Torsten Grehl

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.

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, p = 0.48). 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.

Screening in ALS and FTD patients reveals 3 novel UBQLN2 mutations outside the PXX domain and a pure FTD phenotype.

Mutations in UBQLN2 have recently been shown to cause dominant X-linked amyotrophic lateral sclerosis (ALS) and ALS plus frontotemporal dementia (FTD). Information on their frequency in different populations is still rare, and a pure FTD phenotype has not yet been reported. Moreover, the mutational spectrum of known UBQLN2 mutations is still limited to its PXX repeat region. Based on a screening of 206 ALS and FTD patients, we here report 3 novel UBQLN2 mutations, accounting for 1.2% (2/161) ALS and 2.2% (1/45) FTD patients, including a patient with pure FTD. All mutations were located in highly conserved domains outside the PXX repeat region and not observed in 1450 ethnically matched control X-chromosomes. All affected patients presented with apparently sporadic disease. UBQLN2 mutations are rare in Central European ALS and FTD patients, but contribute significantly to patients with seemingly sporadic disease. UBQLN2 is able to cause any disease on the ALS-FTD continuum, including pure FTD. Because the pathogenic mechanism of UBQLN2 mutations is not limited to its PXX region, UBQLN2 screening in neurodegenerative patients should not be limited to this region.

Low energy dual beam depth profiling: influence of sputter and analysis beam parameters on profile performance using TOF-SIMS

Abstract In the dual beam mode for TOF-SIMS depth profiling, the high energy analysis beam performs the analysis in the center of the sputter crater. Usually, the depth resolution is determined only by the parameters of the low energy sputter beam eroding the sample given that its sputter rate—and therefore, its contribution to the atomic mixing—is large compared to the sputter rate of the analysis beam. Typically, in shallow depth profiling the ratio of the sputter rates R is larger than 100. In microarea depth profiling however, the area to be analyzed is comparatively small. If the analysis current is kept high in order to get a good dynamic range, the sputter rate of the analysis beam is increased and its contribution to the atomic mixing becomes significant. We have investigated the dependence of the depth resolution on the sputter rate ratio for different sputter and analysis beam energies, using delta layer samples of B and Ge in Si. From these results, the value of R, below which the mixing of the analysis beam can degrade the depth resolution, can be estimated. On the other hand, the amount of additional mixing in applications that require low values of R like 3D microanalysis can be derived as well.

Breakdown of adenine nucleotide pool in fatiguing skeletal muscle in McArdle's disease: a noninvasive 31P-MRS and EMG study.

Energy metabolism and electrical muscle activity were studied in the calf muscles of 19 patients with proven McArdles disease and in 25 healthy subjects. Phosphorus magnetic resonance spectroscopy and surface electromyography (S‐EMG) were performed during two isometric muscle contractions of 3 min at 30% maximum voluntary contraction, one performed during normal perfusion and the other during applied ischemia. After about 1 min of ischemic muscle contraction in diseased muscle a significant acceleration in phosphocreatine breakdown was observed, along with a significant decrease in adenosine triphosphate. During both contractions the absence of glycolysis was shown by a significant alkalinization. Furthermore, in patients we observed a greater increase in the S‐EMG amplitude than in control subjects. We conclude that early on during moderate exercise, a small number of muscle fibers reach metabolic depletion, indicated by a reduction in the adenine nucleotide pool. An increasing number of motor units, which are still in a high‐energy state, are continuously recruited to compensate for muscle fatigue. This functional compartmentation may contribute to the pathophysiology of exercise intolerance in McArdles disease. Muscle Nerve 27: 728–736, 2003

Alterations in the hypothalamic melanocortin pathway in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis, the most common adult-onset motor neuron disease, leads to death within 3 to 5 years after onset. Beyond progressive motor impairment, patients with amyotrophic lateral sclerosis suffer from major defects in energy metabolism, such as weight loss, which are well correlated with survival. Indeed, nutritional intervention targeting weight loss might improve survival of patients. However, the neural mechanisms underlying metabolic impairment in patients with amyotrophic lateral sclerosis remain elusive, in particular due to the lack of longitudinal studies. Here we took advantage of samples collected during the clinical trial of pioglitazone (GERP-ALS), and characterized longitudinally energy metabolism of patients with amyotrophic lateral sclerosis in response to pioglitazone, a drug with well-characterized metabolic effects. As expected, pioglitazone decreased glycaemia, decreased liver enzymes and increased circulating adiponectin in patients with amyotrophic lateral sclerosis, showing its efficacy in the periphery. However, pioglitazone did not increase body weight of patients with amyotrophic lateral sclerosis independently of bulbar involvement. As pioglitazone increases body weight through a direct inhibition of the hypothalamic melanocortin system, we studied hypothalamic neurons producing proopiomelanocortin (POMC) and the endogenous melanocortin inhibitor agouti-related peptide (AGRP), in mice expressing amyotrophic lateral sclerosis-linked mutant SOD1(G86R). We observed lower Pomc but higher Agrp mRNA levels in the hypothalamus of presymptomatic SOD1(G86R) mice. Consistently, numbers of POMC-positive neurons were decreased, whereas AGRP fibre density was elevated in the hypothalamic arcuate nucleus of SOD1(G86R) mice. Consistent with a defect in the hypothalamic melanocortin system, food intake after short term fasting was increased in SOD1(G86R) mice. Importantly, these findings were replicated in two other amyotrophic lateral sclerosis mouse models based on TDP-43 (Tardbp) and FUS mutations. Finally, we demonstrate that the melanocortin defect is primarily caused by serotonin loss in mutant SOD1(G86R) mice. Altogether, the current study combined clinical evidence and experimental studies in rodents to provide a mechanistic explanation for abnormalities in food intake and weight control observed in patients with amyotrophic lateral sclerosis. Importantly, these results also show that amyotrophic lateral sclerosis progression impairs responsiveness to classical drugs leading to weight gain. This has important implications for pharmacological management of weight loss in amyotrophic lateral sclerosis.

