Human brain organogenesis: Toward a cellular understanding of development and disease.

The construction of the human nervous system is a distinctly complex although highly regulated process. Human tissue inaccessibility has impeded a molecular understanding of the developmental specializations from which our unique cognitive capacities arise. A confluence of recent technological advances in genomics and stem cell-based tissue modeling is laying the foundation for a new understanding of human neural development and dysfunction in neuropsychiatric disease. Here, we review recent progress on uncovering the cellular and molecular principles of human brain organogenesis in vivo as well as using organoids and assembloids in vitro to model features of human evolution and disease.

Control of neuronal excitation-inhibition balance by BMP-SMAD1 signalling.

Throughout life, neuronal networks in the mammalian neocortex maintain a balance of excitation and inhibition, which is essential for neuronal computation1,2. Deviations from a balanced state have been linked to neurodevelopmental disorders, and severe disruptions result in epilepsy3-5. To maintain balance, neuronal microcircuits composed of excitatory and inhibitory neurons sense alterations in neural activity and adjust neuronal connectivity and function. Here we identify a signalling pathway in the adult mouse neocortex that is activated in response to increased neuronal network activity. Overactivation of excitatory neurons is signalled to the network through an increase in the levels of BMP2, a growth factor that is well known for its role as a morphogen in embryonic development. BMP2 acts on parvalbumin-expressing (PV) interneurons through the transcription factor SMAD1, which controls an array of glutamatergic synapse proteins and components of perineuronal nets. PV-interneuron-specific disruption of BMP2-SMAD1 signalling is accompanied by a loss of glutamatergic innervation in PV cells, underdeveloped perineuronal nets and decreased excitability. Ultimately, this impairment of the functional recruitment of PV interneurons disrupts the cortical excitation-inhibition balance, with mice exhibiting spontaneous epileptic seizures. Our findings suggest that developmental morphogen signalling is repurposed to stabilize cortical networks in the adult mammalian brain.

Propofol rescues voltage-dependent gating of HCN1 channel epilepsy mutants.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels1 are essential for pacemaking activity and neural signalling2,3. Drugs inhibiting HCN1 are promising candidates for management of neuropathic pain4 and epileptic seizures5. The general anaesthetic propofol (2,6-di-iso-propylphenol) is a known HCN1 allosteric inhibitor6 with unknown structural basis. Here, using single-particle cryo-electron microscopy and electrophysiology, we show that propofol inhibits HCN1 by binding to a mechanistic hotspot in a groove between the S5 and S6 transmembrane helices. We found that propofol restored voltage-dependent closing in two HCN1 epilepsy-associated polymorphisms that act by destabilizing the channel closed state: M305L, located in the propofol-binding site in S5, and D401H in S6 (refs. 7,8). To understand the mechanism of propofol inhibition and restoration of voltage-gating, we tracked voltage-sensor movement in spHCN channels and found that propofol inhibition is independent of voltage-sensor conformational changes. Mutations at the homologous methionine in spHCN and an adjacent conserved phenylalanine in S6 similarly destabilize closing without disrupting voltage-sensor movements, indicating that voltage-dependent closure requires this interface intact. We propose a model for voltage-dependent gating in which propofol stabilizes coupling between the voltage sensor and pore at this conserved methionine-phenylalanine interface in HCN channels. These findings unlock potential exploitation of this site to design specific drugs targeting HCN channelopathies.

De novo variants in DENND5B cause a neurodevelopmental disorder.

The Rab family of guanosine triphosphatases (GTPases) includes key regulators of intracellular transport and membrane trafficking targeting specific steps in exocytic, endocytic, and recycling pathways. DENND5B (Rab6-interacting Protein 1B-like protein, R6IP1B) is the longest isoform of DENND5, an evolutionarily conserved DENN domain-containing guanine nucleotide exchange factor (GEF) that is highly expressed in the brain. Through exome sequencing and international matchmaking platforms, we identified five de novo variants in DENND5B in a cohort of five unrelated individuals with neurodevelopmental phenotypes featuring cognitive impairment, dysmorphism, abnormal behavior, variable epilepsy, white matter abnormalities, and cortical gyration defects. We used biochemical assays and confocal microscopy to assess the impact of DENND5B variants on protein accumulation and distribution. Then, exploiting fluorescent lipid cargoes coupled to high-content imaging and analysis in living cells, we investigated whether DENND5B variants affected the dynamics of vesicle-mediated intracellular transport of specific cargoes. We further generated an in silico model to investigate the consequences of DENND5B variants on the DENND5B-RAB39A interaction. Biochemical analysis showed decreased protein levels of DENND5B mutants in various cell types. Functional investigation of DENND5B variants revealed defective intracellular vesicle trafficking, with significant impairment of lipid uptake and distribution. Although none of the variants affected the DENND5B-RAB39A interface, all were predicted to disrupt protein folding. Overall, our findings indicate that DENND5B variants perturb intracellular membrane trafficking pathways and cause a complex neurodevelopmental syndrome with variable epilepsy and white matter involvement.

