P92 PROLIFERATION OF NEURAL STEM/PROGENITOR CELLS IN ADULT RATS AFTER FACIAL NERVE AVULSION
Watabe K, Kawazoe Y, Ojima M, Hayashi Y, Wang W, Takazawa T
ALS Project, Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo, Japan
E‐mail address for correspondence: [email protected]
Background: Neurogenesis occurs throughout life in two specific regions of the adult mammalian brain; the subventricular zone of the lateral ventricle and in the subgranular zone of the hippocampal dentate gyrus. After injury and pathological stimulations, adult neurogenesis has also been demonstrated in regions otherwise considered to be non‐neurogenic.
Objectives: We investigated proliferation of nestin‐immunoreactive (IR) neural stem/progenitor cells (NSPCs) in injured facial nuclei after facial nerve avulsion in adult rats.
Methods: The facial nerve of three‐month‐old Fischer 344 male rats was either avulsed or axotomized (1–4), and facial nuclei were histologically examined at different time points. Some animals were given 5‐bromo‐2'‐deoxyuridine (BrdU) in their drinking water for four weeks after avulsion. Intact and injured facial nuclei from some animals were dissected, trypsinized and cultured in Neurobasal/B27 medium containing fibroblast growth factor‐2 (FGF2) and epidermal growth factor (EGF).
Results: Two weeks after avulsion, motor neuron death became apparent (1–4) and nestin‐IR cells proliferated in lesioned facial nuclei. BrdU‐incorporated nestin‐IR cells were observed in the facial nucleus on the avulsed side. Neither motor neuron death nor proliferation of nestin‐IR cells was evident in facial nuclei after facial nerve axotomy. When intact facial nuclei were dissociated and cultured in the presence of FGF2 and EGF, neurospheres formed and nestin/NG2‐IR NSPCs could be expanded and passaged for over eight months. In vitro proliferation of NSPCs derived from avulsed facial nuclei was much more vigorous than that from intact nuclei. These NSPCs differentiated to TuJ1, MAP2 and neurofilament‐IR neurons, GFAP‐IR astrocytes, and Rip, O4 and O1‐IR oligodendrocytes in the presence of all trans retinoic acid and/or neurotrophic factors that include brain‐derived neurotrophic factor and insulin‐like growth factor‐1.
Discussion and conclusions: We demonstrated that NSPCs proliferate following motor neuron degeneration caused by avulsion, but not axotomy, of the facial nerve in adult rats. These results indicate that the adult rat facial nerve avulsion model is a novel tool to investigate endogenous NSPCs in adult rat brain, and could be used to screen candidate therapeutics to promote in vitro and in vivo neurogenesis against motor neuron injury and motor neuron diseases in adult humans.
References
- Sakamoto T, Watabe K, Ohashi T, et al. NeuroReport 2000;11:1857–60.
- Sakamoto T, Kawazoe Y, Shen J-S, et al. J Neurosci Res. 2003;72:54–64.
- Ikeda K, Aoki M, Kawazoe Y, et al. J Neurosci Res. 2005;82:63–70.
- Watabe K, Hayashi Y, Kawazoe Y. Neuropathology 2005;25:371–80.
P93 NEUROPROTECTIVE EFFECTS OF MULTI‐VITAMIN THERAPY IN A TRANSGENIC MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS
Lee KW, Park JH, Kim MY, Min JH, Cho AS, Hong YH, Sung JJ
Seoul National University Clinical Research Insitute of Seoul National University Hospital, Seoul, South Korea
E‐mail address for correspondence: [email protected]
Background: There is no currently effective treatment for amyotrophic lateral sclerosis (ALS), although this disorder is a progressive neurodegenerative disease resulting in death within several years. Because recent evidence suggests that homocysteine (HC) is highly related to neurodegenerative disorders with ageing, we tried to elucidate the effects of multi‐vitamin therapy on G93A SOD1 transgenic mice.
Objectives: The multi‐vitamin therapy lowers homocysteine levels. We assessed whether multi‐vitamin therapy reduced the risk of major symptoms in G93A SOD1 transgenic mice.
Methods: We treated this murine model (nine multi‐vitamin treated mice, nine control mice) of ALS with multi‐vitamin. The multi‐vitamin therapy was composed of folic acid 1.97 mg/ml, pyridoxine 0.98 mg/ml and cyanocobalamin 0.1 mg/ml. We fed this complex with water to mice from 45 days of age. We performed the rotarod test and measured body weight from post‐natal 10 weeks, weekly
Results: We found that multi‐vitamin reinforcement significantly prolonged average lifespan and delayed disease onset with improvement of motor performance. The lifespan of mice with multi‐vitamin was 138.3 ±8.3 but control was 125.2 ±9.7. We found that multi‐vitamin treatment delayed disease onset and protected against loss of body weight. The onset of disease onset was 111.3 days for mice that took multi‐vitamins but 96.6 days for controls. The body weight average of multi‐vitamin treated mice was heavier than control mice. Especially, 16 weeks of age had the largest difference between multi‐vitamin mice (22.7 g) and control mice (20.8 g). However, body weight reduced from 16 weeks of age in multi‐vitamin treated mice and 14 weeks of age in control mice. Finally, the result of the rotarod test showed that multi‐vitamin treated mice tolerated it longer than control mice, from 12 weeks of age. At 13 weeks of age, multi‐vitamin treated mice tolerated rotarod for 180 s but control mice tolerated it for 139.4 s. However, it did not significantly slow disease progression
Discussion and conclusions: These results suggest that multi‐vitamin can be a potent therapeutic strategy for familial forms of ALS.
P94 L‐CARNITINE SUPPRESSES THE ONSET OF NEUROMUSCULAR DEGENERATION AND INCREASES THE LIFESPAN OF MICE WITH FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS
Kira Y, Nishikawa M, Inoue M
Osaka City University Medical School, Osaka, Japan
E‐mail address for correspondence: [email protected]
Background: Mitochondrial dysfunction has been reported to participate in the pathogenesis of ALS. L‐carnitine, an essential cofactor for the ß‐oxidation of long‐chain fatty acids, effectively inhibits various types of mitochondrial injury.
Objective: We examined the effect of L‐carnitine on disease onset and survival of FALS‐related SOD1G93A transgenic mice (FALS‐Tg).
Method: L‐carnitine was orally administered or subcutaneously injected to FALS‐Tg. We analysed the effect of L‐carnitine on neuromuscular injury, disease progression, and survival.
Results: We confirmed that the oral administration of L‐carnitine prior to disease onset significantly delayed the onset of signs of disease (log‐rank p = 0.0008), delayed deterioration of motor activity, and extended life span (log‐rank p = 0.0001) in FALS‐Tg. More importantly, subcutaneous injection of L‐carnitine increased the life span of FALS‐Tg (46% increase in males, 60% increase in females) even when given after the appearance of signs of disease.
Discussion and conclusions: L‐carnitine may have beneficial, therapeutic effects on the improvement of neuromuscular functions in ALS.
P95 BENEFIT OF PYRUVATE IN G93A SOD1 MUTANT TRANSGENIC MICE
Cho AS, Kim MY, Kim SM, Kim HJ, Sung JJ, Lee KW
Department of Neurology, Seoul National University College of Medicine, Clinical Research Institute of Seoul National University Hospital, Seoul, South Korea
E‐mail address for correspondence: [email protected]
Background: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that produces selective motor neuron death. Currently no effective pharmacological treatment is available for ALS.
Objectives: We investigated the neuroprotective effects of pyruvate in G93A SOD1 transgenic mice, the most widely used animal model of the familial form of ALS.
Methods:
Animal and sample preparation. Transgenic TgN (SOD1‐G93A) 1GUR mice were obtained from the Jackson Laboratory (Bar Harbor, ME).
Survival and disease onset analysis. A resting tremor in G93A SOD1 transgenic mice is the initial symptom of disease onset. Subsequently, the mice developed gait impairment and paralysis of one (or both) hindlimbs.
Immunohistochemistry. Sections were incubated using the free‐floating method (24 ∼ 72 h, 4°C) with one of the following primary antibodies; GFAP, Bcl‐2. Immunoreactivity was visualized using diaminobenzidine after signal amplification.
Motor Function Testing (Rotarod). Testing began by placing each mouse on a rod (diameter = 4 cm) rotated at a constant speed of 16 rpm, and the time that a mouse stayed on the rod before falling off (3 min maximum) was recorded as a measure of motor function competence.
