| Epilepsy is one of the most common chronic nervous system disorders in childhood.The incidence of epilepsy in Chinese children is 151/100000 per year,and the prevalence is approximately 3.45%.Long-term recurrent epileptic seizures during development can cause cognitive deficits,affective disorders,and behavioral abnormalities,and burden individuals,families,and society.At present,antiepileptic drugs(AEDs)are the main treatment for epilepsy,with surgical treatment as adjuvant therapy.Although traditional AEDs have some therapeutic efficacy,they also introduce serious toxicity and side effects,especially cognitive impairment and behavioral disorders.Meanwhile,surgical treatment of epilepsy has limited indications and also risks.Therefore,exploration of new anti-epileptic approaches by further elucidating the mechanism of epilepsy may achieve the purpose of treating epilepsy with minimal side effects,which has become a research hotspot in the context of treating pediatric epilepsy.The pathophysiology of epilepsy is complex,which initiates several biological processes,including autophagy.Autophagy is important for maintaining intracellular homeostasis and coping with stress,and is closely associated with several human diseases.Degradation of intracellular damaged or excessive mitochondria by autophagy is called mitophagy,and the clearance of damaged mitochondria contributes to cell survival.Studies have shown that neuronal injury is associated with the activation of mitophagy.The neuroprotective effect of apolipoprotein J(clusterin)has been widely recognized in recent years,but its mechanism has not been fully understood.Recently Studies have shown that clusterin has the function of anti-mitochondrial-mediated apoptosis and is closely related to autophagy.Our previous study found that clusterin was highly expressed during epilepsy and was closely related to the expression of autophagy-associated proteins.These findings suggest that clusterin may also be associated with mitophagy during epilepsy.The objectives of the present study are to clarify the role of clusterin in brain injury caused by epilepsy,explore its relationship with and its role in mitophagy,clarify its regulatory mechanism,and explore the possibility of using clusterin as a therapeutic target for epilepsy.Part 1 Seizure during development promotes high expression of clusterin and up-regulates NIX-mediated mitophagyObjective:The objectives of the first part of the present study are to clarify the expression of clusterin and mitophagic proteins in a convulsive seizure model and analyze their correlations,and to preliminarily clarify the role of clusterin in the survival of mitophagic neurons.Methods:(1)ICR mice at postnatal day 21(P21)were randomly divided into a control group and seizure group.After being acclimatized for one day,mice in the seizure group were intraperitoneally injected with kainic acid to induce convulsive seizures at P22;meanwhile,mice in the control group were injected with the same volume of normal saline.Mitochondrial proteins were extracted from hippocampal tissue 24 h after the model was established,and the expression of clusterin and mitophagic proteins was measured by Western blotting(WB),then analyze their correlations with each other.On this basis,the kainic-acid-induced seizure model was established as described above with P21 day mice.Sixty successful mice were selected as the seozire group,and 60 mice were injected with normal saline as the control group.Hippocampal tissues were taken at 6 h,12 h,1 d,3 d,5 d,7 d,10 d,14 d,21 d,and 28 d after seizure induction(six mouses in each group at each time point).The expression of clusterin and NIX genes at different time points was measured by RT-PCR,and the expression of clusterin and mitophagic proteins(NIX and LC3)were measured by WB,then analysis of their expression trends and rules.In addition,immunofluorescent staining was used to measure the expression and location of clusterin,NIX,and LC3 in the hippocampus.(2)To verify the above results,we cultured primary hippocampal neurons and established a glutamate-injury model after 7 days.Neurons were divided into the glutamate group(GLu)and the control group(control);the glutamate group was supplemented with 50 μM glutamate,while the control group was supplemented with the same volume of medium.After treatment for 6h,the expression and location of clusterin,NIX,and LC3 in cells was measured by immunofluorescence.(3)To determine whether clusterin plays a role in NIX-mediated mitophagy,primary neurons were used to establish glutamate-injury models and exogenous clusterin was added as an intervention.Cells were divided into the following groups:control;clusterin control(CLU);glutamate(Glu);and clusterin plus glutamate(Glu+CLU).The control group was supplemented with a corresponding volume of medium,the CLU group was supplemented with clusterin(the final concentration was 100 ng/ml),the Glu group was supplemented with glutamate(the final concentration was 100μM)and the Glu+CLU group was supplemented with clusterin before glutamate treatment(the final concentration were 100 ng/ml and 100μM).The cells were collected at 1.5,3,6,12,18,24,36 and 48 h after glutamic acid supplementation.The expression of clusterin and NIX genes was measured by RT-PCR,while the expression patterns of clusterin,NIX,and LC3 in mitochondria were measured by WB.The effect of clusterin on mitophagy was detected by mitophagy detection kit.CCK8 and LDH were used to measure the effect of clusterin on the survival of neurons.Results:(1)The results of the 24 h seizure model demonstrated the following.Compared with the control group,the expression of clusterin was significantly upregulated in seizure group,and mitophagy-associated proteins-NIX,LC3Ⅱ/Ⅰ,PINK1 and Parkin—were also up-regulated;among them,the changes in NIX and LC3Ⅱ/Ⅰ were significant and consistent with the pattern of clusterin.The correlation of the expression of clusterin,NIX,and LC3Ⅱ/Ⅰ in the mitochondria of the seizure group was analyzed,and the results showed a positive correlation among these three factors.