-
Treatment with hydrogen peroxide stimulate cleavage of p35
to p25 in primary neurons. Other insults such as treatment with the excitatory
amino-acid glutamate also caused the production of p25 in cortical neurons
at high concentrations of glutamate. An increase in intracellular calcium
levels, caused by the calcium ionophore ionomycin, stimulated efficient
conversion of p35 to p25. These results indicate that neurotoxicity induces
cleavage of p35 to p25 and suggest a role for calcium in this process.
|
-
To identify the protease that cleaves p35 to p25, they used
the proteolytic cleavage event. In fresh mouse brain lysates,1 mM Ca2+
efficiently stimulates the cleavage of p35. The cleavage product is likely
to be p25, as it has a relative molecular mass of 25K. The p25 was specifically
recognized by the p35 carboxy-terminal- specific antibody, but not by the
p35 amino-terminal-specific antibody.
|
-
To identify the protease activated by calcium, protease inhibitors
with different specificities for their effectiveness in inhibiting the
calcium-stimulated p35 conversion. Calpeptin and calpain inhibitor II,
which inhibit the calcium- dependent cysteine protease calpain, completely
inhibited p35 cleavage, whereas the general cysteine protease inhibitor
leupeptin partially inhibited p35 cleavage. The lack of effect of the cdk5
inhibitor roscovitine indicates that cdk5 activity may not be necessary
for cleavage to occur.
|
-
To investigate further whether calpain cleaves p35 in brain
lysates, fractionats of mouse brain lysates were examined whether the p35
cleavage activity co-segregated with calpain activity. Incubation of the
glycerol gradient fractions with either purified p35 showed that both p35
cleavage activity and calpain activity completely co-segregated in the
same fractions. Depletion of m-calpain from brain lysates using an m-calpain-specific
antibody significantly reduced the Ca2+-induced
cleavage of p35. further indicating that conversion of p35 to p25 may lie
downstream of calpain activation.
|
-
Incubation of purified m-calpain and µ-calpain with
purified p35, cleaved p35 to a 25K fragment.
|
-
In primary cortical neurons, H2O2
or
ionomycin did not stimulate conversion of p35 to p25. Similarly, removing
calcium with either EGTA or BAPTA-AM also prevented conversion of p35.
These results indicate that calcium is necessary for conversion of p35.
The addition of calpeptin, which inhibits calpain activity in neurons also
inhibited the cleavage of endogenous p35 to p25 caused by H2O2.
Similarly, conversion of p35 to p25 induced by ionomycin was completely
reversed by increasing concentrations of calpeptin. Together, these experiments
indicate that activation of calpain by neurotoxic processes through calcium
influx is both necessary and sufficient for conversion of p35 to p25 in
cortical neurons.
|
-
p25 is found to accumulate in brains of Alzheimer's disease
patients but not in normal brains.The Aß peptides are primary constituents
of the amyloid plaques found in the brains of patients with Alzheimer's
disease. They have been shown to aggregate and cause neuronal deaths. Treatment
of primary cortical neurons with 20 µM Aß (1-42) peptide resulted
in conversion of p35 to p25. These results indicate that, in Alzheimer's
disease, the abundant amyloid plaques in the brain can cause calpain activation
and conversion of p35 to p25.
|
