNA was then amplified. Libraries were pooled and sequenced single-end 50 base-pair (bp) on the Illumina HiSeq 2500 platform. Sequencing reads were mapped to the mm10 mouse genome or rn6 rat genome using the spliced aligner STAR (2.5.1b) with default parameters . ReferenTranscriptional drift analysis was performed as previously described  with theMitochondrial DNA copy number was determined in the hippocampus of SAMP8 mice using the kit from Detroit R and D, Inc (MCN 3) according to the manufacturer's instructions.R package ‘gplots’  was usedPCA of the transcriptome data was performed on the log2(FPKM+5) values of the top 10% most expressed genes (cutoff: RowSums(log2(FPKM+5))>=110.41; 2458 genes selected) by the R base function ‘prcomp’ with data mean centered. Metabolome data was imputed to replace the missing value by the minimal value observed across all samples and transformed by the function ‘glog’ from the R package ‘FitAR’  before PCA analysis. The ellipses showed the 70% confidence interval of a multivariate normal-distribution of sample groups estimated by PC1 and PC2 data.Pathway enrichment test of the transcriptome data was analyzed by DAVID Bioinformatics Resources v6.8 . The totPathway enrichment analysis on metabolic pathways was conducted using the MetaboLync platform (Metabolon). Enrichment scores = /.The metabolic network was reconstructed by GAM  with inpRandom Sampling: The dataset that contained the test statistics  between 13 months+CMS121 and 13 months was used for the random sampling experiment. The Gene IDs and the Compound IDs were randomly shuffled 10 times (sampling without replacement) followed by the network analysis using the same parameters and cutoffs. The probability of observing acetyl-CoA in the resulted network was calculated as pnull. Random sampling p=pnull^na*(1-pnull)^nb . In this experiment, pnull = 0.1 and random sampling p=0.01. For the rat primary neuron transcriptome data, random sampling was performed using the same method with data generated from the comparison between CMS121 and control.P values are indicated (for p<0.050).GraphPad Prism six was used for statistical analysis and exact  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:In this paper the authors characterized two neuroprotective compounds, CMS121 and J147. Although these compounds target different upstream pathways, they are capable of preventing age-associated cognitive decline and metabolic changes in the mouse model. Using transcriptomics and metabolomics, the authors identified that the target pathways converge on the TCA cycle, and acetyl-CoA specifically, demonstrating its causative role in neuroprotection. The study not only enhances the translational value of the two compounds but also provides deeper understanding of processes that are effectively modulated in the course of drug administration. This study is also relevant for the broad audience, because it brings an important step in understanding of age-related pathologies, and provides a new concept to develop prevention strategies and cures.Decision letter after peer review:eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Huda Zoghbi as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Lee-way Jin (Reviewer #1); Martin Denzel (Reviewer #2).Thank you for submitting your article "Elevating acetyl-CoA levels reduces aspects of brain aging" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The reviewers find the data valuable and interesting. However, they raised several points that must be addressed in the revised version. As you will see from the reviewers detailed comments appended below, the work requires a number of substantial revisions addressing the concerns. The number of them are to be addressed by additional justifications, explanations or toning down the interpretations. Specifically, the reviewers require to explain and justify an inconsistent experimental design, including the thorough analyses of statistical significance. The reviewers also agree that the manuscript should be experimentally enriched addressing the role of AMPK in mediating these effects. These additional experiments should be performed as thoroughly as possible given the time frame for revisions, i.e. focusing on most important effects and using cultured rat cortical neurons.Essential revisions:The data are complex; the readers will benefit from a diagram illustrating key pathways affected by senescence and by CMS121 and J147. Also, the authors state that "the coordinated responses at the gene expression level with aging represent a compensatory mechanism that is linked to dysregulation at the metabolic level". However, the authors only present correlational data and no evidence for this compensatory mechanism.The description/explanation of the numbers and sexes of mice used is confusing:1) The authors used only female SAMP8 mice because "women are at a higher risk than men of developing AD". However, this is not exactly a study on AD. On the other hand, when it came to AD mice, only male APPswe/PSEN1dE9 mice were used.2) "Eleven 9 months old male SAMP8 mice were used as the baseline control group." It is