P48 ANALYSIS OF GENE EXPRESSION USING WHOLE GENOME ARRAYS IN NORMAL AND SMA AFFECTED TISSUES
van den Heuvel M, Sayin A & Lee S
University of Oxford, Oxford, UK
E‐mail address for correspondence: [email protected]
The loss of motor neurons and muscle atrophy in spinal muscular atrophy is caused by the reduced activity of the survival motor neuron (SMN) gene. This gene codes for a protein that has been shown to be required for assembly of large complexes involved in RNA processing, such as the spliceosome and ribonucleo protein (RNP) particles. Such a function for SMN protein does not explain why, when function is reduced, specifically the motor neuron and/or the muscle are affected.
We have created a Drosophila melanogaster model for SMA by mutating the endogenous smn gene. Reduction of zygotic activity of this gene leads to neuromuscular defects in affected larvae. The characterization of these defects shows that they concentrate at the neuromuscular junction (NMJ) (1). In order to further our understanding of the connection between smn function and the RNA associated processes, we generated RNA samples of smn mutant and normal fly tissues (the genetic background we used as ‘normal’ is as close to the smn mutant background as possible, except for the offending mutation). Using standard protocols and adhering to strict MIAME guidelines, labelled RNA was produced using these RNAs as templates. Each of these samples was then hybridized to Affymetrix Drosophila whole genome arrays (in triplicate or quadruplicate). The resulting hybridization results were normalized employing several different methods. The most reliable of these were used and gene ontology determined for the genes for which expression profiles were changing comparing normal with smn affected. We will discuss the technical background to these experiments as well as the further experimentation to back up the array findings. The most interesting genes that are implicated in this system will be presented.
Reference
- Chan YB, Miguel-Aliaga I, Franks C, et al. Human Molecular Genetics 2003:12:1367–76.
P49 ANALYSIS OF TRANSCRIPT TRANSPORT AND TRANSLATION CONTROL BY THE SURVIVAL MOTOR NEURON PROTEIN
van den Heuvel M, Thomas N & Lee L
University of Oxford, Oxford, UK
E‐mail address for correspondence: [email protected]
The loss of motor neurons and muscle atrophy in spinal muscular atrophy is caused by the reduced activity of the survival motor neuron (SMN) gene. This gene codes for a protein that has been shown to be required for assembly of large complexes involved in RNA processing, such as the spliceosome and ribonucleo protein (RNP) particles. Such a function for SMN protein does not explain why, when function is reduced, specifically the motor neuron and/or the muscle are affected.
We have created a Drosophila melanogaster model for SMA by mutating the endogenous smn gene. Reduction of zygotic activity of this gene leads to neuromuscular defects in affected larvae. The characterization of these defects shows that they concentrate at the neuromuscular junction (NMJ) (1). In order to further our understanding of the connection between smn function and the RNA associated processes, we generated smn mutant fly ovaries. These tissues have been studied over the last decades specifically because they appear to provide an easy accessible (by genetics) system in which several mRNAs are translocated and translationally controlled in very precise ways. It is also clear that these processes are driven by components of a machinery of a set of highly conserved gene functions.
Smn mutant ovaries develop in ways very similar to ovaries mutant for several other gene functions; these encode for proteins known to associate with either smn function or with RNP function. Several mRNA transcripts that are normally localized to particular positions in the egg chamber are dispersed in smn mutant ovaries. In the normal tissues, the translocation of such transcripts is coupled to their translation inhibition. This system allows the transport of the transcripts to a particular position; translocation is controlled to allow translation to take place only when they have arrived at their appropriate place. This generates not only localized protein function but also controlled high concentrations of proteins in these regions. This coupled translocation and translation control process seems disrupted in smn mutant ovaries. Premature translation leading to mis‐localized protein is observed. Our findings will be discussed in the light of a role for smn in transcript control at the NMJ.
Reference
- Chan YB, Miguel-Aliaga I, Franks C, et al. Human Molecular Genetics 2003:12:1367–76.
P50 THE SLOW WALLERIAN DEGENERATION GENE (WLDS) DOES NOT PREVENT APOPTOSIS OF MOTOR NEURON CELL BODIES
Adalbert R1, Nogradi A2, Szabo A2 & Coleman MP1
1The Babraham Institute, Cambridge, UK, and 2University of Szeged, Szeged, Hungary
E‐mail address for correspondence: [email protected]
Background: The slow Wallerian degeneration gene (WldS) delays the degeneration of injured axons ten‐fold and delays axonal death in some non‐injury disorders by several weeks, most notably in the progressive motoneuronopathy mouse (pmn). Interestingly, motor neuron cell death was also delayed in WldS/pmn mice, suggesting either that these motor neurons die secondarily to axon degeneration or that WldS has a direct neuroprotective effect on motor neuron cell bodies as well as on their axons. The question of whether WldS has a direct protective effect on neuronal cell bodies has previously been addressed only in sympathetic neurons in vitro, where WldS did not prevent apoptosis. However, it is not clear whether those data hold for motor neurons in vivo.
Objectives: Thus, we tested the hypothesis that WldS can directly protect motor neurons whose axons have been almost completely removed by intravertebral avulsion. We also tested for a delay in apoptotic motor neuron death following neonatal nerve injury in WldS rats.
Methods: Intravertebral avulsion is one of a number of difficult surgical techniques where the effect of WldS can now be studied following our development of a WldS rat model. L4 ventral roots were avulsed in three‐month‐old rats and motor neuron survival assessed in cresyl violet (Nissl stained) 25 µm cryostat sections 3–4 weeks later. Neonatal nerve injury was made by crushing sciatic nerve in P3 rats and motor neuron survival assessed after 3–14 days.
Results: Eighteen percent and 16% of motor neurons survived in WldS rats three and four weeks, respectively, after intravertebral avulsion. These numbers were similar to wild‐type controls. The motor neuron survival rate after neonatal nerve injury was also little altered. Forty percent of motor neurons survived after three days and 30–35% after 7–14 days.
Conclusions: We conclude that WldS has no direct neuroprotective effect on motor neuron cell bodies in vivo. Thus, the increase in motor neuron survival in WldS/pmn mice most probably shows that motor neuron death in the pmn mutant is secondary to axon death. Our data show how WldS can be used as a tool to test whether various ALS models bring about motor neuron death directly or whether this death is secondary to axon degeneration. The data also support the model of compartmentalised cell death programmes in motor neurons and show how the availability of WldS rats has made possible new surgical studies in this field.
P51 A NEW MOUSE MODEL FOR MOTOR NEURON DISEASE: THE DOUBLE HETEROZYGOTE CRA1/SODG93A MOUSE
Teuchert M, Schwalenstoecker B, Meier S, Wipp T & Ludolph AC
Department of Neurology, University of Ulm, Ulm, Germany
E‐mail address for correspondence: [email protected]
Background: Familial forms of ALS are associated with mutations in the gene for the Cu/Zn superoxide dismutase (SOD1). These mutations gave rise to several animal models, the G93A (Gly ‐>Ala) mutation being most commonly used. Transgenic mice expressing the mutated human Cu/Zn superoxide dismutase (SOD1G93A) develop motor neuron pathology and clinical symptoms similar to those seen in patients with ALS. Another model for motor neuron disease is the Cra1/+ mouse. A missense point mutation in the motor protein dynein, leading to impairment of retrograde axonal transport, results in progressive motor neuron degeneration in Cra1/ heterozygous mice, without major reduction in lifespan.
Objectives: The objective of this study was to compare the phenotype of double heterozygote Cra1/SODG93A mice to hemizygote SOD1G93A mice with regard to motor activity, survival time and weight, expecting a more severe phenotype because of the added defect of dynein. Furthermore, we compared the number of motor neurons in the ventral horn of the spinal cord between both groups.
