Aging impairs the osteocytic regulation of collagen integrity and bone quality.

Poor bone quality is a major factor in skeletal fragility in elderly individuals. The molecular mechanisms that establish and maintain bone quality, independent of bone mass, are unknown but are thought to be primarily determined by osteocytes. We hypothesize that the age-related decline in bone quality results from the suppression of osteocyte perilacunar/canalicular remodeling (PLR), which maintains bone material properties. We examined bones from young and aged mice with osteocyte-intrinsic repression of TGFß signaling (TßRIIocy-/-) that suppresses PLR. The control aged bone displayed decreased TGFß signaling and PLR, but aging did not worsen the existing PLR suppression in male TßRIIocy-/- bone. This relationship impacted the behavior of collagen material at the nanoscale and tissue scale in macromechanical tests. The effects of age on bone mass, density, and mineral material behavior were independent of osteocytic TGFß. We determined that the decline in bone quality with age arises from the loss of osteocyte function and the loss of TGFß-dependent maintenance of collagen integrity.

Dicarboxylic Acid Dietary Supplementation Protects against AKI.

SIGNIFICANCE STATEMENT: In this study, we demonstrate that a common, low-cost compound known as octanedioic acid (DC 8 ) can protect mice from kidney damage typically caused by ischemia-reperfusion injury or the chemotherapy drug cisplatin. This compound seems to enhance peroxisomal activity, which is responsible for breaking down fats, without adversely affecting mitochondrial function. DC 8 is not only affordable and easy to administer but also effective. These encouraging findings suggest that DC 8 could potentially be used to assist patients who are at risk of experiencing this type of kidney damage. BACKGROUND: Proximal tubules are rich in peroxisomes, which are damaged during AKI. Previous studies demonstrated that increasing peroxisomal fatty acid oxidation (FAO) is renoprotective, but no therapy has emerged to leverage this mechanism. METHODS: Mice were fed with either a control diet or a diet enriched with dicarboxylic acids, which are peroxisome-specific FAO substrates, then subjected to either ischemia-reperfusion injury-AKI or cisplatin-AKI models. Biochemical, histologic, genetic, and proteomic analyses were performed. RESULTS: Both octanedioic acid (DC 8 ) and dodecanedioic acid (DC 12 ) prevented the rise of AKI markers in mice that were exposed to renal injury. Proteomics analysis demonstrated that DC 8 preserved the peroxisomal and mitochondrial proteomes while inducing extensive remodeling of the lysine succinylome. This latter finding indicates that DC 8 is chain shortened to the anaplerotic substrate succinate and that peroxisomal FAO was increased by DC 8 . CONCLUSIONS: DC 8 supplementation protects kidney mitochondria and peroxisomes and increases peroxisomal FAO, thereby protecting against AKI.

Proteomic quantification of native and ECM-enriched mouse ovaries reveals an age-dependent fibro-inflammatory signature.

The ovarian microenvironment becomes fibrotic and stiff with age, in part due to increased collagen and decreased hyaluronan. However, the extracellular matrix (ECM) is a complex network of hundreds of proteins, glycoproteins, and glycans which are highly tissue specific and undergo pronounced changes with age. To obtain an unbiased and comprehensive profile of age-associated alterations to the murine ovarian proteome and ECM, we used a label-free quantitative proteomic methodology. We validated conditions to enrich for the ECM prior to proteomic analysis. Following analysis by data-independent acquisition (DIA) and quantitative data processing, we observed that both native and ECM-enriched ovaries clustered separately based on age, indicating distinct age-dependent proteomic signatures. We identified a total of 4,721 proteins from both native and ECM-enriched ovaries, of which 383 proteins were significantly altered with advanced age, including 58 ECM proteins. Several ECM proteins upregulated with age have been associated with fibrosis in other organs, but to date their roles in ovarian fibrosis are unknown. Pathways regulating DNA metabolism and translation were downregulated with age, whereas pathways involved in ECM remodeling and immune response were upregulated. Interestingly, immune-related pathways were upregulated with age even in ECM-enriched ovaries, suggesting a novel interplay between the ECM and the immune system. Moreover, we identified putative markers of unique immune cell populations present in the ovary with age. These findings provide evidence from a proteomic perspective that the aging ovary provides a fibroinflammatory milieu, and our study suggests target proteins which may drive these age-associated phenotypes for future investigation.

