Interference of CLN6 mutants

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 transmembrane, 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

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

Modeling of a dispersive tsunami caused by a submarine landslide based on detailed bathymetry of the continental slope in the Nankai trough, southwest Japan

Tsunamis caused by submarine landslides are not accompanied by seismic waves and thus may appear at the coast without warning. In this study, detailed bathymetric surveys with a multi-narrow beam echo sounder were used to map submarine landslides on the continental shelf near Cape Muroto, in the Nankai trough off southwestern Japan. One of the surveyed submarine landslides was selected to supply dimensions for the simulation of a submarine mass movement by a two-layer flow model in which the upper and lower layers correspond to seawater and turbidity currents, respectively. The time series of seafloor deformation during this simulated landslide was used as the boundary condition to drive a tsunami simulation. The results showed strong directivity effects during tsunami generation in which pushing-dominant (positive) tsunami waves propagated seaward, in the direction of the submarine landslide, and pulling-dominant (negative) tsunami waves propagated landward. Both types of waves were strongly modified by frequency dispersion. For pulling-dominant waves, a tsunami simulation that included dispersion (Boussinesq) terms predicted greater maximum tsunami heights than a non-dispersive tsunami simulation. To avoid underestimation of tsunami heights, we recommend including dispersion terms when modeling tsunamis caused by submarine landslides for disaster planning purposes

Development of an intravital imaging system for the synovial tissue reveals the dynamics of CTLA-4 Ig in vivo

There have been many attempts to visualize the inflamed joints using multiphoton microscopy. However, due to the hypervascular and multilayered structure of the inflamed synovium, intravital imaging of the deep synovial tissue has been difficult. Here, we established original intravital imaging systems to visualize synovial tissue and pathological osteoclasts at the pannus–bone interface using multiphoton microscopy. Combined with fluorescence-labeling of CTLA-4 Ig, a biological agent used for the treatment of rheumatoid arthritis, we identified that CTLA-4 Ig was distributed predominantly within the inflamed synovium and bound to CX3CR1+ macrophages and CD140a+ fibroblasts 6 h after injection, but not to mature osteoclasts. Intravital imaging of blood and lymphatic vessels in the inflamed synovium further showed that extravasated CTLA-4 Ig was immediately drained through lymphatic vessels under acute arthritic conditions, but the drainage activity was retarded under chronic conditions. These results indicate that this intravital synovial imaging system can serve as a platform for exploring the dynamics of immune cells, osteoclasts, and biological agents within the synovial microenvironment in vivo.Hasegawa T., Kikuta J., Sudo T., et al. Development of an intravital imaging system for the synovial tissue reveals the dynamics of CTLA-4 Ig in vivo. Scientific Reports 10, 13480 (2020); https://doi.org/10.1038/s41598-020-70488-y

Augmented survival of out-of-hospital cardiac arrest victims with the use of mobile phones for emergency communication under the DA-CPR protocol getting information from callers beside the victim

Purpose To investigate the impacts of emergency calls made using mobile phones on the quality of dispatcher-assisted cardiopulmonary resuscitation (DA-CPR) and survival from out-of-hospital cardiac arrests (OHCAs) that were not witnessed by emergency medical service (EMS). Methods In this prospective study, we collected data for 2530 DA-CPR-attempted medical emergency cases (517 using mobile phones and 2013 using landline phones) and 2980 non-EMS-witnessed OHCAs (600 using mobile phones and 2380 using landline phones). Time factors and quality of DA-CPR, backgrounds of callers and outcomes of OHCAs were compared between mobile and landline phone groups. Results Emergency calls are much more frequently placed beside the arrest victim in mobile phone group (52.7% vs. 17.2%). The positive predictive value and acceptance rate of DA-CPR in mobile phone group (84.7% and 80.6%, respectively) were significantly higher than those in landline group (79.2% and 70.9%). The proportion of good-quality bystander CPR in mobile phone group was significantly higher than that in landline group (53.5% vs. 45.0%). When analysed for all non-EMS-witnessed OHCAs, rates of 1-month survival and 1-year neurologically favourable survival in mobile phone group (7.8% and 3.5%, respectively) were higher than those in landline phone group (4.6% and 1.9%; p < 0.05). Multiple logistic regression analysis, including other backgrounds, revealed that mobile phone calls were associated with increased 1-month survival in the subgroup of OHCAs receiving bystander CPR (adjusted odds ratio, 1.84; 95% CI, 1.15–2.92). Conclusion Emergency calls made using mobile phones are likely to augment the survival from OHCAs by improving DA-CPR. © 2016 Elsevier Ireland LtdEmbargo Period 12 month

Fixation Strength of Caudal Pedicle Screws after Posterior Lumbar Interbody Fusion with the Modified Cortical Bone Trajectory Screw Method

Study DesignClinical case series.PurposeIn the posterior lumbar interbody fusion (PLIF) procedure in our institute, the cephalad screw trajectory follows a mediolateral and caudocephalad directed path according to the original cortical bone trajectory (CBT) method. However, the starting point of the caudal screw is at the medial border of the pedicle on an articular surface of the superior articular process, and the trajectory takes a mediolateral path parallel to the cephalad endplate. The incidence of caudal screw loosening after PLIF with this modified CBT screw method was investigated, and significant risk factors for caudal screw loosening were evaluated.Overview of LiteratureA biomechanical study of this modified caudal screw trajectory using the finite element method reported about a 20% increase in uniaxial yield pullout load compared with the traditional trajectory. However, there has been no clinical study concerning the fixation strength of this modified caudal screw trajectory.MethodsThe subjects were 193 consecutive patients who underwent single-level PLIF with modified CBT screw fixation. Caudal screw loosening was checked in computed tomography at 6 months after surgery, and screw loosening was defined as a radiolucency of 1 mm or more at the bone-screw interface.ResultsThe incidence of caudal screw loosening after lumbosacral PLIF (46.2%) was significantly higher than that after floating PLIF (6.0%). No significant differences in sex, brand of the instruments, and diameter and length of the caudal screw were evident between patients with and without caudal screw loosening. Patients with caudal screw loosening were significantly older at the time of surgery than patients without caudal screw loosening.ConclusionsFixation strength of the caudal screw after floating PLIF with this modified CBT screw technique was sufficiently acceptable. Fixation strength after the lumbosacral procedure was not