The Effect Of Cyclophilin D-mediated Mitochondrial Permeability Transition Pore Opening On The Formation Of Neuronal Dendritic Protrusions And The Underlying Mechanisms

Background:Neurons are the structural and functional units of the brain.They are highly polarized cells with different membrane domains.A typical neuron consists of a cell body(soma),axon and dendrites.Many tiny protrusions which are called dendritic protrusions are emitted on the dendrites.They can be classified into dendritic spines and filopodia.Dendritic spines are highly specialized protrusions on the dendrites.The length is generally less than 5 ?m.The dendritic spine contains smooth endoplasmic reticulum,polysomes,endosomes,and lysosomes and mitochondria in the head.Filopodia is an elongated protrusion structure on the dendrites.It is generally more than 5?m in length.It is rich in actin and has no obvious globular head.A number of studies have suggested that filopodia are precursors of dendritic spines,while some believe that filopodia are a special type of dendritic spines.Dendritic spines,axon terminals and the gaps between them form synapses,which are the structural basis of neuronal connection.Synaptic plasticity is considered to be an important neurochemical basis for learning and memory,and is divided into functional plasticity related to transmission efficiency and structural plasticity associated with information storage.Changes in the number or morphology of dendritic spines are involved in the regulation of synaptic plasticity,and understanding the cellular mechanisms of dendritic spines or dendritic protrusions can extend-our understanding of normal and abnormal brain functions.Calcium(Ca2+)plays a key role in regulating the morphology and density of dendritic protrusions and is the basis of synaptic plasticity.In addition,synaptic transmission requires high levels of ATP,and the ability of mitochondria to provide ATP and regulate Ca2+ homeostasis plays a key role in neuronal development and synaptic function.The calcium homeostasis in the mitochondria is maintained through Na+-Ca2+ exchanger(mNCX),H+-Ca2+ exchanger(mHCX),mitochondrial calcium uniporter(MCU)complex,and the mitochondrial permeability transition pore(mPTP).Ca2+ enters the mitochondria through the MCU complex,leaves the mitochondria through mNCX,mHCX and mPTP,and the rapid mPTP opening can rapidly release Ca2+.However,the molecular composition of mPTP is still unclear.Recently,many studies have concluded that the dimer or monomer of FIFO-ATP synthase,or its c-subunits ring participates in the formation of mPTP.However,cyclophilin D(CypD)is currently the only genetically defined molecule that regulates mPTP opening.Mitochondrial permeability transition(mPT)refers to the opening of mPTP in the presence of a specific inducer,and the mitochondrial membrane rapidly increases the permeability of solutes with a molecular weight of<1,500 Da,which causes the breakdown of mitochondrial membrane potential,uncoupling of the respiratory chain,decreased ATP synthesis,and influx of solutes,resulting in mitochondrial matrix expansion,mitochondrial swelling,and cytochrome c release and subsequent caspase activation,ultimately leading to cell death.Therefore,it is speculated that inhibition of mPTP opening has a protective effect on cells.In many diseases models(muscular dystrophy,Alzheimer's disease,myocardial infarction),the genetic knockout or drug inhibition of CypD interferes with the opening of mPTP,and has a protective effect.However,in the heart failure model,CypD deficiency has no protective effect,but instead aggravates heart failure.The above two situations seem to be contradictory.Is CypD-mediated mPTP opening harmful or beneficial?More and more studies suggest that CypD-mediated mPTP opening is divided into long-term openning and transient openning.The long-term openning of mPTP leads to membrane potential collapse,oxidative phosphorylation uncoupling,mitochondrial matrix swelling,ATP depletion and increased reactive oxygen species,which ultimately leads to cell death.The transient opening of mPTP can lead to small changes in mitochondrial membrane potential,which does not have any adverse effects on cell viability,and can reduce excessive metabolites and ions(especially Ca2+)in mitochondria,thereby avoid mitochondrial swelling and preven pro-apoptotic factors release,ultimately preserve the integrity of the mitochondria.Therefore,the transient reversible opening of mPTP is a physiological process,and we should pay attention to the physiological role of CypD and its mediated mPTP opening.Aims:1.To clarify the effect of CypD-mediated mPTP opening on the formation of dendritic protrusions;2.Explore the underlying mechanisms of CypD-mediated mPTP opening on calcium and their downstream signaling molecules.Methods:1.Primary neuron culture:neonatal Ppif+/+ and Ppif-/-pups within I day were usedto isolate the cerebral cortex and hippocampus.Neurons were planted in culture medium containing Neurobasal A,B27,L-glutamin and antibiotic,then placed in a cell incubator containing 5%CO2 at 37 ?.The experiment was carried out until 14 days of culture.2.Dsred lentivirus production:HEK 293T cells were transfected with calcium phosphate method.After 24 hours,the supernatant was collected,filtered,and centrifuged.The pellet was the collection of lentivirus particles.3.Treatment of neurons with 90 mM potassium chloride(KCl):incubate neurons with 90 mM KCl solution for 3 min in a 37 ? incubator,discard the 90 mM KCl solution,and wash the cells once with pre-warmed neuron medium,additional neuronal medium was added and incubate the neurons for 10 minutes at 37 ?,which was referred to as 1 cycle of KCl treatment.Repeat the above steps until 4 cycles are completed.4.Measurement of ATP:use the Abeam ATP assay kit and follow the instructions.5.Detection of mPTP open:stain the neurons with calcein AM and then quenched with cobalt chloride.6.Calcium level detection:Fluo-4 AM staining was used to detect calcium in dendrites,and Rhod-2 AM staining was used to detect calcium in mitochondria.7.Western blotting:Western blotting