The mechanism of mitochondrial autophagy regulating Clathrin‐mediated endocytosis in epilepsy

Abstract

Objective

The objective of this study is to determine whether inhibition of mitophagy affects seizures through Clathrin‐mediated endocytosis (CME).

Methods

Pentylenetetrazol (PTZ) was intraperitoneally injected daily to establish a chronic PTZ‐kindled seizure. The Western blot (WB) was used to compare the differences in Parkin protein expression between the epilepsy group and the control group. Immunofluorescence was used to detect the expression of MitoTracker and LysoTracker. Transferrin‐Alexa488 (Tf‐A488) was injected into the hippocampus of mice. We evaluated the effect of 3‐methyladenine (3‐MA) on epilepsy behavior through observation in PTZ‐kindled models.

Results

The methylated derivative of adenine, known as 3‐MA, has been extensively utilized in the field of autophagy research. The transferrin protein is internalized from the extracellular environment into the intracellular space via the CME pathway. Tf‐A488 uses a fluorescent marker to track CME. Western blot showed that the expression of Parkin was significantly increased in the PTZ‐kindled model (p < 0.05), while 3‐MA could reduce the expression (p < 0.05). The fluorescence uptake of MitoTracker and LysoTracker was increased in the primary cultured neurons induced by magnesium‐free extracellular fluid (p < 0.05); the fluorescence uptake of Tf‐A488 was significantly decreased in the 3‐MA group compared with the control group (p < 0.05). Following hippocampal injection of Tf‐A488, both the epilepsy group and the 3‐MA group exhibited decreased fluorescence uptake, with a more pronounced effect observed in the 3‐MA group. Inhibition of mitophagy by 3‐MA from day 3 to day 9 progressively exacerbated seizure severity and shortened latency.

Significance

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.

Summary

Abnormal mitophagy was observed in a chronic pentylenetetrazol‐induced seizure model and a Mg²⁺‐free‐induced spontaneous recurrent epileptiform discharge model. A fluorescent transferrin marker was utilized to track clathrin‐mediated endocytosis. Using an autophagy inhibitor (3‐methyladenine) on primary cultured neurons, we discovered that inhibition of autophagy led to a reduction in fluorescent transferrin uptake, while impairing clathrin‐mediated endocytosis function mediated by mitophagy. Finally, we examined the effects of 3‐methyladenine in an animal model of seizures showing that it exacerbated seizure severity. Ultimately, this study provides insights into potential mechanisms through which mitophagy regulates clathrin‐mediated endocytosis in epilepsy.

Aberrant mitochondrial autophagy facilitates the occurrence of seizures.

Key points.

• Epilepsy is a prevalent neurological disorder, yet its pathogenesis remains elusive. The relationship between epilepsy and mitophagy, a complicated process with dual effects, remains unknown.

• Mitophagy is a process of removing and transferring damaged mitochondria to lysosomes for degradation, thereby regulating the quality of mitochondria and maintaining the stability of the intracellular environment.

• Clathrin‐mediated endocytosis (CME) plays an important role in the endocytosis and recovery of vesicles. It is also the main way for macromolecules to enter cells and is widely involved in the signal transduction of various cellular physiological activities.

• The aberrant mitochondrial autophagy disrupts the vesicular endocytosis of CME and enhances susceptibility to epilepsy.

1. INTRODUCTION

Epilepsy is a chronic neurological disease caused by the abnormal synchronous discharge of neurons with a clinical manifestation 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 , ² , ³ , ⁴ At present, the treatment of epilepsy is mainly drug intervention. Although a wide variety of antiseizure medications 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 dynamic organelles that undergo continuous fusion and division processes 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 and transferring damaged mitochondria to lysosomes for degradation to regulate the quality of mitochondria and maintain the stability of the 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 various neurological diseases, including epilepsy. In the hippocampus of patients with intractable temporal lobe epilepsy, transmission electron microscopy (TEM) revealed the abnormal accumulation of damaged mitochondria. 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 an epileptic network. ¹⁴

Synapse is the contact part between neurons and is important 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 comprises a heavy chain protein with a molecular weight of 192 kDa and a light chain protein with a molecular weight of 33–35 kDa. CME is important in vesicle endocytosis and recovery. It is responsible for the uptake of transmembrane receptors and transporters, for remodeling plasma membrane composition in response to environmental changes, and for regulating cell surface signaling.

