[Pathophysiology of Respiratory Failure in Neuromuscular Diseases].

Based on a recent review by Krohn et al, the respiratory center and its regulatory mechanisms are described. Although the respiratory control centers in the medulla and pons ensure rhythmic respiration, maintaining and regulating respiration involves a complex network of peripheral chemoreceptors, vagal nerves, and central chemoreceptors. This review discusses the pathophysiology of respiratory disorders in neuromuscular diseases and evaluation and treatment methods based on the anatomy of the respiratory network.

Hematopoietic Prostaglandin D Synthase Is Increased in Mast Cells and Pericytes in Autopsy Myocardial Specimens from Patients with Duchenne Muscular Dystrophy.

The leading cause of death for patients with Duchenne muscular dystrophy (DMD), a progressive muscle disease, is heart failure. Prostaglandin (PG) D2, a physiologically active fatty acid, is synthesized from the precursor PGH2 by hematopoietic prostaglandin D synthase (HPGDS). Using a DMD animal model (mdx mice), we previously found that HPGDS expression is increased not only in injured muscle but also in the heart. Moreover, HPGDS inhibitors can slow the progression of muscle injury and cardiomyopathy. However, the location of HPGDS in the heart is still unknown. Thus, this study investigated HPGDS expression in autopsy myocardial samples from DMD patients. We confirmed the presence of fibrosis, a characteristic phenotype of DMD, in the autopsy myocardial sections. Additionally, HPGDS was expressed in mast cells, pericytes, and myeloid cells of the myocardial specimens but not in the myocardium. Compared with the non-DMD group, the DMD group showed increased HPGDS expression in mast cells and pericytes. Our findings confirm the possibility of using HPGDS inhibitor therapy to suppress PGD2 production to treat skeletal muscle disorders and cardiomyopathy. It thus provides significant insights for developing therapeutic drugs for DMD.

Analysis of splicing abnormalities in the white matter of myotonic dystrophy type 1 brain using RNA sequencing.

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by the genomic expansion of CTG repeats, in which RNA-binding proteins, such as muscleblind-like protein, are sequestered in the nucleus, and abnormal splicing is observed in various genes. Although abnormal splicing occurs in the brains of patients with DM1, its relation to central nervous system symptoms is unknown. Several imaging studies have indicated substantial white matter defects in patients with DM1. Here, we performed RNA sequencing and analysis of CTG repeat lengths in the frontal lobe of patients with DM1, separating the gray matter and white matter, to investigate splicing abnormalities in the DM1 brain, especially in the white matter. Several genes showed similar levels of splicing abnormalities in both gray and white matter, with an observable trend toward an increased number of repeats in the gray matter. These findings suggest that white matter defects in DM1 stem from aberrant RNA splicing in both gray and white matter. Notably, several of the genes displaying abnormal splicing are recognized as being dominantly expressed in astrocytes and oligodendrocytes, leading us to hypothesize that splicing defects in the white matter may be attributed to abnormal RNA splicing in glial cells.

An autopsy case of diffuse atypical argyrophilic grain disease (AGD) with presenile onset and three-year course of motor and cognitive impairment.

We report a case of argyrophilic grain disease (AGD) with unique clinical and pathological presentations. A 52-year-old man presented with spastic quadriparesis, bulbar palsy, and mild cognitive decline. His condition deteriorated rapidly and he died of pneumonia three years from onset. Pathologically, neuronal degeneration was involved severely in the amygdala, ambient gyrus, midbrain tegmentum, and reticular formation. The neurons of the temporal lobe, cingulate gyrus, brainstem, and spinal gray matter were also lost moderately. There was diffuse 4-repeat tau-pathology with argyrophilic grains. There were pretangles, globose-type neurofibrillary tangles, and coiled bodies in the cerebral cortices, basal ganglia, thalami, brainstem, and the spinal cord except for the cerebellar cortices. There was no pathologic mutation in MAPT.

Intracytoplasmic sperm injection induces transgenerational abnormalities in mice.

