Metabolite accumulation from oral NMN supplementation drives aging-specific kidney inflammation.

The mitochondrial-rich renal tubule cells are key regulators of blood homeostasis via excretion and reabsorption of metabolic waste. With age, tubules are subject to increasing mitochondrial dysfunction and declining nicotinamide adenine dinucleotide (NAD+) levels, both hampering ATP production efficiency. We tested two mitochondrial interventions in young (6-mo) and aged (26-mo) adult male mice: elamipretide (ELAM), a tetrapeptide in clinical trials that improves mitochondrial structure and function, and nicotinamide mononucleotide (NMN), an NAD+ intermediate and commercially available oral supplement. Kidneys were analyzed from young and aged mice after eight weeks of treatment with ELAM (3 mg/kg/day), NMN (300 mg/kg/day), or from aged mice treated with the two interventions combined (ELAM+NMN). We hypothesized that combining pharmacologic treatments to ameliorate mitochondrial dysfunction and boost NAD+ levels, would more effectively reduce kidney aging than either intervention alone. Unexpectedly, in aged kidneys, NMN increased expression of genetic markers of inflammation (IL-1-beta; and Ccl2) and tubule injury (Kim-1). Metabolomics of endpoint sera showed that NMN-treated aged mice had higher circulating levels of uremic toxins than either aged controls or young NMN-treated mice. ELAM+NMN-treated aged mice accumulated uremic toxins like NMN-only aged mice, but reduced IL-1-beta; and Ccl2 kidney mRNA. This suggests that pre-existing mitochondrial dysfunction in aged kidney underlies susceptibility to inflammatory signaling with NMN supplementation in aged, but not young, mice. These findings demonstrate age and tissue dependent effects on downstream metabolic accumulation from NMN and highlight the need for targeted analysis of aged kidneys to assess the safety of anti-aging supplements in older populations.

Comparative Levels of Urinary Biomarkers of Renal Injury and Inflammation Among Patients With Diabetic Nephropathy With or Without Hyperuricemia.

BACKGROUND: The association between hyperuricemia and development of progressive chronic kidney disease has received increasing attention in recent years. Recent preclinical studies have shown that non-crystalline uric acid can induce renal-specific arteriolopathy, leading to renal injury and tubulointerstitial inflammation. METHODS: We conducted a open-label cross-sectional study of 25 patients with chronic kidney disease stage III (estimated glomerular filtration rate [eGFR], 7.0 mg/dL) levels of serum uric acid. To determine the correlation between hyperuricemia on urinary protein levels and renal disease progression, we retrospectively compared urine protein and eGFR data between the 2 groups. RESULTS: Eleven patients with normal uric acid levels and 14 with hyperuricemia were enrolled. Urinary levels of both kidney injury molecule-1 (KIM-1) and monocyte chemoattractant protein-1 (MCP-1) were significantly higher in patients with hyperuricemia. Among the normouricemic White and African American (AA) subgroups, there was no difference in KIM-1 or MCP-1 levels, whereas KIM-1 levels were significantly higher among hyperuricemic AA patients with hyperuricemia. Urinary protein was significantly higher between Whites and AA patients with serum uric acid level >7.0 mg/dL as well as patients with urinary KIM-1 levels >1000 pg/mg Cr. A trend toward a more rapid decline in eGFR was noted among hyperuricemic AAs; however, this trend was not statistically significant. CONCLUSIONS: Patients with type 2 diabetic nephropathy and persistently elevated serum uric acid levels express higher levels of both KIM-1 and MCP-1 reflective of on-going renal injury and inflammation.

The mitochondrially targeted peptide elamipretide (SS-31) improves ADP sensitivity in aged mitochondria by increasing uptake through the adenine nucleotide translocator (ANT).

