Jinko Sawashita

Lanosterol synthase mutations cause cholesterol deficiency–associated cataracts in the Shumiya cataract rat

The Shumiya cataract rat (SCR) is a hereditary cataractous strain. It is thought that the continuous occurrence of poorly differentiated epithelial cells at the bow area of the lens forms the pathophysiological basis for cataract formation in SCRs. In this study, we attempted to identify the genes associated with cataract formation in SCRs by positional cloning. Genetic linkage analysis revealed the presence of a major cataract locus on chromosome 20 as well as a locus on chromosome 15 that partially suppressed cataract onset. Hypomorphic mutations were identified in genes for lanosterol synthase (Lss) on chromosome 20 and farnesyl diphosphate farnesyl transferase 1 (Fdft1) on chromosome 15, both of which function in the cholesterol biosynthesis pathway. A null mutation for Lss was also identified. Cataract onset was associated with the specific combination of Lss and Fdft1 mutant alleles that decreased cholesterol levels in cataractous lenses to about 57% of normal. Thus, cholesterol insufficiency may underlie the deficient proliferation of lens epithelial cells in SCRs, which results in the loss of homeostatic epithelial cell control of the underlying fiber cells and eventually leads to cataractogenesis. These findings may have some relevance to other types of cataracts, inborn defects of cholesterol synthesis, and the effects of cholesterol-lowering medication.

Ubiquinol-10 Supplementation Activates Mitochondria Functions to Decelerate Senescence in Senescence-Accelerated Mice

AIMnThe present study was conducted to define the relationship between the anti-aging effect of ubiquinol-10 supplementation and mitochondrial activation in senescence-accelerated mouse prone 1 (SAMP1) mice.nnnRESULTSnHere, we report that dietary supplementation with ubiquinol-10 prevents age-related decreases in the expression of sirtuin gene family members, which results in the activation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a major factor that controls mitochondrial biogenesis and respiration, as well as superoxide dismutase 2 (SOD2) and isocitrate dehydrogenase 2 (IDH2), which are major mitochondrial antioxidant enzymes. Ubiquinol-10 supplementation can also increase mitochondrial complex I activity and decrease levels of oxidative stress markers, including protein carbonyls, apurinic/apyrimidinic sites, malondialdehydes, and increase the reduced glutathione/oxidized glutathione ratio. Furthermore, ubiquinol-10 may activate Sirt1 and PGC-1α by increasing cyclic adenosine monophosphate (cAMP) levels that, in turn, activate cAMP response element-binding protein (CREB) and AMP-activated protein kinase (AMPK).nnnINNOVATION AND CONCLUSIONnThese results show that ubiquinol-10 may enhance mitochondrial activity by increasing levels of SIRT1, PGC-1α, and SIRT3 that slow the rate of age-related hearing loss and protect against the progression of aging and symptoms of age-related diseases.

Characterization of the Cheetah Serum Amyloid A1 Gene: Critical Role and Functional Polymorphism of a Cis-Acting Element

Amyloid A (AA) amyloidosis is one of the principal causes of morbidity and mortality in captive cheetahs (Acinonyx jubatus), which are in danger of extinction. For practical conservation of this species, therefore, it is critical to elucidate the etiology of AA amyloidosis, especially to understand the mechanisms of transcriptional regulation of serum amyloid A (SAA), a precursor protein of the AA protein. In this study, the structure and nucleotide sequence of the cheetah SAA1 gene including the 5-flanking promoter/enhancer region was determined. Putative nuclear factor kappa-B (NF-kappaB) and CCAAT/enhancer binding protein beta (C/EBPbeta) cis-acting elements, which play key roles in SAA1 transcriptional induction in response to inflammation, were identified in the 5-flanking region of the cheetah SAA1 gene. Fortuitously, a single nucleotide polymorphism was identified in the captive cheetah cohort in the putative NF-kappaB cis-acting element and had a remarkable effect on SAA1 transcriptional induction. These results provide a foundation not only for clarifying the etiology of AA amyloidosis in the cheetah but also for contriving a strategy for conservation of this species.

ApoA-I deficiency in mice is associated with redistribution of apoA-II and aggravated AApoAII amyloidosis

Apolipoprotein A-II (apoA-II) is the second major apolipoprotein following apolipoprotein A-I (apoA-I) in HDL. ApoA-II has multiple physiological functions and can form senile amyloid fibrils (AApoAII) in mice. Most circulating apoA-II is present in lipoprotein A-I/A-II. To study the influence of apoA-I on apoA-II and AApoAII amyloidosis, apoA-I-deficient (C57BL/6J.Apoa1⁻/⁻) mice were used. Apoa1⁻/⁻ mice showed the expected significant reduction in total cholesterol (TC), HDL cholesterol (HDL-C), and triglyceride (TG) plasma levels. Unexpectedly, we found that apoA-I deficiency led to redistribution of apoA-II in HDL and an age-related increase in apoA-II levels, accompanied by larger HDL particle size and an age-related increase in TC, HDL-C, and TG. Aggravated AApoAII amyloidosis was induced in Apoa1⁻/⁻ mice systemically, especially in the heart. These results indicate that apoA-I plays key roles in maintaining apoA-II distribution and HDL particle size. Furthermore, apoA-II redistribution may be the main reason for aggravated AApoAII amyloidosis in Apoa1⁻/⁻ mice. These results may shed new light on the relationship between apoA-I and apoA-II as well as provide new information concerning amyloidosis mechanism and therapy.

