C3 PATHOLOGICAL VARIATION IN MOTOR NEURON DISEASE
Ince P
No abstract available
C4 IS THERE A CAUSAL ROLE OF STERYL GLUCOSIDE NEUROTOXINS IN ALS?
Shaw CA1, Chung JS1, Wilson JMB1, Cudkowicz ME2, Newhall K2 & Brown RH2
1University of British Columbia, Vancouver, Canada and 2Harvard Medical School, Boston, USA
E‐mail address for correspondence: [email protected]
Background: The causal factor(s) responsible for sporadic neurological diseases are unknown and the stages of disease progression remain undefined and poorly understood. Epidemiological studies of the Guamanian variant of ALS, amyotrophic lateral sclerosis‐Parkinsonism dementia complex (ALS‐PDC) have shown a positive correlation between consumption of washed cycad seed flour and the development of the disease. In vivo studies in which adult male mice consume washed cycad seed flour as part of diet show that treated animals show profound and progressive motor, cognitive, and olfactory behavioural deficits combined with the loss of neurons in each of the respective neural subsets. The expression of these outcomes mirrors precisely the behavioural and pathological deficits in ALS‐PDC. In vitro experiments using isolated cycad fractions have identified the likely neurotoxins as variant steryl glucoside molecules contained in washed cycad flour, specifically β‐sitosterol β‐D‐glucoside (BSSG), campesterol β‐D‐glucoside, and stigmasterol β‐D‐glucoside.
Objectives: To determine if steryl glucoside molecules are causal agents in ALS‐PDC and if similar molecules can be identified in plasma from ALS patients in North America.
Methods: BSSG was fed to mice as part of their diet for 10 weeks. Mice were tested using an established battery of behavioural tests and histology previously used to observe neurological deficits and pathology. Screening for these putative molecules was conducted with human ALS and control patients. HPTLC was used to measure steryl glucoside concentration in 40 ALS and 30 control plasma samples.
Results:In vivo experiments with mice fed BSSG show a significant 25% loss of motor neurons in the lumbar spinal cord with increased caspase‐3 labelling. In human samples, initial results show increased steryl glucoside concentrations in ALS patients compared to controls.
Discussion and conclusions: The identification of steryl glucosides as neurotoxins in the animal model and the preliminary data suggesting elevated levels of these molecules in human neurological disease warrant further investigation of steryl glucoside as causal neurotoxins or as biomarkers of disease status or progression.
C5 MUTANT HEAT SHOCK PROTEIN 27 RESULTS IN THE SELECTIVE AGGREGATION OF SPECIFIC CELLULAR COMPONENTS
Ackerley S, James P, Davies KE & Talbot K
University of Oxford, Oxford, UK
E‐mail address for correspondence: [email protected]
Background: Recently there has been a dramatic increase in the identification of genes in which mutations lead to isolated motor neuron degeneration. The hereditary motor neuropathies (distal spinal muscular atrophies) are a heterogeneous group of pure lower motor neuron degenerative disorders for which several causative genes including heat shock protein 27 (Hsp27) have been identified. Hsp27 is a molecular chaperone which has a number of different roles within cells and has been shown to be essential for the survival of motor neurons.
Objectives: To develop a cell culture model to examine the molecular mechanism whereby mutant Hsp27 leads to motor neuron degeneration.
Methods: We have developed an assay using transfected primary cultures to investigate the effects of wild‐type (wt) and mutant (P182L) Hsp27 on the distribution and transport of a number of cellular components.
Results: We have demonstrated that mutant but not wt Hsp27 abnormally aggregates in the cell bodies of primary cultures. These aggregates of mutant Hsp27 then result in the sequestering of wt Hsp27 within cell bodies. In addition, selective components of the neuronal cytoskeleton such as neurofilaments, but not tubulin, are also found to co‐aggregate with mutant Hsp27. Furthermore, a component of the dynein/dynactin complex which is essential for retrograde transport was also found to aggregate with mutant Hsp27 in cell bodies. The distribution of mitochondria which are known to be transported within axons was not affected by mutant Hsp27, suggesting selective disruption of the transport of certain cellular components.
