Supplementation with ubiquinol 10 has been shown to improve the health of experimental animals and elderly individuals. The present study investigated the effects of lifetime supplementation with ubiquinol 10 on the progression of aging and lifespan in C57BL/6 mice, a standard strain for biomedical and aging research. A diet containing ubiquinol 10 (0.3 % w/w) and a control diet were fed to female C57BL/6J mice from 8 weeks of age until death, and the progression of senescence, lifespan, and physiological and pathological findings were examined. Body weights increased until 72 weeks of age and then decreased gradually in both groups. Food intake was significantly higher in the ubiquinol 10 group until 56 weeks of age. The median, 10th decile (10 % survivors), and maximum lifespans and survival curves did not significantly differ between the ubiquinol 10 and control groups. The grading score of senescence improved only at the age of 48 weeks in ubiquinol 10 group. No significant differences were observed in major physiological markers, such as the glucose tolerance test, serum triglycerides, and cholesterol concentrations. The expression levels of genes regulating aging in the liver markedly decreased with age in both groups. Organ weights did not significantly differ, except for significantly lighter brown adipose tissue in the ubiquinol 10 group. Senile AApoAII amyloidosis was noted in old mice; however, the degree of amyloid deposition was similar in the two groups. The degree of senescence only improved in middle-aged mice (48 weeks of age), and no apparent anti-aging or lifespan-extending effects were observed with lifetime supplementation with ubiquinol 10 in female C57BL/6J mice, which are standard laboratory mice considered to exhibit normal aging processes.

Amyloid resistance is the inability or the reduced susceptibility of an organism to develop amyloidosis. In this study we have analysed the molecular basis of the resistance to systemic AApoAII amyloidosis, which arises from the formation of amyloid fibrils from apolipoprotein A-II (ApoA-II). The disease affects humans and animals, including SAMR1C mice that express the C allele of ApoA-II protein, whereas other mouse strains are resistant to development of amyloidosis due to the expression of other ApoA-II alleles, such as ApoA-IIF. Using cryo-electron microscopy, molecular dynamics simulations and other methods, we have determined the structures of pathogenic AApoAII amyloid fibrils from SAMR1C mice and analysed the structural effects of ApoA-IIF-specific mutational changes. Our data show that these changes render ApoA-IIF incompatible with the specific fibril morphologies, with which ApoA-II protein can become pathogenic in vivo.

Amyloidosis is a group of diseases in which proteins become amyloid, an insoluble fibrillar aggregate, resulting in organ dysfunction. Amyloid deposition has been reported in various animal species. To diagnose and understand the pathogenesis of amyloidosis, it is important to identify the amyloid precursor protein involved in each disease. Although 42 amyloid precursor proteins have been reported in humans, little is known about amyloidosis in animals, except for a few well-described amyloid proteins, including amyloid A (AA), amyloid light chain (AL), amyloid β (Aβ), and islet amyloid polypeptide-derived amyloid. Recently, several types of novel amyloidosis have been identified in animals using immunohistochemistry and mass spectrometry-based proteomic analysis. Certain species are predisposed to specific types of amyloidosis, suggesting a genetic background for its pathogenesis. Age-related amyloidosis has also emerged due to the increased longevity of captive animals. In addition, experimental studies have shown that some amyloids may be transmissible. Accurate diagnosis and understanding of animal amyloidosis are necessary for appropriate therapeutic intervention and comparative pathological studies. This review provides an updated classification of animal amyloidosis, including associated protein misfolding disorders of the central nervous system, and the current understanding of their pathogenesis. Pathologic features are presented together with state-of-the-art diagnostic methods that can be applied for routine diagnosis and identification of novel amyloid proteins in animals.

Spontaneous and age-related amyloidosis has been reported in C57BL/6J mice. However, the biochemical characteristics of age-related amyloidosis remain unclear. Herein, the age-related prevalence of amyloidosis, the types of amyloid fibril proteins, and the effects of amyloid deposition were investigated in renal function in C57BL/6J mice. The results obtained revealed a high incidence of amyloidosis in C57BL/6J mice originating from The Jackson Laboratory as well as the deposition of large amounts of amyloid in the glomeruli of aged mice. The amyloid fibril protein was identified as wild-type apolipoprotein A-II (ApoA-II). Induction of amyloid deposition in 40-week-old mice, equivalent to that of spontaneous development in 80-week-old mice, to rule out the effects of aging, revealed subsequent damage to kidney function by amyloid deposits. Furthermore, amyloid deposition in the mesangial region decreased podocyte density, compromised foot processes, and led to the accumulation of fibroblast growth factor 2 in glomeruli. Collectively, these results suggest that ApoA-II deposition is a general pathology in aged C57BL/6J mice and is dependent on supplier colonies. Therefore, the effects of age-related amyloid deposition need to be considered in research on aging in mice.

