Abnormal intracellular calcium signaling and SNARE-dependent exocytosis contributes to SOD1G93A astrocyte-mediated toxicity in amyotrophic lateral sclerosis.

Motor neurons are progressively and predominantly degenerated in ALS, which is not only induced by multiple intrinsic pathways but also significantly influenced by the neighboring glial cells. In particular, astrocytes derived from the SOD1 mutant mouse model of ALS or from human familial or sporadic ALS patient brain tissue directly induce motor neuron death in culture; however, the mechanisms of pathological astroglial secretion remain unclear. Here we investigated abnormal calcium homeostasis and altered exocytosis in SOD1G93A astrocytes. We found that purinergic stimulation induces excess calcium release from the ER stores in SOD1G93A astrocytes, which results from the abnormal ER calcium accumulation and is independent of clearance mechanisms. Furthermore, pharmacological studies suggested that store-operated calcium entry (SOCE), a calcium refilling mechanism responsive to ER calcium depletion, is enhanced in SOD1G93A astrocytes. We found that oxidant-induced increased S-glutathionylation and calcium-independent puncta formation of the ER calcium sensor STIM1 underlies the abnormal SOCE response in SOD1G93A astrocytes. Enhanced SOCE contributes to ER calcium overload in SOD1G93A astrocytes and excess calcium release from the ER during ATP stimulation. In addition, ER calcium release induces elevated ATP release from SOD1G93A astrocytes, which can be inhibited by the overexpression of dominant-negative SNARE. Selective inhibition of exocytosis in SOD1G93A astrocytes significantly prevents astrocyte-mediated toxicity to motor neurons and delays disease onset in SOD1G93A mice. Our results characterize a novel mechanism responsible for calcium dysregulation in SOD1G93A astrocytes and provide the first in vivo evidence that astrocyte exocytosis contributes to the pathogenesis of ALS.

UCP2 overexpression worsens mitochondrial dysfunction and accelerates disease progression in a mouse model of amyotrophic lateral sclerosis.

Mitochondrial dysfunction leading to deficits in energy production, Ca(2+) uptake capacity, and free radical generation has been implicated in the pathogenesis of familial amyotrophic lateral sclerosis (ALS) caused by mutations in Cu,Zn superoxide dismutase (SOD1). Numerous studies link UCP2, a member of the uncoupling protein family, to protection of neurons from mitochondrial dysfunction and oxidative damage in various mouse models of acute stress and neurodegeneration, including Parkinson's disease. Here, we tested the potential neuroprotective effects of UCP2 and its ability to modulate mitochondrial function, in the G93A mutant SOD1 mouse model of familial ALS. Disease phenotype, mitochondrial bioenergetics, and Ca(2+) uptake capacity were investigated in the central nervous system of double transgenic mice, expressing both human mutant G93A SOD1 and human UCP2 (hUCP2). Unexpectedly, hUCP2 expression accelerated the disease course of SOD1 mutant mice. In addition, we did not observe a classical uncoupling effect of hUCP2 in G93A brain mitochondria, although we did detect a decrease in reactive oxygen species (ROS) production from mitochondria challenged with the respiratory chain inhibitors rotenone and antimycin A. We also found that mitochondrial Ca(2+) uptake capacity was decreased in the double transgenic mice, as compared to G93A mice. In summary, our results indicate that the neuroprotective role of UCP2 in neurodegeneration is disease-specific and that, while a mild uncoupling by UCP2 in brain mitochondria may protect against neurodegeneration in some injury paradigms, the mitochondrial damage and the disease caused by mutant SOD1 cannot be ameliorated by UCP2 overexpression.