In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are 2 major assisted reproductive techniques (ARTs) used widely to treat infertility. Recently, spermatogonial transplantation emerged as a new ART to restore fertility to young patients with cancer after cancer therapy. To examine the influence of germ cell manipulation on behavior of offspring, we produced F1 offspring by a combination of two ARTs, spermatogonial transplantation and ICSI. When these animals were compared with F1 offspring produced by ICSI using fresh wild-type sperm, not only spermatogonial transplantation-ICSI mice but also ICSI-only control mice exhibited behavioral abnormalities, which persisted in the F2 generation. Furthermore, although these F1 offspring appeared normal, F2 offspring produced by IVF using F1 sperm and wild-type oocytes showed various types of congenital abnormalities, including anophthalmia, hydrocephalus, and missing limbs. Therefore, ARTs can induce morphological and functional defects in mice, some of which become evident only after germline transmission.

[Involvement of autonomic nervous system since middle age in elderly patient with giant axonal neuropathy caused by novel genetic mutation].

A 69-year-old man began to experience difficulty with walking at the age of 5 years and started use of a cane at around 13 years, then finally started using a wheelchair at 17 years old. A diagnosis of Charcot-Marie-Tooth disease was previously determined at another hospital, though neither peripheral nerve biopsy nor gene analysis was conducted. He visited our institution at the age of 54 years and irregular outpatient examinations were started, which indicated slowly progressive muscle weakness and sensory disturbance of the limbs, leading to a decline in activities of daily living. Gene analysis at 60 years old identified a novel homozygous missense mutation in the gigaxonin gene, c.1478A>C, p.E493A. Intellectual capacity was preserved and kinky hair was not present, though complications such as vocal cord paralysis, paralytic ileus, and dysarthria were noted starting at age 61. Based on these findings, the patient was diagnosed with a mild form of giant axonal neuropathy.

Incomplete activation of Alyref and Gabpb1 leads to preimplantation arrest in cloned mouse embryos.

Differentiated cell nuclei can be reprogrammed after nuclear transfer (NT) to oocytes and the produced NT embryos can give rise to cloned animals. However, development of NT embryos is often hampered by recurrent reprogramming failures, including the incomplete activation of developmental genes, yet specific genes responsible for the arrest of NT embryos are not well understood. Here, we searched for developmentally important genes among the reprogramming-resistant H3K9me3-repressed genes and identified Alyref and Gabpb1 by siRNA screening. Gene knockout of Alyref and Gabpb1 by the CRISPR/Cas9 system resulted in early developmental arrest in mice. Alyref was needed for the proper formation of inner cell mass by regulating Nanog, whereas Gabpb1 deficiency led to apoptosis. The supplement of Alyref and Gabpb1 mRNA supported efficient preimplantation development of cloned embryos. Alyref and Gabpb1 were silenced in NT embryos partially because of the repressed expression of Klf16 by H3K9me3. Thus, our study shows that the H3K9me3-repressed genes contain developmentally required genes, and the incomplete activation of such genes results in preimplantation arrest of cloned embryos.

Derivation of embryonic stem cells from wild-derived mouse strains by nuclear transfer using peripheral blood cells.

Wild-derived mouse strains have been extensively used in biomedical research because of the high level of inter-strain polymorphisms and phenotypic variations. However, they often show poor reproductive performance and are difficult to maintain by conventional in vitro fertilization and embryo transfer. In this study, we examined the technical feasibility of derivation of nuclear transfer embryonic stem cells (ntESCs) from wild-derived mouse strains for their safe genetic preservation. We used leukocytes collected from peripheral blood as nuclear donors without sacrificing them. We successfully established 24 ntESC lines from two wild-derived strains of CAST/Ei and CASP/1Nga (11 and 13 lines, respectively), both belonging to Mus musculus castaneus, a subspecies of laboratory mouse. Most (23/24) of these lines had normal karyotype, and all lines examined showed teratoma formation ability (4 lines) and pluripotent marker gene expression (8 lines). Two male lines examined (one from each strain) were proven to be competent to produce chimeric mice following injection into host embryos. By natural mating of these chimeric mice, the CAST/Ei male line was confirmed to have germline transmission ability. Our results demonstrate that inter-subspecific ntESCs derived from peripheral leukocytes could provide an alternative strategy for preserving invaluable genetic resources of wild-derived mouse strains.