Deep coverage and quantification of the bone proteome provides enhanced opportunities for new discoveries in skeletal biology and disease.

Dysregulation of cell signaling in chondrocytes and in bone cells, such as osteocytes, osteoblasts, osteoclasts, and an elevated burden of senescent cells in cartilage and bone, are implicated in osteoarthritis (OA). Mass spectrometric analyses provides a crucial molecular tool-kit to understand complex signaling relationships in age-related diseases, such as OA. Here we introduce a novel mass spectrometric workflow to promote proteomic studies of bone. This workflow uses highly specialized steps, including extensive overnight demineralization, pulverization, and incubation for 72 h in 6 M guanidine hydrochloride and EDTA, followed by proteolytic digestion. Analysis on a high-resolution Orbitrap Eclipse and Orbitrap Exploris 480 mass spectrometer using Data-Independent Acquisition (DIA) provides deep coverage of the bone proteome, and preserves post-translational modifications, such as hydroxyproline. A spectral library-free quantification strategy, directDIA, identified and quantified over 2,000 protein groups (with ≥ 2 unique peptides) from calcium-rich bone matrices. Key components identified were proteins of the extracellular matrix (ECM), bone-specific proteins (e.g., secreted protein acidic and cysteine rich, SPARC, and bone sialoprotein 2, IBSP), and signaling proteins (e.g., transforming growth factor beta-2, TGFB2), and lysyl oxidase homolog 2 (LOXL2), an important protein in collagen crosslinking. Post-translational modifications (PTMs) were identified without the need for specific enrichment. This includes collagen hydroxyproline modifications, chemical modifications for collagen self-assembly and network formation. Multiple senescence factors were identified, such as complement component 3 (C3) protein of the complement system and many matrix metalloproteinases, that might be monitored during age-related bone disease progression. Our innovative workflow yields in-depth protein coverage and quantification strategies to discover underlying biological mechanisms of bone aging and to provide tools to monitor therapeutic interventions. These novel tools to monitor the bone proteome open novel horizons to investigate bone-specific diseases, many of which are age-related.

Simultaneous neuronal expression of human amyloid-ß and Tau genes drives global phenotypic and multi-omic changes in C. elegans.

Alzheimer's disease and Alzheimer's related diseases (ADRD) are a class of prevalent age-related neurodegenerative disorders characterized by the accumulation of amyloid- ß (Aß) plaques and Tau neurofibrillary tangles. The intricate interplay between Aß and Tau proteins requires further investigation to better understand the precise mechanisms underlying disease pathology. The nematode Caenorhabditis elegans ( C. elegans ) serves as an invaluable model organism for studying aging and neurodegenerative diseases. Here we performed an unbiased systems analysis of a C. elegans strain expressing both Aß and Tau proteins within neurons. Intriguingly, even at an early stage of adulthood, we observed reproductive impairments and mitochondrial dysfunction consistent with substantial disruptions in mRNA transcript abundance, protein solubility, and metabolite levels. Notably, the simultaneous expression of these two neurotoxic proteins exhibited a synergistic effect, leading to accelerated aging in the model organism. Our comprehensive findings shed new light on the intricate relationship between normal aging processes and the etiology of ADRD. Specifically, we demonstrate the alterations to metabolic functions precede age-related neurotoxicity, offering critical insights into potential therapeutic strategies.

Amyloid ß accelerates age-related proteome-wide protein insolubility.

