Journal: Journal of Biological Chemistry

Article Title: Distinct Mechanistic Roles of Calpain and Caspase Activation in Neurodegeneration as Revealed in Mice Overexpressing Their Specific Inhibitors

doi: 10.1074/jbc.m500939200

Figure Lengend Snippet: FIG. 5. Dual impacts of calpain activation on biochemical properties of tau. A, Western blot analyses for the 136-kDa fragment, MAP2a/b (AP20), total tau (T49), phosphorylated tau (AT8), and Cdk5 activator p25/p35 using protein samples extracted from ipsilateral (i) and contralateral (c) hippocampal formations of nTg and KA-treated hCAST Tg mice 24 h after treatment with PBS or KA. B and C, ratio of AT8 to T49 (B) and ratio of p25 to p35 (C) in the ipsilateral (closed columns) and contralateral (open columns) hippocampi based on densitometric quantification of immunoblot signals (n 3 in each group). The ratios indicate the relative incidence of phosphorylation and limited proteolysis of tau and p35 proteins per molecule at this time point, respectively. The quantification was performed within a linear range. Vertical bars represent S.E. Data for tau phosphorylation are normalized against the mean value given by the PBS-treated mice. D and E, AT8 immunofluo- rescence staining in the CA1 neurons of nTg mice 24 h after PBS (D) or KA (E) treatment. KA-induced calpain activation not only reduced the total amount of tau but also promoted phosphorylation of remaining tau. Scale bar, 50 m (D and E). *, p 0.05; **, p 0.01 versus PBS-treated nTg mice by ANOVA/LSD.

Article Snippet: Other antibodies used in the present study are as follows: mouse monoclonal antibodies to MAP2 (AP20; 1:10 dilution; Roche Diagnostics), -tubulin (DM1A; 1:1,000 dilution; Sigma), synaptophysin (1:50 dilution; Progen Biotechnik), and NeuN (1:2,000 dilution; Chemicon); and rabbit polyclonal antibodies to SV2A (1:100 dilution; Calbiochem-Novabiochem), GluR1 (1:1,000 dilution; Chemicon), mGluR2/3 (1:1,000 dilution; Chemicon), and Cdk5 activator p25/p35 (C19; 1:1,000 dilution; Santa Cruz Biotechnology).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Staining

Journal: Journal of Biological Chemistry

Article Title: Distinct Mechanistic Roles of Calpain and Caspase Activation in Neurodegeneration as Revealed in Mice Overexpressing Their Specific Inhibitors

doi: 10.1074/jbc.m500939200

Figure Lengend Snippet: FIG. 7. A proposed scheme for calpain-mediated neurodegenerative processes focusing on cytoskeletal disorganization. Excitotoxic insults induce calpain and caspase activation, whereas CAST and VIC can suppress activation of these proteases in a specific manner. Reduction of CAST may arise as a consequence of the calpain activation, driving a vicious cycle that further deteriorates the balance of the calpain-calpastatin system. Calpain is activated primarily in the somato-dendritic compartments of neurons and consequently degrades dendritic MAP2 and cytoplasmic MT-unbound tau in a preferential manner. Tau in the neuronal soma also undergoes hyper-phosphorylation catalyzed by Cdk5, which can be activated by a Cdk5 activator fragment (p25) converted from its intact form (p35) as a consequence of calpain-mediated proteolysis. Dendritic cytoskeletons become deranged as well in these locations by the calpain activation, giving rise to pronounced degeneration of dendrites in an acute phase. In contrast, calpain activation is smaller and slower in the axon, and consequently axonal cytoskeletons degenerate more slowly than those in the dendrites, resulting from degradation and hyper-phosphorylation of tau in the soma and subsequent depletion of axonal tau supplied from the soma. Finally, cytoskeletal disorganization in the neuritic processes substantially deteriorates neuronal functions, leading to neuronal death in a subacute phase.

Article Snippet: Other antibodies used in the present study are as follows: mouse monoclonal antibodies to MAP2 (AP20; 1:10 dilution; Roche Diagnostics), -tubulin (DM1A; 1:1,000 dilution; Sigma), synaptophysin (1:50 dilution; Progen Biotechnik), and NeuN (1:2,000 dilution; Chemicon); and rabbit polyclonal antibodies to SV2A (1:100 dilution; Calbiochem-Novabiochem), GluR1 (1:1,000 dilution; Chemicon), mGluR2/3 (1:1,000 dilution; Chemicon), and Cdk5 activator p25/p35 (C19; 1:1,000 dilution; Santa Cruz Biotechnology).

Techniques: Activation Assay, Phospho-proteomics