Aging muscle experiences functional decline in part mediated by impaired mitochondrial ADP sensitivity. Elamipretide (ELAM) rapidly improves physiological and mitochondrial function in aging and binds directly to the mitochondrial ADP transporter ANT. We hypothesized that ELAM improves ADP sensitivity in aging leading to rescued physiological function. We measured the response to ADP stimulation in young and old muscle mitochondria with ELAM treatment, in vivo heart and muscle function, and compared protein abundance, phosphorylation, and S-glutathionylation of ADP/ATP pathway proteins. ELAM treatment increased ADP sensitivity in old muscle mitochondria by increasing uptake of ADP through the ANT and rescued muscle force and heart systolic function. Protein abundance in the ADP/ATP transport and synthesis pathway was unchanged, but ELAM treatment decreased protein s-glutathionylation incuding of ANT. Mitochondrial ADP sensitivity is rapidly modifiable. This research supports the hypothesis that ELAM improves ANT function in aging and links mitochondrial ADP sensitivity to physiological function. ELAM binds directly to ANT and ATP synthase and ELAM treatment improves ADP sensitivity, increases ATP production, and improves physiological function in old muscles. ADP (adenosine diphosphate), ATP (adenosine triphosphate), VDAC (voltage-dependent anion channel), ANT (adenine nucleotide translocator), H+ (proton), ROS (reactive oxygen species), NADH (nicotinamide adenine dinucleotide), FADH2 (flavin adenine dinucleotide), O2 (oxygen), ELAM (elamipretide), -SH (free thiol), -SSG (glutathionylated protein).

Developmental exposure to the pesticide malathion enhances expression of Prdm12, a regulator of nociceptor development, in Xenopus laevis.

The transcription factor Prdm12 exerts important influences on the development of nociceptors, peripheral touch and pain-sensing neurons, and has been implicated in human pain sensation disorders. We examined the consequences of exposing developing Xenopus laevis embryos to the commonly used pesticide malathion on Prdm12 expression. Using qPCR and western blot analysis we observed that malathion treatment for the first six days of tadpole development significantly increased both prdm12 mRNA levels and Prdm12 protein levels compared to controls. Consequently, early exposure to this pesticide has potential to alter nociceptor development.

Elamipretide Improves ADP Sensitivity in Aged Mitochondria by Increasing Uptake through the Adenine Nucleotide Translocator (ANT).

Aging muscle experiences functional decline in part mediated by impaired mitochondrial ADP sensitivity. Elamipretide (ELAM) rapidly improves physiological and mitochondrial function in aging and binds directly to the mitochondrial ADP transporter ANT. We hypothesized that ELAM improves ADP sensitivity in aging leading to rescued physiological function. We measured the response to ADP stimulation in young and old muscle mitochondria with ELAM treatment, in vivo heart and muscle function, and compared protein abundance, phosphorylation, and S-glutathionylation of ADP/ATP pathway proteins. ELAM treatment increased ADP sensitivity in old muscle mitochondria by increasing uptake of ADP through the ANT and rescued muscle force and heart systolic function. Protein abundance in the ADP/ATP transport and synthesis pathway was unchanged, but ELAM treatment decreased protein s-glutathionylation incuding of ANT. Mitochondrial ADP sensitivity is rapidly modifiable. This research supports the hypothesis that ELAM improves ANT function in aging and links mitochondrial ADP sensitivity to physiological function.

The multi-tissue landscape of somatic mtDNA mutations indicates tissue-specific accumulation and removal in aging.

Accumulation of somatic mutations in the mitochondrial genome (mtDNA) has long been proposed as a possible mechanism of mitochondrial and tissue dysfunction that occurs during aging. A thorough characterization of age-associated mtDNA somatic mutations has been hampered by the limited ability to detect low-frequency mutations. Here, we used Duplex Sequencing on eight tissues of an aged mouse cohort to detect >89,000 independent somatic mtDNA mutations and show significant tissue-specific increases during aging across all tissues examined which did not correlate with mitochondrial content and tissue function. G→A/C→T substitutions, indicative of replication errors and/or cytidine deamination, were the predominant mutation type across all tissues and increased with age, whereas G→T/C→A substitutions, indicative of oxidative damage, were the second most common mutation type, but did not increase with age regardless of tissue. We also show that clonal expansions of mtDNA mutations with age is tissue- and mutation type-dependent. Unexpectedly, mutations associated with oxidative damage rarely formed clones in any tissue and were significantly reduced in the hearts and kidneys of aged mice treated at late age with elamipretide or nicotinamide mononucleotide. Thus, the lack of accumulation of oxidative damage-linked mutations with age suggests a life-long dynamic clearance of either the oxidative lesions or mtDNA genomes harboring oxidative damage.

