Spectra and characteristics of somatic mutations induced by ionizing radiation in hematopoietic stem cells.

Spectra and frequencies of spontaneous and X-ray-induced somatic mutations were revealed with mouse long-term hematopoietic stem cells (LT-HSCs) by whole-genome sequencing of clonal cell populations propagated in vitro from single isolated LT-HSCs. SNVs and small indels were the most common types of somatic mutations, and increased up to twofold to threefold by whole-body X-irradiation. Base substitution patterns in the SNVs suggested a role of reactive oxygen species in radiation mutagenesis, and signature analysis of single base substitutions (SBS) revealed a dose-dependent increase of SBS40. Most of spontaneous small deletions were shrinkage of tandem repeats, and X-irradiation specifically induced small deletions out of tandem repeats (non-repeat deletions). Presence of microhomology sequences in non-repeat deletions suggested involvement of microhomology mediated end-joining repair mechanisms as well as nonhomologous end-joining in radiation-induced DNA damages. We also identified multisite mutations and structural variants (SV), i.e., large indels, inversions, reciprocal translocations, and complex variants. The radiation-specificity of each mutation type was evaluated from the spontaneous mutation rate and the per-Gy mutation rate estimated by linear regression, and was highest with non-repeat deletions without microhomology, followed by those with microhomology, SV except retroelement insertions, and multisite mutations; these types were thus revealed as mutational signatures of ionizing radiation. Further analysis of somatic mutations in multiple LT-HSCs indicated that large fractions of postirradiation LT-HSCs originated from single LT-HSCs that survived the irradiation and then expanded in vivo to confer marked clonality to the entire hematopoietic system, with varying clonal expansion and dynamics depending on radiation dose and fractionation.

Massive expansion of multiple clones in the mouse hematopoietic system long after whole-body X-irradiation.

Clonal hematopoiesis (CH) is prevalent in the elderly and associates with hematologic malignancy and cardiovascular disease. Although the risk of developing these diseases increases with radiation doses in atomic-bomb survivors, the causal relationship between radiation exposure and CH is unclear. This study investigated whether radiation exposure induces CH in mice 12-18 months after 3-Gy whole-body irradiation. We found radiation-associated increases in peripheral blood myeloid cells and red blood cell distribution width (RDW). Deep sequencing of bone marrow and non-hematopoietic tissue cells revealed recurrent somatic mutations specifically in the hematopoietic system in 11 of 12 irradiated mice but none in 6 non-irradiated mice. The irradiated mice possessed mutations with variant allele frequencies (VAFs) of > 0.02 on an average of 5.8 per mouse; mutations with VAFs of > 0.1 and/or deletion were prevalent. Examining hematopoietic stem/progenitor cells in two irradiated mice revealed several mutations co-existing in the same clones and multiple independent clones that deliver 60-80% of bone marrow nuclear cells. Our results indicate development of massive CH due to radiation exposure. Moreover, we have characterized mutations in radiation-induced CH.

Kv11 (ether-à-go-go-related gene) voltage-dependent K+ channels promote resonance and oscillation of subthreshold membrane potentials.

