In vivo serial MRI of age-dependent neural progenitor cell migration in the rat brain.

The subventricular zone (SVZ) is a neurogenic niche in the mammalian brain, giving rise to migratory neural progenitor cells (NPC). In rodents, it is well-established that neurogenesis decreases with aging. MRI-based cell tracking has been used to measure various aspects of neurogenesis and NPC migration in rodents, yet it has not yet been validated in the context of age-related decrease in neurogenesis. This validation is critical to using these MRI techniques to study changes in neurogenesis that arise in diseases prevalent in aging populations and their combination with advanced cellular therapeutic approaches aiming to combat neurodegeneration. As such, in this work we used MRI-based cell tracking to measure endogenous neurogenesis and cell migration from the SVZ along the rostral migratory stream to the olfactory bulb, for 12 days duration, in rats aged 9 weeks to 2 years old. To enable the specific detection of NPCs by MRI, we injected micron sized particles of iron oxide (MPIOs) into the lateral ventricle to endogenously label cells within the SVZ, which then appeared as hypo-intensive spots within MR images. In vivo MRI data showed that the rate of NPC migration was significantly different between all ages examined, with decreases in the distance traveled and migration rate as age progressed. The total number of MPIO-labeled cells within the olfactory bulb on day 12, was significantly decreased when compared across ages in ex vivo high-resolution scans. We also demonstrate for the first-time, provocative preliminary data suggesting age-dependent MPIO uptake within the dentate gyrus (DG) as well. Histology to identify doublecortin-positive NPCs, verified the decrease in cell labeling as a function of aging, for both regions. The dramatic reduction of NPC labeling within the SVZ observed with MRI, validates the sensitivity of MRI-based cell tracking to neurogenic potential and demonstrates the importance of understanding the impact of age on the relationship of NPC and disease.

Adaptive gene expression of alternative splicing variants of PGC-1α regulates whole-body energy metabolism.

The transcriptional coactivator PGC-1α has been implicated in the regulation of multiple metabolic processes. However, the previously reported metabolic phenotypes of mice deficient in PGC-1α have been inconsistent. PGC-1α exists as multiple isoforms, including variants transcribed from an alternative first exon. We show here that alternative PGC-1α variants are the main entity that increases PGC-1α during exercise. These variants, unlike the canonical isoform of PGC-1α, are robustly upregulated in human skeletal muscle after exercise. Furthermore, the extent of this upregulation correlates with oxygen consumption. Mice lacking these variants manifest impaired energy expenditure during exercise, leading to the development of obesity and hyperinsulinemia. The alternative variants are also upregulated in brown adipose tissue in response to cold exposure, and mice lacking these variants are intolerant of a cold environment. Our findings thus indicate that an increase in PGC-1α expression, attributable mostly to upregulation of alternative variants, is pivotal for adaptive enhancement of energy expenditure and heat production and thereby essential for the regulation of whole-body energy metabolism.

Caloric restriction primes mitochondria for ischemic stress by deacetylating specific mitochondrial proteins of the electron transport chain.

RATIONALE: Caloric restriction (CR) confers cardioprotection against ischemia/reperfusion injury. However, the exact mechanism(s) underlying CR-induced cardioprotection remain(s) unknown. Recent evidence indicates that Sirtuins, NAD(+)-dependent deacetylases, regulate various favorable aspects of the CR response. Thus, we hypothesized that deacetylation of specific mitochondrial proteins during CR preserves mitochondrial function and attenuates production of reactive oxygen species during ischemia/reperfusion. OBJECTIVE: The objectives of the present study were (1) to investigate the effect of CR on mitochondrial function and mitochondrial proteome and (2) to investigate what molecular mechanisms mediate CR-induced cardioprotection. METHODS AND RESULTS: Male 26-week-old Fischer344 rats were randomly divided into ad libitum-fed and CR (40% reduction) groups for 6 months. No change was observed in basal mitochondrial function, but CR preserved postischemic mitochondrial respiration and attenuated postischemic mitochondrial H(2)O(2) production. CR decreased the level of acetylated mitochondrial proteins that were associated with enhanced Sirtuin activity in the mitochondrial fraction. We confirmed a significant decrease in the acetylated forms of NDUFS1 and cytochrome bc1 complex Rieske subunit in the CR heart. Low-dose resveratrol treatment mimicked the effect of CR on deacetylating them and attenuated reactive oxygen species production during anoxia/reoxygenation in cultured cardiomyocytes without changing the expression levels of manganese superoxide dismutase. Treatment with nicotinamide completely abrogated the effect of low-dose resveratrol. CONCLUSIONS: These results strongly suggest that CR primes mitochondria for stress resistance by deacetylating specific mitochondrial proteins of the electron transport chain. Targeted deacetylation of NDUFS1 and/or Rieske subunit might have potential as a novel therapeutic approach for cardioprotection against ischemia/reperfusion.

