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Morphometric Analyses of Retinal Sections
Published on: February 19, 2012
Neuroprotective effect of AIP on N-methyl-D-aspartate-induced cell death in retinal neurons
1Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, 500 S. Preston St., Louisville, KY 40202, USA.
Abstract:
Excessive activation of glutamate receptors mediates neuronal death, but the intracellular signaling pathways that mediate this type of neuronal death are only partly understood. Previously, we have demonstrated that calcium/calmodulin-dependent protein kinase II-alpha(B) (CaMKII-alpha(B)) containing a nuclear localizing signal but not CaMKII-alpha is altered in retinal neurons exposed to N-methyl-D-aspartate (NMDA). The present study describes a prospective function of CaMKII-alpha(B) in signal transduction leading to apoptosis. The terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labelling (TUNEL) method was used to detect fragmented DNA in fixed tissue sections of rat retina. The TUNEL assay confirmed that cell death occurs in the inner nuclear and ganglion cell layers following injection of 4 mM NMDA. A specific AIP (myristoylated autocamtide-2-related inhibitory peptide) with proven cell permeability inhibits CaMKII activity in vivo. Neuroprotection achieved by 500 microM AIP was complete when administered 2 h before and coincident with the NMDA application. Additionally, 100 microM of AIP protects only partially against the NMDA-induced excitotoxicity. The conformationally active fragment of caspase-3 (17 kDa), known to be involved in neuronal apoptosis was apparent within 30 min and at 2 h postinjection with NMDA. This activation was inhibited by 500 microM AIP when administered 2 h before and coincident with the NMDA application. The results suggest that CaMKII-alpha(B) isoform plays a role in excitotoxicity-induced neuronal apoptosis.
Insights
Calcium/calmodulin-dependent protein kinase II-alpha(B) (CaMKII-alpha(B)) plays a role in N-methyl-D-aspartate (NMDA)-induced neuronal death. Inhibiting CaMKII-alpha(B) with AIP provides neuroprotection against excitotoxicity.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Biology
Background:
- Excessive glutamate receptor activation leads to neuronal death, but intracellular pathways are not fully understood.
- Calcium/calmodulin-dependent protein kinase II-alpha(B) (CaMKII-alpha(B)) is altered in retinal neurons exposed to N-methyl-D-aspartate (NMDA).
Purpose of the Study:
- To investigate the role of CaMKII-alpha(B) in signal transduction pathways leading to NMDA-induced apoptosis.
- To evaluate the neuroprotective effects of a CaMKII inhibitor against NMDA excitotoxicity.
Main Methods:
- Terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labelling (TUNEL) assay to detect DNA fragmentation in rat retina.
- Administration of N-methyl-D-aspartate (NMDA) to induce excitotoxicity.
- Inhibition of CaMKII activity using myristoylated autocamtide-2-related inhibitory peptide (AIP).
- Western blot analysis to detect activated caspase-3.
Main Results:
- NMDA injection caused cell death in the inner nuclear and ganglion cell layers of the rat retina.
- Complete neuroprotection was achieved with 500 microM AIP administered before and during NMDA application.
- Partial protection was observed with 100 microM AIP.
- Activated caspase-3 was detected within 30 minutes to 2 hours post-NMDA injection, and this activation was inhibited by 500 microM AIP.
Conclusions:
- The CaMKII-alpha(B) isoform is implicated in excitotoxicity-induced neuronal apoptosis.
- Inhibition of CaMKII activity offers significant neuroprotection against NMDA-induced excitotoxicity.
- CaMKII-alpha(B) is a potential therapeutic target for conditions involving excitotoxic neuronal death.
