Neuronal DSCAM regulates the peri-synaptic localization of GLAST in Bergmann glia for functional synapse formation.

In the central nervous system, astrocytes enable appropriate synapse function through glutamate clearance from the synaptic cleft; however, it remains unclear how astrocytic glutamate transporters function at peri-synaptic contact. Here, we report that Down syndrome cell adhesion molecule (DSCAM) in Purkinje cells controls synapse formation and function in the developing cerebellum. Dscam-mutant mice show defects in CF synapse translocation as is observed in loss of function mutations in the astrocytic glutamate transporter GLAST expressed in Bergmann glia. These mice show impaired glutamate clearance and the delocalization of GLAST away from the cleft of parallel fibre (PF) synapse. GLAST complexes with the extracellular domain of DSCAM. Riluzole, as an activator of GLAST-mediated uptake, rescues the proximal impairment in CF synapse formation in Purkinje cell-selective Dscam-deficient mice. DSCAM is required for motor learning, but not gross motor coordination. In conclusion, the intercellular association of synaptic and astrocyte proteins is important for synapse formation and function in neural transmission.

The Utility of Pneumatic Cavity Volume in the Treatment of Exudative Otitis Media in Children with Cleft Palate.

OBJECTIVE: Measure the volume of air-containing space in children with cleft palate and assess age-related changes, recurrence rate of otitis media with effusion (OME) after tube removal, and temporal bone development trend based on time of tube placement. DESIGN: Interventional prospective study. SETTING: Cleft Lip and Palate Center at a Tertiary-level institution. PATIENTS/PARTICIPANTS: One hundred sixty-eight ears of 86 patients who visited our center from January 2018 to December 2019. INTERVENTIONS: We performed tympanometry (impedance audiometry) after tube placement. MAIN OUTCOME MEASURES: Recurrence (at least one episode of OME after tympanic membrane closure), tympanic cavity volumes, and timing of tube placement. RESULTS: The mean air-containing cavity volume was 1.62 mL, 2.99 mL, and 3.29 mL in patients aged 1, 2, and 3 years, respectively. A rapid increase in volume was observed around 2 years of age. Twenty-two (42.3%) of the 52 ears with pneumatic cavity volumes <3 mL, and four (14.3%) of the 28 ears with pneumatic cavity volumes ≥3 mL had recurrence. Tubes were placed at ages <1 year and ≥1 year in 28 and 62 ears, respectively. The pneumatic cavity volume tended to be greater in the ears with tube placement at age <1 year. CONCLUSION: This study provided insights into using pneumatic cavity volume measurements to determine the appropriate timing for tube removal. Tubes should be placed as early as possible (before the age of 2 years) for prolonged OME associated with children with cleft palate.

Visualization of trans homophilic interaction of clustered protocadherin in neurons.

Clustered protocadherin (Pcdh) functions as a cell recognition molecule through the homophilic interaction in the central nervous system. However, its interactions have not yet been visualized in neurons. We previously reported PcdhγB2-Förster resonance energy transfer (FRET) probes to be applicable only to cell lines. Herein, we designed γB2-FRET probes by fusing FRET donor and acceptor fluorescent proteins to a single γB2 molecule and succeeded in visualizing γB2 homophilic interaction in cultured hippocampal neurons. The γB2-FRET probe localized in the soma and neurites, and FRET signals, which were observed at contact sites between neurites, eliminated by ethylene glycol tetraacetic acid (EGTA) addition. Live imaging revealed that the FRET-negative γB2 signals rapidly moved along neurites and soma, whereas the FRET-positive signals remained in place. We observed that the γB2 proteins at synapses rarely interact homophilically. The γB2-FRET probe might allow us to elucidate the function of the homophilic interaction and the cell recognition mechanism.

Insufficient Workplace Infection Control and Unhealthy Lifestyle Behaviors Are Related to Poor Self-Rated Health During the COVID-19 Pandemic.

OBJECTIVE: This study investigated whether workers who practiced unhealthy lifestyles but worked under organizations with insufficient control against coronavirus disease 2019 (COVID-19) would pose a synergistic risk of poor self-rated health (SRH). METHODS: A total of 22,637 workers (men, 48.5%) were extracted from an online survey during the COVID-19 pandemic in Japan (December 2020). Multiple logistic regression analyses were performed to estimate adjusted odds ratio (AOR) against poor (poor, fair) SRH. RESULTS: Accumulation of unhealthy lifestyle behaviors (AOR, 1.49 to 4.40; P < 0.05) and insufficient infection control (AOR, 1.80; P < 0.05) were independently related to poor SRH; however, when these factors were combined, SRH was additively worsened (AOR, 2.14 to 7.72; P < 0.05). CONCLUSIONS: This study highlights that not only unhealthy lifestyle practices but also poor organizational management against infection would worsen workers' SRH during the COVID-19 pandemic.