Serum amyloid A expression in the breast cancer tissue is associated with poor prognosis

Background Serum amyloid A (SAA), an acute-phase protein, is expressed primarily in the liver, and recently found also expressed in cancer tissues. However, its expression and prognostic value in breast cancer have not been described. Results SAA protein was found expressed in tumor cells in 44.2% cases and in TAM in 62.5% cases. FISH showed more frequent SAA mRNA expression in TAM than in tumor cells (76% versus 12%, p < 0.001), and a significant association between the frequencies of SAA mRNA expression in TAM and tumor cells (rs = 0.603, p < 0.001). The immunoreactivities of SAA protein in TAM and tumor cells were both associated with lymphovascular invasion and lymph node metastasis. Moreover, SAA-positivity in TAMs was associated with larger tumor-size, higher histological-grade, negative estrogen-receptor and progesterone-receptor statuses, and HER-2 overexpression. It was also linked to worse recurrence-free survival in a multivariable regression model. Methods Immunohistochemistry was applied on the tumor tissues from 208 breast cancer patients to evaluate the local SAA-protein expression with additional CD68 stain to identify the tumor-associated macrophage (TAM) on the serial tissue sections. Fluorescent in situ hybridization (FISH) was conducted on serial tissue sections from 25 of the 208 tumors to examine the expression and location of SAA mRNA. Conclusions Our results suggested that the TAMs may be a pivotal and main source of SAA production in tumor microenvironment of breast cancer. SAA immunoreactivity in TAM is associated with worse recurrence-free survival, and is therefore a biomarker candidate for postoperative surveillance and perhaps a therapeutic target for breast cancer.

Coenzyme Q10 Improves Lipid Metabolism and Ameliorates Obesity by Regulating CaMKII-Mediated PDE4 Inhibition

Our recent studies revealed that supplementation with the reduced form of coenzyme Q10 (CoQ10H2) inhibits oxidative stress and slows the process of aging in senescence-accelerated mice. CoQ10H2 inhibits adipocyte differentiation and regulates lipid metabolism. In the present study, we show that dietary supplementation with CoQ10H2 significantly reduced white adipose tissue content and improved the function of brown adipose tissue by regulating expression of lipid metabolism-related factors in KKAy mice, a model of obesity and type 2 diabetes. In the liver, CoQ10H2 reduced cytoplasmic Ca2+ levels and consequently inhibited the phosphorylation of CaMKII. CoQ10H2 also regulated the activity of the transcription factor C-FOS and inhibited gene expression of PDE4, a cAMP-degrading enzyme, via the CaMKII-MEK1/2-ERK1/2 signaling pathway, thereby increasing intracellular cAMP. This increased cAMP activated AMPK, enhanced oxidative decomposition of lipids, and inhibited de novo synthesis of fatty acids, inhibiting the development and progression of obesity and type 2 diabetes. These results suggest that CoQ10H2 supplementation may be useful as a treatment for metabolic disorders associated with obesity.

Extracellular deposition of mouse senile AApoAII amyloid fibrils induced different unfolded protein responses in the liver, kidney, and heart

Mouse senile amyloidosis is a disorder in which apolipoprotein A-II deposits extracellularly in many organs as amyloid fibrils (AApoAII). In this study, we intravenously injected 1u2009μg of isolated AApoAII fibrils into R1.P1-Apoa2c mice, to induce AApoAII amyloidosis. We observed that the unfolded protein response was induced by deposition of AApoAII amyloid. We found that the mRNA and the protein expression levels of heat shock protein A5 (HSPA5; also known as glucose-regulated protein 78) were increased in the liver with AApoAII amyloid deposits. Immunohistochemistry showed that HSPA5 was only detected in hepatocytes close to AApoAII amyloid deposits. Furthermore, gene transcription of several endoplasmic reticulum (ER) stress-related proteins increased, including eukaryotic translation initiation factor 2 alpha kinase 3 (Eif2ak3), activating transcription factor 6 (Atf6), activating transcription factor 4 (Atf4), X-box-binding protein 1 splicing (Xbp1s), DNA-damage inducible transcript 3 (Ddit3), and autophagy protein 5 (Atg5). Moreover, apoptosis-positive cells were increased in the liver. Similar results were seen in the kidney but not in the heart. Our study indicates that ER stress responses differed among tissues with extracellular AApoAII amyloid fibril deposition. Although upregulated HSPA5 and the activated unfolded protein response might have roles in protecting tissues against aggregated extracellular AApoAII amyloid deposition, prolonged ER stress induced apoptosis in the liver and the kidney.

