The mechanism of mitochondrial autophagy regulating CME in epilepsy

doi:10.21203/rs.3.rs-1640810/v1

Download PDF

Research Article

The mechanism of mitochondrial autophagy regulating CME in epilepsy

https://doi.org/10.21203/rs.3.rs-1640810/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Mitophagy is a process of removing damaged mitochondria, transferring damaged mitochondria to lysosomes for degradation, thereby regulating the quality of mitochondria and maintaining the stability of the intracellular environment. CME(Clathrin-mediated-endocytosis)plays an important role in the endocytosis and recovery of vesicles. It is also the main way for large parts of substances to enter cells, and it is widely involved in the signal transduction of various physiological activities of cells. Therefore, this study used pentylenetetrazol to ignite the abnormal mitochondrial autophagy in the chronic epilepsy model and the epilepsy model without magnesium-induced neurons. Subsequently, the autophagy inhibitor 3-MA was used to further observe that the inhibition of autophagy caused a decrease in the fluorescence uptake of Tf-A488 in the primary cultured neurons and the hippocampus of experimental mice, and the inhibition of mitochondrial autophagy could inhibit the function of CME. Finally, through PTZ epilepsy model and kainic acid model, we observed the effect of 3-MA on epilepsy behavior and its in-body field potential, and finally clarify the possible mechanism of mitochondrial autophagy regulating CME and participating in epilepsy.

Epilepsy is a chronic neurological disease, which is caused by abnormal synchronous discharge of neurons with a clinical manifestaition of frequent spontaneous seizures. It affects about 1% of the global population, making it one of the most common chronic nervous system diseases, of which 80% of the patients live in low and middle-income countries [1–4]. At present, the treatment of epilepsy is mainly drug intervention. Although a wide variety of antiepileptic drugs (AEDs) have been developed in the past few decades, the level of drug resistance is still stable at about 30% [5] Long term recurrent seizures increase the risk of behavioral and cognitive changes and even death of patients by 2–3 times compared with the general population [6, 7]. Adverse outcomes are closely related to the complexity and unknown pathogenesis. Therefore, it is of great clinical significance to explore the potential pathogenesis of epilepsy and promote the development of new antiepileptic drugs.

Mitochondria are a dynamic process of continuous fusion and division to maintain morphology and function [8]. After mitochondrial fusion and division, damaged mitochondria can be separated and become the object of autophagy [9]. Mitochondrial autophagy is a process of clearing damaged mitochondria, transferring damaged mitochondria to lysosomes for degradation, so as to regulate the quality of mitochondria and maintain the stability of intracellular environment. PINK1 / parkin is the most important way to mediate mitochondrial autophagy. Under normal circumstances, Phosphatase and tensin homolog(PTEN) induced putative kinase 1 (Pink1) exists in the outer membrane of mitochondria, will be continuously transferred to the inner membrane of mitochondria, and then rapidly degraded by proteolytic enzymes [10]. When mitochondria are damaged, PINK1 transfers from the inner mitochondrial membrane to the outer membrane, aggregates on the outer membrane, activates E3 ubiquitin protein ligase (parkin) in the cytoplasm, recognizes and precisely determines that it is located in the damaged mitochondria to make it ubiquitinated, and the ubiquitinated parkin is the marker protein of autophagy [11, 12]. More and more studies have shown that autophagy is related to a variety of neurological diseases, including epilepsy; In the hippocampus of patients with intractable temporal lobe epilepsy, abnormal accumulation of damaged mitochondria was found by microscope [13]. Autophagy is also related to synaptic transmission, plasticity, excitotoxicity and other functions. Therefore, it is speculated that abnormal autophagy can enhance abnormal synaptic plasticity and eventually form epileptic network [14].

