Skip to Content
MilliporeSigma
  • Defects in optineurin- and myosin VI-mediated cellular trafficking in amyotrophic lateral sclerosis.

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

Human molecular genetics (2015-04-11)
Vinod Sundaramoorthy, Adam K Walker, Vanessa Tan, Jennifer A Fifita, Emily P Mccann, Kelly L Williams, Ian P Blair, Gilles J Guillemin, Manal A Farg, Julie D Atkin
ABSTRACT

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 trafficking, 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 trafficking 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 trafficking 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 significantly 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 findings also indicate that optineurin-myosin VI dysfunction is a common feature of both sporadic and familial ALS.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Urea, SAJ first grade, ≥98.0%
Sigma-Aldrich
Urea, JIS special grade, ≥99.0%
Sigma-Aldrich
Urea, ≥99.0%
Sigma-Aldrich
MISSION® esiRNA, targeting human OPTN
Sigma-Aldrich
Urea, BioUltra, for molecular biology, 99% (T)
Sigma-Aldrich
Urea, BioXtra, pH 7.5-9.5 (20 °C, 5 M in H2O)
Sigma-Aldrich
Urea, puriss., meets analytical specification of Ph. Eur., BP, USP, 99.0-100.5%, 99.0-101.0% (calc. on dry substance)
Supelco
Urea, 8 M (after reconstitution with 16 mL high purity water)
Sigma-Aldrich
Urea, powder, BioReagent, for molecular biology, suitable for cell culture
Sigma-Aldrich
Urea, ReagentPlus®, ≥99.5%, pellets
Sigma-Aldrich
Urea, ACS reagent, 99.0-100.5%
Sigma-Aldrich
Urea, suitable for electrophoresis
Sigma-Aldrich
Urea, puriss. p.a., ACS reagent, reag. Ph. Eur., ≥99%
Sigma-Aldrich
Urea, meets USP testing specifications
Sigma-Aldrich
Sodium dodecyl sulfate solution, BioUltra, for molecular biology, 10% in H2O
Sigma-Aldrich
Sodium dodecyl sulfate solution, BioUltra, for molecular biology, 20% in H2O
Sigma-Aldrich
Urea-12C, 99.9 atom % 12C
Sigma-Aldrich
Sodium chloride-35Cl, 99 atom % 35Cl
Sigma-Aldrich
Sodium chloride, random crystals, optical grade, 99.9% trace metals basis
Sigma-Aldrich
Sodium dodecyl sulfate, BioUltra, for molecular biology, ≥99.0% (GC)
Sigma-Aldrich
Sodium dodecyl sulfate, tested according to NF, mixture of sodium alkyl sulfates consisting mainly of sodium dodecyl sulfate
Sigma-Aldrich
Bicinchoninic acid disodium salt hydrate, ≥98% (HPLC)
Sigma-Aldrich
Urea solution, 40 % (w/v) in H2O
Sigma-Aldrich
Sodium chloride, JIS special grade, ≥99.5%
Supelco
Sodium dodecyl sulfate, dust-free pellets, suitable for electrophoresis, for molecular biology, ≥99.0% (GC)
SAFC
Sodium chloride solution, 5 M
Sigma-Aldrich
Sodium dodecyl sulfate, ≥99.0%
Sigma-Aldrich
Sodium chloride solution, 1 M
Sigma-Aldrich
Sodium chloride solution, 0.1 M
Sigma-Aldrich
Sodium dodecyl sulfate, SAJ special grade, ≥97.0%