SINE RNA of the imprinted miRNA clusters mediates constitutive type III interferon expression and antiviral protection in hemochorial placentas.

Hemochorial placentas have evolved defense mechanisms to prevent the vertical transmission of viruses to the immunologically underdeveloped fetus. Unlike somatic cells that require pathogen-associated molecular patterns to stimulate interferon production, placental trophoblasts constitutively produce type III interferons (IFNL) through an unknown mechanism. We demonstrate that transcripts of short interspersed nuclear elements (SINEs) embedded in miRNA clusters within the placenta trigger a viral mimicry response that induces IFNL and confers antiviral protection. Alu SINEs within primate-specific chromosome 19 (C19MC) and B1 SINEs within rodent-specific microRNA cluster on chromosome 2 (C2MC) produce dsRNAs that activate RIG-I-like receptors (RLRs) and downstream IFNL production. Homozygous C2MC knockout mouse trophoblast stem (mTS) cells and placentas lose intrinsic IFN expression and antiviral protection, whereas B1 RNA overexpression restores C2MCΔ/Δ mTS cell viral resistance. Our results uncover a convergently evolved mechanism whereby SINE RNAs drive antiviral resistance in hemochorial placentas, placing SINEs as integral players in innate immunity.

UHRF1 is essential for proper cytoplasmic architecture and function of mouse oocytes and derived embryos.

Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is a protein essential for the maintenance of DNA methylation in somatic cells. However, UHRF1 is predominantly localized in the cytoplasm of mouse oocytes and preimplantation embryos, where it may play a role unrelated to the nuclear function. We herein report that oocyte-specific Uhrf1 KO results in impaired chromosome segregation, abnormal cleavage division, and preimplantation lethality of derived embryos. Our nuclear transfer experiment showed that the phenotype is attributable to cytoplasmic rather than nuclear defects of the zygotes. A proteomic analysis of KO oocytes revealed the down-regulation of proteins associated with microtubules including tubulins, which occurred independently of transcriptomic changes. Intriguingly, cytoplasmic lattices were disorganized, and mitochondria, endoplasmic reticulum, and components of the subcortical maternal complex were mislocalized. Thus, maternal UHRF1 regulates the proper cytoplasmic architecture and function of oocytes and preimplantation embryos, likely through a mechanism unrelated to DNA methylation.

Mouse somatic cell nuclear transfer: What has changed and remained unchanged in 25 years.

Somatic cell nuclear transfer (SCNT) is the only reproductive technology used to produce individuals from somatic cells by transferring them to enucleated oocytes. Although more than 25 years have passed since the first mammalian SCNT was reported in sheep, problems such as low birth rates and morphological abnormalities have persisted and limited its practical applications. The mouse is the ideal laboratory animal to unveil these questions due to its established reproductive technologies and extensive knowledge base of its genome and various strains. We investigated the causes of incomplete reprogramming after nuclear transfer of donor somatic cells and found that the loss of imprint in some placenta-specific imprinted genes could induce non-random SCNT abnormalities. By ameliorating aberrantly expressed imprinted genes, we succeeded in increasing the low birth rate and improving morphological abnormalities observed in SCNT fetuses. Furthermore, we sought appropriate mouse strains and cell types as nuclear donors to increase their developmental efficiencies and expand their applications in various fields. Peripheral blood cells are useful as ethical and economical cell species because they can be collected easily, even though SCNT embryos derived from hematopoietic cells show poor developmental abilities after reconstruction. Additionally, it is possible to obtain mice that are reactive to specific antigens of interest by using lymphocytes. Although there are still many limitations to the practical use of SCNT, its utilization is steadily expanding.

