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Following peripheral nerve injury, successful axonal growth and functional recovery require Schwann cell (SC) reprogramming into a reparative phenotype, a process dependent upon c‐Jun transcription factor activation. Unfortunately, axonal regeneration is greatly impaired in aged organisms and following chronic denervation, which can lead to poor clinical outcomes. While diminished c‐Jun expression in SCs has been associated with regenerative failure, it is unclear whether the inability to maintain a repair state is associated with the transition into an axonal growth inhibition phenotype. We here find that reparative SCs transition into a senescent phenotype, characterized by diminished c‐Jun expression and secretion of inhibitory factors for axonal regeneration in aging and chronic denervation. In both conditions, the elimination of senescent SCs by systemic senolytic drug treatment or genetic targeting improved nerve regeneration and functional recovery, increased c‐Jun expression and decreased nerve inflammation. This work provides the first characterization of senescent SCs and their influence on axonal regeneration in aging and chronic denervation, opening new avenues for enhancing regeneration and functional recovery after peripheral nerve injuries. Synopsis Following nerve damage of aged nerves or delayed repair, Schwann cells (SCs) are less able to transition into the reparative phenotype necessary for axonal regrowth. Here, SCs from chronically denervated or aged conditions are characterized to better understand their influence on regeneration. Schwann cells from aged and chronic denervation mice transition into a senescent phenotype. Chronically denervated and aged nerves present a proinflammatory profile and upregulation of Senescent‐associated Secretory Phenotype‐related genes. Secreted factors from senescent SCs inhibit axonal growth of sensory neurons. Senescent cell elimination restores a reparative SC phenotype and decreases injury‐induced nerve inflammation in aged and chronically denervated animals. Elimination of senescent cells improves nerve regeneration in aging and chronic denervation. Graphical Abstract Following nerve damage of aged nerves or delayed repair, Schwann cells (SCs) are less able to transition into the reparative phenotype necessary for axonal regrowth. Here, SCs from chronically denervated or aged conditions are characterized to better understand their influence on regeneration.

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.

Dicarboxylic fatty acids are generated in the liver and kidney in a minor pathway called fatty acid ω-oxidation. The effects of consuming dicarboxylic fatty acids as an alternative source of dietary fat have not been explored. Here, we fed dodecanedioic acid, a 12-carbon dicarboxylic (DC12), to mice at 20% of daily caloric intake for 9 weeks. DC12 increased metabolic rate, reduced body fat, reduced liver fat, and improved glucose tolerance. We observed DC12-specific breakdown products in liver, kidney, muscle, heart, and brain, indicating that oral DC12 escaped first-pass liver metabolism and was utilized by many tissues. In tissues expressing the \"a\" isoform of acyl-CoA oxidase-1 (ACOX1), a key peroxisomal fatty acid oxidation enzyme, DC12 was chain shortened to the TCA cycle intermediate succinyl-CoA. In tissues with low peroxisomal fatty acid oxidation capacity, DC12 was oxidized by mitochondria. In vitro, DC12 was catabolized even by adipose tissue and was not stored intracellularly. We conclude that DC12 and other dicarboxylic acids may be useful for combatting obesity and for treating metabolic disorders.

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.

Introduction Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by an expansion of the CAG trinucleotide repeat in the Huntingtin gene ( HTT ). Immune activation is abundant in the striatum of HD patients. Detection of active microglia at presymptomatic stages suggests that microgliosis is a key early driver of neuronal dysfunction and degeneration. Recent studies showed that deletion of  Tyrobp , a microglial protein, ameliorates neuronal dysfunction in Alzheimer’s disease amyloidopathy and tauopathy mouse models while decreasing components of the complement subnetwork. Objective While TYROBP/DAP12-mediated microglial activation is detrimental for some diseases such as peripheral nerve injury, it is beneficial for other diseases. We sought to determine whether the TYROBP network is implicated in HD and whether  Tyrobp  deletion impacts HD striatal function and transcriptomics. Methods To test the hypothesis that  Tyrobp  deficiency would be beneficial in an HD model, we placed the Q175 HD mouse model on a  Tyrobp -null background. We characterized these mice with a combination of behavioral testing, immunohistochemistry, transcriptomic and proteomic profiling. Further, we evaluated the gene signature in isolated Q175 striatal microglia, with and without  Tyrobp. Results Comprehensive analysis of publicly available human HD transcriptomic data revealed that the TYROBP network is overactivated in the HD putamen. The Q175 mice showed morphologic microglial activation, reduced levels of post-synaptic density-95 protein and motor deficits at 6 and 9 months of age, all of which were ameliorated on the  Tyrobp -null background. Gene expression analysis revealed that lack of  Tyrobp  in the Q175 model does not prevent the decrease in the expression of striatal neuronal genes but reduces pro-inflammatory pathways that are specifically active in HD human brain, including genes identified as detrimental in neurodegenerative diseases, e.g. C1q and members of the Ccr5 signaling pathway. Integration of transcriptomic and proteomic data revealed that astrogliosis and complement system pathway were reduced after  Tyrobp  deletion, which was further validated by immunofluorescence analysis. Conclusions Our data provide molecular and functional support demonstrating that  Tyrobp  deletion prevents many of the abnormalities in the HD Q175 mouse model, suggesting that the  Tyrobp  pathway is a potential therapeutic candidate for Huntington’s disease.

