Cannabinoid receptor 2 agonist AM1241 alleviates epileptic seizures and epilepsy-associated depression via inhibiting neuroinflammation in a pilocarpine-induced chronic epilepsy mouse model.

Increasing evidence suggests that cannabinoid receptor 2 (CB2R) serves as a promising anti-inflammatory target. While inflammation is known to play crucial roles in the pathogenesis of epilepsy, the involvement of CB2R in epilepsy remains unclear. This study aimed to investigate the effects of a CB2R agonist, AM1241, on epileptic seizures and depressive-like behaviors in a mouse model of chronic epilepsy induced by pilocarpine. A chronic epilepsy mouse model was established by intraperitoneal administration of pilocarpine. The endogenous cannabinoid system (eCBs) in the hippocampus was examined after status epilepticus (SE). Animals were then treated with AM1241 and compared with a vehicle-treated control group. Additionally, the role of the AMPK/NLRP3 signaling pathway was explored using the selective AMPK inhibitor dorsomorphin. Following SE, CB2R expression increased significantly in hippocampal microglia. Administration of AM1241 significantly reduced seizure frequency, immobility time in the tail suspension test, and neuronal loss in the hippocampus. In addition, AM1241 treatment attenuated microglial activation, inhibited pro-inflammatory polarization of microglia, and suppressed NLRP3 inflammasome activation in the hippocampus after SE. Further, the therapeutic effects of AM1241 were abolished by the AMPK inhibitor dorsomorphin. Our findings suggest that CB2R agonist AM1241 may alleviate epileptic seizures and its associated depression by inhibiting neuroinflammation through the AMPK/NLRP3 signaling pathway. These results provide insight into a novel therapeutic approach for epilepsy.

Regulation of Erythroid Differentiation via the HIF1α-NFIL3-PIM1 Signaling Axis Under Hypoxia.

Aims: Erythropoiesis is controlled by several factors, including oxygen level under different circumstances. However, the role of hypoxia in erythroid differentiation and the underlying mechanisms are poorly understood. We studied the effect and mechanism of hypoxia on erythroid differentiation of K562 cells and observed the effect of hypoxia on early erythropoiesis of zebrafish. Results: Compared with normal oxygen culture, both hemin-induced erythroid differentiation of K562 cells and the early erythropoiesis of zebrafish were inhibited under hypoxic treatment conditions. Hypoxia-inducible factor 1 alpha (HIF1α) plays a major role in the response to hypoxia. Here, we obtained a stable HIF1α knockout K562 cell line using the CRISPR-Cas9 technology and further demonstrated that HIF1α knockout promoted hemin-induced erythroid differentiation of K562 cells under hypoxia. We demonstrated an HIF1-mediated induction of the nuclear factor interleukin-3 (NFIL3) regulated in K562 cells under hypoxia. Interestingly, a gradual decrease in NFIL3 expression was detected during erythroid differentiation of erythropoietin-induced CD34+ hematopoietic stem/progenitor cells (HSPCs) and hemin-induced K562 cells. Notably, erythroid differentiation was inhibited by enforced expression of NFIL3 under normoxia and was promoted by the knockdown of NFIL3 under hypoxia in hemin-treated K562 cells. In addition, a target of NFIL3, pim-1 proto-oncogene, serine/threonine kinase (PIM1), was obtained by RNA microarray after NFIL3 knockdown. PIM1 can rescue the inhibitory effect of NFIL3 on hemin-induced erythroid differentiation of K562 cells. Innovation and Conclusion: Our findings demonstrate that the HIF1α-NFIL3-PIM1 signaling axis plays an important role in erythroid differentiation under hypoxia. These results will provide useful clues for preventing the damage of acute hypoxia to erythropoiesis.

Neuropilin-2 Signaling Modulates Mossy Fiber Sprouting by Regulating Axon Collateral Formation Through CRMP2 in a Rat Model of Epilepsy.

Programmed neural circuit formation constitutes the foundation for normal brain functions. Axon guidance cues play crucial roles in neural circuit establishment during development. Whether or how they contribute to maintaining the stability of networks in mature brains is seldom studied. Upon injury, neural rewiring could happen in adulthood, of which mossy fiber sprouting (MFS) is a canonical example. Here, we uncovered a novel role of axon guidance molecule family Sema3F/Npn-2 signaling in MFS and epileptogenesis in a rat model of epilepsy. Dentate gyrus-specific Npn-2 knockdown increased seizure activity in epileptic animals along with increased MFS. Hippocampal culture results suggested that Npn-2 signaling modulates MFS via regulating axon outgrowth and collateral formation. In addition, we discovered that Sema3F/Npn-2 signal through CRMP2 by regulating its phosphorylation in the process of MFS. Our work illustrated that Npn-2 signaling in adult epilepsy animals could potentially modulate seizure activity by controlling MFS. MFS constitutes the structural basis for abnormal electric discharge of neurons and recurrent seizures. Therapies targeting Npn-2 signaling could potentially have disease-modifying anti-epileptogenesis effects in epilepsy treatment.

Conditional knockout of ASK1 in microglia/macrophages attenuates epileptic seizures and long-term neurobehavioural comorbidities by modulating the inflammatory responses of microglia/macrophages.

