Mitochondrial translation deficiency impairs NAD+ -mediated lysosomal acidification.

Mitochondrial translation dysfunction is associated with neurodegenerative and cardiovascular diseases. Cells eliminate defective mitochondria by the lysosomal machinery via autophagy. The relationship between mitochondrial translation and lysosomal function is unknown. In this study, mitochondrial translation-deficient hearts from p32-knockout mice were found to exhibit enlarged lysosomes containing lipofuscin, suggesting impaired lysosome and autolysosome function. These mice also displayed autophagic abnormalities, such as p62 accumulation and LC3 localization around broken mitochondria. The expression of genes encoding for nicotinamide adenine dinucleotide (NAD+ ) biosynthetic enzymes-Nmnat3 and Nampt-and NAD+ levels were decreased, suggesting that NAD+ is essential for maintaining lysosomal acidification. Conversely, nicotinamide mononucleotide (NMN) administration or Nmnat3 overexpression rescued lysosomal acidification. Nmnat3 gene expression is suppressed by HIF1α, a transcription factor that is stabilized by mitochondrial translation dysfunction, suggesting that HIF1α-Nmnat3-mediated NAD+ production is important for lysosomal function. The glycolytic enzymes GAPDH and PGK1 were found associated with lysosomal vesicles, and NAD+ was required for ATP production around lysosomal vesicles. Thus, we conclude that NAD+ content affected by mitochondrial dysfunction is essential for lysosomal maintenance.

Doxycycline induces apoptosis via ER stress selectively to cells with a cancer stem cell-like properties: importance of stem cell plasticity.

Tumor heterogeneity can be traced back to a small subset of cancer stem cells (CSCs), which can be derived from a single stem cell and show chemoresistance. Recent studies showed that CSCs are sensitive to mitochondrial targeting antibiotics such as doxycycline. However, little is known about how cancer cells undergo sphere formation and how antibiotics inhibit CSC proliferation. Here we show that under sphere-forming assay conditions, prostate cancer cells acquired CSC-like properties: promoted mitochondrial respiratory chain activity, expression of characteristic CSC markers and resistance to anticancer agents. Furthermore, those CSC-like properties could reversibly change depending on the culture conditions, suggesting some kinds of CSCs have plasticity in tumor microenvironments. The sphere-forming cells (i.e. cancer stem-like cells) showed increased contact between mitochondria and mitochondrial associated-endoplasmic reticulum (ER) membranes (MAM). Mitochondrial targeting doxycycline induced activating transcription factor 4 (ATF4) mediated expression of ER stress response and led to p53-upregulated modulator of apoptosis (PUMA)-dependent apoptosis only in the cancer stem-like cells. We also found that doxycycline effectively suppressed the sphere formation in vitro and blocked CD44v9-expressing tumor growth in vivo. In summary, these data provide new molecular findings that monolayer cancer cells acquire CSC-like properties in a reversible manner. These findings provide important insights into CSC biology and a potential new treatment of targeting mitochondria dependency.

Neural-specific deletion of mitochondrial p32/C1qbp leads to leukoencephalopathy due to undifferentiated oligodendrocyte and axon degeneration.

Mitochondrial dysfunction is a critical step in the pathogenesis of many neurodegenerative diseases. The p32/ C1qbp gene functions as an essential RNA and protein chaperone in mitochondrial translation, and is indispensable for embryonic development. However, little is known about the consequences of mitochondrial dysfunction of p32 deletion in the brain development. Here, we found that mice lacking p32 in the central nervous system (p32cKO mice) showed white matter degeneration accompanied by progressive oligodendrocyte loss, axon degeneration and vacuolation in the mid brain and brain stem regions. Furthermore, p32cKO mice died within 8 weeks of birth. We also found that p32-deficient oligodendrocytes and neurons showed reduced oligodendrocyte differentiation and axon degeneration in primary culture. We show that mitochondrial disruption activates an adaptive program known as the integrated stress response (ISR). Mitochondrial respiratory chain function in oligodendrocytes and neurons is, therefore, essential for myelination and axon maintenance, respectively, suggesting that mitochondrial respiratory chain dysfunction in the central nervous system contributes to leukoencephalopathy.