Neuropathology of spinocerebellar ataxia type 8: Common features and unique tauopathy.

Spinocerebellar ataxia type 8 (SCA8) is a neurodegenerative condition that presents with several neurological symptoms, such as cerebellar ataxia, parkinsonism, and cognitive impairment. It is caused by a CTA/CTG repeat expansion on chromosome 13q21 (ataxin 8 opposite strand [ATXN8OS]). However, the pathological significance of this expansion remains unclear. Moreover, abnormal CTA/CTG repeat expansions in ATXN8OS have also been reported in other neurodegenerative diseases, including progressive supranuclear palsy. In this study, we analyzed all available autopsy cases in Japan to investigate common pathological features and profiles of tau pathology in each case. Severe neuronal loss in the substantia nigra and prominent loss of Purkinje cells, atrophy of the molecular layer, and proliferation of Bergmann glia in the cerebellum were common features. Regarding tauopathy, one case presented with progressive supranuclear palsy-like 4-repeat tauopathy in addition to mild Alzheimer-type 3- and 4-repeat tauopathy. Another case showed 3- and 4-repeat tauopathy accentuated in the brainstem. The other two cases lacked tauopathy after extensive immunohistochemical studies. The present study confirmed common pathological features of SCA8 as degeneration of the substantia nigra in addition to the cerebellum. Our study also confirmed unique tauopathy in two of four cases, indicating the necessity to further collect autopsy cases.

Suppression of endogenous retroviral enhancers in mouse embryos derived from somatic cell nuclear transfer.

Endogenous retroviruses (ERVs) in the mammalian genome play diverse roles in embryonic development. These developmentally related ERVs are generally repressed in somatic cells and therefore are likely repressed in embryos derived from somatic cell nuclear transfer (SCNT). In this study, we sought to identify ERVs that are repressed in SCNT-derived morulae, which might cause previously unexplained embryonic deaths shortly after implantation. Our transcriptome analysis revealed that, amongst ERV families, ERVK was specifically, and strongly downregulated in SCNT-derived embryos while other transposable elements including LINE and ERVL were unchanged. Among the subfamilies of ERVK, RLTR45-int was most repressed in SCNT-derived embryos despite its highest expression in control fertilized embryos. Interestingly, the nearby genes (within 5-50 kb, n = 18; 50-200 kb, n = 63) of the repressed RLTR45-int loci were also repressed in SCNT-derived embryos, with a significant correlation between them. Furthermore, lysine H3K27 acetylation was enriched around the RLTR45-int loci. These findings indicate that RLTR45-int elements function as enhancers of nearby genes. Indeed, deletion of two sequential RLTR45-int loci on chromosome 4 or 18 resulted in downregulations of nearby genes at the morula stage. We also found that RLTR45-int loci, especially SCNT-low, enhancer-like loci, were strongly enriched with H3K9me3, a repressive histone mark. Importantly, these H3K9me3-enriched regions were not activated by overexpression of H3K9me3 demethylase Kdm4d in SCNT-derived embryos, suggesting the presence of another epigenetic barrier repressing their expressions and enhancer activities in SCNT embryos. Thus, we identified ERVK subfamily RLTR45-int, putative enhancer elements, as a strong reprogramming barrier for SCNT (253 words).

TDP-43 Proteinopathy Presenting with Typical Symptoms of Parkinson's Disease.

Accumulation of abnormal transactivation response DNA-binding protein of 43 kDa (TDP-43) independently induces dopaminergic neuronal loss in the substantia nigra without Lewy pathology, and results in typical Parkinson's disease-like motor symptoms.

Birth of mice from meiotically arrested spermatocytes following biparental meiosis in halved oocytes.

