Exploring Dendritic and Spine Structural Profiles in Epilepsy: Insights From Human Studies and Experimental Animal Models.

Dendrites are tree-like structures with tiny spines specialized to receive excitatory synaptic transmission. Spino-dendritic plasticity, driven by neural activity, underlies the maintenance of neuronal connections crucial for proper circuit function. Abnormalities in dendritic morphology are frequently seen in epilepsy. However, the exact etiology or functional implications are not yet known. Therefore, to better comprehend the structure-function significance of this dendritic pathology in epilepsy, it is necessary to identify the common spino-dendritic disturbances present in both human and experimental models. Here, we describe the dendritic and spine structural profiles found across human refractory epilepsy as well as in animal models of developmental, acquired, and genetic epilepsies.

Human iPSC-derived microglia sense and dampen hyperexcitability of cortical neurons carrying the epilepsy-associated SCN2A-L1342P mutation.

Neuronal hyperexcitability is a hallmark of seizures. It has been recently shown in rodent models of seizures that microglia, the brain's resident immune cells, can respond to and modulate neuronal excitability. However, how human microglia interacts with human neurons to regulate hyperexcitability mediated by epilepsy-causing genetic mutation found in human patients remains unknown. The SCN2A genetic locus is responsible for encoding the voltage-gated sodium channel Nav1.2, recognized as one of the leading contributors to monogenic epilepsies. Previously, we demonstrated that the recurring Nav1.2-L1342P mutation identified in patients with epilepsy leads to hyperexcitability in a hiPSC-derived cortical neuron model from a male donor. While microglia play an important role in the brain, these cells originate from a different lineage (yolk sac) and thus are not naturally present in hiPSCs-derived neuronal culture. To study how microglia respond to diseased neurons and influence neuronal excitability, we established a co-culture model comprising hiPSC-derived neurons and microglia. We found that microglia display altered morphology with increased branch length and enhanced calcium signal when co-cultured with neurons carrying the Nav1.2-L1342P mutation. Moreover, the presence of microglia significantly lowers the action potential firing of neurons carrying the mutation. Interestingly, we further demonstrated that the current density of sodium channels in neurons carrying the epilepsy-associated mutation was reduced in the presence of microglia. Taken together, our work reveals a critical role of human iPSCs-derived microglia in sensing and dampening hyperexcitability mediated by an epilepsy-causing mutation present in human neurons, highlighting the importance of neuron-microglia interactions in human pathophysiology.

Mice deficient in complement C3 are protected against recognition memory deficits and astrogliosis induced by status epilepticus.

Introduction: Status epilepticus (SE) can significantly increase the risk of temporal lobe epilepsy (TLE) and cognitive comorbidities. A potential candidate mechanism underlying memory defects in epilepsy may be the immune complement system. The complement cascade, part of the innate immune system, modulates inflammatory and phagocytosis signaling, and has been shown to contribute to learning and memory dysfunctions in neurodegenerative disorders. We previously reported that complement C3 is elevated in brain biopsies from human drug-resistant epilepsy and in experimental rodent models. We also found that SE-induced increases in hippocampal C3 levels paralleled the development of hippocampal-dependent spatial learning and memory deficits in rats. Thus, we hypothesized that SE-induced C3 activation contributes to this pathophysiology in a mouse model of SE and acquired TLE. Methods: In this study C3 knockout (KO) and wild type (WT) mice were subjected to one hour of pilocarpine-induced SE or sham conditions (control; C). Following a latent period of two weeks, recognition memory was assessed utilizing the novel object recognition (NOR) test. Western blotting was utilized to determine the protein levels of C3 in hippocampal lysates. In addition, we assessed the protein levels and distribution of the astrocyte marker glial fibrillary acidic protein (GFAP). Results: In the NOR test, control WT + C or C3 KO + C mice spent significantly more time exploring the novel object compared to the familiar object. In contrast, WT+SE mice did not show preference for either object, indicating a memory defect. This deficit was prevented in C3 KO + SE mice, which performed similarly to controls. In addition, we found that SE triggered significant increases in the protein levels of GFAP in hippocampi of WT mice but not in C3 KO mice. Discussion: These findings suggest that ablation of C3 prevents SE-induced recognition memory deficits and that a C3-astrocyte interplay may play a role. Therefore, it is possible that enhanced C3 signaling contributes to SE-associated cognitive decline during epileptogenesis and may serve as a potential therapeutic target for treating cognitive comorbidities in acquired TLE.

Assessment of tau phosphorylation and ß-amyloid pathology in human drug-resistant epilepsy.

