Dysregulation of stress granule dynamics by DCTN1 deficiency exacerbates TDP-43 pathology in Drosophila models of ALS/FTD.

The abnormal aggregation of TDP-43 into cytoplasmic inclusions in affected neurons is a major pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although TDP-43 is aberrantly accumulated in the neurons of most patients with sporadic ALS/FTD and other TDP-43 proteinopathies, how TDP-43 forms cytoplasmic aggregates remains unknown. In this study, we show that a deficiency in DCTN1, a subunit of the microtubule-associated motor protein complex dynactin, perturbs the dynamics of stress granules and drives the formation of TDP-43 cytoplasmic aggregation in cultured cells, leading to the exacerbation of TDP-43 pathology and neurodegeneration in vivo. We demonstrated using a Drosophila model of ALS/FTD that genetic knockdown of DCTN1 accelerates the formation of ubiquitin-positive cytoplasmic inclusions of TDP-43. Knockdown of components of other microtubule-associated motor protein complexes, including dynein and kinesin, also increased the formation of TDP-43 inclusions, indicating that intracellular transport along microtubules plays a key role in TDP-43 pathology. Notably, DCTN1 knockdown delayed the disassembly of stress granules in stressed cells, leading to an increase in the formation of pathological cytoplasmic inclusions of TDP-43. Our results indicate that a deficiency in DCTN1, as well as disruption of intracellular transport along microtubules, is a modifier that drives the formation of TDP-43 pathology through the dysregulation of stress granule dynamics.

Expression of RNautophagy/DNautophagy-related genes is regulated under control of an innate immune receptor.

Double-stranded RNA (dsRNA) is a molecular pattern uniquely produced in cells infected with various viruses as a product or byproduct of replication. Cells detect such molecules, which indicate non-self invasion, and induce diverse immune responses to eliminate them. The degradation of virus-derived molecules can also play a role in the removal of pathogens and suppression of their replication. RNautophagy and DNautophagy are cellular degradative pathways in which RNA and DNA are directly imported into a hydrolytic organelle, the lysosome. Two lysosomal membrane proteins, SIDT2 and LAMP2C, mediate nucleic acid uptake via this pathway. Here, we showed that the expression of both SIDT2 and LAMP2C is selectively upregulated during the intracellular detection of poly(I:C), a synthetic analog of dsRNA that mimics viral infection. The upregulation of these two gene products upon poly(I:C) introduction was transient and synchronized. We also observed that the induction of SIDT2 and LAMP2C expression by poly(I:C) was dependent on MDA5, a cytoplasmic innate immune receptor that directly recognizes poly(I:C) and induces various antiviral responses. Finally, we showed that lysosomes can target viral RNA for degradation via RNautophagy and may suppress viral replication. Our results revealed a novel degradative pathway in cells as a downstream component of the innate immune response and provided evidence suggesting that the degradation of viral nucleic acids via RNautophagy/DNautophagy contributes to the suppression of viral replication.

Reliability and head-loading effects in imaging diagnosis of vertical subluxation in patients with rheumatoid arthritis.

OBJECTIVES: The purpose of this study was to examine the reproducibility of vertical subluxation (VS) parameters using X-ray, computed tomography (CT), and tomosynthesis (TS) while comparing the head-loading effects. METHODS: The VS parameters of 26 patients (retrospective review) were evaluated. Using the intra-class correlation coefficient, we statistically examined the intra-rater and inter-rater reliabilities of the parameters. Head-loaded and -unloaded imagings were compared using a Wilcoxon signed-rank test. RESULTS: The intra-rater reliability of TS and CT showed intra-class correlation coefficients of ≥0.8 (X-ray range: 0.6-0.8), with similar results for the inter-rater reliabilities. Furthermore, in head-loading imaging, the TS had significantly higher VS scores than that of CT (P < .05). CONCLUSIONS: In comparison with the X-ray, TS and CT were more accurate and reproducible. In terms of head loading, the VS values for TS were worse than those for CT, indicating that TS was more effective than CT in diagnosing VS.

Surgical outcomes of intraoperative O-arm versus C-arm fluoroscopy in occipitocervical fixation: a retrospective analysis.

