Waafi, Afiyfah Kiysa and dr. Hidayat Sujuti, SpM, PhD and Prof. Dr. drh. Aulanni’am, DES (2023) Pengaruh Pemberian Rapamycin Jangka Panjang terhadap Konsentrasi Kalsium Intraseluler dan Aktivitas AKT pada Organotypic Hippocampal Slice Culture Model Epilepsi yang Diinduksi dengan Low-Mg. Magister thesis, Universitas Brawijaya.
Abstract
Epilepsi adalah penyakit neurologis kronis dengan angka kejadian yang semakin meningkat setiap tahunnya. Selain itu, dari seluruh penderita epilepsi, 30% mengalami epilepsi refrakter, yaitu epilepsi yang tidak responsif terhadap pengobatan epilepsi.Terapi epilepsi saat ini mengandalkan obat anti epilepsi (OAE) yang berfokus sebagai antikejang, namun tidak menghambat epileptogenesis. Salah satu penelitian terkait epileptogenesis dan terapi epilepsi yang sedang banyak dilakukan yaitu mengenai peran mTOR (mammalian/mechanistic target of rapamycin) terhadap epilepsi. Hiperaktivitas mTOR telah dibuktikan secara in vivo dan in vitro dapat menyebabkan kejang pada epilepsi. Terdapat 2 kompleks mTOR, yaitu mTORC1 dan mTORC2. Upregulasi dari mTORC1 telah dibuktikan berperan penting dapam epileptogenesis dengan mengganggu pembentukan sirkuit neuronal dan mengganggu jaringan neuronal yang sudah ada. mTORC1 sendiri diaktifkan oleh AKT melalui inhibisinya terhadap TSC1/2. AKT merupan protein Ser/Thr kinase yang berperan dalam banyak fungsi neuronal, seperti plastisitas sinaps, neurogenesis, axon guidance, morfogenesis dendrit, polarisasi neuronal, dan reorganisasi sitoskeleton. Upregulasi ekspresi PI3K/AKT/mTORC1 menyebabkan hipereksitabilitas hipokampus dan menginduksi kejang epileptik pada penelitian-penelitian sebelumnya. Peningkatan aktivitas AKT juga berperan dalam aktivasi kanal kalsium dan meningkatkan influks kalsium sehingga menginduksi potensiasi kanal kalsium tipe L. AKT juga merupakan efektor dari mTORC2, namun penelitian terkait mTORC2 masih terbatas hingga saat ini. Rapamycin sebagai penghambat mTOR, ditemukan sensitif terhadap mTORC1, namun insensitif terhadap mTORC2. Namun, beberapa penelitian membuktikan adanya penghambatan terhadap mTORC2, apabila rapamycin diberikan secara jangka panjang. Pada penelitian ini digunakan organotypic hippocampal slice culture (OHSC) yang diinduksi epilepsi menggunakan media Low-Mg2+ kemudian digunakan rapamycin yang diberikan selama 3, 5, 8, dan 10 hari untuk menghambat mTORC2. Kemudian dilakukan evaluasi terhadap aktivitas AKT menggunakan metode western blot dan kalsium intraseluler menggunakan immunofluorsensi dibawah confocal laser scanning microscope (CLSM). Hasil dari penelitian ini menunjukkan bahwa pemberian media Low-Mg2+ dapat meningkatkan aktivitas dari AKT (p=0.038) dan ekspresi kalsium intraseluler (p<0.001) secara signifikan. Pemberian rapamycin jangka panjang juga dapat menurunkan kalsium intraseluler secara signifikan. Secara statistik, terdapat korelasi bersifat negatif antara pemberian rapamycin jangka panjang dan konsentrasi kalsium intraseluler. Hal tersebut dapat diartikan bahwa semakin lama durasi pemberian rapamycin jangka panjang, semakin rendah ekspresi kalsium intraseluler pada OHSC. Pada penelitian ini, pada pemberian rapamycin selama 10 hari didapatkan ekspresi kalsium intraseluler yang paling rendah dan tidak ada perbedaan yang signifikan antara konsentrasi kalsium intraseluler setelah pemberian rapamycin selama 10 hari dengan kalsium intraseluler pada kelompok kontrol negatif. Pemberian rapamycin jangka panjang juga memberikan efek yang mirip pada aktivitas AKT. Terdapat perbedaan yang signifikan antara aktivitas AKT pada kelompok OHSC yang diberikan rapmycin selama 8 dan 10 hari dengan kelompok kontrol positif. Secara statistik, juga didapatkan korelasi bersifat negatif antara pemberian rapamycin jangka panjang dengan aktivitas AKT. Efek rapamycin jangka panjang terhadap penurunan konsentrasi kalsium intraseluler menunjukkan potensinya sebagai terapi baru untuk epilepsi. Namun, penelitianpenelitian lebih lanjut perlu dilakukan untuk membandingkan efektivitas rapamycin jangka panjang dengan terapi epilepsi yang sudah ada saat ini, serta untuk mengevaluasi efek toksisitas dari rapamycin jangka panjang.