Loss of proteostasis is a highly conserved feature of aging across model organisms and typically results in the accumulation of insoluble protein aggregates. Protein insolubility is a central feature of major age-related neurodegenerative diseases, including Alzheimer's Disease (AD), where hundreds of insoluble proteins associate with aggregated amyloid beta (Aß) in senile plaques. Moreover, proteins that become insoluble during aging in model organisms are capable of accelerating Aß aggregation in vitro. Despite the connection between aging and AD risk, therapeutic approaches to date have overlooked aging-driven protein insolubility as a contributory factor. Here, using an unbiased proteomics approach, we questioned the relationship between Aß and age-related protein insolubility. We demonstrate that Aß expression drives proteome-wide protein insolubility in C. elegans and this insoluble proteome closely resembles the insoluble proteome driven by normal aging, suggesting the possibility of a vicious feedforward cycle of aggregation in the context of AD. Importantly, using human genome-wide association studies (GWAS), we show that the CIP is replete with biological processes implicated not only in neurodegenerative diseases but also across a broad array of chronic, age-related diseases (CARDs). This provides suggestive evidence that age-related loss of proteostasis could play a role in general CARD risk. Finally, we show that the CIP is enriched with proteins that modulate the toxic effects of Aß and that the gut-derived metabolite, Urolithin A, relieves Aß toxicity, supporting its use in clinical trials for dementia and other age-related diseases.

Fructose induced KHK-C can increase ER stress independent of its effect on lipogenesis to drive liver disease in diet-induced and genetic models of NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is a liver manifestation of metabolic syndrome, and is estimated to affect one billion individuals worldwide. An increased intake of a high-fat diet (HFD) and sugar-sweetened beverages are risk-factors for NAFLD development, but how their combined intake promotes progression to a more severe form of liver injury is unknown. Here we show that fructose metabolism via ketohexokinase (KHK) C isoform leads to unresolved endoplasmic reticulum (ER) stress when coupled with a HFD intake. Conversely, a liver-specific knockdown of KHK in mice consuming fructose on a HFD is adequate to improve the NAFLD activity score and exert a profound effect on the hepatic transcriptome. Overexpression of KHK-C in cultured hepatocytes is sufficient to induce ER stress in fructose free media. Upregulation of KHK-C is also observed in mice with genetically induced obesity or metabolic dysfunction, whereas KHK knockdown in these mice improves metabolic function. Additionally, in over 100 inbred strains of male or female mice hepatic KHK expression correlates positively with adiposity, insulin resistance, and liver triglycerides. Similarly, in 241 human subjects and their controls, hepatic Khk expression is upregulated in early, but not late stages of NAFLD. In summary, we describe a novel role of KHK-C in triggering ER stress, which offers a mechanistic understanding of how the combined intake of fructose and a HFD propagates the development of metabolic complications.

A natural variation-based screen in mouse cells reveals USF2 as a regulator of the DNA damage response and cellular senescence.

Cellular senescence is a program of cell cycle arrest, apoptosis resistance, and cytokine release induced by stress exposure in metazoan cells. Landmark studies in laboratory mice have characterized a number of master senescence regulators, including p16INK4a, p21, NF-κB, p53, and C/EBPß. To discover other molecular players in senescence, we developed a screening approach to harness the evolutionary divergence between mouse species. We found that primary cells from the Mediterranean mouse Mus spretus, when treated with DNA damage to induce senescence, produced less cytokine and had less-active lysosomes than cells from laboratory Mus musculus. We used allele-specific expression profiling to catalog senescence-dependent cis-regulatory variation between the species at thousands of genes. We then tested for correlation between these expression changes and interspecies sequence variants in the binding sites of transcription factors. Among the emergent candidate senescence regulators, we chose a little-studied cell cycle factor, upstream stimulatory factor 2 (USF2), for molecular validation. In acute irradiation experiments, cells lacking USF2 had compromised DNA damage repair and response. Longer-term senescent cultures without USF2 mounted an exaggerated senescence regulatory program-shutting down cell cycle and DNA repair pathways, and turning up cytokine expression, more avidly than wild-type. We interpret these findings under a model of pro-repair, anti-senescence regulatory function by USF2. Our study affords new insights into the mechanisms by which cells commit to senescence, and serves as a validated proof of concept for natural variation-based regulator screens.

