Mitochondrial translation failure represses cholesterol gene expression via Pyk2-Gsk3ß-Srebp2 axis.

Neurodegenerative diseases and other age-related disorders are closely associated with mitochondrial dysfunction. We previously showed that mice with neuron-specific deficiency of mitochondrial translation exhibit leukoencephalopathy because of demyelination. Reduced cholesterol metabolism has been associated with demyelinating diseases of the brain such as Alzheimer's disease. However, the molecular mechanisms involved and relevance to the pathogenesis remained unknown. In this study, we show that inhibition of mitochondrial translation significantly reduced expression of the cholesterol synthase genes and degraded their sterol-regulated transcription factor, sterol regulatory element-binding protein 2 (Srebp2). Furthermore, the phosphorylation of Pyk2 and Gsk3ß was increased in the white matter of p32cKO mice. We observed that Pyk2 inhibitors reduced the phosphorylation of Gsk3ß and that GSK3ß inhibitors suppressed degradation of the transcription factor Srebp2. The Pyk2-Gsk3ß axis is involved in the ubiquitination of Srebp2 and reduced expression of cholesterol gene. These results suggest that inhibition of mitochondrial translation may be a causative mechanism of neurodegenerative diseases of aging. Improving the mitochondrial translation or effectiveness of Gsk3ß inhibitors is a potential therapeutic strategy for leukoencephalopathy.

Cardiomyocyte-specific deletion of the mitochondrial transporter Abcb10 causes cardiac dysfunction via lysosomal-mediated ferroptosis.

Heart function is highly dependent on mitochondria, which not only produce energy but also regulate many cellular functions. Therefore, mitochondria are important therapeutic targets in heart failure. Abcb10 is a member of the ABC transporter superfamily located in the inner mitochondrial membrane and plays an important role in haemoglobin synthesis, biliverdin transport, antioxidant stress, and stabilization of the iron transporter mitoferrin-1. However, the mechanisms underlying the impairment of mitochondrial transporters in the heart remain poorly understood. Here, we generated mice with cardiomyocyte-specific loss of Abcb10. The Abcb10 knockouts exhibited progressive worsening of cardiac fibrosis, increased cardiovascular risk markers and mitochondrial structural abnormalities, suggesting that the pathology of heart failure is related to mitochondrial dysfunction. As the mitochondrial dysfunction was observed early but mildly, other factors were considered. We then observed increased Hif1α expression, decreased NAD synthase expression, and reduced NAD+ levels, leading to lysosomal dysfunction. Analysis of ABCB10 knockdown HeLa cells revealed accumulation of Fe2+ and lipid peroxides in lysosomes, leading to ferroptosis. Lipid peroxidation was suppressed by treatment with iron chelators, suggesting that lysosomal iron accumulation is involved in ferroptosis. We also observed that Abcb10 knockout cardiomyocytes exhibited increased ROS production, iron accumulation, and lysosomal hypertrophy. Our findings suggest that Abcb10 is required for the maintenance of cardiac function and reveal a novel pathophysiology of chronic heart failure related to lysosomal function and ferroptosis.

Improving lysosomal ferroptosis with NMN administration protects against heart failure.

Myocardial mitochondria are primary sites of myocardial energy metabolism. Mitochondrial disorders are associated with various cardiac diseases. We previously showed that mice with cardiomyocyte-specific knockout of the mitochondrial translation factor p32 developed heart failure from dilated cardiomyopathy. Mitochondrial translation defects cause not only mitochondrial dysfunction but also decreased nicotinamide adenine dinucleotide (NAD+) levels, leading to impaired lysosomal acidification and autophagy. In this study, we investigated whether nicotinamide mononucleotide (NMN) administration, which compensates for decreased NAD+ levels, improves heart failure because of mitochondrial dysfunction. NMN administration reduced damaged lysosomes and improved autophagy, thereby reducing heart failure and extending the lifespan in p32cKO mice. We found that lysosomal damage due to mitochondrial dysfunction induced ferroptosis, involving the accumulation of iron in lysosomes and lipid peroxide. The ameliorative effects of NMN supplementation were found to strongly affect lysosomal function rather than mitochondrial function, particularly lysosome-mediated ferroptosis. NMN supplementation can improve lysosomal, rather than mitochondrial, function and prevent chronic heart failure.