Microinjection of spermatozoa or spermatids into oocytes is a major choice for infertility treatment. However, the use of premeiotic spermatocytes has never been considered because of its technical problems. Here, we show that the efficiency of spermatocyte injection in mice can be improved greatly by reducing the size of the recipient oocytes. Live imaging showed that the underlying mechanism involves reduced premature separation of the spermatocyte's meiotic chromosomes, which produced much greater (19% vs. 1%) birth rates in smaller oocytes. Application of this technique to spermatocyte arrest caused by STX2 deficiency, an azoospermia factor also found in humans, resulted in the production of live offspring. Thus, the microinjection of primary spermatocytes into oocytes may be a potential treatment for overcoming a form of nonobstructive azoospermia caused by meiotic failure.

Noncanonical imprinting sustains embryonic development and restrains placental overgrowth.

Genomic imprinting regulates parental origin-dependent monoallelic gene expression. It is mediated by either germline differential methylation of DNA (canonical imprinting) or oocyte-derived H3K27me3 (noncanonical imprinting) in mice. Depletion of Eed, an essential component of Polycomb repressive complex 2, results in genome-wide loss of H3K27me3 in oocytes, which causes loss of noncanonical imprinting (LOI) in embryos. Although Eed maternal KO (matKO) embryos show partial lethality after implantation, it is unknown whether LOI itself contributes to the developmental phenotypes of these embryos, which makes it unclear whether noncanonical imprinting is developmentally relevant. Here, by combinatorial matKO of Xist, a noncanonical imprinted gene whose LOI causes aberrant transient maternal X-chromosome inactivation (XCI) at preimplantation, we show that prevention of the transient maternal XCI greatly restores the development of Eed matKO embryos. Moreover, we found that the placentae of Eed matKO embryos are remarkably enlarged in a manner independent of Xist LOI. Heterozygous deletion screening of individual autosomal noncanonical imprinted genes suggests that LOI of the Sfmbt2 miRNA cluster chromosome 2 miRNA cluster (C2MC), solute carrier family 38 member 4 (Slc38a4), and Gm32885 contributes to the placental enlargement. Taken together, our study provides evidence that Xist imprinting sustains embryonic development and that autosomal noncanonical imprinting restrains placental overgrowth.

Highly rigid H3.1/H3.2-H3K9me3 domains set a barrier for cell fate reprogramming in trophoblast stem cells.

The placenta is a highly evolved, specialized organ in mammals. It differs from other organs in that it functions only for fetal maintenance during gestation. Therefore, there must be intrinsic mechanisms that guarantee its unique functions. To address this question, we comprehensively analyzed epigenomic features of mouse trophoblast stem cells (TSCs). Our genome-wide, high-throughput analyses revealed that the TSC genome contains large-scale (>1-Mb) rigid heterochromatin architectures with a high degree of histone H3.1/3.2-H3K9me3 accumulation, which we termed TSC-defined highly heterochromatinized domains (THDs). Importantly, depletion of THDs by knockdown of CAF1, an H3.1/3.2 chaperone, resulted in down-regulation of TSC markers, such as Cdx2 and Elf5, and up-regulation of the pluripotent marker Oct3/4, indicating that THDs maintain the trophoblastic nature of TSCs. Furthermore, our nuclear transfer technique revealed that THDs are highly resistant to genomic reprogramming. However, when H3K9me3 was removed, the TSC genome was fully reprogrammed, giving rise to the first TSC cloned offspring. Interestingly, THD-like domains are also present in mouse and human placental cells in vivo, but not in other cell types. Thus, THDs are genomic architectures uniquely developed in placental lineage cells, which serve to protect them from fate reprogramming to stably maintain placental function.

Clinical trajectory of a patient with filaminopathy who developed arrhythmogenic cardiomyopathy, myofibrillar myopathy, and multiorgan tumors.

