High plasma concentrations of acyl-coenzyme A binding protein (ACBP) predispose to cardiovascular disease: Evidence for a phylogenetically conserved proaging function of ACBP

Autophagy defects accelerate aging, while stimulation of autophagy decelerates aging. Acyl-coenzyme A binding protein (ACBP), which is encoded by a diazepam-binding inhibitor (DBI), acts as an extracellular feedback regulator of autophagy. As shown here, knockout of the gene coding for the yeast orthologue of ACBP/DBI (ACB1) improves chronological aging, and this effect is reversed by knockout of essential autophagy genes (ATG5, ATG7) but less so by knockout of an essential mitophagy gene (ATG32). In humans, ACBP/DBI levels independently correlate with body mass index (BMI) as well as with chronological age. In still-healthy individuals, we find that high ACBP/DBI levels correlate with future cardiovascular events (such as heart surgery, myocardial infarction, and stroke), an association that is independent of BMI and chronological age, suggesting that ACBP/DBI is indeed a biomarker of “biological” aging. Concurringly, ACBP/DBI plasma concentrations correlate with established cardiovascular risk factors (fasting glucose levels, systolic blood pressure, total free cholesterol, triglycerides), but are inversely correlated with atheroprotective high-density lipoprotein (HDL). In mice, neutralization of ACBP/DBI through a monoclonal antibody attenuates anthracycline-induced cardiotoxicity, which is a model of accelerated heart aging. In conclusion, plasma elevation of ACBP/DBI constitutes a novel biomarker of chronological aging and facets of biological aging with a prognostic value in cardiovascular disease.     

Effects of acyl-coenzyme A binding protein (ACBP)/diazepam-binding inhibitor (DBI) on body mass index

In mice, the plasma concentrations of the appetite-stimulatory and autophagy-inhibitory factor acyl-coenzyme A binding protein (ACBP, also called diazepam-binding inhibitor, DBI) acutely increase in response to starvation, but also do so upon chronic overnutrition leading to obesity. Here, we show that knockout of Acbp/Dbi in adipose tissue is sufficient to prevent high-fat diet-induced weight gain in mice. We investigated ACBP/DBI plasma concentrations in several patient cohorts to discover a similar dual pattern of regulation. In relatively healthy subjects, ACBP/DBI concentrations independently correlated with body mass index (BMI) and age. The association between ACBP/DBI and BMI was lost in subjects that underwent major weight gain in the subsequent 3–9 years, as well as in advanced cancer patients. Voluntary fasting, undernutrition in the context of advanced cancer, as well as chemotherapy were associated with an increase in circulating ACBP/DBI levels. Altogether, these results support the conclusion that ACBP/DBI may play an important role in body mass homeostasis as well as in its failure.Subject terms: Obesity, Diagnostic markers     

Acyl coenzyme A binding protein (ACBP): An aging- and disease-relevant “autophagy checkpoint”

Acyl coenzyme A binding protein (ACBP), also known as diazepam-binding inhibitor (DBI), is a phylogenetically ancient protein present in some eubacteria and the entire eukaryotic radiation. In several eukaryotic phyla, ACBP/DBI transcends its intracellular function in fatty acid metabolism because it can be released into the extracellular space. This ACBP/DBI secretion usually occurs in response to nutrient scarcity through an autophagy-dependent pathway. ACBP/DBI and its peptide fragments then act on a range of distinct receptors that diverge among phyla, namely metabotropic G protein-coupled receptor in yeast (and likely in the mammalian central nervous system), a histidine receptor kinase in slime molds, and ionotropic gamma-aminobutyric acid (GABA)_A receptors in mammals. Genetic or antibody-mediated inhibition of ACBP/DBI orthologs interferes with nutrient stress-induced adaptations such as sporulation or increased food intake in multiple species, as it enhances lifespan or healthspan in yeast, plant leaves, nematodes, and multiple mouse models. These lifespan and healthspan-extending effects of ACBP/DBI suppression are coupled to the induction of autophagy. Altogether, it appears that neutralization of extracellular ACBP/DBI results in “autophagy checkpoint inhibition” to unleash the anti-aging potential of autophagy. Of note, in humans, ACBP/DBI levels increase in various tissues, as well as in the plasma, in the context of aging, obesity, uncontrolled infection or cardiovascular, inflammatory, neurodegenerative, and malignant diseases.     