Transgenic type2 diabetes mouse models for in vivo redox measurement of hepatic mitochondrial oxidative stress.

BACKGROUND: Oxidative stress is involved in the progression of diabetes and its associated complications. However, it is unclear whether increased oxidative stress plays a primary role in the onset of diabetes or is a secondary indicator caused by tissue damage. Previous methods of analyzing oxidative stress have involved measuring the changes in oxidative stress biomarkers. Our aim is to identify a novel approach to clarify whether oxidative stress plays a primary role in the onset of diabetes. METHODS: We constructed transgenic type 2 diabetes mouse models expressing redox-sensitive green fluorescent proteins (roGFPs) that distinguished between mitochondria and whole cells. Pancreas, liver, skeletal muscle, and kidney redox states were measured in vivo. RESULTS: Hepatic mitochondrial oxidation increased when the mice were 4 weeks old and continued to increase in an age-dependent manner. The increase in hepatic mitochondrial oxidation occurred simultaneously with weight gain and increased blood insulin levels before the blood glucose levels increased. Administering the oxidative stress inducer acetaminophen increased the vulnerability of the liver mitochondria to oxidative stress. CONCLUSIONS: This study demonstrates that oxidative stress in liver mitochondria in mice begins at the onset of diabetes rather than after the disease has progressed. GENERAL SIGNIFICANCE: RoGFP-expressing transgenic type 2 diabetes mouse models are effective and convenient tools for measuring hepatic mitochondrial redox statuses in vivo. These models may be used to assess mitochondria-targeting antioxidants and establish the role of oxidative stress in type 2 diabetes.

Mitochondrial membrane potential decrease caused by loss of PINK1 is not due to proton leak, but to respiratory chain defects.

Mutations in PTEN-induced putative kinase 1 (PINK1) cause a recessive form of Parkinson's disease (PD). PINK1 is associated with mitochondrial quality control and its partial knock-down induces mitochondrial dysfunction including decreased membrane potential and increased vulnerability against mitochondrial toxins, but the exact function of PINK1 in mitochondria has not been investigated using cells with null expression of PINK1. Here, we show that loss of PINK1 caused mitochondrial dysfunction. In PINK1-deficient (PINK1(-/-)) mouse embryonic fibroblasts (MEFs), mitochondrial membrane potential and cellular ATP levels were decreased compared with those in littermate wild-type MEFs. However, mitochondrial proton leak, which reduces membrane potential in the absence of ATP synthesis, was not altered by loss of PINK1. Instead, activity of the respiratory chain, which produces the membrane potential by oxidizing substrates using oxygen, declined. H(2)O(2) production rate by PINK1(-/-) mitochondria was lower than PINK1(+/+) mitochondria as a consequence of decreased oxygen consumption rate, while the proportion (H(2)O(2) production rate per oxygen consumption rate) was higher. These results suggest that mitochondrial dysfunctions in PD pathogenesis are caused not by proton leak, but by respiratory chain defects.

The tumor suppression theory of aging.

