Seizure-induced impairment in neuronal ketogenesis: Role of zinc-α2-glycoprotein in mitochondria.

Changhong Tan,Xi Liu,Jin Jiang,Wuxue Peng,Hui Wang,Lijuan Mo,Wen Zhou,Yangmei Chen,Xin Wei,Lifen Chen

doi:10.1111/jcmm.15337

Abstract

Ketone bodies (KBs) were known to suppress seizure. Untraditionally, neurons were recently reported to utilize fatty acids and produce KBs, but the effect of seizure on neuronal ketogenesis has not been researched. Zinc‐α2‐glycoprotein (ZAG) was reported to suppress seizure via unclear mechanism. Interestingly, ZAG was involved in fatty acid β‐oxidation and thus may exert anti‐epileptic effect by promoting ketogenesis. However, this promotive effect of ZAG on neuronal ketogenesis has not been clarified. In this study, we performed immunoprecipitation and mass spectrometry to identify potential interaction partners with ZAG. The mechanisms of how ZAG translocated into mitochondria were determined by quantitative coimmunoprecipitation after treatment with apoptozole, a heat shock cognate protein 70 (HSC70) inhibitor. ZAG level was modulated by lentivirus in neurons or adeno‐associated virus in rat brains. Seizure models were induced by magnesium (Mg2+)‐free artificial cerebrospinal fluid in neurons or intraperitoneal injection of pentylenetetrazole kindling in rats. Ketogenesis was determined by cyclic thio‐NADH method in supernatant of neurons or brain homogenate. The effect of peroxisome proliferator–activated receptor γ (PPARγ) on ZAG expression was examined by Western blot, quantitative real‐time polymerase chain reaction (qRT‐PCR) and chromatin immunoprecipitation qRT‐PCR. We found that seizure induced ketogenesis deficiency via a ZAG‐dependent mechanism. ZAG entered mitochondria through a HSC70‐dependent mechanism, promoted ketogenesis by binding to four β‐subunits of long‐chain L‐3‐hydroxyacyl‐CoA dehydrogenase (HADHB) and alleviated ketogenesis impairment in a neuronal seizure model and pentylenetetrazole‐kindled epileptic rats. Additionally, PPARγ activation up‐regulated ZAG expression by binding to promoter region of AZGP1 gene and promoted ketogenesis through a ZAG‐dependent mechanism.

Highlights

Ketone bodies (KBs) are clearly effective in controlling refractory epilepsy both directly and indirectly.[1,2] whether KB insufficiency exists in epilepsy or seizure, and its mechanism remain unclear
We found that neuronal seizure model induced by magnesium (Mg2+)-free artificial cerebrospinal fluid (ACSF) and PTZ-kindled epileptic rats presented impaired ketogenesis, and this impairment could be alleviated by ZAG overexpression
PTZ-kindled rats or Mg2+-free ACSF-treated neurons exhibited impaired ketogenesis characterized by decreased AcAc synthesis and unchanged BHB levels

Summary

| INTRODUCTION

Ketone bodies (KBs) are clearly effective in controlling refractory epilepsy both directly and indirectly.[1,2] whether KB insufficiency exists in epilepsy or seizure, and its mechanism remain unclear. Peroxisome proliferator–activated receptor γ (PPARγ) activation increases ZAG levels in the circulation and adipocytes of patients.[20,21] PPARγ expression is decreased in the hippocampus of mice with kainic acid–induced seizures.[22] PPARγ activation suppresses spontaneous recurrent seizures in subjects with pilocarpine-induced status epilepticus[23] and exerts a synergistic anti-epileptic effect with the ketogenic diet on an in vivo seizure model.[24] Notably, as a transcription factor, PPARγ promotes fatty acid β-oxidation and ketogenesis.[25,26] neuronal ZAG decrease in epilepsy or seizure may be mediated by PPARγ, but whether PPARγ regulates fatty acid β-oxidation and ketogenesis in neurons via ZAG is unclear. We identified that ZAG was translocated into mitochondria via a HSC70-dependent mechanism and regulated HADHB activity and ketogenesis in neuronal mitochondria. We found that PPARγ up-regulated ZAG expression by binding to the promoter region of the AZGP1 gene

| MATERIALS AND METHODS

Findings

| DISCUSSION