OBJECTIVE: Epilepsy can be comorbid with cognitive impairments. Recent evidence suggests the possibility that cognitive decline in epilepsy may be associated with mechanisms typical of Alzheimer's disease (AD). Neuropathological hallmarks of AD have been found in brain biopsies surgically resected from patients with drug-resistant epilepsies. These include hyperphosphorylation of the tau protein (p-tau) that aggregates into neuropil threads (NT) or neurofibrillary tangles (NFT), as well as the presence of ß-amyloid (Aß) deposits. While recent studies agree on these AD neuropathological findings in epilepsy, some contrast in their correlation to cognitive decline. Thus, to further address this question we determined the abundance of p-tau and Aß proteins along with their association with cognitive function in 12 cases of refractory epilepsy. METHODS: Cortical biopsies surgically extracted from the temporal lobes of patients with refractory epilepsy were processed for immunohistology and enzyme-linked immunoassays to assess distribution and levels, respectively, of p-tau (Antibodies: Ser202/Thr205; Thr205; Thr181) and Aß proteins. In parallel, we measured the activation of mechanistic target of rapamycin (mTOR) via p-S6 (Antibodies: Ser240/244; Ser235/236). Pearson correlation coefficient analysis determined associations between these proteins and neurophysiological scores for full-scale intelligence quotient (FSIQ). RESULTS: We found a robust presence of p-tau (Ser202/Thr205)-related NT and NFT pathology, as well as Aß deposits, and p-S6 (Ser240/244; Ser235/236) in the epilepsy biopsies. We found no significant correlations between p-tau (Thr205; Thr181), Aß, or mTOR markers with FSIQ scores, although some correlation coefficients were modest to strong. SIGNIFICANCE: These findings strongly support the existence of hyperphosphorylated tau protein and Aß deposits in patients with human refractory epilepsy. However, their relation to cognitive decline is still unclear and requires further investigation.

The validity of animal models to explore the pathogenic role of the complement system in multiple sclerosis: A review.

Animal models of multiple sclerosis (MS) have been extensively used to characterize the disease mechanisms in MS, as well as to identify potential pharmacologic targets for this condition. In recent years, the immune complement system has gained increased attention as an important effector in the pathogenesis of MS. Evidence from histological, serum, and CSF studies of patients supports an involvement of complement in both relapsing-remitting and progressive MS. In this review, we discuss the history and advances made on the use of MS animal models to profile the effects of the complement system in this condition. The first studies that explored the complement system in the context of MS used cobra venom factor (CVF) as a complement depleting agent in experimental autoimmune encephalomyelitis (EAE) Lewis rats. Since then, multiple mice and rat models of MS have revealed a role of C3 and the alternative complement cascade in the opsonization and phagocytosis of myelin by microglia and myeloid cells. Studies using viral vectors, genetic knockouts and pharmacologic complement inhibitors have also shown an effect of complement in synaptic loss. Antibody-mediated EAE models have revealed an involvement of the C1 complex and the classical complement as an effector of the humoral response in this disease. C1q itself may also be involved in modulating microglia activation and oligodendrocyte differentiation in these animals. In addition, animal and in vitro models have revealed that multiple complement factors may act as modulators of both the innate and adaptive immune responses. Finally, evidence gathered from mice models suggests that the membrane attack complex (MAC) may even exert protective roles in the chronic stages of EAE. Overall, this review summarizes the importance of MS animal models to better characterize the role of the complement system and guide future therapeutic approaches in this condition.

Suppression of Microgliosis With the Colony-Stimulating Factor 1 Receptor Inhibitor PLX3397 Does Not Attenuate Memory Defects During Epileptogenesis in the Rat.

