Regional metabolic signatures in the Ndufs4(KO) mouse brain implicate defective glutamate/α-ketoglutarate metabolism in mitochondrial disease.

Leigh Syndrome (LS) is a mitochondrial disorder defined by progressive focal neurodegenerative lesions in specific regions of the brain. Defects in NDUFS4, a subunit of complex I of the mitochondrial electron transport chain, cause LS in humans; the Ndufs4 knockout mouse (Ndufs4(KO)) closely resembles the human disease. Here, we probed brain region-specific molecular signatures in pre-symptomatic Ndufs4(KO) to identify factors which underlie focal neurodegeneration. Metabolomics revealed that free amino acid concentrations are broadly different by region, and glucose metabolites are increased in a manner dependent on both region and genotype. We then tested the impact of the mTOR inhibitor rapamycin, which dramatically attenuates LS in Ndufs4(KO), on region specific metabolism. Our data revealed that loss of Ndufs4 drives pathogenic changes to CNS glutamine/glutamate/α-ketoglutarate metabolism which are rescued by mTOR inhibition Finally, restriction of the Ndufs4 deletion to pre-synaptic glutamatergic neurons recapitulated the whole-body knockout. Together, our findings are consistent with mTOR inhibition alleviating disease by increasing availability of α-ketoglutarate, which is both an efficient mitochondrial complex I substrate in Ndufs4(KO) and an important metabolite related to neurotransmitter metabolism in glutamatergic neurons.

Transaldolase inhibition impairs mitochondrial respiration and induces a starvation-like longevity response in Caenorhabditis elegans.

Mitochondrial dysfunction can increase oxidative stress and extend lifespan in Caenorhabditis elegans. Homeostatic mechanisms exist to cope with disruptions to mitochondrial function that promote cellular health and organismal longevity. Previously, we determined that decreased expression of the cytosolic pentose phosphate pathway (PPP) enzyme transaldolase activates the mitochondrial unfolded protein response (UPRmt) and extends lifespan. Here we report that transaldolase (tald-1) deficiency impairs mitochondrial function in vivo, as evidenced by altered mitochondrial morphology, decreased respiration, and increased cellular H2O2 levels. Lifespan extension from knockdown of tald-1 is associated with an oxidative stress response involving p38 and c-Jun N-terminal kinase (JNK) MAPKs and a starvation-like response regulated by the transcription factor EB (TFEB) homolog HLH-30. The latter response promotes autophagy and increases expression of the flavin-containing monooxygenase 2 (fmo-2). We conclude that cytosolic redox established through the PPP is a key regulator of mitochondrial function and defines a new mechanism for mitochondrial regulation of longevity.

mTOR inhibitors may benefit kidney transplant recipients with mitochondrial diseases.

Mitochondrial diseases represent a significant clinical challenge. Substantial efforts have been devoted to identifying therapeutic strategies for mitochondrial disorders, but effective interventions have remained elusive. Recently, we reported attenuation of disease in a mouse model of the human mitochondrial disease Leigh syndrome through pharmacological inhibition of the mechanistic target of rapamycin (mTOR). The human mitochondrial disorder MELAS/MIDD (Mitochondrial Encephalopathy with Lactic Acidosis and Stroke-like Episodes/Maternally Inherited Diabetes and Deafness) shares many phenotypic characteristics with Leigh syndrome. MELAS/MIDD often leads to organ failure and transplantation and there are currently no effective treatments. To examine the therapeutic potential of mTOR inhibition in human mitochondrial disease, four kidney transplant recipients with MELAS/MIDD were switched from calcineurin inhibitors to mTOR inhibitors for immunosuppression. Primary fibroblast lines were generated from patient dermal biopsies and the impact of rapamycin was studied using cell-based end points. Metabolomic profiles of the four patients were obtained before and after the switch. pS6, a measure of mTOR signaling, was significantly increased in MELAS/MIDD cells compared to controls in the absence of treatment, demonstrating mTOR overactivation. Rapamycin rescued multiple deficits in cultured cells including mitochondrial morphology, mitochondrial membrane potential, and replicative capacity. Clinical measures of health and mitochondrial disease progression were improved in all four patients following the switch to an mTOR inhibitor. Metabolomic analysis was consistent with mitochondrial function improvement in all patients.

