The SYNGAP1 3'UTR Variant in ALS Patients Causes Aberrant SYNGAP1 Splicing and Dendritic Spine Loss by Recruiting HNRNPK.

Fused in sarcoma (FUS) is a pathogenic RNA-binding protein in amyotrophic lateral sclerosis (ALS). We previously reported that FUS stabilizes Synaptic Ras-GTPase activating protein 1 (Syngap1) mRNA at its 3' untranslated region (UTR) and maintains spine maturation. To elucidate the pathologic roles of this mechanism in ALS patients, we identified the SYNGAP1 3'UTR variant rs149438267 in seven (four males and three females) out of 807 ALS patients at the FUS binding site from a multicenter cohort in Japan. Human-induced pluripotent stem cell (hiPSC)-derived motor neurons with the SYNGAP1 variant showed aberrant splicing, increased isoform α1 levels, and decreased isoform γ levels, which caused dendritic spine loss. Moreover, the SYNGAP1 variant excessively recruited FUS and heterogeneous nuclear ribonucleoprotein K (HNRNPK), and antisense oligonucleotides (ASOs) blocking HNRNPK altered aberrant splicing and ameliorated dendritic spine loss. These data suggest that excessive recruitment of RNA-binding proteins, especially HNRNPK, as well as changes in SYNGAP1 isoforms, are crucial for spine formation in motor neurons.SIGNIFICANCE STATEMENT It is not yet known which RNAs cause the pathogenesis of amyotrophic lateral sclerosis (ALS). We previously reported that Fused in sarcoma (FUS), a pathogenic RNA-binding protein in ALS, stabilizes synaptic Ras-GTPase activating protein 1 (Syngap1) mRNA at its 3' untranslated region (UTR) and maintains dendritic spine maturation. To elucidate whether this mechanism is crucial for ALS, we identified the SYNGAP1 3'UTR variant rs149438267 at the FUS binding site. Human-induced pluripotent stem cell (hiPSC)-derived motor neurons with the SYNGAP1 variant showed aberrant splicing, which caused dendritic spine loss along with excessive recruitment of FUS and heterogeneous nuclear ribonucleoprotein K (HNRNPK). Our findings that dendritic spine loss is because of excess recruitment of RNA-binding proteins provide a basis for the future exploration of ALS-related RNA-binding proteins.

Genetic factors affecting survival in Japanese patients with sporadic amyotrophic lateral sclerosis: a genome-wide association study and verification in iPSC-derived motor neurons from patients.

BACKGROUND: Several genetic factors are associated with the pathogenesis of sporadic amyotrophic lateral sclerosis (ALS) and its phenotypes, such as disease progression. Here, in this study, we aimed to identify the genes that affect the survival of patients with sporadic ALS. METHODS: We enrolled 1076 Japanese patients with sporadic ALS with imputed genotype data of 7 908 526 variants. We used Cox proportional hazards regression analysis with an additive model adjusted for sex, age at onset and the first two principal components calculated from genotyped data to conduct a genome-wide association study. We further analysed messenger RNA (mRNA) and phenotype expression in motor neurons derived from induced pluripotent stem cells (iPSC-MNs) of patients with ALS. RESULTS: Three novel loci were significantly associated with the survival of patients with sporadic ALS-FGF1 at 5q31.3 (rs11738209, HR=2.36 (95% CI, 1.77 to 3.15), p=4.85×10-9), THSD7A at 7p21.3 (rs2354952, 1.38 (95% CI, 1.24 to 1.55), p=1.61×10-8) and LRP1 at 12q13.3 (rs60565245, 2.18 (95% CI, 1.66 to 2.86), p=2.35×10-8). FGF1 and THSD7A variants were associated with decreased mRNA expression of each gene in iPSC-MNs and reduced in vitro survival of iPSC-MNs obtained from patients with ALS. The iPSC-MN in vitro survival was reduced when the expression of FGF1 and THSD7A was partially disrupted. The rs60565245 was not associated with LRP1 mRNA expression. CONCLUSIONS: We identified three loci associated with the survival of patients with sporadic ALS, decreased mRNA expression of FGF1 and THSD7A and the viability of iPSC-MNs from patients. The iPSC-MN model reflects the association between patient prognosis and genotype and can contribute to target screening and validation for therapeutic intervention.