was used to detect the related proteins that control mitochondrial fusion/fission and calcium signaling.8.Cellular immunofluorescence staining was used to detect the number and morphology of dendritic protrusions and the distribution of mitochondria within the dendrites.9.Image acquisition,processing,and analysis:images were taken using a Nikon inverted fluorescence microscope and confocal microscope,and image processing and data analysis were performed using the Nikon NIS Advanced Research Software.10.Statistical analysis:data storage and simple analysis were performed using Microsoft Excel(2007),and the quantitative data were expressed as mean ±standard error of the mean.The normality test and statistical analysis of the data were performed using SPSS(21.0)software.The t-test was used for comparison between the two groups,and the comparison between multiple groups was analyzed by analysis of variance.P<0.05 was considered statistically significant.Results:1.Neuronal depolarization causes CypD-mediated mPTP transient opening.KCl-treated Ppif+/+ neurons showed a significant decrease in dendritic mitochondrial Calcein AM staining,while the staining of CypD knockout Ppif-/-neurons was not significantly reduced(P<0.05).After KCl treatment,both types of neurons exhibited similar patterns of mitochondrial length reduction and similar changes in ATP levels.2.The loss of CypD impedes the formation of dendritic protrusions induced by neuronal depolarization.At baseline levels,there were no significant differences in dendritic protrusions,dendritic spines,and filopodia density between Ppif+/+ and Ppif-/-neurons.Repeated KC1 stimulation can induce a significant increase in the density of dendritic protrusions,especially filopodia,in Ppif+/+ neurons,and dendritic spine changes were not obvious.However there was only a slight increase in the density of dendritic protrusions in Ppif-/-neurons(P<0.05).3.CypD deletion impairs the flexibility of dendritic mitochondrial calcium dynamics during neuronal depolarization.In Ppif+/+ and Ppif-/-neurons,KCl treatment induced a significant increase in Fluo-4 AM staining intensity which then rapidly decreased to baseline levels.However,the rate of Ppif-/-neuronal dendrites was faster than that of Ppif+/+ neurons(P<0.05).After KC1 treatment,the intensity of Rhod-2 AM staining of Ppif+/+ and Ppif-/-neurons was significantly higher than that of baseline;however,the increase of Ppif-/-dendritic mitochondria was more obvious(P<0.01).Further analysis showed that most Ppif+/+ dendritic mitochondria belonged to the calcium"flickering" type;while most Ppif-/-dendritic mitochondria were in a calcium "net loss" state during neuronal depolarization(P<0.05).).4.CypD deletion inhibits the movement of dendritic mitochondria during neuronal depolarization.At baseline,only a small fraction of the dendritic mitochondria in Ppif+/+ and Ppif-/-neurons were movable,but the Ppif+/+ dendrites mitochondria were more active(P<0.05).During depolarization,the Ppif-1-dendritic mitochondria quickly stopped moving;whereas the Ppif+/+ dendritic mitochondria moved in the antegrade and retrograde directions before they reached stability(P<0.05).Moreover,the Ppif+/+ dendritic mitochondria moved faster than Ppif-/-dendritic mitochondria in both antegrade and retrograde directions at baseline and during depolarization(P<0.05).The velocity of mitochondrial movement was reduced during depolarization in the Ppif+/+ and Ppif-/-neurons.5.CypD deletion inhibits the redistribution of dendritic mitochondria in depolarized neurons.At rest conditions,6.32 ±2.76%of Ppif+/+ dendritic protrusions contained mitochondria,and approximately one quarter of Ppif-/-dendritic protrusions contained mitochondrial components.After 1 cycle and 4 cycles of KCl-induced depolarization,the percentage of Ppif+/+ dendritic protrusions containing mitochondria increased significantly to 16.52±3.39%and 23.52±3.82%,respectively,while the percentage of Ppif-/-dendritic protrusions containing mitochondria showed little changes.6.CypD deletion does not affect proteins that control mitochondrial fusion and fission,but inhibits phosphorylation of CaMKII and GluR1 during neuronal depolarization.During neuronal depolarization,the expression levels of mitochondrial fusion protein(OPA1 and MFN2)and mitochondrial fission protein(DLP1)didn't change significantly in Ppif+/+ and Ppif-/-neurons.The phosphorylation level of DLP1(p-Drp1/DLP1)decreased significantly during depolarization,but there was no significant difference between the two types of neurons.KCl treatment induced activation of CaMKII? and ? in both types of neurons,but Ppif+/+ neurons responded more strongly to KCl-induced CaMKII? and ? phosphorylation(P<0.05).After KCI stimulation,the level of phosphorylated GluR1(p-GluR1)in Ppif+/+neurons was much higher than that in Ppif-/-neurons(P<0.05),while the level of total GluR1 did not change.7.CypD deletion attenuates dendritic mitochondrial calcium perturbation and preserves dendritic protrusions in oxidative stress.The H2O2-treated Ppif+/+ dendritic mitochondria showed a significant decrease in calcium levels after treatment with KCI.Further analysis indicated that most of the mitochondria were of the calcium "net loss" type;while most Ppif-/-dendritic mitochondria still had the ability to buffer calcium,although their ability to store calcium was also impaired(P<0.05).The density of dendritic protrusions in Ppif+/+ neurons treated with H2O2 was significantly lower than that of the Ppif-/-neurons at rest and after KCI treatment(P<0.05).Conclusions:1.CypD-mediated transient opening of mPTP could promote the formation of dendritic protrusions induced by neuroal depolarization.2.Under oxidative stress conditions,CypD-mediated long term opening of mPTP could inhibit the formation of dendritic protrusions induced by neuroal depolarization.3.CypD-mediated mPTP opening regulated the formation of dendritic protrusions through the regulation of mitochondrial calcium homeostasis,the movement and distribution of mitochondria in dendrites,as well as the phosphorylation of CaMKII Thr286 and GuR1 Ser831.