P119 FOLDING OF CU/ZN SOD REVEALS STRUCTURAL HOTSPOTS FOR GAIN OF NEUROTOXIC FUNCTION IN ALS: PARALLELS TO PRECURSORS IN AMYLOID DISEASE
Oliveberg M, Nordlund A
Dept Biochemistry and Biophysics, Stockholm University, 106 91 Stockholm, Sweden
Email address for correspondence: [email protected]
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease linked to misfolding of the ubiquitous enzyme Cu/Zn superoxide dismutase (SOD). In contrast to other protein‐misfolding disorders with similar neuropathogenesis, ALS is not always associated with the in vivo deposition of protein aggregates. Thus, under the assumption that all protein‐misfolding disorders share at primary level a similar disease mechanism, ALS constitutes an interesting disease model for identifying the as yet mysterious precursor states from which the cytotoxic pathway emerges. In this study, we have mapped out the conformational repertoire of the apoSOD monomer through analysis of its folding behaviour. The results allow us to target the regions of the SOD structure that are most susceptible to unfold locally under physiological conditions, leading to the exposure of structurally promiscuous interfaces that are normally hidden in the protein's interior. The structure of this putative ALS precursor is strikingly similar to those implicated in amyloid disease (1).
References
- Nordlund A, Oliveberg M. PNAS 2006;103:10218–23.
P120 THE ROLE OF CYSTEINE 111 RESIDUE IN MUTANT COPPER/ZINC SUPEROXIDE DISMUTASE (SOD1)
Watanabe S1, Nagano S1, Bush AI2, Sakoda S1
1Department of Neurolgy, Osaka University Graduate School of Medicine, Suita, Osaka, Japan, 2Department of Pathology, University of Melbourne, Parkville, Victoria, Australia
E‐mail address for correspondence: [email protected]
Background: Mutations in the gene encoding human copper/zinc superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis (FALS). It has been proposed that neuronal cell death might be brought about by oxidative stress through the inappropriate redox activity of Cu associated with mutant SOD1. On the other hand, genetic ablation of copper chaperone for SOD1 (CCS), which delivers copper to the active site of SOD1, cannot rescue the disease. In previous studies, we proved mutant SOD1 possessed higher affinity for Cu than wild‐type by immobilized copper affinity chromatography (Cu‐IMAC) probably at a solvent‐facing surface, not the active site. However, the region in mutant SOD1 related to high affinity for Cu has not been decided.
Objectives: To clarify the role of cysteine 111 residue in mutant human SOD1 by Cu‐IMAC and assess the toxicity of each mutant SOD1 by cell culture system.
Methods: We prepared plasmids containing wild‐type, mutant SOD1 (A4V, G85R, G93A) or mutant SOD1 with substitution of serine for cysteine (Cys) at Cys 111 residue (i.e. A4V/C111S, G85R/C111S, G93A/C111S). The homogenate of COS7 cells transfected with each plasmid was applied to the column pre‐saturated with CuCl2 and proteins were eluted under an imidazole gradient stepwise (Cu‐IMAC). The elution profiles of SOD1 from the Cu‐IMAC column were analysed by SDS‐PAGE and immunoblotting. To investigate the stability of each mutant SOD1, we carried out native‐PAGE and cycloheximide assays. To study the viability of N2a cells transfected with each plasmid, we stained these cells with propidium iodide and analysed by flow cytometry.
Results: All mutant SOD1 with C111S exhibited lower affinity for Cu than that without C111S. C111S substitution accelerated the mobility of mutant SOD1 on native‐PAGE and stabilized mutant SOD1 on cycloheximide assays. Furthermore, the viability of N2a cells transfected with mutant SOD1 was restored by C111S substitution.
Conclusions: These results suggest that aberrant copper may directly bind at Cys 111 residue or modification at this residue may cause conformational instability in mutant SOD1, that would be harmful to neuronal cells.
Acknowledgements: This work was supported by a Grant‐in‐Aid from the Ministry of Education, Culture, Science and Technology of Japan.
P121 INVOLVEMENT OF CYS111 IN THE STABILITY OF HUMAN COPPER/ZINC‐SUPEROXIDE DISMUTASE
Fujiwara N1, Nakano M2, Ookawara T1, Eguchi H1, Yoshiharad1, Taniguchi N3, Suzuki K1
1Department of Biochemistry, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan, 2Department of Biochemistry, Osaka University Medical School & Graduate School of Medicine, Suita, Osaka, Japan, 3Department of Disease Glycomics Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan
E‐mail address for correspondence: [email protected]
Background: Copper/zinc‐superoxide dismutase (Cu/Zn‐SOD)‐immunoreactive inclusion bodies in motor neurons are observed in not only familial but also sporadic ALS patients. Since it is a key enzyme that catalyses the disproponation of superoxide radicals into molecular oxygen and hydrogen peroxide, Cu/Zn‐SOD is thought to be a major target of oxidative stress. Although oxidative stress induces aggregation in mutant and even wild‐type Cu/Zn‐SOD, the implication in the pathogenesis of ALS is still unclear. We have previously reported that different conformational changes may occur in Greek key loop (loop VI, residues 102–115 in human Cu/Zn‐SOD) between wild‐type and FALS mutant SODs during the unfolding process (1). We consider that this loop is highly solvent accessible and easily moveable by various denaturing triggers.