Plasmalemmal Vesicle-Associated Protein Is Associated with Endothelial Cells Sprouting from the Peribiliary Capillary Plexus in Human Cirrhotic Liver.

INTRODUCTION: Plasmalemmal vesicle-associated protein (PLVAP) is an endothelial-specific integral membrane glycoprotein that localizes to caveolae and fenestrae in animal models; however, little is known about PLVAP in endothelial cells (ECs) in hepatic sinusoids during liver cirrhosis (LC). Here, we aimed to elucidate PLVAP localization and expression in the human liver during LC progression. METHODS: PLVAP protein expression was detected in specimens from normal control livers and hepatitis C-related cirrhotic livers using immunohistochemistry, Western blotting, and immunoelectron microscopy. RESULTS: PLVAP mainly localized to the peribiliary capillary plexus (PCP) and was rarely observed in hepatic artery branches and portal venules in control tissue, but was aberrantly expressed in capillarized sinusoids and proliferated capillaries in fibrotic septa within cirrhotic liver tissue. Ultrastructural analysis indicated that PLVAP localized to thin ECs in some caveolae, whereas PLVAP localized primarily to caveolae-like structures and proliferative sinusoid capillary EC vesicles in cirrhotic liver tissue. Western blot analysis confirmed that PLVAP was overexpressed at the protein level in advanced cirrhotic liver tissue. CONCLUSION: PLVAP was strongly expressed in the caveolae of proliferated capillaries directly connected with sinusoids linked with the PCP, suggesting that it plays a role in angiogenesis and sinusoidal remodeling in LC.

An autopsy report of a familial amyotrophic lateral sclerosis case carrying VCP Arg487His mutation with a unique TDP-43 proteinopathy.

We here report an autopsy case of familial amyotrophic lateral sclerosis (ALS) with p.Arg487His mutation in the valosin-containing protein (VCP) gene (VCP), in which upper motor neurons (UMNs) were predominantly involved. Moreover, our patient developed symptoms of frontotemporal dementia later in life and pathologically exhibited numerous phosphorylated transactivation response DNA-binding protein of 43 kDa (p-TDP-43)-positive neuronal cytoplasmic inclusions and short dystrophic neurites with a few lentiform neuronal intranuclear inclusions, sharing the features of frontotemporal lobar degeneration with TDP-43 pathology type A pattern. A review of previous reports of ALS with VCP mutations suggests that our case is unique in terms of its UMN-predominant lesion pattern and distribution of p-TDP-43 pathology. Thus, this case report effectively expands the clinical and pathological phenotype of ALS in patients with a VCP mutation.

Prognosis of amyotrophic lateral sclerosis patients undergoing tracheostomy invasive ventilation therapy in Japan.

OBJECTIVE: The aim of this study is to describe and clarify the factors affecting the prognosis of Japanese patients with amyotrophic lateral sclerosis (ALS) undergoing tracheostomy invasive ventilation (TIV) therapy. METHODS: We conducted a prospective longitudinal observational case-control study using a multicentre registry. ALS patients who started TIV therapy after registration (TIV group) and those who did not receive TIV (non-TIV group) were included. We compared the survival time between the TIV group and the non-TIV group using a propensity score matching analysis and evaluated the prognostic factors in the TIV group. RESULTS: From February 2006 to January 2018, 190 patients in the TIV group and 1093 patients in the non-TIV group were included in this study. The mean age of disease onset and usage rate of gastrostomy and non-invasive ventilation therapy differed between the groups. In the propensity score matching analysis using known prognostic factors, the median overall survival time of the TIV group was significantly greater than that of the non-TIV group (11.33 years vs 4.61 years; p<0.001). Analysis using the Cox proportional hazard model suggested that older age of onset and respiratory onset was an independent factor for poor prognosis after starting TIV therapy. CONCLUSION: We showed that there was a significant difference of approximately 7 years in life expectancy between Japanese ALS patients who did and did not receive TIV therapy.

Freeze-drying enables homogeneous and stable sample preparation for determination of fecal short-chain fatty acids.