PURPOSE: This study aims to compare the effect of using O-arm and C-arm fluoroscopy on the surgical outcomes of occipitocervical fixation. METHODS: The study included patients who underwent occipitocervical fixation using O-arm or C-arm between 2005 and 2021. Of 56 patients, 34 underwent O-arm-assisted surgery (O-group) and 22 underwent C-arm-assisted surgery (C-group). We assessed surgical outcomes, including operative time, intraoperative blood loss, perioperative complications, and bone union. RESULTS: Almost half of the patients had rheumatoid arthritis-related disorders in both groups. Sixteen cases (47.1%) in the O-group and 12 cases (54.5%) in the C-group were fixed from occipito (Oc) to C3, 12 cases (38.2%) in the O-group and 7 cases (31.8%) in the C-group from Oc to C4-7, 5 cases (14.7%) in the O-group, and 3 cases (13.6%) in the C-group from Oc to T2 (p = 0.929). There was no significant difference in operative time (p = 0.239) and intraoperative blood loss (p = 0.595) between the two groups. Dysphagia was the most common complication in both groups (O-group vs. C-group, 11.7% vs. 9.1%). Regarding implant-related complications, occipital plate dislodgement was observed in four cases (18.2%) in the C-group (p = 0.02). The bone union rate was 96.3% in the O-group and 93.3% in the C-group (P = 1). CONCLUSIONS: O-arm use is associated with a reduced rate of occipital plate dislodgment and has a similar complication incidence compared with C-arm-assisted surgery and does not prolong operative time despite the time needed for setting and scanning. Accordingly, an O-arm is safe and useful for occipitocervical fixation surgery.

FUS regulates RAN translation through modulating the G-quadruplex structure of GGGGCC repeat RNA in C9orf72-linked ALS/FTD.

Abnormal expansions of GGGGCC repeat sequence in the noncoding region of the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). The expanded repeat sequence is translated into dipeptide repeat proteins (DPRs) by noncanonical repeat-associated non-AUG (RAN) translation. Since DPRs play central roles in the pathogenesis of C9-ALS/FTD, we here investigate the regulatory mechanisms of RAN translation, focusing on the effects of RNA-binding proteins (RBPs) targeting GGGGCC repeat RNAs. Using C9-ALS/FTD model flies, we demonstrated that the ALS/FTD-linked RBP FUS suppresses RAN translation and neurodegeneration in an RNA-binding activity-dependent manner. Moreover, we found that FUS directly binds to and modulates the G-quadruplex structure of GGGGCC repeat RNA as an RNA chaperone, resulting in the suppression of RAN translation in vitro. These results reveal a previously unrecognized regulatory mechanism of RAN translation by G-quadruplex-targeting RBPs, providing therapeutic insights for C9-ALS/FTD and other repeat expansion diseases.

Nonresponder Considerations for Romosozumab Treatment.

Romosozumab can increase bone mineral density (BMD) in patients with osteoporosis, but some patients do not respond to it. This study aimed to identify risk factors for being a nonresponder to romosozumab treatment. This retrospective observational study included 92 patients. Romosozumab (210 mg) was subcutaneously administered to the participants every 4 weeks over 12 months. We excluded patients who previously underwent treatment for osteoporosis to assess the impact of romosozumab alone. We evaluated the proportion of patients who did not respond to romosozumab treatment to the lumbar spine and hip with increased BMD. Nonresponders were defined as those with a bone density change of < 3% after 12 months of treatment. We compared demographics and biochemical markers between responders and nonresponders. We found that 11.5% of patients were nonresponders at the lumbar spine, and 56.8% were nonresponders at the hip. A risk factor for nonresponse at the spine was low type I procollagen N-terminal propeptide (P1NP) values at 1 month. The cutoff value for P1NP at month 1 was 50 ng/ml. We found that 11.5% and 56.8% of patients experienced no significant improvement in the lumbar spine and hip BMD, respectively. Clinicians should use nonresponse risk factors to inform decisions about romosozumab treatment for patients with osteoporosis.

