The Role Of PGRMC1/GLP-1R/Mfn2-mediated Mitochondrial Dysfunction In Clozapine-induced Neurotoxicity

Background:Schizophrenia is a complex and diverse behavioral and cognitive syndrome.Cognitive impairment is the third core symptom of schizophrenia besides positive and negative symptoms,which seriously affects the patient’s condition and quality of life.Drugs are the main treatment for schizophrenia.Among them,clozapine is the drug of choice for the treatment resistant schizophrenia.However,our research group previously found that clozapine can mediate abnormal glucose and lipid metabolism and neurotoxic side effects by downregulating PGRMC1 protein.Recent studies have indicated that mitochondria play a key role in the maintenance of cognitive function and neuronal function.This study intends to further explore the neurotoxicity induced by clozapine and the role of PGRMC1 in clozapine-induced neurotoxicity.Objective:1.To explore whether clozapine can modulate the PGRMC1/GLP-1R/Mfn2 pathway,leading to mitochondrial dysfunction and resulting in neurotoxicity.2.To further verify the pivotal role of PGRMC1 in the abovementioned neurotoxicity induced by clozapine.Methods:This study can be divided into three parts: drug-receptor binding assay,cell and animal experiments.1.Surface plasmon resonance experiment was conducted in the drugreceptor binding assay to investigate the kinetic parameters of the interaction between the target protein PGRMC1 and clozapine,olanzapine,risperidone,aripiprazole,progesterone and AG205.2.PC12 cells were used in cell experiment to explore the effects of clozapine and PGRMC1 specific inhibitor AG205 on neurotoxicity and mitochondrial function.Lentiviruses were used to overexpress and knockdown the target protein PGRMC1 to verify the key role of PGRMC1 in the above-mentioned neurotoxicity induced by clozapine.(1)CCK-8method was used to explore the effect of clozapine on cell viability under different concentrations and different times of clozapine.Mitochondrial superoxide and mitochondrial membrane potential were used to investigate the effect of different concentrations of clozapine on mitochondrial function.Western blot was used to measure the protein expression of PGRMC1,GLP-1R and Mfn2.(2)CCK-8 method was used to detect the effect of AG205 on cell viability.Mitochondrial superoxide and mitochondrial membrane potential were used to investigate the effect of AG205 on mitochondrial function.Western blot was used to detect the protein expression of PGRMC1,GLP-1R and Mfn2.(3)Lentiviruses were used to overexpress and downregulate the expression of PGRMC1,and western blot was used to detect the expression of PGRMC1.The experimental groups were divided into normal control group(nc group),PGRMC1 overexpression + clozapine group(oe + clz group),PGRMC1 knockdown group(kd group)and blank vector + clozapine(clz group).Further verify the role of PGRMC1 in the mitochondrial dysfunction of PC12 cells induced by clozapine.3.Male adult SD rats were used in animal experiments.The rats were randomly divided into normal control group(nc group),PGRMC1 overexpression + clozapine group(oe + clz group),PGRMC1 knockdown group(kd group)and blank adeno-associated virus(AAV)vector +clozapine group(clz group).Our research group has verified the effects of AAV which can across the blood-brain barrier and overexpress or downregulate the expression of PGRMC1.The blank vehicle or clozapine were administered by intraperitoneal injection for 28 days.Morris water maze was used to investigate the cognitive function of the rats.TUNEL staining was used to detect the neuronal apoptosis of rat hippocampus.Nissl staining was used to detect the morphological changes of neurons in hippocampal CA1 and CA3 areas.Western blot was used to detect the protein expression of PGRMC1,GLP-1R and Mfn2 in rat hippocampus.Results:1.Results of surface plasmon resonance experiment:(1)According to the comparison of KD values,the KD value of the endogenous ligand progesterone is lower than that of clozapine,olanzapine,risperidone,aripiprazole and AG205.The binding affinity of above compounds to PGRMC1 was stronger than that of progesterone.