Amyloidosis refers to a group of degenerative diseases that are characterized by the deposition of misfolded protein fibrils in various organs. Deposited amyloid may be removed by a phagocyte-dependent innate immune system; however, the precise mechanisms during disease progression remain unclear. We herein investigated the properties of macrophages that contribute to amyloid degradation and disease progression using inducible apolipoprotein A-II amyloidosis model mice. Intravenously injected AApoAII amyloid was efficiently engulfed by reticuloendothelial macrophages in the liver and spleen and disappeared by 24 h. While cultured murine macrophages degraded AApoAII via the endosomal-lysosomal pathway, AApoAII fibrils reduced cell viability and phagocytic capacity. Furthermore, the depletion of reticuloendothelial macrophages before the induction of AApoAII markedly increased hepatic and splenic AApoAII deposition. These results highlight the physiological role of reticuloendothelial macrophages in the early stages of pathogenesis and suggest the maintenance of phagocytic integrity as a therapeutic strategy to inhibit disease progression.

The Matsumoto Eosinophilia Shinshu (MES) is a rat model for hereditary blood eosinophilia. The incidence of eosinophilia is 100% in both female and male MES. The primary cause of the eosinophilia in MES is a loss-of-function mutation in the gene encoding the cytochrome b-245, alpha polypeptide (Cyba(mes) mutant allele). CYBA protein is a constituent of the superoxide-generating NADPH oxidase complex, the catalytic subunit of which is either NOX1, NOX2, or NOX4. However, the molecular mechanisms for the loss of CYBA to cause eosinophilia and even which of the three NOX isotypes is causally linked to the disease have been unknown. To resolve the latter issue, we generated F344/N rats knockout for Nox1, Nox2, and Nox4 genes. Also, we bred F344.MES-Cyba(mes) congenic rats that have a similar genetic background to the Nox knockout rats. We found that approximately 20% of female F344/N-Nox2(em1) rats but none of the males developed blood eosinophilia. Also, we observed that all female F344.MES-Cybames and approximately 50% of male congenic rats developed the disorder. These results revealed that loss of NOX2 is the cause of blood eosinophilia in rats. Meanwhile, the data also indicated that in addition to the loss of NOX2 NADPH oxidase, both the genetic background of F344/N strain and gender influence the development of the disorder. These Nox and Cyba mutant rat strains with different eosinophilia incidences should be useful to elucidate molecular mechanisms and factors involved in the development of the disease.

In humans, mutations in the coproporphyrinogen oxidase (CPOX) gene can result in hereditary coproporphyria (HCP), characterized by high levels of coproporphyrin excretion in the urine and feces, as well as acute neurovisceral and chronic cutaneous manifestations. Appropriate animal models for comprehending the precise pathogenesis mechanism of HCP have not been reported that show similarities in terms of gene mutation, reduced CPOX activity, excess coproporphyrin accumulation, and clinical symptoms. As previously discovered, the BALB.NCT-Cpoxnct mouse carries a hypomorphic mutation in the Cpox gene. Due to the mutation, BALB.NCT-Cpoxnct had a drastic increase in coproporphyrin in the blood and liver persistently from a young age. In this study, we found that BALB.NCT-Cpoxnct mice manifested HCP symptoms. Similar to HCP patients, BALB.NCT-Cpoxnct excreted an excessive amount of coproporphyrin and porphyrin precursors in the urine and displayed neuromuscular symptoms, such as a lack of grip strength and impaired motor coordination. Male BALB.NCT-Cpoxnct had nonalcoholic steatohepatitis (NASH)-like liver pathology and sclerodermatous skin pathology. A portion of male mice had liver tumors as well, whereas female BALB.NCT-Cpoxnct lacked these hepatic and cutaneous pathologies. In addition, we discovered that BALB.NCT-Cpoxnct exhibited microcytic anemia. These results indicate that BALB.NCT-Cpoxnct mice serve as the suitable animal model to help gain insight into the pathogenesis and therapy of HCP.