Results: Pyruvate delayed disease progression and prolonged the lifespan of G93A SOD1 transgenic mice, but did not delay disease onset. Pyruvate treated mice had better motor performance. Pyruvate treated and saline treated G93A SOD1 transgenic mice showed similar weight losses. Pyruvate treatment reduced GFAP and Nitrotyrosine immunoreactivities, and increased Bcl‐2 immunoreactivity.
Discussion and conclusions: Our data demonstrate that pyruvate treatment has neuroprotective effects in G93A SOD1 transgenic mice. These neuroprotective effects may be associated with the inhibition of peroxynitrite‐mediated nitration and apoptotic cell death.
P96 GSK‐3β INHIBITOR SUPPRESSES SYMPTOM ONSET AND PROGRESSION IN ALS MOUSE MODEL
Koh SH, Kim Y, Noh M, Yoo A, Kim H, Kim H, Lee K, Lee Y, Kim J, Hwang S, Kim S
Departments of Neurology and Anatomy, College of Medicine, Hanyang University, Seoul, South Korea
E‐mail address for correspondence: [email protected]
Background and objectives: Glycogen synthase kinase (GSK)‐3β has recently been emphasized as an important pathogenic mechanism in neurodegenerative diseases. Although the neuroprotective effects of GSK‐3β inhibitors in Alzheimer's disease have been demonstrated, there has been no report of their effects on amyotrophic lateral sclerosis (ALS). This study was undertaken to evaluate the effects of a GSK‐3β inhibitor on ALS model mice.
Methods: We treated each of 14 ALS mice with 0.5 ml normal saline mixed with 1.8 μg and 3.6 μg, respectively, of GSK‐3β inhibitor per g of mouse and with only 0.5 ml normal saline intraperitoneally every week after 60 days of age (presymptomatic treatment).
Results: The GSK‐3β inhibitor treatments significantly prolonged the time until symptom onset and lifespan, especially in the 1.8 μg/g dosage. The GSK‐3β inhibitor inhibited the activity of GSK‐3β in a concentration‐dependent manner. Although this treatment preserved survival signal and attenuated death and inflammatory signals, the expression levels of intracellular signals were slightly different depending on the dose.
Conclusion: These data suggest that the inhibition of GSK‐3β could be one potential therapeutic candidate for ALS, and that levels of GSK‐3β inhibition might be very important in the therapeutic approach to ALS.
P97 INTRATHECAL INFUSION OF ANTI‐HEPATOCYTE GROWTH FACTOR ANTIBODY EXACERBATES DISEASE PROGRESSION IN A RAT MODEL OF ALS
Warita H1, Aoki M1, Nagai M3, Ishigaki A1, Mizuno H1, Funakoshi H2, Itoyama Y1
1Tohoku University Graduate School of Medicine and Tohoku University Hospital ALS Center, Sendai, Japan, 2Osaka University Graduate School of Medicine, Osaka, Japan, and 3Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan
E‐mail address for correspondence: [email protected]
Background and objectives: Hepatocyte growth factor (HGF) is one of the potent neurotrophic factors for motor neurons. A previous report had shown that overexpression of HGF in the nervous system could attenuate motor neuron loss and prolong the lifespan of transgenic mice with a mutant Cu/Zn superoxide dismutase (SOD1) gene. In addition, expression of HGF and its receptor, c‐Met, was reported in surviving motor neurons in patients with amyotrophic lateral sclerosis (ALS). Thus, we have examined the possible significance of HGF as an endogenous disease‐inhibitory factor using anti‐HGF antibody in a rat model of ALS.
Methods: Expression of the endogenous rat HGF was examined in the lumbar spinal cords of Gly93Ala mutant SOD1 (SOD1G93A) transgenic rats and their non‐transgenic littermates at 10, 14, and 19 weeks old (n = 3 in each group) with enzyme‐linked immunosorbent assay (ELISA). Based on the results, rabbit polyclonal neutralizing antibody specific for rat endogenous HGF (5 µg/g (body weight)) was continuously infused into the subarachnoid space of SOD1G93A transgenic rats (n = 5) for four weeks from the age at the beginning of endogenous HGF induction. As a control, normal rabbit IgG was administered instead of the antibody (n = 5). Careful and daily observation revealed the onset of the disease and the survival endpoint. The endpoint used was the loss of righting reflex within 10 s. At the endpoint, we examined histopathology in the lumbar spinal cord by immunohistochemistry with neuronal and glial markers.
Results: In the SOD1G93A transgenic rats, the progressive induction of endogenous HGF occurred from the presymptomatic stage. At 14 and 19 weeks old, there was a 35% and 107% increase in the rat endogenous HGF levels compared with the age‐matched non‐transgenic littermates. Therefore, the anti‐HGF antibody or normal rabbit IgG was administered to the SOD1G93A transgenic rats between the age of 14 and 18 weeks, continuously. In the anti‐HGF‐treated rats, the earlier onset (p = 0.18), the faster progression (p = 0.03), and the earlier survival endpoint (p = 0.046) of the disease was observed compared with those of control rats. At the endpoint, the anti‐HGF‐treated rats showed the same pathology such as loss of ventral horn neurons, astrocytosis, and microgliosis as in the controls.
Discussion and conclusions: The present results showed that a blocking of endogenous HGF by intrathecal infusion of the specific antibody exacerbated the mutant SOD1‐mediated disease. These results suggest that endogenous HGF may have an inhibitory role against ALS‐like disease progression in this rat model and also in ALS patients. Therefore, the exogenous administration of HGF could be a therapeutic strategy for ALS in the future.
P98 DEVELOPMENT AND CHARACTERIZATION OF A MURINE INTRATHECAL CATHETERIZATION TECHNIQUE FOR DRUG DELIVERY TO THE CENTRAL NERVOUS SYSTEM
Vieira F, de Zutter J, Lincecum J, Scott S
ALS Therapy Development Foundation, Cambridge, MA, USA
E‐mail address for correspondence: [email protected]
Drug discovery and drug target validation in the field of amyotrophic lateral sclerosis research has been limited by the inability to consistently deliver drugs to the brain and spinal cords of animal models of the disease. Until recently intracerebral ventricular (ICV) cannulation has been the only CNS drug delivery technique in use for mice, and preliminary evidence suggests that brain delivery of drugs does not ensure that they reach the spinal cord. The purpose of this study was to establish an intrathecal catheterization‐infusion method for continuous delivery of drugs directly to murine spinal cords. We verified the catheter‐pump localization and determined brain:spinal cord concentration ratios of drugs delivered by this method. Furthermore, we demonstrated that the surgical technique did not affect survival, neurological function, or chronically alter the profile of inflammatory mediators in spinal cord tissues of non‐transgenic mice and SOD1 G93A transgenic mice.
P99 PROTEOMIC ANALYSIS OF NERVOUS SYSTEM OF SOD1‐G93A MICE ADMINISTERED WITH POSSIBLE NEUROPROTECTIVE DRUGS
Kawamata J1, Yamashita H2, Shimohama s1, Takahashi R1
1Kyoto University, Kyoto, Japan, 2Hikone Municipal Hospital, Shiga, Japan
E‐mail address for correspondence: [email protected]
Background: Several drugs are reported to prolong survival of SOD1‐G93A mice. Proteomic analysis of tissue from drug‐administered Tg mice might elucidate change of some key proteins.
Objectives: To identify some key proteins, which might play some roles in the neuroprotective effect of effective drugs.
Methods: Transgenic mice model for FALS, designated B6SJL‐TgN(SOD1‐G93A)1Gur purchased from Jackson Laboratory was used. Minomycin was administered daily by intra‐peritoneal injection. Galanthamin‐suspension was administered orally every day. Mice were sacrificed at three months old. Spinal cord tissues from minomycin or galanthamin‐treated and control mice were thoroughly sonicated in 1 v/w of sample solution buffer consisting of 10 mM Tris HCl (pH 7.5), 9 M urea, 2% NP40, 2% mercaptoethanol, 1% protease inhibitor cocktail (Sigma) and centrifuged at 100,000 g for 1 h, and the supernatant was collected. The first dimension of gel electrophoresis was carried out using an immobilized pH gradient gel with a horizontal electrophoresis apparatus. The second dimension of gel electrophoresis was carried out on a 15% running gel in the presence of SDS. Protein spots on silver‐stained 2‐DE gels were digitized using a flatbed scanner. The image data were analysed using Melanie II software. Proteins with changed density in gels were identified with mass spectrometry.
