bioRxiv Science⌕ Search

Biology subjects

Bons, J.

Publications and source records attributed to Bons, J..

At least 19 recordsLinked to original sources

Oral administration of dibenzoylmethane (DBM) prevents cognitive decline in a C9ORF72-mediated FTD mouse model

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two related neurodegenerative disorders that display overlapping features. The hexanucleotide repeat expansion GGGGCC (G4C2) in the C9ORF72 gene is the most common cause of ALS and FTD, which results in the accumulation of dipeptide-repeat protein aggregates. Regulation of protein synthesis at the level of the initiation factor eIF2 has been suggested as a transversal event contributing to neurodegeneration in ALS and FTD. eIF2 phosphorylation blocks protein synthesis to alleviate protein misfolding overload, but conversely it can reduce the expression of synaptic proteins resulting in neuronal dysfunction. Dibenzoylmethane (DBM) is a small molecule that reverses the translational attenuation mediated by eIF2 phosphorylation which has been shown to alleviate neurodegeneration in prion-infected mice and Tau transgenic animals. Here we investigated the efficacy of the oral administration of DBM in protecting a mouse model of C9ORF72 pathogenesis. Treatment of mice with 0.5% of DBM mixture in powdered food ad libitum was sufficient to prevent cognitive impairment in C9ORF72 mice. Unexpectedly, DBM treatment did not modify the content of poly(GA) and poly(GR) protein inclusion in the hippocampus and brain cortex. Proteomic profiling of brain tissue indicated that DBM administration corrected nearly 70% of the changes in gene expression triggered by expanded G4C2, where the main pathways modified by DBM were related to cytoskeleton organization, ALS, and metabolic processes. Most proteins corrected by DBM in our C9ORF72 model were also altered in the brain of human FTD/ALS patients. Overall, our results reinforce the idea that targeting protein synthesis with small molecules in patients carrying C9ORF72 mutations may result in improved cognitive capacity.

molecular biology↗

Confident Identification and Quantification of Mouse Brain Tissues Reveals Sirtuin 5-Dependent Regulation

Methylmalonylation is a non-enzymatic lysine post-translational modification derived from methylmalonyl-CoA, a reactive intermediate that accumulates during mitochondrial dysfunction and branched-chain amino acid catabolism. Although reported in models of methylmalonic acidemia, its broader distribution and functional relevance remain largely unexplored. Progress has been hindered by a key analytical challenge: methylmalonyl-and succinyl-lysine are isobaric (+100.0160 Da) and generate overlapping mass spectrometric fragmentation spectra, preventing confident identification in conventional proteomic workflows. Here, we establish a straightforward proteomic workflow that overcomes this barrier and enables confident identification and quantification of lysine methylmalonylation by combining antibody-based enrichment with data-independent acquisition mass spectrometry (DIA-MS). Anti-malonyl antibodies were used to enrich methylmalonylated peptides through cross-reactivity. Using synthetic peptide standards containing malonyl-, succinyl-, or methylmalonyl-lysine, we defined distinguishing analytical features including chromatographic retention time, ion mobility, and fragmentation patterns. Applying this approach to mouse brain tissues from Sirtuin-5 (SIRT5) knockout and wild-type mice, we identified 44 methylmalonylated peptides across 41 proteins, enriched in neuronal and myelin-associated proteins (NEFM, NEFL, MBP) and mitochondrial enzymes such as ADT1. Several sites were increased in SIRT5-deficient brains, consistent with regulation by this mitochondrial deacylase. Functional assays demonstrated that methylmalonylation of myelin basic protein (MBP) impairs lipid binding, linking this modification to myelin stability. Together, this workflow enables confident methylmalonylation identification and defines it as a widespread and regulated modification in the brain, providing a framework to study metabolically driven protein acylation in neurobiology and disease. SignificanceLysine methylmalonylation has remained largely unexplored due to its isobaric overlap with succinylation, which prevents confident identification using conventional proteomic workflows. Here, we establish an integrated strategy combining antibody-based enrichment, data-independent acquisition mass spectrometry, and orthogonal analytical features to resolve these modifications with high confidence. Applying this approach to mouse brain tissue reveals a SIRT5-regulated methylmalonylome enriched in mitochondrial and myelin-associated proteins, including myelin basic protein (MBP). Functional assays demonstrate that methylmalonylation impairs MBP lipid binding, linking this modification to myelin stability. Beyond this specific application, our workflow provides a generalizable framework to resolve isobaric post-translational modifications and expands the study of metabolically driven protein acylation in neurobiology and disease.