Acarbose suppresses symptoms of mitochondrial disease in a mouse model of Leigh syndrome.

Mitochondrial diseases represent a spectrum of disorders caused by impaired mitochondrial function, ranging in severity from mortality during infancy to progressive adult-onset disease. Mitochondrial dysfunction is also recognized as a molecular hallmark of the biological ageing process. Rapamycin, a drug that increases lifespan and health during normative ageing, also increases survival and reduces neurological symptoms in a mouse model of the severe mitochondrial disease Leigh syndrome. The Ndufs4 knockout (Ndufs4-/-) mouse lacks the complex I subunit NDUFS4 and shows rapid onset and progression of neurodegeneration mimicking patients with Leigh syndrome. Here we show that another drug that extends lifespan and delays normative ageing in mice, acarbose, also suppresses symptoms of disease and improves survival of Ndufs4-/- mice. Unlike rapamycin, acarbose rescues disease phenotypes independently of inhibition of the mechanistic target of rapamycin. Furthermore, rapamycin and acarbose have additive effects in delaying neurological symptoms and increasing maximum lifespan in Ndufs4-/- mice. We find that acarbose remodels the intestinal microbiome and alters the production of short-chain fatty acids. Supplementation with tributyrin, a source of butyric acid, recapitulates some effects of acarbose on lifespan and disease progression, while depletion of the endogenous microbiome in Ndufs4-/- mice appears to fully recapitulate the effects of acarbose on healthspan and lifespan in these animals. To our knowledge, this study provides the first evidence that alteration of the gut microbiome plays a significant role in severe mitochondrial disease and provides further support for the model that biological ageing and severe mitochondrial disorders share underlying common mechanisms.

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.

Stress-induced senescence in human and rodent astrocytes.

There is an increasing awareness that astrocytes, the most abundant cell type in the central nervous system, are critical mediators of brain homeostasis, playing multifunctional roles including buffering potassium ions, maintaining the blood-brain barrier, releasing growth factors, and regulating neurotransmitter levels. Defects in astrocyte function have been implicated in a variety of diseases including age-related diseases such Alzheimer's disease and Parkinson's disease. However, little is known about the age-related changes that occur in astrocytes and if these cells are able to generate a senescent phenotype in response to stress. In this report we have examined whether astrocytes can initiate a senescence program similar to that described in other cell types in response to a variety of stresses. Our results indicate that after oxidative stress, proteasome inhibition, or exhausted replication, human and mouse astrocytes show changes in several established markers of cellular senescence. Astrocytes appear to be more sensitive to oxidative stress than fibroblasts, suggesting that stress-induced senescence may be more pronounced in the brain than in other tissues.

The NDUFS4 Knockout Mouse: A Dual Threat Model of Childhood Mitochondrial Disease and Normative Aging.

Mice missing the Complex I subunit NADH:Ubiquinone Oxidoreductase Fe-S Protein 4 (NDUFS4) of the electron transport chain are a leading model of the severe mitochondrial disease Leigh syndrome. These mice have enabled a better understanding of mitochondrial dysfunction in human disease, as well as in the discovery of interventions that can potentially suppress mitochondrial disease manifestations. In addition, increasing evidence suggests significant overlap between interventions that increase survival in NDUFS4 knockout mice and that extend life span during normative aging. This chapter discusses the practical aspects of handling and studying these mice, which can be challenging due to their severe disease phenotype. Common procedures such as breeding, genotyping, weaning, or treating these transgenic mice are also discussed.

Proceedings from the annual University of Washington Geroscience Symposium, October 23, 2020.

