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Linsitinib (OSI-906) modulates brain energy metabolism and seizure activity in the lithium-pilocarpine rat model

Linsitinib (OSI-906) modulates brain energy metabolism and seizure activity in the... Background: Epileptic seizure is a process of energy accumulation, bursting, and depletion accompanied by the production, spread, and termination of epileptic discharges. The energy required for a seizure is mainly provided through mitochondrial production of ATP. Mitochondrial diseases often lead to epileptic seizures, and energy deple- tion caused by seizures can lead to mitochondrial dysfunction. The energy metabolism has become a key target for treatment of epileptic diseases. Method: The effect of OSI-906, an insulin receptor (IR)/ insulin-like growth factor 1 receptor (IGF-1R) inhibitor, on behaviors and electroencephalographic activity in the lithium-pilocarpine rats were tested. F-FDG positron emission tomography (PET )/ computed tomography (CT ) was performed to detect the relative whole-brain glucose uptake values. Electron microscopy was performed to observe the ultrastructure of neuronal and mitochondrial damage. The changes in blood glucose at different time points before and after the intervention were tested and the effects of OSI- 906 on IR/IGF-1R and downstream Akt signaling in the context of seizures were evaluated. Results: The OSI-906 treatment applied 3 days before the pilocarpine-induced seizures significantly reduced the sei- zure severity, prolonged the seizure latency and decreased the EEG energy density. MicroPET/CT revealed that 50 mg/ kg of OSI-906 inhibited the F-FDG glucose uptake after epileptic seizures, suggesting that OSI-906, through inhibit- ing IR/IGF-1R and the downstream AKT signaling, may regulate the excessive energy consumption of the epileptic brain. The OSI-906 treatment also reduced the mitochondrial damage caused by epileptic seizures. Conclusion: The IR/IGF-1R inhibitor OSI-906 can significantly reduce the sensitivity and severity of pilocarpine- induced seizures by inhibiting the IR/IGF-1R and the downstream Akt signaling pathway. Keywords: Epilepsy, Mitochondria, Energy metabolism, Insulin receptor, IGF-1 receptor, Akt signaling [1, 2]. An epileptic seizure represents a process of energy Background accumulation, bursting and depletion, accompanied by Epilepsy is one of the most common diseases of the nerv- the production, spread and termination of epileptic dis- ous system, with an electrophysiological feature of highly charges that consequently lead to neuronal injury [3, 4]. synchronous abnormal discharges of neurons, which Early experimental studies demonstrated that the cellular leads to clinical syndromes of transient brain dysfunction adenosine triphosphate (ATP) content in brain is reduced before the onset of epileptic activity [5, 6]. Previous stud- *Correspondence: wangxm238@163.com ies have suggested that the depletion of ATP, which is Department of Neurology, Affiliated Hospital of North Sichuan Medical College, Institute of Neurological Diseases, North Sichuan Medical produced and supplied by the mitochondria, triggers College, Nanchong, China © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Jiang et al. Acta Epileptologica (2021) 3:19 Page 2 of 12 chronic spontaneous seizures, and the excessive energy Materials and Methods depletion during seizures also promotes the progression Animals of seizures [7, 8]. Male Sprague–Dawley rats (weighting 180–200  g) Insulin and insulin-like growth factor-1 (IGF-1) are were housed at the Laboratory Animal Center of North multipotent bioactive substances found in almost all tis- Sichuan Medical College (NSMC 201,810), under stand- sues. In the brain they are involved in energy and glucose ard temperature, humidity and circadian rhythm condi- homeostasis and affect neuronal survival, learning, mem - tions with ad libitum access to food and water. All animal ory, and injury repair [9, 10]. Disorders of the insulin and experiments were performed in accordance of the Ani- insulin receptor (IR) system in the central nervous sys- mal Research Committee of North Sichuan Medical Col- tem (CNS) play vital roles in the occurrence and devel- lege and were approved by the Chinese Animal Welfare opment of nerve injury, neurodegenerative diseases, and Act for the use and care of laboratory animals. neuropsychiatric diseases [11–13]. Previous studies have found that the expression of  IR is significantly increased Experimental groups in the anterior temporal neocortex of patients with The commonly used doses of OSI-906 in in  vivo inter - refractory epilepsy [14]. We have also detected increased ventional studies in rodents range 25–75  mg/kg [16]. levels of IGF-1 and IGF-1 receptor (IGF-1R) in the tem- Therefore, we divided the animals into the vehicle group, poral lobe and hippocampus of various epileptic mod- 25  mg/kg OSI-906 group, 50  mg/kg OSI-906 group, els. Intracerebroventricular injection of neuroprotective and 75  mg/kg OSI-906 group (n = 18 in each group, 5 doses of IGF-1 in other neurological diseases may pro- for electrophysiological recording and 13 for behavio- mote epileptic seizures and the development of epilepsy ral recording and related testing). OSI-906 was diluted by activating IGF-1R and the downstream signaling [15]. to a volume of 2  ml with tartaric acid solution and was Linsitinib (OSI-906) is an oral small-molecule inhibi- administered by gavage once daily for 3 consecutive days, tor that has demonstrated strong anti-tumor effects in while the vehicle group was given 2  ml of tartaric acid tumor  cell models in  vitro, by selectively inhibiting the solution only (Fig. 1). autophosphorylation of both human IGF-1R and IR [16]. OSI-906 reduces the tumor volume and prolongs the sur- Animal model of epilepsy vival of patients by inhibiting the proliferation, survival, The pilocarpine model, which reproduces most of the and energy metabolism of tumor cells through the phos- seizure phenotypes, was used in this study. The electro - phatidylinositol 3-kinase (PI3K)/ protein kinase B (Akt) encephalogram (EEG) data and the neuropathological signaling pathway [17, 18]. However, the effect of OSI- characteristics of temporal lobe epilepsy were recorded 906 in epilepsy has not been studied. as described previously [19, 20]. In brief, the rats were In this study we evaluated the effects of OSI-906 on the injected with lithium chloride (Sigma, USA; 127  mg/ IR/IGF-1R and the downstream PI3K/AKT signaling, the kg,  i.p.) 20  h and atropine sulfate (1  mg/kg, i.p.) 30  min energy metabolism level, and the mitochondrial mor- before the first pilocarpine (35  mg/kg, i.p.) administra - phology in the brain of a rat model with lithium-pilocar- tion. If rats did not develop epileptic seizures 30  min pine-induced epileptic seizures, as well as the effect on after the first injection of pilocarpine, the dose of pilo - lithium-pilocarpine-induced epileptic seizures in the rat carpine was increased by 20% every 10  min until the model. development of level-4–5 seizures. The total number Fig. 1 Schematic of experimental design Jiang  et al. Acta Epileptologica (2021) 3:19 Page 3 of 12 of pilocarpine injections was limited to five per animal. OSI-906 treatment significantly increased blood glucose After 60  min of status epilepticus (SE), diazepam (DZP, and aggravated epileptic seizures, and the mortality rate 10  mg/kg), phenobarbital (25  mg/kg), and atropine sul- reached 100% during the acute epileptic seizure, subse- fate (1  mg/kg) were  injected intraperitoneally to termi- quent relevant tests were not completed in this group. nate the convulsive seizures [21]. The seizure activity was scored according to the Racine’s standard criteria [22]. F‑FDG‑microPET imaging Only rats that achieved recurrent seizures of stage 4 to 5 MicroPET is a non-invasive imaging technique that is were used for subsequent tests. often used to record and analyze the correlation between neural function changes and brain energy metabolism Surgical procedures and electrophysiological recordings