Over-expression of an inactive mutant cathepsin D increases endogenous alpha-synuclein and cathepsin B activity in SH-SY5Y cells

J Neurochem. 2014 Mar;128(6):950-61. doi: 10.1111/jnc.12497. Epub 2013 Nov 13.

Abstract

Parkinson's disease is a neurodegenerative movement disorder. The histopathology of Parkinson's disease comprises proteinaceous inclusions known as Lewy bodies, which contains aggregated α-synuclein. Cathepsin D (CD) is a lysosomal protease previously demonstrated to cleave α-synuclein and decrease its toxicity in both cell lines and mouse brains in vivo. Here, we show that pharmacological inhibition of CD, or introduction of catalytically inactive mutant CD, resulted in decreased CD activity and increased cathepsin B activity, suggesting a possible compensatory response to inhibition of CD activity. However, this increased cathepsin B activity was not sufficient to maintain α-synuclein degradation, as evidenced by the accumulation of endogenous α-synuclein. Interestingly, the levels of LC3, LAMP1, and LAMP2, proteins involved in autophagy-lysosomal activities, as well as total lysosomal mass as assessed by LysoTracker flow cytometry, were unchanged. Neither autophagic flux nor proteasomal activities differs between cells over-expressing wild-type versus mutant CD. These observations point to a critical regulatory role for that endogenous CD activity in dopaminergic cells in α-synuclein homeostasis which cannot be compensated for by increased Cathepsin B. These data support the potential need to enhance CD function in order to attenuate α-synuclein accumulation as a therapeutic strategy against development of synucleinopathy.

Keywords: autophagy; cathepsin D; lysosome; α-synuclein.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Autophagy / drug effects
  • Autophagy / physiology
  • Caspases / metabolism
  • Cathepsin B / metabolism*
  • Cathepsin D / genetics*
  • Cathepsin D / metabolism
  • Cell Line, Tumor
  • Gene Expression / physiology
  • Humans
  • Lentivirus / genetics
  • Lysosomes / metabolism
  • Neuroblastoma
  • Neurodegenerative Diseases / metabolism*
  • Neurons / cytology
  • Neurons / drug effects
  • Neurons / metabolism*
  • Pepstatins / pharmacology
  • Protease Inhibitors / pharmacology
  • alpha-Synuclein / metabolism*

Substances

  • Pepstatins
  • Protease Inhibitors
  • SNCA protein, human
  • alpha-Synuclein
  • Caspases
  • CTSB protein, human
  • Cathepsin B
  • CTSD protein, human
  • Cathepsin D
  • pepstatin