cell biology↗

Investigation of Neurons and Microglia from APOE3 Induced Pluripotent Stem Cells using Data-Independent Acquisitions on a ZenoTOF 7600

Induced pluripotent stem cell (iPSC)-derived neurons and microglia are valuable human models for studying neurodegenerative diseases. Specifically, the apolipoprotein E4 (APOE4) gene is a major genetic risk factor for late-onset Alzheimers disease. Apolipoprotein E (APOE) alleles E2, E3 and E4 can be beneficial, neutral, or increase the risk of Alzheimers disease (AD). Here, we developed a proteomic workflow using data-independent acquisitions to provide a quantitative mass spectrometric proteome analysis, and proteomic screening assays for brain-specific cell types derived from iPSC. Protein groups were quantified in APOE3 neurons and microglia, respectively, with [~]80% overlap. Cell type-specific markers and enriched pathways reflected the specialized functions of each cell type, such as synaptic signaling in neurons and immune and inflammatory responses in microglia. The neuron-specific markers included proteins APP, CALB1, CALB2, DLGs, GAP43, NEFL, MAPs; while microglial markers included proteins AIF1, CDs, MMP9, and ITGAM. Ultimately, the combination of robust iPSC differentiation and sensitive proteomic screening assays described here provides a valuable platform for probing the cellular mechanisms underlying neurological disorders. SignificanceThe quantification of dysregulated proteins and pathways in patient-derived neurons and microglia can provide insights into disease etiology and progression. More broadly, this DIA approach enables deep proteome profiling of unique iPSC-derived cell models, increasing their utility for investigating disease biology and therapeutic development. We focused on iPSC models from two important cell types of the brain, excitatory neurons and microglia. We integrated the proteomes of these two cell types. These tools provide robust biological and mass spectrometric screening tools for future therapeutic interventions using disease-relevant human brain cell types or brain organoid models. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/686128v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@d7dorg.highwire.dtl.DTLVardef@17594d3org.highwire.dtl.DTLVardef@7dd894org.highwire.dtl.DTLVardef@1606db_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPresentation of a proteomic workflow using data-independent acquisitions to monitor and screen proteomes of iPSC-derived brain cell types. C_LIO_LIQuick MS Assays to determine protein profiles of iPSC-derived neurons and microglia. C_LIO_LICharacterization of different cell type proteomes from APOE3 iPSCs. C_LIO_LIRevealing of neuron-specific markers and microglia-specific markers by mass spectrometry. C_LIO_LIStep-by-step instructions for the set-up of the DIA-MS assays C_LI

neuroscience↗

Novel high-resolution ion mobility mass spectrometry for site-specific quantification of the sirtuin-5 regulated kidney succinylome

Protein post-translational modifications (PTMs) dynamically regulate essential biological and cellular processes. Lysine succinylation changes the amino acid charge, potentially affecting protein structures and functions, and dysregulation of protein succinylation may lead to metabolic disorders. Proteome-wide succinylation quantification using proteomic tools remains challenging, especially due to the low abundance of succinylated peptides and the frequent presence of isomeric PTM forms. Ion mobility spectrometry workflows that can differentiate peptidoforms with different PTM distributions represent a powerful strategy to alleviate these challenges. Recently, a new Parallel Accumulation with Mobility Aligned Fragmentation (PAMAF) operating mode for high-resolution ion mobility-mass spectrometry (HRIM-MS) analysis based on the structures for lossless ion manipulation (SLIM) technology was introduced. Here, we first assessed the performance of PAMAF mode for protein succinylation analysis using synthetic succinylated peptides, demonstrating residue-level differentiation of co-eluting isomers and isobars and precise PTM site localization. We leveraged this novel approach to investigate succinylome remodeling in kidney tissues from wild-type and Sirtuin-5 (Sirt5) knock-out mice, a NAD+-dependent lysine de-succinylase. PAMAF acquisitions yielded [~]1,000 confidently identified and accurately quantified succinylated peptides and sites from mouse kidney. Sirt5 regulated succinylation of mitochondrial proteins involved in metabolic processes, including fatty acid oxidation, the tricarboxylic acid cycle, and propionate metabolism.