Exercise interventions are beneficial for reducing the risk of age related diseases, including amyloidosis, but the underlying molecular links remain unclear. Here, we investigated the protective role of interval exercise training in a mouse model of age-related systemic apolipoprotein A-II amyloidosis (AApoAII) and identified potential mechanisms. Mice subjected to 16 weeks of exercise showed improved whole-body physiologic functions and exhibited substantial inhibition of amyloidosis, particularly in the liver and spleen. Exercise activated the hepatic p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway and the downstream transcription factor tumor suppressor p53. This activation resulted in elevated expression and phosphorylation of heat shock protein beta-1 (HSPB1), a chaperone that defends against protein aggregation. In amyloidosis-induced mice, the hepatic p38 MAPK-related adaptive responses were additively enhanced by exercise. We observed that with exercise, greater amounts of phosphorylated HSPB1 accumulated at amyloid deposition areas, which we suspect inhibits amyloid fibril formation. Collectively, our findings demonstrate the exercise-activated specific chaperone prevention of amyloidosis, and suggest that exercise may amplify intracellular stress-related protective adaptation pathways against age-associated disorders, such as amyloidosis.

The Nakano cataract mouse (NCT) manifests a wavy coat for their first hair as a genetic trait. In this study, we explored the molecular genetic basis of the wavy coat. We revealed by crossing experiments that the wavy coat is controlled by a major gene on chromosome 7 of NCT, homozygosity of which is a prerequisite for developing the wavy coat, and by a gene on chromosome 9 with a minor effect to reinforce the manifestation of the trait. In humans, a polymorphism of the protease, serine 53 (PRSS53) gene on the homologous chromosome is known to be associated with curly scalp hair. We then investigated the Prss53 gene and discovered that NCT has an insertion of an intracisternal A particle element in the first intron of the gene. Nevertheless, the expression of the Prss53 is not altered in the NCT skin both in transcript and protein levels. Subsequently, we created C57BL/6J-Prss53em1 knockout mice and found that these mice manifest vague wavy coats. A portion of backcross and intercross mice between the C57BL/6J-Prss53em1 and NCT manifested intense or vague wavy coats. These findings demonstrate the polygenic nature of the wavy coat of NCT and Prss53 knockout mice and highlight the similarity of the trait to the curly hair of humans associated with the PRSS53 alteration.

The BALB.NCT-Cpoxnct is a mutant mouse model for hereditary cataracts. We previously uncovered that the primary cause of the cataracts of BALB.NCT-Cpoxnct is a mutation in the coproporphyrinogen oxidase (Cpox) gene. Because of the mutation, excessive coproporphyrin is accumulated in the BALB.NCT-Cpoxnct lens. In this study, we analyzed the changes in transcriptome and proteins in the lenses of 4- and 12-week-old BALB.NCT-Cpoxnct to further elucidate the molecular etiology of cataracts in this mouse strain. Transcriptome analysis revealed that endoplasmic reticulum (ER) stress was increased in the BALB.NCT-Cpoxnct lens that induced persistent activation of the PERK signaling pathway of the ER stress response. Also, levels of crystallin transcripts and proteins were reduced in the BALB.NCT-Cpoxnct lens. Analysis of proteins disclosed aggregation of crystallins and keratins prior to the manifestation of cataracts in 4-week-old BALB.NCT-Cpoxnct mice. At 12 weeks of age, insoluble crystallins were accumulated in the cataractous BALB.NCT-Cpoxnct lens. Overall, our data suggest the following sequence of events in the BALB.NCT-Cpoxnct lens: accumulated coproporphyrin induces the aggregation of proteins including crystallins. Aggregated proteins increase ER stress that, in turn, leads to the repression of global translation of proteins including crystallins. The decline in the molecular chaperone crystallin aggravates aggregation and insolubilization of proteins. This vicious cycle would eventually lead to cataracts in BALB.NCT-Cpoxnct.

