Disease Outcome and Brain Metabolomics of Cyclophilin-D Knockout Mice in Sepsis.

Sepsis-associated encephalopathy (SAE) is a diffuse brain dysfunction resulting from a systemic inflammatory response to infection, but the mechanism remains unclear. The mitochondrial permeability transition pore (MPTP) could play a central role in the neuronal dysfunction, induction of apoptosis, and cell death in SAE. The mitochondrial isomerase cyclophilin D (CypD) is known to control the sensitivity of MPTP induction. We, therefore, established a cecal ligation and puncture (CLP) model, which is the gold standard in sepsis research, using CypD knockout (CypD KO) mice, and analyzed the disease phenotype and the possible molecular mechanism of SAE through metabolomic analyses of brain tissue. A comparison of adult, male wild-type, and CypD KO mice demonstrated statistically significant differences in body temperature, mortality, and histological changes. In the metabolomic analysis, the main finding was the maintenance of reduced glutathione (GSH) levels and the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio in the KO animals following CLP. In conclusion, we demonstrate that CypD is implicated in the pathogenesis of SAE, possibly related to the inhibition of MPTP induction and, as a consequence, the decreased production of ROS and other free radicals, thereby protecting mitochondrial and cellular function.

miRNA-Based Rapid Differentiation of Purified Neurons from hPSCs Advancestowards Quick Screening for Neuronal Disease Phenotypes In Vitro.

Obtaining differentiated cells with high physiological functions by an efficient, but simple and rapid differentiation method is crucial for modeling neuronal diseases in vitro using human pluripotent stem cells (hPSCs). Currently, methods involving the transient expression of one or a couple of transcription factors have been established as techniques for inducing neuronal differentiation in a rapid, single step. It has also been reported that microRNAs can function as reprogramming effectors for directly reprogramming human dermal fibroblasts to neurons. In this study, we tested the effect of adding neuronal microRNAs, miRNA-9/9*, and miR-124 (miR-9/9*-124), for the neuronal induction method of hPSCs using Tet-On-driven expression of the Neurogenin2 gene (Ngn2), a proneural factor. While it has been established that Ngn2 can facilitate differentiation from pluripotent stem cells into neurons with high purity due to its neurogenic effect, a long or indefinite time is required for neuronal maturation with Ngn2 misexpression alone. With the present method, the cells maintained a high neuronal differentiation rate while exhibiting increased gene expression of neuronal maturation markers, spontaneous calcium oscillation, and high electrical activity with network bursts as assessed by a multipoint electrode system. Moreover, when applying this method to iPSCs from Alzheimer's disease (AD) patients with presenilin-1 (PS1) or presenilin-2 (PS2) mutations, cellular phenotypes such as increased amount of extracellular secretion of amyloid ß42, abnormal oxygen consumption, and increased reactive oxygen species in the cells were observed in a shorter culture period than those previously reported. Therefore, it is strongly anticipated that the induction method combining Ngn2 and miR-9/9*-124 will enable more rapid and simple screening for various types of neuronal disease phenotypes and promote drug discovery.

[PSI(+)] aggregate enlargement in rnq1 nonprion domain mutants, leading to a loss of prion in yeast.

[PIN(+)] is the prion form of the Rnq1 protein of unknown function in Saccharomyces cerevisiae. A glutamine/asparagine (Q/N)-rich C-terminal domain is necessary for the propagation of [PIN(+)], whereas the N-terminal region is non-Q/N-rich and considered the nonprion domain. Here, we isolated numerous single-amino-acid mutations in Rnq1, phenotypically similar to Rnq1Δ100, which inhibit [PSI(+)] propagation in the [PIN(+)] state, but not in the [pin(-)] state, when overproduced. The dynamics of the prion aggregates was analyzed by semi-denaturing detergent-agarose gel electrophoresis and fluorescence correlation spectroscopy. The results indicated that [PSI(+)] aggregates were enlarged in mother cells and, instead, not apparently transmitted into daughter cells. Under these conditions, the activity of Hsp104, a known prion disaggregase, was not affected when monitored for the thermotolerance of the rnq1 mutants. These [PSI(+)]-inhibitory rnq1 mutations did not affect [PIN(+)] propagation itself when over-expressed from a strong promoter, but instead destabilized [PIN(+)] when expressed from the weak authentic RNQ1 promoter. The majority of these mutated residues are mapped to the surface, and on one side, of contiguous α-helices of the nonprion domain of Rnq1, suggesting its involvement in interactions with a prion or a factor necessary for prion development.

Localization of prion-destabilizing mutations in the N-terminal non-prion domain of Rnq1 in Saccharomyces cerevisiae.

[PIN(+)] is the prion form of Rnq1 in Saccharomyces cerevisiae and is necessary for the de novo induction of a second prion, [PSI(+)]. The function of Rnq1, however, is little understood. The limited availability of defective rnq1 alleles impedes the study of its structure-function relationship by genetic analysis. In this study, we isolated rnq1 mutants that are defective in the stable maintenance of the [PIN(+)] prion. Since there is no rnq1 phenotype available that is applicable to a direct selection or screening for loss-of-function rnq1 mutants, we took advantage of a prion inhibitory agent, Rnq1Delta100, to develop a color-based genetic screen. Rnq1Delta100 eliminates the [PSI(+)] prion in the [PIN(+)] state but not in the [pin(-)] state. This allows us to find loss-of-[PIN(+)] rnq1 mutants as white [PSI(+)] colonies. Nine rnq1 mutants with single-amino-acid substitutions were defined. These mutations impaired the stable maintenance of [PIN(+)] and, as a consequence, were also partially defective in the de novo induction of [PSI(+)]. Interestingly, eight of the nine alleles were mapped to the N-terminal region of Rnq1, which is known as the non-prion domain preceding the asparagine and glutamine rich prion domain of Rnq1. Notably, overexpression of these rnq1 mutant proteins restored [PIN(+)] prion activity, suggesting that each of the rnq1 mutants was not completely inactive. These findings indicate that the N-terminal non-prion domain of Rnq1 harbors a potent activity to regulate the maintenance of the [PIN(+)] prion.

