Comparison of Scheimpflug and Anterior Segment Optical Coherence Tomography Imaging Parameters for Japanese Patients With Fuchs Endothelial Corneal Dystrophy With and Without TCF4 Repeat Expansions.

PURPOSE: The aim of this study was to investigate the association between cytosine-thymine-guanine trinucleotide repeat (TNR) expansion in TCF4 and the clinical phenotypes of corneal densitometry or anterior segment morphology in Fuchs endothelial corneal dystrophy. METHODS: This retrospective cross-sectional study included 150 eyes from 75 Japanese consecutive patients with Fuchs endothelial corneal dystrophy. Cytosine-thymine-guanine repeat expansion of leukocyte-derived genomic DNA was analyzed through fragment analysis using polymerase chain reaction and triplet repeat primed polymerase chain reaction. Scheimpflug-based densitometry and anterior segment optical coherence tomography were applied. Corneal densitometry, and corneal and anterior segment morphology parameters were compared between patients with and without TNR expansion of 50 or more (expansion and nonexpansion groups, respectively) using a mixed model. RESULTS: The average age of the patients was 66.8 ± 13.0 years, and the modified Krachmer grading scale was 1, 2, 3, 4, 5, and 6 for 7, 32, 28, 51, 6, and 18 eyes, respectively. Sixteen patients (21%) exhibited ≥50 TNR expansion. No significant differences in sex, age, history of keratoplasty, modified Krachmer grade, and corneal densitometry in either diameter or depth were observed between the 2 groups. No significant differences in anterior segment morphology, including the anterior chamber depth and anterior chamber angle width parameters, were observed using a univariate mixed model, except for central corneal thickness (P = 0.047). However, according to the multivariate mixed model, repeat expansion was not significantly associated with central corneal thickness (P = 0.27). CONCLUSIONS: No significant differences in clinical phenotypes were found between Japanese patients having Fuchs endothelial corneal dystrophy with and without TNR expansion.

p.Asn77Lys homozygous CLN6 mutation in two unrelated Japanese patients with Kufs disease, an adult onset neuronal ceroid lipofuscinosis.

BACKGROUND: The neuronal ceroid lipofuscinosis (NCL) are a group of autosomal recessive neurodegenerative disorders that are characterized by the accumulation of ceroid lipofuscins. The NCLs are categorized into four classes based on the age of onset. Kufs disease is a rare adult-onset NCL caused by mutations in the CLN6 gene, which is rarely observed in the Japanese population. CASE: We previously reported a case study on a patient with Kufs disease, whose parents had a consanguineous marriage. Later, we observed another unrelated patient with Kufs. Here we present the case and mutational gene report in patients with Kufs disease. CONCLUSIONS: Gene analysis results of the first patient revealed a homozygous mutation c231C > G, p.Asn77Lys in exon 3 and a homozygous c.297 + 48 A > T mutation in intron 3 in the CLN6 gene. The Asn amino acid is perfectly conserved among species. In silico analysis showed that the mutation is predicted to be probably damaging. Moreover, the second patient with Kufs disease also had the same homozygous mutations. These data suggest that the missense mutation must be pathogenic. Furthermore, the patients had lived in the same district; therefore, they both potentially inherited the founder effect mutations.

CLN6's luminal tail-mediated functional interference between CLN6 mutants as a novel pathomechanism for the neuronal ceroid lipofuscinoses.

CLN6 (Ceroid Lipofuscinosis, Neuronal, 6) is a 311-amino acid protein spanning the endoplasmic reticulum membrane. Mutations in CLN6 are linked to CLN6 disease, a hereditary neurodegenerative disorder categorized into the neuronal ceroid lipofuscinoses. CLN6 disease is an autosomal recessive disorder and individuals affected with this disease have two identical (homozygous) or two distinct (compound heterozygous) CLN6 mutant alleles. Little has been known about CLN6's physiological roles and the disease mechanism. We recently found that CLN6 prevents protein aggregate formation, pointing to impaired CLN6's anti-aggregate activity as a cause for the disease. To comprehensively understand the pathomechanism, overall anti-aggregate activity derived from two different CLN6 mutants needs to be investigated, considering patients compound heterozygous for CLN6 alleles. We focused on mutant combinations involving the S132CfsX18 (132fsX) prematurely terminated protein, produced from the most frequent mutation in CLN6. The 132fsX mutant nullified anti-aggregate activity of the P299L CLN6 missense mutant but not of wild-type CLN6. Wild-type CLN6's resistance to the 132fsX mutant was abolished by replacement of amino acids 297-301, including Pro297 and Pro299, with five alanine residues. Given that removal of CLN6's C-terminal fifteen amino acids 297-311 (luminal tail) did not affect the resistance, we suggested that CLN6's luminal tail, when unleashed from Pro297/299-mediated conformational constraints, is improperly positioned by the 132fsX mutant, thereby blocking the induction of anti- aggregate activity. We here reveal a novel mechanism for dissipating CLN6 mutants' residual functions, providing an explanation for the compound heterozygosity-driven pathogenesis.

