Cellular pathogenic mechanisms linked to TBK-1 and optineurin in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease associated with the loss of both upper and lower motor neurons in the motor cortex, brain stem and spinal cord. The pathogenic mechanisms underlying ALS remaln largely unknown. The pathological hallmark of ALS is the presence of cytoplasmic inclusions containing misfolded proteins in degenerating motor neurons. These inclusions are present regardless of the site of symptom onset. age or gender of the patient. Moreover, these inclusions are present in both sporadic and familial forms of ALS. Misfolded protein accumulation triggers stress in the endoplasmic reticulum (ER), inducing the unfolded protein response (UPR), and ER stress is now a well described feature of ALS. The UPR aims to restore proteostasis, but if unresolved triggers apoptosis. The UPR is also linked to autophagy, a self- degradative process that could clear protein aggregates. However. the presence of protein Inclusions suggests that both UPR and autophagy could be dysfunctional in ALS. One of the genes most recently identified In ALS encoded, TBK-1, a kinase involved in different forms of selective autophagy including mitophagy and xenophagy. Interestingly, TBK-1 phosphorylates optienurin an autophagy adaptor protein that is also linked genetically to ALS. In this project the effect of ALS-causing mutations in TBK1 on ER-stress, autophagy and the association between optienurin and TBK-1 autophagy were examined. Using site directed mutagenesis kinase and substrate binding deficient ALS·mutant TBK·1 overexpresslng constructs were generated and the effect of expression of the mutants was examined. Preliminary results showed that expresslon of kinase deficient mutants decreased expression of CHOP, a pro-apoptotic transcription factor, normally induced during ER-stress. This suggests that the kinase activity ofTBK-1 is associated with CHOP independent of ALS. Furthermore. a slight but significantly decrease In LC3-11 levels was detected upon ALS·mutant TBK-1 overexpresslon, suggesting AlS-mutants inhibit autophagy. Additionally, ALS-mutant optineurin failed to recruit endogenous TBK-1 to autophagosomes In NSC-34 cells. This thesis therefore provides novel insight into cellular pathways perturbed in ALS· associated mutants of TBK·1.