Discussion: Using a primary cell culture based assay we have demonstrated that mutant but not wt Hsp27 appears to result in the selective aggregation of specific cellular components. To extend these in vitro studies, we have generated transgenic mice overexpressing mutant Hsp27. We are currently in the process of trying to identify further targets, the transport of which may be disrupted by mutant Hsp27. In addition we are further investigating the role of wt Hsp27 in axons. The identification of the specific targets disrupted by mutant Hsp27 may increase our understanding of the mechanisms whereby motor neurons are selectively vulnerable.
C6 HEAT SHOCK RESPONSE, BUT NOT Hsp27, PREVENTS MUTANT SOD1‐DEPENDENT CELL DEATH IN A CULTURE MODEL OF ALS
Krishnan J, Dewil M, Lemmens R, Robberecht W & van den Bosch L
Laboratory of Neurobiology, K.U.Leuven, Leuven, Belgium
E‐mail address for correspondence: [email protected]
Background: Amyotrophic lateral sclerosis (ALS) is characterized by selective loss of motor neurons in the spinal cord, brainstem and motor cortex. Mutations in the free‐radical scavenging copper‐zinc superoxide dismutase (SOD1) enzyme underlie one form of familial ALS. The pathogenic mechanism of these mutations is elusive but is thought to involve oxidative stress and abnormal protein aggregation. These two phenomena are known to induce heat shock proteins (Hsps) which protect stressed cells through their chaperoning and anti‐apoptotic activity.
Objectives: We previously showed that Hsp27 is up‐regulated in the spinal cord of mutant SOD1 mice. This study was conducted to evaluate the possible protective role of Hsp27, using an in vitro model of mutant SOD1 and WT SOD1 overexpressing neuro2a (N2a) mouse neuroblastoma cells.
Methods: N2a cells were exposed to cyclosporine A, lactacystin or a severe heat shock and assessed for cell survival using MTS assay. Transfection with an Hsp27 construct was performed using Amaxa transfection. For endogenous up‐regulation of Hsp27, these cells were exposed to a pre‐conditioning heat shock. Blocking of this endogenous up‐regulation of Hsp27 was achieved with RNAi technology. Western blotting was carried out to assess the expression of SOD1, as well the levels of Hsp27 before and after transfection with siRNA. Immunohistochemistry was also performed to study the efficacy of the siRNA treatment.
Results: Mutant SOD1 dependent differential cell death could be induced by heat shocking the cells, but also by treating the cells with cyclosporine A and lactacystin. Transfection of the cells with an Hsp27 construct did not protect mutant SOD1 cells from differential death. However, pre‐conditioning N2a cells with a mild heat shock was accompanied by a significant up‐regulation of Hsp27 in the mutant SOD1 cells, and protected these cells against subsequent mutant SOD1‐dependent cell death induced by a more severe heat shock. Selective inhibition of this Hsp27 up‐regulation, through the use of Hsp27 siRNA, did not attenuate the protective effect of this treatment.
Conclusions: These results suggest that activation of the heat shock response can provide the necessary defences for cells with mutant SOD1 to withstand other stresses, and that this protection is probably mediated through a complex cohort of molecules of the stress response pathway of which Hsp27 is not an essential component.