The Arabidopsis thaliana ACBP3 regulates leaf senescence by modulating phospholipid metabolism and ATG8 stability

Bulk degradation and nutrient recycling are events associated with autophagy. The core components of the autophagy machinery have been elucidated recently using molecular and genetic approaches. In particular, two ubiquitin-like proteins, ATG8 and ATG12, which conjugate with phosphatidylethanolamine (PE) and ATG5, respectively, forming ATG8-PE and ATG12-ATG5 complexes, were shown to be essential in autophagosome formation. Our recent findings reveal that the Arabidopsis thaliana acyl-CoA-binding protein ACBP3 binds the phospholipid PE in vitro and that ACBP3 overexpression and downregulation correlate with PE composition in rosettes. Furthermore, ACBP3-overexpressors (ACBP3-OEs) display accelerated salicylic acid-dependent leaf senescence resembling the phenotype of Arabidopsis knockout (KO) mutants defective in autophagy-related (ATG) proteins. Consistently, downregulation of ACBP3 (ACBP3-KOs) delays dark-induced leaf senescence. By analysis of transgenic Arabidopsis expressing GFP-ATG8e as well as those co-expressing ACBP3-OE and GFP-ATG8e, we showed that ACBP3-overexpression disrupts autophagosome formation and enhanced degradation of ATG8 under starvation conditions, suggesting that ACBP3 is an important regulator of the ATG8-PE complex via its interaction with PE. Here, a working model for the role of ACBP3 in the regulation of autophagy-mediated leaf senescence is presented.     

Premature aging is associated with higher levels of 8‐oxoguanine and increased DNA damage in the Polg mutator mouse

Mitochondrial dysfunction plays an important role in the aging process. However, the mechanism by which this dysfunction causes aging is not fully understood. The accumulation of mutations in the mitochondrial genome (or “mtDNA”) has been proposed as a contributor. One compelling piece of evidence in support of this hypothesis comes from the Polg ^D257A/D257A mutator mouse (Polg ^mut/mut ). These mice express an error‐prone mitochondrial DNA polymerase that results in the accumulation of mtDNA mutations, accelerated aging, and premature death. In this paper, we have used the Polg ^mut/mut model to investigate whether the age‐related biological effects observed in these mice are triggered by oxidative damage to the DNA that compromises the integrity of the genome. Our results show that mutator mouse has significantly higher levels of 8‐oxoguanine (8‐oxoGua) that are correlated with increased nuclear DNA (nDNA) strand breakage and oxidative nDNA damage, shorter average telomere length, and reduced mtDNA integrity. Based on these results, we propose a model whereby the increased level of reactive oxygen species (ROS) associated with the accumulation of mtDNA mutations in Polg ^mut/mut mice results in higher levels of 8‐oxoGua, which in turn lead to compromised DNA integrity and accelerated aging via increased DNA fragmentation and telomere shortening. These results suggest that mitochondrial play a central role in aging and may guide future research to develop potential therapeutics for mitigating aging process.     

Regulation of autophagosome biogenesis by OFD1‐mediated selective autophagy

Autophagy is a lysosome‐dependent degradation pathway essential to maintain cellular homeostasis. Therefore, either defective or excessive autophagy may be detrimental for cells and tissues. The past decade was characterized by significant advances in molecular dissection of stimulatory autophagy inputs; however, our understanding of the mechanisms that restrain autophagy is far from complete. Here, we describe a negative feedback mechanism that limits autophagosome biogenesis based on the selective autophagy‐mediated degradation of ATG13, a component of the ULK1 autophagy initiation complex. We demonstrate that the centrosomal protein OFD1 acts as bona fide autophagy receptor for ATG13 via direct interaction with the Atg8/LC3/GABARAP family of proteins. We also show that patients with Oral‐Facial‐Digital type I syndrome, caused by mutations in the OFD1 gene, display excessive autophagy and that genetic inhibition of autophagy in a mouse model of the disease, significantly ameliorates polycystic kidney, a clinical manifestation of the disorder. Collectively, our data report the discovery of an autophagy self‐regulated mechanism and implicate dysregulated autophagy in the pathogenesis of renal cystic disease in mammals.     

