Abstract
Introduction:
Cerebral ischemia can trigger cell death in the CA1 region of the hippocampus, an area important for memory. Protein kinase C epsilon (PKCε) activation prior to ischemia protects the CA1 from injury by modulating neurotransmission. The underlying mechanism needs further study.
Hypothesis:
PKCε-induced neuroprotection increases latency until anoxic depolarization (AD) through an activity-regulated cytoskeleton-associated protein (arc)-dependent mechanism of regulating AMPAR currents.
Results:
In vivo
activation of PKCε by the PKCε-activator ψε-Receptor of Activated C Kinase (ψεRACK) increased BDNF 5.99 +/- 0.11 fold and TrkB phosphorylation levels 2.94 +/- 0.32 fold (enhancers of arc protein levels) (n = 4, p < 0.005, t-test). Arc mRNA and protein was increased 143.97 +/-7.68 % and 1.91 +/- 0.22 fold (n = 9, p < 0.005, t-test). Inhibition of arc using antisense oligodeoxynucleotides (arc AS ODNs) in cultured slices blocked PKCε-mediated neuroprotection against lethal oxygen and glucose deprivation (OGD) from 35.91 +/- 5.97 to 74.93 +/- 4.24 % cell death (n = 6, p < 0.005, ANOVA, Bonferroni). ΨεRACK decreased AMPAR-mediated mEPSC amplitude to 12.75 +/- 0.35 pA from 14.80 +/- 0.39 pA (n = 20, p < 0.01, ANOVA, Bonferroni). This effect was arc-dependent. Additionally, ψεRACK treatment increased latency until AD from 29.27 +/- 3.6 min 50.77 +/- 5.08 min (n = 13, p < 0.01, ANOVA, Bonferroni). This increase was arc-dependent, and required AMPAR internalization. Inhibiting internalization reduced AD latency from 54.5 +/- 8.40 min to 22.3 + 5.17 min (n = 6, p <0.005, t-test).
Conclusion:
Arc expression is necessary for neuroprotection afforded by PKCε-activation, modulates excitatory synaptic strength, and increases latency until AD.
Methods:
Western blot:
Proteins (40 μg) were separated on a 12% SDS-PAGE gel.
Immunofluorescence
: 30 μm sections were incubated with 1:500 NeuN and 1:50 arc in PBS with 0.8% triton.
Cultured slices preparation
: sections from P9-11 rats were plated on inserts and cultured for 14 days.
PI measurements of cell death
: Slices were incubated in medium with 2 μg/mL PI.
mEPSC measurements
: Whole-cell voltage clamp was performed.
AD:
OGD, perfusate was switched to glucose free media, and bubbled with a 95% N
2
and 5% CO
2
gas.