cell biology↗

Alzheimer's disease risk factor APOE4 exerts dimorphic effects on female bone

Individuals diagnosed with Alzheimers disease (AD) are at an increased risk of bone fractures. Conversely, a diagnosis of osteoporosis in women is the earliest known predictor for AD. However, mechanisms responsible for the coupled decline in cognitive and skeletal health remain unclear. Proteomic analysis of cortical bone from aged mice revealed neurological disease-associated proteins that are highly enriched in aged mouse bones, including apolipoprotein E (Apoe) and amyloid precursor protein. Further, Apoe localized specifically to bone-embedded osteocytes with expression twice as high in aged female bone as in young or male counterparts. In humans, APOE allele variants carry differing AD risk with age. To investigate APOE allelic roles in bone, we utilized a humanized APOE knock-in mouse model that expresses either the protective APOE2, the neutral APOE3, or the AD risk factor APOE4, and analyzed bone and hippocampus from the same mice. APOE4 exerted strong sex-specific effects on the bone transcriptome and proteome, relative to APOE2 or APOE3. Interestingly, the APOE4-associated perturbation in the female bone proteome was more pronounced than the corresponding alterations observed in the hippocampus. APOE4 protein causes bone fragility in females, but not males, even without changes in cortical bone structure. These bone quality deficits arose from suppression of osteocyte perilacunocanalicular remodeling. We find that APOE4 is a new molecular culprit capable of disrupting osteocyte maintenance of bone quality as early as midlife in a manner that disproportionately affects females. These findings highlight osteocytes as potential targets for early diagnosis of age-related cognitive impairment, and treatment for bone fragility, in females.

molecular biology↗

A multi-omics analysis of human fibroblasts overexpressing an Alu transposon reveals widespread disruptions in aging-associated pathways

During aging and cellular senescence, repetitive elements are frequently transcriptionally derepressed across species and cell types. Among these, the most abundant repeats by copy number in the human genome are Alu retrotransposons. Though Alu elements are often studied for their mutagenic potential, there is increasing appreciation for their contributions to other biological functions, including pro-inflammatory signaling and mitochondrial dysfunction. However, a comprehensive analysis of Alu-driven molecular changes remains to be conducted, and Alus potential contributions to aging features remain incompletely characterized. Here, we show that overexpression of an AluJb transposon in human primary IMR-90 fibroblasts leads to large-scale alterations across the transcriptome, cellular proteome, and secretome. Functional genomics analyses reveal alterations in aging pathways, broadly, and mitochondrial metabolism, proteostasis, cell cycle, and extracellular matrix pathways, more specifically. Our results demonstrate that Alu transcriptional upregulation is sufficient to drive widespread disruptions to cellular homeostasis that mirror aging-associated alterations.

genomics↗

A Fibroblast State Choreographs an Epithelial YAP-dependent Regenerative Program Essential to (Pre)malignancy via ECM-mediated Mechanotransduction

