Long-term Rapamycin Treatment Inhibit AKT Activity and Lower Intracellular Calcium Expression in Organotypic Hippocampal Slice Cultures Model of Epilepsy

 

Machlusil Husna1,2*, Kusworini Handono3, Hidayat Sujuti4, Aulanni’am Aulanni’am5,

Afiyfah Kiysa Waafi6, Rumman Karimah6, Alya Satira6

1Doctoral Program in Medical Science, Faculty of Medicine Universitas Brawijaya, East Java, Indonesia.

2Department of Neurology, Faculty of Medicine Universitas Brawijaya,

Saiful Anwar General Hospital, Malang, East Java, Indonesia.

3Department of Clinical Pathology, Faculty of Medicine Universitas Brawijaya,

Saiful Anwar General Hospital, Malang, East Java, Indonesia.

4Department of Ophthalmology, Faculty of Medicine Universitas Brawijaya,

Saiful Anwar General Hospital, Malang, East Java, Indonesia.

5Department of Biochemistry, Laboratory of Biochemistry,

Faculty of Sciences Universitas Brawijaya, East Malang, Java, Indonesia.

6Master Program in Biomedical Sciences, Faculty of Medicine Universitas Brawijaya,

Malang, East Java, Indonesia.

*Corresponding Author E-mail: machlusilhusna.fk@ub.ac.id

 

ABSTRACT:

Despite the development of anti-epilepsy drugs, drug-refractory epilepsy still becomes a challenging problem along with an increased incidence of epilepsy. To face that challenge and increase patients’ quality of life, treatment of epilepsy must effectively prevent epileptogenesis, not only symptomatic treatment. AKT signaling pathway was proven to have important roles in epilepsy through its function in the synaptic plasticity, neurogenesis, axon guidance, modulation of the glutamate transporter, and activation of the Ca2+ channel. AKT also activated mTOR signaling pathway as activator of mTORC1 and also effector of mTORC2. Several studies showed the ability of long-term rapamycin treatment to inhibit mTORC2. This study used organotypic hippocampal slice cultures (OHSC) and long-term rapamycin treatment was administered for 3, 5, 8, and 10 days at a dose of 20 nM after induction of epilepsy by low-Mg2+ medium administration for 40 minutes. Low-Mg2+ medium administration induced seizure activity in OHSC showed by significant increase in intracellular Ca2+expressionand also significantly increase AKT activity. After administration of long-term rapamycin treatment AKT activity and intracellular Ca2+expression were significantly reduced. The longer the treatment of rapamycin, the lower the AKT activity and intracellular Ca2+expression. Long-term rapamycin treatment has the potential to become a novel epilepsy drug through its ability to attenuate AKT activity and suppress the seizures proven by lower intracellular Ca2+expression.

 

KEYWORDS: AKT activity, Calcium, Epilepsy, Rapamycin, Seizure.

 

 


INTRODUCTION: 

Epilepsy is a chronic neurological condition that affects people of all agesas a result of an abnormal and uncontrolled discharge of neurons causing recurrent seizures1,2.

 

In 2016, around 49.5 million people3 worldwide have epilepsy with a global age-standardized mortality rate of up to 1.74 per 100.000 population4. Currently, the treatments of epilepsy rely on symptomatic strategies, targeting to suppress the seizures5, but do not target epileptogenesis processes. Despite the development of the antiepileptic drug, it only provides satisfactory seizure control for 60-70% patients6, there are still 30% of epileptic patients remain unresponsive (refractory epilepsy)5.

One of the types of epilepsy with the highest probability of refractory epilepsy is temporal lobe epilepsy or TLE with a 70% rate of refractory epilepsy7,8. In pediatric epilepsy, approximately 17-40% of patients do not respond to the monotherapy of antiepileptic drug9. Also, some of the antiepileptic drug has serious complication, such as hepatotoxicity of valproic acid10. The development of a new antiepileptic drug is expected to prevent epileptogenesis, not only symptomatic.

