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P10.3 Treating Krabbe disease: neurophysiological assessment of a mouse model
14th ECCN / 4th ICTMS/DCS values of the scalograms in five selected scales, and averaging across MUAPs to give a single 5-dimensional feature vector per subject. After SVM analysis, the vector is reduced to a single decision parameter, called the Wavelet Index, allowing the subject to be assigned to one of three groups: myogenic, neurogenic or normal. The software implementation of the presented method created a tool supporting EMG examination. The method is characterized by a high probability for the accurate diagnosis of muscle state. Authors received 5 misclassifications out of 800 examined cases (total error of 0.6%). The authors hope that this method gives useful tool for classification of healthy and irregular MUAPs. P10.3 Treating Krabbe disease: neurophysiological assessment of a mouse model D. Ungaro1 , I. Visigalli2 , S. Ungari2 , S. Amadio1 , C. Butera1 , F. Bianchi1 , A. Biffi2 , G. Comi1 , U. Del Carro1 1 San Raffaele Neurology and Neurophysiology Department, Milan, Italy, 2 San Raffaele Telethon Institute for Gene Therapy, Milan, Italy Introduction: Globoid cell leukodystrophy (GLD) or Krabbe disease is a lysosomal storage disorder due to the inherited deficiency of galactocerebrosidase (GALC) activity. The enzymatic defect results in the storage of undegraded metabolites in the nervous systems, leading to progressive dysmyelination and early death of the affected patients. In previous works it has been demonstrated that transplantation of hematopoietic stem cells (HSC) genetically corrected by means of a lentiviral vector (LV) can correct disease manifestations in murine models of neurodegenerative lysosomal diseases. Aim of the study: We performed a neurophysiological study on GLD mice transplanted with HSC transduced with LV encoding the functional GALC gene and to investigated whether this therapy could prevent the development of motor conduction impairments. Methods: The sciatic nerve motor conduction velocity (MCV) and Motor evoked potentials (MEP) by transcranial electric stimulation (TES) were performed to obtain respectively peripheral and central nervous system functional parameters. An unpaired Student’s t test was performed for statistical evaluation of the data. Results: The F wave latency, the motor conduction velocity and MEPs latency showed a significant improvement in treated GLD mice at comparison with untreated affected controls (respectively F wave: 18.1 ms vs 15.65 ms p = 0.02; MCV: 3.5 m/s vs 10.8 m/s p = 0.001 MEPs latency 8.4 ms vs 5.10 ms p = 0.01). Central parameters (cMEP and CCT) were recordable at the 35 post-natal days in all the treated mice and in only 2 of 6 untreated mice (respectively cMEP latency:17.35 vs 13.55 ms p = 0.001; CCT 10.25 vs 8.45 ms p = 0.02). Conclusion: The transplantation of gene-corrected HSC by re-establishing the defective enzymatic activity in the hematopoietic system of GLD mice allows preventing the development of the central and peripheral motor conduction impairment typical of the disease. P10.4 Analysis of ryanodine receptor 1 (RyR1) and voltage-gated Ca2+ channel (VGCC) a1s subunit (Cav 1.1) pre-mRNA splicing and correlation with intracellular calcium signals in myotonic dystrophy type 1 (DM1) and in myotonic dystrophy type 2 (DM2) myotubes M. Santoro1 , R. Piacentini2 , M. Masciullo1 , C. Grassi2 , A. Modoni1 , E. Ricci1 , G. Silvestri1 1 Department of Neuroscience, Center of Neuromuscular Disorders, Catholic University of Sacred Heart, Rome, Italy, 2 Institute of Human Physiology, Catholic University of Sacred Heart, Rome, Italy Introduction: The pathogenesis of myotonic dystrophy type 1 (DM1) and type 2 (DM2) has been related to nuclear accumulation of RNAs containing expanded CUG and CCUG sequences, respectively, which sequester RNA-binding proteins, thus affecting in trans the alternative splicing of several genes. The evidence of increased intracellular Ca2+ ([Ca2+ ]i ) levels documented in DM1 myotubes suggested that an altered Ca2+ homeostasis may contribute to the muscle pathology in myotonic dystrophy. Accordingly, aberrant splicing of ryanodine receptor 1 (RyR1) and sarcoplasmatic/endoplasmatic Ca2+ -ATPase were found in DM1 muscle tissues. However, data regarding RyR1 expression and intracellular Ca2+ handling in DM2 muscle tissues have not been reported. Objectives: To determine whether the expression of RyR1 and voltagegated Ca2+ channel (VGCC) a1s subunit (Cav 1.1) genes in myotubes
