C23 NEUROTOXIC ACTIONS OF SECRETED MUTANT CU/ZN SOD
Julien J‐P
Research Centre of CHUL, Quebec, Canada
E‐mail address for correspondence: [email protected]
Despite a decade of investigation on familial ALS caused by missense mutations in the superoxide dismutase (SOD1) gene, the mechanism of toxicity to motor neurons has remained elusive. The current view is that the toxicity of mutant SOD1 is not related to aberrant copper‐mediated catalysis but rather to the propensity of the abnormal protein to aggregate. Surprisingly, recent studies with chimeric mice expressing SOD1 mutants or with mice bearing excisable mutant SOD1 transgenes demonstrated that the toxicity of SOD1 mutants is not strictly autonomous to motor neurons. However, the mechanism by which the toxicity of mutant SOD1 may be transferred from one cell to another has remained unclear.
Although it is well known that SOD1 is a cytosolic protein without specific translocation sequence, a yeast two‐hybrid screen led us to discover that chromogranins, components of neurosecretory vesicles, are interacting partners of SOD1 mutants linked to ALS, but not of wild‐type SOD1. The existence of such interactions was confirmed by coimmunoprecipitation assays using either lysates from Neuro2a cells cotransfected with chromogranins and SOD1 mutants or from spinal cord of ALS mice. Moreover, confocal and immunoelectron microscopy revealed a partial colocalization of SOD1 mutant with chromogranins in spinal cord of ALS mice. Cell culture studies showed that chromogranins may act as chaperones to promote the secretion of mutant SOD1. Further support for secretion of SOD1 came from fluorescent microscope observation of live cells that revealed considerable distribution of EGFP‐fused SOD1, both wild‐type and mutant SOD1 in endoplasmic reticulum (ER) labelled by ER‐tracker and Golgi markers. Cell‐free translocation assay using recombinant SOD1 and microsomes showed that the apo‐form SOD1 of both wild and mutant types translocated into microsomes in an ATP‐dependent fashion.
We also discovered that extracellular mutant SOD1 can trigger microgliosis and death of motor neurons in culture suggesting a pathogenic mechanism based on toxicity of secreted SOD1 mutant proteins. In this model, it is the burden of extracellular mutant SOD1 in close proximity to motor neurons that would increase the risk of damage. Even though interneurons, motor neurons and astrocytes would be the predominant source of extracellular mutant SOD1 mediated by chromogranin interactions, mutant SOD1 secreted by other pathways in cells such as microglia could also contribute to pathogenesis. Although the deleterious effects of intracellular mutant SOD1 cannot be excluded, our model of toxicity based on secreted mutant SOD1 is compatible with the idea that the disease is not autonomous to motor neurons.
C24 REDOX SYSTEM UP‐REGULATION IN ALS MOTOR NEURONS IS A SURVIVAL MECHANISM UNDER STRESS
Kato S1, Kato M2, Ohama E1, Abe Y3, Nishino T3, Aoki M4, Itoyama Y4, Hirano A5
1Department of Neuropathology, Institute of Neurological Sciences, Faculty of Medicine, Yonago, Japan, 2Division of Pathology, Tottori University Hospital, Yonago, Japan, 3Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan, 4Department of Neuroscience, Division of Neurology, Tohoku University Graduate School of Medicine, Sendai, Japan, 5Division of Neuropathology, Department of Pathology, Montefiore Medical Center, New York NY, USA
E‐mail address for correspondence: [email protected]
Background: In neurons, peroxiredoxin‐II (PrxII) and glutathione peroxidase‐I (GPxI) are extremely important enzymes of the redox system that is a crucial antioxidant enzyme defence system and is synchronously linked to other important cell supporting systems.
Objective: To clarify the common self‐survival mechanism of the motor neurons in amyotrophic lateral sclerosis (ALS), we investigated PrxII/GPxI‐expression dynamics in the motor neurons on the basis of the redox system.