Implications of graded reductions in CLN6's anti-aggregate activity for the development of the neuronal ceroid lipofuscinoses.

CLN6, spanning the endoplasmic reticulum membrane, is a protein of unknown function. Mutations in the CLN6 gene are linked to an autosomal recessively inherited disorder termed CLN6 disease, classified as a form of the neuronal ceroid lipofuscinoses (NCL). The pathogenesis of CLN6 disease remains poorly understood due to a lack of information about physiological roles CLN6 plays. We previously demonstrated that CLN6 has the ability to prevent protein aggregate formation, and thus hypothesized that the abrogation of CLN6's anti-aggregate activity underlies the development of CLN6 disease. To test this hypothesis, we narrowed down the region vital for CLN6's anti-aggregate activity, and subsequently investigated if pathogenic mutations within the region attenuate CLN6's anti-aggregate activity toward four aggregation-prone αB-crystallin (αBC) mutants. None of the four αBC mutants was prevented from aggregating by the Arg106ProfsX truncated CLN6 mutant, the human counterpart of the nclf mutant identified in a naturally occurring mouse model of late infantile-onset CLN6 disease. In contrast, the Arg149Cys and the Arg149His CLN6 mutants, both associated with adult-onset CLN6 disease, blocked aggregation of two out of and all of the four αBC mutants, respectively, indicating that CLN6's anti-aggregate activity is differentially modulated according to the substitution pattern at the same amino acid position. Collectively, we here propose that the graded reduction in CLN6's anti-aggregate activity governs the clinical course of late infantile- and adult-onset NCL.

Genetic variants of SMAD2/3/4/7 are associated with susceptibility to ulcerative colitis in a Japanese genetic background.

PURPOSE: Inflammatory bowel disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC), is attributed to inappropriate inflammatory response in intestinal mucosa. Transforming growth factor ß (TGF-ß)/SMAD signaling plays key role in differentiation of naïve CD4+ T cells to T helper 17 (Th17) cells or regulatory T (Treg) cells. This study aimed to investigate associations between single nucleotide polymorphisms (SNPs) of SMAD family genes and susceptibility to IBD in a Japanese cohort to elucidate genetic determinants of IBD. METHODS: This study included 81 patients with CD, 108 patients with UC, and 199 healthy subjects as controls. A total of 21 SNPs in four genes (SMAD2, SMAD3, SMAD4, and SMAD7) involved in the TGF-ß/SMAD signaling pathway were genotyped by polymerase chain reaction (PCR)-restriction fragment length polymorphism, PCR-direct DNA sequencing, or PCR-high resolution melting curve analysis. RESULTS: Four SNPs (rs13381619, rs9955626, rs1792658, and rs1792671) within SMAD2, one SNP within SMAD3 (rs41473580), two SNPs within SMAD4 (rs7229678 and rs9304407), and one SNP within SMAD7 (rs12956924) were significantly associated with susceptibility only to UC. rs13381619 within SMAD2, rs4147358 within SMAD3, rs9304407 within SMAD4, and rs12956924 within SMAD7 exhibited the strongest association (p < 0.001, p = 0.021, p = 0.005, and p = 0.001, respectively). Furthermore, rs4147358 of SMAD3 altered the expression of a luciferase reporter gene in Jurkat T cell line in vitro. CONCLUSIONS: Genetic variants of several SMAD family of genes might alter the balance of differentiation between Th17 and Treg, resulting in the development of IBD, especially UC.

Identification of CLN6 as a molecular entity of endoplasmic reticulum-driven anti-aggregate activity.