Additionally, it serves as the primary pathway through which most substances enter the cell. ¹⁶ , ¹⁷ It is widely involved in signal transduction of various 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. ¹⁸ During these stages, the involvement of molecules such as adaptor protein‐2 (AP2), Dynamin (Dnm), and heat shock cognate protein 70 (Hsc70) is indispensable. Some studies have found that blocking Dnm weakens the role of neurons in recovering excitatory neurotransmitters such as glutamate, ¹⁹ and many glutamate‐toxic neurotransmitters accumulate in neurons, increasing neuronal excitability. Helbig ²⁰ 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 repeat kinase2 (LRRK2), the mutation interferes with the vesicle Swallowing, which causes PD. ²¹ 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). ²² , ²³ , ²⁴ , ²⁵ , ²⁶

To elucidate the impact of mitophagy on the pathogenesis and progression of epilepsy, we initially assessed mitophagy abnormalities in a chronic PTZ‐kindled seizures model and an Mg²⁺‐free‐induced spontaneous recurrent epileptiform discharge model, thereby implying a potential association between mitophagy and epilepsy. 3‐MA has gained widespread usage as an autophagy inhibitor due to its specific ability to impede the fusion process between autophagic vesicles and lysosomes. The transferrin protein is internalized from the extracellular environment into the intracellular space via the CME pathway. Tf‐A488 uses a fluorescent marker to track CME. Consequently, we used the autophagy inhibitor 3‐MA to investigate further its effects on fluorescence uptake of Tf‐A488 in primary cultured neurons and that inhibition of autophagy led to decreased uptake while inhibiting mitophagy‐impaired CME function. Finally, we examined the influence of 3‐MA on seizure behavior and the local field potentials (LFPs) in the kainic acid (KA)‐induced model. Ultimately, this study provides insights into the plausible mechanism underlying mitophagy‐mediated regulation of CME in epilepsy.

2. MATERIALS AND METHODS

2.1. Animals

Healthy male C57BL/6 mice (weight 20–28 g, age 8–10 W) 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, seizures group, solvent control group, and 3‐MA group. Mice were housed in groups of 5/cage under cycles of 12‐h light/12‐h dark, with ad libitum access to water and food. All animal experiment procedures strictly followed the relevant animal ethics requirements and obtained the approval of the experimental animal ethics committee.

2.2. In vivo seizure models

2.2.1. Kainate model

C57BL/6 mice were deeply anesthetized, and a 0.5 μL microinjector was used to extract kainic acid (KA, 1 nM) into the hippocampus. Stereotaxic injections into the dorsal region of the CA1 area were performed at the following coordinates with respect to bregma: anteroposterior, −1.6 mm; mediolateral, −1.5 mm; and 1.5 mm below the dura. ²⁷ We injected KA over a 1‐min period. At the end of the injection, the microsyringe remained in situ for an additional 5 min and was finally withdrawn slowly.

2.2.2. PTZ model

PTZ‐kindled seizures mouse chronic model is similar to human seizures. It is a classic model of epilepsy pathogenesis and drug efficacy. ²⁸ C57BL/6 mice were intraperitoneally injected with a subthreshold dose of PTZ (35 mg/kg). After the injections, we observed and recorded the evoked behavioral seizures of the mice on the basis of the standard Racine Scale. ²⁹ Mice that showed at least three consecutive level 4 or 5 epileptic seizures after receiving PTZ injections were considered completely kindled, and we classified those mice as the epilepsy group. If the attack is not terminated within 5 min, diazepam (5 mg/kg) was injected intraperitoneally to terminate the attack. Normal group: given the same volume of normal saline instead of PTZ intraperitoneal injection, the feeding conditions were the same as those of the PTZ‐kindled seizures model group. The mice in the model and normal control groups were deeply anesthetized. Mice were decapitated using surgical scissors, and the skull was peeled off with hemostatic forceps. The cortex and hippocampus were dissected on ice, collected, and processed for immunostaining. The mice were randomly divided into the 3‐MA group (3 mg/kg), PTZ‐kindled seizures, and solvent groups. After 30 min, PTZ was injected intraperitoneally according to the subthreshold dose of 35 mg/kg body weight for 15 days. The latency of seizures and the highest level of seizure were recorded daily.