 

Recently, mammalian/ mechanistic target of rapamycin (mTOR) pathway has been studied as a key in the regulation of neuronal function and cellular processes related to epileptogenesis11. Accumulating evidence showed mTOR hyperactivity in in vivo and in vitro models of epilepsy11,12, also in human temporal lobe epilepsy specimens13.There are two complexes of mTOR, mTORC1 and mTORC2. Upregulation of mTORC1 has been proven to be critical in epileptogenesis by interfering with neural circuits formation and disrupting established neural networks leading to hyperexcitability of neurons14.

 

mTORC1 is activated by AKT through its inhibition of TSC1/2 which is a negative regulator of mTORC1. AKT is a Ser/Thr kinase protein involved in various neuronal functions via TSC1/2-mTORC1 signaling, including synaptic plasticity, neurogenesis, axon guidance, dendritic morphology, and neuronal polarization15. AKT was also found to have an important role in cytoskeleton organization by phosphorylating several cytoskeleton proteins which plays a key role in the pathogenesis of epilepsy16. Upregulation of PI3K/AKT/mTORC1 expression is causing hyperexcitability of hippocampal neurons and induced epileptic seizure in kainic acid and pilocarpine-induced epilepsy17,18. Meanwhile, when AKT activity is inhibited by AKT inhibitor, the seizure activity was attenuated18.

 

Expression of the glutamate transporter, one of the excitatory neurotransmitters, is also regulated by the AKT/mTOR pathway. Upregulation of AKT activity will increase glutamate transporter levels leading to hyperexcitability of neurons15. AKT also plays important roles in calcium homeostasis. Activation of Ca2+-channel and Ca2+-influx is modulated by PI3K/AKT pathway19. AKT induces translocation of Ca­v channels and induced potentiation of L-type calcium channels20 thus altering the excitability of neurons by modulating Ca2+-influx and could increase frequency and duration of seizures21.AKT is also a main effector of mTORC2 and it is also thought to be activated in epilepsy15. But despite numerous research about AKT and mTORC1, the study about the role of mTORC2 in epileptogenesis is still limited. Due to its critical role in epilepsy, AKT could be a new target for the novel treatment of epilepsy.

 

mTORC1 is known to be inhibited by rapamycin or sirolimus, a macrolide drug and an antifungal metabolite produced by Streptomyces hygropicus22sensitive to mTORC1 but insensitive to mTORC223. But several studies showed mTORC2 inhibition by long-term administration of rapamycin24. So, in this study, we used long-term rapamycin treatment in order to inhibit mTORC2 and evaluate AKT activity along with intracellular calcium expression in an in vitro model of epilepsy.

 

MATERIALS AND METHODS:

Animals:

Rattus norvegicus strain Wistar 7-10 days of age were used in this study. The sex of the animal did not have influence on this research. We used male rats in this study. All animals were obtained from the Animal Laboratory of Faculty of Medicine, Universitas Brawijaya. All procedures were approved by Health Research Ethics Commission, Faculty of Medicine, Universitas Brawijaya, with ethical approval letter number 98/EC/KEPK/04/2022.

 

Organotypic Hippocampal Slice Culture (OHSC):

OHSC were prepared mainly based on methods described by Blazejczyk, et al (2017)25. The animals were sacrificed by cervical dislocation under sterile conditions and immediately decapitated. Brains were removed and placed into a slicing medium consisting of 24 g HEPES (Sigma, H3375, Lot 011M5434) dissolved in 10 mL EBSS (Sigma, Lot RNBG8086) for 10 minutes on ice with continuous aeration of 95% O2 and 5% CO2. The hippocampi were isolated in a slicing medium under stereo microscope (Olympus). We included the dentate gyrus, CA1, and CA3 regions. The hippocampi were sliced with a manual tissue slicer (Stoeting Tissue Slicer 51425)  for coronal sectioning at 350 μm. The slices were gently separated from each other and placed above millipore membranes (LCR Filter type 0.45 mm PTFE membrane, Merck, Lot: R9JA55940) and millicell cell culture insert (0.4 mm, 30 mm diameter, Merck, Lot: R9JA56969) in 6-wells cell culture plate (Biologix). A culture medium containing Minimum Essential Medium (MEM) (Sigma, M0769-10x1L, Lot SLBS6945), Earle’s Balanced Salt Solution (EBSS) (Sigma, Lot RNBG8086), heat-inactivated horse serum (Abcam ab7484, Lot GR3217249-1), D-Glucose (Abcam ab143108, Lot GR186597-6), amphotericin B (Glibco, Lot 2090190), and penicillin-streptomycin (Gibco, Ref15140-122, Lot 1665606) was given into all wells before the hippocampi slices were placed. The OHSC was then incubated in a humidified atmosphere of 95% air and 5% CO2 at 37oC and culture medium replacement was performed every 2 days. All processes were performed under sterile condition at laminar air flow (Faster).