We report a case of a patient presenting with arrhythmogenic cardiomyopathy, myofibrillar myopathy, and multiorgan tumors. A 41-year-old woman with a history of hypertrophic cardiomyopathy, diagnosed at 6 years of age, developed scoliosis after puberty. Following spinal surgery to address the scoliosis, she developed recurrent severe arrhythmia and heart failure. She developed hypoventilation at age 29 years. Proximal dominant weakness and mild elevation of serum creatine kinase indicated possible myopathy. Myofibrillar myopathy was diagnosed by muscle biopsy at age 30 year. Acute abdomen was repeatedly reported from age 33 years, eventually leading to a diagnosis of gastric polyp and erosive ulcer. A urinary bladder tumor was found at age 35 years, and breast cancer was diagnosed at age 40 years. Whole exome sequencing detected a heterozygous missense mutation in Filamin C. Recent evidences suggest that filamins are associated with tumors, and this case further highlights the clinical spectrum of filaminopathy.

Improved development of mouse somatic cell nuclear transfer embryos by chlamydocin analogues, class I and IIa histone deacetylase inhibitors†.

In mammalian cloning by somatic cell nuclear transfer (SCNT), the treatment of reconstructed embryos with histone deacetylase (HDAC) inhibitors improves efficiency. So far, most of those used for SCNT are hydroxamic acid derivatives-such as trichostatin A-characterized by their broad inhibitory spectrum. Here, we examined whether mouse SCNT efficiency could be improved using chlamydocin analogues, a family of newly designed agents that specifically inhibit class I and IIa HDACs. Development of SCNT-derived embryos in vitro and in vivo revealed that four out of five chlamydocin analogues tested could promote the development of cloned embryos. The highest pup rates (7.1-7.2%) were obtained with Ky-9, similar to those achieved with trichostatin A (7.2-7.3%). Thus, inhibition of class I and/or IIa HDACs in SCNT-derived embryos is enough for significant improvements in full-term development. In mouse SCNT, the exposure of reconstructed oocytes to HDAC inhibitors is limited to 8-10 h because longer inhibition with class I inhibitors causes a two-cell developmental block. Therefore, we used Ky-29, with higher selectivity for class IIa than class I HDACs for longer treatment of SCNT-derived embryos. As expected, 24-h treatment with Ky-29 up to the two-cell stage did not induce a developmental block, but the pup rate was not improved. This suggests that the one-cell stage is a critical period for improving SCNT cloning using HDAC inhibitors. Thus, chlamydocin analogues appear promising for understanding and improving the epigenetic status of mammalian SCNT-derived embryos through their specific inhibitory effects on HDACs.

[A case study of a patient with myotonic dystrophy type 1 whose gait disturbance was improved by gait training with hybrid assistive limbs].

A Japanese woman first noticed dysarthria at the age of 23. She visited a hospital at the age of 32 and was diagnosed as having myotonic dystrophy clinically. She was diagnosed genetically as having myotonic dystrophy type 1 at 47 years old with 160-270 CTG repeats on the DMPK gene. At the age of 48, she needed non-invasive positive pressure ventilation because of hypoxia at night. Her gait function also deteriorated. She could not stand up from the supine position by herself. However, when she stood, she could walk without a cane for a short distance. She was admitted to our hospital to receive rehabilitation against progressive gait disturbance at the age of 53. She received gait training with hybrid assistive limb® (HAL®). We evaluated some parameters such as walking distance of 2-minute walk test (2MWT), gait speed /cadence/stride length of 10-meter walk test (10MWT), before and just after the course. The first course was performed in September 2017 and the second was done in May 2018 so the interval was about six months. After two courses of HAL® gait training, the distance on the 2-minute walk test increased from 111 m to 154 m, the average speed and the cadence of 10MWT improved from 2.01 m/s to 2.78 m/s and from 2.21 steps/s to 3.05 steps/s respectively. The score of the muscular disability quality of life (QOL) rating scale was also improved. The factors including "defecation," "breathing," and "ADL" suggest that the patient's physical abilities improved and she could move easily. Other factors such as "hope", "activity" and "human relationship" suggest that patient's mood improved after the HAL® training.It was suggested that HAL® gait training could improve QOL as well as gait function in patients with progressive neuromuscular disorder.