Unexpected Mutations by CRISPR-Cas9 CTG Repeat Excision in Myotonic Dystrophy and Use of CRISPR Interference as an Alternative Approach.

Myotonic dystrophy type 1 is the most common type of adult-onset muscular dystrophy. This is an autosomal dominant disorder and caused by the expansion of the CTG repeat in the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Messenger RNAs containing these expanded repeats form aggregates as nuclear RNA foci. Then, RNA binding proteins, including muscleblind-like 1, are sequestered to the RNA foci, leading to systemic abnormal RNA splicing. In this study, we used CRISPR-Cas9 genome editing to excise this CTG repeat. Dual cleavage at the 5' and 3' regions of the repeat using a conventional Cas9 nuclease and a double nicking with Cas9 nickase successfully excised the CTG repeat. Subsequently, the formation of the RNA foci was markedly reduced in patient-derived fibroblasts. However, contrary to expectations, a considerable amount of off-target digestions and on-target genomic rearrangements were observed using high-throughput genome-wide translocation sequencing. Finally, the suppression of DMPK transcripts using CRISPR interference significantly decreased the intensity of RNA foci. Our results indicate that close attention should be paid to the unintended mutations when double-strand breaks are generated by CRISPR-Cas9 for therapeutic purposes. Alternative approaches independent of double-strand breaks, including CRISPR interference, may be considered.

Overexpression of LARGE suppresses muscle regeneration via down-regulation of insulin-like growth factor 1 and aggravates muscular dystrophy in mice.

Several types of muscular dystrophy are caused by defective linkage between α-dystroglycan (α-DG) and laminin. Among these, dystroglycanopathy, including Fukuyama-type congenital muscular dystrophy (FCMD), results from abnormal glycosylation of α-DG. Recent studies have shown that like-acetylglucosaminyltransferase (LARGE) strongly enhances the laminin-binding activity of α-DG. Therefore, restoration of the α-DG-laminin linkage by LARGE is considered one of the most promising possible therapies for muscular dystrophy. In this study, we generated transgenic mice that overexpress LARGE (LARGE Tg) and crossed them with dy(2J) mice and fukutin conditional knockout mice, a model for laminin α2-deficient congenital muscular dystrophy (MDC1A) and FCMD, respectively. Remarkably, in both the strains, the transgenic overexpression of LARGE resulted in an aggravation of muscular dystrophy. Using morphometric analyses, we found that the deterioration of muscle pathology was caused by suppression of muscle regeneration. Overexpression of LARGE in C2C12 cells further demonstrated defects in myotube formation. Interestingly, a decreased expression of insulin-like growth factor 1 (IGF-1) was identified in both LARGE Tg mice and LARGE-overexpressing C2C12 myotubes. Supplementing the C2C12 cells with IGF-1 restored the defective myotube formation. Taken together, our findings indicate that the overexpression of LARGE aggravates muscular dystrophy by suppressing the muscle regeneration and this adverse effect is mediated via reduced expression of IGF-1.

Innate immune response precedes Mycobacterium leprae-induced reprogramming of adult Schwann cells.

Recently, we showed a natural reprogramming process during infection with Mycobacterium leprae (ML), the causative organism of human leprosy. ML hijacks the notable plasticity of adult Schwann cells in the peripheral nervous system (PNS), bacteria's preferred nonimmune niche, to reprogram infected cells to progenitor/stem cell-like cells (pSLCs). Whereas ML appear to use this reprogramming process as a sophisticated bacterial strategy to spread infection to other tissues, understanding the mechanisms may shed new insights into the basic biology of cellular reprogramming and the development of new approaches for generating pSLC for therapeutic purposes as well as targeting bacterial infectious diseases at an early stage. Toward these goals, we extended our studies to identify other players that might be involved in this complex host cell reprogramming. Here we show that ML activates numerous immune-related genes mainly involved in innate immune responses and inflammation during early infection before downregulating Schwann cell lineage genes and reactivating developmental transcription factors. We validated these findings by demonstrating the ability of infected cells to secrete soluble immune factor proteins at early time points and their continued release during the course of reprogramming. By using time-lapse microscopy and a migration assay with reprogrammed Schwann cells (pSLCs) cultured with macrophages, we show that reprogrammed cells possess the ability to attract macrophages, providing evidence for a functional role of immune gene products during reprogramming. These findings suggest a potential role of innate immune response and the related signaling pathways in cellular reprogramming and the initiation of neuropathogenesis during ML infection.