in small animals. To determine the effect of IR/IGF-1R The rat was anesthetized with chloral hydrate (350  mg/ inhibitor OSI-906 on glucose uptake in the brain after kg, i.p.) and flurbiprofen axetil was injected (5  mg/kg, the epileptic seizures, a F-FDG-PET-CT scanner (Sie- Beijing Tide Pharmaceutical Co., Ltd., Beijing) through mensInveon Multi-Modality System, Erlangen, Germany) a tail vein to relieve pain. Then, the rat was placed in a was used to detect cerebral glucose uptake at 1 h after the stereotaxic apparatus. A microwire array (4 × 4 array of pilocarpine-induced seizures. First, the rats were fixed platinum-iridium alloy electrodes, each 25  μm in diam- and injected with F-FDG (500 µCi) through the tail vein eter) was implanted in the right dorsal hippocampus as a tracer to detect the relative cerebral glucose metabo- (anterior–posterior –3.6  mm, medial–lateral 2.8  mm, lism rate. Thirty minutes later, they were anesthetized dorsal–ventral –3.5 mm) one week before recording and with isoflurane (induction period: 5% isoflurane + 1  L/ fixed with dental cement as described by Paxinos and min O , maintenance period: 1% isoflurane + 1 L/min O ) 2 2 Watson (2007). Local field potentials (LFPs) were pre - using an inhaled anesthesia system. Then the rats were amplified (× 1000), filtered (0.1–1000  Hz), and digitized scanned in the prone position in a three-dimensional at 4 kHz using an OmniPlex D Neural Data Acquisition model for 8 min (field of vision: 85.62 mm × 125.58 mm), System (Plexon, Dallas, TX). Electrophysiological record- and the static PET data were collected for 7 min. The CT ings were made with the direct current-coupled head signal was mainly used for anatomical description and stages, and all recordings were referenced to the two attenuation correction of F-FDG-PET images. ground screws. PET data analysis Analysis of electrophysiological data The imaging data were imported into the Inveon We employed NeuroExplorer (v4.0; Plexon, USA) and Research Workplace software for analysis, and data were MATLAB software (v7.1, R2009a; MathWorks, Inc., collected in the form of lists. The cerebral metabolism Natick, MA) to calculate the electrophysiological data. rate was reconstructed using the maximum posterior A fast Fourier transform was applied to power spectrum probability algorithm, with a pixel size of 0.4 × 0.4 × 1.2 analysis after digital filtering with 0.1–1000 Hz bandpass. mm . Glucose metabolism in the brain was evaluated as The frequency spectra of continuous variables and the the whole-brain average standard uptake values (SUV) neuronal rate histograms were obtained by the power using the following equation: SUV = Ct/ID*Wt.Ct(MBq/ spectral density analysis of our previous method [23]. We cm ), in which Ct indicates the activity concentration calculated the high-frequency discharge energy during after the decay correction of the measured brain region; seizures including ripples (80–200  Hz). The MATLAB ID (mCi) indicates the injection dose of F-FDG; and Wt function in the Signal Processing Toolbox was used to (kg) represents the weight of the rat [24]. We recorded compute the power of ripple oscillators and magnitude- the maximum and the mean SUV values of normal rats, squared coherence. MATLAB and GraphPad Prism soft- as well as SUV value 1  h post-seizure, and that post the wares (GraphPad Software, Inc., La Jolla, CA) were used OSI-906 intervention (OSI-906 50 mg/kg + SE). for statistical analysis and image processing. Projection electron microscopy Blood glucose monitoring The ultrastructural changes of nucleus and mitochondria Blood glucose was measured by a Roche glucometer in rat hippocampal neurons were observed by projection (Roche, Germany) in venous blood from the tail vein electron microscopy in the vehicle control, SE, and OSI- before treatment, 1 h after the first intragastric adminis - 906 + SE groups (n = 3). All animals were deeply anes- tration of vehicle or OSI-906, 1  h after gastric perfusion thetized with 10% chloral hydrate and then injected with of OSI-906 on the third day, and 1  h after the epileptic fresh normal saline to the heart. The rat was cardinally seizures. Animals were fasted for more than 12 h before perfused with a mixture of glutaraldehyde (2%) and para- OSI-906 intervention. As we found that 75  mg/kg of formaldehyde for heart reperfusion and tissue fixation, Jiang et al. Acta Epileptologica (2021) 3:19 Page 4 of 12 and then the brain was collected. The hippocampal tissue for comparisons between more than two groups; the Stu- was isolated immediately, and the CA1 area of the hip- dent’s t-test was used for comparison between groups of pocampus was cut into 1 mm pieces. Three specimens multiple samples, and the Bonferroni test was used for were taken from each rat and fixed in 4% glutaralde - post-test analysis. The Wilcoxon rank-sum test (Mann– hyde solution. The fixed tissues were sent to the electron Whitney U-test) was performed to compare two groups microscope room of Chongqing Medical University for of ordinal variables. The K-W test was used to compare unified specimen preparation. Images under projec - the classification data of multiple groups, and Fisher’s tion electron microscopy (JEOL JEM-1400, Japan) were exact test was also used to compare classification vari - captured. ables between two groups. P < 0.05 was considered as sta- tistically significant. Western blotting Hippocampal tissues were collected 24  h after pilocar- Results pine administration and stored at -80  °C for western OSI‑906 at 50 mg/kg significantly reduced the severity blotting. The tissues were homogenized in lysate buffer of SE and prevented mortality during SE containing phosphatase and protease inhibitors  (Sigma, We first observed the effect of OSI-906 on the latency St. Louis, MO), centrifuged (Sigma-Aldrich, USA) and of class 4–5 seizures induced by pilocarpine. Compared the supernatant collected. The total protein concentra - with the vehicle control group, the 50  mg/kg OSI-906 tion was determined with the BCA protein assay (Beyo- group showed a significantly prolonged latency of the time Institute of Biotechnology, China). The total protein pilocarpine-induced seizures (Bonferroni’s multiple com- samples were loaded at 40  μg/lane for sodium dodecyl parisons test, P = 0.028) and a decreased rate of first- sulfate polyacrylamide gel (5% spacer gel; 10% separat- dose seizures of stage 4–5 (Fisher’s exact test, P = 0.03). ing gel) electrophoresis, followed by electro-transfer onto In addition, the 25  mg/kg OSI-906 group did not show polyvinylidene fluoride membranes (Millipore). significant difference in the above indicators compared The membranes were blocked with 5% non-fat milk at to the control group (P > 0.05), while the 75  mg/kg OSI- room temperature for 120 min, then incubated with anti- 906 group had opposite effects to the 50 mg/kg OSI-906 p-IR (Tyr1361), anti-IR, anti-pIGF-1R (Tyr1135), anti- group (Table  1 and Fig.  2). At last, 7 rats in the vehicle IGF-1R, anti-p-AKT (Ser473), anti-AKT or anti-β-actin group, 7 rats in the 25  mg/kg OSI-906 group and 6 rats (1:500–1000, Cell Signaling Technology, Inc Boston, in the 50  mg/kg OSI-906 group developed recurrent MA, USA) antibody for 16  h at 4  °C. After TBST wash, stage 4–5 seizures. They were used for subsequent elec - the membranes were incubated with goat anti-rabbit IgG tron microscopy and western blotting assay (n = 5). Rats (1:5000, Cell Signaling Technology, Inc Boston, MA) for that underwent LFP recordings were added for electron 1  h. Electrochemiluminescence system (Bio-Rad, CA, microscopy detection (the side without electrodes). USA) was used to visualize the protein bands with a chemiluminescence kit (Biosharp, Beijing, China). Blot Eec ff t of OSI‑906 on hippocampal epileptiform discharge intensities were analyzed with QuantityOne software in pilocarpine‑induced seizures (Bio-Rad Laboratories, version 4.6.2, Hercules, CA). We recorded LFPs in hippocampal CA1 using in  vivo multichannel microfilament electrodes to investigate the Statistical analysis effect of OSI-906 on the excitability