Structure and inhibition mechanism of the human citrate transporter NaCT.

Citrate is best known as an intermediate in the tricarboxylic acid cycle of the cell. In addition to this essential role in energy metabolism, the tricarboxylate anion also acts as both a precursor and a regulator of fatty acid synthesis1-3. Thus, the rate of fatty acid synthesis correlates directly with the cytosolic concentration of citrate4,5. Liver cells import citrate through the sodium-dependent citrate transporter NaCT (encoded by SLC13A5) and, as a consequence, this protein is a potential target for anti-obesity drugs. Here, to understand the structural basis of its inhibition mechanism, we determined cryo-electron microscopy structures of human NaCT in complexes with citrate or a small-molecule inhibitor. These structures reveal how the inhibitor-which binds to the same site as citrate-arrests the transport cycle of NaCT. The NaCT-inhibitor structure also explains why the compound selectively inhibits NaCT over two homologous human dicarboxylate transporters, and suggests ways to further improve the affinity and selectivity. Finally, the NaCT structures provide a framework for understanding how various mutations abolish the transport activity of NaCT in the brain and thereby cause epilepsy associated with mutations in SLC13A5 in newborns (which is known as SLC13A5-epilepsy)6-8.

Mosaicism-independent mechanisms contribute to Pcdh19-related epilepsy and repetitive behaviors in Xenopus.

Protocadherin19 (PCDH19)-related epilepsy syndrome is a rare disorder characterized by early-onset epilepsy, intellectual disability, and autistic behaviors. PCDH19 is located on the X chromosome and encodes a calcium-dependent single-pass transmembrane protein, which regulates cell-to-cell adhesion through homophilic binding. In human, 90% of heterozygous females, containing PCDH19 wild-type and mutant cells due to random X inactivation, are affected, whereas mutant males, containing only mutant cells, are typically not. The current view, the cellular interference, is that the altered interactions between wild-type and mutant cells during development, rather than loss of function itself, are responsible. However, studies using Pcdh19 knockout mice showed that the complete loss of function also causes autism-like behaviors both in males and females, suggesting that other functions of PCDH19 may also contribute to pathogenesis. To address whether mosaicism is required for PCDH19-related epilepsy, we generated Xenopus tropicalis tadpoles with complete or mosaic loss of function by injecting antisense morpholino oligonucleotides into the blastomeres of neural lineage at different stages of development. We found that either mosaic or complete knockdown results in seizure-like behaviors, which could be rescued by antiseizure medication, and repetitive behaviors. Our results suggest that the loss of PCDH19 function itself, in addition to cellular interference, may also contribute to PCDH19-related epilepsy.

Structural and functional reorganization of inhibitory synapses by activity-dependent cleavage of neuroligin-2.

Recent evidence has demonstrated that the transsynaptic nanoscale organization of synaptic proteins plays a crucial role in regulating synaptic strength in excitatory synapses. However, the molecular mechanism underlying this transsynaptic nanostructure in inhibitory synapses still remains unclear and its impact on synapse function in physiological or pathological contexts has not been demonstrated. In this study, we utilized an engineered proteolysis technique to investigate the effects of acute cleavage of neuroligin-2 (NL2) on synaptic transmission. Our results show that the rapid cleavage of NL2 led to impaired synaptic transmission by reducing both neurotransmitter release probability and quantum size. These changes were attributed to the dispersion of RIM1/2 and GABAA receptors and a weakened spatial alignment between them at the subsynaptic scale, as observed through superresolution imaging and model simulations. Importantly, we found that endogenous NL2 undergoes rapid MMP9-dependent cleavage during epileptic activities, which further exacerbates the decrease in inhibitory transmission. Overall, our study demonstrates the significant impact of nanoscale structural reorganization on inhibitory transmission and unveils ongoing modulation of mature GABAergic synapses through active cleavage of NL2 in response to hyperactivity.

