BackgroundAmyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects motor neurons (MNs). It was shown that human astrocytes with mutations in genes associated with ALS, like C9orf72 (C9) or SOD1, reduce survival of MNs. Astrocyte toxicity may be related to their dysfunction or the release of neurotoxic factors.MethodsWe used human induced pluripotent stem cell-derived astrocytes from ALS patients carrying C9orf72 mutations and non-affected donors. We utilized these cells to investigate astrocytic induced neuronal toxicity, changes in astrocyte transcription profile as well as changes in secretome profiles.FindingsWe report that C9-mutated astrocytes are toxic to MNs via soluble factors. The toxic effects of astrocytes are positively correlated with the length of astrocyte propagation in culture, consistent with the age-related nature of ALS. We show that C9-mutated astrocytes downregulate the secretion of several antioxidant proteins. In line with these findings, we show increased astrocytic oxidative stress and senescence. Importantly, media conditioned by C9-astrocytes increased oxidative stress in wild type MNs.InterpretationOur results suggest that dysfunction of C9-astrocytes leads to oxidative stress of themselves and MNs, which probably contributes to neurodegeneration. Our findings suggest that therapeutic strategies in familial ALS must not only target MNs but also focus on astrocytes to abrogate nervous system injury.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by cell death of upper and lower motor neurons (MNs). The cause of MN cell loss is not completely understood but involves both cell autonomous and non-cell autonomous mechanisms. Numerous molecules have been implicated to be involved in the death of MNs. One such candidate is semaphorin 3A (Sema3A). In ALS patients, Sema3A was shown to be significantly upregulated in the motor cortex and downregulated in the spinal cord. In the mouse, Sema3A was shown to be an axon repellent molecule for MNs. Sema3A could also induce death of different neuronal types that are also repelled by it, including sensory, sympathetic, retinal, and cortical neurons. In contrast, astrocyte-specific knockout of Sema3A results in motor neuron cell death, consistent with the idea that Sema3A is a survival factor for mouse motor neurons. Here, we tested the response of human cortical neurons and spinal cord MNs to Sema3A. We found that Sema3A enhances the survival of spinal cord MNs. In contrast, Sema3A reduces the survival of cortical neurons. Thus, both upregulation of Sema3A in the cortex, or downregulation in the spinal cord of ALS patients is likely to directly contribute to MNs cell loss in ALS patients.