of the hippocam - All data were analyzed using the SPSS 19.0 statistical pal neural circuit during pilocarpine-induced seizures. software (IBM Corp., Armonk, NY), and the values are Compared to the vehicle control group, the 50  mg/kg expressed as mean ± SEM. All data were tested for nor- OSI-906 intervention group showed a significantly pro - mality and if non-normal, a non-parametric test was longed latency of epileptic discharge, a significantly used. One-way analysis of variance (ANOVA) was used shortened discharge duration, and a reduced average Table 1 Eec ff t of OSI-906 on acute epileptic seizures (n = 13) Group Latency of seizure (min) Seizure onset rate at Cumulative seizure rate Mortality the first dose Vehicle 28.55 ± 2.76 7/13 (53.85%) 11/13 (84.62%) 4/13 (30.77%) OSI-906 25 31.30 ± 4.01 6/13 (46.15%) 10/13 (76.92%) 3/13 (23.08%) OSI-906 50 41.33 ± 12.79* 1/13 (7.69%)* 6/13 (46.15%) 0/13 (0%) OSI-906 75 14.46 ± 14.08** 13/13 (100%)* 13/13 (100%)* 13/13 (100%)** Note: The dosing unit of OSI-906 is mg/kg. Compared with the vehicle group, *P < 0.05 and **P < 0.01 Jiang  et al. Acta Epileptologica (2021) 3:19 Page 5 of 12 Fig. 2 Eec ff ts of OSI-906 on acute epileptic seizures. (a) 50 mg/kg OSI-906 extended the seizure latency. (b) 50 mg/kg OSI-906 significantly reduced Racine score, but 75 mg/kg OSI-906 aggravated the severity of seizures. (c&d) First-dose and cumulative stage 4–5 seizure rates. (e) Eec ff ts of OSI-906 on mortality in acute rats. Bars represent mean ± SEM (n = 13 for each group). *P < 0.05 and **P < 0.01 compared to the control group power spectrum of electrical activity (Bonferroni’s mul- experiments to exclude possible confounding factors. The tiple comparisons test, P = 0.001, 0.013, 0.0001, respec- specific blood glucose values are shown in Table 2. tively, Fig.  3c, d). However, there was no such significant difference between the 25  mg/kg OSI-906 intervention OSI‑906 inhibits excessive energy consumption group and the vehicle group. The mortality was 30.77% of the brain caused by seizures in the control group, and zero in the 50  mg/kg OSI-906 Compared to the normal control group, the average group. Surprisingly, the death rate of animals was 100% whole-brain SUV value was significantly increased 1  h after pilocarpine-induced epileptic seizures in the 75 mg/ after the pilocarpine-induced recurrent epileptic seizures kg OSI-906 group. (1.63 ± 0.14 vs 1.98 ± 0.34, n = 5, P = 0.003, Fig. 5). In the 50  mg/kg OSI-906 group, the average whole-brain SUV Eec ff t of OSI‑906 on blood glucose value 1 h after the epileptic seizures was 1.77 ± 0.21, sig- The 25 mg/kg and 50 mg/kg groups had similar changes nificantly lower than the SE group (P = 0.013). However, of blood glucose in comparison to the control group at it was unclear whether the reduced SUVs were attributed different time points of treatment, suggesting that the to the inhibition of seizures or were because OSI-906 effect of OSI-906 on epileptic seizures was not directly lowered the glucose uptake. through the regulation of blood glucose. OSI-906 at 75  mg/kg significantly increased the blood glucose level Ultrastructural changes of the nucleus and mitochondria 1 h after the third gastric perfusion and 1 h after the epi- in neurons leptic seizures, compared to the control group (ANOVA The mitochondria are responsible for aerobic respiration and post hoc analysis using the Bonferroni method, and energy transformation, and make most of the cell’s P < 0.01 and P < 0.0001, respectively, n = 5; Fig. 4). Consid- supply. Repeated or continuous epileptic seizures rely on ering that hyperglycemia or hypoglycemia may affect the mitochondrial function. Meanwhile, excessive energy mitochondrial function and epileptic seizures [25], the consumption caused by epileptic seizures will destroy the 75 mg/kg OSI-906 group was excluded from subsequent mitochondrial structure and function, leading to persistent Jiang et al. Acta Epileptologica (2021) 3:19 Page 6 of 12 Fig. 3 Eec ff ts of OSI-906 on epileptic discharge in acute epileptic seizures. (a) Eec ff ts of OSI-906 on hippocampal electrical activity (the X-axis is 60 min and the Y-axis is ± 1.0 millivolts). (b) Corresponding power spectrograms of each group. (c‑ e) 50 mg/kg OSI-906 shortened the latency of epileptic discharge, and reduced the duration of epileptic discharge and the mean energy density value of brain electrical activity. Data are shown as mean ± SEM. *P < 0.05 and **P < 0.01 compared to the control group; n = 5 each group disorders of brain energy metabolism in patients with key targets in epilepsy research and treatment strategies. epilepsy, thus resulting in a vicious cycle [4, 26]. There - Transmission electron microscopy revealed that the nor- fore, brain energy metabolism and mitochondria are mal rat hippocampal neurons had a clear outline, a large Jiang  et al. Acta Epileptologica (2021) 3:19 Page 7 of 12 Fig. 4 Eec ff t of OSI-906 treatment on blood glucose before and after the seizures. (a) Blood glucose level did not significantly change at 1 h after the first OSI-906 gavage, compared with the control group (P > 0.05). (b&c) OSI-906 at 75 mg/kg significantly increased the blood glucose at fasting or 1 h after gavage on day 3. P = 0.0024 and 0.0002, respectively, vs the control, one-way ANOVA. (d) In the 75 mg/kg OSI-906 group, blood glucose levels were higher after the seizure compared with the control group. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 (n = 5) and round nucleus, uniform chromatin, clear cytoplasm, the mitochondrial cristae were disordered, shortened, and clear and complete double-layer nuclear membrane at swollen. The cristae of mitochondria blurred, were bro - high magnification, and mitochondria with complete inner ken, and disappeared, the matrix was swollen and vacu- and outer membranes as well as cristae formed by inner- olated, and an autophagosome was formed. The nucleus membrane depression. At 24 h after the epileptic seizures, of the neuron was shrunken and deeply stained, and the Table 2 Eec ff ts of OSI-906 on blood glucose (n = 5) Groups Blood glucose 1 h after the Blood glucose before gavage Blood glucose1 h after gavage Blood glucose 1 h first OSI‑906 (mmol/L) on day 3 (mmol/L) on day 3 (mmol/L) after SE (mmol/L) vehicle 5.94 ± 0.31 6.02 ± 0.48 6.26 ± 0.79 15.60 ± 1.09 OSI-906 25 mg/kg 6.24 ± 0.49 6.56 ± 0.54 7.10 ± 0.84 16.78 ± 1.18 OSI-906 50 mg/kg 6.52 ± 0.58 7.22 ± 1.20 8.44 ± 2.18 17.04 ± 1.44 OSI-906 75 mg/kg 6.84 ± 0.90 8.12 ± 2.10** 11.18 ± 4.92*** ≧33.33**** ** P < 0.01, ***P < 0.001, and ****P < 0.0001 vs the vehicle control group Jiang et al. Acta Epileptologica (2021) 3:19 Page 8 of 12 Fig. 5 OSI-906 decreased the glucose uptake 1 h after seizures. (a) F-FDG micro-PET/CT quantification of glucose metabolism in vivo. (b) Compared with the normal control group, the average whole-brain SUV value was significantly increased 1 h after pilocarpine-induced epileptic seizures. Treatment with 50 mg/kg of OSI-906 significantly reduced the value of glucose uptake in the brain (n = 5). Data are shown as mean ± SEM. **P < 0.01, P < 0.05 chromatin in the nucleus was concentrated. Compared signaling pathways involved in the pilocarpine- with the epilepsy model group, the 50  mg/kg OSI-906 induced seizures. intervention group showed less damage to the mitochon- dria and nucleus (Fig. 6). Discussion In the present study, we showed that OSI-906 at 50  mg/ Eec ff ts of OSI‑906 on IR/IGF‑1R and Akt signaling kg significantly reduced the susceptibility to and sever - As shown in Fig.  7, the  pIR/IR, pIGF-1R/IGF-1R, ity of seizures induced by pilocarpine (Table  1 &Fig.  2). and p-AKT/AKT ratios were prominently elevated Synchronous EEG recording also showed that 50  mg/kg at 24  h after the pilocarpine-induced seizures, com- of OSI-906 significantly prolonged the latency of epilep - pared to those in the vehicle group (n = 5, AN OVA , tic discharge, shortened the duration of discharge, and P < 0.01). However, they were markedly suppressed decreased the mean energy density of  brain electrical in the 50  mg/kg OSI-906 group (Fig.  