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Calpain-2 as a Treatment Target in Prenatal Stress-induced Epileptic Spasms in Infant Rats.

Experimental neurobiology (2019-09-09)
Hyeok Hee Kwon, Chiranjivi Neupane, Juhee Shin, Do Hyeong Gwon, Yuhua Yin, Nara Shin, Hyo Jung Shin, Jinpyo Hong, Jin Bong Park, YoonYoung Yi, Dong Woon Kim, Joon Won Kang
ABSTRACT

Stress can induce a serious epileptic encephalopathy that occurs during early infancy. Recent studies have revealed that prenatal stress exposure is a risk factor for the development of infantile spasms. Our previous work demonstrates that prenatal stress with betamethasone-induced alterations to the expression of the K+/Cl- co-transporter (KCC2) in gamma-aminobutyric acid (GABA) interneurons lowers the seizure threshold in exposed animals. Here, we further investigated the mechanisms involved in this KCC2 dysfunction and explored possible treatment options. We stressed Sprague-Dawley rats prenatally and further treated dams with betamethasone on gestational day 15, which increases seizure susceptibility and NMDA (N-Methyl-D-aspartate)-triggered spasms on postnatal day 15. In this animal model, first, we evaluated baseline calpain activity. Second, we examined the cleavage and dephosphorylation of KCC2. Finally, we checked the effect of a calpain inhibitor on seizure occurrence. The phosphorylated-N-methyl-Daspartate Receptor 2B (NR2B):non-phosphorylated NR2B ratio was found to be higher in the cortex of the prenatally stressed betamethasone model. We further found that the betamethasone model exhibited increased phosphorylation of calpain-2 and decreased phosphorylation of KCC2 and Glutamic acid decarboxylase 67 (GAD67). After using a calpain inhibitor in prenatal-stress rats, the seizure frequency decreased, while latency increased. GABAergic depolarization was further normalized in prenatal-stress rats treated with the calpain inhibitor. Our study suggests that calpain-dependent cleavage and dephosphorylation of KCC2 decreased the seizure threshold of rats under prenatal stress. Calpain-2 functions might, thus, be targeted in the future for the development of treatments for epileptic spasms.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Monoclonal Anti-β-Actin antibody produced in mouse, clone AC-74, ascites fluid
Sigma-Aldrich
Anti-GAD67 Antibody, clone 1G10.2, clone 1G10.2, Chemicon®, from mouse
Sigma-Aldrich
N-Methyl-D-aspartic acid, ≥98% (TLC), solid
Sigma-Aldrich
Anti-K+/Cl- Cotransporter (KCC2) Antibody, Upstate®, from rabbit
Sigma-Aldrich
MDL 28170, ≥90% (TLC)
Sigma-Aldrich
Anti-NR2B Antibody, Upstate®, from rabbit
Sigma-Aldrich
Anti-NMDAR2A Antibody, Chemicon®, from rabbit
Supelco
Vials, screw top with phenolic open-top cap, pre-assembled, volume 40 mL, clear glass vial, O.D. × H 29 mm × 82 mm, tan PTFE/silicone septum