Older-aged C57BL/6 mice fed a diet high in saturated fat and sucrose for ten months show decreased resilience to aging.

The ability to respond to physical stress that disrupts normal physiological homeostasis at an older age embraces the concept of resilience to aging. A physical stressor could be used to induce physiological responses that are age-related, since resilience declines with increasing age. Increased fat and sugar intake is a nutritional stress with a high prevalence of obesity in older people. In order to determine the effect of this type of diet on resilience to aging, 18-month-old C57BL/6J male mice were fed a diet high in saturated fat (lard) and sucrose (HFS) for ten months. At the end of the 10-month study, mice fed the HFS diet showed increased cognitive impairment, decreased cardiac function, decreased strength and agility, and increased severity of renal pathology compared to mice fed a rodent chow diet low in saturated fat and sucrose (LFS). The degree of response aligned with decreased resilience to the long-term adverse effects of the diet with characteristics of accelerated aging. This observation suggests additional studies could be conducted to investigate the relationship between an accelerated decline in resilience to aging and enhanced resilience to aging under different dietary conditions.

Aging Increases Susceptibility to Develop Cardiac Hypertrophy following High Sugar Consumption.

Aging and poor diet are independent risk factors for heart disease, but the impact of high-sucrose (HS) consumption in the aging heart is understudied. Aging leads to impairments in mitochondrial function that result in muscle dysfunction (e.g., cardiac remodeling and sarcopenia). We tested whether HS diet (60%kcal sucrose) would accelerate muscle dysfunction in 24-month-old male CB6F1 mice. By week 1 on HS diet, mice developed significant cardiac hypertrophy compared to age-matched chow-fed controls. The increased weight of the heart persisted throughout the 4-week treatment, while body weight and strength declined more rapidly than controls. We then tested whether HS diet could worsen cardiac dysfunction in old mice and if the mitochondrial-targeted drug, elamipretide (ELAM), could prevent the diet-induced effect. Old and young mice were treated with either ELAM or saline as a control for 2 weeks, and provided with HS diet or chow on the last week. As demonstrated in the previous experiment, old mice had age-related cardiac hypertrophy that worsened after one week on HS and was prevented by ELAM treatment, while the HS diet had no detectable effect on hypertrophy in the young mice. As expected, mitochondrial respiration and reactive oxygen species (ROS) production were altered by age, but were not significantly affected by HS diet or ELAM. Our findings highlight the vulnerability of the aged heart to HS diet that can be prevented by systemic targeting of the mitochondria with ELAM.

Long-term treatment with Elamipretide enhances healthy aging phenotypes in mice.

Background: Disruption of metabolic and bioenergetic homeostasis related to mitochondrial dysfunction is a key driver of aging biology. Therefore, targeting mitochondrial function would be a rational approach to slowing aging. Elamipretide (Elam, a.k.a. SS-31) is a peptide known to target mitochondria and suppress mammalian signs of aging. The present study was designed to examine the phenotypic effects of long-term Elam treatment on aging in C57BL/6 mice starting at 18 months of age. Methods: Mice were fed regular chow (RC diet) or a diet high in fat and sugar (HF diet) and treated with 3 mg/kg of Elam or saline subcutaneously 5 days per week for 10 months. Physiological performance assessments were conducted at 28 months of age. Results: Elam improved the physical performance of males but not females, while in females Elam improved cognitive performance and enhanced the maintenance of body weight and fat mass. It also improved diastolic function in both males and females, but to a greater extent in males. The HF diet over 10 months had a negative effect on health span, as it increased body fat and decreased muscle strength and heart function, especially in females. Conclusions: Elam enhanced healthy aging and cardiac function in both male and female mice, although the specific effects on function differed between sexes. In females, the treatment led to better cognitive performance and maintenance of body composition, while in males, performance on a rotating rod was preserved. These overall observations have translational implications for considering additional studies using Elam in therapeutic or preventive approaches for aging and age-related diseases.

Age-related disruption of the proteome and acetylome in mouse hearts is associated with loss of function and attenuated by elamipretide (SS-31) and nicotinamide mononucleotide (NMN) treatment.