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.

Mitochondrial translation inhibition triggers ATF4 activation, leading to integrated stress response but not to mitochondrial unfolded protein response.

Mitochondrial-nuclear communication, known as retrograde signaling, is important for regulating nuclear gene expression in response to mitochondrial dysfunction. Previously, we have found that p32/C1qbp-deficient mice, which have a mitochondrial translation defect, show endoplasmic reticulum (ER) stress response and integrated stress response (ISR) gene expression in the heart and brain. However, the mechanism by which mitochondrial translation inhibition elicits these responses is not clear. Among the transcription factors that respond to mitochondrial stress, activating transcription factor 4 (ATF4) is a key transcription factor in the ISR. Herein, chloramphenicol (CAP), which inhibits mitochondrial DNA (mtDNA)-encoded protein expression, induced eukaryotic initiation factor 2 α subunit (eIF2α) phosphorylation and ATF4 induction, leading to ISR gene expression. However, the expression of the mitochondrial unfolded protein response (mtUPR) genes, which has been shown in Caenorhabditis elegans, was not induced. Short hairpin RNA-based knockdown of ATF4 markedly inhibited the CAP-induced ISR gene expression. We also observed by ChIP analysis that induced ATF4 bound to the promoter region of several ISR genes, suggesting that mitochondrial translation inhibition induces ISR gene expression through ATF4 activation. In the present study, we showed that mitochondrial translation inhibition induced the ISR through ATF4 activation rather than the mtUPR.

Macrophage Migration Inhibitory Factor Deficiency Causes Prolonged Hearing Loss After Acoustic Overstimulation.

HYPOTHESIS: Macrophage migration inhibitory factor plays an important role in noise-induced hearing loss. BACKGROUND: Macrophage migration inhibitory factor is an essential factor in axis formation and neural development. Macrophage migration inhibitory factor is expressed in the inner ear, but its function remains to be elucidated. METHODS: Macrophage migration inhibitory factor-deficient mice (MIF(-/-) mice) were used in this study. Wild-type and MIF(-/-) mice received noise exposure composed of octave band noise. Auditory brainstem response thresholds were examined before (control) and at 0, 12, and 24 hours and 2 weeks after the intense noise exposure. Morphological findings of cochlear hair cells were investigated using scanning electron microscopy. Histopathological examination with hematoxylin and eosin staining and TUNEL assay were also performed. RESULTS: In both the wild-type and MIF(-/-) mice, acoustic overstimulation induced significant hearing loss compared with the control level. Two weeks after the intense noise exposure, the MIF(-/-) mice had an increased hearing threshold compared with the wild-type mice. Scanning electron microscopy demonstrated that the outer hair cells in the MIF(-/-) mice were affected 2 weeks after noise exposure compared with the wild-type mice. TUNEL-positive cells were identified in the organ of Corti of the MIF(-/-) mice. CONCLUSION: The MIF(-/-) mice had prolonged hearing loss and significant loss of cochlear hair cells after intense noise exposure. Macrophage migration inhibitory factor may play an important role in recovery from acoustic trauma. Management of macrophage migration inhibitory factor may be a novel therapeutic option for noise-induced hearing loss.

Determining minimal clinically important differences in Japanese cedar/cypress pollinosis patients.