S105 from DM1 and DM2 patients are associated with alteration of [Ca2+ ]i transients. Methods: The mRNA processing of RyR1 and Cav 1.1 in myotubes from healthy controls, DM1 and DM2 patients was studied by RT-PCR. [Ca2+ ]i transients induced by VGCC or RyR1 activation through stimulation with 100mM KCl or 20mM caffeine were studied by confocal Ca2+ imaging. Results: Abnormal RyR1 splicing was found in DM1 but not in DM2 myotubes. On the other hand, Cav 1.1 mRNA processing was normal in both muscle cultures. In DM1 myotubes the mean amplitude of [Ca2+ ]i transients induced by KCl was greater than controls (6.6±1.4 vs. 5.7±1.9 DF/F, respectively, n = 53, p < 0.05), whereas in DM2 it was lower (3.7±1.4 DF/F, p < 0.01). In both DM1 and DM2 myotubes Ca2+ released through RyRs after caffeine stimulation was lower than controls ( 25 and 30%, respectively, p < 0.01). Conclusion: Our data suggest that [Ca2+ ]i transients induced by KCl and caffeine are altered in both myotonic dystrophies, but only in DM1 these alterations are related with RyR1 aberrant splicing. P10.5 New mutations in muscle voltage-gated sodium channel and their biophysical properties A. Modoni1 , S. Pagliarani2 , G. Silvestri1 , A. D’Amico3 , E. Redaelli4 , E. Wanke4 , G.P. Comi2 , M. Lo Monaco1 1 Dept. of Neuroscience, Universit` a Cattolica del Sacro Cuore, Rome, Italy, 2 Dino Ferrari Center, Department of Neurological Sciences, University of Milan, Foundation I.R.C.C.S. C` a Granda, Ospedale u Research Pediatric Maggiore Policlinico, Milan, Italy, 3 Bambino Ges` a Milano-Bicocca, Dipartimento di Hospital, Rome, Italy, 4 Universit` Biotecnologie e Bioscienze, Milan, Italy Introduction: Muscle voltage-gated sodium channel gene (SCN4A) mutations are usually associated with different clinical phenotypes, including non-distrophic myotonias, hyperkalemic periodic paralysis and hypokalemic periodic paralysis type II. Objective: To describe the clinical and electrophysiological properties of two new genetic SCN4A variants. Methods: SCN4A was sequenced in one subject affected by hypokaliemic periodic paralysis and in one proband presenting clinical myotonia without weakness. Genetic analysis was performed in patients’ parents in both cases. The new variants were cloned and their electrophysiological properties were studied by recording whole-cell Na+ currents from TSA cells transiently transfected with the human SCN4A cDNA. Results: We found two new variants in SCN4A gene. The functional study of the first mutation (p.Gly241Val), clinically presenting with myotonia, showed a hyperpolarizing shift (about 10mV) of the voltage dependence of activation, leading to a significant increase of the window current amplitude. Preliminary studies for the second variant (p.Arg1132Gly), associated with hypokaliemic periodic paralysis, showed no biophysical differences from the wild-type: activation, fast and slow inactivation were not appreciably affected. Otherwise there seems to be a reduction of current density. Conclusions: This study describes the biophysical properties of two new mutations of the human SCN4A. One of these show a hyperpolarizing shift of activation. This effect confirms “gain-of-function” alterations leading to muscle hyperexcitability and myotonic phenotype. The reduction of Na+ current observed in the second variant seems a “loss-of-function” effect that can account, at least in part, for membrane hypoexcitability and muscle weakness, compatible with paralysis phenotype. Cloning and functional studies of new variants are important to distinguish between new pathologic mutations and very rare polymorphisms. P10.6 Dopamine-modulated cross-frequency coupling of local field potential oscillations in the rat motor circuit as1 , M. Valencia1 , M. Alegre1 , J. Artieda1 J. L´ opez-Azc´ arate1 , M.J. Nicol´ Neuroscience Area, Centro de Investigaci´ on M´ edica Aplicada, Universidad de Navarra, Pamplona, Spain
1
Brain oscillations have been classically divided into specific frequency ranges associated with multiple processes. Oscillatory rhythms in these frequencies can interact in several ways. Recently, phase-to-amplitude coupling between neuronal oscillations has received and increasing interest. Study the presence of cross-frequency couplings in the motor circuit of the healthy rat and its modulation with dopamine agonist and antagonist drugs. We recorded local field potentials from motor cortex,
