Mutations in superoxide dismutase-1 (SOD1) cause a form of the fatal paralytic disorder amyotrophic lateral sclerosis (ALS), presumably by a combination of cell-autonomous and non-cell-autonomous processes. Here, we show that expression of mutated human SOD1 in primary mouse spinal motor neurons does not provoke motor neuron degeneration. Conversely, rodent astrocytes expressing mutated SOD1 kill spinal primary and embryonic mouse stem cell-derived motor neurons. This is triggered by soluble toxic factor(s) through a Bax-dependent mechanism. However, mutant astrocytes do not cause the death of spinal GABAergic or dorsal root ganglion neurons or of embryonic stem cell-derived interneurons. In contrast to astrocytes, fibroblasts, microglia, cortical neurons and myocytes expressing mutated SOD1 do not cause overt neurotoxicity. These findings indicate that astrocytes may play a role in the specific degeneration of spinal motor neurons in ALS. Identification of the astrocyte-derived soluble factor(s) may have far-reaching implications for ALS from both a pathogenic and therapeutic standpoint.

Mutation in superoxide dismutase-1 (SOD1), which is a cause of ALS, alters the folding patterns of this protein. Accumulation of misfolded mutant SOD1 might activate endoplasmic reticulum (ER) stress pathways. Here we show that transgenic mice expressing ALS-linked SOD1 mutants exhibit molecular alterations indicative of a recruitment of ER's signaling machinery. We demonstrate by biochemical and morphological methods that mutant SOD1 accumulates inside the ER, where it forms insoluble high molecular weight species and interacts with the ER chaperone immunoglobulin-binding protein. These alterations are age-and regionspecific, because they develop over the course of the disease and occur in the affected spinal cord but not in the nonaffected cerebellum in transgenic mutant SOD1 mice. Our results suggest a toxic mechanism for mutant SOD1 by which this ubiquitously expressed pathogenic protein could affect motor neuron survival and contribute to the selective motor neuronal degeneration in ALS.neurodegeneration ͉ protein misfolding A LS is the most common adult-onset paralytic disease characterized by a loss of motor neurons in the cerebral cortex, brainstem, and spinal cord. Insights into the neurodegenerative mechanisms followed the discovery that dominant mutations in the gene for superoxide dismutase-1 (SOD1) cause familial ALS. Overexpression of SOD1 mutants in rodents recapitulates ALS clinical and pathological hallmarks through a toxic gain of function (1). Many mutations in SOD1 decrease its stability and increase its unfolding rates and propensity to aggregate (2). High molecular weight complexes of SOD1 are observed in mammalian cells and spinal cords of transgenic mice expressing this mutant protein (3). In these animals, intracellular ubiquitin-positive proteinaceous inclusions are also often seen in spinal cord motor neurons and, in some cases, in neighboring astrocytes (3-5). These findings posit that accumulation of misfolded mutant SOD1 could contribute to the demise of motor neurons.Endoplasmic reticulum (ER) stress signaling, otherwise known as the unfolded protein response (UPR), is triggered by an increased load of misfolded proteins in the organelle (6). Herein, we show that transgenic mice expressing mutant SOD1 exhibit age-and region-specific molecular alterations indicative of a broad recruitment of ER signaling pathways, including caspase-12, a prototypical ER cell death effector (7). We also show that mutant SOD1, and to a lesser extent wild-type SOD1 (SOD1 WT ), do accumulate in the ER. Within this organelle, mutant, but not SOD1 WT , forms high molecular weight species and interacts with the ER chaperone immunoglobulin-binding protein (BiP), which is a key component of the ER misfolded protein recognition machinery (6). The preferential accumulation of mutant SOD1 in the ER in the spinal cord cells and the ensuing stress response may represent novel aspects of motor neuron degeneration in this ALS model.