Results: Several spots including GFAP, Vimentin, α internextin, Peroxiredoxin 5, HSP25, and 27 were elevated in mutant mice compared with non‐transgenic mice, whereas NF‐L, malate dehydrogenase, and Stathmin were decreased. Minomycin decreased HSP27 level and galanthamin also decreased HSP25 and 27 in challenged mice compare with sham operated mice.
Conclusion: The neuroprotective effect of minomycin and galanthamin might be mediated with heat shock molecules especially HSP25 and HSP27.
P100 CSF FROM AMYOTROPHIC LATERAL SCLEROSIS PATIENTS DECREASES VOLTAGE ACTIVATED SODIUM CHANNEL NAV1.6 EXPRESSION IN DEVELOPING RAT VENTRAL HORN MOTOr NEURONS
Gunasekaran R, Nalini A, Raju TR
National Institute of Mental Health and Neurosciences, Bangalore‐Karanataka, India
E‐mail address for correspondence: [email protected]
Amyotrophic lateral sclerosis (ALS) is a devastating human neurodegenerative disorder, which causes atrophy and weakness of skeletal muscles leading to death, the aetiology of which is still unknown. Our study has investigated the effects of CSF from ALS patients on voltage activated Nav1.6 sodium channel expression in developing rat ventral horn motor neurons. CSF obtained from ALS and non‐ALS neurological patients was injected into three‐day‐old rat pups in the spinal subarachnoid space at the rate of 1 µl/2.5 min using a micro injector. The rats were sacrificed 48 h after CSF injection and spinal cord sections were processed for immunocytochemistry with Nav1.6 sodium channel antibody and also for cytochrome oxidase labelling. CSF samples from all six ALS patients significantly decreased Nav1.6 sodium channel expression compared to CSF from non‐ALS patients. CSF from all six ALS patients also decreased cytochrome oxidase activity in the rat spinal motor neurons, which could be a sign of degeneration of motor neurons. Nav1.6 sodium channel produces rapidly activating and inactivating currents, which support the action potential generation. Decreased expression of this channel may suppress the electrical activity of the motor neurons, which may lead to degeneration of the neurons.
P101 AMPA AND CANNABINOID RECEPTOR TRAFFICKING IS ALTERED IN MOTOR NEURONS OF ALS MODEL MICE: IMPLICATIONS FOR EXCITOTOXICITY
Zhao P, Beattie EC, Abood ME
California Pacific Medical Center Research Institute, San Francisco, CA, USA
E‐mail address for correspondence: [email protected]
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder involving the selective loss of spinal cord motor neurons. Excitotoxicity mediated by glutamate has been implicated as a cause of this progressive degeneration. In this study we used quantitative immumocytochemistry to comparatively measure the total cellular expression and the plasma membrane localization of AMPA‐type glutamate receptors (AMPARs) and cannabinoid receptors (CB1 and CB2) in spinal cord motor neurons. Wild‐type and ALS model mice SOD1G93A were compared and specific subunit subtypes (GluR1 and GluR2) of AMPARs were followed. Motor neurons from SOD1G93A mice displayed variable expression patterns of glutamate AMPA receptor subunits during disease progression. Prior to disease onset, motor neurons from 40‐day‐old SOD1G93A mice showed increased GluR1 AMPAR surface expression compared to wild‐type, but no change in total cellular expression when GluR1 was used to follow AMPAR localization. GlurR2 total cellular expression is found to be decreased at this time point. This finding is pertinent to excitotoxicity since the presence of properly edited GluR2 subunits in AMPARs blocks this channel from passing calcium while GluR2 lacking GluR1 homomers allows calcium passage. In a later stage of disease progression, in 120‐day‐old SOD1G93A mice, both plasma membrane and total cellular levels of GluR1 increased while total cellular GluR2 levels were decreased. The cannabinoid system in spinal cord has been shown to have protective effects on motor neuron survival, which may involve antioxidant and anti‐excitotoxic functions. Here we measured CB1 and CB2 total cellular expression and plasma membrane localization alongside AMPAR expression at 40, 90, and 120 days. Total cellular level of CB1 was decreased while CB2 total cellular expression was increased at 120 days. Taken together, the changes of GluR1/GluR2 ratio in motor neurons in very young SOD1G93A mice suggest a possible mechanistic role for GluR2‐lacking AMPA receptors in ALS disease initiation and progression. Observed CB1 and CB2 changes late in disease progression suggest an adaptive response to neurodegeneration induced by excitoxicity.
P102 ROLE OF SCHWANN CELL DYSFUNCTION IN MUTANT SOD1‐MEDIATED NEURODEGENERATION
Turner BJ, Ackerley S, Davies KE, Talbot K
University of Oxford, Oxford, UK
E‐mail address for correspondence: [email protected]
Background: Mounting evidence from transgenic mice with cell restrictive, silenced or chimeric expression of mutant SOD1 suggests a potential non‐neuronal contribution to motor neuron degeneration in FALS. Such studies of non‐cell autonomous toxicity to date have focused on adjacent extraneuronal cells that occupy the spinal ventral horns. However, a recent evaluation of transgenic SOD1G93A mice revealed a neuropathological sequence consistent with a distal axonopathy (1), suggesting that peripheral cells such as skeletal myocytes or myelinating glia may influence disease. Schwann cells (SCs) are the major glial population of the PNS and interact intimately with lower motor neurons. Although principally concerned with myelination, SCs also modulate axonal transport, secrete neurotrophic factors and promote neuromuscular synapse regeneration following injury and disease. Thus, SC and motor neuron interactions may be relevant to FALS pathogenesis.
Objectives: To investigate the functional properties of SCs harbouring SOD1 mutations in vitro using primary transgenic SOD1G93A cultures or transfected cell lines and in vivo by generation and characterization of transgenic mice with SC targeted expression of mutant SOD1.
Methods: SCs were initially characterized in transgenic SOD1WT, SOD1G93A and non‐transgenic mice by immunohistochemical analysis of peripheral nerves for S100, myelin basic protein and p75 neurotrophin receptor (p75NTR). Primary SCs were then isolated, purified and cultured from sciatic nerves of the neonatal mice above. In addition, the post‐natal rat SC line (SCTM41) was stably transfected with EGFP‐tagged wild‐type and SOD1 mutant plasmids. Expression of neurotrophins, neuregulins and related receptors was analysed in both cell culture models using real time PCR, antibody arrays and immunoblotting. Conditioned medium and neuronal‐glial co‐culture experiments were also performed using stably transfected motor neuron‐like (NSC‐34) cells. Lastly, transgenic mice that overexpress SOD1WT or SOD1G93A exclusively in myelinating SCs were generated using a myelin protein zero promoter (P0‐Cx32) splice construct and studied longitudinally for motor pathology and survival.
Results: Induction of p75NTR expression was observed in SCs from adult transgenic SOD1G93A mice, consistent with a denervated or pro‐apoptotic phenotype accompanying axonal degeneration. Transgenic P0‐Cx32 SOD1 mice were generated and these will be further characterized.
Conclusions: The characterization of these culture models and generation of transgenic mice with SC targeted expression of mutant SOD1 should clarify the role, if any, of peripheral myelinating glia in FALS aetiology and/or progression.
References
- Fischer LR, Culver DG, Tennant P, et al. Exp Neurol. 2004;185:232–40.
P103 CONDITIONAL KNOCKOUT OF SOD2 IN POSTNATAL MOTOR NEURONS REVEALS IN‐VIVO RESISTANCE TO MITOCHONDRIAL GENERATED SUPEROXIDE RADICALS
Misawa H1, Moriwaki Y1, Kawashima K1, Shimizu T2, Shirasawa T2, Takahashi R3
1Kyoritsu University of Pharmacy, Minato‐ku, Tokyo, Japan, 2Tokyo Metropolitan Institute of Gerontology, Itabashi‐ku, Tokyo, Japan, 3Kyoto Univerisity, Sakyo‐ku, Tokyo, Japan
E‐mail address for correspondence: [email protected]
Background: Oxidative stress has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), yet it remains unclear whether oxidative stress is a major cause or merely a consequence of cellular dysfunction associated with ALS. Mitochondria are both a major source of reactive oxygen species (ROS) production as well as a major target of ROS‐induced cellular injury. Although motor neurons in cell culture are shown to be vulnerable to cell death mediated via calcium influx after exposure to glutamate, it is unclear how motor neurons respond to the overproduction of mitochondrial‐derived ROS in vivo.