Chronic lung injury generates metaplasia which occasionally, but ominously, progresses to squamous dysplasia and squamous lung cancer. To identify mechanisms through which disrupted tissue homeostasis contributes to malignant initiation and progression, we used in vivo and in vitro heterotypic recombinant models of human bronchial epithelial cells (hBECs) and fibroblasts. We demonstrate that injury-associated TGF-{beta} signaling creates a fibroblast state dependent upon HSP47 upregulation. These fibroblasts accumulated collagen, thus elevating tissue stiffness and activating mechanosignaling that sustained YAP-dependent embryonic-like, pro-malignant activities in adjacent hBECs. This Stress/Tension-Instructive Fibroblast (STIF) state, exhibited by stressed fibroblasts in premalignant and malignant lesions across multiple cancer types, was sufficient to reprogram disease-free hBECs to metaplasia and to drive hBECs with compromised tumor suppressor function to dysplasia, yet could be inhibited and reversed. STIFs suffice to activate epithelial phenotypes reminiscent of oncogene-mediated cell transformation and induce (pre)malignancy via increased force transmission, providing novel targets for prevention. Statement of significanceTissue injury creates a regenerative pro-tumorigenic Stress/Tension-Instructive Fibroblast (STIF) state which is sufficient to activate a YAP-dependent, pre-malignant program to induce or unmask pre-cancerous phenotypes in epithelial cells through mechanotransduction. Inhibition of STIF activity or mechanosignaling prevents metaplasia and progression to dysplasia. HighlightsO_LITissue injury creates a pro-tumorigenic Stress/Tension-Instructive Fibroblast (STIF) state in multiple organs that precedes and persists through cancer C_LIO_LISTIF signaling alone, working through fibroblasts and not epithelial cells, is sufficient to activate embryonic-like plasticity and induce epithelial pre-cancerous metaplastic lesions C_LIO_LISTIFs program (pre)malignant phenotypes in adjacent epithelial cells through mechanosignaling by activating YAP prior to tumor formation C_LIO_LIInhibiting STIFs or mechanosignaling prevents/reverts metaplasia and prevents progression to dysplasia C_LI

cancer biology↗

Age-Dependent Increase and Dysregulation of the Splicing Machinery in Acute Myeloid Leukemia of Older Patients

Acute myeloid leukemia (AML) is characterized by age-related resistance to therapy and poor outcomes. Initial remission rates vary from 30-80% with current therapies, but relapses are common. The 5-year survival is 30% overall but less than 10% for those 60 years of age or older. Resistance to current therapies is the central clinical challenge. This study investigated how age influences the AML proteome in patients with increasing age. We analyzed bone marrow or leukapheresis samples from 14 AML patients, including 9 older ([≥] 66 years) and 5 younger ([≤] 58 years) individuals, using proteomic technologies (Data-Independent Acquisitions, DIA-MS). We identified 4,471 protein groups, of which 889 exhibited significant changes and regulation between the two age groups. Proteins upregulated in older patients were predominantly associated with DNA transcription and translation, while downregulated proteins were linked to metabolic, catabolic, and extracellular matrix or structural processes. Significant changes in older samples affected the splicing machinery. Significant alterations in several of these factors, suggests either dysregulated or elevated splicing activity and reduced transcription initiation factor activity in older AML patients. Our study provides novel insights into molecular changes during AML arising in the older adult, highlighting the importance of age-specific therapeutic strategies. Statement of Significance of the StudyThis study is significant as it investigates a devastating and highly aggressive cancer, acute myeloid leukemia (AML), which often presents in patients with very low 5-year survival rates (32%), however which is even lower in patients over 60 years old (10%). This study really focuses on the complications and severe outcome in older patients which is not well understood. This is particularly relevant as older AML patients often do not respond to the available treatments. Understanding the pathways and protein profiles that are dysregulated in older AML patients compared to younger AML patients may in the future help to develop more customized therapeutic interventions or allow for some precision treatments. AML patients were stratified into cohort groups by age (older vs younger) and the proteomic profiling highlighted the particularly strong dysregulation of the splicing machinery in the older AML patients. While alternative splicing is often observed in cancers, here the emphasis is pointed to the fact that the dysregulation of the spliceosome and other pathways are greatly exacerbated in the older AML patients, likely leading to even worse outcomes and poor response to treatments in the older patients.

cancer biology↗

Multiplexed Targeted Spatial Mass Spectrometry Imaging Assays to monitor lipids and NAD+ metabolites in CD38 knockout mice exhibiting improved metabolism.