Stereotaxic injections Tf‐A488

The Tf‐A488 (5 μL) was injected into the unilateral hippocampus of the mice at a rate of 1 μL/min under the stereotaxic apparatus, while PTZ was administered intraperitoneally for 1 week following the protocol described in (2.2.2) to establish the PTZ‐kindled seizures model. After anesthesia, 0.9% normal saline (about 30 mL) and 4% paraformaldehyde (about 40 mL) were injected into the mice for fixation, and the brain was taken out and placed in 4% paraformaldehyde solution for 24 h, with a PBS configuration concentration is 20% sucrose, and carry out gradient dehydration for 24 h. Add sucrose to make the solution concentration 30%, continue dehydrating for 24 h, remove 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 an OCT embedding agent and put it into the slicer for slicing (16 μm) for laser confocal microscopy.

2.3. Field potential recording

KA‐induced models were randomly divided into 3‐MA (3 mg/kg) and control groups. At the same time, they were given 3‐MA and an equal volume of normal saline every day for 10 days, and then the local field potentials (LFPs) were recorded. In the KA‐induced seizures model, LFPs were recorded by implanting a multichannel microwire array (a 4 × 4 array of platinum‐iridium alloy wires, each 25 μm in diameter, Plexon, Dallas, TX, USA) in the right dorsal hippocampus (anteroposterior, −1.8 mm; mediolateral, −1.5 mm; and 1.5 mm below the dura). Electrophysiological epileptiform‐like discharges were defined as spike activity lasting longer than 5 s with high frequency (>5 Hz) and high amplitude (>2 times the baseline). For each recording session, we determined the frequency and duration of seizure‐like events (SLEs).

2.4. Primary neuron culture

Before culture, the surgical instruments were autoclaved and dried, and the glass plates were washed. Remove the 1 cm round glass slide from ethanol, put it into a 24‐well plate, open the cover, irradiate it with UV for 1 h, and then add polylysine solution, at least 400 μL 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 h of birth, remove the cerebral cortex, and put it into 0.25% trypsin. After being cut into pieces, they were placed in a cell incubator (37°C, 5% CO₂) for digestion for 10 min. Add 3 mL inoculum to neutralize trypsin for 5 min, blow‐mix with a 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 72 h, cytarabine (10 μM) was added to the culture medium. Half of the solution was changed with Neurobasal medium every 2 days in a 10 days procedure.

2.5. Mg²⁺‐free‐induced spontaneous recurrent epileptiform discharge model of cortex neurons

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

2.6. Fluorescence quantification

The cultured neurons in Mg²⁺ media were treated with MitoTracker (30 nM) and LysoTracker (50 nM) in the culture medium of the control group and epilepsy group, respectively, for treatment for 30 min (37°C incubator, away from light), suck and discard the above incubation medium for cell fixation and immunofluorescence staining.

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

PBS slowly washed the cell climbing sheet three times. Inhibitor group: 3‐MA (10 mM), treated for 30 min. Add Tf‐A488 (The transferrin protein is internalized from the extracellular environment into the intracellular space via the CME pathway. Tf‐A488 was used as a fluorescent marker to track CME. ³⁰ ) prepared with culture medium in each group, with the final concentration of 50 μg/mL, put it back into the (37°C, 5% CO₂, dark) cell incubator, incubate for 20 min, suck, and discard the culture medium, and carry out cell fixation and immunofluorescence staining.

2.8. Immunofluorescence staining

Immunofluorescence staining was performed with neuron‐specific marker Microtubule‐associated protein‐2 (MAP2). Remove the climbing tablets, wash the cells with PBS twice, add 4% paraformaldehyde, and fix them in the 37°C incubator for 15 min, and wash away the residual fixing solution with PBS 3 times, 5 min each time. Add 0.3% Triton X‐100, place it at room temperature for 15 min, and wash off the residual liquid with PBS three times for 5 min each time. The primary antibody working solution prepared with PBS (4% goat serum + 1:200 MAP2) was added dropwise and incubated overnight at 4°C. Remove the climbing tablets and wash them in PBS buffer 3 times for 10 min each time. The fresh secondary antibody Goat anti guinea pig daylight 633 (1:300) was added and incubated at room temperature for 1 h. PBS was washed three times for 10 min each time. Drip the anti‐fluorescence attenuation sealing agent containing DAPI to seal the film. Images were captured using laser‐scanning confocal microscopy (Nikon) under an Olympus IX 70 inverted microscope equipped with a Fluoview FVX confocal scan head. The fluorescence intensity was analyzed using Image‐Pro Plus software, and colocalization analyses were performed using NIS‐viewer (Nikon).

2.9. 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. 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 (1:1500) 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 (1:4000) for 1 h 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.