Objectives: In this study, we focused on Cys111 in the Greek key loop to elucidate the role of free cysteine residues on a stability of human Cu/Zn‐SOD under oxidative stress.
Methods: We obtained recombinant human 2‐mercaptoethanolated Cu/Zn‐SOD (2‐ME‐SOD) from Ube Industries Ltd. MALDI‐TOF‐mass spectrometry (MS) analyses determined that Cys111 in the 2‐ME‐SOD is specifically 2‐mercaptoethanolated and that additional incubation with 20 mM 2‐ME removes the 2‐ME from the Cys111. This recovered wild‐type Cu/Zn‐SOD is denoted as re‐SOD. In order to compare the stability between 2‐ME‐SOD and re‐SOD, 1) the effect of heat treatment on the secondary (whole) structure of both SODs was assessed by circular dichroism (CD) measurements; 2) the effect of oxidation on both SODs was assessed by SDS‐PAGE and anion exchange column chromatography. In addition, we identified the oxidative modification of Cys111 by HPLC and MALDI‐TOF‐MS (MS‐MS) analyses.
Results: CD measurements revealed that this re‐SOD is more sensitive to heat treatment than the 2‐ME‐SOD. The re‐SOD was also more easily oxidized and produced oligomers/aggregates compared with 2‐ME‐SOD. In addition, we determined that a sulphhydryl group of only Cys111 in the re‐SOD, but not in the 2‐ME‐SOD, is irreversibly oxidized to a sulphinic acid or a sulphonic acid by mild oxidation.
Discussion: Our findings suggest that Cys111 is involved in the propensity for oxidation and aggregation of human Cu/Zn‐SOD and that a masking of the Cys111 residue by a thiol specific modifier, such as 2‐ME, can stabilize ALS mutant SODs.
References
- Fujiwara N, Miyamoto Y, Ogasahara K, et al. J Biol Chem. 2005;280:5061–70.
P122 AGRREGATION PROPENSITY OF SOD1 PROTEIN: IMPACT OF CHARGE MUTATIONS
Byström R, Lindberg MJ, Boknäs N, Andersen PM, Aisenbrey C, Gröbner G, Oliveberg M
Department of Biochemistry & Biophysical Chemistry, University of Umea, Umeå, Sweden
E‐mail address for correspondence: [email protected]
Over 100 mutations in the human gene encoding the cytosolic homodimeric enzyme Cu/Zn superoxide dismutase (SOD) have been linked to the familial neurodegenerative amyotrophic lateral sclerosis (ALS) syndrome. The key event in ALS seems to be the pathological formation of toxic protein aggregates formed by initially unfolded or partly structured SOD protein mutants. However the molecular mechanism behind the neurotoxic effects by these mutants remains elusive.
In a recent study (1) we have mapped out and compared the folding behavior of 15 SOD mutants, each of them correlated to specific patient survival times, ranging from one year for the most aggressive mutant to 17 years for the most mild one after onset of symptoms. All of these mutations move the folding equilibrium towards the denatured monomer, but their effects differ in magnitude and mechanistic origins. Therefore, the mutations can be divided into different categories with either reducing the stability of the monomer (class 1), the strength of the dimer interface (class 2) or both (class 1+2). In general, there is a strong correlation between reduced stability and reduced mean survival times except in those cases where an alteration of the total net negative charge is involved. With a pI of 4.8–5.1, SOD holds a net negative charge under physiological conditions. If a mutation increases the net negative charge of the protein a slower disease progression is observed. In contrast, mutations leading to a decrease of the net negative charge lead to shorter survival times than expected from protein stability data alone. Of all known ALS associated SOD mutants 26% reduce the magnitude of the net negative charge whereas only 7% produce an increase. Our idea is that ALS responds to a combination of low protein stability and reduced magnitude of the net negative charge. We will here present stability data and aggregation propensity for 10 new net charge altering ALS associated SOD1 mutations to further elucidate the question.
One alternative explanation for the behaviour of these mutants might be their interactions with membranes. Since the apo protein is partly unfolded and monomeric under reducing conditions, it exposes both its hydrophobic core and the hydrophobic dimer‐interface to the solvent. Therefore, membrane interactions might be likely to occur for the reduced apo form of different SOD mutants (2). Preliminary studies already indicate interactions of SOD with charged membrane surfaces, which modulate secondary structures and aggregation behaviour of these mutants in a way quite different from the situation of membrane‐free aqueous environment. In particular, the presence of charged membranes seems to trigger aggregation of the A4V mutant already at physiological temperatures, while much higher temperature is required in membrane‐free solution.
