Pretreatment with Apoaequorin Protects Hippocampal CA1 Neurons from Oxygen-Glucose Deprivation

Abstract

Ischemic stroke affects ∼795,000 people each year in the U.S., which results in an estimated annual cost of $73.7 billion. Calcium is pivotal in a variety of neuronal signaling cascades, however, during ischemia, excess calcium influx can trigger excitotoxic cell death. Calcium binding proteins help neurons regulate/buffer intracellular calcium levels during ischemia. Aequorin is a calcium binding protein isolated from the jellyfish Aequorea victoria, and has been used for years as a calcium indicator, but little is known about its neuroprotective properties. The present study used an in vitro rat brain slice preparation to test the hypothesis that an intra-hippocampal infusion of apoaequorin (the calcium binding component of aequorin) protects neurons from ischemic cell death. Bilaterally cannulated rats received an apoaequorin infusion in one hemisphere and vehicle control in the other. Hippocampal slices were then prepared and subjected to 5 minutes of oxygen-glucose deprivation (OGD), and cell death was assayed by trypan blue exclusion. Apoaequorin dose-dependently protected neurons from OGD – doses of 1% and 4% (but not 0.4%) significantly decreased the number of trypan blue-labeled neurons. This effect was also time dependent, lasting up to 48 hours. This time dependent effect was paralleled by changes in cytokine and chemokine expression, indicating that apoaequorin may protect neurons via a neuroimmunomodulatory mechanism. These data support the hypothesis that pretreatment with apoaequorin protects neurons against ischemic cell death, and may be an effective neurotherapeutic.

Apoaequorin differentially modulates fear memory in adult and aged rats

“…Because excitotoxicity and cell death following ischemia can be enhanced by excessive Ca 2+ influx (Choi, 1985; Simon, Swan, Griffiths, & Meldrum, 1984), application of the AQ protein likely confers neuroprotection via its calcium‐binding capabilities. However, Western blots indicate the AQ protein is present in hippocampal tissue 1 hr after infusion, but it is greatly diminished or absent by 1 or 2 days later (Detert et al., 2013). This suggests the neuroprotection conferred by the AQ protein in these experiments may be due its influence on other signaling molecules involved in neuroprotection (e.g., cytokines and/or chemokines; Detert et al., 2013).…”

“…Although these studies suggest that preventing Ca 2+ entry into the cytosol improves cognitive and physiological function during aging, it remains unclear if sequestration of free Ca 2+ ions already located in the intracellular space would also effectively restore cognitive function in aged animals. While synaptic plasticity in aged hippocampal CA1 neurons is enhanced following application of the Ca 2+ chelators BAPTA-AM and EGTA-AM (Ouanounou, Zhang, Charlton, & Carlen, 1999), Previous research from our laboratory indicates that cell death following an in vitro ischemic insult is reduced when AQ is infused into the dorsal hippocampus either 24 or 48 hr prior to the insult (Detert et al, 2013). Because excitotoxicity and cell death following ischemia can be enhanced by excessive Ca 2+ influx (Choi, 1985;Simon, Swan, Griffiths, & Meldrum, 1984), application of the AQ protein likely confers neuroprotection via its calcium-binding capabilities.…”

“…Thirty minutes following the cue test rats were placed back into the training chamber for 10 min to assess context fear memory. Abbreviations: vehicle (Veh); apoaequorin (AQ) stereotaxic coordinates (AP −3.5 mm, L ± 2.6 mm, V −3.0 mm) relative to Bregma (see Figure 1; Detert et al, 2013). Stainless steel screws and acrylic cement were used to secure the cannula to the skull.…”

“…The AQ protein and coelenterazine are components of the photoprotein aequorin, which has a similar protein structure to other EF‐hand calcium‐binding proteins (Inouye et al., 1985), and has traditionally been used as a Ca 2+ indicator (for review, see Blinks, 1978; Shimomura, Kishi, & Inouye, 1993). Our laboratory has previously demonstrated that AQ is neuroprotective when it is bilaterally infused into the dorsal hippocampus either 24 or 48 hr prior to an in vitro ischemic insult (Detert, Adams, Lescher, Lyons, & Moyer, 2013), which normally produces cell death via calcium‐mediated toxicity (Kristian & Siesjo, 1998). Given that age‐related cognitive decline is associated with Ca 2+ dysregulation as well as reduced expression of endogenous calcium‐binding proteins, the goal of the present study was to determine whether administration of the AQ protein would mitigate learning deficits in aged animals.…”

“…Our laboratory has previously demonstrated that AQ is neuroprotective when it is bilaterally infused into the dorsal hippocampus either 24 or 48 hr prior to an in vitro ischemic insult (Detert, Adams, Lescher, Lyons, & Moyer, 2013), which normally produces cell death via calcium-mediated toxicity (Kristian & Siesjo, 1998).…”