αB-crystallin (αBC) is a small heat shock protein. Mutations in the αBC gene are linked to α-crystallinopathy, a hereditary myopathy histologically characterized by intracellular accumulation of protein aggregates. The disease-causing R120G αBC mutant, harboring an arginine-to-glycine replacement at position 120, is an aggregate-prone protein. We previously showed that the R120G mutant's aggregation in HeLa cells was prevented by enforced expression of αBC on the endoplasmic reticulum (ER). To elucidate the molecular nature of the preventive effect on the R120G mutant, we isolated proteins binding to ER-anchored αBC (TMαBC). The ER transmembrane CLN6 protein was identified as a TMαBC's binder. CLN6 knockdown in HeLa cells attenuated TMαBC's anti-aggregate activity against the R120G mutant. Conversely, CLN6 overexpression enhanced the activity, indicating that CLN6 operates as a downstream effector of TMαBC. CLN6 physically interacted with the R120G mutant, and repressed its aggregation in HeLa cells even when TMαBC was not co-expressed. Furthermore, CLN6's antagonizing effect on the R120G mutant was compromised upon treatment with a lysosomal inhibitor, suggesting CLN6 requires the intact autophagy-lysosome system to prevent the R120G mutant from aggregating. We hence conclude that CLN6 is not only a molecular entity of the anti-aggregate activity conferred by the ER manipulation using TMαBC, but also serves as a potential target of therapeutic interventions.

A strategy for the prevention of protein oxidation by drug product in polymer-based syringes.

UNLABELLED: Recently, new and advanced ideas have been presented on the value of polymer-based syringes for improved safety, better strength, reduced aggregation, and the prevention of drug degradation. In this report, our findings on drug degradation from protein oxidation will be presented and discussed. Commonly, dissolved oxygen is one of the factors for causing protein degradation. Due to the nature of higher gas permeability in polymer-based syringes, it was thought to be difficult to control the oxygen level during storage. However, this report demonstrates the appropriateness of combining the use of an oxygen absorber within the secondary packaging as a deoxygenated packaging system. In addition, this report suggests that another factor to enhance protein oxidization is related to radicals on the syringe barrel from sterilization by irradiation. We demonstrate that steam sterilization can minimize protein oxidization, as the protein filled in steam sterilized syringe is much more stable. In conclusion, the main oxidation pathway of a protein has been identified as dissolved oxygen and radical generation within a polymer container. Possible solutions are herewith presented for controlling oxidation by means of applying a deoxygenated packaging system as well as utilizing steam sterilization as a method of sterilization for prefillable polymer syringes. LAY ABSTRACT: There have been many presentations and discussions about the risks associated with glass prefilled syringes. Advanced ideas are being presented on the value of polymer-based syringes for improved safety, better strength, reduced protein aggregation, and the prevention of drug degradation. Drug degradation based on protein oxidation is discussed in this report. Identification of the main factors causing this degradation and possible solutions available by using polymer-based syringes will be presented. The causes of protein oxidation have been identified as dissolved oxygen and radicals generated by the applied method of sterilization. The oxidation reaction created by dissolved oxygen within the drug product can be effectively inhibited by controlling the removal of the oxygen through the use of a deoxygenated packaging system. This packaging system can control the level or complete removal of oxygen from the primary container and the secondary packaging system. Protein oxidation induced by the formation of radicals from sterilization by irradiation is another critical aspect where it was thought that various sterilization methods were acceptable without loosing drug product quality. However, this report is first to demonstrate that gamma sterilized polymer-based syringes accelerated protein oxidation by radical generation; this effect can be prevented by means of steam sterilization.

Assessment of the effects of sterilization methods on protein drug stability by elucidating decomposition mechanism and material analysis.

The effects of different sterilization methods on the stability of highly sensitive protein drugs were assessed by elucidating mechanism involved in the process of protein decomposition. Results demonstrated that the steam sterilized syringes produced less protein oxidation compared with sterilization by the electron beam method. Electron spin resonance analysis showed that while considerably high levels of radicals were observed in the electron beam-sterilized syringes, no radicals were detected with steam sterilization. To identify the factor involved in protein oxidation, stability of the chemical composition of the syringe material was investigated using various analytical methods. Results showed that the syringe material itself was oxidized and two forms of oxidation products were identified with electron beam sterilization. Protein oxidation was shown to increase over time, and this was thought to be as a result of persistent exposure to the oxidized syringe barrel surface, which induced further protein oxidation. These results suggest that compared to electron beam sterilization, steam sterilization is a preferable method for the plastic prefilled syringe system, particularly for biopharmaceutical drug products that are highly sensitive to oxidization.

Prevention of aberrant protein aggregation by anchoring the molecular chaperone αB-crystallin to the endoplasmic reticulum.