C7 ONSET ADMINISTRATION OF MOTEXAFIN GADOLINIUM EXTENDS SURVIVAL IN G93A MICE: POSSIBLE ROLE OF INCREASED ZINC OCCUPANCY
Bhogaraju VK1, Hannigan R3, Magda D2, Miller R2 & Crow JP1
1Department of Pharmacology and Toxicology, University of Arkansas College of Medicine, Little Rock, 2Pharmacyclics, Inc., Sunnyvale, and 3University of Arkansas, Jonesboro, Jonesboro, USA
E‐mail address for correspondence: [email protected]
Background: ALS‐associated SOD1 mutants, including G93A SOD1, have been shown to have lower Zn binding affinity; Zn‐deficient SOD1 has both pro‐oxidant and pro‐aggregatory properties, but the presence of Zn‐deficient SOD1 in vivo has yet to be demonstrated. Motexafin gadolinium (MGd) is a novel drug that has been shown to increase intracellular concentrations of free Zn in cancer cell lines.
Objectives: To measure the in vivo metallation state of G93A SOD1 from spinal cord and to assess the magnitude and mechanism(s) of the neuroprotective effect of MGd.
Methods: Rapidly purify G93A SOD1 mutant from transgenic spinal cord under mild, non‐denaturing conditions in the presence and absence of low abundance Zn isotope (Zn67) and measure Zn67 enrichment via ICP‐MS. Administer MGd at symptom onset to G93A transgenic mice and examine survival and proteomic changes after 10 days (relative to age‐matched vehicle‐treated controls), as well as assess ultimate survival.
Results: G93A SOD1 mutant purified from spinal cord at four time points, from symptom onset to end stage, revealed a progressive increase in Zn67 enrichment, indicating that Zn binding sites in the mutant enzyme were unoccupied at the time of tissue disruption. Onset administration of MGd (1 mg/kg/day IP) to G93A mice resulted in up to a 2.5‐fold increase in survival after onset, relative to vehicle‐treated littermate controls. Preliminary proteomic analysis of cohort mice treated for 10 days revealed decreased expression of the Zn binding protein NF‐L, increased expression of NF‐H, and alterations in important mitochondrial enzymes.
Discussion and conclusions: Using a novel method that we developed for measuring Zn binding site occupancy of Zn‐binding proteins, we offer the first compelling evidence for the presence of Zn‐deficient SOD1 mutant in the spinal cord of transgenic animals. Furthermore, the levels of Zn‐deficient SOD1 mutant increase over time and correlate with disease severity. Using a clinically relevant onset administration paradigm, MGd produces a marked and reproducible increase in survival of G93A mice. Changes associated with the therapeutic effect suggest that could be related, in part, to changes in Zn uptake and handling by neurons, i.e.increases in intracellular free zinc, combined with decreases in the major Zn binding protein in neurons (NF‐L), could result in more available Zn and a net decrease in the amount of toxic Zn‐deficient SOD1 present. In any case, lowering of NF‐L expression and increased NF‐H expression, in and of themselves, have been shown to produce an increase in survival in G93A mice. Overall, these results suggest that MGd could be a useful therapeutic agent in all forms of ALS.
C8 GLUR2 DEFICIENCY ACCELERATES MOTOR NEURON DEGENERATION IN A MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS
Van Damme P, Bogaert E, Callewaert G, Robberecht W & van den Bosch L
K.U.Leuven, Leuven, Belgium
E‐mail address for correspondence: [email protected]
Amyotrophic lateral sclerosis (ALS) is characterized by a progressive degeneration of motor neurons. Familial ALS accounts for 10% of all cases and in about 20% of the familial cases, mutations of the superoxide dismutase 1 (SOD1) gene have been identified. Transgenic mice overexpressing human mutant SOD1G93A develop a progressive motor neuron disorder and provide a good model for familial ALS.