KEY POINTS: Some ion channels are known to behave as inductors and make up the parallel resonant circuit in the plasma membrane of neurons, which enables neurons to respond to current inputs with a specific frequency (so-called 'resonant properties'). Here, we report that heterologous expression of mouse Kv11 voltage-dependent K+ channels generate resonance and oscillation at depolarized membrane potentials in HEK293 cells; expressions of individual Kv11 subtypes generate resonance and oscillation with different frequency properties. Kv11.3-expressing HEK293 cells exhibited transient conductance changes that opposed the current changes induced by voltage steps; this probably enables Kv11 channels to behave like an inductor. The resonance and oscillation of inferior olivary neurons were impaired at the resting membrane potential in Kv11.3 knockout mice. This study helps to elucidate basic ion channel properties that are crucial for the frequency responses of neurons. ABSTRACT: The plasma membranes of some neurons preferentially respond to current inputs with a specific frequency, and output as large voltage changes. This property is called resonance, and is thought to be mediated by ion channels that show inductor-like behaviour. However, details of the candidate ion channels remain unclear. In this study, we mainly focused on the functional roles of Kv11 potassium (K+ ) channels, encoded by ether-á-go-go-related genes, in resonance in mouse inferior olivary (IO) neurons. We transfected HEK293 cells with long or short splice variants of Kv11.1 (Merg1a and Merg1b) or Kv11.3, and examined membrane properties using whole-cell recording. Transfection with Kv11 channels reproduced resonance at membrane potentials depolarized from the resting state. Frequency ranges of Kv11.3-, Kv11.1(Merg1b)- and Kv11.1(Merg1a)-expressing cells were 2-6 Hz, 2-4 Hz, and 0.6-0.8 Hz, respectively. Responses of Kv11.3 currents to step voltage changes were essentially similar to those of inductor currents in the resistor-inductor-capacitor circuit. Furthermore, Kv11 transfections generated membrane potential oscillations. We also confirmed the contribution of HCN1 channels as a major mediator of resonance at more hyperpolarized potentials by transfection into HEK293 cells. The Kv11 current kinetics and properties of Kv11-dependent resonance suggested that Kv11.3 mediated resonance in IO neurons. This finding was confirmed by the impairment of resonance and oscillation at -30 to -60 mV in Kcnh7 (Kv11.3) knockout mice. These results suggest that Kv11 channels have important roles in inducing frequency-dependent responses in a subtype-dependent manner from resting to depolarized membrane potentials.

The first Japanese case of primary familial brain calcification caused by an MYORG variant.

Primary familial brain calcification (PFBC) is a hereditary neurological disorder characterized by idiopathic calcification of the bilateral basal ganglia and other areas of the brain. MYORG has been identified as the first causative gene of autosomal recessive PFBC in Chinese families. There have been several reports of PFBC associated with MYORG (MYORG-PFBC) in individuals of Middle Eastern, European, and Latin American ancestry but to date, there have been no reported Japanese cases. We report the first Japanese case of MYORG-PFBC. The patient was a 43-year-old Japanese woman who experienced mild headaches and cerebellar ataxia including dysarthria. Computed tomography showed calcification in the cerebral white matter, basal ganglia, cerebellum, and brainstem. Using exome sequencing, we identified a homozygous variant in the MYORG gene (NM_020702.4: c.794C>T,p.Thr265Met). Our patient presented dysarthria and extensive calcification affecting the pons, which are specific features of MYORG-PFBC. We report clinical symptoms and imaging findings of a case with p.Thr265Met variant.

Optineurin regulates osteoblastogenesis through STAT1.

A sophisticated and delicate balance between bone resorption by osteoclasts and bone formation by osteoblasts regulates bone metabolism. Optineurin (OPTN) is a gene involved in primary open-angle glaucoma and amyotrophic lateral sclerosis. Although its function has been widely studied in ophthalmology and neurology, recent reports have shown its possible involvement in bone metabolism through negative regulation of osteoclast differentiation. However, little is known about the role of OPTN in osteoblast function. Here, we demonstrated that OPTN controls not only osteoclast but also osteoblast differentiation. Different parameters involved in osteoblastogenesis and osteoclastogenesis were assessed in Optn-/- mice. The results showed that osteoblasts from Optn-/- mice had impaired alkaline phosphatase activity, defective mineralized nodules, and inability to support osteoclast differentiation. Moreover, OPTN could bind to signal transducer and activator of transcription 1 (STAT1) and regulate runt-related transcription factor 2 (RUNX2) nuclear localization by modulating STAT1 levels in osteoblasts. These data suggest that OPTN is involved in bone metabolism not only by regulating osteoclast function but also by regulating osteoblast function by mediating RUNX2 nuclear translocation via STAT1.

Retromolar canals as observed on cone-beam computed tomography: their incidence, course, and characteristics.

OBJECTIVE: The study aims at assessing the incidence, course, and characteristics of retromolar canals. STUDY DESIGN: The cone-beam computed tomography images of 171 subjects were evaluated for the presence, course, and pattern of occurrence of retromolar canals. RESULTS: Three types of retromolar canals namely A, B, & C were detected in 129 subjects. Type A branched off the mandibular canal distal to third molar to open into retomolar fossa, type B coursed between retromolar fossa and radicular portion of third molar, type C coursed from mandibular foramen anteroinferiorly to exit into retromolar fossa. The type B retromolar canal presented features distinguishing it from other types. CONCLUSIONS: Considerable number of individuals presented with retromolar canals emphasizing their significance in surgeries involving the retromolar area. The type B pattern, to the best of our knowledge, hasn't been reported by most researchers and hence, could be considered as an additional type of retromolar canal.