References
- Lindberg MJ, Byström R, Bokna¨s N, Andersen PM, Oliveberg M. PNAS. USA 2005;102:9754–9.
- Fernandez A, Berry RS. Proteins with H-bond packing defects are highly interactive with lipid bilayers: implications for amyloidogenesis. PNAS 2003;100:2391–6.
P123 ARCHAEAL PROTEASOMES DEGRADE MUTANT SUPEROXIDE DISMUTASE‐1 AND REDUCE ITS CELLULAR TOXICITY
Yamada SY, Niwa JN, Ishigaki SI, Takahashi MT, Ito TI, Sone JS, Doyu MD, Sobue GS
Nagoya University Graduate School of Medicine, Nagoya, Aichi Prefecture, Japan
E‐mail address for correspondence: [email protected]
Background: The 20S proteasome is a ubiquitous, barrel‐shaped protease complex responsible for most of cellular proteolysis, and its reduced activity is thought to be associated with accumulations of aberrant or misfolded proteins resulting in a number of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), spinal and bulbar muscular atrophy (SBMA), Parkinson's disease, and Alzheimer's disease. The 20S proteasomes of archaebacteria (archaea) are structurally simple and proteolytically powerful and thought to be an evolutionary precursor to eukaryotic proteasomes.
Objectives: To treat familial ALS and other intractable neurodegenerative diseases caused by mutant and modified proteins, which are not properly degraded by endogeneous proteasomes and, thus, prone to toxic gain of function resulting in neurodegeneration.
Methods: We cloned the Mm proteasome alpha‐ (Accession No.: 1480962) and beta‐subunits (Accession No.: 1479036) from genomic DNA of Mm and generated a mutant alpha‐subunit lacking amino acids 2–13, delta(2–13) alpha‐subunit (delta‐alpha). We also generated a mutant beta‐subunit with Thr1Cys (mβ1). Thr 1 in the beta‐subunit of the archaeal proteasome is essential for proteolysis and Thr 1 mutants lose their proteolytic activities. The experiments were performed in both HEK293 and Neuro2a cells. Transfections were performed using Lipofectamine 2000 (Invitrogen) in the MTS assay, or Effectene Transfection Reagent (Qiagen) in other experiments. To analyse turnover of wild‐type and mutant SOD1, cycloheximide and pulse chasing studies were performed. We chose MTS assay to evaluate the toxicities of mutant SOD1 with or without Mm proteasomes.
Result: We successfully reproduced the archaeal proteasome in a functional state in mammalian cells and here we show that the archaeal proteasome effectively accelerated species‐specific degradation of mutant superoxide dismutase‐1 and the mutant polyglutamine tract‐extended androgen receptor, causative proteins of familial ALS and SBMA, respectively, by cycloheximide and pulse chase studies, and reduced the cellular toxicities of these mutant proteins by MTS assay. Furthermore, we demonstrate that archaeal proteasome can also degrade other neurodegenerative disease‐associated proteins such as alpha‐synuclein and tau.
Discussion: Archaeal proteasomes would open a new therapeutic avenue for diseases in which the toxic‐gain of proteins are causative.
P124 ALTERATION OF NEUROFILAMENT PHOSPHORYLATION IN MOTOR NEURONS EXPRESSING MUTANT SUPEROXIDE DISMUTASE 1
Lee KW, Kim MH, Park KS, Hong YH, Sung JJ, Shin HI, Kim HJJ, Kim MY, Cho AS, Min JH
Seoul National University Clinical Research Institute of Seoul National University Hospital, Seoul, South Korea
E‐mail address for correspondence: [email protected]
Background: Mutations in the copper/zinc superoxide dismutase (SOD1) gene are known to be responsible for familial amyotrophic lateral sclerosis (ALS). Aberrant accumulations of hyperphosphorylated neurofilaments (NFs) at the perikarya and proximal axons are pathological hallmarks of ALS. There is increasing evidence that the phosphorylation of NFs is involved in the pathogenesis of ALS.
Objectives: We examined the effects of mutant SOD1 on the regulation of neurofilament phosphorylation in a motor neuron‐neurofilament hybrid (VSC4.1) cell line.
Methods: Following transfection of human SOD1 cDNA of normal (wild‐type) and mutant (G93A), cells were differentiated by dibutryl cAMP (1mM) for 24 h and aphidicolin (0.025 µg/ml) for 48 h. We then treated pharmacologic inhibitors of extracellular signal‐regulated kinase (ERK) cascade; PD098059, cyclin‐dependent kinase (CDK); Olomucine and glycogen synthase kinase‐3 (GSK‐3); lithium chloride. Also we identified the viability by MTT assay, NFs morphology by immunofluorescence microscopy and protein levels by Western blotting.
Results: Activities of GSK‐3, CDK and ERK did not contribute to the phosphorylation of NFs, but rather significantly increased the state of NF phosphorylation in motoneuronal cells expressing mutant SOD1. Inhibition of ERK cascade in mutant cells led to the hyperphosphorylation of NFs through a persistent activation of c‐jun N‐terminal kinase (JNK).