Cardiomyocyte-specific loss of mitochondrial p32/C1qbp causes cardiomyopathy and activates stress responses.

AIMS: Mitochondria are important organelles, dedicated to energy production. Mitochondrial p32/C1qbp, which functions as an RNA and protein chaperone, interacts with mitochondrial mRNA and is indispensable for mitochondrial function through its regulation of mitochondrial translation in cultured cell lines. However, the precise role of p32/C1qbp in vivo is poorly understood because of embryonic lethality in the systemic p32-deficient mouse. The goal of this study was to examine the physiological function of mitochondrial p32/C1qbp in the heart. METHODS AND RESULTS: We investigated the role of p32 in regulating cardiac function in mice using a Cre-loxP recombinase technology against p32 with tamoxifen-inducible knockdown or genetic ablation during postnatal periods. Cardiomyocyte-specific deletion of p32 resulted in contractile dysfunction, cardiac dilatation and cardiac fibrosis, compared with hearts of control mice. We also found decreased COX1 expression, decreased rates of oxygen consumption and increased oxidative stress, indicating that these mice had cardiac mitochondrial dysfunction provoked by p32-deficiency at early stage. Next, we investigated lifespan in cardiac-specific p32-deficient mice. The mice died beginning at 12 months and their median lifespan was ∼14 months. Cardiac mitochondria in the p32-deficient mice showed disordered alignment, enlargement and abnormalities in their internal structure by electron microscopy. We observed that, in p32-deficient compared with control myocytes, AMPKɑ was constitutively phosphorylated and 4EBP-1 and ribosomal S6K were less phosphorylated, suggesting impairment of mammalian target of rapamycin signalling. Finally, we found that expression levels of mitokines such as FGF21 and of integrated stress response genes were significantly increased. Metabolic analysis demonstrated that the urea cycle was impaired in the p32-deficient hearts. CONCLUSION: These findings support a key role for mitochondrial p32 protein in cardiac myocytes modulating mitochondrial translation and function, and thereby survival.

Serum depletion induced cancer stem cell-like phenotype due to nitric oxide synthesis in oncogenic HRas transformed cells.

Cancer cells rewire their metabolism and mitochondrial oxidative phosphorylation (OXPHOS) to promote proliferation and maintenance. Cancer cells use multiple adaptive mechanisms in response to a hypo-nutrient environment. However, little is known about how cancer mitochondria are involved in the ability of these cells to adapt to a hypo-nutrient environment. Oncogenic HRas leads to suppression of the mitochondrial oxygen consumption rate (OCR), but oxygen consumption is essential for tumorigenesis. We found that in oncogenic HRas transformed cells, serum depletion reversibly increased the OCR and membrane potential. Serum depletion promoted a cancer stem cell (CSC)-like phenotype, indicated by an increase in CSC markers expression and resistance to anticancer agents. We also found that nitric oxide (NO) synthesis was significantly induced after serum depletion and that NO donors modified the OCR. An NOS inhibitor, SEITU, inhibited the OCR and CSC gene expression. It also reduced anchorage-independent growth by promoting apoptosis. In summary, our data provide new molecular findings that serum depletion induces NO synthesis and promotes mitochondrial OXPHOS, leading to tumor progression and a CSC phenotype. These results suggest that mitochondrial OCR inhibitors can be used as therapy against CSC.

The Expression of Ubiquitous Mitochondrial Creatine Kinase Is Downregulated as Prostate Cancer Progression.