 

Epilepsy Induction and Rapamycin Treatment:

OHSC were grouped as follows: vehicle/DMSO (dimethylsulfoxide) treated control group, low-Mg2+ medium OHSC group, and low-Mg2+ medium + long-term rapamycin treatment group. Each group consisted of 5 slices of OHSC for intracellular Ca2+ expression evaluation and 30 slices of OHSC for AKT activity evaluation which needed more slices to obtain higher protein concentration. In this study, we removed Mg2+ from the culture medium by perfusing the OHSC with low-Mg2+ medium (EBSS no calcium, no magnesium, Gibco Lot 2477793) for 40 minutes. In this condition, there was still a residual concentration of Mg2+ up to 0.08 mM due to contamination from other culture materials. Low-Mg2+ medium was given to low-Mg2+ medium OHSC group, and low-Mg2+ medium + long-term rapamycin treatment groups at between 7 and 11 days in vitro26. In low-Mg2+ medium + long-term rapamycin treatment groups, after low-Mg2+ medium, rapamycin (MedChemExpress, HY-10219) was given for 3, 5, 8, and 10 days. Rapamycin was dissolved in DMSO up to 20 Nm25,27 in concentration and administered by bath application.

 

Evaluation of intracellular Ca2+ expression:

Intracellular calcium was evaluated with the immunofluorescence method based on standard procedure stated in Fluo-4 Assay Kit (Calcium) (Abcam: ab228555; Lot GR3249725-1). For preparation, 20 μL of Fluo-4 AM stock solution was diluted in 1x Assay Buffer consisting of 1 mL of 10X F127 Plus and 9 mL of HHBS (Hank’s Balance Salt Solution) buffer. The samples were washed with ice-cold PBS three times and the fluorescence reagent was added in the dark room until the samples were covered in the reagent then incubated in a humidified atmosphere of 95% air and 5% CO2 at 37oC temperature for 1 hour. After incubation, the samples were washed with HHBS three times and immediately observed under a confocal laser scanning microscope (CLSM) at Ex/Em = 490/525 nm intensity. Quantification of intracellular Ca2+ expression was performed using Olympus Fluoview Ver 4.2a software based on fluorescence intensity in each hippocampus.

 

Evaluation of AKT activity:

The evaluation of AKT activity was conducted based on the standard protocol from AKT Activity Assay Kit (RayBioKinaseSTAR, Cat#: 68AT-Akt-S40). OHSC was washed three times in 1X ice-cold PBS then samples were homogenized with cell scrapper in 200 μL ice-cold Kinase Extraction Buffer consisting of 4% sodium dodecyl sulfate (SDS), 50 mM Tris, 2 mM EDTA, and 1% mammalian protease inhibitor. Homogenized samples were centrifuged at 13.000 rpm for 10 minutes at 4oC and the supernatant was transferred to a new tube. Protein concentration measurement was performed using nanodrop spectrophotometer. After protein concentration adjustment, 2 μL of AKT specific antibody were added and rotated slowly for 45 minutes at room temperature.

 

The next step,  50 μL of Protein A Sepharose slurry was added to each assay and continued to rotate slowly for an hour at room temperature. Each assay was centrifuged at 15.000 rpm for 2 minutes and the supernatant was removed. The remaining protein A beads were washed two times with 0.5 mL Kinase Extraction Buffer and one time with 0.5 mL Kinase Assay Buffer. Then, 50 μL of Kinase Assay Buffer and 2 μL of GSK 3α Protein were added to the washed protein A beads before incubating at 30oC for 1 hour. After incubation, Protein A beads were spined down at 15.000 rpm for 2 minutes and the supernatant was separated and15 μL of 3X SDS-PAGE buffer was added. Samples were boiled for 3 minutes and then centrifuged at 15.000 rpm for 2 minutes to spin down the Protein A beads. 20 μL of supernatant was used to SDS-PAGE. Samples were separated by SDS-PAGE using 15% separating gel and 4% stacking gel. After the SDS-PAGE procedure was done, the samples were transferred to a nitrocellulose membrane. Non-specific binding was blocked by incubation in 5% TBS-Skim Milk overnight. For immunodetection, the membrane was incubated in a solution of anti-Phospo-GSK-3α (Ser 21) specific antibody (1:1000) in TBS-Skim milk for 2 hours at room temperature, followed by incubation in a solution of secondary antibody (IgG anti Rabbit-HRP, 1:20.000) for 2 hours at room temperature.