BACKGROUND: Statistically significant results of medical intervention trials are not always clinically meaningful. We sought to estimate the minimal clinically important difference (MCID) (the smallest change in a given endpoint that is meaningful to a patient) during seasonal alteration of Japanese cedar/cypress pollinosis (JCCP). METHODS: Results of a double-blinded, placebo-controlled trial of JCCP patients conducted between 2008 and 2010 were analyzed using an anchor-based method in which a face scale for Japanese rhinoconjunctivitis quality-of-life questionnaire (JRQLQ) was set as an anchor. MICDs were calculated as changes of average scores, including those for naso-ocular symptoms with 5 items in diary cards (T5SS), naso-ocular symptoms with 6 items (T6SS) and QOL with 17 items on the JRQLQ when face scale scores either improved or deteriorated by one point. RESULTS: In 2009 and 2010, 3,698 and 374, respectively, grains/cm(2) of pollens were dispersed. The MCIDs for T5SS in 2009 and 2010 were 1.426 (0.285 per item) and 1.441 (0.288), respectively. The MCIDs for T6SS were 4.115 (0.686) and 3.183 (0.531) in 2009 and 2010, respectively. The MCIDs for QOL were 10.469 (0.616) and 6.026 (0.354) in 2009 and 2010, respectively. CONCLUSIONS: For T5SS in the diary, T6SS and QOL in JRQLQ, unit differences of 1.5 (0.3 per item), 3.6 (0.6) and 8.2 (0.5), respectively, were considered clinically meaningful by JCCP patients. The MCID for symptoms recorded in the diary was stable irrespective of the dispersed pollen level.

Expression of toll-like receptors in chronic otitis media and cholesteatoma.

OBJECTIVE: Otitis media is one of the most common infectious diseases, especially in young children. Multiple factors affect the onset or development of otitis media. Human toll-like receptors recognize associated patterns and play a critical role in innate immune mechanisms. Toll-like receptors are considered to be important factors for clearance of infection and resolution of inflammation in otitis media. The purpose of this study was to evaluate the histological expression of toll-like receptor 2, which recognizes many kinds of pathogen-associated molecular patterns, and toll-like receptor 4, which recognizes lipopolysaccharide on Gram-negative bacteria, in tissue samples from patients with chronic otitis media and middle ear cholesteatoma. METHODS: Human middle ear tissue samples from 12 patients with chronic otitis media (n=7) and acquired middle ear cholesteatoma (n=5) were examined. Normal control middle ear samples without any inflammation were also included (n=7). The expressions of toll-like receptors 2 and 4 in middle ear tissues were examined immunohistochemically. RESULTS: Only one normal control middle ear sample showed weak expression of toll-like receptor 2, and toll-like receptor 4 was not observed in all control samples. On the other hand, both toll-like receptors 2 and 4 were markedly expressed in chronic otitis media and cholesteatoma. There was a significant difference between chronic otitis media and normal controls in the expressions of both toll-like receptors. Significant up-regulation of toll-like receptors 2 and 4 was observed in cholesteatoma as compared with control samples. CONCLUSIONS: Toll-like receptors 2 and 4 were strongly expressed in chronic otitis media and middle ear cholesteatoma. These findings suggest that toll-like receptors may play a principal role in human chronic otitis media and cholesteatoma.

Minimally invasive procedure for accurate diagnosis of mucosa-associated lymphoid tissue lymphoma of the head and neck.

Sonography-guided cutting needle biopsy for the diagnosis of malignant lymphoma has recently come into wide use. However, surgery is sometimes unavoidable for the diagnosis of malignant lymphoma, particularly for low-grade malignant lymphoma such as extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue, because cutting needle biopsy offers limited diagnostic accuracy for low-grade malignant lymphoma. Of course, unnecessary invasive procedures like open biopsy should be avoided wherever possible, given the cosmetic problems and burden on the patient. We tried to diagnose malignant lymphoma using the combination of cutting needle biopsy, flow cytometry and polymerase chain reaction to identify monoclonal rearrangement of immunoglobulin heavy chain genes. We have used this method in two cases in whom malignant lymphoma was suspected in the head and neck region, allowing diagnosis of mucosa-associated lymphoid tissue lymphoma in both cases. One case involved a 23-year-old woman with mucosa-associated lymphoid tissue lymphoma in the parotid glands, and the other involved a 77-year-old man with mucosa-associated lymphoid tissue lymphoma in the thyroid. The combination of cutting needle biopsy, flow cytometry and immunoglobulin heavy chain gene rearrangement testing might offer a useful alternative to open biopsy for the diagnosis of mucosa-associated lymphoid tissue lymphoma. We recommend this procedure, particularly for young women or patients with poor performance status in whom malignant lymphoma is suspected.