7, P < 0.05), sug- activity (Fig. 3). However, OSI-906 at 75 mg/kg increased gesting that 50  mg/kg of OSI-906 could inhibit the the blood glucose and aggravated seizures (Table  2, activation of IR/IGF-1R and the downstream AKT Fig.  4). As an increase or decrease of blood glucose can Jiang  et al. Acta Epileptologica (2021) 3:19 Page 9 of 12 Fig. 6 Ultrastructural changes of nucleus and mitochondria in neurons. (a) Hippocampal neurons and mitochondria in control rats. (b) Ultrastructural changes of nuclei and mitochondria in hippocampal neurons. (c) Compared to the epilepsy model group, the 50 mg/kg OSI-906 group showed less damage to the mitochondria and nuclei. a, b and c are magnified 8,000 times, and a1, b1 and c1 are magnified 20,000 times affect the mitochondrial function and epileptic activity related to its inhibition of seizure activity (Fig. 5). During [25], this group of rats failed to complete the subsequent epileptic seizures, neurons are highly synchronized and tests. discharge abnormally, which leads to the brain energy In a clinical study, OSI-906 was continuously given depletion. This excessive energy depletion depends on in a once-  or twice-daily schedule in patients with the mitochondrial oxidative phosphorylation to produce advanced solid tumors [27]. In this study, the blood glu- a large amount of ATP. Of note, energy depletion hinders cose increased after 75  mg/kg of OSI-906 application glucose transport and oxidative metabolism and destroys for 3 consecutive days, indicating a cumulative effect of the mitochondrial structure and function [4]. As a result, OSI-906. However, there was no significant difference in after the onset of epilepsy, ATP production is compro- blood glucose during single administration, and continu- mised, and pathological changes such as abnormal neu- ous dosing could better reflect the effect of homeostasis ronal ion transport, disorders of neurotransmitter uptake OSI-906 concentration on epileptic activity. The high and release, and blockade of protein signaling, may ulti- dose of OSI-906 (75  mg/kg) inhibits IR/IGF-1R, result- mately result in recurrent spontaneous epileptic seizures, ing in increased blood glucose, which may be the main namely, epileptogenesis [30, 31]. In addition, after long- reason for the opposite outcome to that of 50 mg/kg OSI- term spontaneous seizures, significantly reduced glucose 906. In addition, the blood glucose increased significantly metabolism is observed in multiple brain regions, which in the vehicle group and in different dose groups of OSI- can affect the neuronal function [32–34]. Hence, OSI-906 906 after SE, which may be related to the stress response may inhibit the seizure activity through reduction of the induced by SE. However, the specific mechanisms remain glucose uptake in the brain and inhibition of excessive unclear. energy consumption of the brain caused by seizures. The brain is a high-consumption organ, which consti - We further found that the epileptic seizures resulted tutes only 2% of the human body weight, while consum- in changes in mitochondrial structure, including dis- ing 20% to 25% of the energy supply in the body [28]. The ordered and shortened cristae, partial mitochondrial brain energy metabolism has been shown as a key factor swelling, mitochondrial ridge blurring, fracture and dis- affecting the epileptic seizures, and may be an important appearance, matrix swelling, vacuolization, neuronal target for epilepsy research and treatment [29]. nuclear shriveling, and hyperchromatic concentration, Surprisingly, the F-FDG-PET scanning showed that which were improved by 50  mg/kg of OSI-906 (Fig.  6). 50 mg/kg of OSI-906 treatment significantly reduced the As an alternative energy source, a ketogenic diet may be average whole-brain SUV value after seizures, possibly an effective treatment of epilepsy, which improves the Jiang et al. Acta Epileptologica (2021) 3:19 Page 10 of 12 Fig. 7 Eec ff ts of OSI-906 on IR, IGF-1R and AKT signaling. (a) The representative bands of western blotting. (b-d) The pIR/IR, pIGF-1R/IGF-1R, and p-Akt/Akt ratios were significantly elevated at 24 h after pilocarpine-induced seizures, compared with those in the vehicle group. Compared to those in the seizure group, these ratios were markedly reduced in the 50 mg/kg OSI-906 group. Data are shown as mean ± SEM. n = 5, # ### ANOVA,**P < 0.01, ***P < 0.001, P < 0.05, P < 0.001 neuronal damage caused by decreased energy metabo- seizures (Fig.  7). We speculated that during the pilocar- lism in the brain during epileptic seizures [35–37]. Our pine-induced epileptic status, OSI-906 inhibits the exces- study suggests that the inhibition of epileptic activity by sive energy intake and consumption in the brain, possibly 50  mg/kg of OSI-906 may restrain the excessive energy through inhibiting the IR/IGF-1R and the downstream consumption of the brain by regulating the function of AKT signaling. More studies are needed to fully clarify mitochondria and may have a protective effect on mito - the underlying mechanisms. chondria in the context of epileptic seizures. In our behavioral and electrophysiological studies, we found Conclusion that 50  mg/kg of OSI-906 significantly reduced the sus - In conclusion, OSI-906 at 50  mg/kg inhibits the brain ceptibility to and severity of seizures. energy metabolism and epileptic activity during epilep- The small molecule OSI-906 is a dual receptor inhibitor tic seizure, and has a protective effect on mitochondrial of IR/IGF-1R, which effectively and selectively inhibits damage caused by epileptic seizure. OSI-906 at 50 mg/kg the phosphorylation of IR and IGF-1R and has a strong may inhibit epileptic seizures by inhibiting the IR/IGF- anti-tumor effect in many types of cancer [38]. We found 1R and the  downstream AKT signaling. These results that 50 mg/kg of OSI-906 significantly inhibited the acti - provide new evidence for the use of OSI-906 in the treat- vation of IR, IGF-1R, and AKT signals after epileptic ment of epileptic status. Jiang  et al. Acta Epileptologica (2021) 3:19 Page 11 of 12 8. Rowley S, Patel M. Mitochondrial involvement and oxidative stress in temporal lobe epilepsy. Free Radic Biol Med. 2013;62:121–31. 9. Fernandez AM, Torres-Aleman I. The many faces of insulin-like peptide Abbreviations signalling in the brain. Nat Rev Neurosci. 2012;13:225–39. ATP: Adenosine triphosphate; CNS: Central nervous system; CT: Computed 10. Plum L, Belgardt BF, Bruning JC. Central insulin action in energy and tomography; EEG: Electroencephalogram; IGF-1: Insulin-like growth factor-1; glucose homeostasis. J Clin Invest. 2006;116:1761–6. IR: Insulin receptor; IGF-1R: Insulin-like growth factor-1 receptor; i.p.: Intraperi- 11. Benarroch EE. Insulin-like growth factors in the brain and their potential toneal; LFPs: Local field potentials; PET: Positron emission tomography; SUV: clinical implications. Neurology. 2012;79:2148–53. Standard uptake values. 12. de la Monte SM, Wands JR. Review of insulin and insulin-like growth fac- tor expression, signaling, and malfunction in the central nervous system: Acknowledgements relevance to Alzheimer’s disease. J Alzheimers Dis. 2005;7:45–61. This work was supported by the National Natural Science Foundation of China 13. Ghasemi R, Dargahi L, Haeri A, Moosavi M, Mohamed Z, Ahmadiani A. (No. 81971220) and the Science and Postdoctoral Science Foundation of Brain insulin dysregulation: implication for neurological and neuropsychi- China (No. 20191964). We thank Drs. Y. Chen and Y. Zhao for their assistance in atric disorders. Mol Neurobiol. 