Synapse is not only the contact part between neurons, but also an important part for information transmission. However, the number of synaptic vesicles in nerve endings is very limited. Due to exocytosis, vesicles are continuously consumed. Therefore, endocytosis of neurons is particularly important for vesicle recovery and continuous signal transmission between neurons [15]. Clathrin is a protein complex that plays a key role in vesicle cycle regeneration. It is composed of a heavy chain protein with a molecular weight of 192KDa and a light chain protein with a molecular weight of 33-35kda. Clathrin-mediated-endocytosis (CME) plays an important role in vesicle endocytosis and recovery. It is also the main way for large parts of substances to enter cells [16]. It is widely involved in signal transduction of a variety of physiological activities of cells, including nutrient absorption, cell growth and differentiation, activation and transmission of neural synapses, cell chemotaxis and immune responses. According to the time sequence, it can be roughly divided into the following stages: the formation of clathrin coating, the formation of presynaptic membrane recess, recess constriction and shear, and vesicle de coating [17]. In these stages, it is indispensable to involve these molecules such as adaptor protein-2 (AP2), Dynamin (Dnm) and heat shock cognate protein 70 (Hsc70). Some studies have found that blocking Dnm weakens the role of neurons in recovering excitatory neurotransmitters such as glutamate [18], and a large number of glutamate toxic neurotransmitters accumulates in neurons, increasing neuronal excitability. Helbig [19] reported that clathrin related gene mutations damage CME and lead to neurodevelopmental disorders. Synaptic vesicle circulation is the basis of neurotransmitter transmission. Vesicle endocytosis can recover glutamate in time and prevent continuous excitation of neurons. Vesicular endocytosis disorder is also closely related to Parkinson's disease (PD); Pan found that in the mouse mutant model of Leucine-rich repeatkinase2 (LRRK2), the mutation interferes with the vesicle Swallowing, which causes PD [20]. CME not only participates in the recycling of presynaptic vesicles, but also mediates the endocytosis, transport and circulation of a variety of neurotransmitter receptors to regulate the signal intensity of neurotransmitters, including α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR), N-methyl-D-aspartate receptors (NMDARs), and Gamma aminobutyric acid receptors (GABAR) [21–25].

Animals

Healthy male C57BL / 6 mice (weight 20-28g, age 8-10W) were used in this experiment. All mice were purchased from Changsha tianqin Biotechnology Co., Ltd. (license: scxk (Xiang) 2018-0014) and randomly divided into Four groups: normal control group, epilepsy group, solvent control group and 3-mA group. All animal experiment procedures strictly followed the relevant requirements of animal ethics and obtained the approval of the experimental animal ethics committee. constant temperature (22–26 ℃), independent pure drinking water and standard feed are raised in separate cages (5 / cage).

Behavioural experiments

PTZ kindled mouse chronic model is similar to human seizures. It is a classic model of epilepsy pathogenesis and drug efficacy[26]. C57BL/6 mice were intraperitoneally injected with a subthreshold dose of PTZ (35mg/kg). After each PTZ injection, mice with seizures of grade 4 and above were regarded as chronic kindling model for three consecutive times according to Racine[27] scoring standard. If the attack is not terminated within 5min, diazepam (5mg / kg) shall be injected intraperitoneally to terminate the attack. Normal group: given the same volume of normal saline instead of PTZ intraperitoneal injection, and the feeding conditions were the same as those of epilepsy model group.The mice in the model group and the normal control group were deeply anesthetized. Cut off the neck with surgical scissors, quickly peel off the skull with hemostatic forceps, and separate the hippocampus and cortex on ice for immunoblotting.

The mice were randomly divided into 3-MA group (3mg / kg), epilepsy group and solvent group. After 30 minutes, PTZ was injected intraperitoneally according to the subthreshold dose of 35mg/kg body weight for 15 days. The latency of complete kindling and the highest level of seizure were recorded on a daily basis.