Reprogramming adult Schwann cells to stem cell-like cells by leprosy bacilli promotes dissemination of infection.

Differentiated cells possess a remarkable genomic plasticity that can be manipulated to reverse or change developmental commitments. Here, we show that the leprosy bacterium hijacks this property to reprogram adult Schwann cells, its preferred host niche, to a stage of progenitor/stem-like cells (pSLC) of mesenchymal trait by downregulating Schwann cell lineage/differentiation-associated genes and upregulating genes mostly of mesoderm development. Reprogramming accompanies epigenetic changes and renders infected cells highly plastic, migratory, and immunomodulatory. We provide evidence that acquisition of these properties by pSLC promotes bacterial spread by two distinct mechanisms: direct differentiation to mesenchymal tissues, including skeletal and smooth muscles, and formation of granuloma-like structures and subsequent release of bacteria-laden macrophages. These findings support a model of host cell reprogramming in which a bacterial pathogen uses the plasticity of its cellular niche for promoting dissemination of infection and provide an unexpected link between cellular reprogramming and host-pathogen interaction.

Reprogramming diminishes retention of Mycobacterium leprae in Schwann cells and elevates bacterial transfer property to fibroblasts.

BACKGROUND: Bacterial pathogens can manipulate or subvert host tissue cells to their advantage at different stages during infection, from initial colonization in primary host niches to dissemination. Recently, we have shown that Mycobacterium leprae (ML), the causative agent of human leprosy, reprogrammed its preferred host niche de-differentiated adult Schwann cells to progenitor/stem cell-like cells (pSLC) which appear to facilitate bacterial spread. Here, we studied how this cell fate change influences bacterial retention and transfer properties of Schwann cells before and after reprogramming. RESULTS: Using primary fibroblasts as bacterial recipient cells, we showed that non-reprogrammed Schwann cells, which preserve all Schwann cell lineage and differentiation markers, possess high bacterial retention capacity when co-cultured with skin fibroblasts; Schwann cells failed to transfer bacteria to fibroblasts at higher numbers even after co-culture for 5 days. In contrast, pSLCs, which are derived from the same Schwann cells but have lost Schwann cell lineage markers due to reprogramming, efficiently transferred bacteria to fibroblasts within 24 hours. CONCLUSIONS: ML-induced reprogramming converts lineage-committed Schwann cells with high bacterial retention capacity to a cell type with pSLC stage with effective bacterial transfer properties. We propose that such changes in cellular properties may be associated with the initial intracellular colonization, which requires long-term bacterial retention within Schwann cells, in order to spread the infection to other tissues, which entails efficient bacterial transfer capacity to cells like fibroblasts which are abundant in many tissues, thereby potentially maximizing bacterial dissemination. These data also suggest how pathogens could take advantage of multiple facets of host cell reprogramming according to their needs during infection.

Polarization and myelination in myelinating glia.

Myelinating glia, oligodendrocytes in central nervous system and Schwann cells in peripheral nervous system, form myelin sheath, a multilayered membrane system around axons enabling salutatory nerve impulse conduction and maintaining axonal integrity. Myelin sheath is a polarized structure localized in the axonal side and therefore is supposed to be formed based on the preceding polarization of myelinating glia. Thus, myelination process is closely associated with polarization of myelinating glia. However, cell polarization has been less extensively studied in myelinating glia than other cell types such as epithelial cells. The ultimate goal of this paper is to provide insights for the field of myelination research by applying the information obtained in polarity study in other cell types, especially epithelial cells, to cell polarization of myelinating glia. Thus, in this paper, the main aspects of cell polarization study in general are summarized. Then, they will be compared with polarization in oligodendrocytes. Finally, the achievements obtained in polarization study for epithelial cells, oligodendrocytes, and other types of cells will be translated into polarization/myelination process by Schwann cells. Then, based on this model, the perspectives in the study of Schwann cell polarization/myelination will be discussed.

Histone deacetylase inhibitor trichostatin A enhances myogenesis by coordinating muscle regulatory factors and myogenic repressors.