2.10. Statistical analysis

Use SPSS18.0 for statistical analysis and GraphPad Prism 6.0 for drawing, so the data drawing is expressed as mean ± standard deviation (mean ± SD). The results of the 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.

3. RESULTS

3.1. Increased expression of Parkin in the hippocampus and cortex of PTZ‐kindled seizures

The PTZ was administered intraperitoneally at a subthreshold dosage of 35 mg/kg body weight for 15 consecutive days. Mice that showed at least three consecutive level 4 or 5 epileptic seizures after receiving PTZ injections were considered completely kindled. The expression of Parkin in the PTZ‐kindled seizures model was detected by Western blot. It was found that the expression of Parkin in the hippocampus and cortex of epileptic mice was significantly increased compared with the control group (p < 0.05; Figure 1).

FIGURE 1.

Parkin expression in PTZ‐kindled seizures model (n = 5/group). Immunoblotting showed that the expression of Parkin in the hippocampus of mice in the PTZ‐kindled seizures group was significant. The increase was compared with the control group (0.79 ± 0.05, 0.46 ± 0.08; p = 0.0079) (A). The expression of Parkin in the cortex of the PTZ‐kindled seizures mouse model also increased. Compared with the normal group, the expression level was (0.43 ± 0.05, 0.26 ± 0.04; p = 0.0164) (C). The above results are the statistical results of the Parkin/GAPDH ratio. (B, D) is the result of the statistical histogram. *p < 0.05 versus control group. Student's t‐tests were performed. The data are expressed as mean ± SEM.

3.2. The expression of Parkin in the hippocampus and cortex of mice inhibited by 3‐MA was detected

The increased expression of Parkin in the PTZ‐kindled seizures model suggests that mitochondrial autophagy may be involved. To solve this problem, we designed the following experiments. 3‐MA is an enzyme that can inhibit the formation of autophagosomes and the occurrence of autophagy. After intraperitoneal injection of 3‐MA (3 mg/kg), equal volume of solvent, and normal saline, the brain tissues of each group were taken after observation for 30 min. 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 the 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 a significant difference between the inhibitor group and the control group (p < 0.05; Figure 2).

FIGURE 2.

Parkin was expressed in the mouse model of the 3‐MA group (n = 5/group). Western blotting showed that the expression of Parkin in mouse hippocampus decreased after administration of 3‐MA. Compared with the control group and solvent group, the intensity ratio of Western blotting was (p = 0.0049) (A). The identical trend was observed in the mouse cortex as in the hippocampus, wherein the Western blot intensity ratio was detected (p = 0.0246) (C). Control group and DMSO group. There was no significant difference between the two groups (p > 0.05). The above results are the statistical results of the Parkin/GAPDH ratio. (B, D) is the result of the statistical histogram. *p < 0.05 versus control group. One‐way ANOVA was performed. The data are expressed as mean ± SEM.

3.3. Expression of MitoTracker and LysoTracker in the Mg²⁺‐free‐induced spontaneous recurrent epileptiform discharge model of cortex neurons

To further verify the changes of mitochondrial autophagy in the epilepsy group, we constructed a primary cultured neuron magnesium‐free extracellular fluid‐induced epilepsy model and detected the fluorescence uptake of MitoTracker, LysoTracker 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; Figure 3).

FIGURE 3.

Effects of fluorescent‐labeled MitoTracker and LysoTracker in the Mg²⁺‐free‐induced spontaneous recurrent epileptiform discharge model of cortex neurons. Confocal images of the control group and epilepsy group, MitoTracker (green), LysoTracker (red), DAPI (blue), MAP2 (purple) (A); The fluorescence quantitative statistical histogram of MitoTracker and LysoTracker in the epilepsy group treated with magnesium‐free compared with the control group LysoTracker fluorescence uptake was significantly enhanced (B). (MitoTracker 42.10 ± 1.614, 57.96 ± 2.566, LysoTracker 31.02 ± 1.266, 36.28 ± 1.151; scale = 75 μm). *p < 0.05 versus control group. Student's t‐tests were performed. The data are expressed as mean ± SEM.

3.4. Effect of 3‐MA on CME in cultured rat cortical neurons

The Tf‐A488 complex consists of transferrin and low‐density lipoprotein (LDL), which are both indicative of the CME pathway for cellular internalization. Therefore, Tf‐A488 is commonly used as a CME marker. ³⁰ , ³¹ , ³² The neurons cultured on day 10 were pretreated with 3‐MA (10 mM) for 30 min. Tf‐A488 (50 μg/mL) was introduced to both the control and 3‐MA groups, with a significantly reduced fluorescence intensity observed in the latter (p < 0.05; Figure 4).