2013;47:1045–65. F-FDG-PET testing from the Department of Nuclear Medicine of Southwest 14. Wang L, Liu G, He M, Shen L, Shen D, Lu Y, et al. Increased insulin receptor Medical University. expression in anterior temporal neocortex of patients with intractable epilepsy. J Neurol Sci. 2010;296:64–8. Authors’ information 15. Jiang G, Wang W, Cao Q, Gu J, Mi X, Wang K, et al. Insulin growth factor-1 Not applicable. (IGF-1) enhances hippocampal excitatory and seizure activity through IGF-1 receptor-mediated mechanisms in the epileptic brain. Clin Sci. Authors’ contributions 2015;129(12):1047–60. GHJ performed experiments and participated in data analysis and manu- 16. Mulvihill MJ, Cooke A, Rosenfeld-Franklin M, Buck E, Foreman K, Landfair script writing. SLW, MYC and XMD carried out animal experiments. WWH, D, et al. Discovery of OSI-906: a selective and orally efficacious dual LW and SXW participated in the study and analyzed the results. JMY and inhibitor of the IGF-1 receptor and insulin receptor. Future Med Chem. XMW supervised the design of the research. All authors approved the final 2009;1:1153–71. manuscript. 17. Kruger DT, Alexi X, Opdam M, Schuurman K, Voorwerk L, Sanders J, et al. IGF-1R pathway activation as putative biomarker for linsitinib therapy Funding to revert tamoxifen resistance in ER-positive breast cancer. Int J Cancer. This work was supported by the National Natural Science Foundation of China 2020;146(8):2348–59. (No. 81971220) and the Science and Postdoctoral Science Foundation of 18. Lamhamedi-Cherradi SE, Menegaz BA, Ramamoorthy V, Vishwamitra D, China (No. 2019M653978). Wang Y, Maywald RL, et al. IGF-1R and mTOR Blockade: Novel Resistance Mechanisms and Synergistic Drug Combinations for Ewing Sarcoma. J Availability of data and materials Natl Cancer Inst. 2016;108(12):djw182. Not applicable. 19. Pitkanen A, Kharatishvili I, Karhunen H, Lukasiuk K, Immonen R, Nairismagi J, et al. Epileptogenesis in experimental models. Epilepsia. 2007;48(Suppl Declarations 2):13–20. 20. Raol YH, Brooks-Kayal AR. Experimental models of seizures and epilepsies. Ethical approval and consent to participate Prog Mol Biol Transl Sci. 2012;105:57–82. All animal experiments were performed in accord with the Animal Research 21. Brandt C, Tollner K, Klee R, Broer S, Loscher W. 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Linsitinib (OSI-906) modulates brain energy metabolism and seizure activity in the lithium-pilocarpine rat model

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Abstract

Background: Epileptic seizure is a process of energy accumulation, bursting, and depletion accompanied by the production, spread, and termination of epileptic discharges. The energy required for a seizure is mainly provided through mitochondrial production of ATP. Mitochondrial diseases often lead to epileptic seizures, and energy deple- tion caused by seizures can lead to mitochondrial dysfunction. The energy metabolism has become a key target for treatment of epileptic diseases. Method: The effect of OSI-906, an insulin receptor (IR)/ insulin-like growth factor 1 receptor (IGF-1R) inhibitor, on behaviors and electroencephalographic activity in the lithium-pilocarpine rats were tested. F-FDG positron emission tomography (PET )/ computed tomography (CT ) was performed to detect the relative whole-brain glucose uptake values. Electron microscopy was performed to observe the ultrastructure of neuronal and mitochondrial damage. The changes in blood glucose at different time points before and after the intervention were tested and the effects of OSI- 906 on IR/IGF-1R and downstream Akt signaling in the context of seizures were evaluated. Results: The OSI-906 treatment applied 3 days before the pilocarpine-induced seizures significantly reduced the sei- zure severity, prolonged the seizure latency and decreased the EEG energy density. MicroPET/CT revealed that 50 mg/ kg of OSI-906 inhibited the F-FDG glucose uptake after epileptic seizures, suggesting that OSI-906, through inhibit- ing IR/IGF-1R and the downstream AKT signaling, may regulate the excessive energy consumption of the epileptic brain. The OSI-906 treatment also reduced the mitochondrial damage caused by epileptic seizures. Conclusion: The IR/IGF-1R inhibitor OSI-906 can significantly reduce the sensitivity and severity of pilocarpine- induced seizures by inhibiting the IR/IGF-1R and the downstream Akt signaling pathway. Keywords: Epilepsy, Mitochondria, Energy metabolism, Insulin receptor, IGF-1 receptor, Akt signaling [1, 2]. An epileptic seizure represents a process of energy Background accumulation, bursting and depletion, accompanied by Epilepsy is one of the most common diseases of the nerv- the production, spread and termination of epileptic dis- ous system, with an electrophysiological feature of highly charges that consequently lead to neuronal injury [3, 4]. synchronous abnormal discharges of neurons, which Early experimental studies demonstrated that the cellular leads to clinical syndromes of transient brain dysfunction adenosine triphosphate (ATP) content in brain is reduced before the onset of epileptic activity [5, 6]. Previous stud- *Correspondence: wangxm238@163.com ies have suggested that the depletion of ATP, which is Department of Neurology, Affiliated Hospital of North Sichuan Medical College, Institute of Neurological Diseases, North Sichuan Medical produced and supplied by the mitochondria, triggers College, Nanchong, China © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Jiang et al. Acta Epileptologica (2021) 3:19 Page 2 of 12 chronic spontaneous seizures, and the excessive energy Materials and Methods depletion during seizures also promotes the progression Animals of seizures [7, 8]. Male Sprague–Dawley rats (weighting 180–200  g) Insulin and insulin-like growth factor-1 (IGF-1) are were housed at the Laboratory Animal Center of North multipotent bioactive substances found in almost all tis- Sichuan Medical College (NSMC 201,810), under stand- sues. In the brain they are involved in energy and glucose ard temperature, humidity and circadian rhythm condi- homeostasis and affect neuronal survival, learning, mem - tions with ad libitum access to food and water. All animal ory, and injury repair [9, 10]. Disorders of the insulin and experiments were performed in accordance of the Ani- insulin receptor (IR) system in the central nervous sys- mal Research Committee of North Sichuan Medical Col- tem (CNS) play vital roles in the occurrence and devel- lege and were approved by the Chinese Animal Welfare opment of nerve injury, neurodegenerative diseases, and Act for the use and care of laboratory animals. neuropsychiatric diseases [11–13]. Previous studies have found that the expression of  IR is significantly increased Experimental groups in the anterior temporal neocortex of patients with The commonly used doses of OSI-906 in in  vivo inter - refractory epilepsy [14]. We have also detected increased ventional studies in rodents range 25–75  mg/kg [16]. levels of IGF-1 and IGF-1 receptor (IGF-1R) in the tem- Therefore, we divided the animals into the vehicle group, poral lobe and hippocampus of various epileptic mod- 25  mg/kg OSI-906 group, 50  mg/kg OSI-906 group, els. Intracerebroventricular injection of neuroprotective and 75  mg/kg OSI-906 group (n = 18 in each group, 5 doses of IGF-1 in other neurological diseases may pro- for electrophysiological recording and 13 for behavio- mote epileptic seizures and the development of epilepsy ral recording and related testing). OSI-906 was diluted by activating IGF-1R and the downstream signaling [15]. to a volume of 2  ml with tartaric acid solution and was Linsitinib (OSI-906) is an oral small-molecule inhibi- administered by gavage once daily for 3 consecutive days, tor that has demonstrated strong anti-tumor effects in while the vehicle group was given 2  ml of tartaric acid tumor  cell models in  vitro, by selectively inhibiting the solution only (Fig. 1). autophosphorylation of both human IGF-1R and IR [16]. OSI-906 reduces the tumor volume and prolongs the sur- Animal model of epilepsy vival of patients by inhibiting the proliferation, survival, The pilocarpine model, which reproduces most of the and energy metabolism of tumor cells through the phos- seizure phenotypes, was used in this study. The electro - phatidylinositol 3-kinase (PI3K)/ protein kinase B (Akt) encephalogram (EEG) data and the neuropathological signaling pathway [17, 18]. However, the effect of OSI- characteristics of temporal lobe epilepsy were recorded 906 in epilepsy has not been studied. as described previously [19, 20]. In brief, the