Impacts of the novel coronavirus SARS-CoV-2 on wildlife behaviour via human activities.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the pandemic of the coronavirus disease 2019 (COVID-19), resulting in a global lockdown in 2020. This stagnation in human activities ('anthropause') has been reported to affect the behaviour of wildlife in various ways. The sika deer Cervus nippon in Nara Park, central Japan, has had a unique relationship with humans, especially tourists, in which the deer bow to receive food and sometimes attack if they do not receive it. We investigated how a decrease and subsequent increase in the number of tourists visiting Nara Park affects the number of deer observed in the park and their behaviour (bows and attacks against humans). Compared with the pre-pandemic years, the number of deer in the study site decreased from an average of 167 deer in 2019 to 65 (39%) in 2020 during the pandemic period. Likewise, the number of deer bows decreased from 10.2 per deer in 2016-2017 to 6.4 (62%) in 2020-2021, whereas the proportion of deer showing aggressive behaviour did not change significantly. Moreover, the monthly numbers of deer and their bows both corresponded with the fluctuation in the number of tourists during the pandemic period of 2020 and 2021, whereas the number of attacks did not. Thus, the anthropause caused by the coronavirus altered the habitat use and behaviour of deer that have continuous interactions with humans.

Synthesis and biological evaluation of coumarin derivatives as selective CYP2A6 inhibitors.

Cytochrome P450 2A6 (CYP2A6) inhibitors are expected to be suitable as smoking cessation aids and for cancer prevention. Because the typical coumarin-based CYP2A6 inhibitor methoxsalen also inhibits CYP3A4, unintended drug-drug interactions are still a concern. Therefore, the development of selective CYP2A6 inhibitors is desirable. In this study, we synthesized coumarin-based molecules, determined the IC50 values for CYP2A6 inhibition, verified the possibility of mechanism-based inhibition, and compared the selectivity for CYP2A6 versus CYP3A4. The results demonstrated that we developed CYP2A6 inhibitors that were more potent and selective than methoxsalen.

Nucleic acid uptake occurs independent of lysosomal acidification but dependent on ATP consumption during RNautophagy/DNautophagy.

RNautophagy/DNautophagy (RDA) is an autophagic process that refers to the direct uptake of nucleic acids by lysosomes for degradation. Autophagy relies on lysosomes and lysosomal acidification is crucial for the degradation of intracellular components. However, whether lysosomal acidification interferes with nucleic acid uptake during RDA is unclear. In this study, we focused on vacuolar H+-ATPase (V-ATPase), the major proton pump responsible for maintaining an acidic pH in lysosomes. Our results show that lysosomes take up nucleic acids independently of the intralysosomal acidic pH during RDA. Isolated lysosomes treated with bafilomycin A1, a potent V-ATPase inhibitor, did not degrade, but took up RNA at similar levels as the control lysosomes. Similarly, the knockdown of Atp6v1a, the gene that encodes V-ATPase catalytic subunit A, did not affect the RNA uptake ability of isolated lysosomes. In addition, we demonstrated that nucleic acid uptake by isolated lysosomes necessitates ATP consumption, although V-ATPase is not required for the uptake process. These results broaden our understanding of the mechanisms underlying nucleic acid degradation via autophagy.

In vivo genotoxicity of high-intensity intermediate frequency magnetic fields in somatic cells and germ cells.

Intermediate frequency magnetic fields (IF-MFs) at ~85 kHz are one of the components of wireless power transfer (WPT) systems. However, the available data needed for the assessment of the safety of organisms from IF-MF exposure are scarce. Thus, there is an imminent need to accumulate evidence-based assessment data. In particular, if humans are exposed to IF-MF due to an accident or trouble, they are at increased risk of being exposed to high-intensity IF-MF within a short period. The already existing exposure system was improved to a system that could intermittently expose animals at 3 s intervals. This system allows the exposure of a mouse to high-intensity IF-MF (frequency: 82.3 kHz; induced electric field: 87 V/m, which was 3.8 times the basic restriction level for occupational exposure in the ICNIRP guideline), while regulating the heat generated by the coil. In vivo genotoxicity after IF-MF exposure was assessed using micronucleus (MN) test, Pig-a assay, and gpt assay. The results of MN test and Pig-a assay in hematopoietic cells revealed that neither the reticulocytes nor the mature erythrocytes exhibited significant increases in the IF-MF-exposed group compared with that in the sham-exposed group. In germ cells, MN test and gpt assay outcomes showed that IF-MF exposure did not cause any genetic or chromosomal abnormality. Based on these data, there was no genotoxic effect of our set IF-MF exposure on somatic and germ cells. These findings can contribute to the widespread use of WPT systems as effective data of IF-MF safety assessment.