Despite continued increases in human life expectancy, the factors determining the rate of human biological aging remain unknown. Without understanding the molecular mechanisms underlying aging, efforts to prevent aging are unlikely to succeed. The tumor suppression theory of aging introduced here proposes somatic mutation as the proximal cause of aging, but postulates that oncogenic transformation and clonal expansion, not functional impairment, are the relevant consequences of somatic mutation. Obesity and caloric restriction accelerate and decelerate aging due to their effect on cell proliferation, during which most mutations arise. Most phenotypes of aging are merely tumor-suppressive mechanisms that evolved to limit malignant growth, the dominant age-related cause of death in early and middle life. Cancer limits life span for most long-lived mammals, a phenomenon known as Peto's paradox. Its conservation across species demonstrates that mutation is a fundamental but hard limit on mammalian longevity. Cell senescence and apoptosis and differentiation induced by oncogenes, telomere shortening or DNA damage evolved as a second line of defense to limit the tumorigenic potential of clonally expanding cells, but accumulating senescent cells, senescence-associated secretory phenotypes and stem cell exhaustion eventually cause tissue dysfunction and the majority, if not most, phenotypes of aging.

Rodent diet aids and the fallacy of caloric restriction.

Understanding the molecular mechanisms of normal aging is a prerequisite to significantly improving human health span. Caloric restriction (CR) can delay aging and has served as a yardstick to evaluate interventions extending life span. However, mice given unlimited access to food suffer severe obesity. Health gains from CR depend on control mice being sufficiently overweight and less obese mouse strains benefit far less from CR. Pharmacologic interventions that increase life span, including resveratrol, rapamycin, nicotinamide mononucleotide and metformin, also reduce body weight. In primates, CR does not delay aging unless the control group is eating enough to suffer from obesity-related disease. Human survival is optimal at a body mass index achievable without CR, and the above interventions are merely diet aids that shouldn't slow aging in healthy weight individuals. CR in humans of optimal weight can safely be declared useless, since there is overwhelming evidence that hunger, underweight and starvation reduce fitness, survival, and quality of life. Against an obese control, CR does, however, truly delay aging through a mechanism laid out in the following tumor suppression theory of aging.

Development of Cancer Immunotherapy Targeting the PD-1 Pathway.

Immune checkpoint inhibitors are causing a paradigm shift in cancer treatment. Immune checkpoint molecules such as programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) dampen T cell activation to avoid autoimmunity and the destructive effects of an excessive inflammatory response. Immune checkpoint signaling can be exploited by tumors to escape host immune surveillance, and immune checkpoint inhibitors enhance antitumor immunity by releasing the brakes on the immune system. PD-1 was identified in 1992 by Honjo and colleagues at Kyoto University. Studies in animal models revealed that PD-1 blockade can inhibit tumorigenesis and tumor metastasis. In addition, PD-1 blockade showed fewer adverse effects than CTLA-4 blockade. Based on these findings, a humanized monoclonal antibody against human PD-1 called nivolumab was developed. Since PD-1 blockade targets lymphocytes rather than tumor cells, the therapeutic effects last longer, even if mutations occur during tumorigenesis. Furthermore, because it does not depend on specific tumor antigens, PD-1 blockade can be applied to various kinds of tumors.

Imaging mitochondrial redox environment and oxidative stress using a redox-sensitive fluorescent protein.

Redox-sensitive green fluorescent protein (roGFP) is a fluorescent protein in which two cysteines are placed adjacently in the barrel structure. Disulfide formation (oxidation) increases the absorption at short wavelengths (410 nm) at the expense of absorption at longer wavelengths (490 nm). The fluorescence ratio indicates reduction/oxidation, i.e., the redox potential at specific cellular locations.

Blue light-induced oxidative stress in live skin.