Histone deacetylase inhibitors (HDACIs) are known to promote skeletal muscle formation. However, their mechanisms that include effects on the expression of major muscle components such as the dystrophin-associated proteins complex (DAPC) or myogenic regulatory factors (MRFs) remain unknown. In this study, we investigated the effects of HDACIs on skeletal muscle formation using the C2C12 cell culture system. C2C12 myoblasts were exposed to trichostatin A (TSA), one of the most potent HDACIs, and differentiation was subsequently induced. We found that TSA enhances the expression of myosin heavy chain without affecting DAPC expression. In addition, TSA increases the expression of the early MRFs, Myf5 and MEF2, whereas it suppresses the expression of the late MRF, myogenin. Interestingly, TSA also enhances the expression of Id1, Id2, and Id3 (Ids). Ids are myogenic repressors that inhibit myogenic differentiation. These findings suggest that TSA promotes gene expression in proliferation and suppresses it in the differentiation stage of muscle formation. Taken together, our data demonstrate that TSA enhances myogenesis by coordinating the expression of MRFs and myogenic repressors.

Secretion of N-terminal domain of α-dystroglycan in cerebrospinal fluid.

α-Dystroglycan (α-DG) plays crucial roles in maintaining the stability of cells. We demonstrated previously that the N-terminal domain of α-DG (α-DG-N) is secreted by cultured cells into the culture medium. In the present study, to clarify its function in vivo, we generated a monoclonal antibody against α-DG-N and investigated the secretion of α-DG-N in human cerebrospinal fluid (CSF). Interestingly, we found that a considerable amount of α-DG-N was present in CSF. α-DG-N in CSF was a sialylated glycoprotein with both N- and O-linked glycan. These observations suggest that secreted α-DG-N may be transported via CSF and have yet unidentified effects on the nervous system.

Biological role of dystroglycan in Schwann cell function and its implications in peripheral nervous system diseases.

Dystroglycan is a central component of the dystrophin-glycoprotein complex (DGC) that links extracellular matrix with cytoskeleton, expressed in a variety of fetal and adult tissues. Dystroglycan plays diverse roles in development and homeostasis including basement membrane formation, epithelial morphogenesis, membrane stability, cell polarization, and cell migration. In this paper, we will focus on biological role of dystroglycan in Schwann cell function, especially myelination. First, we review the molecular architecture of DGC in Schwann cell abaxonal membrane. Then, we will review the loss-of-function studies using targeted mutagenesis, which have revealed biological functions of each component of DGC in Schwann cells. Based on these findings, roles of dystroglycan in Schwann cell function, in myelination in particular, and its implications in diseases will be discussed in detail. Finally, in view of the fact that understanding the role of dystroglycan in Schwann cells is just beginning, future perspectives will be discussed.

Defective peripheral nerve myelination and neuromuscular junction formation in fukutin-deficient chimeric mice.

Dystroglycan is a central component of the dystrophin-glycoprotein complex that links the extracellular matrix with cytoskeleton. Recently, mutations of the genes encoding putative glycosyltransferases were identified in several forms of congenital muscular dystrophies accompanied by brain anomalies and eye abnormalities, and aberrant glycosylation of alpha-dystroglycan has been implicated in their pathogeneses. These diseases are now collectively called alpha-dystroglycanopathy. In this study, we demonstrate that peripheral nerve myelination is defective in the fukutin-deficient chimeric mice, a mouse model of Fukuyama-type congenital muscular dystrophy, which is the most common alpha-dystroglycanopathy in Japan. In the peripheral nerve of these mice, the density of myelinated nerve fibers was significantly decreased and clusters of abnormally large non-myelinated axons were ensheathed by a single Schwann cell, indicating a defect of the radial sorting mechanism. The sugar chain moiety and laminin-binding activity of alpha-dystroglycan were severely reduced, while the expression of beta1-integrin was not altered in the peripheral nerve of the chimeric mice. We also show that the clustering of acetylcholine receptor is defective and neuromuscular junctions are fragmented in appearance in these mice. Expression of agrin and laminin as well as the binding activity of alpha-dystroglycan to these ligands was severely reduced at the neuromuscular junction. These results demonstrate that fukutin plays crucial roles in the myelination of peripheral nerve and formation of neuromuscular junction. They also suggest that defective glycosylation of alpha-dystroglycan may play a role in the impairment of these processes in the deficiency of fukutin.

Harmful effects of anti-GalNAc-GD1a antibodies and TNF-alpha on rat dorsal root ganglia.