Methods:Cra1/+ females (background C3), obtained from Ingenium Pharmaceuticals AG, Martinsried, Germany were crossed with SOD1G93A males (background B6), obtained from The Jackson Laboratory, Maine, USA. F1 offspring (Cra1/SODG93A, SOD1G93A) was used for investigations. Cra1/+ and +/+ wild‐type mice served as controls. Motor activity was measured by Rotarod. Survival, weight, and Rotarod data were analysed statistically. For histological investigations, mice were perfused with 4% paraformaldehyde. Cervical and lumbar spinal cord were dissected and processed according to standard protocols. Slices (10 µm) were prepared and stained with cresyl violet (Nissl staining).
Results: Surprisingly, the double heterozygote Cra1/SODG93A mice showed a statistically significant extension of lifespan and improved motor activity compared to hemizygote SOD1G93A mice. Moreover, the loss of body weight of Cra1/SODG93A mice was reduced compared to SOD1G93A mice during disease progression.
Conclusions: Our data suggest that an impairment of retrograde fast axonal transport leads to an attenuation of the mice SODG93A phenotype. Further studies will be necessary to reveal the molecular mechanisms of these findings.
P52 CONTRIBUTION OF BACKGROUND STRAIN TO PHENOTYPE IN G93A MUTANT SOD1 TRANSGENIC MICE
Hong Y, Coltas I, Reese L, Gibson D & Figlewicz DA
University of Michigan, Ann Arbor, MI, USA
E‐mail address for correspondence: [email protected]
Background: Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease of the motor system which occurs in sporadic and familial forms. Mutations in Cu/Zn superoxide dismutase (SOD1) were identified in affected individuals from a subset of familial ALS kindreds; this has led to the creation of transgenic mice which overexpress one of several mutant human SOD1 genes. The G93A mSOD transgenic mice exhibit an autosomal dominant, adult‐onset, progressive neurodegeneration of the motor system which bears a striking resemblance to ALS, both clinically and pathologically. These mice have been extensively utilized to test hypothetical mechanisms of motor neuron death and putative therapeutic agents.
Objectives: We hypothesize that genetic susceptibility combined with specific lifestyle elements, such as exposure to environmental toxins, or diet, contribute to the development of sporadic ALS. There is considerable evidence for the contribution of modifying genes and genetic background to variation in phenotypes in the mouse. We are constructing and characterizing several different mouse congenic lines carrying the human mG93A SOD1 transgene as the first step in identifying genes which can modify the course of motor neuron degeneration.
Methods: The mSOD1 trait has been bred onto DBA, HeJ, and B10 backgrounds. Transgene copy number is verified in all mice in collaboration with G. Alexander et al. (2004) (1). Beginning at 50 days of age, mice are monitored for body weight and motor skills (grip strength, hind limb splay, righting reflex, Rotarod performance, and gait). To examine the progression of cellular and molecular changes which accompany the development of motor symptoms in the three background strains, cells are isolated from frozen spinal cord sections using laser‐capture followed by quantitation of specific gene expression by real‐time RT‐PCR.
Results: We have observed that genetic background, and in some strains, gender, influences the phenotype of motor neuron degeneration in the G93A mice. Some changes in motor function (grip strength and Rotarod performance) already vary significantly between the three strains at 50 days. However, onset of motor symptoms does not correlate with ultimate survival in each mouse strain. We have found the shortest survival and most rapidly progressive disease course, as well as gender dependence of the phenotype, in the HeJ mice. Among genes whose expression levels are being quantitated, we find that specific to late stage motor neurons in HeJ mice is the significant up‐ regulation of the Ca+2 ‐permeable GluR3 AMPA receptor subtype, and decreased expression of the GluR2 editing enzyme, ADAR2.
Discussion and conclusions: Our finding of increased susceptibility in the HeJ mice is similar to what has been reported by Heiman‐Patterson et al.(2) or the G93A phenotype on the SJL mouse background. The up‐regulation of the GluR3 receptor subunit in degenerating motor neurons is consistent with our previous work establishing the role of Ca+2‐permeable AMPA receptors in mutant SOD1‐mediated motor neuron death. It is also consistent with the findings of Spalloni et al.(3) and Rembach et al. (4) in in vitro and in whole spinal cord studies of AMPA receptor subunit expression in G93A transgenic mice.
References
- Alexander GM, Erwin KL, Byers N, Deitch JS, Augelli BJ, Blankenhorn, EP, Heiman-Patterson TD Effect of transgene copy number on survival in the G93A SOD1 transgenic mouse model of ALS brain research. Molecular Brain Research 2004;130:7–15.
- Heiman-Patterson T, Deitch J, Alexander G, Erwins K, Byers N, Toman I, Blankenhorn EP. Genetic loci linked to phenotype in the G93A SOD1 mouse. ALS Other Motor Neuron Disord. 2004;5(Suppl.2):36.
- Spalloni A, Albo F, Ferrari F, Mercuri N, Bernardi G, Zona C, Longone P. Cu/Zn superoxide dismutase (GLY93–wALA) mutation alters AMPA receptor subunit expression and function and potentiates kainate-mediated toxicity in motor neurons in culture. Neurobiology of Disease 2004;15:340–50.
- Rembach A, Turner BJ, Bruce S, Cheah IK, Scott RL, Lopes EC, et al. Antisense peptide nucleic acid targeting GluR3 delays disease onset and progression in the SOD1 G93A mouse model of familial ALS Journal of Neuroscience Research 2004;77:573–82
P53 MOUSE MOTOR NEURON DISEASE CAUSED BY TRUNCATED CU/ZN SUPEROXIDE DISMUTASE
Watanabe Y, Yasui K, Nakano T, Kitayama M, Doi K, Fukada Y, Morita R & Nakashima K
Department of Neurology, Institute of Neurological Sciences, Faculty of Medicine, Tottori University, Yonago, Japan
E‐mail address for correspondence: [email protected]
Background: Mutation of Cu/Zn superoxide dismutase (SOD1) contributes to a portion of the cases of familial amyotrophic lateral sclerosis (FALS). We previously reported on a FALS family whose members had a mutant form of SOD1 characterized by a 2‐base pair (bp) deletion at codon 126 of the SOD1 gene (Leu126delTT). We also reported the possibility that this mutation made the mutant SOD1 protein functionally unstable and, as a result, the amount of mutant protein was considerably low. In order to investigate the cellular consequences of the mutation, we produced a transgenic animal that expressed the same mutated form of SOD1 as found in this family.
Objectives: We produced transgenic mice that were introduced with normal and mutated copies of human SOD1 gene: wild‐type SOD1 (W), wild‐type SOD1 with a FLAG epitope at C terminal (WF), mutated SOD1 with the 2‐bp deletion (D), and SOD1 with the 2‐bp deletion with FLAG (DF). Among these heterozygotes, those that constantly exhibited motor neuron symptoms were inbred to obtain homozygotes. Then heterozygous and homozygous mice were evaluated further.
Methods: Mice were evaluated by clinical assessment (onset of the disease and date of death), biochemical analysis (SOD1 activity, Western analysis and northern analysis) and pathological examination (haematoxylin‐eosin and Klüver‐Barrera stain as well as immunohistochemistry using anti‐GFAP antibody, anti‐SOD1 antibody and anti‐FLAG antibody).
Results: The mice heterozygotic for the human mutated SOD1 (D and DF) showed distinct ALS‐like motor symptoms, whereas the mice heterozygotic for the normal SOD1 (W and WF) mice did not. Homozygotes of D and DF lines showed ALS symptoms at an earlier age and died earlier than the heterozygotes. By northern analysis, the mRNAs for all human SOD1s were confirmed in these lines. All the human SOD1 proteins, except the D mutant, were detectable by immunoblot. The D protein was only confirmed when it was concentrated by immunoprecipitation. Neuropathologically, loss of spinal motor neurons and reactive gliosis were common features in the symptomatic lines. The remaining motor neurons in these mice exhibited Lewy body‐like eosinophilic inclusions that were positive for SOD1. In the case of DF lines, inclusion was also positive for FLAG.
