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Amyotrophic lateral sclerosis (ALS) is a fatal progressive disease characterized by motor neuron degeneration. Approximately 90% of ALS patients are diagnosed with sporadic ALS (sALS) without a known cause, while 10% have familial ALS (fALS) arising from an identified genetic mutation. Previous in vivo and in vitro studies have demonstrated that ALS cerebrospinal fluid (CSF) is neurotoxic, but it is unclear whether differences in neurotoxicity exist between sALS and fALS CSF, and the neurotoxic candidates in CSF have yet to be identified. Here, our goal was to identify the neurotoxic factor(s) in sALS CSF which trigger motor disability and motor neuron degeneration in our CSF-induced animal model.
Adult female C57BL/6J mice underwent laminectomies at cervical levels 4 and 5, and received 3 µl injections into the subarachnoid space of either: 1) CSF from sALS or fALS (SOD1, C9orf72, or TARDBP) patients, 2) filtered sALS CSF, 3) apolipoprotein B-100 (ApoB)-depleted sALS CSF, or 4) human ApoB protein. Control mice received either saline, CSF from healthy individuals (HC) or multiple sclerosis patients, or human CSF proteins. Forelimb motor deficits were assessed at 1 day post injection, then mice were perfused for histological analyses of the spinal cord. All motor and histological assessments were performed blinded. sALS CSF was passed through a tangential flow filtration system to remove components by molecular weight and unbiased global proteomic profiling was performed on CSF pre- and post-filtration. Immunodepletion of ApoB from sALS CSF was performed using ApoB antibody-coated Dynabeads®.
sALS CSF-injected mice exhibited significantly impaired forelimb function associated with increased motor neuron death compared to controls and fALS CSF-injected mice. Filtration studies determined the neurotoxic protein in sALS CSF to be between 300 kDa and 750 kDa in size as removal of components larger than 300 kDa attenuated neurotoxicity, while the 750 kDa filtrate retained neurotoxicity. Proteomic analyses revealed ApoB to be upregulated in sALS CSF compared to HC CSF, downregulated post-filtration, and the only candidate with the appropriate molecular weight (550 kDa). Mice injected with ApoB-depleted sALS CSF did not show motor impairments nor loss of motor neurons, while ApoB protein, but not control proteins, recapitulated the neurotoxic effects of sALS CSF. This study identifies ApoB as the neurotoxic protein in sALS CSF responsible for motor disability and motor neuron degeneration and provides proof-of-concept to support CSF pheresis as a therapeutic strategy for the predominant sporadic form of ALS.