Objectives: Mitochondrial localized superoxide dismutase (SOD2) is thought to play an important role in cellular defense against oxidative damage by ROS. Mice deficient in SOD2 die during embryonic or early postnatal development, which precludes analysis of a pathological role for superoxide in adult tissue. In order to circumvent the early lethality of SOD2 knockout mice, here we generated postnatal motor neuron‐specific SOD2 knockouts, and analysed their pathophysiological alterations in motor neurons.
Methods: To generate postnatal motor neuron‐specific SOD2 knockout mice, we crossed mice homozygous for floxed SOD2 alleles (1) with VAChT‐Cre Slow mice in which Cre expression is restricted in postnatal somatomotor neurons (2). The spatial production of superoxide radicals was investigated by in situ detection of oxidized hydroethidine (HEt). Histological assessment was conducted by staining brain sections with cresyl violet or Fluoro‐Jade B. The following marker antibodies were used to detect oxidative damage(s) in tissue sections: 8‐OHdG, nitrotyrosine, malondialdehyde, SMI‐31, and GFAP. To test whether SOD2‐deficient motor neurons are more vulnerable to nerve injury, we employed unilateral transection of the hypoglossal nerve. Hypoglossal motor neuron survival was then assessed five weeks following transection. We also analysed the structural stability of the transected hypoglossal axons two days post‐operation (Wallerian degeneration).
Results: SOD2 immunoreactivity was specifically lost in a subset of somatomotor neurons, resulting in enhanced superoxide production as revealed by massive oxidation and deposition of HEt in mitochondria. However, extensive histological examination revealed no signs of oxidative damage in animals up to 18 months after birth. Ultrastructural analysis of mitochondria in SOD2‐deficient motor neurons revealed no degenerative changes such as swelling, disorganization of the cristae or vacuolar formation. Although motor neuron survival five weeks after hypoglossal nerve axotomy was unchanged, disorganization of distal nerve axons two days following injury was accelerated in SOD2‐deficient motor neurons.
Discussion and conclusions: These data demonstrate that postnatal motor neurons are surprisingly resistant to oxidative damage from mitochondrial‐derived superoxide radicals, but that such damage may sensitize axons to disorganization following nerve injury.
References
- Ikegami T, et al. Biochem Biophys Res Commun. 2002;296:729–36.
- Misawa H, et al. Genesis 2003;37:44–50.
P104 SOD1 AGGREGATES WITHIN MOTONEURONAL AXONS DO NOT ASSOCIATE WITH MITOCHONDRIA
Tateno M1, Takahashi R2, Araki T1
1National Institute of Neuroscience, NCNP, Tokyo, Japan, 2Kyoto University, Kyoto, Japan
E‐mail address for correspondence: [email protected]
Background and objectives: Conversion of mutant SOD1 proteins into aggregates is thought to be responsible for the selective loss of motor neurons in SOD1‐related ALS, although the mechanisms underlying aggregate‐associated toxicity have not been clarified. Since SOD1 aggregates are detected in purified mitochondria and reported to be associated with an anti‐apoptotic protein, Bcl‐2, in symptomatic mutant SOD1 transgenic mice, dysfunction of mitochondria and/or an enhancement of mitochondria‐dependent apoptosis is implicated in the pathogenic mechanism (1). In the course of study for the localization of SOD1 aggregates in SOD1G93A transgenic mice, we detected a significant amount of SOD1 aggregates within the axons in pre‐symptomatic mice (2). Since a large portion of mitochondria is transported within motor neurons, we investigated whether SOD1 aggregates detected in axons were derived from transported mitochondria and associated with Bcl‐2.
Methods: To isolate intact mitochondria or SOD1 aggregates derived from axons, not from cell bodies/dendrites of motor neurons, ventral white matter from the spinal cords of SOD1G93A and SOD1wt transgenic mice was dissected, homogenized, and subjected to Nycodenz‐density gradient centrifugation. The conditions for homogenization differed in mitochondria‐ and SOD1 aggregation‐isolation.
Results: To compare the distribution of mitochondria and SOD1 aggregates in Nycodenz fractions, we performed Western blotting using antibodies against SOD1 and several mitochondrial markers, and found that their distribution was clearly different. To study an association of SOD1 aggregates and Bcl2, the aggregate‐enriched fraction was subjected to an immunoprecipitation with anti‐SOD1 antibody followed by Western blotting with anti‐Bcl2 antibody. In our system, Bcl‐2 was not found to be co‐immunoprecipitated by anti‐SOD1 antibody.
Discussions and conclusions: Since the distribution of mitochondria and SOD1 aggregates was different in Nycodenz‐density gradient fractionation, a majority of mitochondria transported within axons is unlikely to involve the SOD1 aggregates. This result was supported by the immunoprecipitation analysis, in which association of SOD1 and Bcl‐2 was not detected in the SOD1 aggregate‐enriched Nycodenz fraction. We alternatively detected certain axonal transport‐related proteins which were co‐immunoprecipitated by anti‐SOD1 antibody. From these and other data, we concluded that the SOD1 aggregates within axons are not co‐transported with mitochondria, but transported by unknown systems.
References
- Pasinelli P, Belford M, E, Lennon N, et al. Neuron 2004; 43:19–30.
- Tateno M, Sadakata H, Tanaka M, et al. Hum Mol Genet. 2004;13:2183–96.
P105 ALTERED UBIQUITINATED PROTEIN EXPRESSION IN WOBBLER AND TRANSGENIC SOD1G93A SPINAL CORD: A PROTEOMIC ANALYSIS
Pioro EP1, Zhang J1, Darko S2, Bowser R2
1Cleveland Clinic, Cleveland, OH, USA, 2University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
E‐mail address for correspondence: [email protected]
Background: Ubiquitinated inclusions (UbIs) occur in neurons of patients with ALS, and of certain models of ALS, including wobbler (wr) and transgenic SOD1G93A (TgSOD1G93A) mice. This probably reflects abortive proteolysis of one or more abnormal proteins, although the contents of the inclusions are not known. Although potentially protective by sequestering toxic proteins, the UbIs may inadvertently trap normal proteins which prevent their functioning and results in further cellular injury.
Objectives: To identify by mass spectrometry based proteomics ubiquitinated proteins in the spinal cord of wr and TgSOD1G93A mice expressing a 6 x His‐tagged ubiquitin transgene. The 6 x His tag allows enrichment of ubiquitinated proteins, either within inclusions or not, for subsequent characterization. This may provide new insights into disease pathogenesis and novel therapeutic targets.
Methods: Cervical and lumbar spinal cord was obtained from 56 day‐old mutant wr and wild‐type littermates, as well as 90 day‐old TgSOD1G93A mutant and nonTgSOD1G93A control mice. 6 x His‐tagged ubiquitinated proteins were enriched from these regions by nickel‐coated ProteinChips® and analysed in duplicate by surface enhanced laser desorption/ionization mass spectrometry (SELDI‐TOF‐MS). Evaluation of the spectra was performed using the Ciphergen software (Ciphergen Biosystems, Inc.). Comparisons were made between mutant and control mice of each group (wr vs. wild‐type and TgSOD1G93A vs. nonTgSOD1G93A) and also between groups (wr vs. TgSOD1G93A).
Results: Significant alterations were detected in 17 protein mass peaks of wr vs. wild‐type spinal cord. We also identified increased levels of peptide fragments in the wr sample corresponding to the molecular mass of the Vps54 protein, known to be mutated in wr mice. Nine protein mass peaks were significantly altered in TgSOD1G93A mice compared to their non‐TgSOD1G93A controls, including increased SOD1, which was not altered in the wr sample. Except for two mass peaks that were increased (6.78 and 7.93 kDa) and one that was decreased (12.2 kDa), in both wr and TgSOD1G93A mice all other peak identities were unique to each group. In addition, two protein peaks exhibiting increased levels in TgSOD1G93A mice were decreased in mutant wr mice.
Discussion and conclusions: Our proteomic data reveal alterations of specific ubiquitinated proteins in the spinal cord of the wr and TgSOD1G93A mouse models of ALS. While some of the proteins involved were similar between the wr and mutant SOD1 groups, most were specific to the model, and sometimes altered in opposite directions. This suggests more uniqueness than commonality at the protein level in the pathogenesis of motor neuron degeneration in these two mouse models. We are determining the protein identity of each protein and applying SELDI‐TOF‐MS methods to identify specific contents of isolated ubiquitinated inclusions. Our study indicates that mass spectrometry based proteomics can identify protein alterations and help elucidate disease mechanisms in mouse models of ALS.