Discussion and conclusions: The biochemical and pathological characteristics of these mice were quite similar to those of human FALS patients with the same mutation. It remains to be solved why the mutant SOD1 protein is quite low in quantity, with the existence of hyaline inclusions that are positive for the mutated form of SOD1. This intriguing mouse model will provide an important source of information of the pathogenesis of FALS.
P54 PROTEIN NITRATION IN A TRANSGENIC MOUSE MODEL OF FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS
Casoni F1, Basso M1, Massignan T1, Gianazza E2, Cheroni C3, Salmona M3, Bendotti C3 & Bonetto V1
1Dulbecco Telethon Institute and Mario Negri Institute for Pharmacological Research, Milan, 2University of Milan, Milan, and 3Mario Negri Institute for Pharmacological Research, Milan, Italy
E‐mail address for correspondence: [email protected]
Background: Multiple mechanisms have been proposed to contribute to Cu/Zn superoxide dismutase (SOD1)‐linked familial amyotrophic lateral sclerosis (ALS) pathogenesis, including oxidative stress. Early evidence came from the identification of markers of oxidative stress in the cortex and spinal cord of patients with sporadic and familial ALS. Among these markers, nitrotyrosine (NT) has attracted attention in view of Beckman's theory, which suggests a greater propensity of SOD1 mutants to use peroxynitrite as an enzyme substrate, leading to tyrosine nitration. However, no comprehensive study on the protein targets of nitration in ALS has been reported.
Objectives: To understand the role of protein nitration in ALS pathogenesis and identify the protein targets, we carried out a proteome‐based analysis of spinal cords from transgenic (Tg) SOD1 G93A mice at a presymptomatic stage of the disease.
Methods: Proteins from spinal cord tissue of nine‐week‐old Tg G93A SOD1 mice and age‐matched Tg wild‐type SOD1 mice, were separated by two‐dimensional (2‐D) electrophoresis. For the identification of nitrated proteins, samples were subjected to 2‐D Western blot (WB) analysis probing the membrane with anti‐NT antibody. Nitrated proteins in 2‐D WB were matched to the Coomassie‐stained gel and excised from the gel for the identification using MALDI mass spectrometry.
Results: We found an increased level of NT‐immunoreactivity in spinal cord protein extracts of Tg G93A SOD1 mice at a presymptomatic stage of the disease compared to age‐matched controls. NT‐immunoreactivity is increased in the soluble fraction of spinal cord homogenates and is found as a punctuate staining in motor neuron perikarya of presymptomatic ALS mice. Using a proteome‐based strategy, we identified the nitrated proteins under physiological or pathological conditions and compared their level of specific nitration. Alpha and gamma enolase, ATP synthase beta chain, heat shock cognate 71 kDa protein and actin were over‐nitrated in presymptomatic ALS mice. In addition, we identified by MALDI mass spectrometry 16 sites of nitration in proteins oxidized in vivo. Alpha enolase nitration at Tyr43, also a target of phosphorylation, represents an additional indication of the possible interference of nitration with phosphorylation.
Discussion and conclusions: We propose that protein nitration may have a role in ALS pathogenesis, acting directly by inhibiting the function of specific proteins and indirectly interfering with protein degradation pathways and phosphorylation cascades.
Acknowledgement: This work was supported by Telethon Foundation and Cariplo Foundation.
P55 PROTEOMIC CHARACTERIZATION OF AGGREGATES IN A TRANSGENIC MOUSE MODEL OF FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS
Basso M1, Samengo G1, Massignan T1, Cheroni C2, Salmona M2, Bendotti C2 & Bonetto V1
1Dulbecco Telethon Institute and Istituto di Ricerche Farmacologiche Mario Negri, Milan, and 2Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy
E‐mail address for correspondence: [email protected]
Background: Ubiquitinated and Cu/Zn superoxide dismutase (SOD1)‐immunopositive protein inclusions in spinal cords are a histological and biochemical hallmark of disease progression in familial ALS (FALS). The aggregates are found in the cytoplasm and cannot be dissociated with strong detergents or reducing agents. Furthermore, in murine models of ALS aggregates can be detected even before the onset of clinical symptoms. It is still controversial if the inclusion bodies may have a cytotoxic activity, are secondary inoffensive products or have a protective role by capturing abnormal proteins.
Objectives: To investigate the role of protein aggregation in FALS pathogenesis, we characterized the proteins that co‐aggregate with mutated SOD1 in a transgenic mouse model of FALS, which over‐express human SOD1 carrying the G93A mutation (Tg SOD1 G93A).
Methods: Experiments were carried out on Tg SOD1 G93A mice at a late stage of the disease. Age‐matched Tg mice overexpressing wild‐type human SOD1 and non‐Tg littermates were used as controls. Proteins were separated by two‐dimensional electrophoresis (2‐DE) and the gel maps were compared by computerized image analysis. Two‐thirds of each sample was used for Coomassie‐stained 2‐D gels and one‐third for Western blotting. The most abundant proteins present in aggregates of Tg SOD1 G93A mice were identified by MALDI mass spectrometry.
Results: The total amount of proteins was about two times more in sick mice than in healthy mice. Among them, structural proteins, chaperones, and enzymes involved in energy metabolism were identified. In agreement with previous results accumulation of mutant SOD1 occurred selectively in the spinal cord of SOD1 G93A mice with the observation of multimeric forms. The accumulation of ubiquitinated or polyubiquitinated proteins was only detected in mice carrying the mutation and consistent anti‐ubiquitin immunoreactivity was evident especially at high molecular weights. Carbonylated proteins were also found only in tissues of Tg SOD1 G93A mice.
Discussion and conclusions: Using a proteomic approach we have characterized the proteins present in FALS aggregates. The proteins identified are involved in axonal transport, folding, energetic metabolism, and are highly ubiquitinated or carbonylated only in SOD1 G93A mice. This confirms a large number of impaired proteins in aggregates and suggests a strong oxidative situation and a decreasing efficiency of the ubiquitin proteasome system.
Acknowledgement: This work was supported by Telethon Foundation and Cariplo Foundation in Italy.
P56 DETERGENT INSOLUBLE FORMS OF SOD1 ARE DISTRIBUTED TO BOTH OUTER AND INNER MEMBRANES OF MITOCHONDRIA AND ARE ASSOCIATED WITH DEVELOPMENT OF ALS IN TRANSGENIC MOUSE MODELS
Deng HX, Shi Y, Zhai H, Fu R, Liu E & Siddique T
Northwestern University, Chicago, USA
E‐mail address for correspondence: [email protected]
Background: Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder caused by degeneration of the upper motor neurons of the motor cortex in the brain, and lower motor neurons in the brainstem and spinal cord, resulting in progressive wasting and paralysis of voluntary muscles. Mutations in SOD1 are found in 20% of familial ALS. Transgenic and knockout mouse models demonstrated that mutant SOD1 exhibits a toxic property that is associated with development of ALS. How mutant SOD1 exhibits neuronal toxicity is not known.
Methods: Multiple lines of transgenic mice that overexpress different SOD1 mutations at different levels were developed. These mice were characterized by using clinical, genetic, biochemical and morphological studies including immuno‐electron microscopy. The fractions from outer membrane (OM), inner membrane (IM), intermembrane space (IMS), and matrix were analyzed with F‐SOD1 and different mitochondrial markers. The fractions from OM and IM were washed three times with 0.1% NP40 solution. The soluble SOD1 (mouse and human) in the cytosol was also analyzed as a positive control (S3). Fractions from IMS and matrix were not washed with detergent because such washing would remove both signals of the mitochondrial markers and SOD1; suggesting SOD1 in these fractions is soluble.