Sequential hydrolysis of FAD by ecto-5' nucleotidase CD73 and alkaline phosphatase is required for uptake of vitamin B2 into cells.

Extracellular hydrolysis of flavin-adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to riboflavin is thought to be important for cellular uptake of vitamin B2 because FAD and FMN are hydrophilic and do not pass the plasma membrane. However, it is not clear whether FAD and FMN are hydrolyzed by cell surface enzymes for vitamin B2 uptake. Here, we show that in human cells, FAD, a major form of vitamin B2 in plasma, is hydrolyzed by CD73 (also called ecto-5' nucleotidase) to FMN. Then, FMN is hydrolyzed by alkaline phosphatase to riboflavin, which is efficiently imported into cells. We determined that this two-step hydrolysis process is impaired on the surface of glycosylphosphatidylinositol (GPI)-deficient cells due to the lack of these GPI-anchored enzymes. During culture of GPI-deficient cells with FAD or FMN, we found that hydrolysis of these forms of vitamin B2 was impaired, and intracellular levels of vitamin B2 were significantly decreased compared with those in GPI-restored cells, leading to decreased formation of vitamin B2-dependent pyridoxal 5'-phosphate and mitochondrial dysfunction. Collectively, these results suggest that inefficient uptake of vitamin B2 might account for mitochondrial dysfunction seen in some cases of inherited GPI deficiency.

Postoperative Complications and Survival Rate in Hemodialysis-Dependent Patients Undergoing Cervical Spine Surgery.

Introduction: Spine surgery is challenging in hemodialysis (HD)-dependent patients owing to their poor general condition. However, postoperative complications and the mortality and survival rates have not been specifically evaluated in a wide series. This study aimed to elucidate postoperative complications and the survival rate in cervical spine surgery in HD patients. Methods: This study included 109 HD patients (70 men, 39 women) who had undergone cervical spine surgery between July 1996 and May 2018. Based on radiological diagnosis, we divided them into the destructive spondyloarthropathy (DSA) and non-DSA groups. We investigated the causes of hemodialysis, postoperative complications, postoperative inpatient mortality rate, and survival rate. Results: The DSA and non-DSA groups included 100 surgeries in 89 patients and 21 surgeries in 20 patients, respectively. The mean age at surgery was 62.9 years for the DSA and 55.9 years for the non-DSA group (P=0.97). The DSA group had a shorter hemodialysis period at surgery compared with the non-DSA group (21.7 vs. 26.5 years, P<0.05). The two most common causes of HD in both groups were chronic glomerulonephritis (DSA, 45%; non-DSA, 57.1%) and diabetes (DSA, 11%; non-DSA, 14.5%). Postoperative complications were observed in 23% (23/100) and 19% (4/21) of surgeries in the DSA and non-DSA groups, respectively (P=0.782). The total in-hospital mortality rate was 2.5% (3/121). The 1-, 3-, 5-, and 10-year postoperative survival rates of all patients were 89.6%, 75.5%, 67.1%, and 44.7%, respectively. The survival rates did not depend on the group (DSA vs. non-DSA), pre- and postoperative Japanese Orthopedic Association score for cervical myelopathy, hemodialysis period, sex, and age (P>0.05). However, significantly low survival rates were observed in HD caused by diabetes compared with that by chronic glomerulonephritis (P<0.001) and other causes (P<0.001). Conclusions: Cervical spine surgery in HD patients is associated with postoperative complications. The postoperative survival rate was found to be low if the cause of hemodialysis was diabetes.

Prolonged contextual fear memory in AMPA receptor palmitoylation-deficient mice.