P106 INTRANEURONAL ACCUMULATION OF UBIQUITINATED INCLUSIONS IS ASSOCIATED WITH IMPAIRED PROTEASOME FUNCTION IN WOBBLER MOTOR NEURON DEGENERATION
Zhang J, Kostenko V, Kidd G, Pioro EP
Cleveland Clinic, Cleveland, OH, USA
E‐mail address for correspondence: [email protected]
Background: Ubiquitinated inclusions (UbIs) occur in neurons of patients with ALS, implicating ineffective proteolysis by the ubiquitin‐proteasome system (UPS). Such inclusions, which may be neurotoxic, can contain an aberrant protein that would be harmful if not sequestered from the cell. The recent discovery that a mutation in Vps54, which is involved in endosomal trafficking, causes neuronal degeneration in the wobbler (wr) mouse is of interest because we find UbIs in neurons of cortex (Cx) and cervical spinal cord (CSC). However, how this mutation results in neuronal degeneration and formation of these inclusions is unknown.
Objectives: To characterize intraneuronal UbIs and to assess proteasome activity in wr CSC and Cx, in order to determine whether the UPS is dysfunctional.
Methods: CSC and Cx of BL6NZB wr mice and their wild‐type littermates were examined at presymptomatic (14 days) and early symptomatic (28 days) stages by immunocytochemical and biochemical methods to characterize the UbIs, their associated proteins, and relationship to organelles. We also assessed chymotrypsin‐, trypsin‐, and caspase‐like enzymatic activities of the 20S proteasome.
Results: UbIs visible by light microscopy develop in the CSC and Cx of the wr mouse, but not its wild‐type littermate, between 16 and 18 days of age. However, ubiquitinated proteins identified by size exclusion filter trapping already appear at 14 days. Although closely associated with endoplasmic reticulum, Golgi apparatus, and lysosomes, UbIs are not colocalized with them. Proteasome 20S activity is significantly reduced (p<0.01) in wr relative to wild‐type, primarily for chymotrypsin‐like activity at 28 days (CSC, –18%; Cx, –25%), but also at 14 days (CSC, –19%; Cx, –16%). Lesser, although still significant, reductions occur in the other enzymatic activities at 28 days.
Discussion and conclusions: Accumulation of ubiquitinated proteins precedes disease onset in wr CSC and Cx in a pre‐aggregate state before forming visible inclusions. Although formed inclusions may protect the cell by sequestering harmful proteins, the pre‐aggregate may be detrimental, and inhibit proteasome activity. We found predominantly chymotrypsin‐like but also trypsin‐ and caspase‐like enzyme activities to be significantly reduced in CSC and Cx as early as 14 days, when mice appear normal. This indicates that UPS dysfunction precedes disease onset and probably contributes to accumulation of UbIs and neurodegeneration in the wr mouse.
P107 REDUCTION OR ABSENCE OF NORMAL PRION PROTEIN ACCELERATES DISEASE IN G93A SOD‐1 MICE
Turnbull J, Jiang F, Li WP, del Luca D, Kim J
McMaster University, Hamilton, Canada
E‐mail address for correspondence: [email protected]
Background: Misfolded prion proteins have been implicated in several neurodegenerative diseases of animals and man. However, the normal function of the prion protein has remained more elusive. PrP null mice breed and develop normally, and seemingly demonstrate few abnormalities in adult life. Recently the prion protein has been shown to be neuroprotective in hypoxic/ischaemic injury and epileptogenesis, with anti‐apoptotic and anti‐excitotoxic activity. Since these pathological processes have been implicated in ALS, PrPc may have beneficial action in ALS. However, there is also a possibility that PrPc could play an unexpected detrimental role in ALS, as PrPc overexpression can lead to caspase 3 activation and cell death independent of PrPsc formation, in a process accelerated by proteasomal inhibition (which might occur in ALS).
Objectives: We wished to test whether transgenic mice overexpressing the human G93A SOD1 transgene but lacking one or both copies of the PrP gene demonstrated improved or worsened survival compared to those expressing normal PrP.
Methods: PrP null mice were obtained as a gift from Dr. Jean Manson of the Institute for Animal Heath in Edinburgh. These mice were crossed with heterozygote G93A SOD1 mice and the F1 generation bearing the SOD1 transgene were backcrossed against PrP null mice. In this way three groups of G93A SOD1 mice can be produced: mice homozygous for the deleted PrP gene, mice heterozygous for the deleted PrP gene, and wild‐type PrP. We compared disease onset and survival in the three groups.
Results: Very few SOD1 mice homozygous for the PrP deletion could be bred, and it is likely that there is decreased survival in utero. Mice heterozygous for the PrP deletion (n = 16) had significantly accelerated disease onset compared to wild‐type (n = 8) (103 days vs. 112 days, respectively), and a major reduction in survival (113 days vs. 144 days, p<0.001).
Conclusions: The normal cellular prion protein plays a protective role in the SOD‐1 mouse model of ALS. We are presently looking for loss‐of‐function mutations or deletions in the prion protein in patients with ALS. It is unclear whether interventions aimed at enhancing PrPc levels would be beneficial.
P108 DYSREGULATION OF LIPIDAEMIA IN ANIMAL MODELS AND ALS PATIENTS
Fergani A1, Dupuis L1, Oudart H2, Gonzalez de Aguilar JL1, Fricker B1, Rene F1, Meininger V3, Loeffler JP1
1INSERM U692, Laboratoire de Signalisations Moléculaires et Neurodégénérescence, Université Louis Pasteur, Strasbourg, France, 2DEPE‐UPR9010 CNRS, Strasbourg, France, 3Service de Neurologie, Hôpital de la Pitié Salpétrière, Paris, France
E‐mail address for correspondence: [email protected]
Background: Our work has previously shown that skeletal muscle hypermetabolism is a hallmark of the pathology developped by mutant SOD1 mice, an animal model of amyotrophic lateral sclerosis (ALS).
Objectives: We analyzsed lipid metabolism in mutant SOD1 mice and in a cohort of 369 patients suffering from ALS.
Methods: We used biochemical and physiological analysis of lipid metabolism coupled with gene expression measurements using real time RT‐PCR.
Results: We show here that SOD1(G86R) mice display decreased blood lipids. Hypolipidaemia was not due to intestinal or hepatic dysfunction but rather to increased triglycerides‐rich lipoproteins clearance by peripheral tissues including skeletal muscle. Moreover, mutant SOD1 mice muscles displayed neutral lipid accumulation, increased expression of genes involved in lipoprotein clearance as well as muscle aggregates of mutant SOD1. Contrary to the animal model, ALS patients were hyperlipidaemic but, similar to the situation in mice, a better survival of ALS patients was associated with an abnormally high LDL/HDL ratio.
Conclusions: Altogether, our results show that alterations in lipoprotein metabolism are associated with amyotrophic lateral sclerosis and contribute to neurodegeneration.
P109 NUCLEOCYTOPLASMIC TRANSPORT INVOLVEMENT IN THE SPINAL CORD OF A MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS
Kinoshita Y1, Ito H1, Zhang J2, Wate R1, Ohnishi S1, Fujita K1, Shinde A1, Nakano S1, Kusaka H1
1Department of Neurology, Kansai Medical University, Moriguchi, Osaka, Japan, 2Department of Neurology, Harbin Medical University, Harbin, Heilongjiang, China
E‐mail address for correspondence: [email protected]
Background: Nucleocytoplasmic transport is essential for maintaining cell viability and cellular functions. Transport of proteins and nucleic acids between the nucleus and the cytoplasm occurs through nuclear pore complexes (NPCs). NPCs have been only recently investigated in patients with autoimmune, neoplastic, viral or hereditary disorders. Regarding neurodegenerative diseases, very recently Sheffield et al. reported the first evidence for the involvement of NPCs in Alzheimer's disease.
Objectives: To examine whether dysfunction of the nucleocytoplasmic transport system is involved in the pathomechanisms of ALS.
