Derlin-1 overexpression ameliorates mutant SOD1-induced endoplasmic reticulum stress by reducing mutant SOD1 accumulation.

Unfolded protein responses, including induction of stress sensor kinases, chaperones, and apoptotic mediators, are involved in the familial amyotrophic lateral sclerosis (ALS) model related to mutant Cu/Zn superoxide dismutase (SOD1) and sporadic ALS. We hypothesized that the endoplasmic reticulum-resident factor Derlin-1 plays a pivotal role in the regulation of misfolded proteins evoked by mutant SOD1. We show that Derlin-1 overexpression reduced mutant SOD1-induced cell toxicity and increased cell viability by suppressing the activation of the ER stress pathway factors: immunoglobulin-binding protein, activating transcription factor 6 p50, and C/EBP homologous protein. Interestingly, exogenous Derlin-1 resulted in a decrease in the amount of mutant SOD1, and a lesser decrease in that of wild-type SOD1, in transfected cells. Reduced SOD1 protein expression was observed in the microsomal fraction of wild-type and mutant SOD1 cells. Our results indicate that Derlin-1 regulates the turn over of SOD1 by promoting the proteasomal and autophagosomal degradation of SOD1 protein, but not by decreasing mutant SOD1 mRNA levels. Insights into the effects of Derlin-1 on mutant SOD1 may facilitate advancements in the treatment of motor neuron degeneration associated with ALS.

DJ-1 forms complexes with mutant SOD1 and ameliorates its toxicity.

Mutations in Cu/Zn superoxide dismutase (SOD1) gene cause familial amyotrophic lateral sclerosis (ALS), which could be attributed to the toxic properties of the misfolded protein, oxidative stress, and mitochondrial dysfunction. DJ-1 - a causative agent of familial Parkinson's disease PARK7 - is responsible for inducing antioxidative reaction. In this study, we showed the up-regulation of DJ-1 protein levels in mutant SOD1 transgenic mice through the lifespan were observed in the motor neurons. We demonstrated biochemically DJ-1 formed complexes with mutant SOD1 in the cell lysates. Furthermore, DJ-1 over-expression resulted in increased cell viability and reduced cell toxicity in mutant SOD1-transfected neuronal cells, because of improvement in apoptotic pathway and reduction in oxidative stress levels. We also evaluated DJ-1 levels in CSF collected from sporadic ALS patients and controls subjects. The CSF DJ-1 levels were significantly higher in patients with sporadic ALS than in control subjects. These results show that DJ-1 may be associated with sporadic and familial ALS pathogenesis. Therefore, insight into the effects of DJ-1 on mutant SOD1-mediated toxicity may provide a therapeutic advance for the treatment of motor neuron degeneration in ALS.

Flexor-dominant myopathic phenotype in patients with His46Arg substitution in the Cu/Zn superoxide dismutase gene.

We present the cases of 3 patients with a histidine-to-arginine substitution at position 46 of the Cu/Zn superoxide dismutase gene. Consistent with previous reports, the initial symptom in each patient was unilateral weakness in the distal leg muscles. Remarkably, muscular atrophy in these patients during the early stage of the disease was more specific to the flexor muscle group, with the extensor muscle group remaining intact over long-term observation. More interestingly, biopsy of the affected muscle in the early stage of the disease revealed necrotic and regenerative myofibers with infiltration of lymphocytes, resembling inflammatory myopathy. These novel findings might provide further insights into the pathophysiology of familial amyotrophic lateral sclerosis.

[Laryngotracheal separation and tracheoesophageal diversion for intractable aspiration in ALS--usefulness and indication].

