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Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by motor neuron death, with its sporadic form affecting 90% of those with the disorder. We previously showed that cerebrospinal fluid (CSF) from sporadic ALS (sALS) patients induces human motor neuron death and neurotoxic factors can be removed following filtration through a 5 kDa MWCO filter. Additionally, our in vivo studies had identified apolipoprotein B-100 (ApoB) as the neurotoxic factor responsible for inducing motor disability and ALS-like pathology in a CSF-mediated animal model of sALS. Here, we aimed to determine whether ApoB in sALS CSF also triggers death of human motor neurons in vitro.
Human iPSC-derived motor neurons were cultured for 8 days, then incubated for 24 hours with either: 1) 50% sALS CSF, 2) 50% filtered sALS CSF, 3) 50% ApoB-depleted sALS CSF, 4) human ApoB protein, or 5) human haptoglobin protein in motor neuron maintenance media. Components larger than 5 kDa, 300 kDa, or 750 kDa were eliminated from sALS CSF through a tangential flow filtration system. ApoB was depleted from sALS CSF using ApoB antibody-coated Dynabeads®. Different concentrations of lipid-free human ApoB were also applied directly to motor neurons, comparing the apolipoprotein’s toxicity to media or the control protein haptoglobin. Motor neurons were fixed in 4% paraformaldehyde for ChAT immunocytochemistry. Areas of ChAT+ motor neuron clusters were quantified as a measure of motor neuron death. All analyses were performed blinded.
sALS CSF significantly reduced motor neuron cluster sizes compared to media. However, ApoB-depletion and filtration prevented sALS CSF-induced neurotoxicity. Only motor neurons treated with 750 kDa-filtered sALS CSF exhibited smaller clusters than control, since the filter was too large to exclude ApoB (550 kDa). In addition, the direct application of lipid-free ApoB revealed a dose-dependent effect on human motor neuron death, where smaller motor neuron clusters were only seen following treatment with ApoB concentrations similar to that found in sALS CSF or higher. Our studies collectively show that ApoB in sALS CSF induces human motor neuron death in vitro. Future experiments should aim to elucidate the mechanisms of ApoB-induced neurotoxicity and identify therapeutic targets to prevent motor neuron degeneration in sALS.
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