Dietary restriction (DR) delays aging, but the mechanism remains unclear. We identified polymorphisms in mtd , the fly homolog of OXR1 , which influenced lifespan and mtd expression in response to DR. Knockdown in adulthood inhibited DR-mediated lifespan extension in female flies. We found that mtd / OXR1 expression declines with age and it interacts with the retromer, which regulates trafficking of proteins and lipids. Loss of mtd / OXR1 destabilized the retromer, causing improper protein trafficking and endolysosomal defects. Overexpression of retromer genes or pharmacological restabilization with R55 rescued lifespan and neurodegeneration in mtd -deficient flies and endolysosomal defects in fibroblasts from patients with lethal loss-of-function of OXR1 variants. Multi-omic analyses in flies and humans showed that decreased Mtd/OXR1 is associated with aging and neurological diseases. mtd/OXR1 overexpression rescued age-related visual decline and tauopathy in a fly model. Hence, OXR1 plays a conserved role in preserving retromer function and is critical for neuronal health and longevity. Dietary restriction promotes healthy brain aging, but the mechanism is unknown. Here, the authors show that OXR1 is upregulated by dietary restriction and confers age-related neuroprotection by maintaining retromer-mediated protein and lipid trafficking.

Sirtuin-2 (Sirt2), an NAD+-dependent lysine deacylase enzyme, has previously been implicated as a regulator of glucose metabolism, but the specific mechanisms remain poorly defined. Here, we observed that Sirt2−/− males, but not females, have decreased body fat, moderate hypoglycemia upon fasting, and perturbed glucose handling during exercise compared to wild type controls. Conversion of injected lactate, pyruvate, and glycerol boluses into glucose via gluconeogenesis was impaired, but only in males. Primary Sirt2−/− male hepatocytes exhibited reduced glycolysis and reduced mitochondrial respiration. RNAseq and proteomics were used to interrogate the mechanisms behind this liver phenotype. Loss of Sirt2 did not lead to transcriptional dysregulation, as very few genes were altered in the transcriptome. In keeping with this, there were also negligible changes to protein abundance. Site-specific quantification of the hepatic acetylome, however, showed that 13% of all detected acetylated peptides were significantly increased in Sirt2−/− male liver versus wild type, representing putative Sirt2 target sites. Strikingly, none of these putative target sites were hyperacetylated in Sirt2−/− female liver. The target sites in the male liver were distributed across mitochondria (44%), cytoplasm (32%), nucleus (8%), and other compartments (16%). Despite the high number of putative mitochondrial Sirt2 targets, Sirt2 antigen was not detected in purified wild type liver mitochondria, suggesting that Sirt2’s regulation of mitochondrial function occurs from outside the organelle. We conclude that Sirt2 regulates hepatic protein acetylation and metabolism in a sex-specific manner.

Lysine succinylation, and its reversal by sirtuin-5 (SIRT5), is known to modulate mitochondrial fatty acid β-oxidation (FAO). We recently showed that feeding mice dodecanedioic acid, a 12-carbon dicarboxylic acid (DC12) that can be chain-shortened four rounds to succinyl-CoA, drives high-level protein hypersuccinylation in the peroxisome, particularly on peroxisomal FAO enzymes. However, the ability of SIRT5 to reverse DC12-induced peroxisomal succinylation, or to regulate peroxisomal FAO in this context, remained unexplored. Here, we showed that feeding DC12 strongly recruits SIRT5 into hepatic peroxisomes. Knocking out SIRT5 impaired peroxisomal FAO as evidenced by reduced 14C-DC12 flux in liver homogenates and elevated levels of partially shortened DC12 catabolites in urine. Further, mass spectrometry revealed a trend toward less peroxisomal protein succinylation in SIRT5 knockout liver. This is consistent with a reduced flux of DC12 through the peroxisomal FAO pathway, thereby reducing the production of the succinyl-CoA that chemically reacts with lysine residues to produce protein succinylation. Mass spectrometry comparisons of site-level succinylation in wildtype and SIRT5 knockout liver did not reveal any clear pattern of SIRT5 target sites in the peroxisome after DC12 feeding. However, SIRT5 co-immunoprecipitated with 15 peroxisomal proteins, including the key peroxisomal FAO enzymes acyl-CoA oxidase-1 and enoyl-CoA/3-hydroxyacyl-CoA dehydrogenase (EHHADH). In vitro, recombinant SIRT5 partially desuccinylated chemically modified recombinants ACOX1a, ACOX1b, and EHHADH. Desuccinylation by SIRT5 had no effect on enzyme activity for ACOX1a and EHHADH. For ACOX1b, SIRT5-mediated desuccinylation decreased activity by ~15%. Possible interpretations of these data are discussed.

The renal tubular epithelial cells (RTECs) are particularly vulnerable to acute kidney injury (AKI). While fatty acids are the preferred energy source for RTECs 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. Furthermore, we showed that renal LCAD becomes hyposuccinylated during AKI. Here, we demonstrated that succinylation of recombinant LCAD protein suppresses the production of H2O2. Following 2 distinct models of AKI, cisplatin treatment or renal ischemia/reperfusion injury (IRI), LCAD-/- mice demonstrated renoprotection. 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 2 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.

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 TGFβ signaling (TβRIIocy−/−) that suppresses PLR. The control aged bone displayed decreased TGFβ signaling and PLR, but aging did not worsen the existing PLR suppression in male TβRIIocy−/− 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 TGFβ. We determined that the decline in bone quality with age arises from the loss of osteocyte function and the loss of TGFβ-dependent maintenance of collagen integrity.