C57BL / 6 mice were deeply anesthetized and a 0.5uL microinjector was used to extract kainic acid (KA, 1 nmol) into hippocampus. Use a micro electric drill to drill the skull with the anterior fontanelle as 0 point, backward (AP) 1.6mm, mediolateral (ML) 1.5mm and depth (DV) 1.5mm[28]. After setting the parameters, install a micro syringe for injection for 1min and keep the needle for 5min. After the same procedure as above, Tf-A488 (5ul) was injected into unilateral mouse hippocampus at the speed of 1ul / min under the stereotactic instrument to establish pentylenetetrazol mouse model, which was injected intraperitoneally for 1 week.After anesthesia, 0.9% normal saline (about 30ml) and 4% paraformaldehyde (about 40ml) were injected into the mice for fixation, and the brain was taken out.Place it in 4% paraformaldehyde solution for 24 hours, PBS configuration concentration is 20% sucrose, and carry out gradient dehydration for 24 hours.Add sucrose to make the solution concentration 30%, continue to dehydrate for 24 hours, take out the tissue, put it into a self-made box made of tin foil, add OCT embedding temperature compound, and put it into liquid nitrogen for quick freezing Fix the tissue on the base with OCT embedding agent and put it into the slicer for slicing (16um) For laser confocal microscopy.

Field potential recording

KA epileptic mice were randomly divided into 3-MA group and control group. At the same time, they were given 3-MA and equal volume of normal saline every day for 10 days, and then the local field potential was recorded. The electrodes were connected to the field potential for multi-channel EEG recording. With the baseline stabilized, spontaneous events were recorded continuously for 30 minutes, and epileptic discharges (EDS) with frequency higher than 5Hz, twice higher than the baseline and duration longer than 5S were defined in electrophysiology. Also, each electrophysiological change is accompanied by corresponding behavioral changes, so that the frequency and duration of epileptic discharge could be detected [29].

Primary neuron culture

The day before culture, the surgical instruments were autoclaved and dried, and the glass plates were washed. Take out the 1cm round glass slide from ethanol, put it into a 24 well plate, open the cover and irradiate it with UV for 1h, and then add polylysine solution, at least 400ul per well. Prepare inoculation solution (DMEM of 20% FBS) and culture medium (Neurobasal + B27 + L-Glu + double antibody). Place the prepared inoculum and culture medium in the cell incubator 4H in advance on the day of culture, and loosen the cover. Take SD rats within 24 hours of birth, take out the cerebral cortex and put it into 0.25% trypsin. After being cut into pieces, they were placed in a cell incubator (37 ℃, 5% CO2) for digestion for 10 minutes. Add 3ml inoculum to neutralize trypsin for 5min, blow-mix with pipette, and suck the cell suspension through a 200 mesh filter screen. Add the filtered cell suspension into the inoculation solution and blow it with a pipette. After 72h, cytarabine (10umol/L) was added to the culture medium. Half of the solution was changed with Neurobasal medium every two days in a 10 day’s procedure.

Establishment of magnesium free model of cultured neurons

After removing the whole solution of maintenance medium, it was placed in magnesium free extracellular solution (NaCl 145, KCl 2.5, CaCl₂ 2, HEPES 10, glucose 10, glycine 0.002mmol/L). MgCl₂ (1mmol/L) is added to the above extracellular fluid. The neurons were treated with the above extracellular solution and placed in the (37℃ 5% CO2) incubator for 3h.

Fluorescence quantification

MitoTracker (30nM) and LysoTracke (50nM) to the culture medium of the control group and epilepsy group respectively for treatment for 30min (37℃ incubator, away from light), suck and discard the above incubation medium for cell fixation and immunofluorescence staining.

Transferrin-Alexa488(Tf-A488) was used to detect CME endocytosis in neurons.

PBS slowly washed the cell climbing sheet for 3 times. Inhibitor group: 3-MA (100mm), treated for 30min. Add Tf-A488 prepared with culture medium in each group, with the final concentration of 50ug/ml, put it back into the (37℃, 5% CO2, dark) cell incubator, incubate for 20 minutes, suck and discard the culture medium, and carry out cell fixation and immunofluorescence staining.