Results: Some of the mice showed ALS‐like phenotype and pathology, and others did not when the expression of mutant SOD1 was low. Taking advantage of the single and double transgenic mice that showed different expression level, different phenotypes and pathology, we found that both mutant and wild‐type SOD1 had a similar distribution pattern in the affected and unaffected tissues, and in the different subcellular organelles. However, only the detergent insoluble form of SOD1 (SOD1DIS) is associated with development of ALS. SOD1DIS was distributed to both the outer and inner membrane of mitochondria, leading to mitochondrial damage.
Discussion: Using transgenic mouse models expressing different forms of SOD1 and a crossbreeding strategy, we observed multiple lines of evidence showing that ALS associated toxicity of SOD1 is derived from a detergent‐insoluble form, rather than soluble form of SOD1. ALS‐associated mutant SOD1 is able to convert wild type SOD1 from soluble form to insoluble form, which in turn exacerbates the disease, a property similar to that found in prion disease: SOD1DIS is distributed to both inner and outer membranes of mitochondria. Both mutant and wild‐type SOD1 confer ALS associated toxicity as long as they form SOD1DIS.
P57 MITOCHONDRIAL ALTERATIONS IN ASYMPTOMATIC G93A‐SOD1 TRANSGENIC MICE (SOD1‐TG)
Nobile V1, Santoro B1, Valsecchi C1, Bendotti C2 & Curti D1
1Department of Cellular and Molecular Physiological and Pharmacological Sciences, University of Pavia, Pavia, and 2Laboratory of Molecular Neurobiology, Mario Negri Institute for Pharmacological Research, Milan, Italy
E‐mail address for correspondence: [email protected]
Background: Mitochondria abnormalities are consistently found in human ALS and in SOD1‐TG mice. Mutant SOD1 was found in the mitochondrial matrix of SOD1‐TG mice (1). SOD1 also binds and docks to BCL2 and forms aggregates that preferentially interact with spinal cord mitochondria (2). However, the nature of mitochondrial damage and the selective vulnerability of motor neurons in ALS are still matter of debate. Moreover, mitochondrial alterations are seen also in wobbler motor neuron disease mice (3) at early stages of the pathology and in human sporadic ALS.
Objectives: Evaluation of abnormalities in energy metabolism, calcium buffering ability, modulation of mPTP in spinal cord (and brain) mitochondria from asymptomatic SOD1‐TG mice (5–10 weeks of age).
Methods: Mitochondria isolated on Percoll gradients were challenged with activators or specific inhibitors of mPTP opening (4,5). The ability to accumulate and release calcium was measured indirectly with CG5N and also mitochondrial activities were assayed (3).
Results: Spinal cord mitochondria from 10‐week‐old SOD1‐TG mice showed lower activities of complex IV (p<0.0001) and I (p<0.03) and exhibited a lower threshold for Ca2+‐induced swelling than control ones. Both in 5‐ and 10‐week‐old SOD1‐TG mice, mitochondria had significantly higher capacity to accumulate calcium (in 30 s) and reduced calcium release upon collapsing ΔΨ□with an uncoupler of oxidative phosphorylation. Thapsigargin (5 µM) greatly increased the proportion of mitochondria undergoing mPTP opening (p<0.003) and the efflux of calcium (p<0.002) but it was less effective in SOD1‐TG mitochondria.
Discussion and conclusions: Alteration of mitochondrial calcium handling is present in the spinal cord but also in the brain of asymptomatic SOD1‐TG mice even at five weeks of age. The selective vulnerability of motor neurons in the pathology may depend on their peculiar microenvironment and on intrinsic properties that may confer to mitochondria a crucial role in the modulation of calcium signalling in these cells.
References
- Vijayvergiya C, Beal MF, Buck J, Manfredi G. J Neurosci. 2005;25:2463–70.
- Pasinellli P, Belford ME, Lennon N, et al. Neuron 2004;43:19–30.
- Santoro B, Bigini P, Levandis G, et al. Neurobiol Dis 2004;17:349–57.
- Hansson MJ, Persson T, Friberg H, et al. Brain Res 2003;960:99–111.
- Korge P, Weiss JN. Eur J Biochem. 1999;265:273–80.
P58 THE SELECTIVE CYTOCHROME C OXIDASE DEFICIENCY IN CNS OF G93A‐SOD1 MICE IS NOT RELATED TO CYTOCHROME C
Vielhaber S1, Kudin AP2, Debska‐Vielhaber G1, Danzeisen R3, Ludolph AC3 & Kunz WS2
1University of Magdeburg, Magdeburg, 2University of Bonn, Bonn, and 3University of Ulm, Ulm, Germany
E‐mail address for correspondence: [email protected]‐magdeburg.de
Rodents transgenic for mutant human Cu/Zn SOD (SOD1) develop progressive skeletal muscle atrophy, paralysis and death similar to human cases. The early onset of mitochondrial degeneration suggests that mutant SOD1 causes dysfunction and structural changes in mitochondria, but a detailed mechanism has not yet been established. In the current work we determined the putative involvement of the respiratory chain in brain and muscle mitochondria of day 40 and day 90 G93A‐SOD1 mice. We found in brain mitochondria of day 40 SOD1 mice a selective decrease in complex IV enzyme activity, long before any symptoms arose. In line with this finding brain mitochondrial cytochrome spectra indicated a decrease in cytochromes aa3 but not in cytochromes b and c+c1. Furthermore, day 90 SOD1 (G93A) mice brain and muscle mitochondria showed additionally decreased complex I and aconitase enzyme activities. Interestingly, in comparison to non‐transgenic mice mitochondria‐associated SOD1 activity was significantly increased in day 40 and day 90 SOD1 (G93A) mice but also in age matched transgenic wild‐type SOD1 with intact mitochondrial function. The effect was CNS specific, because it was not observed in muscle mitochondria. Our results suggest an early direct impairment of cytochrome c oxidase (complex IV) in brain mitochondria which seems not to be linked to the increase in SOD1 activity, or to a recently proposed alteration of cytochrome c association with the inner mitochondrial membrane. We propose that brain mitochondria with mutant SOD1 have a defect in complex IV, generating elevated levels of reactive oxygen species leading at later stages of the disease to an oxygen‐radical mediated decline of complex I and aconitase activities. The observed respiratory chain defect in muscle mitochondria of day 90 SOD1 mice reflects most probably an unspecific phenomenon related to muscle denervation, prior to the decline of motor performance.
P59 BIOLOGICAL SIGNIFICANCE OF P38 MITOGEN ACTIVATED PROTEIN KINASE ACTIVATION IN MUTANT SOD1G93A INDUCED MOTOR NEURON DEATH
Dewil M, Lemmens G, Robberecht W & van den Bosch L
Laboratory for Neurobiology, Department of Experimental and Clinical Neurology, KULeuven, Leuven, Belgium
E‐mail address for correspondence: [email protected]
Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the selective loss of motor neurons. In 20% of these familial cases, mutations in the superoxide dismutase 1 gene (SOD1) have been reported. To date, no effective treatment is available and the pathogenic mechanism remains enigmatic. We have previously reported minocycline to significantly attenuate mutant SOD1‐induced motor neuron degeneration and to expand the life span of mutant SOD1 overexpressing mice, a mouse model for human ALS. It is thought that the effect of minocycline is exerted through an inhibition of the activation of the p38 mitogen activated protein kinase (MAPK) pathway, which is known to occur in microglia and motor neurons of mutant SOD1 mice.
Objectives: In the present study, we aimed to characterize the biological relevance of up‐regulation of p38 MAPK for motor neuron degeneration in the mutant SOD1 mouse.