BACKGROUND: The analysis methods for fecal short-chain fatty acids (SCFAs) have evolved considerably. Recently, the role of SCFAs in gastrointestinal physiology and their association with intestinal microbiota and disease were reported. However, the intra-fecal variability and storage stability of SCFAs have not been extensively investigated. The aim of this study was to understand the limitations of the measurement of SCFAs in crude feces and develop a useful pre-examination procedure using the freeze-drying technique. METHODS: SCFAs in crude feces, obtained from healthy volunteers, and freeze-dried feces were determined by derivatization with isobutyl chloroformate, followed by liquid-liquid extraction with hexane, and separation and analysis using gas chromatography-mass spectrometry. RESULTS: Among the SCFAS, the maximum intra-fecal variability was observed for iso-butyrate (coefficient of variation of 37.7%), but the freeze-drying procedure reduced this variability (coefficient of variation of 7.9%). Similar improvements were also observed for other SCFAs. Furthermore, significant decreases in the SCFA amounts were observed with storage at 4 °C for 24 h. CONCLUSIONS: The freeze-drying procedure affords fecal SCFA stability, even with storage at room temperature for 3 d. The freeze-drying procedure allows reliable SCFA measurements without labour-intensive processes. Therefore, the freeze-drying procedure can be applied in basic, clinical, and epidemiological studies.

Correction to: Cavin-1 is linked to lipid droplet formation in human hepatic stellate cells.

In the original publication, the part figures (e, f) of Fig. 2 were wrongly cited as "g, h" in the text and in Fig. 2 caption.

[Intracranial Growing Teratoma Syndrome:Case Reports and Literature Review].

BACKGROUND: Growing teratoma syndrome(GTS)is the progression of a mature teratoma during or following radiochemotherapy for germ cell tumors. We report two surgical cases of GTS. CASE 1: A 24-day-old new-born presented with vomiting and head enlargement. Blood alfa-feto protein(AFP)and beta-human chorionic gonadotropin(ß-hCG)were within or at the upper limits of the normal ranges. Magnetic Resonance Imaging(MRI)demonstrated a large mass in the posterior fossa causing the severe hydrocephalus. Tumor removal was immediately performed. Histological diagnosis given was immature teratoma. While chemotherapy effectively reduced the level of tumor makers, multiple recurrence was noticed on MRI 70 days after the surgery. GTS was suspected and total removal was performed. Histological examination revealed a mature teratoma. The patient is growing normally thereafter, 2.5 years after the onset. CASE 2: A 16-year-old male presented with binasal hemianopsia. Blood AFP and ß-hCG were within or at the upper limits of the normal ranges. MRI demonstrated an intrasellar mass protruding upward. Tumor removal was performed and histological diagnosis given was mixed germ cell tumor. While radiochemotherapy effectively normalized the tumor makers, recurrence was noticed on MRI 190 days after the surgery. Total removal was performed with the diagnosis of GTS. Histological examination revealed a mature teratoma. The patient lives a normal school life thereafter as followed up after a year after the onset. CONCLUSION: It is important to diagnose and perform the surgery early enough to enable total removal of the mass presenting as GTS because total surgical removal is the only treatment for GTS.

Amyotrophic lateral sclerosis of long clinical course clinically presenting with progressive muscular atrophy.

Amyotrophic lateral sclerosis (ALS) primarily affects upper and lower motor neurons. Phosphorylated trans-activation response DNA-binding protein of 43 kDa (TDP-43) inclusion bodies are reportedly a pathological hallmark of sporadic ALS. Here, we present an atypical case of sporadic ALS that progressed very slowly, persisted for 19 years, and clinically appeared to only affect the lower motor neurons; however, upper motor neuron degeneration was detected at autopsy. Furthermore, no inclusion bodies positive for phosphorylated TDP-43, ubiquitin, fused in sarcoma, or superoxide dismutase-1 were detected in the central nervous system. We performed exome-sequencing data analysis but found no genetic disorders. This was therefore an unusual case of lower motor neuron-predominant ALS without TDP-43 pathology or known gene-disease associations. We also reviewed autopsied ALS cases that progressed slowly and had no phosphorylated TDP-43 or ubiquitin-positive inclusions and present the clinicopathological features of such cases. Based on these results, there may be a sporadic ALS subgroup that progresses slowly and shows no accumulation of phosphorylated TDP-43.

A sensitive and efficient procedure for the high-throughput determination of nine urinary metabolites of pyrethroids by GC-MS/MS and its application in a sample of Japanese children.