The chaperone αB-crystallin (αBC) is a member of the small heat shock protein family and its point or truncated mutants cause the muscular disorder α-crystallinopathy. The illness is histologically characterized by accumulation of protein aggregates in muscle cells. Expression of the myopathy-causing R120G mutant of αBC, harboring an arginine-to-glycine mutation at position 120, results in aggregate formation. We demonstrated that tethering αBC to the endoplasmic reticulum (ER) membrane represses the protein aggregation mediated by the R120G mutant. ER-anchored αBC decreased the amount of the R120G mutant through autophagic proteolysis. In contrast, knockdown of ATG5, an E3 ligase essential for autophagy, in ER-anchored αBC-transfected cells restored the quantity of the R120G mutant. In this context, aggregate formation was still suppressed, indicating that ER-anchored αBC profoundly constrains aggregation competency of the R120G mutant separately from downregulating the abundance of the mutant. We have proposed that protein aggregation is prevented by manipulation of the ER microenvironment with αBC, and have shed light on a novel aspect of the ER as a therapeutic target.

Functional evaluation and characterization of a newly developed silicone oil-free prefillable syringe system.

The functionality of a newly developed silicone oil-free (SOF) syringe system, of which the plunger stopper is coated by a novel coating technology (i-coating™), was assessed. By scanning electron microscopy observations and other analysis, it was confirmed that the plunger stopper surface was uniformly covered with the designed chemical composition. A microflow imaging analysis showed that the SOF system drastically reduced both silicone oil (SO) doplets and oil-induced aggregations in a model protein formulation, whereas a large number of subvisible particles and protein aggregations were formed when a SO system was used. Satisfactory container closure integrity (CCI) was confirmed by means of dye and microorganism penetration studies. Furthermore, no significant difference between the break loose and gliding forces was observed in the former, and stability studies revealed that the SOF system could perfectly show the aging independence in break loose force observed in the SO system. The results suggest that the introduced novel SOF system has a great potential and represents an alternative that can achieve very low subvisible particles, secure CCI, and the absence of a break loose force. In particular, no risk of SO-induced aggregation can bring additional value in the highly sensitive biotech drug market.

Involvement of reactive oxygen species in osteoblastic differentiation of MC3T3-E1 cells accompanied by mitochondrial morphological dynamics.

Bone remodeling is regulated by local factors that regulate bone-forming osteoblasts and bone-resorbing osteoclasts, in addition to hormonal activity. Recent studies have shown that reactive oxygen species (ROS) act as an intracellular signal mediator for osteoclast differentiation. However the role of ROS on osteoblast differentiation is poorly understood. Here, we investigated the impact of ROS on osteoblastic differentiation of MC3T3-E1 cells. Osteogenic induction resulted in notable enhancement of mineralization and expression of osteogenic marker gene alkaline phosphatase, which were accompanied by an increase in ROS production. Additionally, we found that mitochondrial morphology dynamically changed from tubular reticulum to fragmented structures during the differentiation, suggesting that mitochondrial morphological transition is a novel osteoblast differentiation index. The antioxidant N-acetyl cysteine prevented not only ROS production but also mineralization and mitochondrial fragmentation. It is therefore suggested that the ROS-dependent signaling pathways play a role in osteoblast differentiation accompanied by mitochondrial morphological transition.

Protective role of the endoplasmic reticulum protein mitsugumin23 against ultraviolet C-induced cell death.

The endoplasmic reticulum (ER) operates in adaptive responses to various stresses, dictating cell fate. Here we show that knockdown of the ER protein mitsugumin23 (MG23) enhances cell death induced by ultraviolet C (UVC), which causes DNA damage. The small heat shock protein αB-crystallin (αBC) is identified as a MG23 binding molecule and its knockdown facilitates death of UVC-exposed cells. Conversely, αBC lowered UVC sensitivity when expressed as an ER-anchored form. Taken together, the results suggest that MG23 plays a protective role against UVC by accumulating αBC in the close vicinity of the ER.

Mitochondrial development of the in vitro hepatic organogenesis model with simultaneous cardiac mesoderm differentiation from murine induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells, resembling embryonic stem (ES) cells in many phenomena, including differentiation potential, colony morphology, and the expression of specific representative markers, were generated from differentiated somatic cells by exogenous expression of several transcriptional factors. In recent, the mitochondria of iPS cells were also reported to be rejuvenated to that of ES cells, however it is not known if the mitochondria have same potential for differentiation as ES cells. We have established the murine ES cell-derived in vitro hepatic organogenesis model, consisting of not only hepatocytes but also endothelial networks together with cardiac mesoderm differentiation, previously. By measuring oxygen concentration and pH in the culture medium, respectively corresponding to the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), we compared the metabolic patterns and bio-energetic profiles of both iPS and ES cells during the hepatic differentiation. The bio-energetic profiles of the in vitro hepatic organogenesis from iPS cells accorded with each differentiation steps, from proliferation stage as the initiation, spontaneously beating cardiac differentiation in the next, and finally liver tissue-formation, as well as that from ES cells. Both iPS and ES cells were differentiated into liver-like tissue with similar mitochondrial development.