(2)The results of RT-PCR showed that the expression of clusterin and NIX mRNAs in the hippocampus of the control group was nearly unchanged across different time points;the mRNA expression of clusterin and NIX in the hippocampus was up-regulated significantly after seizure induction and showed a regularity of expression,which peaked at 6 h after seizure induction and returned to normal levels after 5 d.furthermore,we found that the expression of clusterin mRNA in seizure group was lower than that in normal control group at 14,21 and 28 days after seizure;And there was no significant difference in the expression of NIX mRNA in seizure group compared with the normal control group at 7,14,21 and 28 days after seizure;.(3)The results of WB showed that the expression of clusterin,NIX,and LC3II/I in hippocampal mitochondria of the control group did not change across different time points;the expression of clusterin,NIX,and LC3II/I in hippocampal mitochondria was up-regulated significantly after seizure induction and showed a regularity of expression,which peaked at 12 h after seizure induction;The expression of clusterin、LC3II/I returned to normal levels at 10d after seizure and the expression of NIX returned to normal levels at 7d after seizure.furthermore,we found that the expression of clusterin in seizure group was lower than that in normal control group at 21 and 28 days after seizure.(4)Immunofluorescent analysis showed that the expression of clusterin,NIX,and LC3 were up-regulated and co-expressed in the CA3 region of the hippocampus in the seizure group.(5)In the glutamate injury model of primary neurons,immunofluorescence showed that clusterin,NIX,and LC3 were highly expressed in the cells of the Glu group at the same sites.(6)The results of the glutamate-injury model of primary neurons showed that the expression of clusterin,NIX,and LC3 genes did not change over any time periods in the control group.In contrast,in the Glu group,the expression of clusterin and NIX mRNAs peaked after 3 h of glutamate treatment.In the Glu+CLU group of primary neurons,the peak expression of clusterin and NIX still appeared at 3 h.There was no difference between the Glu group and the Glu+CLU group,both groups began to decline after glutamate treatment for 9 hours.(7)In the clusterin plus glutamic acid group of primary neurons,WB showed that the expression of clusterin,NIX,and LC3 proteins did not change over any time periods in the control group.In the Glu group,the expression of clusterin、NIX and LC3 protein peaked after 6h of glutamate treatment.However,the peak expression of NIX and LC3 in mitochondria of the Glu+CLU group appeared at 3 h,which was earlier than that of the Glu group.(8)Detection of mitophagy in the four group of primary neurons showed that,compared with the control group,the level of mitophagy was significantly up-regulated in the Glu group,while the addition of exogenous clusterin in the Glu+CLU group further increased the level of mitophagy.(9)The results of CCK8 and LDH showed that,compared with the glutamate group,the Glu+CLU group had significantly higher cell survival rate,while the CLU group was similar to the control group in cell survival.Conclusions:(1)The expression of autophagy-associated proteins in mitochondria is up-regulated after seizures and t the expression of NIX,LC3Ⅱ/Ⅰ was positively correlated with the expression of clusterin,suggesting that clusterin may be more closely related to NIX-mediated mitophagy.(2)The expression of clusterin and NIX genes and the expression of NIX,LC3Ⅱ/Ⅰ,and clusterin proteins in mitochondria after seizure induction were temporally and spatially similar.(3)Exogenous clusterin increased the expression NIX and LC3 in mitochondria in glutamate injury model,but did not affect the expression of clusterin or NIX.(4)clusterin significantly up-regulated the level of mitophagy in glutamate injury model.