Triglyceride level and soft drink consumption predict nonalcoholic fatty liver disease in nonobese male adolescents.

AIM: Differential metabolic risk factors of nonalcoholic fatty liver disease (NAFLD) in nonobese male adolescents were analyzed examining relationships between NAFLD and clinical parameters of metabolic syndrome, including exercise and soft drink consumption, in male adolescents. METHODS: In total, 134 male university students (nonobese, n = 78; obese, n = 56) who underwent the first-year health checkup were divided into the NAFLD and non-NAFLD groups based on abdominal ultrasonography (AUS) findings. Relationships between NAFLD and metabolic parameters, including body mass index (BMI) and AUS score, were examined in nonobese students. RESULTS: Metabolic factors associated with hypertension, abdominal fat, liver damage, dyslipidemia, and impaired glucose tolerance were significantly less common in nonobese students than in obese students. The aforementioned factors and soft drink consumption were significantly more common in the NAFLD group than in the non-NAFLD group. The univariate and multivariate analyses of nonobese students showed that the triglyceride level (odds ratio [OR], 1.06; 95% confidence interval [CI], 1.01-1.10, p = 0.001) was higher and soft drink consumption (OR, 36.8; 95% CI, 3.69-368, p < 0.001) was more common in the NAFLD group than the non-NAFLD group. CONCLUSIONS: Triglyceride level and soft drink consumption could aid in the detection of NAFLD in nonobese male adolescents. Our findings could provide useful information related to NAFLD and metabolic syndrome in nonobese adolescents.

Distinctive chaperonopathy in skeletal muscle associated with the dominant variant in DNAJB4.

DnaJ homolog, subfamily B, member 4, a member of the heat shock protein 40 chaperones encoded by DNAJB4, is highly expressed in myofibers. We identified a heterozygous c.270 T > A (p.F90L) variant in DNAJB4 in a family with a dominantly inherited distal myopathy, in which affected members have specific features on muscle pathology represented by the presence of cytoplasmic inclusions and the accumulation of desmin, p62, HSP70, and DNAJB4 predominantly in type 1 fibers. Both Dnajb4F90L knockin and knockout mice developed muscle weakness and recapitulated the patient muscle pathology in the soleus muscle, where DNAJB4 has the highest expression. These data indicate that the identified variant is causative, resulting in defective chaperone function and selective muscle degeneration in specific muscle fibers. This study demonstrates the importance of DNAJB4 in skeletal muscle proteostasis by identifying the associated chaperonopathy.

Mice with R2509C-RYR1 mutation exhibit dysfunctional Ca2+ dynamics in primary skeletal myocytes.

Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum (SR) of the skeletal muscle and plays a critical role in excitation-contraction coupling. Mutations in RYR1 cause severe muscle diseases, such as malignant hyperthermia, a disorder of Ca2+-induced Ca2+ release (CICR) through RYR1 from the SR. We recently reported that volatile anesthetics induce malignant hyperthermia (MH)-like episodes through enhanced CICR in heterozygous R2509C-RYR1 mice. However, the characterization of Ca2+ dynamics has yet to be investigated in skeletal muscle cells from homozygous mice because these animals die in utero. In the present study, we generated primary cultured skeletal myocytes from R2509C-RYR1 mice. No differences in cellular morphology were detected between wild type (WT) and mutant myocytes. Spontaneous Ca2+ transients and cellular contractions occurred in WT and heterozygous myocytes, but not in homozygous myocytes. Electron microscopic observation revealed that the sarcomere length was shortened to ∼1.7 µm in homozygous myocytes, as compared to ∼2.2 and ∼2.3 µm in WT and heterozygous myocytes, respectively. Consistently, the resting intracellular Ca2+ concentration was higher in homozygous myocytes than in WT or heterozygous myocytes, which may be coupled with a reduced Ca2+ concentration in the SR. Finally, using infrared laser-based microheating, we found that heterozygous myocytes showed larger heat-induced Ca2+ transients than WT myocytes. Our findings suggest that the R2509C mutation in RYR1 causes dysfunctional Ca2+ dynamics in a mutant-gene dose-dependent manner in the skeletal muscles, in turn provoking MH-like episodes and embryonic lethality in heterozygous and homozygous mice, respectively.

