Thomas Meyer

Point mutations of the p150 subunit of dynactin (DCTN1) gene in ALS

The authors report mutation screening of the p150 subunit of dynactin (DCTN1) and the cytoplasmic dynein heavy chain (DNCHC1) genes in 250 patients with ALS and 150 unrelated control subjects. Heterozygous missense mutations of the DCTN1 gene were detected in one apparently sporadic case of ALS (T1249I), one individual with familial ALS (M571T), two patients with familial ALS, and two unaffected relatives in the same kindred (R785W). The allelic variants of the DCTN1 gene may represent a previously unknown genomic risk factor for ALS.

Heterozygous R1101K mutation of the DCTN1 gene in a family with ALS and FTD.

A heterozygous R1101K mutation of the p150 subunit of dynactin (DCTN1) is reported in a family with amyotrophic lateral sclerosis (ALS) and co‐occurrence of frontotemporal dementia (FTD). Two members of our kindred were affected with motor neuron disease and two with dementia in an autosomal dominant pattern of inheritance. We excluded the involvement of the ALS and FTD‐linked genes for copper/zinc superoxide dismutase (SOD1) and tau. The R1101K sequence alteration of the DCTN1 gene may predispose subjects to ALS and FTD. Ann Neurol 2005;58:777–780

The RNA of the glutamate transporter EAAT2 is variably spliced in amyotrophic lateral sclerosis and normal individuals

Impaired re-uptake of synaptic glutamate, and a reduced expression of the glutamate transporter EAAT2 have been found in the motor cortex of patients with amyotrophic lateral sclerosis (ALS). Two splice forms of the EAAT2 RNA resulting from retention of intronic sequences (EAAT2/Int) and deletion of one protein coding exon (EAAT2/C1) have been reported to account for the EAAT2 protein loss in ALS. In this study we investigated the presence of two known (EAAT2/C1; EAAT2/Int) and three novel (EAAT2/C2-4) EAAT2 RNA in motor cortex of 17 ALS cases and 11 controls. Reverse transcription and PCR were carried out to amplify the complementary DNA of the complete and variably spliced EAAT2 transcripts. Nested PCR was followed to generate amplicons specific for EAAT2/C1-4 and EAAT2/Int. EAAT2/Int was detected in 59% of ALS specimens as compared to 36% of controls showing a trend but no statistical significance of a more frequent expression in ALS (Type I error 24.6%). EAAT2/C1-4 were found to be equally expressed in ALS patients and controls. Our results indicate that the involvement of EAAT2 transcripts in ALS is unlikely to be primary, and more complex than previously recognized. Alterations of quantitative expression of distinct EAAT2 splice forms in ALS cannot be excluded from this study and remain to be investigated.

Phase II/III randomized trial of TCH346 in patients with ALS

Background: TCH346 exerts antiapoptotic effects by binding to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and blocking the apoptotic pathway in which GAPDH is involved. Apoptosis is considered to be a key pathogenic mechanism in neurodegenerative diseases including ALS. Methods: Patients were randomly assigned in a double-blind fashion to receive either placebo or one of four doses of TCH346 (1.0, 2.5, 7.5, or 15 mg/day) administered orally once daily for at least 24 weeks. The primary outcome measure was the rate of change in the revised ALS functional rating scale (ALSFRS-R). The trial design included a 16-week lead-in phase to determine each patients rate of disease progression. The between treatment comparison was adjusted for the individual pretreatment rates of progression. The study was powered to detect a 25% reduction in the rate of decline of the ALSFRS-R as compared with placebo. Secondary outcome measures included survival, pulmonary function, and manual muscle testing (MMT). Results: Five hundred ninety-one patients were enrolled at 42 sites in Europe and North America. There were no differences in baseline variables. There were no significant differences between placebo and active treatment groups in the mean rate of decline of the ALSFRS-R or in the secondary outcome measures (survival, pulmonary function, and MMT). Conclusion: The trial revealed no evidence of a beneficial effect of TCH346 on disease progression in patients with ALS.

Ataxin-2 intermediate-length polyglutamine expansions in European ALS patients

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disease primarily affecting motor neurons. We recently identified intermediate-length polyglutamine (polyQ) expansions (27-33 Qs) in ataxin 2 as a genetic risk factor for sporadic ALS in North American ALS patients. To extend these findings, we assessed the ataxin 2 polyQ repeat length in 1294 European ALS patients and 679 matched healthy controls. We observed a significant association between polyQ expansions and ALS (>30 Qs; P= 6.2 × 10(-3)). Thus, intermediate-length ataxin 2 polyQ repeat expansions are associated with increased risk for ALS also in the European cohort. The specific polyQ length cutoff, however, appears to vary between different populations, with longer repeat lengths showing a clear association. Our findings support the hypothesis that ataxin 2 plays an important role in predisposing to ALS and that polyQ expansions in ataxin 2 are a significant risk factor for the disease.