Discussion: We suggest a neuroprotective role of NFs by acting as a phosphorylation sink. The mutant SOD1 may alter the signalling process implicated in the phosphorylation of NFs. Also, activation of JNK might contribute to the aberrant hyperphosphorylation of NFs in ALS.
P125 IN VITRO MODELS OF MUTANT SOD1‐MEDIATED AXONAL DEGENERATION
Fischer LR, Wang MS, Asress SA, Fan GG, Huang X, Glass JD
Center for Neurodegenerative Disease, Emory University, Atlanta, GA, USA
E‐mail address for correspondence: [email protected]
Background: Denervation and distal axonal degeneration begin very early in the hindlimbs of SOD1‐G93A mice, long before loss of ventral root axons and lumbar motor neurons (1–3). Protection of the distal axon may therefore represent an important therapeutic target. However, the mechanism by which mutant SOD1 causes axonal degeneration remains unknown. In vitro models of mutant SOD1‐mediated axonal degeneration are needed in order to better understand this phenomenon and to determine whether protection of the axon can prevent death of the motor neuron.
Objective: To develop organotypic dorsal root ganglion and spinal cord cultures expressing mutant SOD1 protein, that reproduce in vitro the axonal degeneration observed in the SOD1‐G93A mouse.
Methods: Dorsal root ganglion (DRG) cultures were generated from E15 rats or from P4 SOD1‐G93A mice. Rat DRG were infected with one of three lentiviruses: wild‐type SOD1, SOD1‐G93A, or GFP only (control). Axonal outgrowth was measured over four to six weeks in culture. Additionally, organotypic spinal cord slice cultures were prepared from P7 SOD1‐G93A mice. Motor axons were extended from the slice cultures by co‐culturing with HEK293 cells secreting the growth factor, pleiotrophin. Axons from both systems were examined for pathologic features using a variety of morphological and immunocytochemical techniques.
Results: Lentivirus‐infected rat DRG and DRG from SOD1‐G93A mice both extended long axons that did not differ in length or morphology from control DRG when followed for four to six weeks in vitro. Mutant SOD1 expression throughout the axon was verified by immunocytochemistry. Motor axons were extended from both mutant and wild‐type spinal cord cultures, and followed over four weeks in vitro. Mutant SOD1 motor neurons and axons were positive for phosphorylated neurofilament, and axons exhibited prominent, neurofilament‐positive swellings.
Discussion: We have established organotypic models of sensory and motor axons that will be used to investigate the axonal pathology associated with mutant SOD1. To date, simply expressing mutant SOD1 in cultured axons does not appear to be sufficient to cause axonal degeneration. However, we have not yet tested whether mutant SOD1 axons may be more susceptible to environmental perturbation, such as oxidative stress. In this system we can also test whether the toxicity of mutant SOD1 is due to expression in the axon itself (cell autonomous), or may be associated with expression in non‐neuronal cells such as Schwann cells (non‐cell‐autonomous). Experiments are currently underway to address these hypotheses.
References
- Fischer LR, Culver DG, Tennant P, et al. Exper Neurol. 2004;185:232–40.
- Schaefer AM, Sanes JR, Lichtman JW. J Comp Neurol. 2005;49:209–19.
- Pun S, Santos AF, Saxena S, et al. Nat Neuro. 2006;9:408–19.
P126 GENE EXPRESSION IN G93A‐SOD1/BV2 CELLS
Lu S, Chen LC, Lee NM
The Forbes Norris ALS Research Center, San Francisco, CA, USA & California Pacific Medical Center Research Institute, San Francisco, CA, USA
E‐mail address for correspondence: [email protected]
Background: Gene expression studies of mutant SOD1 mice in our laboratory have shown that in this animal model of ALS activity, many genes associated with microglia are differentially regulated. These results, together with those of other studies, suggest that neuroinflammation may be an early event in ALS.
Objectives: To obtain more insight into the role of microglia in ALS, we analysed expression of the same panel of genes in BV‐2 cells, a microglia cell line, that were transfected with a mutant SOD1 gene, G93A‐SOD1.
Methods: Both non‐transfected BV‐2 cells and BV‐2 cells transfected with the wild‐type human SOD1 gene served as controls. We then made three types of comparisons of gene expression patterns: non‐transfected vs. wild‐type transfected; mutant vs. non‐transfected; and mutant vs. wild‐type transfected.
Results: Significant changes (up‐ or down‐regulation) were observed between mutant and wild‐type‐transfected or non‐transfected for several classes of genes, including those involved in oxidative stress neurotoxicity, neuroinflammation, zinc regulation and apoptosis, as well as in cell markers for microglia. However, changes were also observed when wild‐type transfected cells were compared with untransfected cells.
Conclusions: Overall, our results indicate that in vivo changes in expression in microglia related genes involve cell types other than microglia, i.e. other cells affect microglia activation in ALS, and genes of these other cells may also be differently regulated.