Skeletal maturity assessment with the use of cone-beam computerized tomography.

OBJECTIVE: The aim of the study was to compare cervical vertebrae maturity assessed with the use of cone-beam computerized tomography (CBCT) with the hand-wrist maturation method and cervical vertebrae maturation assessed with the use of lateral cephalography for the assessment of skeletal maturity. STUDY DESIGN: Assessment of skeletal maturation was done using skeletal maturity indicators (SMI) from hand-wrist radiography, cervical vertebrae maturity index (CVMI) from CBCT and lateral cephalography (cephalo-CVMI). The Spearman correlation coefficient was used for statistical analysis. RESULTS: We observed a significant relationship between CBCT-CVMI and cephalo-CVMI as well as between CBCT-CVMI and SMI stages. The Spearman correlation coefficient value between CBCT-CVMI and cephalo-CVMI was 0.975 (P < .0001) and between CBCT-CVMI and SMI was 0.961(P < .0001). CONCLUSIONS: Cervical vertebrae maturity assessment with CBCT provided a reliable assessment of pubertal growth spurt, and therefore CBCT can be used to assess skeletal maturity.

Maturation of BRI2 generates a specific inhibitor that reduces APP processing at the plasma membrane and in endocytic vesicles.

Processing of the amyloid-ß (Aß) precursor protein (APP) has been extensively studied since it leads to production of Aß peptides. Toxic forms of Aß aggregates are considered the cause of Alzheimer's disease (AD). On the other end, BRI2 is implicated in APP processing and Aß production. We have investigated the precise mechanism by which BRI2 modulates APP cleavages and have found that BRI2 forms a mature BRI2 polypeptide that is transported to the plasma membrane and endosomes where it interacts with mature APP. Notably, immature forms of APP and BRI2 fail to interact. Mature BRI2 inhibits APP processing by α-, ß- and γ-secretases on the plasma membrane and in endocytic compartments. Thus, BRI2 is a specific inhibitor that reduces secretases' access to APP in the intracellular compartments where APP is normally processed.

Assessment of the blood supply to the lingual surface of the mandible for reduction of bleeding during implant surgery.

OBJECTIVES: The aim of this study was to assess the frequency of the foramina and their canals on the lingual surface of the mandible using computed tomography (CT), which was carried out for dental implant planning. MATERIAL AND METHODS: First, the visibility of the lingual canals of the CT image was verified by dissecting five cadavers. CT images of 200 patients, who had decided on implant treatment, were used in this study. The visibility of the foramina and their canals on the lingual surface of the mandible were assessed. RESULTS: The foramina were divided into two groups by the positions of the mandible, the medial lingual foramen and the lateral lingual foramen. At least one foramen was found in all patients. In the medial group, a higher level of mental spine was seen in 190 patients, the same level of mental spine was observed in 99 patients and a lower level of mental spine was observed in 114 patients. The lateral lingual foramina were found in 160/200 patients and 88/200 patients presented bilaterally. CT can predict the position and the size of the foramina and their canals on the lingual surface of the mandible. All the patients had more than one foramen in the middle of the lingual surface of the mandible on the CT image. CONCLUSION: The frequency of the lingual foramina in the medial region was 100% and that in the lateral region was 80%. It would also be useful to emphasize the significant variation in the precise location of these lingual foramina, and that these can only be visualized presurgically with volumetric imaging modalities, such as CT or Cone beam 3D systems.

The familial dementia BRI2 gene binds the Alzheimer gene amyloid-beta precursor protein and inhibits amyloid-beta production.