FIGURE 4.

In the primary cortical cultures from rats, Tf‐A488 (green) and MAP2 (red) have a common overlap on neurons (yellow) and DAPI (blue) nuclear staining. The arrow shows positive cells (A). The fluorescence quantitative statistical histogram of Tf‐A488 fluorescence demonstrated a significant reduction in the uptake of Tf‐A488 fluorescence in the 3‐MA group compared to the control group (B) (37.89 ± 2.631, 14.96 ± 1.440; scale = 75 μm; p = 0.0007). *p < 0.05 versus control group. Student's t‐tests were performed. The data are expressed as mean ± SEM.

3.5. Effect of injection of 3‐MA on CME in mice

Adult male mice were stereotaxically injected with Tf‐A488 in the hippocampus. 30 min before PTZ intraperitoneal injection, 3‐MA and the same normal saline and solvent volume were given, respectively. Frozen sections of brain tissue were taken after 1 week. There was a significant difference in the fluorescence intensity of Tf‐A488 between the 3‐MA group and the PTZ‐kindled seizures model group, as well as between the normal group and the PTZ‐kindled seizures model group (p < 0.05; Figure 5).

FIGURE 5.

Expression of Tf‐A488 (green) in the hippocampus of PTZ‐kindled seizures model (A) (n = 5/group). According to the fluorescence statistics of each group in the hippocampal DG area, the fluorescence uptake of the 3‐MA group was significantly lower than that of the PTZ‐kindled seizures model group, and the fluorescence uptake of Tf‐A488 was lower than that of the normal group (B) (scale = 75 μm; *p = 0.0052, ^# p = 0.0047). One‐way ANOVA was performed. The data are expressed as mean ± SEM.

3.6. Effect of 3‐MA on behavior in PTZ‐kindled and KA‐induced seizures model

3‐MA attenuated the fluorescence uptake of Tf‐A488 in neurons and mouse PTZ‐kindled seizures model, indicating that it may affect the endocytosis function of vesicles. However, it remains to be determined whether the endocytic function plays a role in inducing behavioral changes in mice. To investigate this, We designed the following experimental procedure: 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 PTZ‐kindled seizures model was observed for 15 days. The results showed that the 3‐MA group increased the degree of epileptic attack compared with the control group on the 3–9th days; The latency of the 3‐MA group (9.37 ± 1.65) was also significantly shorter than that of the 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 an obvious seizure‐like event appeared in the LFPs of mice, and the LFPs were recorded continuously for 30 min. The results showed that the times and total duration of seizure‐like events in the 3‐MA group were significantly higher than those in the control group (p < 0.05; Figure 6).

FIGURE 6.

Statistical chart of seizure degree and latency in PTZ‐kindled and KA‐induced seizures model (n = 7/group). Compared with the control group, according to the Racine scoring standard, the degree of seizure in the 3‐MA group was significantly higher (3–9 days), and the latency in the 3‐MA group was significantly shorter, which were (9.37 ± 1.65, 13.5 ± 1.8, 13.25 ± 2.0) compared with the control group and solvent group respectively (A) Repeated measures ANOVA analysis were performed. The data are expressed as mean ±SEM; Representative diagram of LFPs seizure‐like event of KA‐induced seizures in 3‐MA group and control group (B); Compared with the control group, 3‐MA group significantly increased the attack frequency and total duration (C, D) (*p < 0.05). Student's t‐tests were performed. The data are expressed as mean ±SEM.

4. DISCUSSION

Our results showed that the expression of Parkin in the chronic seizures model ignited by PTZ and the increase of MitoTracker and LysoTracker 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. 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 the 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 PTZ‐kindled seizures model showed that the inhibition of autophagy increased the susceptibility to seizures and shortened the latency of seizures through behavior (PTZ) and LFPs (KA), indicating that autophagy is slightly elevated in the PTZ‐kindled seizures 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.