We analyzed the effects of aging on protein abundance and acetylation, as well as the ability of the mitochondrial-targeted drugs elamipretide (SS-31) and nicotinamide mononucleotide (NMN) to reverse aging-associated changes in mouse hearts. Both drugs had a modest effect on restoring the abundance and acetylation of proteins that are altered with age, while also inducing additional changes. Age-related increases in protein acetylation were predominantly in mitochondrial pathways such as mitochondrial dysfunction, oxidative phosphorylation, and TCA cycle signaling. We further assessed how these age-related changes associated with diastolic function (Ea/Aa) and systolic function (fractional shortening under higher workload) measurements from echocardiography. These results identify a subset of protein abundance and acetylation changes in muscle, mitochondrial, and structural proteins that appear to be essential in regulating diastolic function in old hearts.

An Analysis of Metabolic Changes in the Retina and Retinal Pigment Epithelium of Aging Mice.

Purpose: The purpose of this study was to present our hypothesis that aging alters metabolic function in ocular tissues. We tested the hypothesis by measuring metabolism in aged murine tissues alongside retinal responses to light. Methods: Scotopic and photopic electroretinogram (ERG) responses in young (3-6 months) and aged (23-26 months) C57Bl/6J mice were recorded. Metabolic flux in retina and eyecup explants was quantified using U-13C-glucose or U-13C-glutamine with gas chromatography-mass spectrometry (GC-MS), O2 consumption rate (OCR) in a perifusion apparatus, and quantifying adenosine triphosphatase (ATP) with a bioluminescence assay. Results: Scotopic and photopic ERG responses were reduced in aged mice. Glucose metabolism, glutamine metabolism, OCR, and ATP pools in retinal explants were mostly unaffected in aged mice. In eyecups, glutamine usage in the Krebs Cycle decreased while glucose metabolism, OCR, and ATP pools remained stable. Conclusions: Our examination of metabolism showed negligible impact of age on retina and an impairment of glutamine anaplerosis in eyecups. The metabolic stability of these tissues ex vivo suggests age-related metabolic alterations may not be intrinsic. Future experiments should focus on determining whether external factors including nutrient supply, oxygen availability, or structural changes influence ocular metabolism in vivo.

A replication-linked mutational gradient drives somatic mutation accumulation and influences germline polymorphisms and genome composition in mitochondrial DNA.

Mutations in mitochondrial DNA (mtDNA) cause maternally inherited diseases, while somatic mutations are linked to common diseases of aging. Although mtDNA mutations impact health, the processes that give rise to them are under considerable debate. To investigate the mechanism by which de novo mutations arise, we analyzed the distribution of naturally occurring somatic mutations across the mouse and human mtDNA obtained by Duplex Sequencing. We observe distinct mutational gradients in G→A and T→C transitions delimited by the light-strand origin and the mitochondrial Control Region (mCR). The gradient increases unequally across the mtDNA with age and is lost in the absence of DNA polymerase γ proofreading activity. In addition, high-resolution analysis of the mCR shows that important regulatory elements exhibit considerable variability in mutation frequency, consistent with them being mutational 'hot-spots' or 'cold-spots'. Collectively, these patterns support genome replication via a deamination prone asymmetric strand-displacement mechanism as the fundamental driver of mutagenesis in mammalian DNA. Moreover, the distribution of mtDNA single nucleotide polymorphisms in humans and the distribution of bases in the mtDNA across vertebrate species mirror this gradient, indicating that replication-linked mutations are likely the primary source of inherited polymorphisms that, over evolutionary timescales, influences genome composition during speciation.

Elamipretide (SS-31) treatment attenuates age-associated post-translational modifications of heart proteins.