Fibronectin type III domain‐containing 5 improves aging‐related cardiac dysfunction in mice

Aging is an important risk factor for cardiovascular diseases, and aging‐related cardiac dysfunction serves as a major determinant of morbidity and mortality in elderly populations. Our previous study has identified fibronectin type III domain‐containing 5 (FNDC5) and its cleaved form, irisin, as the cardioprotectant against doxorubicin‐induced cardiomyopathy. Herein, aging or matched young mice were overexpressed with FNDC5 by adeno‐associated virus serotype 9 (AAV9) vectors, or subcutaneously infused with irisin to uncover the role of FNDC5 in aging‐related cardiac dysfunction. To verify the involvement of nucleotide‐binding oligomerization domain‐like receptor with a pyrin domain 3 (NLRP3) and AMP‐activated protein kinase α (AMPKα), Nlrp3 or Ampkα2 global knockout mice were used. Besides, young mice were injected with AAV9‐FNDC5 and maintained for 12 months to determine the preventive effect of FNDC5. Moreover, neonatal rat cardiomyocytes were stimulated with tumor necrosis factor‐α (TNF‐α) to examine the role of FNDC5 in vitro. We found that FNDC5 was downregulated in aging hearts. Cardiac‐specific overexpression of FNDC5 or irisin infusion significantly suppressed NLRP3 inflammasome and cardiac inflammation, thereby attenuating aging‐related cardiac remodeling and dysfunction. In addition, irisin treatment also inhibited cellular senescence in TNF‐α‐stimulated cardiomyocytes in vitro. Mechanistically, FNDC5 activated AMPKα through blocking the lysosomal degradation of glucagon‐like peptide‐1 receptor. More importantly, FNDC5 gene transfer in early life could delay the onset of cardiac dysfunction during aging process. We prove that FNDC5 improves aging‐related cardiac dysfunction by activating AMPKα, and it might be a promising therapeutic target to support cardiovascular health in elderly populations.     

α‐synuclein suppresses microglial autophagy and promotes neurodegeneration in a mouse model of Parkinson’s disease

The cell‐to‐cell transfer of α‐synuclein (α‐Syn) greatly contributes to Parkinson''s disease (PD) pathogenesis and underlies the spread of α‐Syn pathology. During this process, extracellular α‐Syn can activate microglia and neuroinflammation, which plays an important role in PD. However, the effect of extracellular α‐Syn on microglia autophagy is poorly understood. In the present study, we reported that extracellular α‐Syn inhibited the autophagy initiation, as indicated by LC3‐II reduction and p62 protein elevation in BV2 and cultured primary microglia. The in vitro findings were verified in microglia‐enriched population isolated from α‐Syn‐overexpressing mice induced by adeno‐associated virus (AAV2/9)‐encoded wildtype human α‐Syn injection into the substantia nigra (SN). Mechanistically, α‐Syn led to microglial autophagic impairment through activating toll‐like receptor 4 (Tlr4) and its downstream p38 and Akt‐mTOR signaling because Tlr4 knockout and inhibition of p38, Akt as well as mTOR prevented α‐Syn‐induced autophagy inhibition. Moreover, inhibition of Akt reversed the mTOR activation but failed to affect p38 phosphorylation triggered by α‐Syn. Functionally, the in vivo evidence showed that lysozyme 2 Cre (Lyz2 ^cre)‐mediated depletion of autophagy‐related gene 5 (Atg5) in microglia aggravated the neuroinflammation and dopaminergic neuron losses in the SN and exacerbated the locomotor deficit in α‐Syn‐overexpressing mice. Taken together, the results suggest that extracellular α‐Syn, via Tlr4‐dependent p38 and Akt‐mTOR signaling cascades, disrupts microglial autophagy activity which synergistically contributes to neuroinflammation and PD development.     

PAQR3 controls autophagy by integrating AMPK signaling to enhance ATG14L‐associated PI3K activity

The Beclin1–VPS34 complex is recognized as a central node in regulating autophagy via interacting with diverse molecules such as ATG14L for autophagy initiation and UVRAG for autophagosome maturation. However, the underlying molecular mechanism that coordinates the timely activation of VPS34 complex is poorly understood. Here, we identify that PAQR3 governs the preferential formation and activation of ATG14L‐linked VPS34 complex for autophagy initiation via two levels of regulation. Firstly, PAQR3 functions as a scaffold protein that facilitates the formation of ATG14L‐ but not UVRAG‐linked VPS34 complex, leading to elevated capacity of PI(3)P generation ahead of starvation signals. Secondly, AMPK phosphorylates PAQR3 at threonine 32 and switches on PI(3)P production to initiate autophagosome formation swiftly after glucose starvation. Deletion of PAQR3 leads to reduction of exercise‐induced autophagy in mice, accompanied by a certain degree of disaggregation of ATG14L‐associated VPS34 complex. Together, this study uncovers that PAQR3 can not only enhance the capacity of pro‐autophagy class III PI3K due to its scaffold function, but also integrate AMPK signal to activation of ATG14L‐linked VPS34 complex upon glucose starvation.     