Immunofluorescence staining

Immunofluorescence staining was performed with neuron specific marker Microtubule-associated protein-2 (MAP2).Take out the climbing tablets, wash the cells with PBS twice, add 4% paraformaldehyde and fix in the 37 ℃ incubator for 15min, and wash away the residual fixing solution with PBS for 3 time, 5 minutes each time.Add 0.3% Triton X-100, place it at room temperature for 15 minutes, and wash off the residual liquid with PBS for 3 times for 5 minutes each time.The primary antibody working solution prepared with PBS (4% goat serum + 1:200MAP2) was added dropwise and incubated overnight at 4℃.Take out the climbing tablets and wash them in PBS buffer for 3 times for 10 minutes each time.The fresh secondary antibody Goat anti guinea pig dylight 633 (1:300) was added and incubated at room temperature for 1 hour. PBS was washed three times for 10 minutes each time. Drip the anti fluorescence attenuation sealing agent containing DAPI to seal the film.

Western Blot

RIPA buffer and protease inhibitors were used to homogenize all samples. The protein concentration of the supernatant was measured with a BCA kit according to the manufacturer's instructions. a total of 50 µg protein from each sample was separated via SdS-PAGE (5% separating, 10% stacking gel), and then proteins were transferred to PVdF membranes (250 mA for 90 min). After blocking for 1 h at room temperature in skimmed milk (8%), PVDF membranes were incubated with primary antibodies Parkin(1:1,500) and anti-GAPDH (1:1500) overnight at 4˚C. The blots were washed with PBS with 1% Tween-20 (PBST) three times and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:4,000) for 1h at room temperature.After exposure to light, the Super ECL Plus luminescent agent was fully contacted with the PVDF film. The Fusion FX5 gel imager was used for exposure, and the OD value of the strip was analyzed by Fusion software.

Statistical analysis

Use SPSS18.0 for data statistical analysis, and Graphpad Prism 6.0 software for drawing, so the data drawing is expressed as mean ± standard deviation (mean ± SD). The results of Western blot and neuron immunofluorescence experiment were used to count the differences between the two groups by Student's t test, and the results of Western blot and fluorescent dye stereo injection were used to evaluate the differences between multiple groups by one-way ANOVA. Behavioral science uses repeated ANOVA to compare the data obtained from multiple measurements of the same observation index at different time points among multiple groups. P < 0.05 indicates that the difference was statistically significant.

To detect the expression of parkin in hippocampus and cortex of epileptic mice

The expression of parkin in PTZ kindled epileptic mouse model was detected by Western blot. It was found that the expression of parkin in hippocampus and cortex of epileptic mice was significantly increased compared with the control group (P < 0.05) (Fig. 1).

The expression of parkin in hippocampus and cortex of mice inhibited by 3-MA was detected

The increased expression of parkin in PTZ kindled epilepsy model suggests that mitochondrial autophagy may be involved.In order to solve this problem, we designed the following experiments. 3-MA is an enzyme that can inhibit the formation of autophagosome and the occurrence of autophagy. After intraperitoneal injection of 3-MA (3mg/kg), equal volume of solvent and normal saline, the brain tissues of each group were taken after observation for 30min. The expression of Parkin in the hippocampus and cortex of mice was detected by Western blot. The experimental results showed that the expression of Parkin in the hippocampus and cortex of 3-MA group was lower than that of the control group. It was further confirmed that 3-MA could inhibit autophagy half an hour after intraperitoneal injection. There was significant difference between the inhibitor group and the control group (P < 0.05) (Fig. 2).

Expression of Mito tracker and lyso tracker in cultured neuron epilepsy model

In order to further verify the changes of mitochondrial autophagy in the epilepsy group, we constructed primary cultured neuron magnesium free extracellular fluid induced epilepsy model, and detected the fluorescence uptake of Mito Tracker、Lyso Tracker in the epilepsy group and the control group by fluorescence staining. The results showed that they were increased in the epilepsy group (P < 0.05) (Fig. 3).