 

The chemiluminescent detection method was used. Detection reagent consisting of Peroxide Solution (Thermo Scientific, Lot#VB295971) and Luminol Enhancer Solution (Thermo Scientific, Lot# VB299180) with a 1:1 ratio was added to the membrane in a dark room and gently shaken for + 30 minutes, then immediately placed in GE Healthcare’s Image QuantTM LAS 500. Quantification of protein band thickness was performed using ImageJ software.

 

Statistical Analysis:

Statistical analysis was performed using SPSS for windows 25 software (IBM). Differences in intracellular Ca2+ expression and AKT activity over long-term rapamycin treatment were analyzed with One-Way ANOVA followed by Tukey test. Correlation between long-term rapamycin treatment and intracellular Ca2+ expression and AKT activity was analyzed using Pearson’s correlation, as well as the correlation between AKT activity and intracellular Ca2+ expression. p < 0.05 was considered statistically significant.

 

RESULTS:

Perfusion of Low-Mg2+ medium increased intracellular Ca2+ expression and AKT activity:

Intracellular Ca2+ expression was used to observe the epileptiform activity. After incubation of OHSC in a low-Mg2+ medium, the intracellular Ca2+ expression was significantly increased. As shown under CLSM a brighter fluorescence image compares with the control group. Figure 1a shows a comparation between OHSC from vehicle-treated control group and low-Mg2+ medium group under CLSM. It also shows  that intracellular Ca2+ expression was increased mainly in DG and CA3 area of hippocampus. The quantification result for intracellular Ca2+ expression average (figure 1b) of the low-Mg2+ medium group was higher (637,245 + 117,288 AU (Arbitrary Unit)) than the vehicle-treated control (216,722 + 48,868 AU, p <0.001).

 

To test whether low-Mg2+ medium activated AKT in OHSC, AKT activity was evaluated by observing the phosphorylation target of AKT, GSK3α (p-GSK3α). Following intracellular Ca2+ expression increase, the low-Mg2+ medium can also increase AKT activity. The western blot band in low-Mg2+ medium group was thicker than the vehicle-treated control group (Figure 2a). This is in line with the quantification results of p-GSK3 (Figure 2b) which increased significantly after low-Mg2+ medium administration (214619,8 + 36623,85 AU) compared with the vehicle-treated control group (168542,3 + 34546,72 AU, p = 0.038).

 

Long-term rapamycin treatment lower intracellular Ca2+ expression:

Long-term rapamycin treatment can significantly lower intracellular Ca2+ expression. Figure 1a shows comparation of immunofluorescence results between all long-term rapamycin treatment group, low-Mg2+ medium group, and vehicle-treated control. The longer the rapamycin treatment, the fainter the immunofluorescence result. Figure 1b shows the average of intracellular Ca2+ expression of all groups and the comparation test result. Statistically, there was a negative correlation between long-term rapamycin treatment and intracellular Ca2+ expression (Figure 1c). This means that the longer the duration of long-term rapamycin treatment, the lower the intracellular Ca2+ expression, and in this study, 10 days of rapamycin treatment have the lowest intracellular Ca2+ expression average. There was no significant difference in intracellular Ca2+ expression between 10 days rapamycin treatment group and vehicle-treated control. The average intracellular Ca2+ expression from the two groups were 226,577 + 49,954 AU and 216,722 + 48,868 AU respectively.