Mass spectrometry imaging (MSI) is a rapidly advancing technology that provides mapping of the spatial molecular landscape of tissues for a variety of analytes. Matrix-assisted laser desorption/ionization (MALDI)-MSI is commonly employed, however, confident in situ identification and accurate quantification of analytes remain challenging. We present a novel imaging methodology combining trapped ion mobility spectrometry (TIMS)-based parallel accumulation-serial fragmentation (PASEF) with MALDI ionization for targeted imaging parallel reaction monitoring (iprm-PASEF). We investigated the spatial distribution of lipids and metabolites in liver tissues from male wild-type and CD38 knockout mice (CD38-/-). CD38, an enzyme involved in nicotinamide adenine dinucleotide (NAD) metabolism, significantly influences liver metabolic function and contributes to age-related NAD decline. Although CD38 deletion previously was linked to improved metabolic phenotypes, the underlying spatial metabolic mechanisms are poorly understood. The spatial iprm-PASEF workflow enabled confident identification and differentiation of lipid isomers at the MS2 fragment ion level and confirmed increased NAD+ and decreased adenosine diphosphate ribose (ADPR), a by-product of NAD+ hydrolysis, in CD38-/- livers. This approach provided confident, specific, and robust MS2-based identification and quantification of fragment ions in spatial MSI experiments. Additionally, the innovative iprm-PASEF opens unprecedented opportunities for spatial metabolomics and lipidomics, offering spatially resolved insights into molecular mechanisms.

cell biology↗

Odd-chain dicarboxylic acid feeding recapitulates the biochemical phenotype of glutaric aciduria type-1 in mice

Glutaric aciduria type-1 (GA1) is an inherited mitochondrial neurometabolic disorder with a poorly understood pathogenesis and unmet medical needs. GA1 can be diagnosed via its hallmark biochemical signature consisting of glutaric aciduria, 3-hydroxyglutaric aciduria, and increased plasma glutarylcarnitine. These glutaryl-CoA-derived metabolites are thought to originate solely in the mitochondria. Here, we demonstrate that wild-type mice fed an 11-carbon odd-chain dicarboxylic acid (undecanedioic acid, DC11) recreates the biochemical phenotype of GA1. Odd-chain dicarboxylic acids like DC11 are not present in food but can arise from several endogenous processes, such as lipid peroxidation and fatty acid {omega}-oxidation. DC11 is chain-shortened in peroxisomes to glutaryl (DC5)-CoA, which then gives rise to the GA1-like pattern of DC5 metabolites in urine, tissues, and blood. Glutaric acid released from peroxisomes during DC11 chain-shortening can enter mitochondria, be activated to CoA by the enzyme succinyl-CoA:glutarate-CoA transferase (SUGCT), and become substrate for glutaryl-CoA dehydrogenase (GCDH), the enzyme that is mutated in GA1. Our data provide proof-of-concept that the generation of dicarboxylic acids by {omega}-oxidation, which is stimulated during the same catabolic states known to trigger acute encephalopathy in GA1, may exacerbate disease by increasing the glutaryl-CoA substrate load in mitochondria.

biochemistry↗

Proteomic Analysis of Breast Cancer Subtypes Identifies Stromal Contributions that Dictate Aggressive Malignant Behavior

Breast cancer manifests as multiple subtypes with distinct patient outcomes and treatment strategies. Here, we optimized proteomic analysis of Formalin-Fixed Paraffin-Embedded (FFPE) specimens from patients diagnosed with five breast cancer subtypes, luminal A, luminal B, Her2, triple negative (TNBC) and metaplastic breast cancers (MBC), and from disease-free individuals undergoing reduction mammoplasty (RM). We identified and quantified [~]6,000 protein groups (with >2 peptides per protein) with significant changes in over 26% of proteins comparing each cancer subtype with control RM. Stringent statistical filters allowed us to deeply mine 576 significant conserved protein changes shared by all subtypes and protein changes unique to each subtype. The most aggressive subtype, MBC, revealed exacerbated stromal stress responses, as illustrated by a collagenolytic extracellular matrix (ECM) and immune participation biased towards neutrophils and eosinophils. Immunostaining of breast tissue sections confirmed differences across subtypes, in particular, a strong upregulation of SERPINH1, neutrophil-specific myeloperoxidase and eosinophil cationic protein in MBC. In summary, we present deep proteomic, digitalized protein abundance profiles, generated from FFPE breast cancer tissues, that revealed significant changes in ECM and cellular proteins. Statement of Significance of the StudyThis study is significant as it discovered deep proteomic signatures for the highly aggressive and malignant metaplastic breast cancer (MCB) which is now considered a fifth subtype based upon its remarkable intra-tumoral heterogeneity that illustrates its unique cell plasticity. To efficiently analyze formalin-fixed paraffin-embedded (FFPE) breast tissues from patients with different breast cancer subtypes and disease-free individuals, we optimized a novel workflow in which we combined paraffinization and Folch extraction. We identified and confidently quantified [~]6,000 protein groups. We were able to find robust changes in extracellular matrix (ECM) with cancer, even though no ECM enrichments were performed. Interestingly, despite the relatively small human cohort size (42 patients), distinct protein signatures emerged throughout all cancer subtypes - common and unique - with remarkable statistical significance for many cancer-relevant proteins and pathways. This Pilot study indicates the hypothesis that an altered stroma can dictate epithelial tumor cell fate. We also observed that MBC was characterized by an especially immunosuppressed tumor environment. We do note the limitation of the relatively small cohort size of our study, and in the future additional patient cohorts will be needed to further validate our findings.