Selfish prion of Rnq1 mutant in yeast.

[PIN(+)] is a prion form of Rnq1 in Saccharomyces cerevisiae and is necessary for the de novo induction of a second prion, [PSI(+)]. We previously isolated a truncated form of Rnq1, named Rnq1Delta100, as a [PSI(+)]-eliminating factor in S. cerevisiae. Rnq1Delta100 deletes the N-terminal non-prion domain of Rnq1, and eliminates [PSI(+)] in [PIN(+)] yeast. Here we found that [PIN(+)] is transmissible to Rnq1Delta100 in the absence of full-length Rnq1, forming a novel prion variant [RNQ1Delta100(+)]. [RNQ1Delta100(+)] has similar [PIN(+)] properties as it stimulates the de novo induction of [PSI(+)] and is eliminated by the null hsp104Delta mutation, but not by Hsp104 overproduction. In contrast, [RNQ1Delta100(+)] inherits the inhibitory activity and hampers the maintenance of [PSI(+)] though less efficiently than [PIN(+)] made of Rnq1-Rnq1Delta100 co-aggregates. Interestingly, [RNQ1Delta100(+)] prion was eliminated by de novo [PSI(+)] induction. Thus, the [RNQ1Delta100(+)] prion demonstrates selfish activity to eliminate a heterologous prion in S. cerevisiae, showing the first instance of a selfish prion variant in living organisms.

Requirement of RNA binding of mammalian eukaryotic translation initiation factor 4GI (eIF4GI) for efficient interaction of eIF4E with the mRNA cap.

Eukaryotic mRNAs possess a 5'-terminal cap structure (cap), m(7)GpppN, which facilitates ribosome binding. The cap is bound by eukaryotic translation initiation factor 4F (eIF4F), which is composed of eIF4E, eIF4G, and eIF4A. eIF4E is the cap-binding subunit, eIF4A is an RNA helicase, and eIF4G is a scaffolding protein that bridges between the mRNA and ribosome. eIF4G contains an RNA-binding domain, which was suggested to stimulate eIF4E interaction with the cap in mammals. In Saccharomyces cerevisiae, however, such an effect was not observed. Here, we used recombinant proteins to reconstitute the cap binding of the mammalian eIF4E-eIF4GI complex to investigate the importance of the RNA-binding region of eIF4GI for cap interaction with eIF4E. We demonstrate that chemical cross-linking of eIF4E to the cap structure is dramatically enhanced by eIF4GI fragments possessing RNA-binding activity. Furthermore, the fusion of RNA recognition motif 1 (RRM1) of the La autoantigen to the N terminus of eIF4GI confers enhanced association between the cap structure and eIF4E. These results demonstrate that eIF4GI serves to anchor eIF4E to the mRNA and enhance its interaction with the cap structure.

Identification of four novel mutations in F5 associated with congenital factor V deficiency.

Coagulation factor V (FV) deficiency is a rare bleeding disorder characterized by low coagulant and antigen levels of FV with bleeding symptoms ranging from mild to severe. Only a limited number of mutations have been reported because of the large size of the factor V gene (F5) as well as the low prevalence. In this study, we have identified four novel mutations in F5 in five unrelated patients with congenital FV deficiency. All the patients, including two with undetectable FV activity, were asymptomatic and were found to have prolonged prothrombin time and activated partial thromboplastin time during preoperative screening or routine examinations. All four mutations found in this study are either missense or in-frame deletion. This is in contrast with previous reports of a high frequency of mutations introducing premature termination codons in inherited FV deficiency. Missense mutations of F5 might produce a mild phenotype and are not frequently diagnosed. Although FV deficiency is a very rare disorder with a predicted incidence of one in 1 million, this study suggests that the numbers of F5 mutations, especially missense mutations, are higher than estimated.

A regulatory role of the Rnq1 nonprion domain for prion propagation and polyglutamine aggregates.

Prions are infectious, self-propagating protein conformations. Rnq1 is required for the yeast Saccharomyces cerevisiae prion [PIN(+)], which is necessary for the de novo induction of a second prion, [PSI(+)]. Here we isolated a [PSI(+)]-eliminating mutant, Rnq1Delta100, that deletes the nonprion domain of Rnq1. Rnq1Delta100 inhibits not only [PSI(+)] prion propagation but also [URE3] prion and huntingtin's polyglutamine aggregate propagation in a [PIN(+)] background but not in a [pin(-)] background. Rnq1Delta100, however, does not eliminate [PIN(+)]. These findings are interpreted as showing a possible involvement of the Rnq1 prion in the maintenance of heterologous prions and polyQ aggregates. Rnq1 and Rnq1Delta100 form a sodium dodecyl sulfate-stable and Sis1 (an Hsp40 chaperone protein)-containing coaggregate in [PIN(+)] cells. Importantly, Rnq1Delta100 is highly QN-rich and prone to self-aggregate or coaggregate with Rnq1 when coexpressed in [pin(-)] cells. However, the [pin(-)] Rnq1-Rnq1Delta100 coaggregate does not represent a prion-like aggregate. These findings suggest that [PIN(+)] Rnq1-Rnq1Delta100 aggregates interact with other transmissible and nontransmissible amyloids to destabilize them and that the nonprion domain of Rnq1 plays a crucial role in self-regulation of the highly reactive QN-rich prion domain of Rnq1.