Here we report that chromogranins, components of neurosecretory vesicles, interact with mutant forms of superoxide dismutase (SOD1) that are linked to amyotrophic lateral sclerosis (ALS), but not with wild-type SOD1. This interaction was confirmed by yeast two-hybrid screen and by co-immunoprecipitation assays using either lysates from Neuro2a cells coexpressing chromogranins and SOD1 mutants or lysates from spinal cord of ALS mice. Confocal and immunoelectron microscopy revealed a partial colocalization of mutant SOD1 with chromogranins in spinal cord of ALS mice. Mutant SOD1 was also found in immuno-isolated trans-Golgi network and in microsome preparations, suggesting that it can be secreted. Indeed we report evidence that chromogranins may act as chaperone-like proteins to promote secretion of SOD1 mutants. From these results, and our finding that extracellular mutant SOD1 can trigger microgliosis and neuronal death, we propose a new ALS pathogenic model based on the toxicity of secreted SOD1 mutants.

Through undefined mechanisms, dominant mutations in (Cu/Zn) superoxide dismutase-1 (mSOD1) cause the non-cell-autonomous death of motoneurons in inherited amyotrophic lateral sclerosis (ALS). Microgliosis at sites of motoneuron injury is a neuropathological hallmark of ALS. Extracellular mSOD1 causes motoneuron injury and triggers microgliosis in spinal cord cultures, but it is unclear whether the injury results from extracellular mSOD1 directly interacting with motoneurons or is mediated through mSOD1-activated microglia. To dissociate these potential mSOD1-mediated neurotoxic mechanisms, the effects of extracellular human mSOD1 G93A or mSOD1 G85R were assayed using primary cultures of motoneurons and microglia. The data demonstrate that exogenous mSOD1 G93A did not cause detectable direct killing of motoneurons. In contrast, mSOD1 G93A or mSOD1 G85R did induce the morphological and functional activation of microglia, increasing their release of pro-inflammatory cytokines and free radicals. Furthermore, only when microglia were co-cultured with motoneurons did extracellular mSOD1 G93A injure motoneurons. The microglial activation mediated by mSOD1 G93A was attenuated using toll-like receptors (TLR) 2, TLR4 and CD14 blocking antibodies, or when microglia lacked CD14 expression. These data suggest that extracellular mSOD1 G93A is not directly toxic to motoneurons but requires microglial activation for toxicity, utilizing CD14 and TLR pathways. This link between mSOD1 and innate immunity may offer novel therapeutic targets in ALS.

There is emerging evidence for the existence of secretory pathways for superoxide dismutase (SOD1) mutants linked to amyotrophic lateral sclerosis (ALS) and for neurotoxicity of extracellular mutant SOD1. This evidence led us to test immunization protocols aiming to reduce the burden of extracellular SOD1 mutants in nervous tissue of mice models of ALS, by using bacterially purified recombinant SOD1 mutant protein as an immunogen. First, a vaccination was tested on a G37R SOD1 mouse strain with lateonset disease exhibiting levels of mutant SOD1 protein at 4-fold higher than normal SOD1 levels. Repeated injections of adjuvant/ SOD1 mutant with a final booster injection before symptoms at 6 months of age were effective in delaying disease onset and extending the life span of G37R SOD1 mice by >4 weeks. Western blot analysis with a monoclonal antibody specific to mutant SOD1 forms provided evidence of clearance of SOD1 species in the spinal cord of vaccinated G37R SOD1 mice. In contrast, this vaccination approach failed to confer significant protection in G93A SOD1 mice with extreme overexpression of mutant SOD1. Nonetheless, a passive immunization through intraventricular infusion of purified anti-human SOD1 antibody with osmotic minipump succeeded in alleviating disease symptoms and prolonging the life span of G93A SOD1 mice. From these results, we propose that immunization strategies should be considered as potential avenues for treatment of familial ALS caused by SOD1 mutations.vaccination ͉ immunotherapy ͉ antibody A myotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized with limb paralysis and atrophy. Since the discovery of genetic mutations in superoxide dismutase 1 (SOD1) in 20% of familial ALS patients (1) and the development of ALS model mice overexpressing SOD1 mutants (2), diverse pathogenic pathways to motor neuron death have been elucidated, such as protein misfolding and aggregation, proteasome impairment, inflammation, reactive oxygen species, excitotoxicity, and mitochondrial dysfunction (3, 4). Based on these hypotheses, multiple approaches for treatment have been tested in the ALS mice, including pharmacological approaches (5-10) and virus-mediated delivery of molecules (11-16). Recent lines of evidence indicate that the toxicity of SOD1 mutants is not strictly autonomous to motor neurons. Neuron-specific expression of mutant SOD1 failed to provoke motor neuron disease (17, 18), and chimeric mouse studies with SOD1 mutants demonstrated that neurodegeneration was delayed or eliminated when motor neurons expressing mutant SOD1 were surrounded by wild-type (WT) healthy cells (19). Moreover, the report by Clement et al. (19) showed evidence of damage to WT motor neurons that were surrounded by cells expressing mutant SOD1. These results emphasized the importance of motor neuron milieu, but they did not provide insight into the mechanism by which the toxicity of mutant SOD1 may be transferred from one cell to another.Although it is well known that SOD1 is a cytosolic ...