Yukiko Kuroda

Oxidative stress–associated mitochondrial dysfunction in corticosteroid‐treated muscle cells

We analyzed the effects of corticosteroid on mitochondrial membrane potentials (ΔΨm), generation of reactive oxygen species (ROS), and apoptosis in a human rhabdomyosarcoma cell line, RD, and a dopaminergic neuroblastoma cell line, SH‐SY5Y. The cell lines were cultured in the presence or absence of dexamethasone and superoxide dismutase (SOD) for up to 1 week. Dexamethasone treatment increased ΔΨm, ROS generation, and apoptosis in proliferating RD cells. Treatment with SOD attenuated ROS generation and apoptosis, but not ΔΨm. The increase in ΔΨm seemed to be the primary effect of dexamethasone on proliferating RD cells, which is probably mediated by mitochondrial transcription. In differentiated RD cells, but not differentiated SH‐SY5Y cells, dexamethasone treatment showed a delayed effect of interfering with the ΔΨm and increasing ROS generation and apoptosis. Since these changes disappeared in the presence of SOD, dexamethasone primarily induced ROS generation, resulting in apoptosis. We speculate that this mechanism provides the basis of a pathophysiological model of corticosteroid myopathy. Muscle Nerve 30:49–54,2004

Immediate effect of spinal magnetic stimulation on camptocormia in Parkinson's disease

Objectives Spinal cord stimulation is a potential therapeutic option for the treatment of Parkinsons disease (PD)-associated symptoms. Repetitive trans-spinal magnetic stimulation (rTSMS) is a non-invasive and safe alternative for stimulation of spinal pathways that has not been studied for therapeutic efficacy in PD. We assessed the benefits of rTSMS on camptocormia, an often treatment-resistant postural abnormality observed in PD patients. Methods We compared rTSMS to sham stimulation in PD patients with camptocormia in a single-centre, randomised, single-blind, crossover, placebo-controlled study. PD patients with camptocormia were administered a single trial of rTSMS (a train of 40 stimuli) or sham treatment followed 1 week later by the alternate treatment. Primary outcome measure was thoracolumbar spine flexion angle in the standing position immediately after the trial. Results Of 320 PD patients examined, 37 had concomitant camptocormia and were randomly assigned to either the rTSMS first group (n=19) or sham first group (n=18). Flexion angle in the standing position decreased by a mean of 10.9° (95% CI 8.1 to 13.65) after rTSMS but remained unchanged after sham stimulation (mean, −0.1°; 95% CI −0.95 to 0.71). The flexion angle while sitting (secondary outcome) decreased by 8.1° (95% CI 5.89 to 10.25) after rTSMS, whereas sham treatment had no significant effect (mean, −0.8°; 95% CI −1.62 to 0.05). Conclusions We found an immediate beneficial effect of rTSMS on camptocormia in PD patients. Although the effect was transient, this successful trial justifies further studies to test if repeated rTSMS treatments can induce longer term improvements in camptocormia associated with PD. Clinical trial registration UMIN Clinical Trials Registry: UMIN000011495.

Parkin interacts with Klokin1 for mitochondrial import and maintenance of membrane potential

Parkin is a multifunctional protein, including maintaining mitochondrial homeostasis. Recent evidence suggests that Parkin is recruited from the cytoplasm to damaged mitochondria with low membrane potential. We found that intracellular localization of Parkin changed with cellular growth phase. Parkin was preferentially localized in the mitochondria of cultured cells. The mitochondria with large amounts of Parkin showed preserved membrane potentials even during treatment with carbonyl cyanide m-chlorophenylhydrazone. Here we report a novel protein named Klokin 1 that transports Parkin to the mitochondria. Klokin 1 was localized to the mitochondria and enhanced mitochondrial expression of Parkin. Klokin 1 enhanced cell viability in Parkin-silenced cells. Klokin 1 expression was enhanced in the brains of Parkin-deficient mice but not in an autopsied PARK2 brain. Our findings indicate that mitochondrial Parkin prevents mitochondrial depolarization and that Klokin 1 may compensate for Parkin deficiency.