AMPA receptor‐mediated excitotoxicity has been implicated in the selective degeneration of motor neurons in ALS. Excessive stimulation of AMPA receptors is particularly toxic to motor neurons in vitro due to Ca2+ influx through AMPA receptors. One of the factors underlying this selective vulnerability of motor neurons is the presence of a large proportion of Ca2+‐permeable (i.e. GluR2‐lacking) AMPA receptors. However, the precise role of GluR2‐lacking AMPA receptors in motor neuron degeneration remains to be defined. We therefore studied the impact of GluR2‐deficiency on motor neuron death. Cultured motor neurons from GluR2 deficient embryos displayed an increased AMPA receptor Ca2+ permeability, an increased Ca2+ influx upon AMPA receptor stimulation and an increased vulnerability to AMPA receptor‐mediated excitotoxicity. To investigate the role of GluR2 in motor neuron degeneration in vivo, we crossbred mutant SOD1G93A mice with GluR2 knockout mice. GluR2 deficiency clearly accelerated the motor neuron degeneration and shortened the lifespan of mutant SOD1G93A mice. These findings indicate that GluR2 plays a pivotal role in the vulnerability of motor neurons in vitro and in vivo and that therapies that limit Ca2+ entry through AMPA receptors might be beneficial in ALS.
C9 ALTERED EXCITABILITY OF CORTICAL NEURONS IN A TRANSGENIC MODEL OF FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS
Zona C1, Pieri M1 & Carunchio I2
1University of Rome ‘Tor Vergata’, Rome, and 2I.R.C.C.S. Fondazione Santa Lucia, Rome, Italy
E‐mail address for correspondence: [email protected]
Background: Abnormal balance between intracortical inhibitory and excitatory mechanisms has been found to contribute to the genesis of motor cortex hyperexcitability in amyotrophic lateral sclerosis (ALS). Transcranial magnetic stimulation (TMS) has shown that both increased excitability of cortical motor neurons and reduced intracortical inhibition contribute to observed motor cortex hyperexcitability in ALS patients.
Objectives: The purpose of the present study is to investigate if cortical neurons excitability is altered in a transgenic model of ALS and if this hyperexcitability is due to intrinsic properties of the single cortical neuron.
Methods: We have grown cortical neurons in cultures from transgenic mice expressing G93A mutant SOD1. We employed non‐transgenic mice (control), transgenic mice expressing high levels of the human wild‐type protein (SOD1), and transgenic mice expressing high levels of the human mutated protein (Gly93→Ala, G93A) to study and compare cortical excitability and passive membrane properties. Embryos at day 15 of gestation were used. All transgenic progenies were screened for the presence of the human SOD1 enzyme. Cells were used between 10 and 12 days in vitro and for the electrophysiological recordings we used the whole‐cell configuration of the patch‐clamp technique.
Results: The passive membrane properties, the pattern of repetitive firing and the action potential properties were examined in cortical neurons. In all tested cells, the injection of −0.1 nA current (200 ms) from the membrane potential of −60 mV evoked a hyperpolarizing response that reached a steady‐state used to calculate the membrane time constant and input resistance of each cell. The resting membrane potential, the input resistance, the membrane time constant and the cell capacitance of control, SOD1 and G93A cortical neurons did not present significantly different values. The injection of +200 pA failed to elicit multiple actions potential in all the neurons. In all tested cells, the time interval between the first two spikes of a train was decreased when the amplitude of the train evoking current was increased. For the injected currents equal to +200 pA, the firing frequency in the G93A culture was significantly greater (37±10 Hz, n = 14; p<0.05) than that in the control (25.7±8.3 Hz, n = 17) and in the SOD1 (29±7.6 Hz, n = 16). The threshold for the action potential onset was decreased significantly (p<0.05) in the G93A neurons (−36.6±4.7 mV) compared to control (−31.7±3.4 mV) and SOD1 (−32.4±4.3 mV).
Discussion and conclusions: These data show that the membrane passive properties of cortical neurons are unaffected by the overexpression of the human transgene, either wild‐type or G93A. On the contrary, the firing frequency and the action potential onset were altered in G93A cortical neurons, indicating an intrinsic neuronal hyperexcitability that may contribute to the pathogenesis of the disease.
Acknowledgements: This work is supported by a grant from MIUR (Firb: RBAU01A7T4), Italy.