Methods: Lumbar spinal cord sections from G93A SOD1 transgenic female mice at 8–20 weeks of age, and from age‐matched female wild‐type littermates were investigated immunohistochemically. The following primary antibodies were used: monoclonal antibodies against nucleoporin p62 (Nup62), against karyopherin alpha2/Rch‐1 (importin alpha), against histone H1, and against caspase‐3, and polyclonal antibodies against the importin beta family (karyopherin beta1 (importin beta), karyopherin beta2 (transportin), karyopherin beta3 (importin 5)), and against beta‐catenin.
Results: The antibody against Nup62, one of the structural proteins of NPC, labeled the nuclear rim because of regular contours of most of the lumbar anterior horn cells in the control mice. By contrast, this antibody demonstrated the nuclear irregularity with disrupted contours of the surviving anterior horn cells of the transgenic mice, irrespective of their age. Using antibodies against the importin beta family, the major carrier proteins of nucleocytoplasmic transport, and those against their adapter protein, importin alpha, the immunoreactivities were decreased within the nuclei and increased within the cytoplasm of a subset of the remaining anterior horn cells of the transgenic mice. In addition, Lewy body‐like hyaline inclusions were invariably identified with these antibodies. Furthermore, the immunoreactivities for histone H1 and beta‐catenin, the representative cargo proteins transported by importin beta family‐dependent and family‐independent nucleocytoplasmic transport pathways, respectively, were demonstrated similarly to those for importin beta family and importin alpha. The altered distributions of these proteins were not associated with caspase‐3 expression. Chronological quantitative analysis of sections from the transgenic mice revealed a progressive decrease in the proportion of the anterior horn cells exhibiting a more intense reactivity for these carrier and cargo proteins in the nucleus than in the cytoplasm. In contrast, we found that anterior horn cells with the immunoreactivity in their cytoplasm being more pronounced than that in their nucleus were obviously increased in number along with the disease progression.
Conclusions: Our present results imply that dysfunctional nucleocytoplasmic transport could be involved in the pathomechanisms underlying ALS.
P110 ANALYSIS OF THE ROLE OF DYNEIN MUTATIONS IN ATTENUATING THE PHENOTYPE OF MUTANT‐SOD1 TRANSGENIC MICE
Morsi el‐Kadi A1, Soura V1, Chia R2, Jackson G2, Greensmith L2, Fisher EMC2, Hafezparast M1
1University of Sussex, Brighton, East Sussex, UK, 2Institute of Neurology, London, UK
E‐mail address for correspondence: [email protected]
Background: Cu/Zn SOD1G93A transgenic mice develop an ALS‐like phenotype, which is characterized by motor neuron degeneration and muscle paralysis. Previously, we demonstrated that a missense point mutation in the gene encoding the heavy chain subunit of cytoplasmic dynein causes degeneration of motor neurons in the legs at odd angles (Loa) mouse (1). We also showed that double mutant (Loa/SOD1G93A) transgenic mice have a significant improvement in SOD1G93A ‐mediated disease accompanied by a reduction in motor neuron degeneration (2).
It is possible that the improvement in the phenotype of the double mutants is due to interaction of SOD1G93A with the dynein complex.
Objectives: 1) To analyse the dynein complex in wild‐type and Loa mice and examine whether mutant SOD1 interacts with the dynein complex. 2) To elucidate at the molecular level the role of the Loa mutation in attenuating the phenotype of SOD1G93A transgenic mice.
Methods: We have been using protein‐protein interaction assays, such as immunoprecipitation and pull‐down assays followed by Western blotting and mass spectrometry to dissect the dynein complex in Loa mice and test the possibility of its interaction with SOD1G93A in vivo and in vitro.
Results: Our preliminary pull‐down assays using bacterially expressed wild‐type and several mutant SOD1 proteins show that there is a potential interaction between mutant SOD1 and the dynein complex from wild‐type and Loa brain homogenates with a possible higher affinity towards mutant dynein. In addition, using an anti‐dynein antibody and brain tissue extracts from double mutant (Loa/SOD1G93A) transgenic mice, we have co‐immunoprecipitated SOD1G93A protein with the dynein complex. Nano‐spray liquid chromatography/mass spectrometry (LC/MS) of the dynein complex from brains of wild‐type and Loa mice immunopreciptitated by anti‐dynein antibodies have identified some interesting interacting proteins including GAPDH, whose importance in neuronal apoptosis and neurodegenerative disorders is emerging.
Discussion: Our work is in progress towards verifying these findings.
References
- Hafezparast M, Klocke R, Ruhrberg C, et al. Science 2003; 300: 808–12.
- Kieran D, Hafezparast M, Bohnert S, et al. J Cell Biol. 2005; 169:561–7.
P111 ROLE OF ASK1 IN FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS‐LINKED MUTANT SOD1 TOXICITY
Sasabe J1, Chiba T1, Yamada M1, Nishimoto I2, Aiso S1, Matsuoka M2
1Department of Anatomy, 2Department of Pharmacology, KEIO University, School of Medicine, Shinanomachi, Shinjuku‐ku, Tokyo, Japan
E‐mail address for correspondence: [email protected]
Background: Some familial ALS (FALS) cases are associated with dominantly inherited missense mutations in the Cu/Zn‐superoxide dismutase‐1 (SOD1) gene. A recent in vitro study suggested that the SOD1 mutant‐induced death is mediated by the Fas receptor and apoptosis signal‐regulating kinase 1 (ASK1) (1).
Objectives: To characterize the role of ASK1 in the pathogenesis of FALS, we crossed ASK1 knockout mice with G93A‐SOD1 transgenic (Tg) mice (G93A Tg/ASK1‐/‐).
Methods: G93A‐SOD1 Tg mice were crossed with ASK1 knockout mice (2). Motor performance of the mice was evaluated in the rotarod test as previously described (3). NSC34 motoneuronal cells were transfected with pEF‐BOS vector, wild‐type‐SOD1, or G93A‐SOD1 cDNA and phosphorylation states of c‐Jun N‐terminal kinase (JNK) and p38 MAPK were detected with immunoblot analysis.
Results: There was no difference in the disease onset between the control mice (G93A/ASK1+/+ mice) and G93A/ASK1‐/‐ mice. Mean survival of G93A/ASK1‐/‐ mice, however, was significantly longer than that of the control mice. Transfection with the G93A‐SOD1 gene led to earlier and continuous JNK activation while it did not lead to significant activation of p38 MAPK in NSC34 cells.
Discussion and conclusions: Enetic disruption of ASK1 from ALS mice improved motor performance and prolonged survival without delaying disease onset, suggesting that ASK1 is involved in the disease progression of FALS. Mutant SOD1 induced the JNK activation rather than the activation of p38 MAPK in NSC34 cells. Thus, the Fas/ASK1/JNK pathway might be involved in motoneuronal death in FALS. Therefore, molecules constituting the signaling pathway would be new targets of a remedy for ALS.
References
- Raoul C, Estevez AG, Nishimune H, et al. Neuron 2002;35:1067–83.
- Tobiume K, Matsuzawa A, Takahaxhi T, et al. EMBO Rep. 2001;2:222–8.
- Chiba T, Hashimoto Y, Tajima H, et al. J Neurosci Res. 2004;78:542–52.
P112 COMPARATIVE STUDIES OF MOTOR NEURON DISEASE IN MICE TRANSGENIC FOR HUMAN WTSOD1 AND MUTATED SOD1
Graffmo KS1, Jonsson PA2, Bergemalm D2, Zetterström P2, Marklund SL2, Brännström T1
1Department of Medical Biosciences/Pathology, 2Department of Medical Biosciences/Clinical Chemistry, Umea, Sweden
E‐mail address for correspondence: [email protected]
Background: Superoxide dismutase 1 (SOD1) is associated with familial amyotrophic lateral sclerosis (ALS) and may well play an important role in sporadic ALS.
Objective: Mice transgenic for mutated D90A, G127X, G93A, G93A del, G85R and wild ‐type human SOD1 as well as non‐transgenic control mice C57BL/BJBom were compared to reveal common traits and differences in development and progress of motor neuron disease.
Methods: Mice were sacrificed at preselected intervals and at terminal stage. Three levels of the spinal cord as well as the brainstem, cerebellum and brain were studied. Genotyping and SOD activity analysis were performed. Histopathology and immunohistochemistry using polypeptide antibodies directed against exon 1 and 5 of human SOD1 were performed.
Results: Blood levels and activity of SOD1 seem to be unrelated to disease progression and lifespan. In all transgenic mice, small SOD1 positive inclusions were found in the ventral horns from young age, and accumulated throughout life. SOD1 positive aggregates also accumulated in both glial cells and in white matter of the spinal cord. Vacuolization, gliosis and hyperchromatic cells were also found, but the extent varied between strains. In terminal mice SOD1 is also present in axons and in peripheral nerves.