Four pyrethroids (PYRs), metofluthrin, profluthrin, tefluthrin, and transfluthrin, which were newly developed and have relatively high vapor activity at ambient temperature, are now playing a key role in safely controlling insects in our daily lives. We developed a sensitive and high-throughput determination method for urinary metabolites derived from the newly developed PYR, e.g., 2,3,5,6-tetrafluoro-1,4-benzenedimethanol (HOCH2-FB-Al), 2,3,5,6-tetrafluorobenzyl alcohol (FB-Al), and other PYR metabolites such as trans-chrysanthemumdicarboxylic acid (trans-CDCA) and 3-phenoxybenzoic acid (3PBA). After high temperature acid hydrolysis of 2 mL urine sample in 24-deep well plate, the PYR metabolites were extracted by semi-automated liquid-liquid extraction with tert-butyl methyl ether. N,O-Bis (trimethylsilyl) trifluoroacetamide containing 1% trimethylchlorosilane or 1,1,1,3,3,3-hexafluoroisopropanol were used for the derivatization of PYR metabolites, and the derivatized metabolites were analyzed separately by GC-MS/MS equipped with dual injector system (DB-5MS and mid- to high-polarity phase Rtx-65 columns). The derivatization and evaporation conditions were mainly optimized for improving sensitivity and reproducibility. The mean within-run day precisions were less than 18.4% (relative standard deviation, %RSD) with low detection limits ranging from 0.01 µg/L for HOCH2-FB-Al to 0.06 µg/L for trans-CDCA. This method was successfully applied to urine samples obtained from 50 3-year-old children with high detection frequencies (e.g., 82% for HOCH2-FB-Al and 84% for FB-Al). This method may be a pivotal tool for developing risk assessment from PYR exposure in the general population.

Opening the ventricle during surgery diminishes survival among patients with newly diagnosed glioblastoma treated with carmustine wafers: a multi-center retrospective study.

Carmustine wafers (CW) were approved in Japan for newly diagnosed and recurrent malignant gliomas during 2013. The ventricle is often opened during surgery to achieve maximum resection. While not generally recommended in such situations, CW might be safely achieved by occluding an opened ventricle using gelform or collagen sheets. However, whether CW implantation actually confers a survival benefit for patients who undergo surgery with an open ventricle to treat glioblastoma remains unclear. Clinical, imaging, and survival data were collected in this multicenter retrospective study of 122 consecutive patients with newly diagnosed glioblastoma to determine adverse events and efficacy. Overall, 54 adverse events of all grades developed in 35 (28.6%) patients, with the most common being new seizures (16%). Adverse events did not significantly differ between patients with opened and closed ventricles during surgery. The 10- and 21.7-month, median, progression-free (PFS) and overall survival (OS), respectively did not significantly differ according to resection rates. However, median PFS and OS were significantly longer among patients with closed, than open ventricles (12.8 vs. 7.4 months; p = 0.0039 and 26.9 vs. 18.6 months; p = 0.011, respectively). Implanting CW into the resection cavity during concomitant radiochemotherapy with temozolomide seems to yield better survival rates without increased adverse events. Occlusion of the ventricular opening during surgery might be safe for CW implantation, but less so for treating patients with newly diagnosed glioblastoma.

Maximum Tongue Pressure is Associated with Swallowing Dysfunction in ALS Patients.

Maximum tongue pressure (MTP) measurement is a convenient, less invasive assessment that has been developed to quantify tongue strength; however, it is unclear whether MTP is useful for the detection of swallowing disorders in amyotrophic lateral sclerosis (ALS) patients. The purpose of this study was to clarify the relationship between MTP and the characteristics of swallowing disorders on videofluorography and to determine the usefulness of tongue pressure measurement for the assessment of swallowing function in ALS patients. Twenty-five ALS patients were evaluated according to the ALS functional rating scale-revised (ALSFRS-R), and their ability to swallow yogurt was observed via videofluorography. MTP was measured using a device (TPM-01, JMS, Hiroshima) equipped with a balloon probe. Then, the relationships between the ALSFRS-R score, swallowing function, and MTP were analyzed. MTP was significantly lower in the subjects with reduced tongue function (p = 0.002) or with pharyngeal residue (p = 0.006) than in the subjects with normal characteristics. Bolus formation and oral transit time and pharyngeal transit time were significantly prolonged among those with reduced MTP. MTP at a cut-off value of 21.0 kPa was associated with a full score on the bulbar-related items of the ALSFRS-R. MTP may serve as a new diagnostic tool for the early detection of swallowing dysfunction in ALS patients, because of its good relationship with their swallowing characteristics.