The University of Washington Nathan Shock Center of Excellence in the Biology of Aging in conjunction with the Healthy Aging and Longevity Research Institute held its annual geroscience symposium virtually on October 23, 2020. The symposium was divided into three sessions: (I) organ aging and growth signaling, (II) neurodegeneration and metabolism, and (III) innovative approaches in geroscience and aging research. Nine speakers affiliated with the University of Washington and three invited guest speakers, predominantly trainee, and junior faculty presented their research. Here, we summarize research presented during the symposium. A geroscience special issue, of which this is a part, collects submissions from symposium presenters as well as trainees supported by the Biological Mechanisms of Healthy Aging training program.

Evidence that C/EBP-ß LAP Increases Fat Metabolism and Protects Against Diet-Induced Obesity in Response to mTOR Inhibition.

Aging and obesity are common risk factors for numerous chronic pathologies, and the compounding effects of old age and increased adiposity pose a serious threat to public health. Starting from the assumption that aging and obesity may have shared underpinnings, we investigated the antiobesogenic potential of a successful longevity intervention, the mTORC1 inhibitor rapamycin. We find that rapamycin prevents diet-induced obesity in mice and increases the activity of C/EBP-ß LAP, a transcription factor that regulates the metabolic shift to lipid catabolism observed in response to calorie restriction. Independent activation of C/EBP-ß LAP with the antiretroviral drug adefovir dipivoxil recapitulates the anti-obesogenic effects of rapamycin without reducing signaling through mTORC1 and increases markers of fat catabolism in the liver. Our findings support a model that C/EBP-ß LAP acts downstream of mTORC1 signaling to regulate fat metabolism and identifies a novel drug that may be exploited to treat obesity and decrease the incidence of age-related disease.

Antiaging diets: Separating fact from fiction.

Caloric restriction has been known for nearly a century to extend life span and delay age-associated pathology in laboratory animals. More recently, alternative "antiaging" diet modalities have been described that provide new mechanistic insights and potential clinical applications. These include intermittent fasting, fasting-mimicking diets, ketogenic diets, time-restricted feeding, protein restriction, and dietary restriction of specific amino acids. Despite mainstream popularization of some of these diets, many questions remain about their efficacy outside of a laboratory setting. Studies of these interventions support at least partially overlapping mechanisms of action and provide insights into what appear to be highly conserved mechanisms of biological aging.

The AGE Presents Introduction to Geroscience video lecture series.

The AGE Presents Introduction to Geroscience video lecture series is a collection of high-quality didactic video lectures and associated teaching materials focused on foundational topics in aging biology. The videos are made freely available on YouTube and are targeted toward an audience familiar with concepts learned in the first year of a college undergraduate biology/biomedical major. Members of the American Aging Association also receive the original lecture slides and lecture notes, with additional course materials to be developed in the future. We expect that these lectures will enhance understanding of geroscience among the general public while also providing tools that educators can use in the classroom for high school, undergraduate, and graduate level curricula.

University of Washington Nathan Shock Center: innovation to advance aging research.

The University of Washington Nathan Shock Center of Excellence in the Basic Biology of Aging provides leadership and resources to support the geroscience community locally, nationally, and internationally. Services are provided through our Resource Cores and funds are available annually to support pilot projects by external investigators. Aging-related studies involving proteomics, metabolomics, invertebrate model organisms, and bioinformatics/artificial intelligence are supported by our Cores. The UW Nathan Shock Center also serves as the administrative home for a Geropathology Research Resource. In addition, the Center works in conjunction with the University of Washington Healthy Aging and Longevity Research Institute to organize and support an annual Seminar Series in the Biology of Aging, an annual 1-day Geroscience Symposium, didactic training for the Biological Mechanisms of Healthy Aging Training Program, and other strategic initiatives. Our Center also supports the American Aging Association Annual Meeting, and we have recently partnered with the American Aging Association and the JAX Aging Center to create a set of video lectures on select topics in geroscience as part of the AGE Presents Video Lecture Series.