It has been demonstrated that elamipretide (SS-31) rescues age-related functional deficits in the heart but the full set of mechanisms behind this have yet to be determined. We investigated the hypothesis that elamipretide influences post-translational modifications to heart proteins. The S-glutathionylation and phosphorylation proteomes of mouse hearts were analyzed using shotgun proteomics to assess the effects of aging on these post-translational modifications and the ability of the mitochondria-targeted drug elamipretide to reverse age-related changes. Aging led to an increase in oxidation of protein thiols demonstrated by increased S-glutathionylation of cysteine residues on proteins from Old (24 months old at the start of the study) mouse hearts compared to Young (5-6 months old). This shift in the oxidation state of the proteome was almost completely reversed by 8 weeks of treatment with elamipretide. Many of the significant changes that occurred were in proteins involved in mitochondrial or cardiac function. We also found changes in the mouse heart phosphoproteome that were associated with age, some of which were partially restored with elamipretide treatment. Parallel reaction monitoring of a subset of phosphorylation sites revealed that the unmodified peptide reporting for Myot S231 increased with age, but not its phosphorylated form and that both phosphorylated and unphosphorylated forms of the peptide covering cMyBP-C S307 increased, but that elamipretide treatment did not affect these changes. These results suggest that changes to thiol redox state and phosphorylation status are two ways in which age may affect mouse heart function, which can be restored by treatment with elamipretide.

Acarbose has sex-dependent and -independent effects on age-related physical function, cardiac health, and lipid biology.

With an expanding aging population burdened with comorbidities, there is considerable interest in treatments that optimize health in later life. Acarbose (ACA), a drug used clinically to treat type 2 diabetes mellitus (T2DM), can extend mouse life span with greater effect in males than in females. Using a genetically heterogeneous mouse model, we tested the ability of ACA to ameliorate functional, pathological, and biochemical changes that occur during aging, and we determined which of the effects of age and drug were sex dependent. In both sexes, ACA prevented age-dependent loss of body mass, in addition to improving balance/coordination on an accelerating rotarod, rotarod endurance, and grip strength test. Age-related cardiac hypertrophy was seen only in male mice, and this male-specific aging effect was attenuated by ACA. ACA-sensitive cardiac changes were associated with reduced activation of cardiac growth-promoting pathways and increased abundance of peroxisomal proteins involved in lipid metabolism. ACA further ameliorated age-associated changes in cardiac lipid species, particularly lysophospholipids - changes that have previously been associated with aging, cardiac dysfunction, and cardiovascular disease in humans. In the liver, ACA had pronounced effects on lipid handling in both sexes, reducing hepatic lipidosis during aging and shifting the liver lipidome in adulthood, particularly favoring reduced triglyceride (TAG) accumulation. Our results demonstrate that ACA, already in clinical use for T2DM, has broad-ranging antiaging effects in multiple tissues, and it may have the potential to increase physical function and alter lipid biology to preserve or improve health at older ages.

SS-31 and NMN: Two paths to improve metabolism and function in aged hearts.

The effects of two different mitochondrial-targeted drugs, SS-31 and NMN, were tested on Old mouse hearts. After treatment with the drugs, individually or Combined, heart function was examined by echocardiography. SS-31 partially reversed an age-related decline in diastolic function while NMN fully reversed an age-related deficiency in systolic function at a higher workload. Metabolomic analysis revealed that both NMN and the Combined treatment increased nicotinamide and 1-methylnicotinamide levels, indicating greater NAD+ turnover, but only the Combined treatment resulted in significantly greater steady-state NAD(H) levels. A novel magnetic resonance spectroscopy approach was used to assess how metabolite levels responded to changing cardiac workload. PCr/ATP decreased in response to increased workload in Old Control, but not Young, hearts, indicating an age-related decline in energetic capacity. Both drugs were able to normalize the PCr/ATP dynamics. SS-31 and NMN treatment also increased mitochondrial NAD(P)H production under the higher workload, while only NMN increased NAD+ in response to increased work. These measures did not shift in hearts given the Combined treatment, which may be owed to the enhanced NAD(H) levels in the resting state after this treatment. Overall, these results indicate that both drugs are effective at restoring different aspects of mitochondrial and heart health and that combining them results in a synergistic effect that rejuvenates Old hearts and best recapitulates the Young state.

Late-life restoration of mitochondrial function reverses cardiac dysfunction in old mice.