Alzheimer disease (AD), the most common senile dementia, is characterized by amyloid plaques, vascular amyloid, neurofibrillary tangles, and progressive neurodegeneration. Amyloid is mainly composed by amyloid-beta (A(beta)) peptides, which are derive from processing of the beta-amyloid precursor protein (APP), better named amyloid-beta precursor protein (A(beta)PP), by secretases. The A(beta)PP intracellular domain (AID), which is released together with A(beta), has signaling function, since it modulates apoptosis and transcription. Despite its biological and pathological importance, the mechanisms regulating A(beta)PP processing are poorly understood. As cleavage of other gamma-secretase substrates is regulated by membrane bound proteins, we have postulated the existence of integral membrane proteins that bind A(beta)PP and regulate its processing. Here, we show that BRI2, a type II membrane protein, interacts with A(beta)PP. Interestingly, 17 amino acids corresponding to the NH2-terminal portion of A(beta) are necessary for this interaction. Moreover, BRI2 expression regulates A(beta)PP processing resulting in reduced A(beta) and AID levels. Altogether, these findings characterize the BRI2-A(beta)PP interaction as a regulatory mechanism of A(beta)PP processing that inhibits A(beta) production. Notably, BRI2 mutations cause familial British (FBD) and Danish dementias (FDD) that are clinically and pathologically similar to AD. Finding that BRI2 pathogenic mutations alter the regulatory function of BRI2 on A(beta)PP processing would define dysregulation of A(beta)PP cleavage as a pathogenic mechanism common to AD, FDD, and FBD.

Amyloid beta protein precursor (AbetaPP), but not AbetaPP-like protein 2, is bridged to the kinesin light chain by the scaffold protein JNK-interacting protein 1.

Proteolytic processing of amyloid beta protein precursor (AbetaPP) generates peptides that regulate normal cell signaling and are implicated in Alzheimer's disease pathogenesis. AbetaPP processing also occurs in nerve processes where AbetaPP is transported from the cell body by kinesin-I, a microtubule motor composed of two kinesin heavy chain and two kinesin light chain (Klc) subunits. AbetaPP transport is supposedly mediated by the direct AbetaPP-Klc1 interaction. Here we demonstrate that the AbetaPP-Klc1 interaction is not direct but is mediated by JNK-interacting protein 1 (JIP1). The phosphotyrosine binding domain of JIP1 binds the cytoplasmic tail of AbetaPP, whereas the JIP1 C-terminal region interacts with the tetratrico-peptide repeats of Klc1. We also show that JIP1 does not bridge the AbetaPP gene family member AbetaPP-like protein 2, APLP2, to Klc1. These results support a model where JIP1 mediates the interaction of AbetaPP to the motor protein kinesin-I and that this JIP1 function is unique for AbetaPP relative to its family member APLP2. Our data suggest that kinesin-I-dependent neuronal AbetaPP transport, which controls AbetaPP processing, may be regulated by JIP1.

Pathological analysis of muscle hypertrophy and degeneration in muscular dystrophy in gamma-sarcoglycan-deficient mice.

While calf muscle hypertrophy is a striking diagnostic finding in sarcoglycanopathy, as it is in Duchenne and Becker muscular dystrophies, its pathogenetic mechanism remains unknown. gamma-Sarcoglycan, one of the subunits of the sarcoglycan complex, is the protein responsible for gamma-sarcoglycanopathy. To elucidate the pathogenetic mechanisms of muscle hypertrophy and degeneration in muscular dystrophy, we utilized a mutant mouse as a model animal. In this study, we generated gamma-sarcoglycan-deficient (gsg-/-) mice by gene targeting. The gsg-/- mice described here, similar to the gsg-/- mice reported previously (J Cell Biol 142 (1998) 1279), demonstrated skeletal and cardiac muscle degeneration. The limb, shoulder, and pelvic muscles of the gsg-/- mice exhibited progressive muscle hypertrophy and weakness with age, and the findings were similar to those seen in other mouse models for limb-girdle and Duchenne muscular dystrophy. We found that the number of muscle fibers increased with age, and most of the fibers in the hypertrophic muscle were centrally nucleated regenerating fibers. Therefore, muscle hypertrophy of the gsg-/- mice may result from an increase of the number of muscle fibers and probable fiber branching and may not be due to the pseudohypertrophy caused by fibrous and fat tissue replacement, as has been long supposed in muscular dystrophy. The muscle pathology became more 'dystrophic' in mice over 1 year of age when there was a marked variation in fiber size with interstitial fibrosis.