(5)clusterin improved the survival of neurons in glutamate injury model.Part 2 The mechanism of clusterin regulates NIX-mediated mitophagy in inhibiting neuronal injuryObjective:The objective of the second part of the present study is to investigate the neuronal protective mechanism of clusterin in regulating NIX-mediated mitophagyMethods:(1)the glutamate-injury model was developed by using the mouse neuronal cell line HT22,and the following four groups were established:control;clusterin control(CLU);glutamate(Glu);and clusterin plus glutamate(Glu+CLU).The control group was supplemented with a corresponding volume of medium,the clusterin control group was supplemented with clusterin(the final concentration was 1 μg/ml),the glutamate d group was supplemented with glutamate(the final concentration was 50mM),and the clusterin plus glutamate group was supplemented with clusterin before glutamate treament(the final concentration were 1 μg/ml and 50mM).The levels of mitophagy in each group were assessed by a mitophagy assay kit after glutamate treatment for 6h.Western blot was used to detect the expression of clusterin,NIX and LC3 in mitochondria of each group.Mitochondrial DNA-encoded genes were measured by PCR，and the mitochondrial number per unit cell/tissue was characterized by the ratio of Nd2 to GAPDH.Mitochondrial red/green probes were used for staining,flow cytometry was used to measure the fluorescence intensity in the two groups,and the ratio of red(active mitochondria)to green(all mitochondria)fluorescence was used to represent the ratio of active mitochondria(overall mitochondrial quality index)in cells.CCK8 and LDH were used to analyze the survival of the cells in each group.An apoptosis detection kit was used to detect apoptosis in the above groups of cells.(2)Furthermore,a lentiviral vector was used to construct stable cell lines with over-expression and silencing of clusterin.six groups were included in the glutamate-injury model:normal cells+glutamate(N+Glu),virus with empty vector+glutamate(C+Glu),and clusterin virus+glutamate(CLU+Glu);normal cells+glutamate(N+Glu),virus with empty vector+glutamate(siC+Glu);and clusterin-silencing virus+glutamate(siCLU+Glu).After glutamate treatment for 6h,mitophagy assay kit was used to measure the level of mitophagy in each group.The expression of clusterin,NIX,and LC3 in mitochondria was measured by WB.(3)Then,the mitophagy inhibitor mdivi-1 was used to determine whether the effect of clusterin on cell survival was achieved by mitophagy.The cells were divided into the following four groups:glutamate;glutamate+clusterin;glutamate+mdivi-1 and mdivi-1+glutamate+clusterin group the mitophagy inhibitor mdivi-1 was added in advance).CCK8 and LDH were used to measure the survival of the cells in each group.(4)A lentiviral vector was used to construct a NIX-silencing stable cell line,which was then used for the development of the glutamate-injury model and subsequent clusterin intervention,in order to determine whether clusterin affected mitophagy level through NIX.The cells were divided into the following four groups:virus with empty vector+glutamate;virus with empty vector+glutamate+clusterin;NIX-silencing virus+glutamate;and NIX-silencing virus+glutamate+clusterin.WB was used to measure the expression of clusterin,NIX,and LC3 in the mitochondria of each group,and a mitophagy assay kit was used to measure the level of autophagy in the mitochondria of each group.(5)Glutamate damage model was established by overexpression of clusterin cell line,and the interaction of clusterin with NIX and LC3 was measured by an immunoprecipitation assay.Then the effect of clusterin on the binding ability of NIX and LC3 to mitochondria was measured by immuno-colocalization.The following four groups were established as in(1)above,double-labeling immunofluorescence was used to assess the binding of NIX and LC3 to mitochondria.Results:(1)Compared with the control group,the mitophagy level of the glutamate group and the clusterin plus glutamate group was significantly up-regulated;Compared with the glutamate group,the mitophagy level of the clusterin plus glutamate group was significantly up-regulated,but there was no significant differences between the control group and the clusterin control group.(2)The results of PCR showed that,Compared with the control group,the mitochondrial gene Nd2 were down-regulated in the glutamate group and the clusterin plus glutamate group;compared with the glutamate group,the mitochondrial gene Nd2 was down-regulated in the clusterin plus glutamate group,while there was no significant difference between the control group and the clusterin control group.These results suggest that clusterin can increase the level of NIX-mediated mitophagy in cells.(3)The results of flow-cytometry analysis after mitochondrial staining showed that,Compared with the control group,the red/green ratio was down-regulated in the glutamate group and the clusterin plus glutamate group were down-regulated;compared with the glutamate group,the red/green ratio in the clusterin plus glutamate group was up-regulated,while there was no significant difference between the control group and the clusterin control group,suggesting that clusterin can decrease mitochondrial damage in the glutamate model.(4)The results of CCK8 and LDH showed that,Compared with the control group,glutamate increased LDH release and mortality;Compared with the the glutamate group,the results of the clusterin plus glutamate group suggested that clusterin could improve the survival of cells,but there was no significant difference between the control group and the clusterin control group.(5)The results of CCK8 and LDH showed that the mitophagy inhibitor mdivi-1 significantly reversed the effect of clusterin on cell survival.(6)Compared with the control group(the empty vector group and the normal group),the expression of NIX and LC3II in mitochondria and the levels of mitophagy were all increased in the clusterin-overexpressing cell line with glutamate-induced injury.Compared the control group(the empty vector group and the normal group),the expression of NIX and LC3II in mitochondria and the levels of mitophagy were significantly lower in the glutamate-injury model developed from the clusterin-silencing cell line.