Time-course changes of hippocalcin expression in the mouse hippocampus following pilocarpine-induced status epilepticus

Hippocalcin participates in the maintenance of neuronal calcium homeostasis. In the present study, we examined the time-course changes of neuronal degeneration and hippocalcin protein level in the mouse hippocampus following pilocarpine-induced status epilepticus (SE). Marked neuronal degeneration was observed in the hippocampus after SE in a time-dependent manner, although neuronal degeneration differed according to the hippocampal subregions. Almost no hippocalcin immunoreactivity was detected in the pyramidal neurons of the cornu ammonis 1 (CA1) region from 6 h after SE. However, many pyramidal neurons in the CA2 region showed hippocalcin immunoreactivity until 24 h after SE. In the CA3 region, only a few hippocalcin immunoreactive cells were observed at 12 h after SE, and almost no hippocalcin immunoreactivity was observed in the pyramidal neurons from 24 h after SE. Hippocalcin immunoreactivity in the polymorphic cells of the dentate gyrus was markedly decreased from 6 h after SE. In addition, hippocalcin protein level in the hippocampus began to decrease from 6 h after SE, and was significantly decreased at 24 h and 48 h after pilocarpine-induced SE. These results indicate that marked reduction of hippocalcin level may be closely related to neuronal degeneration in the hippocampus following pilocarpine-induced SE.

Kainic Acid Treatment Increases Ca²⁺-mediated Neurotoxicity in the Mouse Hippocampus / 체질인류학회지

Kainic acid (KA)-induced neuronal cell death is associated with intracellular Ca²⁺ influx. However, it is unknown whether Lyn/Btk pathway is involved in the Ca²⁺-mediated neurotoxicity and neuronal death induced by KA. In the present study, we investigated the altered expression of Ca²⁺-controlled proteins in KA-treated hippocampus. Mice were sacrificed at 24 h after KA (20 mg/kg) systemic injection. We conducted Electroencephalographic (EEG) recording and examined hippocampal alterations by Western blotting and immunostaining in control mice or KA-treated mice. EEG tests showed that KA-treated mice increased seizure frequency and severity compared with control mice during KA-induced seizures. We found that KA decreases hippocalcin and calpain-mediated proteolysis in the hippocampus. In particular, the phosphorylation of Lyn and Btk was increased in KA-treated hippocampus compared to those of control mice. Our findings identify tyrosine kinases such as Lyn/Btk as a critical regulator of Ca²⁺-mediated neurotoxicity in KA-induced seizures.

Gingko biloba extract (EGb 761) attenuates ischemic brain injury-induced reduction in Ca2+ sensor protein hippocalcin / 한국실험동물학회지

Gingko biloba extract 761 (EGb 761) protects neuronal cells from ischemic brain injury via a number of neuroprotective mechanisms. Hippocalcin is a calcium sensor protein that regulates intracellular calcium concentrations and apoptotic cell death. We investigated whether EGb 761 regulates hippocalcin expression in cerebral ischemia. Male Sprague-Dawley rats were treated with vehicle or EGb 761 (100 mg/kg) prior to middle cerebral artery occlusion (MCAO), and cerebral cortex tissues were collected 24 h after MCAO. A proteomic approach demonstrated reduction in hippocalcin expression in vehicle-treated animals during MCAO, whereas EGb 761 treatment prevented injury-induced decreases in hippocalcin expression. RT-PCR and Western blot analyses indicated that EGb 761 attenuates injury-induced decrease in hippocalcin. These results suggest that the maintenance of hippocalcin during cerebral ischemia contributes to the neuroprotective role of EGb 761.

Expression of a cDNA for hippocalcin from rat brain

A hippocalcin cDNA from rat brain cDNA library was amplified by polymerase chain reation(PCR) and cloned using TA Cloning technique. For this PCR cloning, 29mer and 28mer oligonucleotide primers containing BamHl and EcoRl sites at the 5' end and 3' end, respectively were used. The nucleotide sequence of hippocalcin cDNA c1one was determined, and the complete amino acid sequence was deduced. Recombinant clone contained a cDNA insert of 610 base pairs with 582 nucleotides of open reading frame including the temination codon, 23 nucleotide of 5'-untranslated region, and 5nucleotides of 3'-nutran,slated region. The open reading frame encoded a polypepetid comprising 193 amino acids with molecular weight of 22kDa. The cDNA insert was subcloned into pVLI393 Baculovirus transfer vector. The recombinant hippocalcin was expressed in insect cell(Sf9 cell) using expression vector pVL1393. The hippocalcin expressed was purified as a single band on polyacrylamide gel electrophoresis following hydrophobic phenyl HPLC and TSKgel G3000SW gel filtration HPLC. Molecular size of rat brain hippocalcin protein expressed in this system was estimated to be 22kDa. Myristoylated hippocalcin migrated faster than nonmyristoyated form on SDS-polyacrylamide gel. Less than 10% of total hippocalcin expressed was myristoylated in this baculovirus expression system.