Neuroprotective Effects And Mechanisms Of Hippocampal Aminoprocalcitonin In Refractory Status Epilepticus

【Background】Refractory status epilepticus（RSE）is a neurological emergency evolved from status epilepticus（SE）.Seizures in RSE are self-sustaining and cannot be terminated with benzodiazepines.RSE not only causes a mortality as high as 35%,but also induces severe neuronal death in the brain which results in high incidence of neurological deficits in survivors.Only 21%RSE survivors could recover their neurological functions to the baseline level.However,there is no available neuroprotective therapy that is effective in RSE.The survival of neurons relies on sufficient energy supply,and sustaining seizure activity in RSE represents a metabolic threat that pushes brain energy consumption and supply to its limits.It has been reported that mitochondrial dysfunction and resultant ATP depletion and energy failure determine the neuronal dying process after RSE.Thus,identifying and targeting the key molecule that modulates neuronal energy homeostasis in RSE bears the potentials to provide novel neuroprotective strategy for RSE.Aminoprocalcitonin（NPCT）is the peptide cleaved from the amino terminal of procalcitonin（PCT）,with the amino acid sequence of NPCT highly conserved among all vertebrates.Although the parent peptide PCT is a serum biomarker of infection routinely checked in clinical practices,knowledges about NPCT remain enigmatic.Recently,we found that hippocampal NPCT was overexpressed in RSE and had potent regulations on the neuronal oxidative phosphorylation（OXPHOS）.This inspires us to hypothesize that NPCT might be involved in neuronal death after RSE via regulating neuronal energy status.【Objectives】The aims of the present study are as follows.1.To investigate the distribution and sketch a quantitative atlas of NPCT throughout the rat brain,and to elucidate the expression details of NPCT in the hippocampus.2.To establish the animal model of diazepam（DZP）-resistant RSE and to investigate the temporal and spatial expression changes of hippocampal NPCT in RSE.3.To explore the biological functions and cellular effects of NPCT in primary hippocampal neurons.4.To establish the in vitro 0-Mg²⁺seizure model and to elaborate on the neuroprotective effects of NPCT against neuronal death in this model.5.To further elucidate the neuroprotective effects and related mechanisms of hippocampal NPCT against neuronal death in RSE models in vivo.【Methods】1.Immunohistochemistry,immunofluorescence staining,and immunoelectron microscopy were used to investigate the distribution and expression characteristics of NPCT in the rat brain.Immunohistochemistry score was used to quantitatively describe the abundance of NPCT in representative brain regions and nuclei.Pre-absorption test was carried out to examine the specificity of the immunohistochemistry experiments.2.Rat lithium-pilocarpine SE model was established.The association between resistance to DZP and seizure duration was determined by behavior scale,electroencephalogram（EEG）monitoring,and survival analyses,to establish DZP-resistant RSE model.Real-time quantitative PCR（q PCR）,western blot,immunofluorescence staining,and immunoelectron microscopy were performed to characterize the spatial and temporal changes of hippocampal NPCT in RSE.3.Primary hippocampal neurons were stimulated with exogenous NPCT.The neurotrophic effects of NPCT were investigated with spine visualization based on plasmid transfection,immunofluorescence staining,western blot,and sholl analysis.The effects of NPCT on neuronal mitochondrial OXPHOS was investigated using RNA-sequencing,Seahorse XFe,and a series of mitochondrial function assays.4.A polyclonal NPCT immunoneutralization antibody was developed to antagonize NPCT both in vivo and in vitro.In the in vitro 0-Mg²⁺seizure model,the expression of NPCT was investigated with q PCR and western blot.Neuronal death and mitochondrial membrane potential were examined after immunoneutralization or exogenous supplementation of NPCT via PI/Hoechst double staining,phase-contrast observation,and JC-1 staining,and CCK-8 assay.5.In the rat RSE model,NPCT was neutralized both peripherally and intracerebroventricularly.Hippocampal neuronal death and astrocytic and microglial activation were investigated with Fluoro-Jade C staining and immunofluorescence staining.Survival of animals,EEG spectrum,hippocampal ATP levels,and the activities of mitochondrial respiratory chain complexes were further investigated.