Involvement of mitochondria in myasthenia gravis complicated with dermatomyositis and rheumatoid arthritis: a case report

We report a 57-year-old male with myasthenia gravis complicated with dermatomyositis and rheumatoid arthritis without evidence of thymoma. He showed prominent muscle wasting and weakness in the four extremities and trunk in addition to swallowing disturbance. He showed intolerance to exercise on a bicycle ergometer, and muscle biopsy specimens demonstrated ragged-red fibers. An anti-acetylcholine receptor (AChR) antibody was detected in his serum but no anti-mitochondrial M2 component antibody was found. In contrast, results of immunohistochemical study indicated that his serum sample reacted to muscle mitochondria as well as AChR. These results indicate the presence of an unidentified anti-mitochondrial antibody that may be involved in the development of mitochondrial dysfunction in skeletal muscle of the present patient.

Superoxide dismutase as a target of clioquinol-induced neurotoxicity.

Subacute myelo-optico-neuropathy (SMON) is a progressive neurological disorder affecting the spinal cord, peripheral nerves and optic nerves. Although it has been assumed that SMON was caused by intoxication of clioquinol, the mechanism underlying clioquinol-induced neurotoxicity is not fully understood. This study aimed to clarify the relevance of oxidative stress to clioquinol-induced neurotoxicity and the cause of the enhanced oxidative stress. Clioquinol induced cell death in human-derived neuroblastoma cell line, SH-SY5Y, in a dose-dependent manner. This process was accompanied by activation of caspase-3 and enhanced production of reactive oxygen species (ROS). We examined whether clioquinol inhibited the activity of superoxide dismutase-1 (SOD1), based on its metal chelating properties. Clioquinol inhibited activities of purified SOD1 in a dose-dependent manner. Cytosolic SOD activities were also inhibited in SH-SY5Y cells treated with clioquinol. Finally, addition of exogenous SOD1 to the culture significantly reduced enhanced ROS production and cell death induced by clioquinol in SH-SY5Y cells. These findings suggested that enhanced oxidative stress caused by inhibition of SOD1 undelay clioquinol-induced neurotoxicity and was relevant to the pathogenesis of SMON.

A homozygous mutation of VWA3B causes cerebellar ataxia with intellectual disability

Background Hereditary cerebellar ataxia constitutes a heterogeneous group of neurodegenerative disorders, occasionally accompanied by other neurological features. Genetic defects remain to be elucidated in approximately 40% of hereditary cerebellar ataxia cases in Japan. We attempted to identify the gene responsible for autosomal recessive cerebellar ataxia with intellectual disability. Methods The present study involved three patients in a consanguineous Japanese family. Neurological examination and gene analyses were performed in all family members. We performed genome-wide linkage analysis including single nucleotide polymorphism arrays, copy-number variation analysis and whole exome sequencing. To clarify the functional alteration resulting from the identified mutation, we performed cell viability assay of cultured cells expressing mutant protein. Results One homozygous region shared among the three patients on chromosomes 2p16.1–2q12.3 was identified. Using whole exome sequencing, six homozygous variants in genes in the region were detected. Only one variant, VWA3B c.A1865C, results in a change of a highly conserved amino acid (p.K622T) and was not present in control samples. VWA3B encodes a von Willebrand Factor A Domain-Containing Protein 3B with ubiquitous expression, including the cerebellum. The viability of cultured cells expressing the specific K622T mutation was proved to decrease through the activation of apoptotic pathway. Conclusions Mutated VWA3B was found to be likely associated with cerebellar degeneration with intellectual disability. Although a rare cause of cerebellar degeneration, these findings indicate a critical role for VWA3B in the apoptosis pathway in neuronal tissues.

Expanding phenotype and clinical heterogeneity in patients with identical mutation of the parkin gene.