Methods and sesults: We found the p38 MAPK to be abnormally activated in the ventral part of the spinal cord of the mutant SOD1G93A mice, where significantly increased levels of phosphorylated p38 MAPK were found. The therapeutic effect of minocycline was accompanied by a significant attenuation of this activation. Activated phospho‐p38 MAPK was exclusively present in microglial cells and motor neurons. In vitro studies showed minocycline to inhibit microglial activation by lipopolysaccharide. In addition, both minocycline and SB203580, a specific p38 MAPK inhibitor, inhibited mutant SOD1‐induced apoptosis in cultured motor neurons. We therefore tested whether semapimod, a specific p38 MAPK inhibitor that readily crosses the blood‐brain barrier, affected mutant SOD1‐induced motor neuron death. Mutant SOD1G93A mice were treated with semapimod or placebo from 70 days of age. Treatment with semapimod effectively inhibited p38 MAPK activity, as phosphorylation of tau at residue Thr231 was significantly reduced. It significantly enhanced survival of mutant SOD1 G93A mice and attenuated motor neuron death in these animals. However, its effect was clearly smaller than that of minocycline.
Conclusion: Our data suggest the p38 MAPK pathway plays a significant, albeit limited, role in mutant SOD1 induced motor neuron degeneration. As the effect of treatment of mutant SOD1 mice with semapimod is smaller than that observed with minocycline, the effect of the latter drug is likely to be mediated by other effects than inhibition of the p38 MAPK pathway alone.
P60 NEUROPATHOLOGICAL CHANGES AND P38 MAPK EXPRESSION IN MOTOR‐RELATED AREAS OF THE CENTRAL NERVOUS SYSTEM OF TGSOD1G93A MICE
Lo Coco D, Veglianese P & Bendotti C
Mario Negri Institute for Pharmacological Research, Milan, Italy
E‐mail address for correspondence: [email protected]
Background and objectives: Amyotrophic lateral sclerosis (ALS) is characterized by the selective loss of upper and lower motor neurons; however, many patients with the familial form of ALS (FALS), including those carrying the superoxide dismutase (SOD1) mutations, present only mild or absent upper motor neuron involvement at both clinical examination and autopsy. Transgenic mice carrying the mutant SOD1 recapitulate many of the features of human disease and are therefore extensively studied to investigate the pathogenesis of ALS and to test novel potential pharmacological treatments. In these mice the disease signs have been associated with the degeneration of spinal motor neurons, whereas there is limited knowledge about the involvement of the other upper motor‐related areas of the CNS, including red nucleus and reticular formation, both part of the oligosynaptic corticospinal pathway. We have recently shown that both the accumulation of phosphorylated neurofilaments (pNF) and activated p38 mitogen activated protein kinase (p‐p38MAPK) are early signs of the degeneration process in the spinal motor neurons of transgenic mice expressing SOD1 with G93A mutation (tgSOD1G93A) and this effect persisted during the progression of the disease. P‐p38MAPK was also increased in spinal motor neurons of human sporadic ALS. Thus, we decided to analyse the extent and distribution pattern of these neuropathological markers in the spinal cord, brainstem and sensorimotor cortex of tgSOD1G93A mice during the progression of the disease.
Methods: Immunohistochemical analysis followed by light or confocal microscopy was performed in frozen sections of tgSOD1G93A mice at different stages of the disease and age matched non‐transgenic mice, using different antibodies directed to astrocytes, microglia, pNF and activated p38 MAPK.
Results: We found extensive alterations characterized by motor neuron death, pNF accumulation, reactive gliosis and p38MAPK activation in the cervical spinal cord of this mouse model during the progression of the disease that closely parallels the degenerative changes found in the lumbar segment. In the other motor‐related areas, such as red nucleus and sensorimotor cortex, no degenerative changes, reactive gliosis or p38MAPK activation could be observed during the course of the disease.
Conclusion: Our findings show that in this mouse model, similarly to human FALS, the primary disease is driven by the loss of lower motor neurons. Moreover, p38MAPK activation is specifically and selectively expressed in degenerating areas only, confirming its relevant role in motor neuron death. Even with the important species differences in the organization of the corticospinal transmission to motor neurons between humans and rodents, these data further confirm that tgSOD1G93A mice represent a useful model highly necessary to understand FALS and to investigate many of the fundamental mechanisms of lower motor neuron degeneration.
Acknowledgement: This work was supported by Telethon, Italian Ministry of Health, MIUR.
P61 NOGOA UP‐REGULATION IN ALS MOTOR NEURONS: INSIGHTS INTO A POTENTIAL FUNCTION IN MOTOR NEURON SURVIVAL
René F1, Dupuis L1, Gonzalez de Aguilar JL1, Pehar M2, Cassina P3, Mohr M4, Dimou L5, Schwab M5, Loeffler JP1 & Barbeito L2
1INSERM U692, Faculté de Médecine, Strasbourg, France, 2Instituto Clemente Estable, Montevideo, 3Faculdad de Medicina, Montevideo, Uruguay, 4Service d'Anatomo‐Pathologie, CHU Hautepierre, Strasbourg, France, and 5Brain Research Institute, Zurich, Switzerland
E‐mail address for correspondence: [email protected]
Background: Amyotrophic lateral sclerosis (ALS) is a motor neuron disease of unknown origin. About 2% of ALS cases are linked to mutations in the gene encoding copper/zinc superoxide dismutase (SOD1). Expression of mutant forms of SOD1 (SOD1m) in transgenic mice leads to motor neuron death and an ALS‐like phenotype. Using an unbiased subtractive suppressive hybridization screen, we have identified a clone encoding the neurite outgrowth inhibitor Nogo that is specifically up‐regulated in the lumbar spinal cord of asymptomatic G86R transgenic mice. In spinal cord of 90‐days‐old G86R mice, NogoA mRNA levels are significantly increased compared to the wild‐type mice.
Objectives and methods: In order to identify the cell type(s) overexpressing NogoA in the spinal cord of G86R mice and ALS patients, we have performed immunohistochemistry using specific NogoA antibody. In addition, by using a double labelling protocol we have studied the localization of NogoA and its receptor (NgR).
Results: In G86R transgenic mice, in addition to a basal immunoreactivity in glial cells also found in wild‐type mice, NogoA is strongly induced in a subset of motor neurons. This result has been confirmed in spinal cord of ALS patients. Furthermore in NogoA positive motor neurons, NgR immunoreactivity was decreased whereas in motor neurons immunoreactive to NgR, NogoA staining was faint or absent.
Conclusion: These results suggest a relationship between NogoA overexpression and NgR down‐regulation. The possible implication of this cross‐talk in motor neurons death is currently under investigation in in vitro models
P62 ROLE OF THE NEURITE OUTGROWTH INHIBITOR NOGO‐A IN SKELETAL MUSCLE: IMPLICATIONS FOR ALS
Loeffler JP1, Jokic N1, Lin S2, Dimou L3, Fergani A1, Dupuis L1, Ruegg MA2, Schwab ME3 & Gonzalez de Aguilar JL1
1INSERM U‐692, Université Louis Pasteur, Strasbourg, France, 2Department of Pharmacology and Neurobiology, Biozentrum, Basel, and 3Brain Research Institute, Zurich, Switzerland
E‐mail address for correspondence: [email protected]
Background: The pathogenesis of ALS still remains unclear. Growing evidence suggests that initial alterations in skeletal muscle, preceding the onset of disease symptoms, are related to a loss of neuromuscular junction integrity, axonal degeneration and muscle denervation, rather than motor neuron death. Our previous studies showed a characteristic expression pattern of the three major isoforms of the neurite outgrowth inhibitor Nogo (including Nogo‐A, ‐B and ‐C) in skeletal muscles of ALS patients and SOD1(G86R) mice. We found that the increased levels of Nogo‐A and Nogo‐B, which had been barely detectable in muscles of control subjects, correlate significantly with the severity of motor impairment of ALS patients, as determined by the clinically validated ALS functional rating scale.