Materials: We have recently produced affinity‐purified rabbit polyclonal antibodies against PrxII and GPxI, and have successfully applied it to the paraffin sections. Histological and immunohistochemical studies were carried out on specimens from ALS autopsies and ALS animal models: 40 patients with sporadic ALS (SALS) and seven patients with superoxide dismutase 1 (SOD1)‐mutated familial ALS (FALS) from three different families (frame‐shift 126 mutation, L106V and A4V) as well as four different strains of the SOD1‐mutated ALS animal models (H46R/G93A rats and G1H/G1L‐G93A mice).
Results: Almost all of the normal neurons in humans and animals expressed the redox system‐related enzymes PrxII/GPxI. Although the number of motor neurons in ALS decreased along with disease progression, the number of neurons negative for redox system‐related enzymes increased with ALS disease progression. Noticeably, certain residual motor neurons showing redox system up‐regulation were commonly found during the clinical course of ALS. In SALS patients, motor neurons showing redox system up‐regulation were present three years after the onset and these up‐regulating neurons thereafter decreased in number dramatically, along with the disease progression. In the SOD1‐mutated motor neurons in humans and animals, like SALS, certain residual motor neurons without inclusions also showed redox system up‐regulation during their clinical course. In addition, some SOD1‐mutated motor neurons formed the inclusions where the coaggregation of the redox system‐related enzymes with the SOD1 occurred, thereby amplifying the cytoplasmic depletion of these enzymes and resulting in the disruption of the redox system. At the terminal stage of ALS, the breakdown of this redox system up‐regulation mechanism in neurons was observed.
Discussion and conclusions: We claim that the residual ALS neurons showing redox system up‐regulation would be less susceptible to ALS stress and protect themselves from ALS neuronal death, whereas the breakdown of this redox system would accelerate the process of neuronal death. Our data lead to the development of a new therapy based on redox system up‐regulaion for the treatment of ALS, which for over 130 years has had an unknown etiology.
C25 SUPEROXIDE DISMUTASE 1 FROM THE SPINAL CORD OF G93A RATS BINDS TO THE INNER MEMBRANE OF MITOCHONDRIA AND INCREASES MITOCHONDRIAL ROS PRODUCTION
Goldsteins G, Ahtoniemi T, Jaronen M, Keksa‐Goldsteine V, Koistinaho J
Department of Neurobiology, A.I. Virtanen Institute for Molecular Science, University of Kuopio, Kuopio, Finland
E‐mail address for correspondence: [email protected]
Mutations in the human Cu/Zn superoxide dismutase (SOD1) gene have been found in 20% of familial amyotrophic lateral sclerosis (FALS) cases. Although the nature of the toxic gain of function in mutant SOD1 has not been identified, it is believed that altered free radical and reactive oxygen species (ROS) generation may be a leading contributing factor to the destruction of motor neurons. Apparent destabilization of the SOD1 molecule, causing enhanced aggregation, is yet another characteristic hallmark of mutant molecule toxicity.
In recent studies mitochondrial localization of mutant SOD1 has been implicated in disease pathology. Although several mechanisms explaining how SOD1 aggregation may cause mitochondrial dysfunction have been proposed, there is a lack of conclusive proof of mutant SOD1 toxicity in mitochondria.
We analysed the molecular features of cytosolic SOD1 extracted from the spinal cord, cortex, cerebellum and liver of G93A SOD1 rats employing modified immunoblotting. This allows us to distinguish the degree of denaturation by binding to a hydrophobic membrane. The results obtained showed clear SOD1 destabilization in the spinal cord. SOD1 stability decreased with disease progression and the amount of destabilized SOD1 peaked at 16 weeks, shortly before the onset of the disease. The binding of destabilized SOD1 to the mitoplasts, isolated from wild‐type rat liver, was increased. In parallel, ROS production was significantly elevated in mitoplasts exposed to destabilized SOD1.
The data obtained shed light on the early events in the pathological chain where SOD1, destabilized by mutations, acquires an increased ability to bind to the inner membrane of mitochondria, which in turn directly increases ROS production.
