Skip to Content
Merck
  • Amyotrophic lateral sclerosis patient iPSC-derived astrocytes impair autophagy via non-cell autonomous mechanisms.

Amyotrophic lateral sclerosis patient iPSC-derived astrocytes impair autophagy via non-cell autonomous mechanisms.

Molecular brain (2017-06-15)
Martin Madill, Katya McDonagh, Jun Ma, Alice Vajda, Paul McLoughlin, Timothy O'Brien, Orla Hardiman, Sanbing Shen
ABSTRACT

Amyotrophic lateral sclerosis, a devastating neurodegenerative disease, is characterized by the progressive loss of motor neurons and the accumulation of misfolded protein aggregates. The latter suggests impaired proteostasis may be a key factor in disease pathogenesis, though the underlying mechanisms leading to the accumulation of aggregates is unclear. Further, recent studies have indicated that motor neuron cell death may be mediated by astrocytes. Herein we demonstrate that ALS patient iPSC-derived astrocytes modulate the autophagy pathway in a non-cell autonomous manner. We demonstrate cells treated with patient derived astrocyte conditioned medium demonstrate decreased expression of LC3-II, a key adapter protein required for the selective degradation of p62 and ubiquitinated proteins targeted for degradation. We observed an increased accumulation of p62 in cells treated with patient conditioned medium, with a concomitant increase in the expression of SOD1, a protein associated with the development of ALS. Activation of autophagic mechanisms with Rapamycin reduces the accumulation of p62 puncta in cells treated with patient conditioned medium. These data suggest that patient astrocytes may modulate motor neuron cell death by impairing autophagic mechanisms, and the autophagy pathway may be a useful target in the development of novel therapeutics.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, ≥96% (agarose gel electrophoresis)
Sigma-Aldrich
TWEEN® 20, for molecular biology, viscous liquid
Sigma-Aldrich
Anti-SOD1 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
Sigma-Aldrich
Human STEMCCA Constitutive Polycistronic (OKSM) Lentivirus Reprogramming Kit, The Human STEMCCA Constitutive Polycistronic Lentivirus Kit contains high titer polycistronic lentivirus & Polybrene transfection reagent that have been validated for the generation of human iPS cells from human foreskin fibroblasts.
Sigma-Aldrich
Triton X-100, for molecular biology
Sigma-Aldrich
Anti-β-Tubulin III antibody produced in rabbit, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
Anti-β-Actin−Peroxidase antibody, Mouse monoclonal, clone AC-15, purified from hybridoma cell culture
Sigma-Aldrich
RIPA Buffer