 

Long-term rapamycin treatment lower AKT activity:

Long-term rapamycin treatment had a similar effect on AKT activity. Administration of rapamycin for 3, 5, 8, and 10 days also lowered AKT activity. Figure 2a shows chemiluminescence result of all groups. Long-term rapamycin treatment could lower the AKT activity up to normal conditions. It shows in Figure 2b, there was no significant difference between AKT activity in all long-term rapamycin treatment groups and vehicle-treated control group, even the AKT activity in 10 days rapamycin treatment OHSC was lower than vehicle-treated OHSC. Whereas, when compared with the low-Mg2+ treated OHSC group, the AKT activity was found to be significantly decreased with long-term administration of rapamycin for 8 and 10 days, but not in 3 and 5 days of treatment. Correlation analysis also showed a negative correlation, which means the longer the treatment of rapamycin, the lower the AKT activity (Figure 2c).

 

 

a

 

b

 

c

Figure 1. a-c. Evaluation and Analysis of Intracellular Ca2+ expression between groups.  Intracellular Ca2+ expression of OHSC between groups under confocal laser scanning microscope (CLSM). Intracellular Ca2+ expression is represented as green fluorescence color. The longer the duration of rapamycin treatment, the lower the intracellular Ca2+ expression

(a). Comparison of intracellular Ca2+ expression between groups. The intracellular Ca2+ expression was significantly higher in the Low-Mg2+ medium group compared with vehicle-treated control and significantly lower since 3 days of treatment compared with Low-Mg2+ medium group (b). Correlation analysis result showed negative correlation between rapamycin treatment duration and intracellular Ca2+ expression. It means the longer the duration of rapamycin treatment, the lower the intracellular Ca2+ expression (c).*Compare to low Mg2+ medium group, p < 0.05, #compared to vehicle-treated control, p < 0.05

 

Correlation of intracellular Ca2+ expression and AKT Activity:

Long-term rapamycin treatment caused both decrease in intracellular Ca2+ expression and AKT activity in the OHSC model of epilepsy. Between both of them, there was a significant correlation with a coefficient of determination (R2) of 0.321. It means that 32.1 % of the decrease in intracellular Ca2+ expression was related to a decrease in AKT. That result also showed that the decrease of intracellular Ca2+ expression is not only influenced by a decrease in AKT activity but there are other influencing factors. In this study, rapamycin (20 nM) can lower AKT activity in minimum 8 days of treatment and it can lower intracellular Ca2+ expression started from day 3 of treatment. The AKT activity did not significantly differ in all rapamycin treatment groups when compared to the vehicle-treated OHSC group. However, only 10 days of treatment with rapamycin could lower the intracellular Ca2+ expression until there were no significant differences with vehicle-treated control group. It means that 10 days of rapamycin treatment is the most effective group to reduce both AKT activity and intracellular Ca2+ expression significantly against low-Mg2+ medium group until close to normal condition as represented in vehicle-treated group.

 

 

a

 

b

 

c

Figure 2. a-c. Evaluation and Analysis of AKT activity. Western blot results of AKT activity. GSK3α is phosphorylated by AKT, so it represents AKT activity (a). Comparison of AKT activity between groups. Significant inhibition of AKT activity was found in 8 and 10 days of rapamycin treatment (b). Correlation analysis result showed negative correlation between rapamycin treatment duration and AKT activity. It means the longer the duration of rapamycin treatment, the lower the AKT activity (c). *Compared to low Mg2+ medium group, p < 0.05.

 

DISCUSSION:

OHSC is increasingly being used as in vitro model of epilepsy. This model recapitulates important features of epilepsy, including the latent period between the trauma obtained during brain slicing and the emergence of spontaneous electrical seizure, axon sprouting, and seizure-dependent cell death. Epilepsy progression in OHSC can be observed with imaging, electrical recordings, or by assays of biochemical markers of epileptiform activity and cell death28. Results of this study support previous studies29,30 that have proven that low-Mg2+ medium administration can cause spontaneous seizures in the hippocampus. Also, it can induce a paroxysmal depolarization shift (PDS) followed by an increase in intracellular Ca2+ expression31. Even when the OHSC is returned to normal culture medium intracellular Ca2+ oscillations can be maintained30.