Objectives: We wished to determine the impact of knocking down Nogo‐A on the progression of ALS pathology in SOD1(G86R) mice and gain insight into the role of Nogo up‐regulation in skeletal muscle.
Methods: We crossbred mice knockout for Nogo‐A with mice overexpressing the ALS‐related mutation G86R, and followed the survival time. We also performed in vivo skeletal muscle transfection of a vector expressing Nogo‐A in Thy‐1/YFP mice, and looked at the morphology of the neuromuscular junction (NMJ).
Results: Double‐transgenic G86R/Nogo‐A(−/−) mice survived longer than G86R/Nogo‐A(+/+) mice. Transient expression of Nogo‐A in skeletal muscle fibres was sufficient to injure the NMJ by inducing dismantlement of the post‐synaptic structures and loss of pre‐synaptic terminals.
Conclusions: The ectopic expression of Nogo‐A in skeletal muscle initiates a cascade of events leading to loss of NMJs and denervation. This deleterious effect may be relevant to ALS since mice suffering from an ALS‐like pathology and lacking Nogo‐A live longer.
P63 ALTERNATIVE SPLICING OF PRO‐ AND ANTI‐APOPTOTIC GENES IN AMYOTROPHIC LATERAL SCLEROSIS
Crosio C1, Casciati A2, Rodriguez M2, Ferri A3, Cozzolino M2, Iaccarino C1, Nencini M2, Rotilio G4 & Carri' MT4
1Dipartimento di Scienze Fisiologiche Biochimiche e Cellulari, Università di Sassari, Sassari, 2Fondazione S.Lucia IRCCS c/o CERC ‘Centro Europeo di Ricerca sul Cervello’, Rome, 3First Neuroscienze CNR, Sez. Psicobiologia e Psicofarmacologia, Rome, and 4Dipartimento di Biologia, Università di Roma & quot;Tor Vergata & quot;, Rome, Italy
E‐mail address for correspondence: [email protected]
Background: Alternative splicing is emerging as a major mechanism of functional regulation in the human genome and it has been involved in many different neurological diseases, including ALS and SMA (spinal muscular atrophy). Change in the balance of pro‐ and anti‐apoptotic Bcl2 family molecules has been described in the motor neurons of human ALS patients as well as in mice expressing mutant SOD1 G93A, the most widely ALS mouse model used (1). Moreover, different members of the Bcl2 family undergo alternative splicing and different isoforms of the same gene could act respectively as pro‐ or anti‐apoptotic.
Objectives: In the present study our aim was to assess if during the progression of ALS there was any change in the splicing isoforms of apoptotic genes that could explain the selective vulnerability of motor neurons.
Methods:RNase protection assay. Total RNA was prepared from spinal cord, brain and muscle, of non‐, G93A‐SOD1 and WT‐SOD1 transgenic mice by the guanidinium‐thiocyanate method. Total RNA was hybridized with a molar excess of 32P‐labelled mouse multiprobe mAPO (Ambion), BFL1, BclX, MnSOD or β‐actin riboprobes.
Immunofluorescence. Anaesthetized mice were perfused with paraformaldehyde 4% in PB and the spinal cord removed. Tissues were sectioned (10 µm) on a cryostat. Immunofluorescence was performed according to Ferrr et al. (2).
Results: RNase protection assays on RNA extracted from spinal cord of ALS mice demonstrate that there was not a detectable accumulation of splicing variants for Bcl2, BclX, BclW, Bad, Bax or Bak. Most interestingly we observe the up‐regulation of the anti‐apoptotic Bfl1 gene in G93A‐SOD1 mice spinal cord, but not in non‐Tg or WT‐SOD1‐Tg, either in the muscle or brain of any genotype. Bfl1 is an antiapoptotic Bcl2 family member and mouse A1 homologue; its expression has been demonstrated to be induced upon activation of the NFkB pathway. The transcriptional induction we observe is not due to a general NFkB up‐regulation since we could not detect any increase in BclX or MnSOD mRNA. By immunofluorescence experiments and confocal analysis we demonstrated that Bfl1 localizes in the motor neurons of G93A‐Tg mice. It accumulates both in the nucleus and cytosol, where it can form punctate structures.
Conclusions: In the mouse model for ALS we did not observe alterations in the alternative splicing of the apoptotic genes analysed (BCL‐2, BCL‐X, BCL‐W, BAD, BAX and BAK). On the contrary, we observed a transcriptional induction of the anti‐apoptotic Bfl1 gene, in the spinal cord of G93A‐SOD1 transgenic mice.
References
- Akhtar RS, Ness JM, Roth KA. Bcl-2 family regulation of neuronal development and neurodegeneration. Biochim Biophys Acta. 2004;1644:189–203.
- Ferri A, Nencini M, Casciati A, Cozzolino M, Angelini DF, Longone P, et al. Cell death in amyotrophic lateral sclerosis: interplay between neuronal and glial cells. Faseb J. 2004;18:1261–3.
P64 CONDITIONING EFFECT OF TARGET TISSUE INJURY ON ADULT MOTOR NEURONAL SURVIVAL
Johnson IP, Aperghis MA & Goldspink G
University College London, London, UK
E‐mail address for correspondence: [email protected]
Background: While the critical role played by target tissue in the survival of developing motor neurons is well known, much less is known about its role in the survival of mature motor neurons. This information is vital for the rational design of potential therapies aimed at promoting motor neuronal survival in the adult.
Objective: To determine the conditioning effects of target tissue injury on the survival of axotomised adult motor neurons.
Methods: Adult (6 m) Sprague Dawley rats (n = 6 per group) were given three 20 µl injections into the snout of either 1) physiological saline, 2) 0.5% w/v of the myotoxic anaesthetic bupivicaine in saline, or 3) distilled water. After seven days the right facial nerve was avulsed by sustained traction at the stylomastoid foramen in a procedure that produced axotomy at the nerve‐rootlet junction. After a further 28 days, rats were perfuse‐fixed with 4% paraformaldehyde in 0.1M phosphate buffer. Total numbers of motor neurons in the left and right facial nuclei were estimated in the confocal microscope using a modified optical dissector method.
Results: Approximately 75% of motor neurons were lost by one month after avulsion alone. This was associated with occasional staining of motor neurons by markers of apoptosis such as cleaved caspase 3 and TUNEL. Motor neuron loss in the saline, bupivicaine and distilled water‐injected groups was 55%, 49% and 32%, respectively. Compared to the avulsion only group, the neuroprotection afforded by all three forms of muscle damage was significant (p = 0.05, Mann Whitney U‐test). Histological examination of snout muscle after four days in all three injected groups showed a qualitative correlation between the degree of lymphocyte infiltration of the muscle and the degree of motor neuron protection conferred.
Discussion: Our results indicate that mechanical damage of muscle due to the injection procedure alone is sufficient to reduce motor neuron loss from 75% to approximately 50%. Adding myotoxic damage has no significant effect on this neuroprotection, but adding osmotic shock reduces motor neurone loss to 32%. The rapid and non‐selective cell death induced in muscle tissue by distilled water is probably responsible for the marked lymphocytic infiltration. This peripheral inflammatory reaction may have modified the subsequent retrograde response of motor neurons to avulsion in a direction that favoured their survival.