Skin damage from exposure to sunlight induces aging-like changes in appearance and is attributed to the ultraviolet (UV) component of light. Photosensitized production of reactive oxygen species (ROS) by UVA light is widely accepted to contribute to skin damage and carcinogenesis, but visible light is thought not to do so. Using mice expressing redox-sensitive GFP to detect ROS, blue light could produce oxidative stress in live skin. Blue light induced oxidative stress preferentially in mitochondria, but green, red, far red or infrared light did not. Blue light-induced oxidative stress was also detected in cultured human keratinocytes, but the per photon efficacy was only 25% of UVA in human keratinocyte mitochondria, compared to 68% of UVA in mouse skin. Skin autofluorescence was reduced by blue light, suggesting flavins are the photosensitizer. Exposing human skin to the blue light contained in sunlight depressed flavin autofluorescence, demonstrating that the visible component of sunlight has a physiologically significant effect on human skin. The ROS produced by blue light is probably superoxide, but not singlet oxygen. These results suggest that blue light contributes to skin aging similar to UVA.

Motor dysfunction and altered synaptic transmission at the parallel fiber-Purkinje cell synapse in mice lacking potassium channels Kv3.1 and Kv3.3.

Micelacking both Kv3.1 and both Kv3.3 K+ channel alleles display severe motor deficits such as tremor, myoclonus, and ataxic gait. Micelacking one to three alleles at the Kv3.1 and Kv3.3 loci exhibit in an allele dose-dependent manner a modest degree of ataxia. Cerebellar granule cells coexpress Kv3.1 and Kv3.3 K+ channels and are therefore candidate neurons that might be involved in these behavioral deficits. Hence, we investigated the synaptic mechanisms of transmission in the parallel fiber-Purkinje cell system. Action potentials of parallel fibers were broader in mice lacking both Kv3.1 and both Kv3.3 alleles and in mice lacking both Kv3.1 and a single Kv3.3 allele compared with those of wild-type mice. The transmission of high-frequency trains of action potentials was only impaired at 200 Hz but not at 100 Hz in mice lacking both Kv3.1 and Kv3.3 genes. However, paired-pulse facilitation (PPF) at parallel fiber-Purkinje cell synapses was dramatically reduced in a gene dose-dependent manner in mice lacking Kv3.1 or Kv3.3 alleles. Normal PPF could be restored by reducing the extracellular Ca2+ concentration indicating that increased activity-dependent presynaptic Ca2+ influx, at least in part caused the altered PPF in mutant mice. Induction of metabotropic glutamate receptor-mediated EPSCs was facilitated, whereas longterm depression was not impaired but rather facilitated in Kv3.1/Kv3.3 double-knockout mice. These results demonstrate the importance of Kv3 potassium channels in regulating the dynamics of synaptic transmission at the parallel fiber-Purkinje cell synapse and suggest a correlation between short-term plasticity at the parallel fiber-Purkinje cell synapse and motor performance.

Real-time monitoring of oxidative stress in live mouse skin.

Oxidative stress is involved in many age-associated diseases, as well as in the aging process itself. The development of interventions to reduce oxidative stress is hampered by the absence of sensitive detection methods that can be used in live animals. We generated transgenic mice expressing ratiometric redox-sensitive green fluorescent protein (roGFP) in the cytosol or mitochondria of several tissues, including skin epidermal keratinocytes. Crossbreeding into hairless albino mice allowed noninvasive optical measurement of skin oxidative state. Topical application of hydrogen peroxide emulsion shifted the keratinocyte redox state toward oxidation within minutes and could be observed in real time by fluorescence ratio imaging. Exposing skin to 365 nm UVA radiation oxidized roGFP localized in keratinocyte mitochondria, but not when roGFP was localized in the cytosol. This suggests that significant amounts of the endogenous photosensitizers that mediate UVA-induced oxidative stress are located in the mitochondria. UVR is the major environmental cause of skin aging and UVA-mediated oxidative stress has been associated with the development of wrinkles in humans. Direct measurements of redox state in defined cell compartments of live animals should be a powerful and convenient tool for evaluating treatments that aim to modulate oxidative stress.

Increased mitochondrial functions in human glioblastoma cells persistently infected with measles virus.