To evaluate the usefulness of laryngotracheal separation or tracheoesophageal diversion (LTS/TED), we investigated changes in medical management after LTS/TED. We performed LTS/TED for intractable aspiration in patients with amyotrophic lateral sclerosis (ALS) and patients with other neurological diseases. Most of the subjects had already received a tracheostomy, and all the patients with ALS had tracheostomy positive pressure ventilation. However, they remained at risk of intractable aspiration, had experienced frequent suctioning of aspiration material, and could not eat. In all cases, LTS/TED was performed safely within 3 hours and without any complications. After LTS/TED, there was no aspiration of saliva in any of the patients. In most subjects, the frequency of suctioning by medical staff and caregivers was much reduced. The frequency of aspiration pneumonia was also extremely lowered. Follow-up study demonstrated that complete control over aspiration was achieved in all of the patients. Some of them were completely self-sufficient in their ability to eat. Some of the other patients were at least able to enjoy taste. Nutritional status was significantly improved. All the patients, family caregivers, and medical staff involved in this study were satisfied with the outcome. These results indicate that LTS/TED is a very useful procedure in several aspects: it benefits patients who have a fear of aspiration; it reduces the burden on patients and family caregivers; it promotes their quality of life; and it limits the aspiration-associated demands on medical staff. Based on the results, we have proposed an indication of LTS/TED for dysphasia in ALS.

Leber's hereditary optic neuropathy with dystonia in a Japanese family.

We investigated a Japanese family with generalized dystonia attributed to striatal degeneration, which occurred in childhood, and late-onset optic neuropathy. We determined the entire nucleotide sequence of mitochondrial DNA (mtDNA) from the proband and compared our findings with the 2001 Revised Cambridge Reference Sequence. The mtDNA of the proband showed a total of 42 nucleotide changes. We identified two A3203G and G14459A mutations, which were completely absent in a population of 200 healthy Japanese, by estimating the frequency of each nucleotide change. The nucleotide G14459A mutation occurs in NADH dehydrogenase subunit 6, and has been suggested previously as the disease-causing mutation in Hispanic, African-American and Caucasian families of Leber's hereditary optic neuropathy (LHON) and/or dystonia. The significance of the A3203G mutation remains unknown. To our knowledge, this is the first case of LHON with dystonia that revealed a mtDNA mutation in a Japanese family.

Effective repetitive dystrophin gene transfer into skeletal muscle of adult mdx mice using a helper-dependent adenovirus vector expressing the coxsackievirus and adenovirus receptor (CAR) and dystrophin.

BACKGROUND: The helper-dependent adenovirus (HDAd) vector is less immunogenic and has a larger cloning capacity of up to 37 kb enough to carry the full-length dystrophin cDNA. However, high and long-term expression of dystrophin transduced to mature muscle still remains difficult. One of the main reasons for this is that the expression of the coxsackievirus and adenovirus receptor (CAR) is very low in mature muscle. METHODS: We have constructed two different HDAd vectors. One contains the LacZ and the murine full-length dystrophin expression cassette (HDAdLacZ-dys), and the other is a new, improved vector containing the CAR and the dystrophin expression cassette (HDAdCAR-dys). RESULTS: We initially demonstrated high dystrophin expression and prevention of the dystrophic pathology in mdx muscle injected during the neonatal phase with HDAdLacZ-dys. Furthermore, we demonstrated that repeated injections of HDAdCAR-dys into mature muscle led to approximately nine times greater dystrophin-positive fibers in number than a single injection, thereby recovering the expression of dystrophin-associated proteins. This data has also shown that HDAdCAR-dys enabled administration of adenovirus (Ad) vector to the host with pre-existing immunity to the same serotype of Ad. CONCLUSIONS: Repetitive injections of the HDAd vector containing the CAR and the dystrophin expression cassette could improve the efficiency of subsequent dystrophin gene transfer to mature mdx muscle. This result suggests that our new HDAd vector will provide a novel gene therapy strategy for Duchenne muscular dystrophy, raising the prospects for gene therapy of other hereditary myopathies.

Bcl-2 expression using retrograde transport of adenoviral vectors inhibits cytochrome c-release and caspase-1 activation in motor neurons of mutant superoxide dismutase 1 (G93A) transgenic mice.