Effect of 3-MA on CME in cultured neurons

The neurons cultured on day 10 were pretreated with 3-MA (100 mm) for 30 minutes. Tf-A488 was added to each group.Compared with the control group, the fluorescence intensity of tf-a488 decreased significantly (P < 0.05) (Fig. 4).

Effect of injection of 3-MA on CME in mice

Adult male mice were stereotaxically injected with Tf-A488 in the hippocampus. 30 minutes before PTZ intraperitoneal injection, 3-MA and the same volume of normal saline and solvent were given respectively, once a day, Frozen sections of brain tissue were taken after 1 week. The fluorescence intensity of Tf-A488 in 3-MA group was significantly lower than that in epilepsy group, and that in normal group was lower than that in epilepsy group (P < 0.05) (Fig. 5)

Effect of 3-MA on behavior in epileptic mice

3-MA attenuated the fluorescence uptake of Tf-488 in neurons and mouse epilepsy models, indicating that it may affect the endocytosis function of vesicles. However, whether the effect of endocytosis function can cause behavioral changes in mice. Half an hour before PTZ modeling, each group of mice was injected with 3-MA, the same volume of normal saline and the solvent required for 3-MA. The chronic kindled epilepsy model was observed for 15 days. The results showed that 3-MA group increased the degree of epileptic attack compared with the control group on the 3-9th day; The latency of 3-MA group (9.37 ± 1.65) was also significantly shorter than that of control group (13.5 ± 1.8) and solvent group (13.25 ± 2.0) (P < 0.05). After KA modeling, 3-MA was injected intraperitoneally every day for 1 week. Hippocampal field potentials were recorded in different groups of mice. It was found that obvious EDS appeared in the EEG of mice and EDS was recorded within 30 minutes. The results showed that the times and total duration of EDs in 3-MA group were significantly higher than those in the control group (P < 0.05) (Fig. 6)

Parkin is an E3 ubiquitin ligase. Continuous research has found that parkin plays an important role in the pathogenesis of neurodegenerative diseases and can regulate mitochondrial autophagy. When mitochondria are damaged, parkin will aggregate onto mitochondria and mediate classical mitochondrial autophagy. Knockout of mouse Parkin gene resulted in a large number of neuron loss, accumulation of damaged mitochondria, disorder or interruption of mitochondrial autophagy to varying degrees, and selective damage to neurons [30]. In epilepsy, some scholars have proposed that parkin mediated abnormal mitochondrial autophagy is an important reason for myoclonic epilepsy and ragged red fibers (MERRF) [31].

Mitochondria are constantly in a dynamic balance process of fusion and division. After mitochondrial fusion, division will produce a mitochondria with normal function and a mitochondria with lower membrane potential. The latter is likely to become the object of autophagy because the membrane potential is reduced and cannot fuse with normal mitochondria [32]. Mitochondrial autophagy is to make the damaged mitochondria specifically wrapped into autophagosomes and degraded after fusion with lysosomes, It is the selective removal and degradation of these damaged mitochondria, that plays a key role in controlling the quality of mitochondria. Mitochondrial autophagy can be roughly divided into four stages: The permeability changes after mitochondrial damage; The depolarized mitochondria form autophagosomes (3-MA can inhibit this process); Autophagosomes combine with lysosomes to form autophagic lysosomes; Lysosomes degrade mitochondria [33, 34].Studies have shown that under stress, parkin induced mitochondrial autophagy has a protective effect on neurons and can prevent cell death [35]. Studies have shown that under stress, parkin induced mitochondrial autophagy has a protective effect on neurons and can prevent cell death (Trempe and Fon 2013). Our results showed that the expression of parkin in the chronic epilepsy model ignited by PTZ and the increase of Mito tracker and lyso tracker in the neuronal epilepsy model induced by magnesium free extracellular fluid of primary cultured neurons, suggesting that autophagy may be involved in the occurrence of epilepsy.