Fructose Induced KHK-C Increases ER Stress and Modulates Hepatic Transcriptome to Drive Liver Disease in Diet-Induced and Genetic Models of NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is a liver manifestation of metabolic syndrome, and is estimated to affect one billion individuals worldwide. An increased intake of a high-fat diet (HFD) and sugar-sweetened beverages are risk-factors for NAFLD development, but how their combined intake promotes progression to a more severe form of liver injury is unknown. Here we show that fructose metabolism via ketohexokinase (KHK) C isoform increases endoplasmic reticulum (ER) stress in a dose dependent fashion, so when fructose is coupled with a HFD intake it leads to unresolved ER stress. Conversely, a liver-specific knockdown of KHK in C57BL/6J male mice consuming fructose on a HFD is adequate to improve the NAFLD activity score and exert a profound effect on the hepatic transcriptome. Overexpression of KHK-C in cultured hepatocytes is sufficient to induce ER stress in fructose free media. Upregulation of KHK-C is also observed in genetically obesity ob/ob, db/db and lipodystrophic FIRKO male mice, whereas KHK knockdown in these mice improves metabolic function. Additionally, in over 100 inbred strains of male or female mice hepatic KHK expression correlates positively with adiposity, insulin resistance, and liver triglycerides. Similarly, in 241 human subjects and their controls, hepatic Khk expression is upregulated in early, but not late stages of NAFLD. In summary, we describe a novel role of KHK-C in triggering ER stress, which offers a mechanistic understanding of how the combined intake of fructose and a HFD propagates the development of metabolic complications.

Ketohexokinase-C regulates global protein acetylation to decrease carnitine palmitoyltransferase 1a-mediated fatty acid oxidation.

BACKGROUND & AIMS: The consumption of sugar and a high-fat diet (HFD) promotes the development of obesity and metabolic dysfunction. Despite their well-known synergy, the mechanisms by which sugar worsens the outcomes associated with a HFD are largely elusive. METHODS: Six-week-old, male, C57Bl/6 J mice were fed either chow or a HFD and were provided with regular, fructose- or glucose-sweetened water. Moreover, cultured AML12 hepatocytes were engineered to overexpress ketohexokinase-C (KHK-C) using a lentivirus vector, while CRISPR-Cas9 was used to knockdown CPT1α. The cell culture experiments were complemented with in vivo studies using mice with hepatic overexpression of KHK-C and in mice with liver-specific CPT1α knockout. We used comprehensive metabolomics, electron microscopy, mitochondrial substrate phenotyping, proteomics and acetylome analysis to investigate underlying mechanisms. RESULTS: Fructose supplementation in mice fed normal chow and fructose or glucose supplementation in mice fed a HFD increase KHK-C, an enzyme that catalyzes the first step of fructolysis. Elevated KHK-C is associated with an increase in lipogenic proteins, such as ACLY, without affecting their mRNA expression. An increase in KHK-C also correlates with acetylation of CPT1α at K508, and lower CPT1α protein in vivo. In vitro, KHK-C overexpression lowers CPT1α and increases triglyceride accumulation. The effects of KHK-C are, in part, replicated by a knockdown of CPT1α. An increase in KHK-C correlates negatively with CPT1α protein levels in mice fed sugar and a HFD, but also in genetically obese db/db and lipodystrophic FIRKO mice. Mechanistically, overexpression of KHK-C in vitro increases global protein acetylation and decreases levels of the major cytoplasmic deacetylase, SIRT2. CONCLUSIONS: KHK-C-induced acetylation is a novel mechanism by which dietary fructose augments lipogenesis and decreases fatty acid oxidation to promote the development of metabolic complications. IMPACT AND IMPLICATIONS: Fructose is a highly lipogenic nutrient whose negative consequences have been largely attributed to increased de novo lipogenesis. Herein, we show that fructose upregulates ketohexokinase, which in turn modifies global protein acetylation, including acetylation of CPT1a, to decrease fatty acid oxidation. Our findings broaden the impact of dietary sugar beyond its lipogenic role and have implications on drug development aimed at reducing the harmful effects attributed to sugar metabolism.