A rapid functional decline type of amyotrophic lateral sclerosis is linked to low expression of TTN.

OBJECTIVE: To classify the patterns of functional decline in patients with sporadic amyotrophic lateral sclerosis (ALS) and explore the genetic backgrounds that modified these patterns. METHODS: We included 465 patients with sporadic ALS in the analysis and clustered the longitudinal functional scores in the registered patients, using a mixture approach of a non-linear mixed-effects model. We conducted a genome-wide analysis of 572 983 single nucleotide polymorphisms (SNPs). We then assessed the association between the clusters of longitudinal functional scores and SNPs. RESULTS: We identified the following four clusters of longitudinal functional decline in the cases: a rapid decline cluster, an intermediate decline cluster, a sigmoidal decline cluster and a moderate decline cluster. We identified seven SNPs associated with the rapid decline cluster, using a recessive model (p=3.47-8.34×10(-8)). The OR for the probabilities of the rapid decline cluster ranged from 5.5 to 5.84. Homozygosity for the minor alleles in the seven SNPs, which constituted a linkage disequilibrium (LD) block, was associated with decreased expression of TTN (encoding Titin, a large sarcomere protein) in the expression quantitative trait loci database of a large-scale Japanese genetic variation database (p=8.6×10(-10)-1.1×10(-7)). TTN expression in immortalised lymphocyte lines was decreased in patients who were homozygous for the minor alleles compared with those who were homozygous for the major alleles (n=19 in each group, p=0.002). CONCLUSIONS: We detected an LD block associated with a rapid functional decline in patients with sporadic ALS, which is linked to decreased expression of TTN.

Co-registered positron emission tomography/computed tomography and gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid magnetic resonance imaging features of multiple angiosarcoma of the liver.

Hepatic angiosarcoma is a very rare disease, accounting for only 2% of primary liver malignancy. An 82-year-old man was admitted to our hospital because of jaundice and weight loss. Computed tomography (CT) and magnetic resonance imaging (MRI) showed diffuse and multiple space-occupying lesions. On gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA)-enhanced MRI, the tumor was not enhanced intensely in the arterial phase following contrast injection, and was then gradually enhanced homogeneously. In the delayed phase and hepatobiliary phase, the tumor was completely washed out. Whole-body (18) F-fluorodeoxyglucose positron emission tomography (FDG-PET)/CT fusion scanning confirmed metabolic activity with maximum uptake value of 3.64 in the lesions. A liver biopsy showed spindle-shaped tumor cells proliferating along sinusoids, with elongated and hyperchromatic nuclei. Immunohistochemical studies showed tumor cells positive for von Willebrand factor and CD34. These findings were consistent with angiosarcoma of the liver. This case report is the first description of co-registered FDG-PET/CT images and Gd-EOB-DTPA-enhanced MRI of primary hepatic angiosarcoma.

Aquaporin-1 is associated with arterial capillary proliferation and hepatic sinusoidal transformation contributing to portal hypertension in primary biliary cirrhosis.

Although aquaporins (AQPs) in normal hepatobiliary system have been studied, little is known about AQP localization and changes in the hepatic microvascular system including sinusoids in cholestatic liver. The present study aimed to clarify the localization of AQP-1 in the microvessels in normal human liver and in primary biliary cirrhosis (PBC). Human normal liver (control) and PBC liver specimens were obtained. Immunohistochemistry, Western blotting, in situ hybridization (ISH) and electron microscopic examination for AQP-1 were conducted. In control liver and stages I-II PBC liver, AQP-1 immunoreactivity was mainly localized in portal venules, hepatic arterioles and bile ducts in the portal tract, but was hardly detected in the sinusoids. However, AQP-1 expression was enhanced in the proliferated bile ductules in PBC. In stages III-IV PBC liver tissues, AQP-1 was aberrantly expressed in proliferated arterial capillaries opening into the sinusoids at the peripheral edge of regenerating hepatic nodules and in the fibrotic septa. Overexpression of AQP-1 at protein and mRNA levels was demonstrated by Western blot and ISH, respectively. Angiogenetic and fibrotic responses are probably induced by AQP-1, leading to enhanced pouring of arterial blood into the sinusoids; thus, contributing to progression of portal hypertension in PBC.