P127 ROLE OF ASTROCYTES IN AMYOTROPHIC LATERAL SCLEROSIS
Bongioanni P1, Scorciapino ML2, Pillai R2, Sirigu AR2, Saba F2, Cabras S2, Reali C2, Metelli MR3, de Tata V3, Sogos V2, Rossi B1
1Neurorehabilitation Unit, Department of Neuroscience, Azienda Ospedaliero‐Universitaria Pisana, Pisa, Italy, 2Department of Cytomorphology, University of Cagliari, Cagliari, Italy, 3Department of Experimental Pathology, University of Pisa, Pisa, Italy
E‐mail address for correspondence: [email protected]
Background: Astrocytes represent the largest glial population in the central nervous system (CNS). They provide metabolic and trophic support to neurons, modulate the microenvironment regulating ion homeostasis, produce a variety of soluble and membrane‐associated growth factors, contribute to the formation of the blood‐brain barrier (BBB) and interact with endothelial cells. After damage to the CNS, glia are thought to support neural growth and metabolism and scavenge agents toxic to neurons. In particular, neurotoxic events in the CNS are often characterized by an early and pronounced activation of astrocytes and microglia. These cells maintain normal functioning of the nervous system both controlling the extracellular environment and supplying metabolites and growth factors. Preservation of the normal relationship between neurons and glia is critical to maintain normal nervous system function.
Since a strong glial reaction surrounds motor neurons in amyotrophic lateral sclerosis (ALS) patients, recent studies suggested the involvement of reactive astrocytes in the pathogenesis of ALS. Astrocytes, together with endothelial cells, play a key role in BBB, acting as an intermediary between blood and neurons.
Objectives: In order to better understand the role of glial cells in neurodegeneration processes in ALS, we studied the effect of ALS patients' sera on cultured astrocytes.
Methods: Primary cultures of human foetal astrocytes were treated with sera from ALS patients (or healthy donors as controls). Cell response was evaluated by expression levels of different immunoregulatory molecules: TNF‐α, IL‐1 α and IL‐1 β, IL‐6, IL‐10, RANTES, CD137 (belonging to the family of TNF receptors) and inducible nitric oxide synthase (iNOS).
Results: Our results showed an increase of pro‐inflammatory molecules, according to different disease severity. A significant increase of chemokine RANTES (CCL5) was observed in all samples, suggesting a recruitment of T‐lymphocytes and macrophages Moreover, some sera induced toxicity and cell death in cultured astrocytes.
Discussion: These findings, although preliminary, suggest that astrocytes may be activated by ALS patient sera, supporting therefore the hypothesis that these cells might play a role in ALS pathogenesis.
P128 LONG TERM CULTURE OF ADULT RAT SPINAL CORD CELLS IN A NOVEL SERUM‐FREE MEDIUM AND ON A SYNTHETIC ORGANOSILANE SUBSTRATE: MORPHOLOGICAL, IMMUNOCYTOCHEMICAL AND ELECTROPHYSIOLOGICAL CHARACTERIZATION
Das M, Bhargava N, Kang JF, Hickman JJ
Hybrid System Laboratory, Nanoscience Technology Center, University of Central Florida, 4th Floor, Research Pavilion, Research Parkway, Suite 400, Orlando, Florida, USA
E‐mail address for correspondence: [email protected]
Background: Culturing adult mammalian spinal cord neurons is a challenging problem due to the inability of the adult neurons to survive in culture after undergoing the trauma of dissection. Hence, most of the cell culture studies have been performed using embryonic spinal cord tissue because embryonic neurons survive better in cell culture. Thus although the embryonic cell culture model is popular, it is not the most appropriate model to study age related disorders, injuries and degenerative diseases of the spinal cord. An adult spinal cord culture model could address this problem.
In this study, we have developed such a model system using a synthetic organosilane substrate and a novel serum‐free medium (1) to culture the neurons and the glial cells of the adult rat spinal cord. We have characterized the adult neurons and glial cells morphologically, immunocytochemically and electrophysiologically.
Objectives: 1) Preparing an organosilane substrate to grow the cells; 2) Developing cell isolation process from adult rat spinal cord; 3) Developing serum‐free medium to grow the cells; and 4) Immunocytochemical and electrophysiological characterization.
Methods: Cell culture, surface chemistry, tissue preparation, immunocytochemistry, patch‐clamp electrophysiology.
Results: In this work, we documented by morphological analysis, immunocytochemistry and electrophysiology, the development of a culture system that promotes the growth and long‐term survival of dissociated adult rat spinal cord neurons and glial cells. This system comprises a patternable, non‐biological, cell growth promoting organosilane substrate N‐1[3‐(trimethoxysilyl)propyl]‐diethylenetriamine (DETA), coated on a glass surface and an empirically derived novel serum‐free medium, supplemented with specific growth factors (1). The culture consisted of 60% neurons and 40% glia.