P65 VEGF RESTORES DECREASED LEVELS OF AKT IN AN ANIMAL MODEL FOR ALS
Dewil M1, Carmeliet P2, van den Bosch l1 & Robberecht W1
1Laboratory for Neurobiology, Department of Experimental and Clinical Neurology, KULeuven, Leuven, and 2Centre for Transgene Technology & Gene Therapy, Flanders Interuniversity Institute for Biotechnology (VIB), University of Leuven, Leuven, Belgium
E‐mail address for correspondence: [email protected]
Background: Amyotrophic lateral sclerosis is one of the most devastating neurodegenerative disorders. The pathogenesis of this disease that results in a relentless loss of motor function remains unknown. We recently reported that treatment of mutant SOD1G93A overexpressing rats with VEGF resulted in a significant increase in survival compared to the control group. The precise mechanism of the neuroprotective effect of VEGF in ALS remains to be elucidated, but modulation of the phosphoinositol‐3‐kinase/Akt cascade is one of the most likely targets.
Objectives: In the present study, we aimed to investigate the pathophysiologic role of Akt in ALS and the influence of VEGF on this important anti‐apoptotic protein.
Methods and results: Western blot analysis showed that protein kinase B or Akt is lost very early on in the disease. Immunohistological studies showed that this loss is confined to motor neurons, supporting the pathogenetic relevance of the potent anti‐apoptotic kinase Akt. In vitro transfection experiments with constitutively active Akt revealed the neuroprotective effect of Akt in mutant dependent cyclosporin A‐induced cell death in G37R overexpressing N2A cells. To evaluate the relevance of the Akt pathway in the neuroprotective effect of VEGF, Western blot analysis was performed in brainstem homogenates of VEGF‐treated SOD1G93A rats. The results showed that treatment with VEGF resulted in a significant increase in the levels of both total and phosphorylated active Akt both after short treatment of 14 days and after chronic treatment.
Conclusions: Our data support the relevance of Akt to the selective loss of motor neurons in ALS and provide evidence for its role as a target of VEGF. Therefore, further development of therapeutic strategies targeting the phosphoinositol‐3‐kinase/Akt pathway may be useful.
P66 OVEREXPRESSION OF HEAT SHOCK PROTEIN 27 RESCUES MOTOR NEURONS AND IMPROVES MUSCLE FUNCTION FOLLOWING NERVE INJURY IN NEONATAL MICE
Sharp P, Krishnan M, Pullar O, Navarrete R, Wells D & De Belleroche J
Imperial College London, London, UK
E‐mail address for correspondence: [email protected]
Background: Heat shock proteins (HSPs) are a family of ubiquitously expressed proteins that are up‐regulated in response to a range of stresses and play an important role in cellular defence mechanisms. The 27‐kDa heat shock protein HSP27 has been shown to have potent neuroprotective effects against oxidative stress and apoptotic cell death. Our previous work has shown that overexpression of HSP27 in transgenic mice significantly reduced cell death in the CNS, possibly through the attenuation of Caspase‐3 induction. In motor neurons, HSP27 is also implicated as a survival promoting factor, since exogenous delivery of HSP27 following neonatal nerve injury, enhances motor neuron survival. However, most importantly, it remains to be established whether HSP27 overexpression in vivo is able to restore muscle function following neonatal nerve injury.
Objectives: Using transgenic mice that overexpress the human form of HSP27 (hHSP27), we examined whether hHSP27 expression can protect developing motor neurons from injury‐induced cell death and improve long term muscle function.
Methods: At birth, the sciatic nerve in one hind limb was crushed in both hHSP27 transgenic mice and wild‐type mice. Twenty to 25 weeks later, in vivo isometric tension recordings of the EDL muscle were carried out to assess muscle function and motor unit number. The spinal cords and EDL muscles were subsequently removed and processed for sciatic motor neuron counts and morphometric analysis, respectively.
Results: The expression of hHSP27 significantly improved the long‐term survival of sciatic motor neurons following neonatal nerve injury. In the wild‐type group only 29.8%±5.9% (mean±SEM; n = 6) of injured motor neurons survived, compared to 56.4%±2.3% (mean±SEM; n = 6) in hHSP27 transgenic mice. Importantly, this improvement in survival was reflected by a substantial improvement in muscle function. For example, in the reinnervated EDL muscle of wild‐type mice the number of motor units was only 29.1%±1.0% (mean±SEM; n = 6) compared to 55.1%±5.6% (mean±SEM; n = 6) in transgenic animals. Furthermore, this increase in motor unit number was associated with improved muscle weight, muscle force, contractile speeds and histochemical markers of EDL muscle activity. Morphometric analysis also revealed an almost normal distribution of muscle fibre sizes in the operated EDL muscle of transgenic mice, whereas in wild‐type mice there was a marked increase in the proportion of small atrophic muscle fibres.
Discussion and conclusions: These results show that inducing neonatal motor neurons to express HSP27 protects them against nerve injury‐induced cell death. Moreover, HSP27 expression also supported the ability of injured neonatal motor neurons to re‐establish functional neuromuscular contacts. Overall, these findings provide further evidence for the therapeutic potential of HSP27 in the treatment of motor neuron diseases.
P67 RILUZOLE/NIMODIPINE/MINOCYCLINE TRITHERAPY ON RAT ALS MODEL: CONFLICTING RESULTS
Dagonnier M, Manto M & Pochet R
Laboratory Histology, Neuroanatomy and Neuropathology, Fac Mececine, Université Libre de Bruxelles, Brussels, Belgium
E‐mail address for correspondence: [email protected]
Background: Tritherapy, using a cocktail of three drugs (riluzole, nimodipine and minocycline), has been suggested to have beneficial effects on a mice model for ALS (1). In this work we have tested on the rat ALS model (transgenic rat overexpressing human mutated SOD1 gene) the effect of such a tritherapy on survival time and on clinical scores.
Methods: Rats were divided in five groups (seven rats per group); group I did not receive any drugs, groups II, III and IV received nimodipine, riluzole and minocycline, respectively, and group V a cocktail of the three drugs. Rats were weighed every day. Treatment started at 60 days of age until death. A clinical score using four items (tail test, mobility in the cage, mobility on the table and amyotrophy) with a maximum of 7 points was measured three times/week. EMGs were performed on day 60 for 13 rats, on days 80, 100 and 120 for all animals. Motor neurons from the frontal cortex were counted on paraffin sections after cresyl violet coloration. Sciatic nerve sections (2 micrometres thick) were used for axonal area and axon density measurements. Statistics were made using the Sigma Stat program.
Results: The quantity of drug ingested was measured: rats from group II ingested 25–35 mg/kg/day of nimodipine, group III 18–25 mg/kg/day of riluzole, group IV 64–80 mg/kg/day of minocycline and group V 20–28 mg/kg/day nimodipine, 20–28 mg/kg/day riluzole and 54–68 mg/kg/day minocycline. The analysis of the survival curve indicated that there is no difference between males and females, and no increase in survival rate after any of the treatments. On the contrary, tritherapy and riluzole decreased it. Analysis of the mortality distribution confirmed this result. For male but not female rats, a decrease in weight appeared on day 60 for the rats treated either with riluzole alone or with tritherapy. The axons numbers were significantly diminished after tritherapy. Axon areas were also significantly diminished after tritherapy, riluzole and minocycline treatment. In contrast, nimodipine treatment increased axon areas. Motor neuron counts did not show any significant variation, neither did the electromyography results. Only riluzole improved the clinical scores.
Conclusions: Our results indicated that although riluzole could improve the clinical score and therefore the quality of life, it had adverse effects on the survival rate. This result is important and we believe allows revisiting the beneficial effects of riluzole in human ALS.
Reference
- Kriz J, Growing G, Julien JP. Efficient three-drug cocktail for disease induced by mutant superoxide dismutase. Ann Neurol. 2003;53:429–36.