The clinical characteristics of five (22%) of 23 patients with Guillain-Barré syndrome (GBS), whose serum contained immunoglobulin G (IgG) antibodies to the ganglioside N-acetylgalactosaminyl GD1a (GalNAc-GD1a), included pure motor weakness of the axonal type. These patients had a relatively good prognosis, but displayed higher serum tumor necrosis factor-alpha (TNF-alpha) titers than the other GBS patients. We examined the effect of serum from these patients with IgG anti-GalNAc-GD1a antibodies on neurites from cultured rat dorsal root ganglia (DRG) and found it to damage the myelin in well-elongated DRG neurites and monolayer cultures of Schwann cells and neurons. In the regeneration model, serum from these patients delayed neurite extension and inhibited Schwann cell proliferation. Neurons in cultured monolayers showed vacuolation and decreased rapidly in number. Schwann cells were also vacuolated and readily detached from the substratum. The effects of IgG anti-GalNAc-GD1a antibodies purified from one of the patients, rabbit serum after immunization with GalNAc-GD1a, and recombinant TNF-alpha were also examined. IgG anti-GalNAc-GD1a antibodies mainly inhibited the regeneration and preservation of neurons, while TNF-alpha mainly induced morphological changes in well-proliferated Schwann cells and myelin.

Association study of brain-derived neurotrophic factor gene polymorphism and alcoholism.

OBJECTIVE: Brain-derived neurotrophic factor (BDNF) influences dopamine and serotonin neurotransmitters that are heavily linked to addiction. A quantitative trait loci study indicated that genes localized to 11p13, where the BDNF gene is mapped (11p13-15), increase the risk for severe alcohol withdrawal. Moreover, a recent study using a pooled-sample microarray suggested that the BDNF gene locus was included in the loci that were shown to be associated with drug abuse. These lines of evidence suggested that BDNF might play some role in the development of or vulnerability to alcoholism and/or clinical characteristics of alcoholic individuals. METHODS: The alcoholic subjects consisted of 377 male Japanese patients. A structured interview was used to obtain social background, drinking history, history of violence while intoxicated, history of alcohol withdrawal, and family history of alcoholism. The control group consisted of 336 nonalcoholic male subjects. Genotyping of the G196A polymorphism of the BDNF gene was done by polymerase chain reaction (PCR)-restriction fragment length polymorphism method. RESULTS: Genotype and allele distributions of the BDNF gene polymorphism did not differ significantly between alcoholic and control subjects. However, comparing clinical characteristics across G196A genotypes, we found that alcoholic subjects with violent tendencies and a history of delirium tremens had a significantly higher frequency of AA genotypes and A allele frequencies than those without them. Moreover, alcoholic subjects with the A allele had earlier onset of the disease than those without it. CONCLUSIONS: These results indicate that BDNF gene polymorphism might modify phenotypes of alcoholism.

Influence of genetic variations of ethanol-metabolizing enzymes on phenotypes of alcohol-related disorders.

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase-2 (ALDH2) play central roles in the metabolism of ethanol and its metabolite, acetaldehyde, in the liver. In ADH2, one nucleotide replacement causes either a super-active beta 2 subunit encoded by the ADH2*2 allele or a less active beta 1 subunit (ADH2*1 allele). In the same way, a G/A replacement at codon 487 of the ALDH2 gene produces an inactive form of the enzyme. Because the geno-types of these genes may explain individual differences in concentration and elimination of ethanol and acetaldehyde in the blood after drinking, they could be used as models to elucidate the contribution of these substances to the development of addiction and various types of organ damage. We have examined the influence of genetic variations of these enzymes on alcohol-related disorders in the Japanese. The results revealed that (1) the less active allele of the ADH2 gene (ADH2*1) is associated with an increased risk for alcohol dependence, alcohol-induced persistent amnestic disorder, alcohol withdrawal syndrome, and cancer of the upper GI tract; (2) the inactive allele of the ALDH2 gene (ALDH2*2) is associated with a decreased risk for alcohol dependence, and an increased risk for alcoholic polyneuropathy and cancer in the same region; and (3) these genetic variations modify clinical features of alcohol dependence. Possible mechanisms of altered risk for these disorders are discussed.

Prolonged central sensory conduction time in alcoholics with hypoactive aldehyde dehydrogenase-2.

People who have a Glu487Lys mutation (single nucleotide polymorphism) in the aldehyde dehydrogenase-2 (ALDH2) gene are slow to metabolize the alcohol breakdown product acetaldehyde. The P13/14-N20 interval of the median nerve somatosensory evoked potential was significantly longer in alcoholic patients with a hypoactive ALDH2 (n = 27) than in those with an active ALDH2 (n = 43). This suggests that acetaldehyde accumulation due to hypoactive ALDH2 is associated with a prolongation of the central sensory conduction time between pons and primary sensory cortex. The present result indicates that an elevated blood concentration of acetaldehyde must cause the central sensory tract involvement and that acetaldehyde is one of factors producing brain damage in alcoholics.