Heat-hypersensitive mutants of ryanodine receptor type 1 revealed by microscopic heating.

Thermoregulation is an important aspect of human homeostasis, and high temperatures pose serious stresses for the body. Malignant hyperthermia (MH) is a life-threatening disorder in which body temperature can rise to a lethal level. Here we employ an optically controlled local heat-pulse method to manipulate the temperature in cells with a precision of less than 1 °C and find that the mutants of ryanodine receptor type 1 (RyR1), a key Ca2+ release channel underlying MH, are heat hypersensitive compared with the wild type (WT). We show that the local heat pulses induce an intracellular Ca2+ burst in human embryonic kidney 293 cells overexpressing WT RyR1 and some RyR1 mutants related to MH. Fluorescence Ca2+ imaging using the endoplasmic reticulum-targeted fluorescent probes demonstrates that the Ca2+ burst originates from heat-induced Ca2+ release (HICR) through RyR1-mutant channels because of the channels' heat hypersensitivity. Furthermore, the variation in the heat hypersensitivity of four RyR1 mutants highlights the complexity of MH. HICR likewise occurs in skeletal muscles of MH model mice. We propose that HICR contributes an additional positive feedback to accelerate thermogenesis in patients with MH.

Humanized substitutions of Vmat1 in mice alter amygdala-dependent behaviors associated with the evolution of anxiety.

The human vesicular monoamine transporter 1 (VMAT1) harbors unique substitutions (Asn136Thr/Ile) that affect monoamine uptake into synaptic vesicles. These substitutions are absent in all known mammals, suggesting their contributions to distinct aspects of human behavior modulated by monoaminergic transmissions, such as emotion and cognition. To directly test the impact of these human-specific mutations, we introduced the humanized residues into mouse Vmat1 via CRISPR/Cas9-mediated genome editing and examined changes at the behavioral, neurophysiological, and molecular levels. Behavioral tests revealed reduced anxiety-related traits of Vmat1 Ile mice, consistent with human studies, and electrophysiological recordings showed altered oscillatory activity in the amygdala under anxiogenic conditions. Transcriptome analyses further identified changes in gene expressions in the amygdala involved in neurodevelopment and emotional regulation, which may corroborate the observed phenotypes. This knock-in mouse model hence provides compelling evidence that the mutations affecting monoaminergic signaling and amygdala circuits have contributed to the evolution of human socio-emotional behaviors.

Targeting Neurons with Functional Oxytocin Receptors: A Novel Set of Simple Knock-In Mouse Lines for Oxytocin Receptor Visualization and Manipulation.

The neuropeptide oxytocin (Oxt) plays important roles in modulating social behaviors. Oxt receptor (Oxtr) is abundantly expressed in the brain and its relationship to socio-behavioral controls has been extensively studied using mouse brains. Several genetic tools to visualize and/or manipulate Oxtr-expressing cells, such as fluorescent reporters and Cre recombinase drivers, have been generated by ES-cell based gene targeting or bacterial artificial chromosome (BAC) transgenesis. However, these mouse lines displayed some differences in their Oxtr expression profiles probably because of the complex context and integrity of their genomic configurations in each line. Here, we apply our sophisticated genome-editing techniques to the Oxtr locus, systematically generating a series of knock-in mouse lines, in which its endogenous transcriptional regulations are intactly preserved and evaluate their expression profiles to ensure the reliability of our new tools. We employ the epitope tagging strategy, with which C-terminally fused tags can be detected by highly specific antibodies, to successfully visualize the Oxtr protein distribution on the neural membrane with super-resolution imaging for the first time. By using T2A self-cleaving peptide sequences, we also induce proper expressions of tdTomato reporter, codon-improved Cre recombinase (iCre), and spatiotemporally inducible Cre-ERT2 in Oxtr-expressing neurons. Electrophysiological recordings from tdTomato-positive cells in the reporter mice support the validity of our tool design. Retro-orbital injections of AAV-PHP.eB vector into the Cre line further enabled visualization of recombinase activities in the appropriate brain regions. Moreover, the first-time Cre-ERT2 line drives Cre-mediated recombination in a spatiotemporally controlled manner on tamoxifen (TMX) administration. These tools thus provide an excellent resource for future functional studies in Oxt-responsive neurons and should prove of broad interest in the field.