The Study Of Endoplasmic Reticulum Calcium Leakage Involved In Excitatory Load-Driven Neurotoxicity And Its Mechanism

Part Ⅰ.The study of the anti-excitotoxicity of SERCA2bObjective:Epilepsy is one of the common diseases of the nervous system.The mechanisms underlying epileptogenesis have not yet been fully elucidated,and effective prevention strategies are still lacking.Glutamate-mediated excitotoxicity is an important factor in epileptogenesis,and the endoplasmic reticulum plays a pivotal role in regulating the calcium-dependent glutamatergic pathway.SERCA2 is the only calcium pump on the endoplasmic reticulum and transports cytoplasmic calcium ions to the endoplasmic reticulum in reverse concentration by consuming ATP to maintain calcium homeostasis in the internal environment.We previously identified four de novo mutations in SERCA2b（NM＿170665:p.Gly23Arg、p.Asp567Tyr、p.Gly860Ser、p.Ile1014Val）in one case of infantile spasms and three cases of epilepsy combined with mental retardation,and bioinformatics analysis suggested that these mutations may be pathogenic.Therefore,this section intends to explore the functional alterations of these 4 SERCA2b mutants and their effects on the glutamatergic excitatory pathway.Methods:（1）HT22 cells overexpressing SERCA2b（G23R,D567Y,G860S,I1014V）mutants were induced using low concentrations of glutamate to establish an excitotoxic cell model.（2）CCK-8,MTT,LDH release,TUNEL staining,WB and q PCR were taken to detect damage and endoplasmic reticulum stress levels in SERCA2b mutant cells.（3）The calcium pump activity and cytoplasmic calcium scavenging ability of SERCA2b mutants were detected by calcium imaging and phosphor fixation method.（4）To detect the expression,degradation and solubility of SERCA2b mutant proteins using immunofluorescence,WB and q PCR.（5）To detect morphological and functional alterations of SERCA2b mutant mitochondria using electron microscopy,WB,calcium imaging,reactive oxygen species,and mitochondrial membrane potential.（6）Recovery of endoplasmic reticulum calcium depletion and excitotoxicity in mutant cells was examined by enhancing（CDN1163）or inhibiting（Thapsigargin）the pump transport/leakage capacity of SERCA2b mutants,confirming that restoration of SERCA2b function antagonizes excitotoxicity.Results:（1）Cell death and endoplasmic reticulum stress were both increased in SERCA2b overexpression mutants.（2）Pump activity was reduced in all SERCA2b mutants,and only the G860S mutant had decreased calcium storage.（3）Some SERCA2b mutants have reduced protein expression and solubility.（4）Mitochondria of SERCA2b mutants were all subject to calcium overload and dysfunction.（5）Increased pump transport capacity of SERCA2b mutants（G23R,D567Y,I1014V）can antagonize excitotoxicity.（6）Inhibition of calcium leakage from SERCA2b mutants（G860S）can attenuate excitotoxicity.Conclusion:SERCA2b mutants exacerbate endoplasmic reticulum calcium depletion-mediated cellular excitotoxicity.The mechanism of damage is mainly related to the heterogeneity of SERCA2b mutant sites.Restoration of SRECA2b calcium transport function or inhibition of calcium leak is an important strategy against glutamate-induced excitotoxicity.Part Ⅱ:The studies of RyR2 involved in excitotoxicityObjective:RyR2 is a major channel for endoplasmic reticulum calcium release in neurons and is widely expressed in brain and cardiac tissues.We identified a de novo mutation in RyR2-P2260L in a patient with infantile spasms.Previous studies have shown that different RyR2variants can lead to endoplasmic reticulum calcium seepage,increased calcium ion sensitivity in the endoplasmic reticulum lumen,and a decreased threshold for calcium release induced by calcium overload.The aim of this study was to explore whether RyR2-P2260L de novo mutations lead to endoplasmic reticulum calcium leakage and are involved in neuronal excitotoxicity and epileptogenesis.Methods:（1）P2259L（corresponding to the P2260L mutation in the human genome）point mutation knock-in mouse model(RyR2^PL/+)was constructed using CRISPR-Cas9 technology,and the phenotypes of RyR2^PL/PLmice were analyzed by behavioral analysis,histomorphological analysis,EEG monitoring,and cardiac ultrasound.（2）A drug-induced acute status epilepticus model was constructed,and the acute seizure susceptibility of RyR2^PL/PLmice and the susceptibility after SE were analyzed by behavioral observation and EEG monitoring.（3）To examine the membrane electrophysiological properties of RyR2^PL/PLneurons using membrane clamp technique and analyze the possible reasons for their increased seizure sensitivity.（4）Calcium imaging was used to confirm that calcium leakage occurs in the endoplasmic reticulum of RyR2^PL/PLneurons under excitatory load.（5）To intervene RyR2^PL/PLneurons with RyR2 stabilizer（S107）,CCK-8,LDH release,ROS,and WB were taken to detect the recovery of neuronal damage and endoplasmic reticulum stress level,and to clarify the anti-excitatory damage effect of RyR2stabilizer.（6）Intervention of RyR2^PL/PLmice with RyR2 stabilizer,behavioral observation and Nissl staining were used to analyze the sensitivity and recovery of neuronal damage after persistent epilepsy,and the neuroprotective effect of inhibiting RyR2-P2259L calcium leakage was confirmed.Results:（1）RyR2^PL/PLmice have normal growth,development,and neurobehavioral phenotypes.（2）RyR2^PL/PLmice have increased acute seizure sensitivity and post-SE sensitivity.（3）RyR2^PL/PLneuronal membranes have increased AHP in electrophysiological features.（4）Calcium leakage occurs in the endoplasmic reticulum of RyR2^PL/PLneurons.（5）Inhibition of RyR2-P2259L calcium leakage antagonizes neuronal excitotoxicity.（6）Inhibition of RyR2-P2259L calcium leakage reduces seizure sensitivity and post-SE injury in RyR2^PL/PLmice.Conclusion:RyR2-P2259L-mediated calcium leakage exacerbates neurotoxicity in neurons driven by excitatory load,and inhibition of RyR2-P2259L calcium leakage reduces susceptibility to seizures and damage after SE in mice.Including 26 figures,2 tables and 166 references.