 

Alteration in homeostasis of intracellular calcium plays an important role in development of seizures in epilepsy. Some of the mechanism of action of the available antiepileptic drug is to block the calcium channel and control the transmembrane currents32. Previous studies showed an elevation of intracellular Ca2+ concentration when the OHSC was induced by magnesium-free solution to create spontaneous PDS. One of the neuronal functions of Mg2+ is to block N-methyl-D-aspartate (NMDA) receptor thus preventing the Ca2+ from entering the calcium channel. Low-Mg2+ concentration will induce Ca2+-influx and activate NMDA receptor33. Long-term activation of intracellular Ca2+ can induce persistent plasticity changes in hippocampal neuron culture that eventually induce epileptogenesis34,35.

 

There are several Ca2+ responsive elements (CaRE) in BDNF promoters, so an increase in the concentration of free Ca2+ in the cytosol could significantly increase the BDNF transcription rate36. BDNF will activate the PI3K/AKT/mTOR signaling pathways increasing AKT activity. Then, AKT will inhibit TSC1/2 leading to the elimination of Rheb suppression which will eventually activate mTORC1. mTORC1 will facilitate mTORC2 activation through the activation of ribosomes where mTORC1 plays a role in stimulating ribosomal assembly. Its activation will further activate AKT activity as its effector. When it is activated by mTORC2, AKT will phosphorylate various proteins, including PAK (p21-activated kinase), Girdin (GIV/APE, and Integrin β3, which have roles in stimulating cell migration, modulation of actin cytoskeleton, soma size, and dendritic growth15.

 

This study showed long-term rapamycin treatment significantly reduce intracellular Ca2+expression supporting the antiseizure effect of long-term rapamycin. Studies about the effect of rapamycin on intracellular Ca2+ expression are still limited. What has been widely studied is the effect of rapamycin on the frequency and duration of seizures using electroencephalography (EEG). In vitro study27 using a post-traumatic OHSC model showed the ability of rapamycin (20 nM) to decrease epileptiform activity from day 6 as evidenced by a decrease in seizure frequency, but not followed by changes in seizure duration. The administration of rapamycin 6 mg/kgBW/day for 14 days in vivo didn’t lower the frequency and duration of seizures significantly37. However, treatment of rapamycin at the same dose for 6 weeks was able to effectively reduce the development of seizures38. So, the antiseizure effect of rapamycin depends on the duration of treatment. Long-term rapamycin treatment can reduce both seizure frequency and duration38.

 

Long-term rapamycin significantly lower AKT activity with a negative correlation against treatment duration. A previous study using genetically modified mice model of cortical dysplasia with the characteristics of epilepsy, it is proved that intraperitoneal administration of rapamycin 10 mg/kg for 2 weeks can significantly reduce AKT activity. The decrease in AKT activity was also followed by a decrease in seizure activities observed by EEG39. In vitro study using cell lines also showed strong or partial inhibition of AKT phosphorylation after rapamycin administration23. This study showed that intracellular Ca2+ expression decrease earlier than AKT activity. We hypothesize that rapamycin might lower all types of Ca2+-channel, not only the L-type Ca2+-channel, so the intracellular Ca2+ decreased earlier than AKT activity. Since the AKT is the main effector of mTORC2, we hypothesize that mTORC2 also plays a role in epilepsy through AKT activation. Previous studies showed long-term rapamycin may inhibit mTORC2 activity depended on duration of treatment39,40,41.The long-term rapamycin treatment effect on the reduction of AKT activity and intracellular Ca2+ expression showed the potential of long-term rapamycin as a novel therapy for epilepsy, not only has an antiseizure effect but long-term rapamycin treatment also inhibits the epileptogenesis. Further studies are needed to compare the effectiveness of long-term rapamycin treatment and current antiepileptic drugs. Also, it is needed to evaluate the toxicity of long-term rapamycin in future studies.

 

CONFLICT OF INTEREST:

None of the authors has any conflict of interest to disclose.

 

ACKNOWLEDGMENTS:

The authors would like to thank to Laboratory of Biomedicine and Laboratory of Parasitology, Faculty of Medicine Universitas Brawijaya, and Central Laboratory of Biological Sciences, Universitas Brawijaya for their supports and contributions in data collection and analysis.

 

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Received on 25.01.2023            Modified on 07.03.2023

Accepted on 18.05.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(3):1232-1239.

DOI: 10.52711/0974-360X.2024.00192