DJ‐1 depletion prevents immunoaging in T‐cell compartments

Decline in immune function during aging increases susceptibility to different aging‐related diseases. However, the underlying molecular mechanisms, especially the genetic factors contributing to imbalance of naïve/memory T‐cell subpopulations, still remain largely elusive. Here, we show that loss of DJ‐1 encoded by PARK7/DJ‐1, causing early‐onset familial Parkinson’s disease (PD), unexpectedly diminished signs of immunoaging in T‐cell compartments of both human and mice. Compared with two gender‐matched unaffected siblings of similar ages, the index PD patient with DJ‐1 deficiency showed a decline in many critical immunoaging features, including almost doubled non‐senescent T cells. The observation was further consolidated by the results in 45‐week‐old DJ‐1 knockout mice. Our data demonstrated that DJ‐1 regulates several immunoaging features via hematopoietic‐intrinsic and naïve‐CD8‐intrinsic mechanisms. Mechanistically, DJ‐1 depletion reduced oxidative phosphorylation (OXPHOS) and impaired TCR sensitivity in naïve CD8 T cells at a young age, accumulatively leading to a reduced aging process in T‐cell compartments in older mice. Our finding suggests an unrecognized critical role of DJ‐1 in regulating immunoaging, discovering a potent target to interfere with immunoaging‐ and aging‐associated diseases.     

Receptor‐mediated clustering of FIP200 bypasses the role of LC3 lipidation in autophagy

Autophagosome formation requires multiple autophagy‐related (ATG) factors. However, we find that a subset of autophagy substrates remains robustly targeted to the lysosome in the absence of several core ATGs, including the LC3 lipidation machinery. To address this unexpected result, we performed genome‐wide CRISPR screens identifying genes required for NBR1 flux in ATG7^KO cells. We find that ATG7‐independent autophagy still requires canonical ATG factors including FIP200. However, in the absence of LC3 lipidation, additional factors are required including TAX1BP1 and TBK1. TAX1BP1''s ability to cluster FIP200 around NBR1 cargo and induce local autophagosome formation enforces cargo specificity and replaces the requirement for lipidated LC3. In support of this model, we define a ubiquitin‐independent mode of TAX1BP1 recruitment to NBR1 puncta, highlighting that TAX1BP1 recruitment and clustering, rather than ubiquitin binding per se, is critical for function. Collectively, our data provide a mechanistic basis for reports of selective autophagy in cells lacking the lipidation machinery, wherein receptor‐mediated clustering of upstream autophagy factors drives continued autophagosome formation.     

A Microarray-Based Genetic Screen for Yeast Chronological Aging Factors

Model organisms have played an important role in the elucidation of multiple genes and cellular processes that regulate aging. In this study we utilized the budding yeast, Saccharomyces cerevisiae, in a large-scale screen for genes that function in the regulation of chronological lifespan, which is defined by the number of days that non-dividing cells remain viable. A pooled collection of viable haploid gene deletion mutants, each tagged with unique identifying DNA “bar-code” sequences was chronologically aged in liquid culture. Viable mutants in the aging population were selected at several time points and then detected using a microarray DNA hybridization technique that quantifies abundance of the barcode tags. Multiple short- and long-lived mutants were identified using this approach. Among the confirmed short-lived mutants were those defective for autophagy, indicating a key requirement for the recycling of cellular organelles in longevity. Defects in autophagy also prevented lifespan extension induced by limitation of amino acids in the growth media. Among the confirmed long-lived mutants were those defective in the highly conserved de novo purine biosynthesis pathway (the ADE genes), which ultimately produces IMP and AMP. Blocking this pathway extended lifespan to the same degree as calorie (glucose) restriction. A recently discovered cell-extrinsic mechanism of chronological aging involving acetic acid secretion and toxicity was suppressed in a long-lived ade4Δ mutant and exacerbated by a short-lived atg16Δ autophagy mutant. The identification of multiple novel effectors of yeast chronological lifespan will greatly aid in the elucidation of mechanisms that cells and organisms utilize in slowing down the aging process.     