rats were In this study we evaluated the effects of OSI-906 on the injected with lithium chloride (Sigma, USA; 127  mg/ IR/IGF-1R and the downstream PI3K/AKT signaling, the kg,  i.p.) 20  h and atropine sulfate (1  mg/kg, i.p.) 30  min energy metabolism level, and the mitochondrial mor- before the first pilocarpine (35  mg/kg, i.p.) administra - phology in the brain of a rat model with lithium-pilocar- tion. If rats did not develop epileptic seizures 30  min pine-induced epileptic seizures, as well as the effect on after the first injection of pilocarpine, the dose of pilo - lithium-pilocarpine-induced epileptic seizures in the rat carpine was increased by 20% every 10  min until the model. development of level-4–5 seizures. The total number Fig. 1 Schematic of experimental design Jiang  et al. Acta Epileptologica (2021) 3:19 Page 3 of 12 of pilocarpine injections was limited to five per animal. OSI-906 treatment significantly increased blood glucose After 60  min of status epilepticus (SE), diazepam (DZP, and aggravated epileptic seizures, and the mortality rate 10  mg/kg), phenobarbital (25  mg/kg), and atropine sul- reached 100% during the acute epileptic seizure, subse- fate (1  mg/kg) were  injected intraperitoneally to termi- quent relevant tests were not completed in this group. nate the convulsive seizures [21]. The seizure activity was scored according to the Racine’s standard criteria [22]. F‑FDG‑microPET imaging Only rats that achieved recurrent seizures of stage 4 to 5 MicroPET is a non-invasive imaging technique that is were used for subsequent tests. often used to record and analyze the correlation between neural function changes and brain energy metabolism Surgical procedures and electrophysiological recordings in small animals. To determine the effect of IR/IGF-1R The rat was anesthetized with chloral hydrate (350  mg/ inhibitor OSI-906 on glucose uptake in the brain after kg, i.p.) and flurbiprofen axetil was injected (5  mg/kg, the epileptic seizures, a F-FDG-PET-CT scanner (Sie- Beijing Tide Pharmaceutical Co., Ltd., Beijing) through mensInveon Multi-Modality System, Erlangen, Germany) a tail vein to relieve pain. Then, the rat was placed in a was used to detect cerebral glucose uptake at 1 h after the stereotaxic apparatus. A microwire array (4 × 4 array of pilocarpine-induced seizures. First, the rats were fixed platinum-iridium alloy electrodes, each 25  μm in diam- and injected with F-FDG (500 µCi) through the tail vein eter) was implanted in the right dorsal hippocampus as a tracer to detect the relative cerebral glucose metabo- (anterior–posterior –3.6  mm, medial–lateral 2.8  mm, lism rate. Thirty minutes later, they were anesthetized dorsal–ventral –3.5 mm) one week before recording and with isoflurane (induction period: 5% isoflurane + 1  L/ fixed with dental cement as described by Paxinos and min O , maintenance period: 1% isoflurane + 1 L/min O ) 2 2 Watson (2007). Local field potentials (LFPs) were pre - using an inhaled anesthesia system. Then the rats were amplified (× 1000), filtered (0.1–1000  Hz), and digitized scanned in the prone position in a three-dimensional at 4 kHz using an OmniPlex D Neural Data Acquisition model for 8 min (field of vision: 85.62 mm × 125.58 mm), System (Plexon, Dallas, TX). Electrophysiological record- and the static PET data were collected for 7 min. The CT ings were made with the direct current-coupled head signal was mainly used for anatomical description and stages, and all recordings were referenced to the two attenuation correction of F-FDG-PET images. ground screws. PET data analysis Analysis of electrophysiological data The imaging data were imported into the Inveon We employed NeuroExplorer (v4.0; Plexon, USA) and Research Workplace software for analysis, and data were MATLAB software (v7.1, R2009a; MathWorks, Inc., collected in the form of lists. The cerebral metabolism Natick, MA) to calculate the electrophysiological data. rate was reconstructed using the maximum posterior A fast Fourier transform was applied to power spectrum probability algorithm, with a pixel size of 0.4 × 0.4 × 1.2 analysis after digital filtering with 0.1–1000 Hz bandpass. mm . Glucose metabolism in the brain was evaluated as The frequency spectra of continuous variables and the the whole-brain average standard uptake values (SUV) neuronal rate histograms were obtained by the power using the following equation: SUV = Ct/ID*Wt.Ct(MBq/ spectral density analysis of our previous method [23]. We cm ), in which Ct indicates the activity concentration calculated the high-frequency discharge energy during after the decay correction of the measured brain region; seizures including ripples (80–200  Hz). The MATLAB ID (mCi) indicates the injection dose of F-FDG; and Wt function in the Signal Processing Toolbox was used to (kg) represents the weight of the rat [24]. We recorded compute the power of ripple oscillators and magnitude- the maximum and the mean SUV values of normal rats, squared coherence. MATLAB and GraphPad Prism soft- as well as SUV value 1  h post-seizure, and that post the wares (GraphPad Software, Inc., La Jolla, CA) were used OSI-906 intervention (OSI-906 50 mg/kg + SE). for statistical analysis and image processing. Projection electron microscopy Blood glucose monitoring The ultrastructural changes of nucleus and mitochondria Blood glucose was measured by a Roche glucometer in rat hippocampal neurons were observed by projection (Roche, Germany) in venous blood from the tail vein electron microscopy in the vehicle control, SE, and OSI- before treatment, 1 h after the first intragastric adminis - 906 + SE groups (n = 3). All animals were deeply anes- tration of vehicle or OSI-906, 1  h after gastric perfusion thetized with 10% chloral hydrate and then injected with of OSI-906 on the third day, and 1  h after the epileptic fresh normal saline to the heart. The rat was cardinally seizures. Animals were fasted for more than 12 h before perfused with a mixture of glutaraldehyde (2%) and para- OSI-906 intervention. As we found that 75  mg/kg of formaldehyde for heart reperfusion and tissue fixation, Jiang et al. Acta Epileptologica (2021) 3:19 Page 4 of 12 and then the brain was collected. The hippocampal tissue for comparisons between more than two groups; the Stu- was isolated immediately, and the CA1 area of the hip- dent’s t-test was used for comparison between groups of pocampus was cut into 1 mm pieces. Three specimens multiple samples, and the Bonferroni test was used for were taken from each rat and fixed in 4% glutaralde - post-test analysis. The Wilcoxon rank-sum test (Mann– hyde solution. The fixed tissues were sent to the electron Whitney U-test) was performed to compare two groups microscope room of Chongqing Medical University for of ordinal variables. The K-W test was used to compare unified specimen preparation. Images under projec - the classification data of multiple groups, and Fisher’s tion electron microscopy (JEOL JEM-1400, Japan) were exact test was also used to compare classification vari - captured. ables between two groups. P < 0.05 was considered as sta- tistically significant. Western blotting Hippocampal tissues were collected 24  h after pilocar- Results pine administration and stored at -80  °C for western OSI‑906 at 50 mg/kg significantly reduced the severity blotting. The tissues were homogenized in lysate buffer of SE and prevented mortality during SE containing phosphatase and protease inhibitors  (Sigma, We first observed the effect of OSI-906 on the latency St. Louis, MO), centrifuged (Sigma-Aldrich, USA) and of class 4–5 seizures induced by pilocarpine. Compared the supernatant collected. The total protein concentra - with the vehicle control group, the 50  mg/kg OSI-906 tion was determined with the BCA protein assay (Beyo- group showed a significantly prolonged latency of the time Institute of Biotechnology, China). The total protein pilocarpine-induced seizures (Bonferroni’s multiple com- samples were loaded at 40  μg/lane for sodium dodecyl parisons test, P = 0.028) and a decreased rate of first- sulfate polyacrylamide gel (5% spacer gel; 10% separat- dose seizures of stage 4–5 (Fisher’s exact test, P = 0.03). ing gel) electrophoresis, followed by electro-transfer onto In addition, the 25  mg/kg OSI-906 group did not show polyvinylidene fluoride membranes (Millipore). significant difference in the above indicators compared The membranes were blocked with 5% non-fat milk at to the control group (P > 0.05), while the 75  mg/kg OSI- room temperature for 120 min, then incubated with anti- 906 group had opposite effects to the 50 mg/kg OSI-906 p-IR (Tyr1361), anti-IR, anti-pIGF-1R (Tyr1135), anti- group (Table  1 and Fig.  2). At last, 7 rats in the vehicle IGF-1R, anti-p-AKT (Ser473), anti-AKT or anti-β-actin group, 7 rats in the 25  mg/kg OSI-906 group and 6 rats (1:500–1000, Cell Signaling Technology, Inc Boston, in the 50  mg/kg OSI-906 group developed recurrent MA, USA) antibody for 16  h at 4  °C. After TBST wash, stage 4–5 seizures. They were used for subsequent elec - the membranes were incubated with goat anti-rabbit IgG tron microscopy and western blotting assay (n = 5). Rats (1:5000, Cell Signaling Technology, Inc Boston, MA) for that underwent LFP recordings were added for electron 1  h. Electrochemiluminescence system (Bio-Rad, CA, microscopy detection (the side without electrodes). USA) was used to visualize the protein bands with a chemiluminescence kit (Biosharp, Beijing, China). Blot Eec ff t of OSI‑906 on hippocampal epileptiform discharge intensities were analyzed with QuantityOne software in pilocarpine‑induced seizures (Bio-Rad Laboratories, version 4.6.2, Hercules, CA). We recorded LFPs in hippocampal CA1 using in  vivo multichannel microfilament electrodes to investigate the Statistical analysis effect of OSI-906 on the excitability of the hippocam - All data were analyzed using the SPSS 19.0 statistical pal neural circuit during pilocarpine-induced seizures. software (IBM Corp., Armonk, NY), and the values are Compared to the vehicle control group, the 50  mg/kg expressed as mean ± SEM. All data were tested for nor- OSI-906 intervention group showed a significantly pro - mality and if non-normal, a non-parametric test was longed latency of epileptic discharge, a significantly used. One-way analysis of variance (ANOVA) was used shortened discharge duration, and a reduced average Table 1 Eec ff t of OSI-906 on acute epileptic seizures (n = 13) Group Latency of seizure (min) Seizure onset rate at Cumulative seizure rate Mortality the first dose Vehicle 28.55 ± 2.76 7/13 (53.85%) 11/13 (84.62%) 4/13 (30.77%) OSI-906 25 31.30 ± 4.01 6/13 (46.15%) 10/13 (76.92%) 3/13 (23.08%) OSI-906 50 41.33 ± 12.79* 1/13 (7.69%)* 6/13 (46.15%) 0/13 (0%) OSI-906 75 14.46 ± 14.08** 13/13 (100%)* 13/13 (100%)* 13/13 (100%)** Note: The dosing unit of OSI-906 is mg/kg. Compared with the vehicle group, *P < 0.05 and **P < 0.01 Jiang  et al. Acta Epileptologica (2021) 3:19 Page 5 of 12 Fig. 2 Eec ff ts of OSI-906 on acute epileptic seizures. (a) 50 mg/kg OSI-906 extended the seizure latency. (b) 50 mg/kg OSI-906 significantly reduced Racine score, but 75 mg/kg OSI-906 aggravated the severity of seizures. (c&d) First-dose and cumulative stage 4–5 seizure rates. (e) Eec ff ts of OSI-906 on mortality in acute rats. Bars represent mean ± SEM (n = 13 for each group). *P < 0.05 and **P < 0.01 compared to the control group power spectrum of electrical activity (Bonferroni’s mul- experiments to exclude possible confounding factors. The tiple comparisons test, P = 0.001, 0.013, 0.0001, respec- specific blood glucose values are shown in Table 2. tively, Fig.  3c, d). However, there was no such significant difference between the 25  mg/kg OSI-906 intervention OSI‑906 inhibits excessive energy consumption group and the vehicle group. The mortality was 30.77% of the brain caused by seizures in the control group, and zero in the 50  mg/kg OSI-906 Compared to the normal control group, the average group. Surprisingly, the death rate of animals was 100% whole-brain SUV value was significantly increased 1  h after pilocarpine-induced epileptic seizures in the 75 mg/ after the pilocarpine-induced recurrent epileptic seizures kg OSI-906 group. (1.63 ± 0.14 vs 1.98 ± 0.34, n = 5, P = 0.003, Fig. 5). In the 50  mg/kg OSI-906 group, the average whole-brain SUV Eec ff t of OSI‑906 on blood glucose value 1 h after the epileptic seizures was 1.77 ± 0.21, sig- The 25 mg/kg and 50 mg/kg groups had similar changes nificantly lower than the SE group (P = 0.013). However, of blood glucose in comparison to the control group at it was unclear whether the reduced SUVs were attributed different time points of treatment, suggesting that the to the inhibition of seizures or were because OSI-906 effect of OSI-906 on epileptic seizures was not directly lowered the glucose uptake. through the regulation of blood glucose. OSI-906 at 75  mg/kg significantly increased the blood glucose level Ultrastructural changes of the nucleus and mitochondria 1 h after the third gastric perfusion and 1 h after the epi- in neurons leptic seizures, compared to the control group (ANOVA The mitochondria are responsible for aerobic respiration and post hoc analysis using the Bonferroni method, and energy transformation, and make most of the cell’s P < 0.01 and P < 0.0001, respectively, n = 5; Fig. 4). Consid- supply. Repeated or continuous epileptic seizures rely on ering that hyperglycemia or hypoglycemia may affect the mitochondrial function. Meanwhile, excessive energy mitochondrial function and epileptic seizures [25], the consumption caused by epileptic seizures will destroy the 75 mg/kg OSI-906 group was excluded from subsequent mitochondrial structure and function, leading to persistent Jiang et al. Acta Epileptologica (2021) 3:19 Page 6 of 12 Fig. 3 Eec ff ts of OSI-906 on epileptic discharge in acute epileptic seizures. (a) Eec ff ts of OSI-906 on hippocampal electrical activity (the X-axis is 60 min and the Y-axis is ± 1.0 millivolts). (b) Corresponding power spectrograms of each group. (c‑ e) 50 mg/kg OSI-906 shortened the latency of epileptic discharge, and reduced the duration of epileptic discharge and the mean energy density value of brain electrical activity. Data are shown as mean ± SEM. *P < 0.05 and **P < 0.01 compared to the control group; n = 5 each group disorders of brain energy metabolism in patients with key targets in epilepsy research and treatment strategies. epilepsy, thus resulting in a vicious cycle [4, 26]. There - Transmission electron microscopy revealed that the nor- fore, brain energy metabolism and mitochondria are mal rat hippocampal neurons had a clear outline, a large Jiang  et al. Acta Epileptologica (2021) 3:19 Page 7 of 12 Fig. 4 Eec ff t of OSI-906 treatment on blood glucose before and after the seizures. (a) Blood glucose level did not significantly change at 1 h after the first OSI-906 gavage, compared with the control group (P > 0.05). (b&c) OSI-906 at 75 mg/kg significantly increased the blood glucose at fasting or 1 h after gavage on day 3. P = 0.0024 and 0.0002, respectively, vs the control, one-way ANOVA. (d) In the 75 mg/kg OSI-906 group, blood glucose levels were higher after the seizure compared with the control group. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 (n = 5) and round nucleus, uniform chromatin, clear cytoplasm, the mitochondrial cristae were disordered, shortened, and clear and complete double-layer nuclear membrane at swollen. The cristae of mitochondria blurred, were bro - high magnification, and mitochondria with complete inner ken, and disappeared, the matrix was swollen and vacu- and outer membranes as well as cristae formed by inner- olated, and an autophagosome was formed. The nucleus membrane depression. At 24 h after the epileptic seizures, of the neuron was shrunken and deeply stained, and the Table 2 Eec ff ts of OSI-906 on blood glucose (n = 5) Groups Blood glucose 1 h after the Blood glucose before gavage Blood glucose1 h after gavage Blood glucose 1 h first OSI‑906 (mmol/L) on day 3 (mmol/L) on day 3 (mmol/L) after SE (mmol/L) vehicle 5.94 ± 0.31 6.02 ± 0.48 6.26 ± 0.79 15.60 ± 1.09 OSI-906 25 mg/kg 6.24 ± 0.49 6.56 ± 0.54 7.10 ± 0.84 16.78 ± 1.18 OSI-906 50 mg/kg 6.52 ± 0.58 7.22 ± 1.20 8.44 ± 2.18 17.04 ± 1.44 OSI-906 75 mg/kg 6.84 ± 0.90 8.12 ± 2.10** 11.18 ± 4.92*** ≧33.33**** ** P < 0.01, ***P < 0.001, and ****P < 0.0001 vs the vehicle control group Jiang et al. Acta Epileptologica (2021) 3:19 Page 8 of 12 Fig. 5 OSI-906 decreased the glucose uptake 1 h after seizures. (a) F-FDG micro-PET/CT quantification of glucose metabolism in vivo. (b) Compared with the normal control group, the average whole-brain SUV value was significantly increased 1 h after pilocarpine-induced epileptic seizures. Treatment with 50 mg/kg of OSI-906 significantly reduced the value of glucose uptake in the brain (n = 5). Data are shown as mean ± SEM. **P < 0.01, P < 0.05 chromatin in the nucleus was concentrated. Compared signaling pathways involved in the pilocarpine- with the epilepsy model group, the 50  mg/kg OSI-906 induced seizures. intervention group showed less damage to the mitochon- dria and nucleus (Fig. 6). Discussion In the present study, we showed that OSI-906 at 50  mg/ Eec ff ts of OSI‑906 on IR/IGF‑1R and Akt signaling kg significantly reduced the susceptibility to and sever - As shown in Fig.  7, the  pIR/IR, pIGF-1R/IGF-1R, ity of seizures induced by pilocarpine (Table  1 &Fig.  2). and p-AKT/AKT ratios were prominently elevated Synchronous EEG recording also showed that 50  mg/kg at 24  h after the pilocarpine-induced seizures, com- of OSI-906 significantly prolonged the latency of epilep - pared to those in the vehicle group (n = 5, AN OVA , tic discharge, shortened the duration of discharge, and P < 0.01). However, they were markedly suppressed decreased the mean energy density of  brain electrical in the 50  mg/kg OSI-906 group (Fig.  7, P < 0.05), sug- activity (Fig. 3). However, OSI-906 at 75 mg/kg increased gesting that 50  mg/kg of OSI-906 could inhibit the the blood glucose and aggravated seizures (Table  2, activation of IR/IGF-1R and the downstream AKT Fig.  4). As an increase or decrease of blood glucose can Jiang  et al. Acta Epileptologica (2021) 3:19 Page 9 of 12 Fig. 6 Ultrastructural changes of nucleus and mitochondria in neurons. (a) Hippocampal neurons and mitochondria in control rats. (b) Ultrastructural changes of nuclei and mitochondria in hippocampal neurons. (c) Compared to the epilepsy model group, the 50 mg/kg OSI-906 group showed less damage to the mitochondria and nuclei. a, b and c are magnified 8,000 times, and a1, b1 and c1 are magnified 20,000 times affect the mitochondrial function and epileptic activity related to its inhibition of seizure activity (Fig. 5). During [25], this group of rats failed to complete the subsequent epileptic seizures, neurons are highly synchronized and tests. discharge abnormally, which leads to the brain energy In a clinical study, OSI-906 was continuously given depletion. This excessive energy depletion depends on in a once-  or twice-daily schedule in patients with the mitochondrial oxidative phosphorylation to produce advanced solid tumors [27]. In this study, the blood glu- a large amount of ATP. Of note, energy depletion hinders cose increased after 75  mg/kg of OSI-906 application glucose transport and oxidative metabolism and destroys for 3 consecutive days, indicating a cumulative effect of the mitochondrial structure and function [4]. As a result, OSI-906. However, there was no significant difference in after the onset of epilepsy, ATP production is compro- blood glucose during single administration, and continu- mised, and pathological changes such as abnormal neu- ous dosing could better reflect the effect of homeostasis ronal ion transport, disorders of neurotransmitter uptake OSI-906 concentration on epileptic activity. The high and release, and blockade of protein signaling, may ulti- dose of OSI-906 (75  mg/kg) inhibits IR/IGF-1R, result- mately result in recurrent spontaneous epileptic seizures, ing in increased blood glucose, which may be the main namely, epileptogenesis [30, 31]. In addition, after long- reason for the opposite outcome to that of 50 mg/kg OSI- term spontaneous seizures, significantly reduced glucose 906. In addition, the blood glucose increased significantly metabolism is observed in multiple brain regions, which in the vehicle group and in different dose groups of OSI- can affect the neuronal function [32–34]. Hence, OSI-906 906 after SE, which may be related to the stress response may inhibit the seizure activity through reduction of the induced by SE. However, the specific mechanisms remain glucose uptake in the brain and inhibition of excessive unclear. energy consumption of the brain caused by seizures. The brain is a high-consumption organ, which consti - We further found that the epileptic seizures resulted tutes only 2% of the human body weight, while consum- in changes in mitochondrial structure, including dis- ing 20% to 25% of the energy supply in the body [28]. The ordered and shortened cristae, partial mitochondrial brain energy metabolism has been shown as a key factor swelling, mitochondrial ridge blurring, fracture and dis- affecting the epileptic seizures, and may be an important appearance, matrix swelling, vacuolization, neuronal target for epilepsy research and treatment [29]. nuclear shriveling, and hyperchromatic concentration, Surprisingly, the F-FDG-PET scanning showed that which were improved by 50  mg/kg of OSI-906 (Fig.  6). 50 mg/kg of OSI-906 treatment significantly reduced the As an alternative energy source, a ketogenic diet may be average whole-brain SUV value after seizures, possibly an effective treatment of epilepsy, which improves the Jiang et al. Acta Epileptologica (2021) 3:19 Page 10 of 12 Fig. 7 Eec ff ts of OSI-906 on IR, IGF-1R and AKT signaling. (a) The representative bands of western blotting. (b-d) The pIR/IR, pIGF-1R/IGF-1R, and p-Akt/Akt ratios were significantly elevated at 24 h after pilocarpine-induced seizures, compared with those in the vehicle group. Compared to those in the seizure group, these ratios were markedly reduced in the 50 mg/kg OSI-906 group. Data are shown as mean ± SEM. n = 5, # ### ANOVA,**P < 0.01, ***P < 0.001, P < 0.05, P < 0.001 neuronal damage caused by decreased energy metabo- seizures (Fig.  7). We speculated that during the pilocar- lism in the brain during epileptic seizures [35–37]. Our pine-induced epileptic status, OSI-906 inhibits the exces- study suggests that the inhibition of epileptic activity by sive energy intake and consumption in the brain, possibly 50  mg/kg of OSI-906 may restrain the excessive energy through inhibiting the IR/IGF-1R and the downstream consumption of the brain by regulating the function of AKT signaling. More studies are needed to fully clarify mitochondria and may have a protective effect on mito - the underlying mechanisms. chondria in the context of epileptic seizures. In our behavioral and electrophysiological studies, we found Conclusion that 50  mg/kg of OSI-906 significantly reduced the sus - In conclusion, OSI-906 at 50  mg/kg inhibits the brain ceptibility to and severity of seizures. energy metabolism and epileptic activity during epilep- The small molecule OSI-906 is a dual receptor inhibitor tic seizure, and has a protective effect on mitochondrial of IR/IGF-1R, which effectively and selectively inhibits damage caused by epileptic seizure. OSI-906 at 50 mg/kg the phosphorylation of IR and IGF-1R and has a strong may inhibit epileptic seizures by inhibiting the IR/IGF- anti-tumor effect in many types of cancer [38]. We found 1R and the  downstream AKT signaling. These results that 50 mg/kg of OSI-906 significantly inhibited the acti - provide new evidence for the use of OSI-906 in the treat- vation of IR, IGF-1R, and AKT signals after epileptic ment of epileptic status. Jiang  et al. Acta Epileptologica (2021) 3:19 Page 11 of 12 8. 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Journal

Acta Epileptologica – Springer Journals

Published: Aug 24, 2021

Keywords: Epilepsy; Mitochondria; Energy metabolism; Insulin receptor; IGF-1 receptor; Akt signaling

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