C-terminal sequence of amyloid-resistant type F apolipoprotein A-II inhibits amyloid fibril formation of apolipoprotein A-II in mice

Significance Apolipoprotein (apo) A-II is the most important protein associated with senile amyloidosis. Because some variants of apoA-II protein have been found among inbred strains of mice, we hypothesized that investigating amyloidogenesis of the variants would improve our understanding of the molecular and biological mechanisms of senile amyloidosis. Here, we demonstrate that mice with type F apoA-II (APOA2F) protein were absolutely resistant to senile amyloidosis. Moreover, a selective C-terminal APOA2F peptide inhibited fibril formation of amyloidogenic apoA-II in vitro and prevented senile amyloidosis in mice. We propose an inhibitory model in which the C-terminal APOA2F peptide prevents further fibril extension by blocking the active ends of seeds. This approach could provide a novel therapeutic option for the treatment of senile amyloidosis. In murine senile amyloidosis, misfolded serum apolipoprotein (apo) A-II deposits as amyloid fibrils (AApoAII) in a process associated with aging. Mouse strains carrying type C apoA-II (APOA2C) protein exhibit a high incidence of severe systemic amyloidosis. Previously, we showed that N- and C-terminal sequences of apoA-II protein are critical for polymerization into amyloid fibrils in vitro. Here, we demonstrate that congenic mouse strains carrying type F apoA-II (APOA2F) protein, which contains four amino acid substitutions in the amyloidogenic regions of APOA2C, were absolutely resistant to amyloidosis, even after induction of amyloidosis by injection of AApoAII. In vitro fibril formation tests showed that N- and C-terminal APOA2F peptides did not polymerize into amyloid fibrils. Moreover, a C-terminal APOA2F peptide was a strong inhibitor of nucleation and extension of amyloid fibrils during polymerization. Importantly, after the induction of amyloidosis, we succeeded in suppressing amyloid deposition in senile amyloidosis-susceptible mice by treatment with the C-terminal APOA2F peptide. We suggest that the C-terminal APOA2F peptide might inhibit further extension of amyloid fibrils by blocking the active ends of nuclei (seeds). We present a previously unidentified model system for investigating inhibitory mechanisms against amyloidosis in vivo and in vitro and believe that this system will be useful for the development of novel therapies.

Caloric restriction reduces the systemic progression of mouse AApoAII amyloidosis.

In mouse senile amyloidosis, apolipoprotein (Apo) A-II is deposited extracellularly in many organs in the form of amyloid fibrils (AApoAII). Reduction of caloric intake, known as caloric restriction (CR), slows the progress of senescence and age-related disorders in mice. In this study, we intravenously injected 1 μg of isolated AApoAII fibrils into R1.P1-Apoa2c mice to induce experimental amyloidosis and investigated the effects of CR for the next 16 weeks. In the CR group, AApoAII amyloid deposits in the liver, tongue, small intestine and skin were significantly reduced compared to those of the ad libitum feeding group. CR treatment led to obvious reduction in body weight, improvement in glucose metabolism and reduction in the plasma concentration of ApoA-II. Our molecular biological analyses of the liver suggested that CR treatment might improve the symptoms of inflammation, the unfolded protein response induced by amyloid deposits and oxidative stress. Furthermore, we suggest that CR treatment might improve mitochondrial functions via the sirtuin 1-peroxisome proliferator-activated receptor γ coactivator 1α (SIRT1-PGC-1α) pathway. We suggest that CR is a promising approach for treating the onset and/or progression of amyloidosis, especially for systemic amyloidosis such as senile AApoAII amyloidosis. Our analysis of CR treatment for amyloidosis should provide useful information for determining the cause of amyloidosis and developing effective preventive treatments.

Apolipoprotein A-II induces acute-phase response associated AA amyloidosis in mice through conformational changes of plasma lipoprotein structure

During acute-phase response (APR), there is a dramatic increase in serum amyloid A (SAA) in plasma high density lipoproteins (HDL). Elevated SAA leads to reactive AA amyloidosis in animals and humans. Herein, we employed apolipoprotein A-II (ApoA-II) deficient (Apoa2−/−) and transgenic (Apoa2Tg) mice to investigate the potential roles of ApoA-II in lipoprotein particle formation and progression of AA amyloidosis during APR. AA amyloid deposition was suppressed in Apoa2−/− mice compared with wild type (WT) mice. During APR, Apoa2−/− mice exhibited significant suppression of serum SAA levels and hepatic Saa1 and Saa2 mRNA levels. Pathological investigation showed Apoa2−/− mice had less tissue damage and less inflammatory cell infiltration during APR. Total lipoproteins were markedly decreased in Apoa2−/− mice, while the ratio of HDL to low density lipoprotein (LDL) was also decreased. Both WT and Apoa2−/− mice showed increases in LDL and very large HDL during APR. SAA was distributed more widely in lipoprotein particles ranging from chylomicrons to very small HDL in Apoa2−/− mice. Our observations uncovered the critical roles of ApoA-II in inflammation, serum lipoprotein stability and AA amyloidosis morbidity, and prompt consideration of therapies for AA and other amyloidoses, whose precursor proteins are associated with circulating HDL particles.