We explored a possible mechanism of the neuro-protective effects of exogenous human Bcl-2 expression on motor neurons of transgenic mice expressing human Cu/Zn superoxide dismutase with a G93A mutation (G93A mice), using retrograde transport and a Cre-loxP recombination system employing adenoviral vectors. We examined the cellular localization of cytochrome c and caspase-1 using immunohistochemical study, in motor neurons of hypoglossal nuclei of G93A mice at 15 weeks after inoculation with the adenoviral vectors, at which time over-expressed exogenous Bcl-2 declined to reach the baseline of intrinsic Bcl-2. We found that a significant number of neurons showed more faint and punctate immunostaining against cytochrome c and significantly less neurons showed immunoreactivity against activated caspase-1, compared with those of mice without inoculation. These results suggest that transient exogenous Bcl-2 expression at the early stage of the disease protects against motor neuronal degeneration in G93A mice by retarding translocation of cytochrome c into the cytosol, and regulating caspase-1 for a substantial period.

A unique case of sporadic Creutzfeldt-Jacob disease presenting as progressive supranuclear palsy.

We report a Japanese case of sporadic Creutzfeldt-Jakob disease (CJD) presenting as progressive supranuclear palsy. For 2 years after onset, neurological deficits had slowly progressed but neither myoclonus nor periodic synchronous discharge was observed. Diffusion-weighted image (DWI) showed unique high signal lesions in the bilateral frontal cortex, left parietooccipital and occipital cortices, but there was nearly no change eight months later. Needle biopsy revealed deposition of prion protein of a patchy/perivacuolar type with spongiform degeneration. Thus, the phenotype of sporadic CJD seems variable and DWI should be performed, even in atypical cases lacking the characteristics of CJD.

Association between interleukin-6 gene polymorphism and human T-cell leukemia virus type I associated myelopathy.

We studied cytokine gene polymorphisms in the promoter region, including interleukin (IL)-6, IL-1beta, and IL-10, in Japanese patients with human T-cell leukemia virus type I (HTLV-I) associated myelopathy (HAM) (n = 65), asymptomatic HTLV-I carriers (n = 143), and HTLV-I seronegative, normal controls (n = 160). There was a significant difference between HAM patients and HTLV-I carriers in the distribution of IL-6 promoter polymorphism at position -634 (chi(2) = 9.90, p = 0.0071). The IL-6 genotype was also significantly different between HAM patients and normal controls (chi(2) = 11.53, p = 0.0033), while a similar distribution was observed in IL-1beta and IL-10 polymorphisms among HAM patients, carriers, and normal controls. The results suggest that IL-6 gene region may contribute to susceptibility to HAM, and that aberrant cytokine productions could be involved in the development of HAM.

Effect on motor neuron survival in mutant SOD1 (G93A) transgenic mice by Bcl-2 expression using retrograde axonal transport of adenoviral vectors.

We investigated the effect of exogenous Bcl-2 on motor neurons in transgenic mice expressing human Cu/Zn superoxide dismutase with a G93A mutation (G93A mice), using adenoviral vectors with a cassette for Bcl-2 (AxCALNLBcl-2) and Cre recombinase (AxCANCre) to express Bcl-2 by Cre-loxP recombination. We were able to detect Bcl-2 in the hypoglossal nuclei of G93A mice for at least 8 weeks after inoculation with AxCALNLBcl-2 followed by inoculation with AxCANCre into the tongue of 10-week-old G93A mice. We examined the morphological changes of motor neurons in the hypoglossal nuclei of each mouse at 25 weeks of age, at which time the G93A mice manifested signs of neural degeneration. We found that the number of motor neurons was significantly higher in the G93A mice with both vectors than in those with AxCALNLBcl-2 alone or without inoculation. Further, we observed an obvious reduction of vacuole formations and reactive astrocytes in and around the hypoglossal nuclei of G93A mice with both vectors. These results suggest that expression of Bcl-2 introduced by our system has a protective effect on degeneration of motor neurons in G93A mice.