C26 CELL‐PERMEABLE PEPTIDE ANTIOXIDANTS AS A NOVEL THERAPEUTIC APPROACH IN A MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS
Petri S3, Kiaei M1, Damiano M1, Hiller A1, Wille E1, Manfredi G1, Calingasan NY1, Szeto HH2, Beal MF1
1Department of Neurology and Neuroscience, 2Department of Pharmacology, Weill Medical College of Cornell University, New York, USA, 3Department of Neurology, Hannover Medical School, Hannover, Germany
E‐mail address for correspondence: [email protected]
Background: Reactive oxygen species (ROS) play a major role in the pathogenesis of neurodegenerative diseases. They are important contributors to necrotic and apoptotic cell death. A major proportion of cellular ROS are generated at the inner mitochondrial membrane by the respiratory chain.
Objectives: In the present study we investigated a novel peptide antioxidant (SS‐31) targeted to the inner mitochondrial membrane for its therapeutic effect in neuronal cells stably transfected with either wild‐type or mutant SOD1 and in the G93A mouse model of amyotrophic lateral sclerosis (ALS).
Results: SS‐31 protected against cell death induced by hydrogen peroxide in vitro in neuronal cells stably transfected with either wild‐type or mutant SOD1. In G93A ALS transgenic mice, daily intraperitoneal injections of SS‐31 started at 30 days of age led to a significant improvement in survival and motor performance. Furthermore, compared to vehicle‐treated G93A mice, SS‐31‐injected mice showed reduced cell loss in the lumbar spinal cord at 110 days of age. We also found a decrease in immunostaining for markers of oxidative stress (4‐hydroxynonenal, 3‐nitrotyrosine) in the lumbar spinal cord of SS‐31 injected animals.
Conclusion: Our data support the assumption that direct targeting of ROS production at the inner mitochondrial membrane and therefore preventing further mitochondrial damage is an interesting new approach to treat neuronal degeneration induced by oxidative stress.
C27 TARGETING OXIDATIVE STRESS AS A POTENTIAL THERAPY FOR ALS
Barber SC, Higginbottom A, Mead RJ, Shaw PJ
University of Sheffield, Sheffield, UK
E‐mail address for correspondence: [email protected]
Background: There is now overwhelming evidence that ALS pathogenesis is, in part, mediated by oxidative damage, although the precise mechanism(s) by which this occurs remains unknown. Given the current lack of effective treatments for ALS, a valid rationale is to investigate the efficacy of anti‐oxidants as potential therapies, either individually or as a cocktail.
Objectives: To develop a high‐throughput cellular assay of motor neuronal oxidative stress that can be used to screen a drug library for candidate drugs.
Methods: NSC34 mouse motor neuronal cells were transfected with either wild‐type or mutant (G37R, H48Q, G93A and I113T) human SOD1 and stable single cell clones were produced. Dichlorofluorescein fluorescence (normalized to cell number to allow comparisons between cell lines) was used to measure reactive oxygen species (ROS) levels in NSC34 cells under basal conditions and during oxidative stress induced by serum withdrawal. Potential anti‐oxidant drugs were tested by addition at the time of serum withdrawal. Drug toxicity was tested by measuring ethidium fluorescence from dead cells.
Results: Under basal conditions, NSC34 cells stably expressing four ALS‐linked human SOD1 mutations had significantly higher levels of ROS than either untransfected NSC34 cells, or cells stably expressing empty vector or wild‐type human SOD1. Untransfected NSC34 cells deprived of serum also exhibited a significant increase in ROS, typically three‐fold, providing a simpler assay system. Ebselen, a peroxiredoxin mimic previously shown to increase NSC34 cell survival during serum withdrawal, protected against the observed increase in ROS following serum withdrawal (EC50 = 4.8 µM). Using these models, we are screening known anti‐oxidant compounds in a targeted approach, and also the Spectrum Collection of 2000 bioactive compounds including known drugs and natural products (MicroSource Discovery Systems) to identify potential therapeutic candidates. Lead drugs identified from this screen will be described.
Discussion and conclusions: The higher oxidative stress levels observed in NSC34 cell lines expressing mutant SOD1 compared to the control cell lines emphasizes the role of oxidative stress in ALS pathogenesis, and provides an in vitro assay in which anti‐oxidant strategies can be tested. This simple assay can be used to screen large numbers of potential drugs, and the most effective targets will be tested and further developed in more complex models of ALS.