17-a-estradiol late in life extends lifespan in aging UM-HET3 male mice; nicotinamide riboside and three other drugs do not affect lifespan in either sex.

In genetically heterogeneous mice produced by the CByB6F1 x C3D2F1 cross, the "non-feminizing" estrogen, 17-α-estradiol (17aE2), extended median male lifespan by 19% (p < 0.0001, log-rank test) and 11% (p = 0.007) when fed at 14.4 ppm starting at 16 and 20 months, respectively. 90th percentile lifespans were extended 7% (p = 0.004, Wang-Allison test) and 5% (p = 0.17). Body weights were reduced about 20% after starting the 17aE2 diets. Four other interventions were tested in males and females: nicotinamide riboside, candesartan cilexetil, geranylgeranylacetone, and MIF098. Despite some data suggesting that nicotinamide riboside would be effective, neither it nor the other three increased lifespans significantly at the doses tested. The 17aE2 results confirm and extend our original reports, with very similar results when started at 16 months compared with mice started at 10 months of age in a prior study. The consistently large lifespan benefit in males, even when treatment is started late in life, may provide information on sex-specific aspects of aging.

Proepithelin regulates prostate cancer cell biology by promoting cell growth, migration, and anchorage-independent growth.

The growth factor proepithelin has recently emerged as an important regulator of transformation in several physiological and pathological systems. In this study, we determined the biological roles of proepithelin in prostate cancer cells using purified human recombinant proepithelin as well as proepithelin-depletion strategies. Proepithelin promoted the migration of androgen-dependent and -independent human prostate cancer cells; androgen-independent DU145 cells were the more responsive. In these cells, proepithelin additionally stimulated wound closure, invasion, and promotion of cell growth in vitro. These effects required the activation of both the Akt and mitogen-activated protein kinase pathways. We have analyzed proepithelin expression levels in different available prostate cancer microarray studies using the Oncomine database and found a statistically significant increase in proepithelin mRNA expression levels in prostate cancers compared with nonneoplastic controls. Notably, depletion of endogenous proepithelin by siRNA and antisense strategies impaired the ability of DU145 cells to grow and migrate after serum withdrawal and inhibited anchorage-independent growth. Our results provide the first evidence for a role of proepithelin in stimulating the migration, invasion, proliferation, and anchorage-independent growth of prostate cancer cells. This study supports the hypothesis that proepithelin may play a critical role as an autocrine growth factor in the establishment and initial progression of prostate cancer. Furthermore, proepithelin may prove to be a useful clinical marker for the diagnosis of prostate tumors.

The importance of diversity and outreach in geroscience research: Insights from the Annual Biomedical Research Conference for Minority Students.

US academic science lacks racial, ethnic, sex, gender, disability, and socioeconomic diversity. Addressing this problem is essential to drive scientific progress but is confounded by broad misunderstandings regarding diverse groups. Increasing representation in science is particularly relevant in geroscience, where our research to maximize healthy human lifespan must also address existing racial and socioeconomic health disparities. The American Aging Association (AGE) is committed to addressing these issues as part of its larger mission to advance and promote geroscience research. Over the last three years, AGE has sponsored an exhibition booth staffed by trainee leaders to promote our society and research at the Annual Biomedical Research Conference for Minority Students (ABRCMS), an ideal venue to interact with diverse students from across the country. Through our interactions with students, advocates, and representatives from other institutions and societies, we have learned a great deal about how to engage and promote the success of diverse students in the sciences. Here, we share these insights that are helping shape our own outreach efforts. In addition to interacting with ABRCMS attendees, we also learned a great deal about how societies like AGE can partner with other organizations to advance our shared goals and the importance of reaching students early in their academic journey to promote their success. Finally, we consider how to grow our outreach efforts beyond ABRCMS to reach those in disadvantaged areas and support students navigating academic science.

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.

Proepithelin is an autocrine growth factor for bladder cancer.