PHF6-mediated transcriptional control of NSC via Ephrin receptors is impaired in the intellectual disability syndrome BFLS.

The plant homeodomain zinc-finger protein, PHF6, is a transcriptional regulator, and PHF6 germline mutations cause the X-linked intellectual disability (XLID) Börjeson-Forssman-Lehmann syndrome (BFLS). The mechanisms by which PHF6 regulates transcription and how its mutations cause BFLS remain poorly characterized. Here, we show genome-wide binding of PHF6 in the developing cortex in the vicinity of genes involved in central nervous system development and neurogenesis. Characterization of BFLS mice harbouring PHF6 patient mutations reveals an increase in embryonic neural stem cell (eNSC) self-renewal and a reduction of neural progenitors. We identify a panel of Ephrin receptors (EphRs) as direct transcriptional targets of PHF6. Mechanistically, we show that PHF6 regulation of EphR is impaired in BFLS mice and in conditional Phf6 knock-out mice. Knockdown of EphR-A phenocopies the PHF6 loss-of-function defects in altering eNSCs, and its forced expression rescues defects of BFLS mice-derived eNSCs. Our data indicate that PHF6 directly promotes Ephrin receptor expression to control eNSC behaviour in the developing brain, and that this pathway is impaired in BFLS.

Dopamine D2 receptors in hilar mossy cells regulate excitatory transmission and hippocampal function.

Hilar mossy cells (MCs) are principal excitatory neurons of the dentate gyrus (DG) that play critical roles in hippocampal function and have been implicated in brain disorders such as anxiety and epilepsy. However, the mechanisms by which MCs contribute to DG function and disease are poorly understood. A defining feature of MCs is the promoter activity of the dopamine D2 receptor (D2R) gene (Drd2), and previous work indicates a key role for dopaminergic signaling in the DG. Additionally, the involvement of D2R signaling in cognition and neuropsychiatric conditions is well known. Surprisingly, though, the function of MC D2Rs remains largely unexplored. In this study, we show that selective and conditional removal of Drd2 from MCs of adult mice impaired spatial memory, promoted anxiety-like behavior, and was proconvulsant. To determine the subcellular expression of D2Rs in MCs, we used a D2R knockin mouse which revealed that D2Rs are enriched in the inner molecular layer of the DG, where MCs establish synaptic contacts with granule cells (GCs). D2R activation by exogenous and endogenous dopamine reduced MC to dentate GC synaptic transmission, most likely by a presynaptic mechanism. In contrast, exogenous dopamine had no significant impact on MC excitatory inputs and passive and active properties. Our findings support that MC D2Rs are essential for proper DG function by reducing MC excitatory drive onto GCs. Lastly, impairment of MC D2R signaling could promote anxiety and epilepsy, therefore highlighting a potential therapeutic target.

A Frameshift Variant of GluN2A Identified in an Epilepsy Patient Results in NMDA Receptor Mistargeting.

N-methyl-D-aspartate receptors (NMDARs) are crucial for neuronal development and synaptic plasticity. Dysfunction of NMDARs is associated with multiple neurodevelopmental disorders, including epilepsy, autism spectrum disorder, and intellectual disability. Understanding the impact of genetic variants of NMDAR subunits can shed light on the mechanisms of disease. Here, we characterized the functional implications of a de novo mutation of the GluN2A subunit (P1199Rfs*32) resulting in the truncation of the C-terminal domain. The variant was identified in a male patient with epileptic encephalopathy, multiple seizure types, severe aphasia, and neurobehavioral changes. Given the known role of the CTD in NMDAR trafficking, we examined changes in receptor localization and abundance at the postsynaptic membrane using a combination of molecular assays in heterologous cells and rat primary neuronal cultures. We observed that the GluN2A P1199Rfs*32-containing receptors traffic efficiently to the postsynaptic membrane but have increased extra-synaptic expression relative to WT GluN2A-containing NMDARs. Using in silico predictions, we hypothesized that the mutant would lose all PDZ interactions, except for the recycling protein Scribble1. Indeed, we observed impaired binding to the scaffolding protein postsynaptic protein-95 (PSD-95); however, we found the mutant interacts with Scribble1, which facilitates the recycling of both the mutant and the WT GluN2A. Finally, we found that neurons expressing GluN2A P1199Rfs*32 have fewer synapses and decreased spine density, indicating compromised synaptic transmission in these neurons. Overall, our data show that GluN2A P1199Rfs*32 is a loss-of-function variant with altered membrane localization in neurons and provide mechanistic insight into disease etiology.

