Necrotic neuronal death is recently known to be mediated by the
calpain-
cathepsin cascade from simpler organisms to primates. The main event of this cascade is
calpain-mediated lysosomal
rupture and the resultant release of lysosomal
cathepsins into the cytoplasm. However, the in-vivo substrate of
calpain for inducing lysosomal destabilization still remains completely unknown. The recent proteomics data using the post-ischemic hippocampal CA1 tissues and
glaucoma-suffered retina from the primates suggested that
heat shock protein (
Hsp) 70.1 might be the in-vivo substrate of activated
mu-calpain at the lysosomal membrane of neurons.
Hsp70.1 is known to stabilize lysosomal membrane by recycling damaged
proteins and protect cells from oxidative stresses. Here, we studied the molecular interaction between activated
mu-calpain and the lysosomal
Hsp70.1 in the monkey hippocampal CA1 neurons after the
ischemia-reperfusion insult. Immunofluorescence histochemistry showed a colocalization of the activated
mu-calpain and upregulated
Hsp70.1 at the lysosomal membrane of the post-ischemic CA1 neurons. In-vitro cleavage assay of hippocampal
Hsp70.1 by Western blotting demonstrated that
Hsp70.1 in the CA1 tissue is an in-vivo substrate of activated
mu-calpain, and that carbonylated
Hsp70.1 in the CA1 tissue by artificial oxidative stressors such as hydroxynonenal (HNE) or
hydrogen peroxide is much more vulnerable to the
calpain cleavage. These data altogether suggested that
Hsp70.1 can become a target of the carbonylation by HNE, and
Hsp70.1 is a modulator of
calpain-mediated lysosomal
rupture/permeabilization after the
ischemia-reperfusion injury.