Events of status epilepticus (SE) trigger the development of temporal lobe epilepsy (TLE), a type of focal epilepsy that is commonly drug-resistant and is highly comorbid with cognitive deficits. While SE-induced hippocampal injury, accompanied by gliosis and neuronal loss, typically disrupts cognitive functions resulting in memory defects, it is not definitively known how. Our previous studies revealed extensive hippocampal microgliosis that peaked between 2 and 3 weeks after SE and paralleled the development of cognitive impairments, suggesting a role for reactive microglia in this pathophysiology. Microglial survival and proliferation are regulated by the colony-stimulating factor 1 receptor (CSF1R). The CSF1R inhibitor PLX3397 has been shown to reduce/deplete microglial populations and improve cognitive performance in models of neurodegenerative disorders. Therefore, we hypothesized that suppression of microgliosis with PLX3397 during epileptogenesis may attenuate the hippocampal-dependent spatial learning and memory deficits in the rat pilocarpine model of SE and acquired TLE. Different groups of control and SE rats were fed standard chow (SC) or chow with PLX3397 starting immediately after SE and for 3 weeks. Novel object recognition (NOR) and Barnes maze (BM) were performed to determine memory function between 2 and 3 weeks after SE. Then microglial populations were assessed using immunohistochemistry. Control rats fed with either SC or PLX3397 performed similarly in both NOR and BM tests, differentiating novel vs. familiar objects in NOR, and rapidly learning the location of the hidden platform in BM. In contrast, both SE groups (SC and PLX3397) showed significant deficits in both NOR and BM tests compared to controls. Both PLX3397-treated control and SE groups had significantly decreased numbers of microglia in the hippocampus (60%) compared to those in SC. In parallel, we found that PLX3397 treatment also reduced SE-induced hippocampal astrogliosis. Thus, despite drastic reductions in microglial cells, memory was unaffected in the PLX3397-treated groups compared to those in SC, suggesting that remaining microglia may be sufficient to help maintain hippocampal functions. In sum, PLX3397 did not improve or worsen the memory deficits in rats that sustained pilocarpine-induced SE. Further research is required to determine whether microglia play a role in cognitive decline during epileptogenesis.

Increased expression of Fragile X mental retardation protein in malformative lesions of patients with focal cortical dysplasia.

OBJECTIVE: Focal cortical dysplasia (FCD) accounts for nearly half of all cases of medically refractory epilepsy in the pediatric and adult patient populations. This neurological disorder stems from localized malformations in cortical brain tissue due to impaired neuronal proliferation, differentiation, and migration patterns. Recent studies in animal models have highlighted the potential role of the Fragile X mental retardation protein (FMRP) levels in FCD. The purpose of this study was to investigate the status of FMRP activation in cortical brain tissues surgically resected from patients with FCD. In parallel, this study also investigated protein levels within the PI3K/AKT/mTOR and canonical Wnt signaling pathways. METHODS: Pathologic tissue from malformative lesions of FCD patients with medically refractory epilepsy was compared to relatively normal control non-epileptic tissue from patients with intracranial neoplasms. A series of western blotting assays were performed to assess key proteins in the PI3K/AKT/mTOR, canonical Wnt signaling pathways, and FMRP. RESULTS: There was suppression of S235/236-phosphorylated S6, GSK3α, and GSK3ß protein levels in samples derived from FCD patients, compared to non-epileptic controls. FCD samples also had significantly greater levels of total and S499-phosphorylated FMRP. CONCLUSION: These findings support our hypothesis that malformative lesions associated with FCD are characterized by high levels of FMRP activation along with dysregulation of both PI3K/AKT/mTOR and canonical Wnt signaling. These novel clinical findings extend previous work in animal models, further suggesting a potential unforeseen role of GSK3α and GSK3ß in the pathophysiology of FCD and refractory epilepsy.

Emerging Roles for Microglial Phagocytic Signaling in Epilepsy.

Microglia are the resident immune cells and professional phagocytes of the central nervous system. However, little is known about the contribution of their phagocytic signaling to the neuropathology and pathophysiology of epilepsy. Here, we summarize and discuss the implications of recent evidence supporting that aberrant microglia phagocytic activity and alterations in phagocytosis signaling molecules occur in association with microglia-neuronal contacts, neuronal/synaptic loss, and spontaneous recurrent seizures in human and preclinical models of epilepsy. This body of evidence provides strong support that the microglial contribution to epileptogenic networks goes beyond inflammation, and suggests that phagocytic signaling molecules may be novel therapeutic targets for epilepsy.

Early treatment with C1 esterase inhibitor improves weight but not memory deficits in a rat model of status epilepticus.