The E3 ubiquitin ligase Parkin recognizes and precisely localizes to damaged mitochondria for ubiquitination. Upon activation, Parkin ubiquitinates mitochondrial proteins to generate ubiquitin chains, which PINK1 subsequently phosphorylates. The accumulation of phosphorylated ubiquitin chains on damaged mitochondria triggers further recruitment and activation of Parkin, establishing a positive feedback loop that drives mitochondrial autophagy. ³³ , ³⁴ 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 the mouse Parkin gene resulted in significant neuronal loss, accumulation of damaged mitochondria, disorder or interruption of mitochondrial autophagy to varying degrees, and selective damage to neurons. ³⁵ In epilepsy, some scholars have proposed that Parkin‐mediated abnormal mitochondrial autophagy is an important reason for myoclonic epilepsy and ragged red fibers (MERRF). ³⁶

Mitochondria are constantly in a dynamic balance process of fusion and division. After mitochondrial fusion, the division will produce mitochondria with normal function and 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. ³⁷ Mitochondrial autophagy is to make the damaged mitochondria specifically wrapped into autophagosomes and degraded after fusion with lysosomes. The selective removal and degradation of these damaged mitochondria plays a key role in controlling the quality of mitochondria. There exists a close correlation between epilepsy and mitochondrial autophagy. ³⁸ In the event of excessive mitochondrial fission occurring under physiological or pathological circumstances, an accumulation of impaired mitochondria will occur abnormally. Only by upholding homeostasis within the body's internal milieu can the functionality of mitochondrial autophagy be harnessed to timely and effectively eliminate these damaged mitochondria; otherwise, it may lead to severe detriment to the body, subsequently triggering pathological reactions. ³⁹ 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; and lysosomes degrade mitochondria. ⁴⁰ , ⁴¹ Studies have shown that Parkin‐induced mitochondrial autophagy protects neurons and can prevent cell death under stress. Studies have shown that under stress, Parkin‐induced mitochondrial autophagy protects neurons and can prevent cell death. ⁴²

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. ⁴³ Among them, CME plays a key role in vesicle circulation and regeneration. The indispensable molecules in CME in mammals mainly include AP2, Dnm, and Hsc70. 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. ⁴² Therefore, AP2 functional defect in neurons is the basis of epilepsy. ⁴³ Dnml is a key member in the process of CME. In recycling presynaptic membrane vesicles, dnm l is assembled in different structures and gathered at the nerve endings. By hydrolyzing the energy the GTP driving mechanism provides, Dnm1 cuts the pits formed after clathrin coating and separates them from the cell membrane. Von Spiczak et al observed that epilepsy occurred in up to 80% of 21 patients with Dnm1 mutation. ⁴⁴ The abnormal expression of Dnm1 in the cortex of epilepsy may be the basis of the abnormal expression of Dnm1 in the seizure.

Epilepsy is a prevalent neurological disorder, yet its pathogenesis remains elusive. The relationship between epilepsy and mitophagy, a complex process with dual effects, remains unknown. This study proposes that aberrant mitophagy plays a significant role in seizure generation by potentially impairing clathrin‐mediated endocytosis (CME). 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, thus providing novel insights into the development of epilepsy. However, it should be noted that there are limitations in the overall experimental design. Tf‐A488, as the primary indicator to observe CME, primarily focuses on endocytosis, while abnormal autophagy exacerbates susceptibility to epilepsy. Further investigation is required to explore whether dysregulation of synaptic vesicle recycling or postsynaptic neurotransmitter transport disorders contribute to excitatory/inhibitory imbalance and participate in epileptic seizures.

AUTHORS’ CONTRIBUTIONS

XJZ and YY conceptualized and designed the study; XJZ, YY, and ZZT performed experiments; HQZ and JY helped with data interpretation; ZL, ZCX, and XJZ performed statistical analyses and wrote the manuscript. All authors have read and approved the final version of the manuscript.

FUNDING INFORMATION

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, gzwkj2023‐004 and gzwkj2022‐009], and the Science and Technology Project in Zunyi [grant nos. HZ‐(2021)64].

CONFLICT OF INTEREST STATEMENT

None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. The original unedited version of this data, prior to the journal's peer review, was published in a preprint server at: https://doi.org/10.21203/rs.3.rs‐1640810/v1.

ETHICS STATEMENT

This study was approved by the ethics committee of the Affiliated Hospital of Zunyi Medical University (Nos. KLLY‐2018‐166).

Zhou X, Yang Y, Tai Z, Zhang H, Yang J, Luo Z, et al. The mechanism of mitochondrial autophagy regulating Clathrin‐mediated endocytosis in epilepsy. Epilepsia Open. 2024;9:1252–1264. 10.1002/epi4.12945

DATA AVAILABILITY STATEMENT

The data can be shared upon reasonable request to the corresponding author.