Eiline Cai

Introduction. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons.1 About 90% of diagnosed patients die within 5 years of onset.¹ Currently, two medications have been approved for ALS treatment – riluzole and edaravone.^1,2 Riluzole increases survival only by about 3 months, and edaravone is targeted specifically to ALS patients of early onset and rapid progression.^1,2 The most commonly implicated genetic cause of ALS is a GGGGCC (G4C2) repeat in the C9orf72 gene, which induces cytosolic TDP-43 aggregates.^3,4 TDP-43 aggregates are then imported into the mitochondria, leading to mitochondrial dysfunction and neuronal death.^5,6 Lon protease (LonP1) typically degrades damaged proteins in the mitochondria as part of mitochondrial proteostasis.⁷ Additionally, LonP1 has been shown to contribute to degradation of cytosolic protein aggregates in yeast cells.⁸ The same mechanism may extend to human cells. The role of mitochondrial proteostasis in the import of cytosolic TDP-43 aggregates suggests a potential novel therapy for ALS. Methods. Tetracycline (Tet) inducible HEK293 cell lines expressing TDP-43 were used to prepare cell lysates.⁹ Coimmunoprecipitation assay with anti-TDP-43 antibody was used to determine TDP-43 interaction with LonP1.⁹ Immuno-electron microscopy was used to observe the mitochondria.⁹ Cell lysates were transfected with LonP1-expressing plasmid and induced by Tet to determine the effect of increased LonP1 expression.⁹ Cell lysates were transduced with a lentivirus expressing shRNA against LonP1 to determine the effect of decreasing LonP1 expression.⁹ Fractionation experiments were used to isolate mitochondrial and cytoplasmic protein.⁹  Western blotting was used to quantify protein expression.⁹ To examine in vivo impact of decreased LonP1, TDP-43 transgenic flies were crossed with flies expressing siRNA against LonP1.⁹  TDP-43 expression was induced by heat shock.9 Quantitative analysis was used to determine mitochondrial damage.⁹  Motor function was measured under the Elav-Gal4/Tub-Gal80ts driver.⁹  Results. TDP-43 interacts with LonP1 within the mitochondria.⁹ Increased LonP1 expression resulted in decreased TDP-43 protein within the mitochondria and decreased cell cytotoxicity.⁹ Decreased LonP1 expression resulted in increased TDP-43 protein within the mitochondria and increased cell cytotoxicity.⁹  Decreased LonP1 in vivo resulted in increased mitochondrial TDP-43 and damage as well as faster progression of locomotor dysfunction.⁹  Conclusion. Cytosolic TDP-43 aggregates are imported into the mitochondria, where LonP1 can degrade the TDP-43 aggregates. In doing so, LonP1 plays a protective role against mitochondrial damage and locomotor deficits. Therefore, increasing LonP1 expression may be a potential therapy for ALS.

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