BACKGROUND: Mitochondria play crucial roles in cell signaling events, interorganellar communication, aging, cell proliferation and apoptosis, and mitochondrial impairment has been shown to accelerate or modulate cancer progression. Ubiquitous mitochondrial creatine kinase (uMtCK) is predominantly localized in the intermembrane space of mitochondria and catalyzes the reversible exchange of high-energy phosphate between adenosine tri-phosphate (ATP) and phosphocreatine. However, little is known about its expression and function in human prostate cancer progression. METHOD: We investigated the expression of uMtCK in 148 prostate carcinoma tissues and matched normal tissue by immunohistochemistry. The expression and localization of uMtCK and hexokinase II, a marker of glycolysis, were examined in prostate carcinoma cell lines using western blot and immunofluorescence. RESULTS: MtCK expression was significantly lower in high Gleason grade carcinoma compared with normal prostate or low grade carcinoma. Western blot further revealed that uMtCK was highly expressed in LNCaP and 22Rv1 cell lines, as well as in the normal prostate cell line RWPE-1. However, uMtCK expression was almost absent in PC3 and DU145 cell lines, in correlation with absent or mutant p53 expression, respectively. In contrast, hexokinase II was overexpressed in PC3 cells. Moreover, in the low uMtCK expressing cell lines, glycolytic ATP production was increased, whereas mitochondrial ATP production was decreased. CONCLUSIONS: These data suggest that uMtCK is downregulated as prostate cancer progresses in correlation with a metabolic switch in ATP usage.

Dihydro-orotate dehydrogenase is physically associated with the respiratory complex and its loss leads to mitochondrial dysfunction.

Some mutations of the DHODH (dihydro-orotate dehydrogenase) gene lead to postaxial acrofacial dysostosis or Miller syndrome. Only DHODH is localized at mitochondria among enzymes of the de novo pyrimidine biosynthesis pathway. Since the pyrimidine biosynthesis pathway is coupled to the mitochondrial RC (respiratory chain) via DHODH, impairment of DHODH should affect the RC function. To investigate this, we used siRNA (small interfering RNA)-mediated knockdown and observed that DHODH knockdown induced cell growth retardation because of G2/M cell-cycle arrest, whereas pyrimidine deficiency usually causes G1/S arrest. Inconsistent with this, the cell retardation was not rescued by exogenous uridine, which should bypass the DHODH reaction for pyrimidine synthesis. DHODH depletion partially inhibited the RC complex III, decreased the mitochondrial membrane potential, and increased the generation of ROS (reactive oxygen species). We observed that DHODH physically interacts with respiratory complexes II and III by IP (immunoprecipitation) and BN (blue native)/SDS/PAGE analysis. Considering that pyrimidine deficiency alone does not induce craniofacial dysmorphism, the DHODH mutations may contribute to the Miller syndrome in part through somehow altered mitochondrial function.

Protein instability and functional defects caused by mutations of dihydro-orotate dehydrogenase in Miller syndrome patients.

Miller syndrome is a recessive inherited disorder characterized by postaxial acrofacial dysostosis. It is caused by dysfunction of the DHODH (dihydroorotate dehydrogenase) gene, which encodes a key enzyme in the pyrimidine de novo biosynthesis pathway and is localized at mitochondria intermembrane space. We investigated the consequence of three missense mutations, G202A, R346W and R135C of DHODH, which were previously identified in patients with Miller syndrome. First, we established HeLa cell lines stably expressing DHODH with Miller syndrome-causative mutations: G202A, R346W and R135C. These three mutant proteins retained the proper mitochondrial localization based on immunohistochemistry and mitochondrial subfractionation studies. The G202A, R346W DHODH proteins showed reduced protein stability. On the other hand, the third one R135C, in which the mutation lies at the ubiquinone-binding site, was stable but possessed no enzymatic activity. In conclusion, the G202A and R346W mutation causes deficient protein stability, and the R135C mutation does not affect stability but impairs the substrate-induced enzymatic activity, suggesting that impairment of DHODH activity is linked to the Miller syndrome phenotype.

Mitochondrial p32/C1QBP is highly expressed in prostate cancer and is associated with shorter prostate-specific antigen relapse time after radical prostatectomy.