Guidelines for the preclinical in vivo evaluation of pharmacological active drugs for ALS/MND: report on the 142nd ENMC international workshop

A transgenic animal model for anterior horn cell loss was established in 1994. This model is based on the insertion of a high copy number of disease‐causing human Cu/Zn SOD mutations into the intact mouse genome. It serves to establish hypotheses for the pathogenesis of anterior horn cell death, but also to test potential pharmacological approaches to therapy in human ALS. Today, more than 100 – published and unpublished – compounds have been tested in this animal model, a large part of them being reported as successful. However, it proved to be difficult to translate these therapeutic successes in the animal model into human trials. Also, a number of disease‐modifying strategies were difficult to reproduce, even by the same group. On the other hand, the step from mice to men means a huge investment for the sponsors of clinical trials and the scientific community. Therefore, establishment of standard methods for drug testing in ALS models is mandatory. In this workshop, clinical and preclinical researchers established in the field of ALS/MND met in Holland in March 2006 in order to establish guidelines for the community for drug testing in mouse models.

The role of excitotoxicity in ALS : what is the evidence?

Abstract It is well accepted that excitotoxic mechanisms contribute to the pathogenesis of acute neuronal death in stroke, epilepsy, or brain trauma. It is less widely acknowledged that excitotoxic mechanisms play a role in the pathogenesis of chronic neurological disorders, in particular neurodegenerative diseases. However, evidence is accumulating that this mechanism is indeed part of the pathogenesis of late-onset neurodegenerative diseases. One of the clinical examples may be amyotrophic lateral sclerosis, a disease in which antiexcitotoxic strategies have neuroprotective effects in both, an established animal model and in man. In addition, there is accumulating neuropathological, pathobiochemical and pathophysiological evidence which indicates that excitotoxic mechanisms are part of the pathogenesis of the human disease and consequently part of the mechanisms explaining selective vulnerability (“pathoclisis”) in the human motor system.

A Randomized, Double Blind, Placebo-Controlled Trial of Pioglitazone in Combination with Riluzole in Amyotrophic Lateral Sclerosis

Background Pioglitazone, an oral anti-diabetic that stimulates the PPAR-gamma transcription factor, increased survival of mice with amyotrophic lateral sclerosis (ALS). Methods/Principal Findings We performed a phase II, double blind, multicentre, placebo controlled trial of pioglitazone in ALS patients under riluzole. 219 patients were randomly assigned to receive 45 mg/day of pioglitazone or placebo (one: one allocation ratio). The primary endpoint was survival. Secondary endpoints included incidence of non-invasive ventilation and tracheotomy, and slopes of ALS-FRS, slow vital capacity, and quality of life as assessed using EUROQoL EQ-5D. The study was conducted under a two-stage group sequential test, allowing to stop for futility or superiority after interim analysis. Shortly after interim analysis, 30 patients under pioglitazone and 24 patients under placebo had died. The trial was stopped for futility; the hazard ratio for primary endpoint was 1.21 (95% CI: 0.71–2.07, pu200a=u200a0.48). Secondary endpoints were not modified by pioglitazone treatment. Pioglitazone was well tolerated. Conclusion/Significance Pioglitazone has no beneficial effects on the survival of ALS patients as add-on therapy to riluzole. Trial Registration Clinicaltrials.gov NCT00690118.

Induction of apoptosis by Toll-like receptor-7 agonist in tissue cultures.

Toll‐like receptor (TLR)‐7 agonists represent a new group of immune response modifiers, which include imiquimod and resiquimod (R‐848). Topically applied imiquimod is used for the treatment of both external and perianal genital warts, and benign and malignant epithelial lesions. Based on the induction of interferons and other cytokines in vitro and in vivo, regression of epithelial lesions probably depends on induction of both innate and cellular immune responses. As clinical remission is not always associated with inflammation, other mechanisms may also be involved. Using two different assays for detection of apoptosis (TUNEL test and gel analysis of DNA fragmentation), we observed induction of apoptosis by imiquimod in human epithelial cell lines (HeLa S3) and keratinocytes (HaCaT, A431 cells), as well as in mouse fibroblasts (McCoy cells). These findings suggest that the mode of action of imiquimod to eliminate virus‐infected, dysplastic or neoplastic epithelial cells may also include the induction of apoptotic processes.

Novel missense and truncating mutations in FUS/TLS in familial ALS

Background: Mutations in the FUS/TLS gene have been associated with familial amyotrophic lateral sclerosis (FALS). Methods: We analyzed the presence and frequency of C-terminal FUS/TLS mutations in a German amyotrophic lateral sclerosis (ALS) cohort, including 133 patients with sporadic ALS (SALS) and 58 patients with FALS by sequence analysis of exons 13–15. Results: We identified 2 novel heterozygous FUS/TLS mutations in 4 German ALS families including the novel missense mutation K510R and the truncating mutation R495X. The truncating mutation was associated with an aggressive disease course whereas the K510R mutation showed a mild phenotype with disease duration ranging from 6 to 8 years. No mutation was detected in 133 patients with SALS. Conclusions: Mutations in FUS/TLS account for 7% (4 of 58) of FALS in our German cohort.