Advancements in automation for plasma proteomics sample preparation.

Automation is necessary to increase sample processing throughput for large-scale clinical analyses. Replacement of manual pipettes with robotic liquid handler systems is especially helpful in processing blood-based samples, such as plasma and serum. These samples are very heterogenous, and protein expression can vary greatly from sample-to-sample, even for healthy controls. Detection of true biological changes requires that variation from sample preparation steps and downstream analytical detection methods, such as mass spectrometry, remains low. In this mini-review, we discuss plasma proteomics protocols and the benefits of automation towards enabling detection of low abundant proteins and providing low sample error and increased sample throughput. This discussion includes considerations for automation of major sample depletion and/or enrichment strategies for plasma toward mass spectrometry detection.

In Vivo Fast Photochemical Oxidation of Proteins Using Enhanced Multiplexing Proteomics.

In vivo fast photochemical oxidation of proteins (IV-FPOP) is a hydroxyl radical protein footprinting method used to study protein structure and protein-protein interactions. Oxidatively modified proteins by IV-FPOP are analyzed by mass spectrometry (MS), and the extent of oxidation is quantified by label-free MS. Peptide oxidation changes yield useful information about protein structure, due to changes in solvent accessibility. However, the sample size necessary for animal studies requires increased sample preparation and instrument time. Here, we report the combined application of IV-FPOP and the enhanced multiplexing strategy combined precursor isotopic labeling and isobaric tagging (cPILOT) for higher-throughput analysis of oxidative modifications in C. elegans. Key differences in the performance of label-free MS and cPILOT were identified. The addition of oxygen (+16) was the most abundant modification identified among all known possible FPOP modifications. This study presents IV-FPOP coupled with enhanced multiplexing strategies such as cPILOT to increase throughput of studies seeking to examine oxidative protein modifications.

Evaluating Combined Precursor Isotopic Labeling and Isobaric Tagging Performance on Orbitraps To Study the Peripheral Proteome of Alzheimer's Disease.

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is an enhanced multiplexing strategy currently capable of analyzing up to 24 samples simultaneously. This capability is especially helpful when studying multiple tissues and biological replicates in models of disease, such as Alzheimer's disease (AD). Here, cPILOT was used to study proteomes from heart, liver, and brain tissues in a late-stage amyloid precursor protein/presenilin-1 (APP/PS-1) human transgenic double-knock-in mouse model of AD. The original global cPILOT assay developed on an Orbitrap Velos instrument was transitioned to an Orbitrap Fusion Lumos instrument. The advantages of faster scan rates, lower limits of detection, and synchronous precursor selection on the Fusion Lumos afford greater numbers of isobarically tagged peptides to be quantified in comparison to the Orbitrap Velos. Parameters such as LC gradient, m/z isolation window, dynamic exclusion, targeted mass analyses, and synchronous precursor scan were optimized leading to >600 000 PSMs, corresponding to 6074 proteins. Overall, these studies inform of system-wide changes in brain, heart, and liver proteins from a mouse model of AD.

Proteomic identification of virulence-related factors in young and aging C. elegans infected with Pseudomonas aeruginosa.