Diastolic dysfunction is a prominent feature of cardiac aging in both mice and humans. We show here that 8-week treatment of old mice with the mitochondrial targeted peptide SS-31 (elamipretide) can substantially reverse this deficit. SS-31 normalized the increase in proton leak and reduced mitochondrial ROS in cardiomyocytes from old mice, accompanied by reduced protein oxidation and a shift towards a more reduced protein thiol redox state in old hearts. Improved diastolic function was concordant with increased phosphorylation of cMyBP-C Ser282 but was independent of titin isoform shift. Late-life viral expression of mitochondrial-targeted catalase (mCAT) produced similar functional benefits in old mice and SS-31 did not improve cardiac function of old mCAT mice, implicating normalizing mitochondrial oxidative stress as an overlapping mechanism. These results demonstrate that pre-existing cardiac aging phenotypes can be reversed by targeting mitochondrial dysfunction and implicate mitochondrial energetics and redox signaling as therapeutic targets for cardiac aging.

Proteomic analysis of the glutathione-deficient LEGSKO mouse lens reveals activation of EMT signaling, loss of lens specific markers, and changes in stress response proteins.

PURPOSE: To determine global protein expression changes in the lens of the GSH-deficient LEGSKO mouse model of age-related cataract for comparison with recently published gene expression data obtained by RNA-Seq transcriptome analysis. METHODS: Lenses were separated into epithelial and cortical fiber sections, digested with trypsin, and labeled with isobaric tags (10-plex TMTTM). Peptides were analyzed by LC-MS/MS (Orbitrap Fusion) and mapped to the mouse proteome for relative protein quantification. RESULTS: 1871 proteins in lens epithelia and 870 proteins in lens fiber cells were quantified. 40 proteins in LEGSKO epithelia, 14 proteins in LEGSKO fiber cells, 22 proteins in buthionine sulfoximine (BSO)-treated LEGSKO epithelia, and 55 proteins in BSO-treated LEGSKO fiber cells had significantly (p<0.05, FDR<0.1) altered protein expression compared to WT controls. HSF4 and MAF transcription factors were the most common upstream regulators of the response to GSH-deficiency. Many detoxification proteins, including aldehyde dehydrogenases, peroxiredoxins, and quinone oxidoreductase, were upregulated but several glutathione S-transferases were downregulated. Several cellular stress response proteins showed regulation changes, including an upregulation of HERPUD1, downregulation of heme oxygenase, and mixed changes in heat shock proteins. NRF2-regulated proteins showed broad upregulation in BSO-treated LEGSKO fiber cells, but not in other groups. Strong trends were seen in downregulation of lens specific proteins, including ß- and γ-crystallins, lengsin, and phakinin, and in epithelial-mesenchymal transition (EMT)-related changes. Western blot analysis of LEGSKO lens epithelia confirmed expression changes in several proteins. CONCLUSIONS: This dataset confirms at the proteomic level many findings from the recently determined GSH-deficient lens transcriptome and provides new insight into the roles of GSH in the lens, how the lens adapts to oxidative stress, and how GSH affects EMT in the lens.

Reduced Glutathione Level Promotes Epithelial-Mesenchymal Transition in Lens Epithelial Cells via a Wnt/ß-Catenin-Mediated Pathway: Relevance for Cataract Therapy.

The epithelial-mesenchymal transition (EMT) process plays a pivotal role in the pathogenesis of posterior capsular opacification because of remnant lens epithelial cell proliferation, migration, and transformation after cataract surgery. The latter, we hypothesize, may result in posterior capsule wrinkling and opacification because of a profound change in the lens growth environment via a 1000-fold reduction of extracellular glutathione (GSH) levels. To test this hypothesis, we investigated the EMT process in cell culture and GSH biosynthesis deficiency mouse models. Our data indicate a dramatic increase of pro-EMT markers, such as type I collagen, α-smooth muscle actin, vimentin, and fibronectin, under conditions of lens GSH depletion. Further study suggests that decreased GSH triggers the Wnt/ß-catenin signal transduction pathway, independent of transforming growth factor-ß. Equally important, the antioxidants N-acetyl cysteine and GSH ethyl ester could significantly attenuate the EMT signaling stimulated by decreased GSH levels. These findings were further confirmed by mock cataract surgery in both gamma glutamyl-cysteine ligase, catalytic subunit, and gamma glutamyl-cysteine ligase, modifier subunit, knockout mouse models. Remarkably, increased EMT marker expression, ß-catenin activation, and translocation into the nucleus were found in both knockout mice compared with the wild type, and such increased expression could be significantly attenuated by N-acetyl cysteine or GSH ethyl ester treatment. This study, for the first time we believe, links oxidative stress to lens fibrosis and posterior capsular opacification formation via EMT-mediated mechanisms.