(7)Silencing the expression of NIX reversed the effect of clusterin on the increase of mitophagy and significantly decreased the overall mitophagy level,as well as the expression of LC3 on mitochondrial membrane.(8)The results of immunoprecipitation showed that clusterin could bind with NIX and LC3,and clusterin-interacting sites were present on both NIX and LC3.(9)Immunofluorescence showed that,compared with the glutamate group,clusterin plus glutamate increased the aggregation of NIX and LC3 around mitochondria and increased their binding with mitochondria.Conclusions:(1)clusterin enhances the survival of neurons mainly through NIX-mediated mitophagy.(2)clusterin can interact with NIX and LC3 and regulate mitophagy by affecting their binding on mitochondria.Part 3 Early clusterin intervention can protect against seizure-induced brain injury through NIX-mediated mitophagyObjective:The above studies suggest that clusterin can promote mitophagy and inhibit neuronal damage.This third part of the present study aimed to verify the protective role of clusterin against convulsive seizure-induced brain injury in animal models,in order to provide evidence of clusterin as a potential treatment for neural injury.Methods:(1)Newborn seven-day-old ICR mice were divided into the following three groups:sham-operated group(Sham);viral control group(AAVctrl+KA);and clusterin virus group(AAVclu+KA).The Sham group was injected with artificial cerebrospinal fluid without any further treatments,the AAVctrl+KA group was injected with adeno-associated virus containing the empty vector,and the AAVclu+KA group was injected with adeno-associated virus over-expressing clusterin.Seizure models were developed in the latter two groups two weeks after the above injections.The hippocampus was taken at 6 h,12 h,1 d,3 d,7 d,14 d,and 28 d after model development.Then,the expression of clusterin,NIX,and LC3 in mitochondria was measured by RT-PCR,and the expression of clusterin,NIX,and LC3 in mitochondria was measured by WB.Immunohistochemistry was conducted on brain sections after 6 h of treatment to study the effect of clusterin on the binding between NIX and LC3.After 28 d,the whole brain of each mouse was fixed and dehydrated,and frozen sections were used to for immunohistochemical detection of neuronal marker proteins and Nissl staining.(2)At 7 d,14 d,and 21 d after seizure,the ability of neural reflexes in mice was measured by the climbing-rod test and suspension test,and the effect of clusterin intervention on cognitive and emotional functions after seizure was measured by the open-field test at 24 d and 25 d after seizure.The Y-maze test was conducted at 22 d and 23 d after seizure,and the dark-box test was conducted at 26-28 d after seizure,in order to determine whether clusterin could improve the degree of memory and cognitive impairment after seizure.Results:(1)The results of RT-PCR showed that the expression of clusterin and NIX genes in the Sham group was nearly unchanged across all time points,while the expression of clusterin and NIX in AAVctrl+KA group and the AAVclu+KA group peaked at 6 h after seizure.The expression of both genes in the AAVclu+KA group and AAVctrl+KA group was higher than that in the sham-operated group,while the overall expression level in the AAVclu+KA group was higher than that of AAVctrl+KAgroup.(2)The expression of clusterin,NIX,and LC3 in mitochondria of the sham-operated group did not change significantly across all time points.The expression of NIX and LC3 in the hippocampus of the AAVclu+KA group peaked at 6 h after the model was developed,while the peak level in AAVctrl+KA group appeared at 12 h.clusterin caused the expression of NIX and LC3 on the mitochondrial membrane to peak earlier,and the expression at each time point was higher than that in AAVctrl+KA group and the sham-operated group.(3)clusterin enhanced the binding of LC3 to NIX,suggesting that clusterin may promote mitophagy by promoting the binding of LC3 to NIX.(4)Nissl staining showed that clusterin reduced neuronal injury.(5)TIMM staining showed that clusterin improved the sprouting of neurons in CA3 and DG regions.(6)Compared with AAVctrl+KA group,the time of forelimb suspension in the AAVclu+KA group was significantly longer,but there was no significant difference between the two groups in the climbing-rod test.(7)The Y-maze test showed that,compared with AAVctrl+KA group,the frequency and duration of entering the food arm in the AAVclu+KA group were significantly increased.(8)The open-field test showed that,compared with AAVctrl+KA group,the AAVclu+KA group had shorter delay time,higher opening score.(9)Compared with AAVctrl+KA group,the frequency and duration of entering the dark box in the AAVclu+KA group were significantly decreased.Conclusions:(1)clusterin can enhance the expression of NIX and LC3 in mitochondria,promote the binding of NIX and LC3,and promote mitophagy.(2)clusterin can interfere with long-term neuronal loss,mossy-fiber sprouting,and neurobehavioral and cognitive damages caused by neonatal seizure-induced brain injury.That is,clusterin intervention can inhibit neuronal loss and abnormal sprouting of hippocampal mossy fibers,and reduce long-term neurobehavioral and cognitive damage. |
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