(2)According to the comparison of KD values,the order of binding affinity with PGRMC1:risperidone > AG205 > aripiprazole > clozapine > olanzapine >progesterone.(3)Combining KD values,the highest effective plasma concentration and lipophilicity,order of binding affinity with PGRMC1:clozapine > aripiprazole > risperidone > olanzapine.2.Results of cell experiment:(1)The damage effects of clozapine on PC12 cells were dose-related and time-related.With the increase of dose and time,cell viabilities decreased gradually.After 24 h intervention with clozapine,with the increase of clozapine concentration,the production of mitochondrial superoxide increased and mitochondrial membrane potential decreased.24 h intervention with 20μM and 40μM clozapine could downregulate the protein expression of PGRMC1 and GLP-1R,and80μM clozapine could downregulate the protein expression of PGRMC1,GLP-1R and Mfn2.(2)The cell viabilities of PC12 cells were decreased with the increase of AG205 concentration and time.Under the same intervention time of AG205 for 24 h,with the increase of concentration,the production of mitochondrial superoxide increased and mitochondrial membrane potential decreased.With the increase of AG205 concentration,the protein expression of PGRMC1 showed a downward trend.24 h intervention with 10μM and 20μM AG205 could downregulate the protein expression of Mfn2,40μM and 80μM AG205 could downregulate the protein expression of GLP-1R and Mfn2.(3)Overexpression and knockdown of the target protein PGRMC1 by lentiviruses: compared with the nc group,the mitochondrial superoxide generation increased and the mitochondrial membrane potential decreased in the kd group and the clz group.Compared with the clz group,the overexpression of PGRMC1 could improve the mitochondrial dysfunction induced by clozapine.Compared with the nc group,the protein expression of PGRMC1 and GLP-1R decreased in the kd group,and that of Mfn2 showed a downward trend;compared with the nc group,the protein expression of GLP-1R decreased significantly in the clz group,and that of PGRMC1 and Mfn2 showed a downward trend.Compared with the clz group,the protein expression of PGRMC1,GLP-1R and Mfn2 were significantly up-regulated in the oe +clz group.3.Results of animal experiment:(1)Hidden platform phase: on the fifth day,compared with the nc group,the latency of the clz group was prolonged.Compared with the clz group,the latency of oe + clz group reduced on the 4th and 5th day.Probe trial on the sixth day: compared with the nc group,the number of target quadrant crossings,swimming time spent in the target quadrant and the percentage of swimming time spent in the target quadrant were reduced significantly in the kd group and clz group.Compared with the clz group,the above three indicators significantly increased in the oe + clz group.(2)The results of TUNEL staining: greenstained positive apoptotic neurons were found in kd group and clz group,and almost no positive cells were found in nc group and oe + clz group.(3)Results of Nissl staining: in the nc group,neurons were evenly arranged,with regular cell morphology and complete structure,while in kd group and clz group,the neurons were disordered in arrangement and abnormal in cell morphology,and the number of intracellular Nissl bodies decreased.Compared with clz group,the neuronal damage was slightly improved in oe + clz group.(4)Compared with the nc group,the protein expression of PGRMC1,GLP-1R and Mfn2 was significantly reduced in kd group,and the protein expression of GLP-1R and Mfn2 significantly decreased in clz group,and that of PGRMC1 showed a downward trend.Compared with clz group,the protein expression of PGRMC1,GLP-1R and Mfn2 was significantly increased in the oe + clz group.Conclusion:The study takes mitochondrial function as the breakthrough point,focuses on the PGRMC1/GLP-1R/Mfn2 pathway,and investigates the role of this pathway in clozapine-induced neurotoxicity both in vitro and in vivo:clozapine can cause mitochondrial dysfunction through down-regulating the PGRMC1/GLP-1R/Mfn2 pathway,resulting in cognitive impairment,while overexpression of PGRMC1 can alleviate the clozapine-induced neurotoxicity.