English Abstract
Epilepsy is a chronic neurological condition that affects people of all ages as a result of an abnormal and uncontrolled discharge of neurons causing recurrent seizures. In 2016, around 49.5 million people worldwide have epilepsy with a global age-standardized mortality rate of up to 1.74 per 100.000 population. Currently, the treatments of epilepsy rely on symptomatic strategies, targeting to suppress the seizures, but do not target epileptogenesis processes. Despite the development of the antiepileptic drug, it only provides satisfactory seizure control for 60-70% patients, there are still 30% of epileptic patients remain unresponsive (refractory epilepsy). 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 epilepsy. 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 epileptogenesis. Accumulating evidence showed mTOR hyperactivity in in vivo and in vitro models of epilepsy, also in human temporal lobe epilepsy specimens. 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 neurons. 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 signalling, including synaptic plasticity, neurogenesis, axon guidance, dendritic morphology, and neuronal polarization. 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 epilepsy. Upregulation of PI3K/AKT/mTORC1 expression is causing hyperexcitability of hippocampal neurons and induced epileptic seizure in kainic acid and pilocarpine-induced epilepsy. Meanwhile, when AKT activity is inhibited by AKT inhibitor, the seizure activity was attenuated mTORC1 is known to be inhibited by rapamycin or sirolimus, a macrolide drug and an antifungal metabolite produced by Streptomyces hygropicus sensitive to mTORC1 but insensitive to mTORC2. But several studies showed mTORC2 inhibition by long-term administration of rapamycin. So, in this study, we used longterm rapamycin treatment in order to inhibit mTORC2 and evaluate AKT activity along with intracellular calcium expression in an in vitro model of epilepsy using organotypic hippocampal slice cultures (OHSC). In this study, low-Mg2+ medium is used to induce epilepsy in OHSC. Then, to inhibit AKT activity, rapamycin (20 nM) was given for 3,5,8, and 10 days. AKT activity was evaluated by western blot method and intracellular calcium was evaluated by immunofluorescence method using confocal laser scanning microscope (CLSM).x This study shows that perfusion of Low-Mg2+ medium could increase intracellular Ca2+ expression (p<0.001) and AKT activity (p=0.038) significantly. Administration of long-term rapamycin could significantly lower intracellular Ca2+ expression. Statistically, there was a negative correlation between long-term rapamycin treatment and intracellular Ca2+ expression. 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. Long-term rapamycin treatment had a similar effect on AKT activity. 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. 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. 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. 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.
| Item Type: | Thesis (Magister) |
|---|---|
| Identification Number: | 042306 |
| Divisions: | S2/S3 > Magister Ilmu Biomedis, Fakultas Kedokteran |
| Depositing User: | Unnamed user with username nova |
| Date Deposited: | 08 Jul 2024 08:13 |
| Last Modified: | 28 Aug 2024 09:35 |
| URI: | http://repository.ub.ac.id/id/eprint/221115 |
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