The recycling of presynaptic vesicles is very important for many physiological processes. If this process is blocked, it will lead to nervous system diseases such as epilepsy [36].Among them, CME plays a key role in vesicle circulation and regeneration. The indispensable molecules involved in CME in mammals mainly include AP2, Dnm, Hsc70 and so on. AP2 plays a key role in regulating excitability / inhibition by controlling the endocytosis of presynaptic vesicular glutamate transporter (VGLUT) and vesicular GABA transporter (VGAT) and the number of postsynaptic glutamate and GABAA receptors [35]; Therefore, AP2 functional defect in neurons is the basis of epilepsy [36]. Dnml is a key member in the process of CME. In the recycling of presynaptic membrane vesicles, dnm l is assembled in different structures and gathered at the nerve endings. By hydrolyzing the energy provided by the GTP driving mechanism, Dnm1 cuts the pits formed after clathrin coating and separates them from the cell membrane. Von Observed that epilepsy occurred in up to 80% of 21 patients with Dnm1 mutation [37]; The abnormal expression of Dnm1 in the cortex of epilepsy may be the basis of the abnormal expression of Dnm1 in the seizure. Therefore, we injected Tf-A488 stereotaxically into the hippocampus of mice. The results showed that the fluorescence intensity of Tf-A488 in the epilepsy group was lower than that in the normal control group, indicating that CME was involved in the formation of epilepsy; The decrease was more obvious in 3-MA group; In the cultured neurons, the fluorescence intensity of Tf-A488 in the 3-MA group was also weakened compared with the control group. Finally, the application of autophagy inhibitor 3-MA in the two epilepsy models showed that the inhibition of autophagy increased the susceptibility to seizures and shortened the latency of seizures through behavior (PTZ) and field potential (KA). It can be inferred that autophagy is slightly elevated in the epilepsy model, which plays a protective role on neurons and promotes the clearance of damaged mitochondria, which is of great significance for maintaining the energy supply of cells. Inhibition of autophagy leads to the abnormal accumulation of damaged mitochondria, aggravates mitochondrial damage, affects the dynamic balance, structure and function of mitochondria, and further increases the excitability and sensitivity of neurons. Therefore, it is speculated that the aggravation of seizures by 3-MA may be related to the failure to remove damaged mitochondria in time and effectively after inhibiting mitochondrial autophagy, affecting the vesicle endocytosis function of CME and increasing the susceptibility to epilepsy.

Funding The present study was supported by grants from the national natural Science Foundation of china (grant nos. 82101527), the Guizhou Provincial Science and Technology Foundation [grant nos. gzwkj2021-017 and gzwkj2022-009] and Science and Technology Project in Zunyi [grant nos. HZ-（2021）64].

Conflict of interest All authors declare no conflict of interest.