S105 from DM1 and DM2 patients are associated with alteration of [Ca2+ ]i transients. Methods: The mRNA processing of RyR1 and Cav 1.1 in myotubes from healthy controls, DM1 and DM2 patients was studied by RT-PCR. [Ca2+ ]i transients induced by VGCC or RyR1 activation through stimulation with 100mM KCl or 20mM caffeine were studied by confocal Ca2+ imaging. Results: Abnormal RyR1 splicing was found in DM1 but not in DM2 myotubes. On the other hand, Cav 1.1 mRNA processing was normal in both muscle cultures. In DM1 myotubes the mean amplitude of [Ca2+ ]i transients induced by KCl was greater than controls (6.6±1.4 vs. 5.7±1.9 DF/F, respectively, n = 53, p < 0.05), whereas in DM2 it was lower (3.7±1.4 DF/F, p < 0.01). In both DM1 and DM2 myotubes Ca2+ released through RyRs after caffeine stimulation was lower than controls ( 25 and 30%, respectively, p < 0.01). Conclusion: Our data suggest that [Ca2+ ]i transients induced by KCl and caffeine are altered in both myotonic dystrophies, but only in DM1 these alterations are related with RyR1 aberrant splicing. P10.5 New mutations in muscle voltage-gated sodium channel and their biophysical properties A. Modoni1 , S. Pagliarani2 , G. Silvestri1 , A. D’Amico3 , E. Redaelli4 , E. Wanke4 , G.P. Comi2 , M. Lo Monaco1 1 Dept. of Neuroscience, Universit` a Cattolica del Sacro Cuore, Rome, Italy, 2 Dino Ferrari Center, Department of Neurological Sciences, University of Milan, Foundation I.R.C.C.S. C` a Granda, Ospedale u Research Pediatric Maggiore Policlinico, Milan, Italy, 3 Bambino Ges` a Milano-Bicocca, Dipartimento di Hospital, Rome, Italy, 4 Universit` Biotecnologie e Bioscienze, Milan, Italy Introduction: Muscle voltage-gated sodium channel gene (SCN4A) mutations are usually associated with different clinical phenotypes, including non-distrophic myotonias, hyperkalemic periodic paralysis and hypokalemic periodic paralysis type II. Objective: To describe the clinical and electrophysiological properties of two new genetic SCN4A variants. Methods: SCN4A was sequenced in one subject affected by hypokaliemic periodic paralysis and in one proband presenting clinical myotonia without weakness. Genetic analysis was performed in patients’ parents in both cases. The new variants were cloned and their electrophysiological properties were studied by recording whole-cell Na+ currents from TSA cells transiently transfected with the human SCN4A cDNA. Results: We found two new variants in SCN4A gene. The functional study of the first mutation (p.Gly241Val), clinically presenting with myotonia, showed a hyperpolarizing shift (about 10mV) of the voltage dependence of activation, leading to a significant increase of the window current amplitude. Preliminary studies for the second variant (p.Arg1132Gly), associated with hypokaliemic periodic paralysis, showed no biophysical differences from the wild-type: activation, fast and slow inactivation were not appreciably affected. Otherwise there seems to be a reduction of current density. Conclusions: This study describes the biophysical properties of two new mutations of the human SCN4A. One of these show a hyperpolarizing shift of activation. This effect confirms “gain-of-function” alterations leading to muscle hyperexcitability and myotonic phenotype. The reduction of Na+ current observed in the second variant seems a “loss-of-function” effect that can account, at least in part, for membrane hypoexcitability and muscle weakness, compatible with paralysis phenotype. Cloning and functional studies of new variants are important to distinguish between new pathologic mutations and very rare polymorphisms. P10.6 Dopamine-modulated cross-frequency coupling of local field potential oscillations in the rat motor circuit as1 , M. Valencia1 , M. Alegre1 , J. Artieda1 J. L´ opez-Azc´ arate1 , M.J. Nicol´ Neuroscience Area, Centro de Investigaci´ on M´ edica Aplicada, Universidad de Navarra, Pamplona, Spain
1
Brain oscillations have been classically divided into specific frequency ranges associated with multiple processes. Oscillatory rhythms in these frequencies can interact in several ways. Recently, phase-to-amplitude coupling between neuronal oscillations has received and increasing interest. Study the presence of cross-frequency couplings in the motor circuit of the healthy rat and its modulation with dopamine agonist and antagonist drugs. We recorded local field potentials from motor cortex,