183 ment underlying the damage can probably be related to an excessive presynaptic blocking of cholinergic junctions in long-term denervated muscle fibers so that the patient’s subclinical defective neuromuscular transmission status was precipitated by the last BTX administration by a sort of long-time toxin overdose [9] in selected muscles and with a low safety margin [7] with mild inflammatory muscular features. Generalized weakness is expected in patients affected by concomitant motor neuron diseases treated for spasticity [10] making these patients at risk of BTX therapy but our patient did not meet any of the motor neuron disease criteria. The dosage of the antibodies against the toxin unfortunately was not performed. Its interest is conceptually and practically relevant mostly in nonresponding subjects [11, 12]. Besides, in our patient the total dosage of BTX and treatment intervals were constant and according to the safety rules [13]. Different unusual complications of BTX therapy apart from transitory generalized weakness [7, 10, 14] are seldom reported in the literature: a flu-like syndrome with low-grade fever that could be due either to concomitant undiagnosed viral infections or to a more direct effect of BTX on pyrogen compounds [15] and occasional seizures within 3 months from the onset of BTX therapy are reported [15]. Autonomic side effects such as constipation are also rarely described as well as a dry mouth, accommodation difficulties, and reduced sweating; these symptoms are related to a BTX bloodstream diffusion [16] and recently described mostly by BTXtype B application [17]. Our patient was not retreated with BTX in consideration of the risk-benefit therapy ratio and because of his still partial and incomplete recovery at 6 months, even though his focal spasticity was again marked and severe (Ashworth modified scale: 4); thus far no further concomitant pathology had been detected.

FK506 attenuates thymic output in patients with myasthenia gravis.

Introduction Myasthenia gravis (MG) is an antibody-mediated, T-cell-dependent autoimmune disease. The symptoms are caused by high-affinity IgG against the muscle acetylcholine receptor (AChR) at the neuromuscular junction. The production of these antibodies in B-cells depends on AChR-specific CD4+ T-cells and the thymus gland seems to play a significant role in the pathogenesis of MG. Altered thymic T-cell export seems to be associated with a pathological mechanism in myasthenia gravis. Tacrolimus (FK506) has recently been used to treat MG. Material and methods We examined the effects of tacrolimus on thymic T-cell export in patients with MG. Sixteen patients with nonthymomatous and/or thymectomized MG were treated with oral administrations of tacrolimus. To assess the effect of tacrolimus on the thymic output, we assayed the levels of T-cell receptor excision circle (TREC), a molecular marker of thymus emigrants. Results T-cell receptor excision circle was not significantly different from those in age-matched controls before tacrolimus therapy, but they were partially decreased 4 months after tacrolimus therapy. T-cell receptor excision circle levels were significantly decreased in the thymomatous group (p < 0.05), but not in the nonthymomatous group. Tacrolimus treatment significantly attenuated TREC levels in cultured CD4–CD8+ cells (p < 0.05), but total cell counts were not significantly changed. Conclusions These results indicate that TREC levels may become a marker of the curative effect of tacrolimus therapy for thymomatous MG, and that tacrolimus suppresses not only activating T-lymphocytes, but also naïve T-cells.

Heterogeneous Muscular Involvement in Inclusion Body Myositis

In patients with inclusion body myositis (IBM), selectivity of involvement of certain muscle groups is common, but the mechanism remains unclear. We report 2 patients with IBM showing selective distribution of weakness in limb muscles. The muscle biopsy revealed the typical features of IBM. Patient 1 underwent the second muscle biopsy because clear abnormality was not seen in the first biopsy specimen performed 15 years ago. Patient 2 was given a diagnosis after the first muscle biopsy. In cryostat sections of biopsy specimens from both patients, some fascicules showed prominent inflammatory features and others showed a normal appearance. In the present patients, the degree of inflammation was heterogeneous between individual muscles and individual fascicules in a muscle, resulting in the diagnostic difficulty. * IBM : inclusion body myositis s-IBM : sporadic inclusion body myositis PM : polymyositis RF : rectus femoris SA : sartorius GR : gracilis BF : biceps femoris EMG : electromyography