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Keywords:

  • apoptosis;
  • cerebellar granule cells;
  • rat;
  • serum and potassium deprivation

Abstract

Serum and potassium deprivation-induced neuronal death on the primary culture of rat cerebellar granule neurons is being widely used as an in vitro model of neurodegeneration and neuronal apoptosis. In our experiments, serum and potassium deprivation for 12 h induced neuronal death in ≈ 20% of cerebellar granule neurons as demonstrated by Trypan Blue assay. Neuronal death was accompanied by a transient increase in the intralysosomal cathepsin L activity, which preceded neuronal death. During this time, the lysosomal membrane integrity remained preserved and no leakage of cathepsin L into the cytosol was seen. Ultrastructural analysis revealed the appearance of multiple vacuoles and autophagosomes in the cytoplasmatic compartment of serum- and potassium-deprived granule neurons. Addition of selective cathepsin L inhibitors or of the autophagy inhibitor 3-methyladenine provided partial protection against serum and potassium deprivation-induced death. Our data also show that combining cathepsin L inhibitors and caspase-3 inhibitors leads to a synergistic neuroprotective effect against serum and potassium deprivation. The results of the current study suggest that activation of the autophagosomal–lysosomal compartment plays an important role in neuronal death induced by serum and potassium deprivation in cultured cerebellar granule cells.