Association of aldehyde dehydrogenase-2 polymorphism with alcoholic polyneuropathy in humans.

Persons who have the Glu-487-->Lys mutation (single nucleotide polymorphism) of the aldehyde dehydrogenase-2 (ALDH2) gene have less ability to metabolize the alcohol breakdown product acetaldehyde. In order to clarify whether acetaldehyde is associated with the pathogenesis of alcoholic polyneuropathy, we compared nerve conduction data as well as clinical signs and symptoms of neuropathy between alcoholics with ALDH2*2 (Lys-487) heterozygotes and those with ALDH2*1 (Glu-487) homozygotes. Alcoholics with ALDH2*2 heterozygotes showed significantly lower sensory nerve action potential amplitudes of the sural and median nerves than those with ALDH2*1 homozygotes, suggesting that the accumulation of acetaldehyde due to ALDH2 inactivity is associated with alcoholic polyneuropathy.

Therapeutic factors causing hallucination in Parkinson's disease patients, especially those given selegiline.

Selegiline protects nigral dopaminergic neurons and is recommended for the treatment of patients in the early stage of Parkinson's disease (PD). We treated 112 PD patients and noted that those given selegiline had a high incidence of hallucination. Our objective was to determine which clinical therapeutic factors cause such hallucinations. The Kruskal-Wallis and chi-square test showed that in 94 patients, the severity of the hallucinations was significantly related to the duration of illness, Hoehn and Yahr stage, doses of levodopa and cabergoline, whether or not selegiline was used, and whether or not medication for constipation was required. In addition, patients who were treated with a low dose of levodopa (< or =300 mg/day), who had a low Hoehn and Yahr stage, and a short duration of illness (< or =8 years) together with a high dose of selegiline or cabergoline also tended to have hallucinations. MRI findings were not related to the incidence of hallucination. When selegiline is given to patients who have PD of long duration and a high Hoehn and Yahr stage, and who already are receiving levodopa and a dopamine agonist, the doses of levodopa and the dopamine agonists given, as well as the presence of constipation, may be related to the incidence of hallucination.

Benign type of central pontine myelinolysis in alcoholism--clinical, neuroradiological and electrophysiological findings.

Nine alcoholic patients with central pontine myelinolysis (CPM),who showed a favorable prognosis, are reported. The majority of them had taken part in binge drinking and had a subsequent consciousness disturbance for 18.1+/-10.9 (mean+/-SD) days. None of the patients had had acute correction of hyponatremia. Truncal ataxia and gait instability were present in most of the patients after recovery from the disturbance of consciousness. Most of them eventually gained independence, and magnetic resonance imaging showed that their pontine lesions tended to shrink. Electrophysiological studies detected prolonged latency between the I and III waves in auditory brainstem responses and between N11 and P13/14 onsets in the somatosensory evoked potentials. These clinical, radiological and electrophysiological findings should be of use in diagnosing CPM.

Association of dystroglycan and laminin-2 coexpression with myelinogenesis in peripheral nerves.

To provide clues to the biological functions of dystroglycan-laminin-2 complex in peripheral nerves, we investigated the expressions of beta-dystroglycan and laminin-alpha(2) chain in rat sciatic nerve during axonal degeneration and regeneration and during development, as well as in rat dorsal root ganglia. In normal conditions, immunoreactivity of the cytoplasmic domain of beta-dystroglycan was associated with the Schwann cell abaxonal membrane. The immunoreactivities of both beta-dystroglycan and the laminin-alpha(2) chain decreased in Schwann cells losing axons during axonal degeneration and progressively increased in remyelinating Schwann cells during axonal regeneration. Interestingly, during axonal degeneration, the abaxonal membrane losing contact with the basal lamina lost the association with beta-dystroglycan immunoreactivity. During development, expression of both beta-dystroglycan and laminin-alpha(2) chain strikingly increased during postnatal 7 days, which is a critical period when basal lamina assembly and myelin formation rapidly progress. These results suggest that coexpression of dystroglycan and laminin-2 is associated with myelinogenesis in peripheral nerves. These two proteins may function as an anchorage between the abaxonal membrane and the basal lamina, enabling myelin forma-tion to progress. Beta-dystroglycan and laminin-2 were also coexpressed in satellite cells in dorsal root ganglia, suggesting that interaction of these two proteins plays some role in physiological functions of these cells.