CHCHD2 P14L, found in amyotrophic lateral sclerosis, exhibits cytoplasmic mislocalization and alters Ca2+ homeostasis.

CHCHD2 and CHCHD10, linked to Parkinson's disease and amyotrophic lateral sclerosis-frontotemporal dementia (ALS), respectively, are mitochondrial intermembrane proteins that form a heterodimer. This study aimed to investigate the impact of the CHCHD2 P14L variant, implicated in ALS, on mitochondrial function and its subsequent effects on cellular homeostasis. The missense variant of CHCHD2, P14L, found in a cohort of patients with ALS, mislocalized CHCHD2 to the cytoplasm, leaving CHCHD10 in the mitochondria. Drosophila lacking the CHCHD2 ortholog exhibited mitochondrial degeneration. In contrast, human CHCHD2 P14L, but not wild-type human CHCHD2, failed to suppress this degeneration, suggesting that P14L is a pathogenic variant. The mitochondrial Ca2+ buffering capacity was reduced in Drosophila neurons expressing human CHCHD2 P14L. The altered Ca2+-buffering phenotype was also observed in cultured human neuroblastoma SH-SY5Y cells expressing CHCHD2 P14L. In these cells, transient elevation of cytoplasmic Ca2+ facilitated the activation of calpain and caspase-3, accompanied by the processing and insolubilization of TDP-43. These observations suggest that CHCHD2 P14L causes abnormal Ca2+ dynamics and TDP-43 aggregation, reflecting the pathophysiology of ALS.

Neck weakness is a potent prognostic factor in sporadic amyotrophic lateral sclerosis patients.

OBJECTIVE: To clarify the emergence of muscle weakness in regions of the body that affect survival, and deterioration in activities of daily living (ADL) in amyotrophic lateral sclerosis (ALS) patients. METHODS: We conducted a multicentre-based prospective cohort study of patients with ALS. We enrolled 401 sporadic patients with ALS. Death or the introduction of invasive ventilation was defined as the primary endpoint, and the time to five clinical markers of ADL deterioration associated with bulbar paralysis or limb weakness were defined as ADL milestones. Muscle weakness was assessed in the neck flexor muscles; the bilateral abductors of the shoulders; the bilateral wrist extensor muscles; the bilateral flexor muscles of the hips; and the bilateral ankle dorsiflexion muscles. We performed Cox proportional hazards regression analyses for the primary endpoint and the five ADL milestones, adjusting for known covariate prognostic factors for ALS. RESULTS: The Medical Research Council (MRC) score for the neck flexors was the most significant prognostic factor for the primary endpoint (HR 0.74, p<0.001), loss of speech (HR 0.66, p<0.001), and loss of swallowing function (HR 0.73, p<0.001), and was one of the significant prognostic factors for loss of upper limb function, difficulty turning in bed, and loss of walking ability (p=0.001, 0.002, and 0.008, respectively). The MRC score for the neck flexors was also a significant prognostic factor for covariates of the previously reported prognostic factors. CONCLUSIONS: Neck weakness is an independent prognostic factor for survival and deterioration in ADL in Patients with ALS.

Influence of food restriction combined with voluntary running on bone morphology and strength in male rats.

Athletes, in particular endurance athletes and dancers, are chronically exposed to a state of low energy availability due to insufficient dietary energy intake and massive exercise energy expenditure. Low energy availability sometimes causes bone fragility, thereby increasing the risk of bone disorders. Although the decrease in energy availability shows no sexual dimorphism, epidemiological studies have reported that bone disorders are less frequent in male athletes than in female athletes. We hypothesized that bone tissue was not affected by low energy availability in males. The purpose of this study was to examine the influence of food restriction combined with voluntary running training on bone morphology and strength in adult male rats. Fourteen-week-old male Sprague-Dawley rats were divided randomly into four groups: control (C) group, food restriction (R) group, exercise (Ex) group, and food restriction plus exercise (REx) group. For the R and REx groups, 30 % food restriction was carried out in comparison with the C group. Bone strength, bone mineral density (BMD), bone architecture, and bone turnover rate were measured after a 13-week experimental period. Bone strength was not significantly lower in the REx group compared with the C group. BMD and trabecular bone volume showed no difference among groups. These findings indicate that bone morphology and strength were little affected by food restriction combined with exercise training in adult male rats.