cancer biology↗

Inhibition of the metalloprotease ADAM19 as a Novel Senomorphic Strategy to Ameliorate gut permeability and senescence markers by modulating senescence-associated secretory phenotype (SASP)

Accumulation of DNA damage can accelerate aging through cellular senescence. Previously, we established a Drosophila model to investigate the effects of radiation-induced DNA damage on the intestine. In this model, we examined irradiation-responsive senescence in the fly intestine. Through an unbiased genome-wide association study (GWAS) utilizing 156 strains from the Drosophila Genetic Reference Panel (DGRP), we identified meltrin {beta} (the drosophila orthologue of mammalian ADAM19) as a potential modulator of the senescence-associated secretory phenotype (SASP). Knockdown of meltrin {beta} resulted in reduced gut permeability, DNA damage, and expression of the senescence marker {beta}-galactosidase (SA-{beta}-gal) in the fly gut following irradiation. Additionally, inhibition of ADAM19 in mice using batimastat-94 reduced gut permeability and inflammation in the gut. Our findings extend to human primary fibroblasts, where ADAM19 knockdown or pharmacological inhibition decreased expression of specific SASP factors and SA-{beta}-gal. Furthermore, proteomics analysis of the secretory factor of senescent cells revealed a significant decrease in SASP factors associated with the ADAM19 cleavage site. These data suggest that ADAM19 inhibition could represent a novel senomorphic strategy.

molecular biology↗

Substrate Stiffness Dictates Unique Doxorubicin-induced Senescence-associated Secretory Phenotypes and Transcriptomic Signatures in Human Pulmonary Fibroblasts

Cells are subjected to dynamic mechanical environments which impart forces and induce cellular responses. In age-related conditions like pulmonary fibrosis, there is both an increase in tissue stiffness and an accumulation of senescent cells. While senescent cells produce a senescence-associated secretory phenotype (SASP), the impact of physical stimuli on both cellular senescence and the SASP is not well understood. Here, we show that mechanical tension, modeled using cell culture substrate rigidity, influences senescent cell markers like SA-{beta}-gal and secretory phenotypes. Comparing human primary pulmonary fibroblasts (IMR-90) cultured on physiological (2 kPa), fibrotic (50 kPa), and plastic (approximately 3 GPa) substrates, followed by senescence induction using doxorubicin, we identified unique high-stiffness-driven secretory protein profiles using mass spectrometry and transcriptomic signatures, both showing an enrichment in collagen proteins. Consistently, clusters of p21+ cells are seen in fibrotic regions of bleomycin induced pulmonary fibrosis in mice. Computational meta-analysis of single-cell RNA sequencing datasets from human interstitial lung disease confirmed these stiffness SASP genes are highly expressed in disease fibroblasts and strongly correlate with mechanotransduction and senescence-related pathways. Thus, mechanical forces shape cell senescence and their secretory phenotypes.