not clear how male "baseline" mice were used (in which experiments) and how their values were compared to female baseline.3) The Materials and methods indicates 4 mice per treatment group; however, it is indicated in Figure 1, n=5-6/group, and in Figure 2, n=11-18/group. Please clarify. A group number of 4 for behavioral tests is not likely feasible.4) Subsection “Increases in acetyl-CoA levels are neuroprotective and are associated with inhibition of ACC1 by CMS121 and J147”: there is no Figure 1I, 1J and 1K.5) Materials and methods: "Because the SAMP8 mice are an inbred strain, the effect of the compounds was only assessed in older SAMP8 mice after the four months of treatment." Please explain.6) Subsection “CMS121 and J147 enhance acetyl-CoA levels and histone acetylation in neurons”: "acetylation of H3K9 (in APPswe/PSEN1dE9 mice) was increased by the compounds". However, the data and figure legend are confusing. First, comparing Figure 6—figure supplement 1 (D and E) and (F and G) – the Ac-H3K9 level of Tg-AD animals in the latter is much smaller than the former (comparing the green bars), even they appears to be from similar animals. Second, in the legend to Figure 6—figure supplement 1, it is described that tissue in D and E was from 18 months and tissue in F and G was from 23 months. Why was that?The transcriptome was done in the hippocampus while the metabolome was analysed in the cortex. This and how it affects the analysis of the data need to be explained.The SAMP8 model needs to be interpreted more carefully regarding normal WT aging. In this context, it would be helpful to compare SAMP8 aging data with published data from WT mice during aging. This would solidify the model. Further, I wonder to what extent the changes in SAMP8 are representative for changes in AD?The Western blots in Figure 6 show only minor changes. For example, the blot in Figure 6—figure supplement 1 A does not support the change documented in Figure 6C regarding Ac-H3K9. In Figure 6H, pACC1 levels go up with age, while compounds change the signal in the same direction. Thus, these data need to be interpreted more carefully.The authors have previously identified ATP synthase as a target of J147, with downstream AMPK activation. Is this effect on ATP synthase responsible for the observed effects on the ETC, ACC1 and acetylation? This would be a plausible explanation but is only mentioned in the Introduction. It is therefore essential to experimentally test the role of AMPK in this context. Are the changes caused by the compounds regarding TCA metabolite levels, ACC1 phosphorylation, H3K9 acetylation, and neuroprotection caused by increased AMPK activity?A color code label is missing for Figure 3 G and H.The authors state in Figure 3A and B that the majority of the changed genes are the same. It would be great to back this up by showing the overlap. What proportion of these genes are the upregulated fraction?The text does not properly explain the neurotoxicity assay in Figure 6E.Generally, the text should follow the order of the figures. In the manuscript, the reader has to jump between sections, making it harder to read. The results in Figures 4, 5, and 6, for example, are presented in a way that is a bit confusing. It would be better to arrange the figures to the order of the text or change the text so that it is not necessary to jump between the figures.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Huda Zoghbi as the Senior Editor.Thank you for submitting your article "Elevating acetyl-CoA levels reduces aspects of brain aging" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The reviewers were largely satisfied with the revisions they have requested. However, there is one remaining issue that needs to be addressed experimentally. The authors show that the compounds appear to act through ACC1 inhibition. Inhibition of ACC1 has two consequences: elevated acetyl-CoA and the reduced flux in downstream metabolism. Which of the two effects of ACC1 inhibition is responsible for the observed phenotype should be clarified by experimentally elevating Acetyl-CoA levels by acetate supplementation. Essential revisions:The data are complex; the readers will benefit from a diagram illustrating key pathways affected by senescence and by CMS121 and J147.A diagram summarizing the findings has been added as Figure 7.Also, the authors state that "the coordinated responses at the gene expression level with aging represent a compensatory mechanism that is linked to dysregulation at the metabolic level". However, the authors only present correlational data and no evidence for this compensatory mechanism.The sentence has been modified to "We next tested the hypothesis that the coordinated responses at the gene expression level with aging are associated with dysregulation at the metabolic level".The description/explanation of the numbers and sexes of mice used is confusing:1) The authors used only female SAMP8 mice because "women are at a higher risk than men of developing AD". However, this is not exactly a study on AD. On the other hand, when it came to AD mice, only male APPswe/PSEN1dE9 mice were