SOD1 positive inclusions are also seen in the motor areas of the brain and brainstem. Large aggregates were found in the cerebellum and in white matter of the brainstem. Hyperchromasia, vacuolization and gliosis were found in parts of the brain, the extent varied between the different mouse strains.
Discussion: Aggregation of SOD1 in the central nervous tissue appears to be related to severity of disease. Degenerative features such as vacuolization and gliosis precede phenotypic alterations. Changes are seen not only in motor areas but also in higher centres of the telencephalon. Our studies indicate that the SOD1 gain‐of‐function is due to aggregation.
P113 HUMAN SOD1 LACKING NATIVE AS WELL AS CARRYING NON‐NATIVE DISULPHIDE BONDS ENRICHED IN SPINAL CORD OF MURINE ALS MODELS
Zetterstrom P1, Stewart HG2, Jonsson PA1, Bergemalm D1, Graffmo KS1, Brännström T1, Andersen PM2, Oliveberg M3, Marklund SL1
1Department of Medical Biosciences, 2Department of Pharmacology and Clinical Neuroscience Umeå, Sweden, 3Department of Biochemistry and Biophysics, University of Stockholm, Stockholm, Sweden
E‐mail address for correspondence: [email protected]
Background: Over 100 mutant SOD1s associated with ALS cause disease by an unknown cytotoxic mechanism. SOD1 is present in higher levels in most peripheral organs than in motor areas of the CNS. The murine transgenic ALS models express variable amounts of human SOD1 in spinal cords, from one‐half (G127X, C‐terminal truncation), equal (G85R) to 17‐fold (G93AGur) and 20‐fold (D90A) higher than endogenous murine SOD1 levels. The G127X and G85R SOD1s in the spinal cord are disulphide‐reduced, inactive, probably misfolded and enriched relative to other organs, suggesting inefficient degradation of such SOD1 forms. Mis/unfolding of proteins leads to exposure of the hydrophobic core and increased binding in hydrophobic interaction chromatography (HIC). One hundred per cent of the G127X and 80% of G85R in the spinal cords binds to HIC columns. In the more stable SOD1 models, G93A and D90A, subfractions of the SOD1 in spinal cord extracts are bound to the HIC columns, 5% and 1%, respectively. Much less of the SOD1 in brain and peripheral organs is bound in the HIC. In the G93A mice there was no change in the binding proportion from 20 days until they were moribund at 124 days. The hydrophobic soluble SOD1, long‐term enriched in spinal cords, could be responsible for the cytotoxicity.
Objectives: The disulphide status of SOD1 is of great importance for the folding of the protein. Reduction of the intrasubunit disulphide bond results in a destabilization of SOD1 that could lead to HIC affinity. This study attempts to reveal the disulphide status of the hydrophobic subfractions of SOD1.
Methods: Brain, spinal cord, liver and kidney homogenates from transgenic mice were centrifuged at 20,000 g for 20 min at 4°C, the supernatants were collected and subjected to octyl‐sepharose HIC. Multiple SOD1 variants expressed in E. coli with different cysteins mutated, were denatured with gunidinium chloride, reduced with TCEP and metal‐deprived with DTPA. They were then boiled in sample buffer and allowed to oxidize and disulphide shuffle. Reduced and non‐reduced Western immunoblots were used to analyse eluates for human SOD1.
Results: All recombinant SOD1s, tissue homogenates and HIC binding fractions show a single band of the expected molecular weight on reduced SDS gels. On non‐reduced gels, tissue homogenates contain both disulphide reduced and oxidized protein as seen before. The recombinant proteins show that SOD1 can disulphide shuffle to form non‐native disulphide bonds with all four cysteins involved. HIC binding SOD1 includes both disulphide‐reduced SOD1 and species with non‐native disulphide bonds.
Discussion: Disulphide shuffling in the reduced cytosol is surprising and might hint that HIC binding SOD1 can be of other cellular origin than cytosolic. These hydrophobic subfractions might be cytotoxic directly via reaction with critical components of the cell, or via aggregation.
P114 EVIDENCE FOR ZINC‐DEFICIENT SOD BEING THE TOXIC FORM RESPONSIBLE FOR ALS
Beckman JS, Nylin K, Robinson KM, Roberts B
Linus Pauling Institute, OSU, Corvallis, OR, USA
E‐mail address for correspondence: [email protected]
We have shown that the ALS‐associated mutant SODs have a decreased affinity for zinc, making them more prone to becoming zinc‐deficient. Furthermore, the copper in zinc‐deficient Cu,(–) SOD catalyses the nitric oxide‐dependent formation of peroxynitrite and nitration of tyrosine, which activates apoptosis in motor neurons. Using a novel mass spectrometric assay, we have directly measured zinc‐deficient SOD from the disease affected spinal cords of transgenic rats. Zinc‐deficient SOD was about one‐third of the Cu/Zn SOD present and four times greater than the endogenous rat SOD. Zinc‐deficient SOD was not present in the non‐disease affected brains of the same animals nor in wild‐type SOD overexpressing rats. In addition, we have solved the X‐ray structure of zinc‐deficient SOD, which provides insights into the increased redox activity of the active site copper as well as to why zinc‐deficient SOD is more prone to aggregation. Inhibition of aggregation by forming heterodimers with Cu/Zn SOD increases the toxicity of zinc‐deficient SOD, which may explain in part the dominant action of ALS mutants. Paradoxically, growing evidence indicates that aggregation of SOD may be a protective mechanism to remove zinc‐deficient SOD. In summary, the loss of zinc from either wild‐type SOD or ALS mutant SOD offers a reasonable explanation for how this key antioxidant enzyme can be involved in sporadic and familial ALS.
P115 THE TOXIC EFFECT OF MUTANT SOD1 ON MOTOR NEURON OUTGROWTH IN THE ZEBRAFISH
Lemmens R, van Hoecke A, Hersmus N, Carmeliet P, van den Bosch L, Robberecht W
KULeuven, Leuven, Belgium
E‐mail address for correspondence: [email protected]
Background: ALS is a devastating neurodegenerative disease that leads to paralysis and death due to progressive loss of upper and lower motor neurons. The mechanism leading to this degeneration is not understood. While most of the cases of ALS are sporadic (SALS), almost 10% are familial (FALS). Approximately 20% of familial ALS is linked to mutations in the Cu/Zn superoxide dismutase (SOD1), of which over 60 different mutations are described. It is assumed that these mutations cause a toxic gain of function rather than a decreased dismutase activity.
Several animal and cell culture models have focused on the pathogenic role of mutant SOD1 in FALS. In spite of these strenuous efforts, the mechanisms underlying its effects remain obscure.
Objectives: This study explores the toxicity of mutant SOD1 on zebrafish motor neurons. In zebrafish it is known that motor neurons originate in two different waves. First, three primary motor neurons per spinal cord hemisegment appear. These neurons send out their axons which follow a distinct pathway. A few hours later, 30 secondary motor neurons per spinal cord hemisegment are generated. These will produce axons that will follow the pathway paved by the primary motor neurons.
Methods: Transient overexpression of wild‐type and different mutant human SOD1 was induced by the microinjection of mRNA. The effect on motor neuron biology was evaluated by determining defects in primary motor neurons, in particular branching and measuring axonal outgrowth of 30‐h‐old embryos.
Results: Embryos injected with three different SOD1 mutant (G93A, G37R and A4V) mRNAs showed more branched and shorter axons compared to their SOD1WTmRNA injected littermates. Furthermore, using different concentrations of RNA, we proved this effect on axonal outgrowth to be dose dependent.
Discussion and conclusions: We conclude that mutant SOD1 is toxic for embryonic zebrafish motor neurons. This new model for acute SOD1 neurotoxicity creates a broad range of opportunities. The effect of mutant SOD1 on zebrafish movement behaviour will be evaluated. Injecting mutant SOD1 mRNA in the many mutants obtained from large mutagenesis screens will facilitate the detection of modifying genes. In the future we are also planning to use this model to perform a small compound screening for the testing of a wide variety of compounds on mutant SOD1 neurotoxicity.
The zebrafish experimental framework integrates the strengths of different existing animal and cell culture models and has the potential to generate new insights in ALS pathogenesis and treatment.