Neurons were characterized by immunoreactivity for neurofilament150, MAP‐2, ISLET‐1, choline acetyltransferase (ChAT) and MO‐1 antibodies. The glial cells were characterized by immunoreactivity for the GFAP antibody. Electrophysiology experiments indicated that 60% of the cells expressed voltage dependent inward and outward currents similar to neurons and were able to generate single, double and multiple (repetitive firing) action potentials. Forty per cent of the cells either showed inward, outward or very small inward and huge outward currents and these recordings are similar to the recording documented for the glial cells in the literature. The cultures were maintained for 8–10 weeks.
Discussion and conclusions: These are the first studies to demonstrate that adult rat spinal cord cells can be cultured in a completely defined serum‐free medium and on a synthetic silane substrate. This culture system will be a useful tool to study adult mammalian spinal neuron repair, myelination and degeneration, as well as to screen different novel and putative drug candidates for spinal cord repair and degenerative diseases of the spinal cord.
References
- Das M, Bhargava N, Gregory CA, et al. In Vitro Cell Dev Biol Anim. 2005:41:343–8.
P129 AMPA RECEPTORS IN CORTICAL NEURONS ARE ALTERED IN A TRANSGENIC MODEL OF FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS
Pieri M1, Zampetti E2, Carunchio I1, Zona C1
1Department of Neuroscience, University of Tor Vergata and CERC, Fondazione S. Lucia, Rome, Italy, 2Department of Electronic Engineering, University of Tor Vergata, Rome, Italy
E‐mail address for correspondence: [email protected]
Background: There are many evidences implicating glutamatergic toxicity as a contributing factor in the selective neuronal injury occurring in amyotrophic lateral sclerosis (ALS). In spinal motor neurons it has been observed that in a mouse model of ALS the alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor is almost three‐fold more permeable to Na+ and K+ than the control whereas the Ca2+ permeability does not differ. In addition, altered motor cortex excitability has been reported in ALS patients and preliminary results from our laboratory have indicated an intrinsic cortical neuron hyperexcitability in the G93A mouse model of ALS.
Objectives: The aim of this study is to establish if AMPA receptors of cortical neurons in a model of a familial form of ALS, expressing high levels of the human mutated protein Cu/Zn superoxide dismutase (Gly93→Ala, G93A) present altered biophysical properties.
Methods: We have investigated the electrophysiological properties of AMPA receptors in cortical neurons in culture of non‐transgenic mice (control), of transgenic mice expressing high levels of the human wild‐type protein (SOD1) and of G93A mice, using the whole cell configuration of the patch‐clamp recording technique.
Kainate‐induced currents were measured from eight to 14 days in vitro. Spectral density analysis of steady‐state current noise evoked by kainate agonist and the calculation of single channel conductance has also been performed in the three types of neurons. After plotting variance versus current, the data was fitted with the equation: var(t) = iI(t)− I(t)2/N, where i is the single channel current, I(t) is the mean macroscopic current for a given data segment, and N is the number of channels. The maximal Popen of the channels underlying each kainate‐evoked response was then calculated as Popen = ISS/Ni, where ISS is the steady‐state kainate evoked current. Finally, the single channel conductance was calculated by γ = i/VM, where VM is the membrane potential.
Results: The electrophysiological experiments indicate that the single cell current density and the reversal potential were not modified in G93A cortical neurons compared to control and SOD1. On the contrary, by the spectral density analysis, preliminary data appear to indicate changed values of single channel conductance in the three different cell populations. In fact, the G93A cortical neurons appear to express a different number of receptors with a different single channel current (i) compared to control and SOD1 neurons.
Conclusions: These results show that the AMPA receptors in the three different populations of cortical neurons have different biophysical properties. Since the receptor's permeability and kinetic properties strictly depend on the subunit composition, these results indirectly suggest that the AMPA receptor subunit composition is altered in cortical neurons with the G93A mutant SOD1. As yet it is unknown what significance the altered expression of AMPA receptors has in relation to the ALS pathology. Further experiments are necessary to study the connection between them.
Acknowledgements: This work is supported by a grant from MIUR (Firb: RBAU01A7T4), Italy.
P130 IVERMECTIN INHIBITS AMPA RECEPTOR‐MEDIATED EXCITOTOXICITY IN CULTURED MOTOR NEURONS AND EXTENDS THE LIFE SPAN OF A TRANSGENIC MOUSE MODEL OF ALS
Andries M, van Damme P, Robberecht W, van den Bosch L
Department of Neurobiology, Campus Gasthuisberg, Leuven, Belgium
E‐mail address for correspondence: [email protected]
Background: Excitotoxicity mediated by AMPA receptors contributes to selective motor neuron death in ALS.
Objectives: We wanted to determine the effect of P2 receptor‐influencing substances on AMPA receptor‐mediated motor neuron death and on the survival of SOD1 (G93A)‐mice.
Methods: We studied the selective vulnerability of motor neurons to AMPA receptor‐mediated excitotoxicity in vitro using purified motor neurons, grown on a pre‐established glial feeder layer. After one week in culture they were exposed for 30 min to 300 µM kainate in a modified Krebs solution. The survival of motor neurons was quantified by counting them in a marked area under phase contrast before pre‐incubation, before the exposure and 24 h after the exposure.