cell biology↗

Loss of long-chain acyl-CoA dehydrogenase protects against acute kidney injury

Proximal tubular epithelial cells (PTECs) are particularly vulnerable to acute kidney injury (AKI). While fatty acids are the preferred energy source for PTECs via fatty acid oxidation (FAO), FAO-mediated H2O2 production in mitochondria has been shown to be a major source of oxidative stress. We have previously shown that a mitochondrial flavoprotein, long-chain acyl-CoA dehydrogenase (LCAD), which catalyzes a key step in mitochondrial FAO, directly produces H2O2 in vitro. Further we have established that loss of a lysine deacylase, Sirtuin 5 (Sirt5-/-), induces hypersuccinylation and inhibition of mitochondrial FAO genes to stimulate peroxisomal FAO and to protect against AKI. However, the role of LCAD has yet to be determined. Mass spectrometry data acquisition revealed that LCAD is hypersuccinylated in Sirt5-/- kidneys after AKI. Following two distinct models of AKI, cisplatin treatment or renal ischemia/reperfusion (IRI), LCAD knockout mice (LCAD-/-) demonstrated renoprotection against AKI. Specifically, LCAD-/- kidneys displayed mitigated renal tubular injury, decreased oxidative stress, preserved mitochondrial function, enhanced peroxisomal FAO, and decreased ferroptotic cell death. LCAD deficiency confers protection against two distinct models of AKI. This suggests a therapeutically attractive mechanism whereby preserved mitochondrial respiration as well as enhanced peroxisomal FAO by loss of LCAD mediates renoprotection against AKI.

pathology↗

A comprehensive multi-omics signature of doxorubicin-induced cellular senescence in the postmenopausal human ovary.

A major aging hallmark is the accumulation of cellular senescence burden. Over time senescent cells contribute to tissue deterioration through chronic inflammation and fibrosis driven by the Senescence-Associated Secretory Phenotype (SASP). The human ovary is one of the first organs to age, and prominent age-related fibroinflammation within the ovarian microenvironment is consistent with the presence of senescent cells, but these cells have not been characterized in the human ovary. We thus established a doxorubicin-induced model of cellular senescence to establish a "senotype" (gene/protein signature of a senescence cell state) for ovarian senescent cells. Explants of human postmenopausal ovarian cortex and medulla were treated with doxorubicin for 24 hours followed by culture for up to 10 days in a doxorubicin-free medium. Tissue viability was confirmed by histology, lack of apoptosis, and continued glucose consumption by explants. Single nuclei sequencing and proteomics revealed an unbiased signature of ovarian senescence. We identified distinct senescence profiles for the cortex and medulla, driven predominantly by epithelial and stromal cells. Proteomics uncovered subregional differences in addition to 120 proteins common to the cortex and medulla SASP. Integration of transcriptomic and proteomic analyses revealed 26 shared markers, defining a senotype of doxorubicin-induced senescence unique to the postmenopausal ovary. A subset of these proteins: Lumican, SOD2, MYH9, and Periostin were mapped onto native tissue to reveal compartment-specific localization. This senotype will help determine the role of cellular senescence in ovarian aging, inform biomarker development to identify, and therapeutic applications to slow or reverse ovarian aging, senescence, and cancer.

molecular biology↗

Mass Spectrometry Imaging of the Subchondral Bone in Osteoarthritis Reveals Tissue Remodeling of Extracellular Matrix Proteins that Precede Cartilage Loss

Osteoarthritis (OA) is a degenerative condition of the skeletal extracellular matrix (ECM) marked by the loss of articular cartilage and changes to subchondral bone homeostasis. Treatments for OA beyond full joint replacement are lacking primarily due to gaps in molecular knowledge of the biological drivers of disease. Mass Spectrometry Imaging (MSI) enables molecular spatial mapping of the proteomic landscape of tissues. Histologic sections of human tibial plateaus from knees of human OA patients and cadaveric controls were treated with collagenase III to target ECM proteins prior to MS Imaging of bone and cartilage proteins using a timsTOF fleX mass spectrometer. Spatial MSI data of the knee were processed and automatically segmented identifying distinct areas of knee joint damage. ECM peptide markers were compared between i) the medial halves of OA patient joints and the medial side of non-OA (cadaveric) joints, and ii) between the same medial OA tissues and their corresponding, less OA impacted, lateral joint halves. Distinct peptide signatures distinguished OA medial tissues from the cadaveric medial and OA lateral tissues (AUROC >0.85). Overall, 31 peptide candidates from ECM proteins, including Collagen alpha-1(I), Collagen alpha-1(III), and surprisingly, Collagen alpha-1(VI) and Collagen alpha-3(VI), exhibited significantly elevated abundance in diseased tissues. Highly specific hydroxyproline-containing collagen peptides, mainly from collagen type I, dominated OA subchondral bone directly under regions of lost cartilage. The identification of specific protein markers for subchondral bone remodeling in OA advances our molecular understanding of disease progression in OA and provides potential new biomarkers for OA detection and disease grading.