Measles virus (MV) is known for its ability to cause an acute infection with a potential of development of persistent infection. However, knowledge of how viral genes and cellular factors interact to cause or maintain the persistent infection has remained unclear. We have previously reported the possible involvement of mitochondrial short chain enoyl-CoA hydratase (ECHS), which is localized at mitochondria, in the regulation of MV replication. In this study we found increased functions of mitochondria in MV-persistently infected cells compared with uninfected or acutely infected cells. Furthermore, impairment of mitochondrial functions by treatment with mitochondrial inhibitors such as ethidium bromide (EtBr) or carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) induced the cytopathic effects of extensive syncytial formation in persistently infected cells. These findings suggest that mitochondria are one of the subcellular organelles contributing to regulate persistent infection of MV. Recent studies showed mitochondria provide an integral platform for retinoic acid-inducible protein (RIG-I)-like cytosolic receptors (RLRs) signaling and participate in cellular innate antiviral immunity. Our findings not only reveal a role of mitochondria in RLR mediated antiviral signaling but also suggest that mitochondria contribute to the regulation of persistent viral infection.

Taurine ameliorates impaired the mitochondrial function and prevents stroke-like episodes in patients with MELAS.

OBJECTIVE: Post-transcriptional taurine modification at the first anticodon ("wobble") nucleotide is deficient in A3243G-mutant mitochondrial (mt) tRNA(Leu(UUR)) of patients with myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Wobble nucleotide modifications in tRNAs have recently been identified to be important in the accurate and efficient deciphering of codons. We herein examined whether taurine can alleviate mitochondrial dysfunction in patient-derived pathogenic cells and prevent clinical symptoms in MELAS patients. METHODS AND RESULTS: The addition of taurine to the culture media ameliorated the reduced oxygen consumption, decreased the mitochondrial membrane potential, and increased the oxidative stress in MELAS patient-derived cells. Moreover, high dose oral administration of taurine (0.25 g/kg/day) completely prevented stroke-like episodes in two MELAS patients for more than nine years. CONCLUSION: Taurine supplementation may be a novel potential treatment option for preventing the stroke-like episodes associated with MELAS.

Mitochondrial DNA alterations in colorectal cancer cell lines.

Somatic mutations of mitochondrial DNA (mtDNA) have been reported in different types of cancers and are suggested to play roles in metastasis, cancer development and response to anticancer agents. To predict potential roles of mtDNA alterations in colorectal cancer, we determined the entire mtDNA sequence of eleven human-derived colorectal cancer cell lines and compared with the revised Cambridge Reference Sequence to identify nucleotide alterations. Four homoplasmic and six heteroplasmic alterations were found to be novel. Among them, homoplasmic G6709A (MT-CO1) and G14804A (MT-CYB) alterations cause amino acid changes in the highly conserved residues. Heteroplasmic G1576A (MT-RNR1) and G2975A (MT-RNR2) alterations are expected to make the stem structure of mitochondrial ribosomal RNAs unstable. These nucleotide alterations are candidates that could play important roles in cancer.

Astaxanthin protects mitochondrial redox state and functional integrity against oxidative stress.

Mitochondria combine the production of energy with an efficient chain of reduction-oxidation (redox) reactions but also with the unavoidable production of reactive oxygen species. Oxidative stress leading to mitochondrial dysfunction is a critical factor in many diseases, such as cancer and neurodegenerative and lifestyle-related diseases. Effective antioxidants thus offer great therapeutic and preventive promise. Investigating the efficacy of antioxidants, we found that a carotenoid, astaxanthin (AX), decreased physiologically occurring oxidative stress and protected cultured cells against strong oxidative stress induced with a respiratory inhibitor. Moreover, AX improved maintenance of a high mitochondrial membrane potential and stimulated respiration. Investigating how AX stimulates and interacts with mitochondria, a redox-sensitive fluorescent protein (roGFP1) was stably expressed in the cytosol and mitochondrial matrix to measure the redox state in the respective compartments. AX at nanomolar concentrations was effective in maintaining mitochondria in a reduced state. Additionally, AX improved the ability of mitochondria to remain in a reduced state under oxidative challenge. Taken together, these results suggest that AX is effective in improving mitochondrial function through retaining mitochondria in the reduced state.