Transcriptome of the GSH-Depleted Lens Reveals Changes in Detoxification and EMT Signaling Genes, Transport Systems, and Lipid Homeostasis.

Purpose: To understand the effects of glutathione (GSH)-deficiency on genetic processes that regulate lens homeostasis and prevent cataractogenesis. Methods: The transcriptome of lens epithelia and fiber cells was obtained from C57BL/6 LEGSKO (lens GSH-synthesis knockout) and buthionine sulfoximine (BSO)-treated LEGSKO mice and compared to C57BL/6 wild-type mice using RNA-Seq. Transcriptomic data were confirmed by qPCR and Western blot/ELISA on a subset of genes. Results: RNA-Seq results were in excellent agreement with qPCR (correlation coefficients 0.87-0.94 and P < 5E-6 for a subset of 36 mRNAs). Of 24,415 transcripts mapped to the mouse genome, 441 genes showed significantly modulated expression. Pathway analysis indicated major changes in epithelial-mesenchymal transition (EMT) signaling, visual cycle, small molecule biochemistry, and lipid metabolism. GSH-deficient lenses showed upregulation of detoxification genes, including Aldh1a1, Aldh3a1 (aldehyde dehydrogenases), Mt1, Mt2 (metallothioneins), Ces1g (carboxylesterase), and Slc14a1 (urea transporter UT-B). Genes in canonical EMT pathways, including Wnt10a, showed upregulation in lens epithelia samples. Severely GSH-deficient lens epithelia showed downregulation of vision-related genes (including crystallins). The BSO-treated LEGSKO lens epithelia transcriptome has significant correlation (r = 0.63, P < 0.005) to that of lens epithelia undergoing EMT. Protein expression data correlated with transcriptomic data and confirmed EMT signaling activation. Conclusions: These results show that GSH-deficiency in the lens leads to expression of detoxifying genes and activation of EMT signaling, in addition to changes in transport systems and lipid homeostasis. These data provide insight into the adaptation and consequences of GSH-deficiency in the lens and suggest that GSH plays an important role in lenticular EMT pathology.

Lens glutathione homeostasis: Discrepancies and gaps in knowledge standing in the way of novel therapeutic approaches.

Cataract is the major cause of blindness worldwide. The WHO has estimated around 20 million people have bilateral blindness from cataract, and that number is expected to reach 50 million in 2050. The cataract surgery is currently the main treatment approach, though often associated with complications, such as Posterior Capsule Opacification (PCO)-also known as secondary cataract. The lens is an avascular ocular structure equipped with an unusually high level of glutathione (GSH), which plays a vital role in maintaining lens transparency by regulating lenticular redox state. The lens epithelium and outer cortex are thought to be responsible for providing the majority of lens GSH via GSH de novo synthesis, assisted by a continuous supply of constituent amino acids from the aqueous humor, as well as extracellular GSH recycling from the gamma-glutamyl cycle. However, when de novo synthesis is impaired, in the presence of low GSH levels, as in the aging human lens, compensatory mechanisms exist, suggesting that the lens is able to uptake GSH from the surrounding ocular tissues. However, these uptake mechanisms, and the GSH source and its origin, are largely unknown. The lens nucleus does not have the ability to synthesize its own GSH and fully relies on transport from the outer cortex by yet unknown mechanisms. Understanding how aging reduces GSH levels, particularly in the lens nucleus, how it is associated with age-related nuclear cataract (ARNC), and how the lens compensates for GSH loss via external uptake should be a major research priority. The intent of this review, which is dedicated to the memory of David C. Beebe, is to summarize our current understanding of lens GSH homeostasis and highlight discrepancies and gaps in knowledge that stand in the way of pharmacologically minimizing the impact of declining GSH content in the prevention of age-related cataract.