Investigation of the molecular causes underlying physical abnormalities in Diamond‐Blackfan anemia patients with RPL5 haploinsufficiency

Diamond-Blackfan anemia (DBA) is a genetic disorder caused by mutations in genes encoding ribosomal proteins and characterized by erythroid aplasia and various physical abnormalities. Although accumulating evidence suggests that defective ribosome biogenesis leads to p53-mediated apoptosis in erythroid progenitor cells, little is known regarding the underlying causes of the physical abnormalities. In this study, we established induced pluripotent stem cells from a DBA patient with RPL5 haploinsufficiency. These cells retained the ability to differentiate into osteoblasts and chondrocytes. However, RPL5 haploinsufficiency impaired the production of mucins and increased apoptosis in differentiated chondrocytes. Increased expression of the pro-apoptotic genes BAX and CASP9 further indicated that RPL5 haploinsufficiency triggered p53-mediated apoptosis in chondrocytes. MDM2, the primary negative regulator of p53, plays a crucial role in erythroid aplasia in DBA patient. We found the phosphorylation level of MDM2 was significantly decreased in RPL5 haploinsufficient chondrocytes. In stark contrast, we found no evidence that RPL5 haploinsufficiency impaired osteogenesis. Collectively, our data support a model in which RPL5 haploinsufficiency specifically induces p53-mediated apoptosis in chondrocytes through MDM2 inhibition, which leads to physical abnormalities in DBA patients

Reduction of lipid accumulation rescues Bietti’s crystalline dystrophy phenotypes

眼の難病クリスタリン網膜症の発症メカニズムを解明 --治療薬の有力候補発見により創薬研究の進展に期待--. 京都大学プレスリリース. 2018-03-27.Bietti’s crystalline dystrophy (BCD) is an intractable and progressive chorioretinal degenerative disease caused by mutations in the CYP4V2 gene, resulting in blindness in most patients. Although we and others have shown that retinal pigment epithelium (RPE) cells are primarily impaired in patients with BCD, the underlying mechanisms of RPE cell damage are still unclear because we lack access to appropriate disease models and to lesion-affected cells from patients with BCD. Here, we generated human RPE cells from induced pluripotent stem cells (iPSCs) derived from patients with BCD carrying a CYP4V2 mutation and successfully established an in vitro model of BCD, i.e., BCD patient-specific iPSC-RPE cells. In this model, RPE cells showed degenerative changes of vacuolated cytoplasm similar to those in postmortem specimens from patients with BCD. BCD iPSC-RPE cells exhibited lysosomal dysfunction and impairment of autophagy flux, followed by cell death. Lipidomic analyses revealed the accumulation of glucosylceramide and free cholesterol in BCD-affected cells. Notably, we found that reducing free cholesterol by cyclodextrins or δ-tocopherol in RPE cells rescued BCD phenotypes, whereas glucosylceramide reduction did not affect the BCD phenotype. Our data provide evidence that reducing intracellular free cholesterol may have therapeutic efficacy in patients with BCD

Human AK2 links intracellular bioenergetic redistribution to the fate of hematopoietic progenitors

AK2 is an adenylate phosphotransferase that localizes at the intermembrane spaces of the mitochondria, and its mutations cause a severe combined immunodeficiency with neutrophil maturation arrest named reticular dysgenesis (RD). Although the dysfunction of hematopoietic stem cells (HSCs) has been implicated, earlier developmental events that affect the fate of HSCs and/or hematopoietic progenitors have not been reported. Here, we used RD-patient-derived induced pluripotent stem cells (iPSCs) as a model of AK2-deficient human cells. Hematopoietic differentiation from RD-iPSCs was profoundly impaired. RD-iPSC-derived hemoangiogenic progenitor cells (HAPCs) showed decreased ATP distribution in the nucleus and altered global transcriptional profiles. Thus, AK2 has a stage-specific role in maintaining the ATP supply to the nucleus during hematopoietic differentiation, which affects the transcriptional profiles necessary for controlling the fate of multipotential HAPCs. Our data suggest that maintaining the appropriate energy level of each organelle by the intracellular redistribution of ATP is important for controlling the fate of progenitor cells

Insulin-producing cells derived from ‘induced pluripotent stem cells’ of patients with fulminant type 1 diabetes: vulnerability to cytokine insults and increased expression of apoptosis-related genes

Aims/Introduction: The present study was carried out to generate induced pluripotent stem cells (iPSCs) from patients with fulminant type 1 diabetes, and evaluate the cytokine‐induced apoptotic reactions of β‐like insulin‐producing cells differentiated from the iPSCs.Materials and Methods: iPSCs were generated from fibroblasts of patients with fulminant type 1 diabetes by inducing six reprogramming factors. Insulin‐producing cells were differentiated from the iPSCs in vitro. The proportion of cleaved caspase‐3‐positive or terminal deoxynucleotidyl transferase 2′‐deoxyuridine, 5′‐triphosphate nick end labeling‐positive cells among insulin (INS)‐positive cells derived from fulminant type 1 diabetes iPSC and control human iPSC lines was evaluated under treatment with tumor necrosis factor‐α, interleukin‐1β and interferon‐γ. Ribonucleic acid sequencing was carried out to compare gene expressions in INS‐positive cells derived from fulminant type 1 diabetes iPSC and control human iPSC lines.Results: Two iPSC clones were established from each of three patients with fulminant type 1 diabetes. The differentiation of insulin‐producing cells from fulminant type 1 diabetes iPSC was confirmed by immunofluorescence analysis and KCl‐induced C‐peptide secretion. After treatment with pro‐inflammatory cytokines, these INS‐positive cells showed higher expression of cleaved caspase‐3 than those derived from control human iPSCs. Altered expression levels of several apoptosis‐related genes were observed in INS‐positive cells derived from the fulminant type 1 diabetes iPSCs by ribonucleic acid sequencing.Conclusions: We generated iPSCs from patients with fulminant type 1 diabetes and differentiated them into insulin‐producing cells. This in vitro disease model can be used to elucidate the disease mechanisms of fulminant type 1 diabetes