To further investigate the relevance of the in vitro findings for ALS, we treated SOD1 (G93A)‐mice with ivermectin. Treatment was started at 50 days of age and continued until time of death. At 120 days randomly selected control and ivermectin‐treated mice were killed for pathological evaluation of neuronal loss in the lumbar spinal cord and of axon loss in the ventral roots.
Results: PPADS, a non‐specific P2 receptor antagonist, did not have a significant effect on the motor neuron death after a short exposure to 300 µM kainate. Because the P2X4 receptor, a subtype widely expressed in the brain, is insensitive to PPADS, we also tested ivermectin and cibacron blue 3G‐A, allosteric modulators of this receptor subtype. After preincubation of the motor neuron cultures with these substances a complete protection against AMPA‐mediated excitotoxicity was seen. The effect was indirect because addition during kainate stimulation alone had no effect, and the number and Ca2+‐permeability of the AMPA receptors were not influenced by preincubation with ivermectin. Preincubation with low concentrations of ATP (100 µM), the natural agonist of the P2X4 receptor, also protected the motor neurons against a subsequent excitotoxic stimulation, while higher concentrations of ATP (1mM) were toxic. Ivermectin increased the toxicity of low ATP concentrations, indicating that ivermectin can potentiate the effect of ATP on its receptor. To further investigate the relevance of these findings for ALS, we treated SOD1 (G93A)‐mice with ivermectin. This resulted in an extension of the life span of these transgenic animals by 9.1%. This protective effect was confirmed by histological evaluation of the lumbar spinal cord (65% increase of neurons/section for ivermectin‐treated animals) and of the ventral roots (60% increase of area occupied by axons in the ivermectin‐treated animals).
Conclusions: We demonstrated that ivermectin induced a protective mechanism in motor neurons, in vivo and in vitro, against excitotoxic assaults. Our in vitro data suggest that this is due to the interaction of ivermectin with ATP and the P2X4 receptor.
P131 AMPA RECEPTORS AS A TARGET FOR PHARMACOLOGICAL NEUROPROTECTION: MOLECULAR MECHANSIMS
Krampfl K, Petri S, Schlesinger F, Jahn K, Jin L, Dengler R
Medizinische Hochschule Hannover, Hannover, Germany
E‐mail address for correspondence: [email protected]
Background: The term excitotoxicity was coined for the overstimulation of neurons via glutamate receptor channels. AMPA‐type glutamate receptors (AMPA‐R) are the most abundant excitatory transmitter receptors of the central nervous system. They play a major role in the cascade of cell damage in neurodegenerative diseases such as amyotrophic lateral sclerosis. Neurons that are vulnerable to excitotoxicity express specific subtypes of AMPA‐R as defined by the combination of subunits, by alternative splicing, and by nuclear editing of subunit mRNAs. The investigation of the neurophysiological properties of specific AMPA‐R allows for the detection of factors of vulnerability on the molecular level.
Objectives: To define molecular targets for pharmacological neuroprotection via modulation of the synaptic input to motor neurons and to test pharmacological effects of candidate compounds at the cellular and molecular level.
Methods: We combined in situ hybridization techniques with immunohistochemistry to reveal the specific receptor pattern of vulnerable neurons. In our neurophysiological studies, we combined the characterization of AMPA‐R subtypes that were recombinantly expressed with the analysis of native receptors at the single channel level. Synaptic currents, networks of cultured motor neurons and the brain slice technique were studied by patch‐clamp and calcium imaging techniques. The data from these studies gave a basis for the testing of compounds that are candidates for pharmacological neuroprotection.
Results: We investigated the molecular mechanism of action of a broad variety of compounds interacting with AMPA‐R (e.g. RPR119990, RPR117824, memantine, topiramate, IEM1460, ZK187638, ZK200775, derivates of valproic acid). The receptor channel subtype specificity of the compounds was characterized as well as the molecular mechanism of action on the single channel and on the synaptic level. Competitive and non‐competitive block mechanisms were observed. The molecular drug profiles were compared with data from synaptic recordings and calcium imaging to allow for predictions of the in vivo mechanisms of action.
Discussion:In vitro and in vivo models of neurodegeneration are used to develop more effective strategies for pharmacological neuroprotection. Antagonizing glutamatergic neurotransmission may be a therapeutic strategy in neurodegenerative diseases. The investigation of the synaptic integration of vulnerable neurons and of the molecular mechanisms of pharmacological AMPA‐R activation and modulation is crucially required to reveal pharmacological strategies that specifically interfere with the pathophysiological cascade of excitotoxicity.
Acknowledgement: This study was supported by the DFG, the DGM, Sanofi‐Aventis, and Schering.