Spinal cord organotypic slice cultures for the study of regenerating motor axon interactions with 3D scaffolds

Numerous in-vitro techniques exist for investigating the influence of 3D substrate topography on sensory axon growth. However, simple and cost-effective methods for studying post-natal motor axon interactions with such substrates are lacking. Here, spinal cord organotypic slice cultures (OSC) from post-natal day 7-9 rat pups were presented with spinal nerve roots, or blocks of fibrin hydrogel or 3D microporous collagen scaffolds to investigate motor axon-substrate interactions. By 7-14 days, axons from motor neuronal pools extended into the explanted nerve roots, growing along Schwann cell processes and demonstrating a full range of axon-Schwann cell interactions, from simple ensheathment to concentric wrapping by Schwann cell processes and the formation of compact myelin within a basal lamina sheath. Extensive motor axon regeneration and all stages of axon-Schwann interactions were also supported within the longitudinally orientated microporous framework of the 3D collagen scaffold. In stark contrast, the simple fibrin hydrogel only supported axon growth and cell migration over its surface. The relative ease of demonstrating such motor axon regeneration through the microporous 3D framework by immunofluorescence, two-photon microscopy and transmission electron microscopy strongly supports the adoption of this technique for assaying the influence of substrate topography and functionalization in regenerative bioengineering.

Impaired aerobic glycolysis in muscle phosphofructokinase deficiency results in biphasic post-exercise phosphocreatine recovery in 31P magnetic resonance spectroscopy

Using 31P magnetic resonance spectroscopy, energy metabolism in calf muscles of two patients with biochemically and genetically proven muscular phosphofructokinase deficiency, and an asymptomatic heterozygote was monitored during isometric foot plantarflexion performed under aerobic and anaerobic conditions and in the aerobic recovery phases. In the heterozygote only a moderate alteration from normal was found in terms of an elevated ATP demand during exercise. In the homozygote, hexose phosphates, indicated as phosphomonoesters, increased dramatically during contraction. Phosphomonoester accumulation resulted in consumption of free inorganic phosphate (P(i)). During ischemic exercise the absence of glycolytic ATP formation resulted in a linear time course of phosphocreatine breakdown and a moderate alkalinization. During the recovery, phosphocreatine resynthesis showed a biphasic time course, indicating that mitochondrial function itself was not directly affected. At first glance, the early depletion of P(i) below initial resting levels and the rate of phosphate splitting from sugar phosphates seemed to become the limiting factor for the rate of the oxidative phosphorylation and creatine kinase reaction. However, the actual concentrations of P(i) and ADP estimated at the onset of delay were too high to exclusively explain the dramatic delay in PCr resynthesis. For this reason, a reduced turnover of the citric acid cycle was assumed, which was caused by the complete absence of glycolysis in PFK deficiency patients. Furthermore, results from PFK deficiency patients were compared with previous findings from myophosphorylase deficiency patients in the literature.

Spatial atmospheric atomic layer deposition of InxGayZnzO for thin film transistors.

We have investigated the nucleation and growth of InGaZnO thin films by spatial atmospheric atomic layer deposition. Diethyl zinc (DEZ), trimethyl indium (TMIn), triethyl gallium (TEGa), and water were used as Zn, In, Ga and oxygen precursors, respectively. The vaporized metal precursors have been coinjected in the reactor. The metal composition of InGaZnO has been controlled by varying the TMIn or TEGa flow to the reactor, for a given DEZ flow and exposure time. The morphology of the films changes from polycrystalline, for ZnO and In-doped ZnO, to amorphous for In-rich IZO and InGaZnO. The use of these films as the active channel in TFTs has been demonstrated and the influence of In and Ga cations on the electrical characteristics of the TFTs has been studied.

Depression and QOL in patients with ALS: how do self-ratings and ratings by relatives differ?

BackgroundAmyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease affecting the motor nervous system and currently lacking effective means of treatment. The focus of ALS treatment therefore lies in palliative treatment from a multidisciplinary team. Published findings regarding affective components and patients’ perceived quality-of-life (QoL) as well as comparative reports of family members/caregivers remain equivocal.MethodsIn this study, 41 ALS patients and their relatives were enrolled in a study employing the 12-item ALS-Depression-Inventory (ADI-12) and the Munich quality-of-life dimensions list (MLDL). The ALS-functional rating scale (ALSFRS-R) was used to evaluate physical disabilities.ResultsThe ADI-12 depression scale data identified nine patients with depressive disorders; the patients had satisfactory QoL outcomes on the MLDL. The results did not differ significantly between ALS patients and their relatives.ConclusionsThus, in agreement with other studies, QoL and emerging depression do not automatically coincide with patients’ physical impairments of the patients. This “well-being paradox” is currently not well understood, and further studies are needed to optimize the treatment of patients through the course of disease progression.