Adenovirus-mediated murine interferon-gamma receptor transfer enhances the efficacy of IFN-gamma in vivo.

Interferon gamma (IFN-gamma) plays an important role in immune response, apoptosis, and anti-tumor activity. Its biological activity depends on expression of IFN-gamma receptor (IFN-gammaR). To address whether increased expression of IFN-gammaR is associated in vivo with a higher biological response by IFN-gamma, we constructed an adenovirus vector including murine IFN-gammaR (Ad-mIFN-gammaR). We confirmed the appropriate function of mIFN-gammaR derived from Ad-mIFN-gammaR based on the observation of signal transduction and transcription. We also found that elevated expression of mIFN-gammaR increases sensitivity to recombinant murine IFN-gamma (rmIFN-gamma) in vitro in target cells. Furthermore, we demonstrated that the growth rate of tumors transfected with Ad-mIFN-gammaR is suppressed in response to rmIFN-gamma in vivo and that such growth suppression is partly due to apoptosis. To our knowledge, this is the first report of adenovirus-mediated IFN-gammaR gene transfer being effective in augmenting the biological activity of IFN-gamma, and the strategy employed in the present study will be useful in studying other kinds of cytokine receptors and applications to gene therapy for cancer and infectious diseases.

Bilateral pallidal stimulation for cervical dystonia. An optimal paradigm from our experiences.

We sought to identify optimal paradigms of bilateral globus pallidus internus (GPi) stimulation in 3 subsequent patients with severe cervical dystonia. At low frequency stimulation (50-60 Hz) with wide pulse width (500 micros) and high amplitude (4.5-8.0 V), we observed immediate and consistent improvement of dystonia and dystonia-associated pain. Stimulation of the posteroventral portion of the GPi led to pronounced alleviation of dystonia; stimulation of the anterodorsal portion or at the dorsal border of the GPi resulted in significant worsening of symptoms. The therapeutic benefit obtained by using the optimal stimulation parameters continues and has lasted for at least 1 year in each patient.

Impact of posterior GPI pallidotomy on leg tremor in Parkinson's disease.

Although stereotactic thalamotomy is the mainstay in the surgical treatment of tremor in patients with Parkinson's disease (PD), this surgery is not favored and is even a matter of potential concern in the treatment of leg tremor since it carries a significant risk of injury to the internal capsule. In this study we have carried out a quantitative assessment of leg tremor alleviation in 12 patients with PD after MRI-/microelectrode-guided stereotactic ablation of the posterior part of the globus pallidus internus (GPi). The results showed that posterior GPi pallidotomy combined with drug therapy is a satisfactorily effective therapeutic strategy to treat parkinsonian leg tremor.

[A case of spontaneous intracranial hypotension without any history of positional headache].

A 52-year-old woman was admitted to our hospital with sudden diplopia, right ophthalmalgia, and occipital pain. The nature of her headache was continuous, and changing her position provided no relief. Three weeks before her admission, she experienced continuous cough and rhinorrhea during a few days. On admission, her neurological examination revealed right abducens paralysis, and right lateral gaze aggravated diplopia. Brain magnetic resonance imaging scan demonstrated diffuse pachymeningeal enhancement by gadolinium. Radioisotope cisternography showed a leak of cerebrospinal fluid from near the third lumbar vertebra. The first lumbar puncture revealed an initial pressure of 0 mm H2O. She was given a diagnosis of spontaneous intracranial hypotension due to a leak of the cerebrospinal fluid. During her hospitalization, she complained of vertigo and nausea. We found horizontal gaze nystagmus to left side and sensorineural deafness in her left ear, which improved in a few days. After treatment by a lumbar epidural continuous infusion of saline and a lumbar epidural blood patch, her headache and abnormal MRI findings had improved. The continuous cough before her admission may be the cause of the leak of cerebrospinal fluid. We had difficulty in diagnosis only by the history and clinical examination, because she had no history of orthostatic headache.