The molecular mechanisms that distinguish immunosenescence from general age-related decline are poorly understood. We addressed this by exposing Day 1 and Day 5 adults of Caenorhabditis elegans to Pseudomonas aeruginosa strain PA01, an opportunistic pathogen. Day 5 adult C. elegans exhibited greater vulnerability to infection as compared to Day 1 C. elegans. Using TMT6-plex isobaric labeling and reductive dimethylation, we identified 55 proteins whose levels were altered following infection of Day 1 and Day 5 adults. Proteins whose levels changed in response to infection at both ages were strongly enriched for locomotory functions underscoring the importance of pathogen avoidance mechanisms. In Day 1 C. elegans, proteins with reproductive functions were highly enriched, whereas, Day 5 worms showed elevated levels of factors representing stress response pathways such as unfolded protein response (UPR) and metabolic functions. We also found that PA01 infection is associated with elevated protein carbonylation, an irreversible marker for oxidative stress. We explored the function of UNC-60, a cytoskeletal protein whose levels were changed by both age and infection, and found that mutants of unc-60 have reduced lifespan. Overall, our data provide novel insights into the relationship between age and immunosenescence in metazoans. SIGNIFICANCE: There are gaps in our knowledge pertaining to how aging influences an organism's response to pathogen exposure. In C. elegans, pathogen exposure to P. aeruginosa PA01 results in shortened lifespan, which is more pronounced in Day 5, compared to Day 1 adult worms. The proteome has age-specific responses to this exposure, and notably affects development, reproduction, metabolism, protein folding/unfolding, locomotion, and response to stress. This study addresses the molecular links between aging and immunosenescence in invertebrates.

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT).

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies that allow simultaneous measurement of multiple samples have become widespread and greatly reduce experimental costs and times. Our laboratory developed a technique called combined precursor isotopic labeling and isobaric tagging (cPILOT), which enhances sample multiplexing of traditional isotopic labeling or isobaric tagging approaches. Global cPILOT can be applied to samples originating from cells, tissues, bodily fluids, or whole organisms and gives information on relative protein abundances across different sample conditions. cPILOT works by 1) using low pH buffer conditions to selectively dimethylate peptide N-termini and 2) using high pH buffer conditions to label primary amines of lysine residues with commercially-available isobaric reagents (see Table of Materials/Reagents). The degree of sample multiplexing available is dependent on the number of precursor labels used and the isobaric tagging reagent. Here, we present a 12-plex analysis using light and heavy dimethylation combined with six-plex isobaric reagents to analyze 12 samples from mouse tissues in a single analysis. Enhanced multiplexing is helpful for reducing experimental time and cost and more importantly, allowing comparison across many sample conditions (biological replicates, disease stage, drug treatments, genotypes, or longitudinal time-points) with less experimental bias and error. In this work, the global cPILOT approach is used to analyze brain, heart, and liver tissues across biological replicates from an Alzheimer's disease mouse model and wild-type controls. Global cPILOT can be applied to study other biological processes and adapted to increase sample multiplexing to greater than 20 samples.

Dataset of proteomics analysis of aging C. elegans exposed to Pseudomonas aeruginosa strain PA01.

Here, we present the proteomics dataset of young and middle-aged Caenorhabditis elegans (C. elegans) exposed to Pseudomonas aeruginosa (P. aeruginosa strain PA01), which is related to the article "Proteomic Identification of Virulence-Related Factors in Young and Aging C. elegans infected with Pseudomonas aeruginosa" (C. D. King et. al, in-revisions). This dataset was generated to better understand the effects of aging on molecular mechanisms involved in host response to pathogen exposure. Protein from C. elegans of different age and exposure to P. aeruginosa PA01 or control E. coli OP50 were extracted and tryptically digested. Peptides were labeled with the reagents tandem mass tag (TMT6-plex), separated, and detected by using offline strong-cation exchange and online liquid chromatography - mass spectrometry (SCX - LC - MS/MS & MS3). A separate mixture of peptides were labeled on N-terminal amines and lysines with dimethylation. Dimethylated peptides were analyzed using LC - MS/MS and a portion of the results were used to verify fold-change direction for TMT6-plex experiments. Raw data can be found online at www.CHORUSproject.org, a cloud-based data repository (see specifications table for details).