used.In order to keep the study affordable and viable, we focused on one gender only. In the past, we have used male mice from the APPswe/PSEN1dE9 strain. Recently, as we started working with the SAMP8 strain, we chose female mice, given that women are at a higher risk than men of developing AD. In order to avoid confusion, we have removed the sentence above from the manuscript.2) "Eleven 9 months old male SAMP8 mice were used as the baseline control group." It is not clear how male "baseline" mice were used (in which experiments) and how their values were compared to female baseline.This was a typo and has been corrected to "female SAMP8 mice".3) The Materials and methods indicates 4 mice per treatment group; however, it is indicated in Figure 1, n=5-6/group, and in Figure 2, n=11-18/group. Please clarify. A group number of 4 for behavioral tests is not likely feasible.The number of mice in this sentence reflects the mice that died during the course of our study"Six SAMP8 mice fed with control diet, four SAMP8 mice fed with CMS121 diet and four SAMP8 mice fed with J147 diet died throughout the course of this study". The numbers stated in the figure legends are correct.4) Subsection “Increases in acetyl-CoA levels are neuroprotective and are associated with inhibition of ACC1 by CMS121 and J147”: there is no Figure 1I, 1J and 1K.This was a typo and has been amended. It should read Figure 6I, 6J and 6K.5) Materials and methods: "Because the SAMP8 mice are an inbred strain, the effect of the compounds was only assessed in older SAMP8 mice after the four months of treatment." Please explain.Because we are assessing the effects of our compounds on the aging phenotype, the control group was comprised of 9 months old SAMP8 mice, prior to compound treatment.This sentence has been clarified and now reads "The effect of the compounds was assessed in older SAMP8 mice after the four months of treatment and any age-related changes are defined by comparison to 9 months old SAMP8 mice.".6) Subsection “CMS121 and J147 enhance acetyl-CoA levels and histone acetylation in neurons”: "acetylation of H3K9 (in APPswe/PSEN1dE9 mice) was increased by the compounds". However, the data and figure legend are confusing. First, comparing Figure 6—figure supplement 1 (D and E) and (F and G) – the Ac-H3K9 level of Tg-AD animals in the latter is much smaller than the former (comparing the green bars), even they appears to be from similar animals. Second, in the legend to Figure 6—figure supplement 1, it is described that tissue in D and E was from 18 months and tissue in F and G was from 23 months. Why was that?These tissues were generated as part of two separate studies with Tg-AD mice that we had carried out previously. The tissues were already available in our laboratory. Therefore, the differences observed in H3K9 acetylation between mice from the two independent studies might be explained by the age difference. This has been clarified in the Materials and methods and legend of Figure 6—figure supplement 1.The transcriptome was done in the hippocampus while the metabolome was analysed in the cortex. This and how it affects the analysis of the data need to be explained.There was not enough tissue to carry out both the transciptomics and metabolomics with the same area of the brain. Based upon data for pathway identification, the global effects of the compounds on these parameters with aging still hold valid despite being from two different parts of the brain, both of which are affected in aging and AD. This is because network analysis using genes and metabolites (GAM) identified acetyl-CoA as a central metabolite regulated by CMS121 and J147 in both cortex and hippocampus, and this finding was further validated in primary neurons, HT22 cells and fibroblasts. In addition, both transcriptomics and metabolomics also identified mitochondrial dysfunction as a feature of aging in SAMP8 mice in both tissues. We have added this information to the Discussion.The SAMP8 model needs to be interpreted more carefully regarding normal WT aging. In this context, it would be helpful to compare SAMP8 aging data with published data from WT mice during aging. This would solidify the model. Further, I wonder to what extent the changes in SAMP8 are representative for changes in AD?There are a large number of publications detailing how the phenotype developed by SAMP8 mice with aging is directly relevant to brain aging, and why they should be considered as a model of dementia and sporadic AD . Some of the pathological traits developed by the SAMP8 mice with aging that are also found in AD include: progressive decline in brain function with early deterioration in learning and memory; increased oxidative stress; inflammation; vascular impairment; gliosis; Aβ accumulation and tau hyperphosphorylation . It is very difficult to compare changes at the molecular level between different strains of mice due to the genetic variability inherent to each strain. There is no ideal mouse model for AD at the moment, but the SAMP8 mice appear to be one of the best to study the overlap of aging and AD. This information and respective references