Thijs RD, Surges R, O'Brien TJ, Sander JW. Epilepsy in adults[J]. Lancet, 2019, 393(10172): 689-701.
Kavehei O, Hamilton TJ, Truong ND, Nikpour A. Opportunities for Electroceuticals in Epilepsy [J]. Trends Pharmacol Sci, 2019, 40(10): 735-746.
Ngugi AK, Bottomley C, Fegan G, et al. Premature mortality in active convulsive epilepsy in rural kenya: Causes and associated factors [J]. Neurology, 2014, 82(7):582-589.
Mallok A, Vaillant JD, Soto MT, et al. Ozone protective effects against ptz-induced generalized seizures are mediated by reestablishment of cellular redox balance and a1 adenosine receptors [J]. Neurological research, 2015, 37(3):204-210.
Arnold S. Cenobamate: new hope for treatment-resistant epilepsy[J]. Lancet Neurol, 2020, 19(1): 23-24.
Halász P, Ujma PP, Fabó D, Bódizs R, Szűcs A. Epilepsy as a derailment of sleep plastic functions may cause chronic cognitive impairment - A theoretical review [J]. Sleep Med Rev, 2019, 45: 31-41.
Verma A, Kumar A. Sudden unexpected death in epilepsy: some approaches for its prevention and medico-legal consideration[J]. Acta Neurol Belg, 2015, 115(3): 207-212.
Twig G, Elorza A, Molina AJ, et al. Fission and selective fusion govern mitochondrial segregation and elimination by autophagy[J]. EMBO J, 2008, 27(2): 433-46.
El-Hattab AW, Suleiman J, Almannai M, Scaglia F. Mitochondrial dynamics: Biological roles, molecular machinery, and related diseases[J]. Mol Genet Metab, 2018, 125(4): 315-321.
Deas E, Plun-Favreau H, Gandhi S, et al. PINK1 cleavage at position A103 by the mitochondrial protease PARL[J]. Hum Mol Genet, 2011, 20(5): 867-79.
Nardin A, Schrepfer E, Ziviani E. Counteracting PINK/Parkin Deficiency in the Activation of Mitophagy: A Potential Therapeutic Intervention for Parkinson's Disease[J]. Curr Neuropharmacol, 2016, 14(3): 250-9.
Heo JM, Ordureau A, Paulo JA, Rinehart J, Harper JW. The PINK1-PARKIN Mitochondrial Ubiquitylation Pathway Drives a Program of OPTN/NDP52 Recruitment and TBK1 Activation to Promote Mitophagy[J]. Mol Cell, 2015, 60(1): 7-20.
Wu M, Liu X, Chi X, et al. Mitophagy in Refractory Temporal Lobe Epilepsy Patients with Hippocampal Sclerosis[J]. Cell Mol Neurobiol, 2018, 38(2): 479-486.
Giorgi FS, Biagioni F, Lenzi P, Frati A, Fornai F. The role of autophagy in epileptogenesis and in epilepsy-induced neuronal alterations[J]. J Neural Transm (Vienna), 2015, 122(6): 849-862.
Sudhof TC. The synaptic vesicle cycle[J]. Annu Rev Neurosci, 2004, 27: 509-547.
McMahon HT, Boucrot E. Molecular mechanism and physiological functions of clathrin-mediated endocytosis[J]. Nat Rev Mol Cell Biol, 2011, 12(8): 517-533.
Schmid SL. Clathrin-coated vesicle formation and protein sorting: an integrated process [J]. Annu Rev Biochem, 1997, 66: 511-548.
Fan F, Funk L, Lou X. Dynamin 1- and 3-Mediated Endocytosis Is Essential for the Development of a Large Central Synapse In Vivo [J]. J Neurosci, 2016, 36(22): 6097-6115.
Helbig I, Lopez-Hernandez T, Shor O, et al. A Recurrent Missense Variant in AP2M1 Impairs Clathrin-Mediated Endocytosis and Causes Developmental and Epileptic Encephalopathy [J]. Am J Hum Genet, 2019, 104(6): 1060-1072.
Pan PY, Li X, Wang J, et al. Parkinson's Disease-Associated LRRK2 Hyperactive Kinase Mutant Disrupts Synaptic Vesicle Trafficking in Ventral Midbrain Neurons [J]. J Neurosci, 2017, 37(47): 11366-11376.
Schubert KO, Föcking M, Prehn JH, Cotter DR. Hypothesis review: are clathrin-mediated endocytosis and clathrin-dependent membrane and protein trafficking core pathophysiological processes in schizophrenia and bipolar disorder [J]. Mol Psychiatry, 2012, 17(7): 669-681.