Long-lasting fear-related disorders depend on the excessive retention of traumatic fear memory. We previously showed that the palmitoylation-dependent removal of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors prevents hyperexcitation-based epileptic seizures and that AMPA receptor palmitoylation maintains neural network stability. In this study, AMPA receptor subunit GluA1 C-terminal palmitoylation-deficient (GluA1C811S) mice were subjected to comprehensive behavioral battery tests to further examine whether the mutation causes other neuropsychiatric disease-like symptoms. The behavioral analyses revealed that palmitoylation-deficiency in GluA1 is responsible for characteristic prolonged contextual fear memory formation, whereas GluA1C811S mice showed no impairment of anxiety-like behaviors at the basal state. In addition, fear generalization gradually increased in these mutant mice without affecting their cued fear. Furthermore, fear extinction training by repeated exposure of mice to conditioned stimuli had little effect on GluA1C811S mice, which is in line with augmentation of synaptic transmission in pyramidal neurons in the basolateral amygdala. In contrast, locomotion, sociability, depression-related behaviors, and spatial learning and memory were unaffected by the GluA1 non-palmitoylation mutation. These results indicate that impairment of AMPA receptor palmitoylation specifically causes posttraumatic stress disorder (PTSD)-like symptoms.

O-Arm Assisted Cervicothoracic Spine Pedicle Screw Placement Accuracy Is Higher Than C-Arm Fluoroscopy.

OBJECTIVE: We compared the accuracy of C-arm fluoroscopy versus O-arm-assisted pedicle screw (PS) placement in the cervicothoracic spinal junction (CTSJ). METHODS: Patients who underwent PS placement in the CTSJ (C7-T4) at our hospital were included in this study. Of 37 patients who underwent PS placement in the CTSJ, 20 underwent intraoperative C-arm fluoroscopy-assisted surgery (C Group) and 17 underwent intraoperative O-arm-assisted surgery (O Group). In total, 159 PSs were placed-73 in the C Group and 86 in the O Group. The accuracy of PS placement was compared between the C Group and O Group using the classification proposed by Gertzbein and Robbins to analyze pedicle violation. RESULTS: PS accuracy was higher in the O Group than C Group; PS placement evaluated as grade A, representing no perforation, was 95.3% (82/86) for the O Group, whereas it was 78.1% (57/73) for the C Group. There was a clear statistically significant difference in accuracy of PS placement between the groups (P = 0.0013). CONCLUSIONS: O-arm-assisted surgery improved the accuracy of PS placement in the CTSJ.

Functional and molecular characterization of a non-human primate model of autism spectrum disorder shows similarity with the human disease.

Autism spectrum disorder (ASD) is a multifactorial disorder with characteristic synaptic and gene expression changes. Early intervention during childhood is thought to benefit prognosis. Here, we examined the changes in cortical synaptogenesis, synaptic function, and gene expression from birth to the juvenile stage in a marmoset model of ASD induced by valproic acid (VPA) treatment. Early postnatally, synaptogenesis was reduced in this model, while juvenile-age VPA-treated marmosets showed increased synaptogenesis, similar to observations in human tissue. During infancy, synaptic plasticity transiently increased and was associated with altered vocalization. Synaptogenesis-related genes were downregulated early postnatally. At three months of age, the differentially expressed genes were associated with circuit remodeling, similar to the expression changes observed in humans. In summary, we provide a functional and molecular characterization of a non-human primate model of ASD, highlighting its similarity to features observed in human ASD.

Exercise Therapy Using the Lumbar-Type Hybrid Assistive Limb Ameliorates Locomotive Function after Lumbar Fusion Surgery in an Elderly Patient.

