Lauren Fitzgerald

 Introduction. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of upper and lower motor neurons and the most common adult-onset motor neuron disease.¹ The mean age of onset is 55 years, and symptoms include denervation, muscle wasting, and weakness.^2,3 Death from respiratory failure typically occurs 3-5 years after diagnosis.^3,4 ALS is treated symptomatically due to lack of disease-modifying treatments.⁴ The pathogenic mechanism of neuron damage in ALS is largely unknown, but cytoplasmic TDP-43 deposition is present in 97% of ALS patients.^1,5 TDP-43 is a predominantly nuclear RNA/DNA-binding protein regulating RNA transcription, editing, and transportation, and also forming cytoplasmic stress granules.² TDP-43 deposition involves prion-like “template seeding” where misfolded aggregates add monomers in the same form. Aggregates are ubiquitin-positive and would normally be removed by the ubiquitin-proteasome system (UPS).^5,6 Determining the mechanisms by which TDP-43 aggregate cause neuronal death in ALS is the focus of this study. Methods. To analyze toxicity of seeded aggregation, samples from spinal cords of post-mortem ALS patients were used to inoculate human motor neuron stem cell cultures. Seeded-aggregation effects were studied in isolation and with proteasome inhibitor MG132. Immunocytochemistry, image analysis software, and statistical analysis were applied.⁶ To analyze the role of the PTK2-TBK1-SQSTM1 autophagy-lysosomal pathway, DNA vectors using GFP-tagged human TDP-43 or GFP alone were injected into mouse N2a cells or primary cortical neurons. Proteasome activity assays, cytotoxicity assays, autophagy analysis, immunostaining, and statistical analysis were performed.⁵ Results. When neuronal cells were inoculated with ALS spinal cord extract alone, cells with cytoplasmic TDP-43 aggregates increased daily, but cell viability remained normal. When MG132 was added with spinal cord extract, cells with cytoplasmic TDP-43 aggregates and the proportion that were caspase positive increased significantly.⁶ TDP-43 overexpression increased poly-ubiquitinated protein levels, impaired chymotrypsin-like proteasome activity, and increased phosphorylation/activation of PTK2. PTK2 inhibition decreased cytoplasmic, poly-ubiquitinated TDP-43. In the presence of proteasome inhibition, PTK2 inhibition improved cell viability and decreased apoptosis. The effect of PTK2 inhibition is mediated by decreasing the phosphorylation and activation of SQSTM1 via TBK1 kinase. This is shown by the following results: 1) phosphorylation-dead SQSTM1 decreased poly-ubiquitination and caspase activation, and 2) TBK1 inhibition significantly decreased PTK2 overexpression-induced SQSTM1 deactivation.⁵ Conclusion. UPS impairment causes TDP-43 seeded-aggregation toxicity, and TDP-43 overexpression causes UPS impairment. These two effects may amplify each other. Inhibiting SQSTM1 phosphorylation along the PTK2-TBK1-SQSTM1 pathway could control poly-ubiquitinated aggregation and neuronal death from UPS impairment in ALS.

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