ALS is a fatal paralytic disorder characterized by a progressive loss of spinal cord motor neurons. Herein, we show that NADPH oxidase, the main reactive oxygen species-producing enzyme during inflammation, is activated in spinal cords of ALS patients and in spinal cords in a genetic animal model of this disease. We demonstrate that inactivation of NADPH oxidase in ALS mice delays neurodegeneration and extends survival. We also show that NADPH oxidase-derived oxidant products damage proteins such as insulin-like growth factor 1 (IGF1) receptors, which are located on motor neurons. Our in vivo and in vitro data indicate that such an oxidative modification hinders the IGF1͞Akt survival pathway in motor neurons. These findings suggest a non-cell-autonomous mechanism through which inflammation could hasten motor neuron death and contribute to the selective motor neuronal degeneration in ALS.LS is the most common adult-onset paralytic disease and is characterized by a loss of motor neurons in the cerebral cortex, brainstem, and spinal cord (1). It is invariably fatal, usually within 3-5 years after onset (1). Insights into its neurodegenerative mechanisms followed the discovery that dominant mutations in the gene for superoxide dismutase 1 (SOD1) cause familial ALS (2, 3). Overexpression of SOD1 mutants in rodents emulate clinical and pathological hallmarks of ALS through a toxic gain of function (4). Development of chimeric mice provided animals with a mixture of neuronal and nonneuronal cells expressing wild-type or mutant SOD1 (5); investigation of these animals suggested that nonneuronal cells influence the fate of spinal cord motor neurons (5). Corroborating this hypothesis is the demonstration that reduction of mutant SOD1, selectively in microglia, extended survival in transgenic SOD1 G37R mice (6). In light of the latter results, microglia are now thought to contribute to the non-cell-autonomous killing of motor neurons.Among the microglia-derived mediators that could promote neurodegeneration are reactive oxygen species (ROS) produced by the enzyme NADPH oxidase complex (7). Although this ROSgenerating multimeric oxidase is indispensable for protecting the host against invading microorganisms in infectious disorders (8), its inappropriate activation may be harmful in noninfectious neurodegenerative disorders. Such bystander cytotoxicity is thought to lead to the death of developing oligodendrocytes in periventricular leukomalacia (9), one of the most important causes of cerebral palsy. In light of these facts, we undertook the study of NADPH oxidase in both human ALS and one of its genetic models. Our results for both human and mouse postmortem tissues indicate that spinal cord microgliosis in ALS is accompanied with an upregulation of NADPH oxidase. Furthermore, by using mutant deficient mice in functional NADPH oxidase as well as in neuronlike cell culture systems, we provide compelling evidence that supports the concept that this microglial ROS-generating enzymatic complex promotes spinal cord motor neuro...

Some cases of familial amyotrophic lateral sclerosis (ALS) are caused by mutations in the gene encoding cytosolic, copper-zinc superoxide dismutase (SOD1). We report here that rats that express a human SOD1 transgene with two different ALS-associated mutations (G93A and H46R) develop striking motor neuron degeneration and paralysis. As in the human disease and transgenic ALS mice, pathological analysis demonstrates selective loss of motor neurons in the spinal cords of these transgenic rats. In spinal cord tissues, this is accompanied by activation of apoptotic genes known to be activated by mutant SOD1 protein in vitro and in vivo. These animals provide additional support for the proposition that motor neuron death in SOD1-related ALS reflects one or more acquired, neurotoxic properties of the mutant SOD1 protein. The larger size of this rat model as compared with the ALS mice will facilitate studies involving manipulations of spinal fluid (implantation of intrathecal catheters for chronic therapeutic studies; CSF sampling) and spinal cord (e.g., direct administration of viral- and cell-mediated therapies).

Recent reports suggest that functional or structural defect of vascular components are implicated in amyotrophic lateral sclerosis (ALS) pathology. In the present study, we examined a possible change of the neurovascular unit consisting of endothelium (PCAM-1), tight junction (occludin), and basement membrane (collagen IV) in relation to a possible activation of MMP-9 in ALS patients and ALS model mice. We found that the damage in the neurovascular unit was more prominent in the outer side and preferentially in the anterior horn of ALS model mice. This damage occurred prior to motor neuron degeneration and was accompanied by MMP-9 up-regulation. We also found the dissociation between the PCAM-1-positive endothelium and GFAP-positive astrocyte foot processes in both humans and the animal model of ALS. The present results indicate that perivascular damage precedes the sequential changes of the disease, which are held in common between humans and the animal model of ALS, suggesting that the neurovascular unit is a potential target for therapeutic intervention in ALS.