Rationale: Reactive astrogliosis is the most common pathological alteration in temporal lobe epilepsy patients and epilepsy animal models. overexpression of adenosine kinase (ADK, which enables astrocytes to function as a metabolic reservoir, effectively clearing adenosine from the brain) facilitated by reactive astrogliosis results in enhanced adenosine clearance by astrocytes, ultimately contributing to the development of epilepsy.
Calponin-3 is an actin-binding protein that regulates the activity of ATPase. Increased levels were found in patients with temporal lobe epilepsy and in rats with pilocarpine induced epilepsy. However, its role in the development of epilepsy remains unclear.
Methods: The protocols were approved by the ethical committee of First Affiliated Hospital of Kunming Medical University (2020L62) and met the requirements of the Chinese Animal Ethics Committee (kmmu20220926). Serum and brain tissues of epileptic patients, Kainic acid (KA) and pentattrazene (PTZ) induced epilepsy mouse models were used to detect the expression level and cellular localization of calponin-3. Calponin-3 in the hippocampus of C57BL/6 mice was up-regulated, and calponin-3 in the hippocampus of KA-induced epilepsy mice was down-regulated. The effect of calponin-3 expression change on the occurrence and development of epilepsy was observed in mice with specific knockout of calponin-3 in astrocytes. By conducting 24-hour video monitoring and electroencephalogram (EEG) in mice to observe epileptic activities. Ultimately, both in vivo and in vitro, immunofluorescence and Western blot were used to explore the relevant molecular mechanisms.
Results: In both epileptic patients and animal models, we observed that calponin-3 significantly increased in the hippocampal tissues and serum, predominantly localized in astrocytes. Behavioral and electrophysiological studies revealed that the upregulation of calponin-3 increased the epilepsy susceptibility in mice, while downregulation of calponin-3 in KA induced epileptic mice had a protective effect on spontaneous recurrent seizures (SRS). In transgenic mice that specifically knocked out calponin-3 in astrocytes (GFAP-CNN3-KO), it was further confirmed that targeted knocking out calponin-3 in astrocytes had a protective effect on epileptogenesis. More importantly, upregulation of calponin-3 leads to
reactive astrogliosis, with significant increases in ADK and equilibrative nucleoside transporters1(ENT1) expression. However, GFAP-CNN3-KO mice inhibited reactive astrocyte hyperplasia and reduced ADK and ENT1 expression. Moreover, knockdown of calponin-3 in human astrocytes by siRNA inhibited their proliferative ability and decreased the expression of ADK and ENT1.
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Conclusions: Our findings uncover that increased calponin-3 in astrocytes promotes epileptogenesis via accelerating reactive astrogliosis and aberrant adenosine metabolism, making it a potential target for epilepsy treatment.
Funding: This work was supported by the grant of 82160261 from the National Natural Science Foundation of China.