It is difficult to diagnose immunoglobulin heavy chain amyloidosis (AH amyloidosis) without proteomic analysis due to no useful diagnostic antibodies. The aim of this study was to develop diagnostic antibodies available to immunohistochemistry and immunoblotting. Two rabbit anti-heavy chain variable region antibodies were generated and evaluated in immunohistochemical studies performed on 11 AH amyloidosis patients and 64 patients with other systemic amyloidoses. Additionally, immunoblotting was performed using extracted amyloid protein from one patient and serum samples from two patients with AH amyloidosis. Immunohistochemical analysis generated a positive outcome in 10 of 11 AH amyloidosis patients (sensitivity 90.9%). While positive staining was also observed in 9 of 64 non-AH amyloidosis patients (specificity 85.9%), substitution of the blocking agent reversed the positive reactivity in 5 of 9 patients. Amyloid protein band was clearly detected via immunoblotting analysis, and protein bands with similar molecular weights of amyloid protein were observed in serum samples from patients with AH amyloidosis. The two antibodies may represent a powerful diagnostic tool for AH amyloidosis. In addition, our data revealed the existence of amyloidogenic variable region fragments in the serum of patients, suggesting their potential as diagnostic markers for AH amyloidosis.

Curcumin is a polyphenol compound that exhibits multiple physiological activities. To elucidate the mechanisms by which curcumin affects systemic amyloidosis, we investigated amyloid deposition and molecular changes in a mouse model of amyloid apolipoprotein A-II (AApoAII) amyloidosis, in which mice were fed a curcumin-supplemented diet. Curcumin supplementation for 12 weeks significantly increased AApoAII amyloid deposition relative to controls, especially in the liver and spleen. Liver weights and plasma ApoA-II and high-density lipoprotein concentrations were significantly elevated in curcumin-supplemented groups. RNA-sequence analysis revealed that curcumin intake affected hepatic lipid metabolism via the peroxisome proliferator-activated receptor (PPAR) pathway, especially PPARα activation, resulting in increased Apoa2 mRNA expression. The increase in liver weights was due to activation of PPARα and peroxisome proliferation. Taken together, these results demonstrate that curcumin is a PPARα activator and may affect expression levels of proteins involved in amyloid deposition to influence amyloidosis and metabolism in a complex manner.

AIMS:Glavonoid-rich oil (GRO) derived from ethanol extraction of licorice (Glycyrrhiza glabra Linne) root has been reported to have beneficial effects on health. In this study, we aimed to determine the effect of long-term administration of GRO on metabolic disorders and to elucidate the molecular mechanism.MAIN METHODS:Female obese, type 2 diabetic KK-Ay mice were fed diets supplemented with 0.3% or 0.8% GRO (w/w) for 4-12 weeks. Mice were euthanized and autopsied at 20 weeks old. The effects of GRO on lipid and glucose metabolism were evaluated by measuring physiological and biochemical markers using mRNA sequencing, quantitative reverse-transcription PCR, and western blot analyses.KEY FINDINGS:Compared to mice fed the control diet, GRO-supplemented mice had reduced body and white adipose tissue weights, serum levels of triglycerides and cholesterol, and improved glucose tolerance, while food intake was not affected. We found remarkable reductions in the gene expression levels of stearoyl-coenzyme A desaturase 1 (Scd1) and pyruvate dehydrogenase kinase isoenzyme 4 (Pdk4) in the liver, in addition to decreased expression of fatty acid synthase (Fasn) in inguinal white adipose tissue (iWAT). These results suggest that GRO supplementation improves lipid profiles via reduced de novo lipogenesis in the liver and white adipose tissue. Glucose metabolism may also be improved by increased glycolysis in the liver.SIGNIFICANCE:Our analysis of long-term supplementation of GRO in obese and diabetic mice should provide novel insight into preventing insulin resistance and metabolic syndromes.

Amyloidosis refers to a group of degenerative diseases that are characterized by the deposition of misfolded protein fibrils in various organs. Deposited amyloid may be removed by a phagocyte-dependent innate immune system; however, the precise mechanisms during disease progression remain unclear. We herein investigated the properties of macrophages that contribute to amyloid degradation and disease progression using transmissible apolipoprotein A-II amyloidosis model mice. Intravenously injected AApoAII amyloid was efficiently engulfed by reticuloendothelial macrophages in the liver and spleen and disappeared by 24 h. While cultured murine macrophages degraded AApoAII via the endosomal-lysosomal pathway, AApoAII fibrils reduced cell viability and phagocytic capacity. Furthermore, the depletion of reticuloendothelial macrophages prior to the induction of AApoAII markedly increased hepatic and splenic AApoAII deposition. These results highlight the physiological role of reticuloendothelial macrophages against inter-individual amyloid propagation and suggest the maintenance of phagocytic integrity as a therapeutic strategy to inhibit disease progression.