BACKGROUND: Apoptosis signal-regulating kinase 1 (ASK1) not only causes neuronal programmed cell death via the mitochondrial pathway but also is an essential component of the signalling cascade during microglial activation. We hypothesize that ASK1 selective deletion modulates inflammatory responses in microglia/macrophages(Mi/Mϕ) and attenuates seizure severity and long-term cognitive impairments in an epileptic mouse model. METHODS: Mi/Mϕ-specific ASK1 conditional knockout (ASK1 cKO) mice were obtained for experiments by mating ASK1flox/flox mice with CX3CR1creER mice with tamoxifen induction. Epileptic seizures were induced by intrahippocampal injection of kainic acid (KA). ASK1 expression and distribution were detected by western blotting and immunofluorescence staining. Seizures were monitored for 24 h per day with video recordings. Cognition, social and stress related activities were assessed with the Y maze test and the three-chamber social novelty preference test. The heterogeneous Mi/Mϕ status and inflammatory profiles were assessed with immunofluorescence staining and real-time polymerase chain reaction (q-PCR). Immunofluorescence staining was used to detect the proportion of Mi/Mϕ in contact with apoptotic neurons, as well as neuronal damage. RESULTS: ASK1 was highly expressed in Mi/Mϕ during the acute phase of epilepsy. Conditional knockout of ASK1 in Mi/Mϕ markedly reduced the frequency of seizures in the acute phase and the frequency of spontaneous recurrent seizures (SRSs) in the chronic phase. In addition, ASK1 conditional knockout mice displayed long-term neurobehavioral improvements during the Y maze test and the three-chamber social novelty preference test. ASK1 selective knockout mitigated neuroinflammation, as evidenced by lower levels of Iba1+/CD16+ proinflammatory Mi/Mϕ. Conditional knockout of ASK1 increased Mi/Mϕ proportion in contact with apoptotic neurons. Neuronal loss was partially restored by ASK1 selective knockout. CONCLUSION: Conditional knockout of ASK1 in Mi/Mϕ reduced seizure severity, neurobehavioral impairments, and histological damage, at least via inhibiting proinflammatory microglia/macrophages responses. ASK1 in microglia/macrophages is a potential therapeutic target for inflammatory responses in epilepsy.

CRMP2 modulates mossy fiber sprouting in dentate gyrus of pilocarpine induced rat model of epilepsy.

As a hallmark of epilepsy, mossy fiber sprouting was regarded as an ideal mode to study neural rewiring upon injury. The process of mossy fiber sprouting constitutes key steps for neural circuit formation, including axon collateral formation and outgrowth, reversed pathfinding and synapse connection. The canonical function of CRMP2 is to promote neurite/axon outgrowth via binding to tubulin heterodimers, which is mainly regulated by its phosphorylation state. CRMP2 expression and phosphorylation were reported to change in medial temporal epilepsy patients and animal modes of epilepsy. As a novel anti-epilepsy drug, Lacosamide is able to impair CRMP2 mediated tubulin polymerization. Previous studies suggested possible roles of CRMP2 in mossy fiber sprouting. Here, we provide direct evidence to support the role of CRMP2 in the process of mossy fiber sprouting in an animal model of epilepsy. We found that CRMP2 phosphorylation was downregulated specifically in the hippocampus during latent phase of epileptic rats. In addition, with the reduction of CRMP2 expression levels in dentate gyrus by CRMP2 shRNA, we observed decreased mossy fiber sprouting in these CRMP2 knockdown rats. Our results demonstrated that CRMP2 modulates mossy fiber sprouting in dentate gyrus of pilocarpine induced rat model of epilepsy.

Inhibition of Gasdermin D-Mediated Pyroptosis Attenuates the Severity of Seizures and Astroglial Damage in Kainic Acid-Induced Epileptic Mice.

Objective: Our study aimed to explore whether gasdermin D (GSDMD)-mediated pyroptosis is involved in the mechanism of kainic acid-induced seizures. Methods: C57BL/6 mice were randomly divided into sham and epilepsy groups. The epilepsy group was intrahippocampally injected with kainic acid to induce status epilepticus (SE), and the sham group was injected with an equal volume of saline. Dimethyl fumarate (DMF) was used as the GSDMD N-terminal fragments (GSDMD-N) inhibitor and suspended in 0.5% sodium carboxymethyl cellulose (CMC) for orally administration. The epilepsy group was divided into SE + CMC and SE + DMF groups. In the SE + DMF group, DMF was orally administered for 1 week before SE induction and was continued until the end of the experiment. An equal volume of CMC was administered to the sham and SE + CMC groups. Recurrent spontaneous seizures (SRSs) were monitored for 21 days after SE. Western blot analysis and immunofluorescent staining was performed. Results: The expression of GSDMD increased at 7-21 days post-SE, and GSDMD-N expression was significantly elevated 7 days after SE in both ipsilateral and contralateral hippocampus. GSDMD-positive cells were co-labeled with astrocytes, but not neurons or microglia. Astroglial damage occurs following status epilepticus (SE). Damaged astrocytes showed typical clasmatodendrosis in the CA1 region containing strong GSDMD expression at 7-21 days post-SE, accompanied by activated microglia. In the SE + DMF group, the expression of GSDMD-N was significantly inhibited compared to that in the SE + CMC group. After administration of DMF, SRSs at 7-21 days after SE were significantly decreased, and the number of clasmatodendritic astrocytes, microglia, and the expression of inflammatory factors such as IL-1ß and IL-18 were significantly attenuated. Conclusion: GSDMD-mediated pyroptosis is involved in the mechanism of kainic acid-induced seizures. Our study provides a new potential therapeutic target for seizure control.