P116 KNOCKING DOWN THE ALS2 GENE IN ZEBRAFISH LEADS TO DEVELOPMENTAL ABNORMALITIES AND SWIMMING DEFICITS
Gros‐Louis F1, McDearmid J2, Drapeau P2, Rouleau GA1
1Centre for the Study of Brain Disease, CHUM Research Centre, Notre‐Dame Hospital, Montreal, Quebec, Canada, 2Montreal University, Department of Pathology and Cellular Biology, Montreal, Quebec, Canada
E‐mail address for correspondence: [email protected]
Background: The ALS2 gene encodes a protein, alsin, which contains multiple motifs with homology to the guanine‐nucleotide exchange factor. To date, there are ten reported mutations in the ALS2 gene, which all lead to either 1) amyotrophic lateral sclerosis (ALS), where both upper and lower motor neurons are affected, or 2) primary lateral sclerosis (PLS), or 3) infantile onset ascending hereditary spastic paraplegia (IAHSP), where only upper motor neurons are affected. Furthermore, the molecular features and function of alsin have not been experimentally determined and the pathological mechanisms by which a loss of alsin function leads to a selective dysfunction and degeneration of motor neurons in three distinct motor neuron diseases remain unknown.
Objective: To generate a zebrafish model of ALS2 in order to address the function and molecular features of alsin, as well as its involvement in neuronal dysfunction.
Methods: The ALS2 zebrafish ortholog was identified using the Ensembl's gene homology prediction program (http://www.ensembl.org, build Zv3). In an attempt to address the function of alsin in zebrafish, we performed a loss‐of‐function study using antisense morpholino oligonucleotide (AMO)‐mediated gene knockdown. AMO sequences were designed to be complimentary to the region of translational initiation of the zebrafish ALS2 ortholog, in order to inhibit protein translation and block endogenous alsin production. The appropriate AMOs were injected into one‐ to two‐cell zebrafish embryos and the resulting larvae were monitored for the presence/absence of a response to touch phenotype.
Results: Zebrafish larvae resulting from the injection of antisense morpholino oligos directed against the start codon of the zALS2 gene presented with an obvious developmental and behavioural phenotype, including swimming impairment after proper touch response and background adaptation. This phenotypic observation was absent in control animals and thus seems to be a direct result of the knocking down of zebrafish alsin expression.
Discussion: According to the preliminary results presented here, the ALS2 knocked‐down zebrafish would be an excellent animal model to study the pathophysiological mechanisms associated with the neuronal dysfunction observed in ALS2.
P117 NAD+ AND WALLERIAN DEGENERATION REVISITED: DISSECTING THE FUNCTIONAL DOMAINS OF THE WLDS PROTEIN
Conforti L1, Morreale G1, Beirowski B1, Wilbrey A1, Laser H2, Sorci L3, Bridge K1, Magni G3, Coleman M1
1The Babraham Institute, Cambridge, UK, 2International University Bremen, Germany, 3Institute of Biochemical Biotechnologies, University of Ancona, Italy
E‐mail address for correspondence: [email protected]
Background: The slow Wallerian degeneration protein, WldS, is a potent and unique neuroprotective factor for axons and synapses. It delays axon degeneration after injury by 10‐fold, and in pmn mice it delays onset of symptoms and increases lifespan by 40%. It also protects neuromuscular junctions in young G93ASOD1 transgenic mice, a widely used model of familial ALS. Its potential effect in sporadic human ALS remains unknown.
Objectives: Understanding the WldS neuroprotective mechanism may reveal novel targets for therapy. WldS is a fusion protein containing 70 N‐terminal amino acids (N70) of multiubiquitination factor Ube4b and full‐length nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1). It was recently proposed that Nmnat1 activity is solely responsible for axon protection, but the relevance of these data in vivo was not tested. Other studies highlight the importance of the ubiquitin proteasome system (UPS) in regulating Wallerian degeneration, so we tested whether the N‐terminal sequence of WldS is truly dispensable in vivo.
Methods: Using the same approach that previously led us to identify the WldS gene, we generated and characterized five lines of Nmnat1 overexpressing transgenic mice. We confirmed that Nmnat1 was overexpressed in the nuclei of lumbar spinal cord motor neurons and in dorsal root ganglia, both of whose axons project to the sciatic nerve, and that total Nmnat activity closely matched that of WldS mice. We then cut sciatic nerves to assess Wallerian degeneration by electron microscopy and other methods. We also pulled down a binding partner for N70 from mouse brain, and confirmed its physiological relevance by immunoprecipitation and colocalization studies.
Results: All five Nmnat1 overexpressing transgenic lines failed to reproduce the WldS phenotype. In contrast, even a low dose of WldS consistently slowed Wallerian degeneration in transected sciatic nerves. In our hands, transgenically overexpressed Nmnat1, exogenous NAD+ and resveratrol were similarly ineffective in vitro. Interestingly, the N70 domain required for neuroprotection directly binds to valosin containing protein (VCP; p97/Cdc48), an AAA‐ATPase with diverse cellular roles including a pivotal role in the UPS. Transgenic mice have been generated to test whether VCP mediates the WldS mechanism.
Conclusion: Nmnat1 does not protect injured axons in vivo when expressed at similar levels to WldS, and we find its protective effect in vitro, if any, to be far weaker than that of WldS. More N‐terminal sequence in WldS is therefore required for the protective phenotype. VCP, a binding partner for N70, is a good candidate to mediate this phenotype, given its critical role in the UPS. As axons and synapses degenerate early in ALS, it is essential to complete our understanding of how they degenerate and how agents such as WldS and its downstream mediators might intervene.
P118 PHYSIOLOGICAL ANALYSIS OF THE PHENOTYPE OF A YAC TRANSGENIC MOUSE MODEL OF SPINAL BULBAR MUSCULAR ATROPHY (SBMA)
Nirmalananthan N1, Dick JRT1, Thomas PS Jr2, Hanna MG1, La Spada AR2, Greensmith L1
1Institute of Neurology, University College London, London, UK, 2Department of Laboratory Medicine, University of Washington Medical Center, Seattle, Washington, USA
E‐mail address for correspondence: [email protected]
Background and objectives: Spinal and bulbar muscular atrophy (SBMA) is a progressive human motor neuron neurodegenerative disorder due to a polyglutamine expansion in the androgen receptor (AR) gene. An SBMA YAC transgenic mouse model (AR100) has previously been generated expressing the full length human AR gene containing 100 CAG repeats, leading to a neuromuscular phenotype (1). Here we characterize the phenotype of both homozygous and heterozygous AR100 transgenic mice by in vivo physiological analysis of muscle function and motor unit survival at a late symptomatic stage of disease.
Methods: At 13 and 17 months, respectively, homozygote and heterozygote mice became clearly symptomatic showing marked locomotor deficits and were prepared for in vivo analysis of hindlimb muscle function. The mice were anaesthetized and the distal tendons of the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles were attached to isometric force transducers and motor unit survival and maximum muscle force elicited by direct stimulation of the sciatic nerve was assessed in both hindlimbs. The fatiguability of EDL was assessed by recording the response to repetitive nerve stimulation. Muscles were then removed for analysis of muscle phenotype and innervation and the spinal cord processed for analysis of motor neuron survival.
Results: Compared to wild‐type control mice, TA muscles were approximately 50% weaker and EDL approximately 40% weaker in homozygote and heterozygote AR100 mice at 13 and 17 months, respectively (p<0.01). There was no significant difference in muscle force between homozygote and heterozygote mice, indicating that the disease process is accelerated in homozygote mice. There was also a significant reduction in the number of motor units innervating EDL so that 25 and 22 motor units survived in homozygotes and heterozygotes, respectively, compared to 30 in wild‐type mice (p<0.05). Moreover, the EDL muscle phenotype was altered so that this normally fast fatiguable muscle became fatigue‐resistant in AR100 homozygote and heterozygote mice – a change in muscle phenotype associated with motor neuron degeneration. Histological analysis reveals marked muscle denervation.
Conclusions: Our results indicate that both homozygote and heterozygote AR100 mice develop significant muscle weakness, a loss of motor units accompanied by a change in muscle phenotype, reflecting the observed neuromuscular phenotype previously described (1). These changes are more severe and of earlier onset in the homozygote mice. Our results provide strong physiological evidence that these mice are a representative model of human SBMA.
References
- Sopher BL, Thomas PS Jr, LaFevre-Bernt MA et al. Neuron 2004;41:687–99.