Till date, there had been no reported case of dialysis-related amyloidosis (DRA) associated with a β2-microglobulin variant. We report here a 41-year-old haemodialysis patient with systemic amyloidosis, exhibiting macroglossia and swelling salivary glands, uncommon clinical manifestations for DRA. Molecular analysis showed that the patient had a new variant of β2-microglobulin (V27M). Extracted amyloid protein was predominantly composed of variant β2-microglobulin. In vitro analysis revealed that this variant β2-microglobulin had a strong amyloidogenic propensity, probably owing to the decreased stability caused by a bulky methionine residue. Our data clearly show that V27M variant is amyloidogenic and this mutation results in unusual clinical manifestations. To date, only one amyloidogenic β2-microglobulin variant (D76N) has been reported in non-dialysis patients. It is noteworthy that the V27M and D76N variants show substantial differences in both clinical phenotypes and pathomechanical features. This is the first case of DRA associated with a naturally occurring β2-microglobulin variant.

Amyloidosis is a group of diseases characterized by protein misfolding and aggregation to form amyloid fibrils and subsequent deposition within various tissues. Previous studies have indicated that amyloidosis is often associated with oxidative stress. However, it is not clear whether oxidative stress is involved in the progression of amyloidosis. We administered the oxidative stress inhibitors tempol and apocynin via drinking water to the R1.P1-Apoa2c mouse strain induced to develop mouse apolipoprotein A-II (AApoAII) amyloidosis and found that treatment with oxidative stress inhibitors led to reduction in AApoAII amyloidosis progression compared to an untreated group after 12 weeks, especially in the skin, stomach, and liver. There was no effect on ApoA-II plasma levels or expression of Apoa2 mRNA. Detection of the lipid peroxidation markers 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) revealed that the antioxidative effects of the treatments were most obvious in the skin, stomach, and liver, which contained higher levels of basal oxidative stress. Moreover, the unfolded protein response was reduced in the liver and was associated with a decrease in oxidative stress and amyloid deposition. These results suggest that antioxidants can suppress the progression of AApoAII amyloid deposition in the improved microenvironment of tissues and that the effect may be related to the levels of oxidative stress in local tissues. This finding provides insights for antioxidative stress treatment strategies for amyloidosis.

Rapid expansion of aged population is predicted worldwide. To cope with problems expected from this situation and extend the period of active and healthy life of people as much as possible, it is important to elucidate not only the biological mechanisms of "aging", but also the etiology of various "age-related diseases". To attain this goal, extensive studies using excellent animal models are indispensable. Senescence-accelerated mouse (SAM) is a series of inbred mouse strains that includes SAMP1, SAMP6, SAMP8, SAMP10, and SAMR1. SAMP strains exhibit accelerated senescence and short lifespan. In addition, each strain shows specific age-related disease phenotypes which are similar to symptoms observed in humans, such as senile amyloidosis (SAMP1), senile osteoporosis (SAMP6), and age-dependent deficits in learning and memory (SAMP8), making SAM mice useful for an aging research. In this review, we introduce the characteristics and application of SAM in geriatrics and aging biology.

Oxidative damage in endothelial cells is proposed to play an important role in endothelial dysfunction and atherogenesis. We previously reported that the reduced form of coenzyme Q10 (CoQ10H2) effectively inhibits oxidative stress and decelerates senescence in senescence-accelerated mice. Here, we treated human umbilical vein endothelial cells (HUVECs) with H2O2 and investigated the protective effect of CoQ10H2 against senescence, oxidative damage, and reduction in cellular functions. We found that CoQ10H2 markedly reduced the number of senescence-associated β-galactosidase-positive cells and suppressed the expression of senescence-associated secretory phenotype-associated genes in H2O2-treated HUVECs. Furthermore, CoQ10H2 suppressed the generation of intracellular reactive oxygen species (ROS) but promoted NO production that was accompanied by increased eNOS expression. CoQ10H2 prevented apoptosis and reductions in mitochondrial function and reduced migration and tube formation activity of H2O2-treated cells. The present study indicated that CoQ10H2 protects endothelial cells against senescence by promoting mitochondrial function and thus could delay vascular aging.