Daytime nap and nighttime breastfeeding are associated with toddlers\u27 nighttime sleep

The purpose of the present study is to examine the association between toddlers\u27 sleep arrangements and their nighttime sleep duration and other sleep variables. For this investigation, we performed a study in which child activity and sleep levels were recorded using actigraphy. The parents of 1.5-year-old toddlers (n = 106) were asked to attach an actigraphy unit to their child’s waist with an adjustable elastic belt and complete a sleep diary for 7 consecutive days. Questionnaires were used to assess the sleep arrangements of the toddlers. There was a significant negative correlation between nap duration and nighttime sleep duration, suggesting that longer nap sleep induces shorter nighttime sleep duration. Among the sleep arrangements, such as nighttime breastfeeding or co-sleeping, only nighttime breastfeeding predicted shorter nighttime sleep duration. Our findings indicate that shorter naps induce a longer nighttime sleep in 1.5-year-old toddlers while nighttime breastfeeding decreases their nighttime sleep duration

Sleep maturation influences cognitive development of preterm toddlers

Our recent study on full-term toddlers demonstrated that daytime nap properties affect the distribution ratio between nap and nighttime sleep duration in total sleep time but does not affect the overall total amount of daily sleep time. However, there is still no clear scientific consensus as to whether the ratio between naps and nighttime sleep or just daily total sleep duration itself is more important for healthy child development. In the current study, to gain an answer to this question, we examined the relationship between the sleep properties and the cognitive development of toddlers born prematurely using actigraphy and the Kyoto scale of psychological development (KSPD) test. 101 premature toddlers of approximately 1.5 years of age were recruited for the study. Actigraphy units were attached to their waist with an adjustable elastic belt for 7 consecutive days and a child sleep diary was completed by their parents. In the study, we found no significant correlation between either nap or nighttime sleep duration and cognitive development of the preterm toddlers. In contrast, we found that stable daily wake time was significantly associated with better cognitive development, suggesting that sleep regulation may contribute to the brain maturation of preterm toddlers

Anti-Aβ Drug Screening Platform Using Human iPS Cell-Derived Neurons for the Treatment of Alzheimer's Disease

Background:Alzheimer's disease (AD) is a neurodegenerative disorder that causes progressive memory and cognitive decline during middle to late adult life. The AD brain is characterized by deposition of amyloid β peptide (Aβ), which is produced from amyloid precursor protein by β- and γ-secretase (presenilin complex)-mediated sequential cleavage. Induced pluripotent stem (iPS) cells potentially provide an opportunity to generate a human cell-based model of AD that would be crucial for drug discovery as well as for investigating mechanisms of the disease. Methodology/Principal Findings:We differentiated human iPS (hiPS) cells into neuronal cells expressing the forebrain marker, Foxg1, and the neocortical markers, Cux1, Satb2, Ctip2, and Tbr1. The iPS cell-derived neuronal cells also expressed amyloid precursor protein, β-secretase, and γ-secretase components, and were capable of secreting Aβ into the conditioned media. Aβ production was inhibited by β-secretase inhibitor, γ-secretase inhibitor (GSI), and an NSAID; however, there were different susceptibilities to all three drugs between early and late differentiation stages. At the early differentiation stage, GSI treatment caused a fast increase at lower dose (Aβ surge) and drastic decline of Aβ production. Conclusions/Significance:These results indicate that the hiPS cell-derived neuronal cells express functional β- and γ-secretases involved in Aβ production; however, anti-Aβ drug screening using these hiPS cell-derived neuronal cells requires sufficient neuronal differentiation

Preterm toddlers have low nighttime sleep quality and high daytime activity.

A number of studies have been made on the sleep characteristics of children born preterm in an attempt to develop methods to address the sleep problems commonly observed among such children. However, the reported sleep characteristics from these studies vary depending on the observation methods used, i.e., actigraphy, polysomnography and questionnaire. In the current study, to obtain reliable data on the sleep characteristics of preterm-born children, we investigated the difference in sleep properties between 97 preterm and 97 term toddlers of approximately 1.5 years of age using actigraphy. Actigraphy units were attached to the toddlers’ waists with an adjustable elastic belt for 7 consecutive days, and a child sleep diary was completed by their parents. In the study, we found that preterm toddlers had more nocturnal awakenings and more daytime activity, suggesting that preterm-born children may have a different process of sleep development in their early development

Haptic Interface with Twin-Driven MR Fluid Actuator for Teleoperation Endoscopic Surgery System

Magnetorheological fluids (MRFs) are composite materials made of ferromagnetic particles, medium oils, and several types of additives. We have developed an actuation system for the fine haptic control of leader-follower robots. In this study, we developed a haptic interface with two link-type twin-driven MR fluid actuators and two MR fluid brakes for a teleoperation endoscopic surgery system and conducted evaluation tests for a remote operational task with a leader-follower robot system. For evaluations, we adopted the NASA-TLX questionnaire as a subjective assessment method. According to the experimental results, the total success rates were 0.462, 0.333, and 0.591, for the first haptic, middle no-haptic, and second haptic phases, respectively. The force information of the haptic forceps helped users to perceive grasping sensation on their fingers. Statistical analyses on the answers to the questionnaire indicate no significant differences. However, a decreasing tendency in the mental stress in the complicated manipulation tasks for fragile objects is observed