Overexpression of Arabidopsis Acyl-CoA Binding Protein ACBP3 Promotes Starvation-Induced and Age-Dependent Leaf Senescence

In Arabidopsis thaliana, a family of six genes (ACBP1 to ACBP6) encodes acyl-CoA binding proteins (ACBPs). Investigations on ACBP3 reported here show its upregulation upon dark treatment and in senescing rosettes. Transgenic Arabidopsis overexpressing ACBP3 (ACBP3-OEs) displayed accelerated leaf senescence, whereas an acbp3 T-DNA insertional mutant and ACBP3 RNA interference transgenic Arabidopsis lines were delayed in dark-induced leaf senescence. Acyl-CoA and lipid profiling revealed that the overexpression of ACBP3 led to an increase in acyl-CoA and phosphatidylethanolamine (PE) levels, whereas ACBP3 downregulation reduced PE content. Moreover, significant losses in phosphatidylcholine (PC) and phosphatidylinositol, and gains in phosphatidic acid (PA), lysophospholipids, and oxylipin-containing galactolipids (arabidopsides) were evident in 3-week-old dark-treated and 6-week-old premature senescing ACBP3-OEs. Such accumulation of PA and arabidopsides (A, B, D, E, and G) resulting from lipid peroxidation in ACBP3-OEs likely promoted leaf senescence. The N-terminal signal sequence/transmembrane domain in ACBP3 was shown to be essential in ACBP3-green fluorescent protein targeting and in promoting senescence. Observations that recombinant ACBP3 binds PC, PE, and unsaturated acyl-CoAs in vitro and that ACBP3 overexpression enhances degradation of the autophagy (ATG)-related protein ATG8 and disrupts autophagosome formation suggest a role for ACBP3 as a phospholipid binding protein involved in the regulation of leaf senescence by modulating membrane phospholipid metabolism and ATG8 stability in Arabidopsis. Accelerated senescence in ACBP3-OEs is dependent on salicylic acid but not jasmonic acid signaling.     

Ganoderic acid D prevents oxidative stress‐induced senescence by targeting 14‐3‐3ε to activate CaM/CaMKII/NRF2 signaling pathway in mesenchymal stem cells

Stem cell senescence is an important cause of aging. Delaying senescence may present a novel way to combat aging and age‐associated diseases. This study provided a mechanistic insight into the protective effect of ganoderic acid D (GA‐D) against human amniotic mesenchymal stem cell (hAMSCs) senescence. GA‐D, a Ganoderma lucidum‐derived triterpenoid, markedly prevented hAMSCs senescence via activating the Ca²⁺ calmodulin (CaM)/CaM‐dependent protein kinase II (CaMKII)/nuclear erythroid 2‐related factor 2 (Nrf2) axis, and 14‐3‐3ε was identified as a target of GA‐D. 14‐3‐3ε‐encoding gene (YWHAE) knockdown in hAMSCs reversed the activation of the CaM/CaMKII/Nrf2 signals to attenuate the GA‐D anti‐aging effect and increase senescence‐associated β‐galactosidase (SA‐β‐gal), p16 and p21 expression levels, including reactive oxygen species (ROS) production, thereby promoting cell cycle arrest and decreasing differentiation potential. YWHAE overexpression maintained or slightly enhanced the GA‐D anti‐aging effect. GA‐D prevented d‐galactose‐caused aging in mice by significantly increasing the total antioxidant capacity, as well as superoxide dismutase and glutathione peroxidase activity, and reducing the formation of malondialdehyde, advanced glycation end products, and receptor of advanced glycation end products. Consistent with the protective mechanism of GA‐D against hAMSCs senescence, GA‐D delayed the senescence of bone‐marrow mesenchymal stem cells in this aging model in vivo, reduced SA‐β‐gal and ROS production, alleviated cell cycle arrest, and enhanced cell viability and differentiation via regulating 14‐3‐3ε and CaM/CaMKII/Nrf2 axis. Therefore, GA‐D retards hAMSCs senescence by targeting 14‐3‐3ε to activate the CaM/CaMKII/Nrf2 signaling pathway. Furthermore, the in vivo GA‐D anti‐aging effect may involve the regulation of stem cell senescence via the same signal axis.