Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which pathogenesis and death of motor neurons are triggered by non-cell-autonomous mechanisms. We showed earlier that exposing primary rat spinal cord cultures to conditioned media derived from primary mouse astrocyte conditioned media (ACM) that express human SOD1G93A (ACM-hSOD1G93A) quickly enhances Nav channel-mediated excitability and calcium influx, generates intracellular reactive oxygen species (ROS), and leads to death of motoneurons within days. Here we examined the role of mitochondrial structure and physiology and of the activation of c-Abl, a tyrosine kinase that induces apoptosis. We show that ACM-hSOD1G93A, but not ACM-hSOD1WT, increases c-Abl activity in motoneurons, interneurons and glial cells, starting at 60 min; the c-Abl inhibitor STI571 (imatinib) prevents this ACM-hSOD1G93A-mediated motoneuron death. Interestingly, similar results were obtained with ACM derived from astrocytes expressing SOD1G86R or TDP43A315T. We further find that co-application of ACM-SOD1G93A with blockers of Nav channels (spermidine, mexiletine, or riluzole) or anti-oxidants (Trolox, esculetin, or tiron) effectively prevent c-Abl activation and motoneuron death. In addition, ACM-SOD1G93A induces alterations in the morphology of neuronal mitochondria that are related with their membrane depolarization. Finally, we find that blocking the opening of the mitochondrial permeability transition pore with cyclosporine A, or inhibiting mitochondrial calcium uptake with Ru360, reduces ROS production and c-Abl activation. Together, our data point to a sequence of events in which a toxic factor(s) released by ALS-expressing astrocytes rapidly induces hyper-excitability, which in turn increases calcium influx and affects mitochondrial structure and physiology. ROS production, mediated at least in part through mitochondrial alterations, trigger c-Abl signaling and lead to motoneuron death.
Highlights
Amyotrophic lateral sclerosis (ALS) is a fatal paralytic disorder caused by the progressive degeneration of upper and lower motoneurons during adulthood, and results in death by respiratory failure, usually within 3–5 years of diagnosis
Immunostaining assays as well as western blots of whole lysate extracts from the spinal cord and brain were used to determine the expression of native c-Abl proteins and of c-Abl that is phosphorylated on tyrosine 412 (Tyr412; a site that enhances c-Abl catalytic activity; Hantschel and Superti-Furga, 2004)
In agreement with a previous study (Katsumata et al, 2012), we found a robust increase in the phosphorylation of c-Abl in the brain and spinal cord of symptomatic hSOD1G93A mice (P120), compared to non-transgenic littermates; unlike this earlier study, our samples did not show an increase in the levels of expression of native c-Abl (Supplementary Figure S1)
Summary
Amyotrophic lateral sclerosis (ALS) is a fatal paralytic disorder caused by the progressive degeneration of upper and lower motoneurons during adulthood, and results in death by respiratory failure, usually within 3–5 years of diagnosis. Despite substantial progress in the identification of pathogenic changes, as well as of the cell types that contribute to them, no cure exists for this profoundly debilitating disease, and the mechanisms that underlie motoneurons death in ALS remain largely unknown; we do not know even whether the neuronal abnormalities are a primary or secondary event, or whether they result from a compensatory mechanism (van Zundert et al, 2012) In part this is because classical approaches for studying neuron-glial interactions use a co-culture system wherein neurons are grown on a feeder layer of astrocytes, masking the temporal interplay between original and secondary pathogenic events
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