were added and discussed more thoroughly in the revised text.The Western blots in Figure 6 show only minor changes. For example, the blot in S5A does not support the change documented in Figure 6C regarding Ac-H3K9. In Figure 6H, pACC1 levels go up with age, while compounds change the signal in the same direction. Thus, these data need to be interpreted more carefully.The data presented in Figure 6C is a result of unbiased band quantification. It should be noted that values of H3K9 acetylation were normalized to total H3 protein, which may be visually misleading. We agree with the reviewers that the data should be interpreted carefully and that is what we tried to do. One possible explanation for the increases in ACC1 phosphorylation in SAMP8 mice with aging could be that this increase represents a compensatory mechanism. This information has now been discussed in the text.The authors have previously identified ATP synthase as a target of J147, with downstream AMPK activation. Is this effect on ATP synthase responsible for the observed effects on the ETC, ACC1 and acetylation? This would be a plausible explanation but is only mentioned in the Introduction.In our previous study , we showed that decreasing ATP synthase activity activated AMPK and increased ACC1 phosphorylation. Because ATP synthase has only been identified as the target of J147 and not CMS121, it was not the focus of our study. However, we did carry out experiments to address the role of AMPK in the signaling pathway identified, and these new data were added to the revised text, as detailed next.It is therefore essential to experimentally test the role of AMPK in this context. Are the changes caused by the compounds regarding TCA metabolite levels, ACC1 phosphorylation, H3K9 acetylation, and neuroprotection caused by increased AMPK activity?We have now tested the role of AMPK in the mode of action of CMS121 and J147. Using fibroblasts  that do not express AMPK (knock out) we show that, similar to what we had found with J147 , the absence of AMPK also reduces the protection against oxytosis by CMS121. In addition, we show that the absence of AMPK prevents the phosphorylation (inhibition) of ACC1 as well as the consequent increases in acetyl-CoA levels. These new data are described in the text and appended as Figure 6—figure supplement 2. Altogether, these results support our conclusion that ACC1 may be a novel target for AD therapy.A color code label is missing for Figure 3 G and H.The label has been added to the figures.The authors state in Figure 3A and B that the majority of the changed genes are the same. It would be great to back this up by showing the overlap. What proportion of these genes are the upregulated fraction?A diagram showing the overlap of genes was added to Figure 3B. In addition, this information was made clearer in the text, "The top KEGG pathway targeted by both aging and CMS121 was oxidative phosphorylation (OP). […] Surprisingly, the expression of all these genes went up between 9 and 13 months in the SAMP8 mice and this was prevented by CMS121 and J147 .". The figure legend was also altered accordingly.The text does not properly explain the neurotoxicity assay in Figure 6E.c-, leading to depletion of intracellular glutathione (GSH), production of ROS and cell death ".This was made clearer in the text, "The primary phenotypic screening model used in our laboratory to identify CMS121 and J147   relies on the lethal induction of mitochondrial ROS in the mouse hippocampal neuronal cell line HT22 , a programmed cell death pathway called oxytosis with physiological features similar to those implicated in the nerve cell damage seen in aging and AD . Oxytosis can be triggered by glutamate which inhibits cystine uptake via system XGenerally, the text should follow the order of the figures. In the manuscript, the reader has to jump between sections, making it harder to read. The results in Figures 4, 5, and 6, for example, are presented in a way that is a bit confusing. It would be better to arrange the figures to the order of the text or change the text so that it is not necessary to jump between the figures.The text in the Results section has been reorganized to match the order of the respective figures. Some graphs in Figure 6 have also been rearranged to follow their description in the text.The reviewers were largely satisfied with the revisions they have requested. However, there is one remaining issue that needs to be addressed experimentally. The authors show that the compounds appear to act through ACC1 inhibition. Inhibition of ACC1 has two consequences: elevated Ac-CoA and the reduced flux in downstream metabolism. Which of the two effects of ACC1 inhibition is responsible for the observed phenotype should be clarified by experimentally elevating Acetyl-CoA levels by acetate supplementation.As per your suggestion, we have now experimentally elevated acetyl-CoA levels by supplementing cells with acetate, a cellular precursor of acetyl-CoA . This was protective in our model, as shown in Figure 6R, supporting our overall conclusions. These new data have been added to the manuscript.