【Results】1.The distribution of NPCT in the brain（1）NPCT was widely distributed throughout the rat brain,with particularly high expressions in the hypothalamus,thalamus,hippocampus,and olfactory brain structures.NPCT was scarcely expressed in nerve structures.（2）Hippocampal NPCT was exclusively expressed in nearly all neurons and a proportion of astrocytes,while it did not exist in microglia.NPCT was contained in both pre-synaptic and post-synaptic domains.2.The alteration of hippocampal NPCT in rat DZP-resistant RSE models（1）In the lithium-pilocarpine models,prolonged seizure duration contributed to increased EEG and behavioral resistance to DZP and decreased survival.（2）The transcription and translation of hippocampal NPCT was increased during RSE,and NPCT was specifically overexpressed in CA3 pyramidal neurons.The increase of NPCT expression coincided with the emergence of resistance to DZP.（3）NPCT was re-distributed in synapses in CA1 and CA3 during RSE.The proportion of synapses that contain NPCT in post-synaptic domains significantly decreased and the proportion of synapses devoid of NPCT significantly increased.3.The neurotrophic effects of NPCT and its regulations on neuronal OXPHOS（1）NPCT simultaneously increased the expression of NR1,NR2B,and GABRG2subunits.NPCT increased the densities of both excitatory synapses and inhibitory synapses without altering the excitation and inhibition balance.（2）NPCT promoted dendrite outgrowth and increased the complexity of dendritic arborization.NPCT also increased the density of dendritic spine.（3）The influences of NPCT on neuronal transcriptome were mainly enriched in OXPHOS and mitochondrial respiratory complexes.NPCT could facilitate the activities of mitochondrial respiratory complexes I,IV,V and thus increased intracellular ATP levels.（4）NPCT enhanced the maximal respiration capacity and respiration spare capacity of hippocampal neurons,and it directly suppressed the expression of caspase-3,which increased the neuronal resistance to environmental stress.4.The neuroprotective effects of NPCT in in vitro 0-Mg²⁺seizure model（1）After the establishment of the 0-Mg²⁺seizure model,neuronal mitochondrial dysfunction occurred before neuronal death.（2）In 0-Mg²⁺seizure model,the transcription of the NPCT-encoding gene Calca was significantly enhanced,and the alternative splicing of Calca shifted towards more NPCT production.The expression and secretion of NPCT protein also increased in the 0-Mg²⁺seizure model.（3）Immunoneutralization of NPCT deteriorated neuronal death after modelling,while exogenous NPCT supplementation protected mitochondrial functions though it could not reverse the death outcomes.5.The neuroprotective effects of NPCT in rat DZP-resistant RSE models and its influences on seizures during RSE（1）Peripheral immunoneutralization of NPCT in rat DZP-resistant RSE models significantly increased 14-day mortality and hippocampal neuronal death,while it had no effect on the activation of hippocampal astrocytes and microglia.（2）Intracerebroventricular immunoneutralization of NPCT significantly decreased the EEG power during RSE,and induced rapid EEG failure and higher mortality after DZP administration.（3）Intracerebroventricular immunoneutralization of NPCT specifically increased the death of CA3 pyramidal neurons,but did not influence the activation of hippocampal astrocytes and microglia.（4）Intracerebroventricular immunoneutralization of NPCT in rat RSE models interfered with the responses of hippocampal mitochondrial respiratory complex I,IV,V,and deteriorated ATP depletion after RSE.【Conclusions】Our study revealed the extensive distribution of NPCT throughout the rat brain and its synaptic localization in the hippocampus.We established DZP-resistant RSE models and found that NPCT was overexpressed in response to RSE.We uncovered that NPCT could be a new member of neurotrophic factors and it was a neuropeptidergic regulator of neuronal mitochondrial respiratory complexes,increasing neuronal ATP supply and boosting the maximal respiration capacity and respiration spare capacity.In in vitro 0-Mg²⁺seizure models and rat DZP-resistant RSE models,NPCT exerted neuroprotective effects and preserved neuronal OXPHOS.Our study provided new insight into the mechanisms of neuronal death after RSE and proposed a new therapeutic target for neuroprotection in RSE.