cell biology↗

Exosomes Released from Senescent Cells and Circulatory Exosomes Isolated from Human Plasma Reveal Aging-associated Proteomic and Lipid Signatures

Senescence emerged as significant mechanism of aging and age-related diseases, offering an attractive target for clinical interventions. Senescent cells release a senescence-associated secretory phenotype (SASP), including exosomes that may act as signal transducers between distal tissues, propagating secondary or bystander senescence and signaling throughout the body. However, the composition of exosome SASP remains underexplored, presenting an opportunity for novel unbiased discovery. We present a detailed proteomic and lipidomic analysis of exosome SASP using mass spectrometry from human plasma from young and older individuals and from tissue culture of senescent primary human lung fibroblasts. We identified [~]1,300 exosome proteins released by senescent cells induced by three different senescence inducers. In parallel, a human plasma cohort from young (20-26 years) and old (65-74 years) individuals revealed over 1,350 exosome proteins and 171 plasma exosome proteins were regulated when comparing old vs young individuals. Of the age-regulated plasma exosome proteins, we observed 52 exosome SASP factors that were also regulated in exosomes from the senescent fibroblasts, including serine protease inhibitors (SERPINs), Prothrombin, Coagulation factor V, Plasminogen, and Reelin. 247 lipids were identified in exosome samples. Following senescence induction, identified phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins increased significantly indicating cellular membrane changes. Interestingly, significantly changed proteins were related to extracellular matrix remodeling and inflammation, both potentially detrimental pathways that can damage surrounding tissues and even induce secondary senescence. Our findings reveal mechanistic insights and potential senescence biomarkers, enabling a better approach to surveilling the senescence burden in the aging population and offering therapeutic targets for interventions.

cell biology↗

Ketogenic diet administration later in life improves memory and regulates the synaptic cortical proteome through the cAMP/PKA signaling pathway in aging mice.

Aging is a complex biological process that compromises brain function and neuronal network activity, leading to cognitive decline and synaptic dysregulation. In recent years, a cyclic Ketogenic Diet (KD) has emerged as a potential treatment to ameliorate cognitive decline by improving memory in aged mice after long-term administration. However, the molecular mechanisms that govern such changes remains unclear. Additionally, whether short-term cyclic KD administration later in life preserves memory has not been addressed in detail. Accordingly, here we investigated how a short-term cyclic KD starting at 20-23 months-old regulates brain function of aged mice. Behavioral testing and long-term potentiation (LTP) recordings revealed that a cyclic KD improves working memory and hippocampal LTP in 24-27 months-old mice after 16 weeks of treatment. Moreover, the diet also promotes higher dendritic arborization complexity and dendritic spine density in the prefrontal cortex. Furthermore, to elucidate the molecular mechanisms underlying the memory improvements elicited by a cyclic KD, cortical synaptosomes of aged mice fed with this diet for 1 year were analyzed by mass spectrometry. Bioinformatics analysis revealed that long-term cyclic KD administration predominantly modulates the presynaptic compartment by inducing changes in the cAMP/PKA signaling pathway, the synaptic vesicle cycle, and the actin/microtubule cytoskeleton. To test these findings in vivo, synaptic proteins from cortices of 24-27-month-old mice fed with control or cyclic KD for 16 weeks were analyzed by western blot. Interestingly, increased Brain Derived Neurotrophic Factor abundance, MAP2 phosphorylation and PKA activity were observed. Overall, we show that a cyclic KD regulates brain function and memory even when it is administered at late mid-life and significantly triggers several molecular features of long-term administration, including the PKA signaling pathway and cytoskeleton dynamics, thus promoting synaptic plasticity at advanced age.

neuroscience↗