P68 AM1241 SLOWS DISEASE PROGRESSION IN A MOUSE MODEL OF ALS
Kim K, Moore DH & Abood M
California Pacific Medical Center Research Institute, San Francisco, USA
E‐mail address for correspondence: [email protected]
Objectives: Effective treatment for amyotrophic lateral sclerosis (ALS) remains elusive. Two of the primary hypotheses underlying motor neuron vulnerability are susceptibility to excitotoxicity and oxidative damage, including inflammatory damage due to microglial activation. CB2 receptor activation blocks β‐amyloid induced microglial activation (1). We have previously shown that the cannabinoid Δ9‐THC (which acts on CB1 and CB2 receptors) inhibits both excitotoxic and oxidative damage in spinal cord cultures and that Δ9‐THC slows progression and improves survival in the ALS mouse model (hSODG93A transgenic mice) even when administered after the onset of disease signs (2). AM1241 is a CB2 selective agonist that has been shown to be effective in models of inflammation and hyperalgesia (3). Thus, we evaluated the efficacy of AM1241 in the ALS mouse model.
Methods: hSOD1G93A mice (B6SJL‐TgN [SOD1‐G93A] 1Gur) were treated daily beginning on day 75 when tremors were first observed with 1 mg/kg AM1241 intraperitoneally or vehicle. Motor function was evaluated using a rotarod. The survival endpoint used was the loss of righting reflex within 3 s. The investigator was blinded to the treatment protocol.
Results: Three conditions of ALS, the loss of motor function, paralysis scoring and weight loss, were analyzed using a mathematical model. Loss of motor function (as assessed by performance on a rotarod) was delayed by 12.5 days in male mice by AM1241. In female mice, AM1241 extended performance by three days. Paralysis was scored on a scale from 5 to 0; 5 was healthy, 1 was paralysis. AM1241 extended by five days the time to reach a score of 2.5. AM1241 did not affect weight loss or survival (129.8±1.7 days, vehicle; 129.1±7.0 days, AM1241, n = 16).
Conclusions: The data presented here indicate that AM1241, a CB2 selective agonist, slowed disease progression in ALS mice. As AM1241 was well tolerated by the animals, CB2‐selective compounds may be the basis for developing new drugs for the treatment of ALS.
References
- Ramirez, et al. J Neurosci. 2005;25:1904–13.
- Raman, et al. Amyotroph Lateral Scler Other Motor Neuron Disord. 2004;5:33–9.
- Ibrahim, et al. Proc Natl Acad Sci. USA 2003;16:10529–33.
P69 DISULFIRAM AS AN INHIBITOR OF SOD1 AND CASPASE‐3 IN G86R TRANSGENIC ALS MICE: CLINICAL AND IMMUNOHISTOCHEMICAL EFFECTS
Barnes MA, Janssen WG, Gordon J, Morrison JH & Lange DJ
Mount Sinai School of Medicine, New York, USA
E‐mail address for correspondence: [email protected]
Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective and progressive degeneration of upper and lower motor neurons in the spinal cord, brainstem, and cerebral cortex. About 10% of ALS cases are familial (FALS). Among the familial cases, 20% are caused by mutations of the Cu/Zn superoxide dismutase (SOD1). The relationship between motor neuron degeneration and the SOD1 mutation is uncertain, but apoptosis is believed to be the mechanism of cell death. The Caspase‐3/caspase system is the final common pathway in apoptosis. It is hypothesized that inhibiting the activation of the pathway or the pathway itself would slow cell death and any diseases caused by programmed cell death. Antabuse (Disulfiram) is a potent inhibitor of SOD1 and Caspase‐3 as well as other stimulators of the cell death pathway such as TNF‐α.
Objective:
1.To determine if disulfiram treatment prolonged life in G86R transgenic ALS mice.
2. To determine if disulfiram influenced the appearance of SOD1 and Caspase‐3 in G86R/SOD transgenic ALS mice.
Methods: Four groups of ten G86R mice were fed disulfiram‐containing pellets beginning at 50 days after birth. Four different doses (investigator blinded) were administered equivalent to the human dose of 500 mg; 250 mg, 125 mg, and placebo. Two transgenic mice treated with 250 mg and two transgenic untreated mice were sacrificed at 95 days, just prior to the usual time that weakness begins. The spinal cords were removed and tissue sections from one treated and one untreated mouse were selected for single‐label immunohistochemistry with antibody to active and proCaspase‐3, SOD1. The tissue was washed, mounted on slides and imaged using the Zeiss LSM laser scanning confocal microscope.
Results: All animals were asymptomatic at day 50 when treatment began. Death was the measured endpoint. The mean duration of life for each group was 118 days (placebo), 121 days (125 mg), 144 days (250 mg) and 113 (500 mg). Statistical significance was not achieved but the 250 mg dose showed a trend for increased longevity. Animals receiving 250 mg sacrificed at day 95 showed no expression of Caspase‐3, yet it was expressed in the untreated mice. Inactive caspase (activated Caspase‐3 precursor) was present in both groups. We observed a difference in cell morphology between the untreated and treated groups when stained with SOD1 but we were unable to draw conclusions.
Conclusion: As Caspase‐3 was present in the untreated pre‐symptomatic mice but not in the treated asymptomatic mice, our data support the hypothesis that Caspase‐3 activation may be inhibited by disulfiram. Further studies are needed to determine how Caspase‐3 inactivation, neuronal loss, and clinical weakness are related.
P70 THE EFFECT OF L‐CARNOSINE AND ZINC IONS ON SURVIVAL OF G93A/SOD1 MICE
Ignacio SG, Smith AP & Lee NM
The Forbes Norris ALS/MDA Research Center, California Pacific Medical Center‐Research Institute, San Francisco CA, USA
E‐mail address for correspondence: [email protected]
Background: Mutations of Cu/Zn superoxide dismutase‐1 (SOD1) are found in patients with a familial form of amyotrophic lateral sclerosis (ALS). Transgenic mice overexpressing human mutant SOD1 genes, such as G93A, exhibit an ALS‐like disease of motor neurons. SOD1 contains sites that bind both copper and zinc, and several studies have reported that administration of excess zinc accelerates death in G93A/SOD1 animals, while more moderate doses may delay death. The dipeptide L‐carnosine has zinc‐binding as well as anti‐oxidant properties. This suggested to us that L‐carnosine, given alone or together with zinc, might delay death in G93A animals.
Objectives: We tested the effect of administering L‐carnosine, with or without zinc ions, on survival of G93A/SOD1 mice. Our rationale was that the presence of L‐carnosine may regulate zinc levels in vivo, preventing them from becoming either too high or too low.
Methods: Beginning at age 6–9 weeks, G93A/SOD1 mice were given both L‐carnosine and zinc chloride in their drinking water at a daily dose calibrated to correspond to an intake of 3000 mg/kg and 32 mg/kg, respectively, body weight. Motor function was assessed by grading the animals twice weekly on the following scale: 5, healthy; 4, weak hind legs; 3, limping; 2, hind leg paralysis; and 1, death. At the onset of paralysis or when the mice exhibited 10% loss of total body weight the drug was administered daily by i.p. injection, and concentrations of L‐carnosine and zinc adjusted according to the total body weight. The animals remained on this regimen until death. The survival endpoint used was the loss of righting reflex within 10 s.
Results: L‐carnosine (3000 mg/kg/day) in the presence of zinc (32 mg/kg/day) had a small but insignificant effect on the age at which animals entered stage 2 and on the age at which they died. The sources of water (tap or distilled) had no effect on any of the results. An analysis of the tap water used as vehicle indicated that it had undetectable (less than 0.05 mg/l) levels of zinc.
Conclusions: This is a work in progress. Our studies suggest that L‐carnosine in the presence of zinc may have a slight beneficial effect on motor neuron disease in G93A/SOD1 mice. However, further studies are necessary to establish whether it could be effective in prolonging survival of these animals.
Acknowledgements: This study was supported in part by the Forbes Norris ALS Research Foundation.