The growth factor proepithelin functions as an important regulator of proliferation and motility. Proepithelin is overexpressed in a great variety of cancer cell lines and clinical specimens of breast, ovarian and renal cancer, as well as glioblastomas. Using recombinant proepithelin on 5637 transitional cell carcinoma-derived cells, we have shown previously that proepithelin plays a critical role in bladder cancer by promoting motility of bladder cancer cells. In this study, we used the ONCOMINE database and gene microarray analysis tool to analyze proepithelin expression in several bladder cancer microarray studies. We found a statistically significant increase in proepithelin messenger RNA expression in bladder cancers vis-à-vis non-neoplastic tissues, and this was associated with pathologic and prognostic parameters. Targeted downregulation of proepithelin in T24 transitional carcinoma cells with small hairpin RNA inhibited both Akt and mitogen-activated protein kinase pathways, severely reduced the ability of T24 cells to proliferate in the absence of serum and inhibited migration, invasion and wound healing. In support of these in vitro results, we discovered that proepithelin expression was significantly upregulated in invasive bladder cancer tissues compared with normal urothelium. In addition, proepithelin was secreted in the urine, where it was detectable by immunoblotting and enzyme-linked immunosorbent assay. Collectively, these results support the hypothesis that proepithelin may play a critical role as an autocrine growth factor in the establishment and progression of bladder cancer and suggest that proepithelin may prove a novel biomarker for the diagnosis and prognosis of bladder neoplasms.

Hepatic S6K1 Partially Regulates Lifespan of Mice with Mitochondrial Complex I Deficiency.

The inactivation of ribosomal protein S6 kinase 1 (S6K1) recapitulates aspects of caloric restriction and mTORC1 inhibition to achieve prolonged longevity in invertebrate and mouse models. In addition to delaying normative aging, inhibition of mTORC1 extends the shortened lifespan of yeast, fly, and mouse models with severe mitochondrial disease. Here we tested whether disruption of S6K1 can recapitulate the beneficial effects of mTORC1 inhibition in the Ndufs4 knockout (NKO) mouse model of Leigh Syndrome caused by Complex I deficiency. These NKO mice develop profound neurodegeneration resulting in brain lesions and death around 50-60 days of age. Our results show that liver-specific, as well as whole body, S6K1 deletion modestly prolongs survival and delays onset of neurological symptoms in NKO mice. In contrast, we observed no survival benefit in NKO mice specifically disrupted for S6K1 in neurons or adipocytes. Body weight was reduced in WT mice upon disruption of S6K1 in adipocytes or whole body, but not altered when S6K1 was disrupted only in neurons or liver. Taken together, these data indicate that decreased S6K1 activity in liver is sufficient to delay the neurological and survival defects caused by deficiency of Complex I and suggest that mTOR signaling can modulate mitochondrial disease and metabolism via cell non-autonomous mechanisms.

Corrigendum: Hepatic S6K1 Partially Regulates Lifespan of Mice with Mitochondrial Complex I Deficiency.

[This corrects the article on p. 113 in vol. 8, PMID: 28919908.].

Transient rapamycin treatment can increase lifespan and healthspan in middle-aged mice.

The FDA approved drug rapamycin increases lifespan in rodents and delays age-related dysfunction in rodents and humans. Nevertheless, important questions remain regarding the optimal dose, duration, and mechanisms of action in the context of healthy aging. Here we show that 3 months of rapamycin treatment is sufficient to increase life expectancy by up to 60% and improve measures of healthspan in middle-aged mice. This transient treatment is also associated with a remodeling of the microbiome, including dramatically increased prevalence of segmented filamentous bacteria in the small intestine. We also define a dose in female mice that does not extend lifespan, but is associated with a striking shift in cancer prevalence toward aggressive hematopoietic cancers and away from non-hematopoietic malignancies. These data suggest that a short-term rapamycin treatment late in life has persistent effects that can robustly delay aging, influence cancer prevalence, and modulate the microbiome.