Defects in optineurin- and myosin VI-mediated cellular trafficking in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder primarily affecting motor neurons. Mutations in optineurin cause a small proportion of familial ALS cases, and wild-type (WT) optineurin is misfolded and forms inclusions in sporadic ALS patient motor neurons. However, it is unknown how optineurin mutation or misfolding leads to ALS. Optineurin acts an adaptor protein connecting the molecular motor myosin VI to secretory vesicles and autophagosomes. Here, we demonstrate that ALS-linked mutations p.Q398X and p.E478G disrupt the association of optineurin with myosin VI, leading to an abnormal diffuse cytoplasmic distribution, inhibition of secretory protein traf fi cking, endoplasmic reticulum (ER) stress and Golgi fragmentation in motor neuron-like NSC-34 cells. We also provide further insight into the role of optineurin as an autophagy receptor. WT optineurin associated with lysosomes and promoted autophagosome fusion to lysosomes in neuronal cells, implying that it mediates traf fi cking of lysosomes during autophagy in association with myosin VI. However, either expression of ALS mutant optineurin or small interfering RNA-mediated knockdown of endogenous optineurin blocked lysosome fusion to autophagosomes, resulting in autophagosome accumulation. Together these results indicate that ALS-linked mutations in optineurin disrupt myosin VI-mediated intracellular traf fi cking processes. In addition, in control human patient tissues, optineurin displayed its normal vesicular localization, but in sporadic ALS patient tissues, vesicles were present in a signi fi cantly decreased proportion of motor neurons. Optineurin binding to myosin VI was also decreased in tissue lysates from sporadic ALS spinal cords. This study therefore links several previously described pathological mechanisms in ALS, including defects in autophagy, fragmentation of the Golgi and induction of ER stress, to disruption of optineurin function. These fi ndings also indicate that optineurin – myosin VI dysfunction is a common feature of both sporadic and familial ALS. ALS we sequenced DNA extracted from patient tissues for common TDP-43, FUS and SOD1 mutations and the C9ORF72 repeat expansion. We found a known sporadic TDP-43 mutation, p.G294A, c.880G>C mutation (rs80356721) in patient P3 (46). We did not detect mutations in C9ORF72 , TDP-43, FUS or SOD1 in any of the other patients. These patients are therefore most likely to be sporadic cases, given the lack of common disease-causative mutations and family these demonstrate that the association between myosin and optineurin disrupted in sporadic of optineurin as an adaptor for myosin VI is inhibited in both sporadic and familial