BACKGROUND: Status epilepticus (SE) is a prolonged and continuous seizure that lasts for at least 5 min. An episode of SE in a healthy system can lead to the development of spontaneous seizures and cognitive deficits which may be accompanied by hippocampal injury and microgliosis. Although the direct mechanisms underlying the SE-induced pathophysiology remain unknown, a candidate mechanism is hyperactivation of the classical complement pathway (C1q-C3 signaling). We recently reported that SE triggered an increase in C1q-C3 signaling in the hippocampus that closely paralleled cognitive decline. Thus, we hypothesized that blocking activation of the classical complement pathway immediately after SE may prevent the development of SE-induced hippocampal-dependent learning and memory deficits. METHODS: Because C1 esterase inhibitor (C1-INH) negatively regulates activation of the classical complement pathway, we used this drug to test our hypothesis. Two groups of male rats were subjected to 1 hr of SE with pilocarpine (280-300 mg/kg, i.p.), and treated with either C1-INH (SE+C1-INH, 20 U/kg, s.c.) or vehicle (SE+veh) at 4, 24, and 48 h after SE. Control rats were treated with saline. Body weight was recorded for up to 23 days after SE. At two weeks post SE, recognition and spatial memory were determined using Novel Object Recognition (NOR) and Barnes maze (BM), respectively, as well as locomotion and anxiety-like behaviors using Open Field (OF). Histological and biochemical methods were used to measure hippocampal injury including cell death, microgliosis, and inflammation. RESULTS: One day after SE, both SE groups had a significant loss of body weight compared to controls (p<0.05). By day 14, the weight of SE+C1-INH rats was significantly higher than SE+veh rats (p<0.05), and was not different from controls (p>0.05). At 14 days post-SE, SE+C1-INH rats displayed higher mobility (distance travelled and average speed, p<0.05) and had reduced anxiety-like behaviors (outer duration, p<0.05) than control or SE+veh rats. In NOR, control rats spent significantly more time exploring the novel object vs. the familiar (p<0.05), while rats in both SE groups spent similar amount of time exploring both objects. During days 1-4 of BM training, the escape latency of the control group significantly decreased over time (p<0.05), whereas that of the SE groups did not improve (p>0.05). Compared to vehicle-treated SE rats, SE+C1-INH rats had increased levels of C3 and microglia in the hippocampus, but lower levels of caspase-3 and synaptic markers. CONCLUSIONS: These findings suggest that acute treatment with C1-INH after SE may have some protective, albeit limited, effects on the physiological recovery of rats' weight and some anxiolytic effects, but does not attenuate SE-induced deficits in hippocampal-dependent learning and memory. Reduced levels of caspase-3 suggest that treatment with C1-INH may protect against cell death, perhaps by regulating inflammatory pathways and promoting phagocytic/clearance pathways.

Human Microglia Seize the Chance to be Different.

Human microglia regional heterogeneity and phenotypes determined by multiplexed single-cell mass cytometry Böttcher C, Schlickeiser S, Sneeboer MAM, et al. Nat Neurosci. 2019;22(1):78-90. Microglia, the specialized innate immune cells of the central nervous system, play crucial roles in neural development and function. Different phenotypes and functions have been ascribed to rodent microglia, but little is known about human microglia (huMG) heterogeneity. Difficulties in procuring huMG and their susceptibility to cryopreservation damage have limited large-scale studies. Here we applied multiplexed mass cytometry for a comprehensive characterization of postmortem huMG (103-104 cells). We determined expression levels of 57 markers on huMG isolated from up to 5 different brain regions of 9 donors. We identified the phenotypic signature of huMG, which was distinct from peripheral myeloid cells but was comparable to fresh huMG. We detected microglia regional heterogeneity using a hybrid workflow combining Cytobank and R/Bioconductor for multidimensional data analysis. Together, these methodologies allowed us to perform high-dimensional, large-scale Immunophenotyping of huMG at the single-cell level, which facilitates their unambiguous profiling in health and disease.

Repeated Use of the Psychoactive Substance Ethylphenidate Impacts Neurochemistry and Reward Learning in Adolescent Male and Female Mice.

Schedule II prescription psychostimulants, such as methylphenidate (MPH), can be misused as nootropic drugs, i.e., drugs that enhance focus and cognition. When users are unable to obtain these prescribed medications, they may seek out novel psychoactive substances (NPSs) that are not yet scheduled. An example of a NPS reportedly being abused is ethylphenidate (EPH), a close analog of MPH but with a higher preference for the dopamine transporter compared with the norepinephrine transporter. Therefore, based upon this pharmacological profile and user self-reports, we hypothesized that repeated EPH exposure in adolescent mice may be rewarding and alter cognition. Here, we report that repeated exposure to 15 mg/kg EPH decreased spatial cognitive performance as assessed by the Barnes maze spatial learning task in adolescent male C57Bl/6 mice; however, male mice did not show alterations in the expression of mature BDNF - a protein associated with increased cognitive function - in key brain regions. Acute EPH exposure induced hyperlocomotion at a high dose (15 mg/kg, i.p.), but not a low dose (5 mg/kg, i.p.). Interestingly, mice exhibited significant conditioned place preference at the low EPH dose, suggesting that even non-stimulating doses of EPH are rewarding. In both males and females, repeated EPH exposure increased expression of deltaFosB - a marker associated with increased risk of drug abuse - in the dorsal striatum, nucleus accumbens, and prefrontal cortex. Overall, our results suggest that repeated EPH use in adolescence is psychostimulatory, rewarding, increases crucial brain markers of reward-related behaviors, and may negatively impact spatial performance.