The number of elderly people who undergo lumbar fusion surgery (LFS) has been increasing. Postoperative rehabilitation for them can be problematic due to lumbar stiffness. This is the first case report describing exercise therapy using the lumbar-type hybrid assistive limb (HAL) after multiple LFS in an elderly patient. An 83-year-old man underwent LFS at L4-S1. Additional fusion surgery at L2-3 for adjacent segment disease was performed 2 years after the primary surgery. Although the patient's leg pain declined, he had severe locomotive dysfunction at 3 months after his final surgery. He started exercise therapy using the lumbar-type HAL including sit-to-stand training and squat training 4 months after his final surgery. He performed 3 sets of 20 sit-to-stand and 20 squat repetitions with adequate rests in between sets. The HAL training was safely conducted every day for 12 weeks without adverse event. Timed up and go test (TUG), 1-minute sit to stand test (1MSTS), one-leg stand test (OLST), and Berg balance scale (BBS) were assessed as locomotive function measurement. Before HAL therapy, TUG, 1MSTS, OLST, and BBS were 18.1 sec, 20 times, less than 1 sec, and 47, respectively. He could not walk without assistance. After the exercise therapy with the lumbar-type HAL, his locomotive function dramatically improved. TUG, 1MSTS, OLST, and BBS were 12.2 sec, 25 times, 3.9 sec, and 52, respectively. Moreover, the patient could walk 60 meters continuously without assistance. The unique characteristics of the lumbar-type HAL to prevent the lumbar overload and assist the voluntary hip joint motion during exercise therapy may be effective for this patient with lumbar stiffness after LFS. Sit-to-stand training and squat training using the lumbar-type HAL are promising options to improve locomotive function in elderly patients after LFS.

No evidence for genotoxicity in mice due to exposure to intermediate-frequency magnetic fields used for wireless power-transfer systems.

Time varying magnetic fields (MFs) are used for the wireless power-transfer (WPT) technology. Especially, 85 kHz band MFs, which are included in the intermediate frequency (IF) band (300 Hz - 10 MHz), are commonly used WPT system for charging electric vehicles. Those applications of WPT technology have elicited public concern about health effects of IF-MF. However, existing data from health risk assessments are insufficient and additional data are needed. We assessed the genotoxic effects of IF-MF exposure on erythroid differentiation in mice. A high-intensity IF-MF mouse exposure system was constructed to induce an average whole-body electric field of 54.1 V/m. Blood samples were obtained from male mice before and after a 2-week IF-MF exposure (1 h/day, total: 10 h); X-irradiated mice were used as positive controls. We analyzed the blood samples with the micronucleus (MN) test and the Pig-a mutation assay. No significant differences were seen between IF-MF-exposed and sham-exposed mice in the frequencies of either MN or Pig-a mutations in mature erythrocytes and reticulocytes. IF-MF exposure did not induce genotoxicity in vivo under the study conditions (2.36× the basic restriction for occupational exposure, 22.9 V/m, in the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines). The absence of significant biological effects due to IF-MF exposure supports the practical application of this technology.

Early life stress from allergic dermatitis causes depressive-like behaviors in adolescent male mice through neuroinflammatory priming.

Allergic dermatitis (AD), associated with pruritus and itchiness, is one of the major stressful conditions early in life. AD also influences the incidence of neuropsychiatric disorders and developmental disorders through neuro-immune interactions. To the best of our knowledge, there is no report that assesses the effects of early childhood dermatitis on psychiatric disorders later in life using an animal model. Here, we developed an oxazolone (Ox)-induced AD model in the early life of male C57BL/6J mice whose ears were challenged by Ox repeatedly from postnatal days (PD) 2 to PD30. On PD30, the Ox-treated ears were remarkably thickened and showed epidermal hyperplasia coupled with increased expression of T helper 2 cytokines, interleukin (IL)-4, and IL-13 in the ear tissue. Additionally, serum immunoglobulin E levels and serum corticosterone levels were higher in the Ox-treated mice than those in the control mice. Although Ox-treated PD40 mice showed neither behavioral abnormalities nor increases in pro-inflammatory cytokine expression in the brain, this study revealed that they experienced downregulation of CD200R1 expression in the amygdala under basal conditions and that additional lipopolysaccharide (LPS) administration induced enhanced neuroinflammatory reaction as the priming effect was accompanied by an increase of Iba-1-positive microglia in the amygdala and hippocampus. Furthermore, the Ox-treated PD40 mice showed depressive-like behaviors 24 h after LPS administration, whereas the control mice did not. Interestingly, the expression of indoleamine 2,3-dioxygenase and kynurenine 3-monooxygenase, key rate-limiting enzymes of the kynurenine metabolism pathway, was upregulated in the hippocampus, prefrontal cortex, and amygdala of the Ox-treated mice 4 h after LPS administration. Based on these results, we suggest that early life stress from AD aggravates susceptibility to systemic inflammation in the adolescent brain, leading to depressive behaviors with abnormal kynurenine metabolism.