Mitochondria are key organelles for ATP production and apoptosis. Therefore, impairment of mitochondria can modulate or accelerate cancer progression. p32, originally identified as a pre-mRNA splicing factor SF2/ASF-associated protein, is localized predominantly in the mitochondrial matrix and involved in mitochondria respiration. Recently, p32 was implicated in apoptosis and resultantly cancer progression. However, little is known about the expression and function of p32 in human tumors including prostate cancer. Here, we investigated the expression of p32 in 148 prostate carcinoma tissues by immunohistochemistry and found a positive correlation of p32 expression to clinicopathological parameters including follow-up data. p32 is highly expressed in prostate tumor samples and its expression is significantly associated with the Gleason score, pathological stage and relapse. For localized cancers, high p32 is a strong and independent predictor of clinical recurrence in multivariate analysis (P=0.01). In addition, p32 is overexpressed in the prostate cancer cell lines examined. The selective knockdown of p32 by RNA interference inhibits the growth of prostate cancer cell lines but not of a non-cancerous cell line. The p32 RNA interference decreases cyclin D1, increases p21 expression and causes a G1/S cell cycle arrest in prostate cancer cells. These data suggest that p32 is critical for prostate cancer cell proliferation and may be a novel marker of clinical progression in prostate cancer.

A comparative study of food habits and body shape perception of university students in Japan and Korea.

BACKGROUND: Abnormal body weight, dietary concerns, and unhealthy weight loss behaviors are increasingly being observed in young females in Japan. Our previous research has shown that the irregular lifestyles of female Japanese and Chinese students are significantly related to their desire to be thinner. In the present study, we compare the food habits and body shape preferences of female university students in South Korea and Japan to explore body shape perceptions in those populations. METHODS: A total of 265 female university students aged 19-25 years participated in this study. University students in Korea (n = 141) and university students in Japan (n = 124) completed a self-reported questionnaire. Data were analyzed using SPSS statistical software. Descriptive statistics were used to identify the demographic characteristics of the students and parametric variables were analyzed using the Student's t-test. Chi-square analyses were conducted for non-parametric variables. RESULTS: Comparison of body mass index (BMI) distributions in Japan and Korea showed the highest value in the normal category (74%) together with a very low obesity rate (1.2%). Significant differences were observed between the two countries in terms of eating patterns, with more Japanese eating breakfast daily and with Japanese students eating meals more regularly than Korean students. A difference was also observed in frequency of meals, where Korean students reported eating meals two times per day (59%) and the majority of Japanese students reported eating meals three times per day (81%). Although most subjects belonged to the normal BMI category, their ideal BMI classification was the underweight category (BMI: 18.4 +/- 3.4). CONCLUSION: Few studies have compared the health related practices of Japanese and Korean university students. The present results suggest the necessity of nutrition and health promotion programs for university students, especially programs emphasizing weight management.

Nutritional knowledge, food habits and health attitude of Chinese university students--a cross sectional study.

BACKGROUND: We have previously shown that irregular lifestyle of young Japanese female students are significantly related to their desire to be thinner. In the present study, we examined the nutritional knowledge and food habits of Chinese university students and compared them with those of other Asian populations. METHODS: A self-reported questionnaire was administered to 540 students, ranging in age from 19-24 years. Medical students from Beijing University (135 men and 150 women) in Northern China and Kunming Medical College in southern China (95 men and 160 women) participated in this study. The parametric variables were analyzed using the Student's t-test. Chi-square analyses were conducted for non-parametric variables. RESULTS: Our results showed that 80.5% of students had a normal BMI and 16.6 % of students were underweight with the prevalence of BMI>30 obesity being very low in this study sample. Young Chinese female students had a greater desire to be thinner (62.0%) than males (47.4%). Habits involving regular eating patterns and vegetable intake were reported and represent practices that ought to be encouraged. CONCLUSIONS: The university and college arenas represent the final opportunity for the health and nutritional education of a large number of students from the educator's perspective. Our findings suggest the need for strategies designed to improve competence in the area of nutrition.