P132 ANTI‐APOPTOTIC ACTIVITY MAINTENANCE OF BRAIN DERIVED NEUROTROPHIC FACTOR AND THE C FRAGMENT OF THE TETANUS TOXIN FUSION PROTEINS
Ciriza J1, MartÍn‐Burriel I1, GarcÍa‐Ojeda ME2, Agulhon C2, Miana‐Mena FJ1, Muñoz MJ1, Yague G1, Zaragoza P1, Brûlet P2, Osta R1
1LAGENBIO‐INGEN, Zaragoza, Spain, 2Institut Pasteur, Paris, France
E‐mail address for correspondence: [email protected]
Background: Neurotrophic factors have been widely suggested as a treatment for multiple diseases including motor neuron pathologies, such as amyotrophic lateral sclerosis (ALS). However, clinical trials in which growth factors have been systematically administered to ALS patients have not been effective, owing in part to the short half‐life of these factors and their low concentrations at target sites. A possible treatment strategy is the use of the atoxic C fragment of the tetanus toxin (TTC) as a neurotrophic factor carrier to the motor neurons. However, in this case, the activity of trophic factors should be tested because their fusion to proteins could alter their folding and conformation, thus undermining their neuroprotective properties.
Objectives: We explored the brain derived neurotrophic factor (BDNF) activity maintenance after genetic fusion with the C fragment of the tetanus toxin (BDNF‐TTC).
Methods: Recombinant proteins (TTC, BDNF‐TTC and BDNF) were purified from Escherichia coli strain Bl21‐C43. Eluted proteins were evaluated by Coomassie blue staining and Western blot analysis. Apoptosis in mouse neuroblastoma Neuro 2A cells (2.5×105) was induced by serum deprivation for 48 and 72 h. Recombinant proteins were added at doses of 10, 50 and 250 ng/ml culture. Quantification of apoptosis was performed using an Annexin V staining protocol to determine the exposure of phosphatidylserine in the cell surface. Akt activation assay was developed with cultured primary cortical neurons incubated with recombinant proteins for 1 h at 37°C. Fluorescence intensity from activated Akt was measured by confocal microscopy.
Results: The functionality of BDNF and BDNF‐TTC proteins was assessed analysing the effect of these proteins in the inhibition of apoptosis induced in Neuro2A cells by serum deprivation. Forty‐eight hour treatment with BDNF showed significant differences from the control for all the doses studied. The other proteins did not show any effect at this time although BDNF‐TTC displayed a tendency for apoptosis inhibition, but this difference was not statistically significant. The three recombinant proteins (BDNF, BDNF‐TTC and TTC) were able to inhibit apoptosis after serum deprivation in Neuro2A after 72 h. Immunodetection of Akt in cultured primary cortical neurons, after incubation with the different proteins, displayed evident differences of signal between the cultures treated with the three proteins and the control. The strongest AKT signal was observed with BDNF, followed by the fusion protein and finally with TTC alone.
Conclusion: We demonstrated that BDNF fused with TTC induces the neuronal survival Akt kinase pathway in cortical culture neurons and maintains its previously described antiapoptotic neuronal activity in vitro.
Acknowledgement: This work was supported by Instituto de Salud Carlos III‐Fondo de Investigación Sanitaria (PI020840).
P133 EFFECTS OF ACTIVATED PROTEIN C ON INSULIN‐LIKE GROWTH FACTOR (IGF‐I), IGF‐I RECEPTOR AND AKT, AND ITS NEUROPROTECTIVE EFFECTS AFTER SPINAL CORD ISCHAEMIA
Yamauchi T1, Sakurai M2, Abe K3, Masai T1, Sawa Y4
1Sakurabashi Watanabe Hospital, Osaka, Japan, 2National Hospital Organization Sendai Medical Center, Sendai, Japan, 3Okayama Graduate School of Medicine, Okayama, Japan, 4Osaka Graduate School of Medicine, Osaka, Japan
E‐mail address for correspondence: [email protected]
Background and purpose: Activated protein C (APC) has beneficial effects on ischemia reperfusion injury in neurons. However, the possible mechanism of such beneficial effect is not fully understood. The aim of this study was to investigate the effects and possible mechanisms of APC on ischemic spinal cord damage.
Methods: After induction of spinal cord ischemia, APC (group A) or vehicle (group I) was injected intravenously. Severity of ischemic damage was analysed by counting the number of motor neurons. To investigate the mechanisms by which APC prevents ischemic spinal cord damage, we performed immunoreactivity and Western blotting of insulin‐like growth factor 1 (IGF‐1), IGF‐1 receptor and phosphorylated serine‐threonine kinase (p‐Akt).
Results: APC eased the functional deficits and increased the number of motor neurons after ischemia. Immunoreactivity of IGF‐1 in group A was stronger than in group I at 8 h after reperfusion but was at the same level at 1 day. Induction of IGF‐1 receptor and the downstream factor p‐Akt was stronger and more prolonged in group A.
Conclusions: These results indicate that induction of IGF‐1, IGF‐1 receptor and p‐Akt might partially explain the neuroprotective effects of APC after transient spinal cord ischemia in rabbit.