Loebrich S, Benoit MR, Konopka JA, Cottrell JR, Gibson J, Nedivi E. CPG2 Recruits Endophilin B2 to the Cytoskeleton for Activity-Dependent Endocytosis of Synaptic Glutamate Receptors [J]. Curr Biol, 2016, 26(3): 296-308.
Fiuza M, Rostosky CM, Parkinson GT, et al. PICK1 regulates AMPA receptor endocytosis via direct interactions with AP2 α-appendage and dynamin [J]. J Cell Biol, 2017, 216(10): 3323-3338.
Yu A, Shibata Y, Shah B, Calamini B, Lo DC, Morimoto RI. Protein aggregation can inhibit clathrin-mediated endocytosis by chaperone competition [J]. Proc Natl Acad Sci USA, 2014, 111(15): E1481-1490.
Savas JN, Ribeiro LF, Wierda KD, et al. The Sorting Receptor SorCS1 Regulates Trafficking of Neurexin and AMPA Receptors [J]. Neuron, 2015, 87(4): 764-780.
Girard B, Tuduri P, Moreno MP, et al. The mGlu7 receptor provides protective effects against epileptogenesis and epileptic seizures[J]. Neurobiol Dis, 2019, 129: 13-28.
Racine RJ. Modification of seizure activity by electrical stimulation. II. Motor seizure[J]. Electroencephalogr Clin Neurophysiol, 1972, 32(3): 281-294.
Häussler U, Rinas K, Kilias A, Egert U, Haas CA. Mossy fiber sprouting and pyramidal cell dispersion in the hippocampal CA2 region in a mouse model of temporal lobe epilepsy[J]. Hippocampus, 2016, 26(5): 577-588.
Zheng F, Yang Y, Lu S, et al. CD36 Deficiency Suppresses Epileptic Seizures[J]. Neuroscience, 2017, 367: 110-120.
Noda S, Sato S, Fukuda T, et al. Loss of Parkin contributes to mitochondrial turnover anddopaminergic neuronal loss in aged mice[J]. Neurobiol Dis, 2020, 136: 104717.
Villanueva-Paz M, Povea-Cabello S, Villalón-García I, et al. Parkin-mediated mitophagy and autophagy flux disruption in cellular models of MERRF syndrome[J]. Biochim Biophys Acta Mol Basis Dis, 2020, 1866(6): 165726.
Scarffe LA, Stevens DA, Dawson VL, Dawson TM. Parkin and PINK1: much more than mitophagy[J]. Trends Neurosci, 2014, 37(6): 315-324.
Kim I, Lemasters JJ. Mitochondrial degradation by autophagy (mitophagy) in GFP-LC3 transgenic hepatocytes during nutrient deprivation[J]. Am J Physiol Cell Physiol, 2011, 300(2): C308-317.
Xie R, Nguyen S, McKeehan WL, Liu L. Acetylated microtubules are required for fusion of autophagosomes with lysosomes[J]. BMC Cell Biol, 2010, 11: 89.
Trempe JF, Fon EA. Structure and Function of Parkin, PINK1, and DJ-1, the Three Musketeers of Neuroprotection [J]. Front Neurol. 2013. 4: 38.
Robinson PJ. Neuroscience. How to fill a synapse[J]. Science, 2007, 316(5824): 551-553.
von SS, Helbig KL, Shinde DN, et al. DNM1 encephalopathy: A new disease of vesicle fission[J]. Neurology. 2017. 89(4): 385-394.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

The mechanism of mitochondrial autophagy regulating CME in epilepsy

Status:

Version 1

Abstract

Figures

Introduction

Experimental Procedures

Animals

Behavioural experiments

Field potential recording

Primary neuron culture

Establishment of magnesium free model of cultured neurons

Fluorescence quantification

Immunofluorescence staining

Western Blot

Statistical analysis

Results

To detect the expression of parkin in hippocampus and cortex of epileptic mice

The expression of parkin in hippocampus and cortex of mice inhibited by 3-MA was detected

Expression of Mito tracker and lyso tracker in cultured neuron epilepsy model

Effect of 3-MA on CME in cultured neurons

Discussion

Declarations

References

Additional Declarations

Status:

Version 1