ANK2 loss-of-function variants are associated with epilepsy, and lead to impaired axon initial segment plasticity and hyperactive network activity in hiPSC-derived neuronal networks.

PURPOSE: To characterize a novel neurodevelopmental syndrome due to loss-of-function (LoF) variants in Ankyrin 2 (ANK2), and to explore the effects on neuronal network dynamics and homeostatic plasticity in human-induced pluripotent stem cell-derived neurons. METHODS: We collected clinical and molecular data of 12 individuals with heterozygous de novo LoF variants in ANK2. We generated a heterozygous LoF allele of ANK2 using CRISPR/Cas9 in human-induced pluripotent stem cells (hiPSCs). HiPSCs were differentiated into excitatory neurons, and we measured their spontaneous electrophysiological responses using micro-electrode arrays (MEAs). We also characterized their somatodendritic morphology and axon initial segment (AIS) structure and plasticity. RESULTS: We found a broad neurodevelopmental disorder (NDD), comprising intellectual disability, autism spectrum disorders and early onset epilepsy. Using MEAs, we found that hiPSC-derived neurons with heterozygous LoF of ANK2 show a hyperactive and desynchronized neuronal network. ANK2-deficient neurons also showed increased somatodendritic structures and altered AIS structure of which its plasticity is impaired upon activity-dependent modulation. CONCLUSIONS: Phenotypic characterization of patients with de novo ANK2 LoF variants defines a novel NDD with early onset epilepsy. Our functional in vitro data of ANK2-deficient human neurons show a specific neuronal phenotype in which reduced ANKB expression leads to hyperactive and desynchronized neuronal network activity, increased somatodendritic complexity and AIS structure and impaired activity-dependent plasticity of the AIS.

Deficit of PKHD1L1 in the dentate gyrus increases seizure susceptibility in mice.

Epilepsy is a chronic neurological disorder featuring recurrent, unprovoked seizures, which affect more than 65 million people worldwide. Here, we discover that the PKHD1L1, which is encoded by polycystic kidney and hepatic disease1-like 1 (Pkhd1l1), wildly distributes in neurons in the central nervous system (CNS) of mice. Disruption of PKHD1L1 in the dentate gyrus region of the hippocampus leads to increased susceptibility to pentylenetetrazol-induced seizures in mice. The disturbance of PKHD1L1 leads to the overactivation of the mitogen-activated protein kinase (MAPK)/extracellular regulated kinase (ERK)-Calpain pathway, which is accompanied by remarkable degradation of cytoplasmic potassium chloride co-transporter 2 (KCC2) level together with the impaired expression and function of membrane KCC2. However, the reduction of membrane KCC2 is associated with the damaged inhibitory ability of the vital GABA receptors, which ultimately leads to the significantly increased susceptibility to epileptic seizures. Our data, thus, indicate for the first time that Pkhd1l1, a newly discovered polycystic kidney disease (PKD) association gene, is required in neurons to maintain neuronal excitability by regulation of KCC2 expression in CNS. A new mechanism of the clinical association between genetic PKD and seizures has been built, which could be a potential therapeutic target for treating PKD-related seizures.

Role and mechanism of EphB3 in epileptic seizures and epileptogenesis through Kalirin.

BACKGROUND: The EphB receptor tyrosine kinase family participates in intricate signaling pathways that orchestrate neural networks, guide neuronal axon development, and modulate synaptic plasticity through interactions with surface-bound ephrinB ligands. Additionally, Kalirin, a Rho guanine nucleotide exchange factor, is notably expressed in the postsynaptic membrane of excitatory neurons and plays a role in synaptic morphogenesis. This study postulates that Kalirin may act as a downstream effector of EphB3 in epilepsy. This investigation focuses on understanding the link between EphB3 and epilepsy. MATERIALS AND METHODS: Chronic seizure models using LiCl-pilocarpine (LiCl/Pilo) and pentylenetetrazol were developed in rats. Neuronal excitability was gauged through whole-cell patch clamp recordings on rat hippocampal slices. Real-time PCR determined Kalirin's mRNA expression, and Western blotting was employed to quantify EphB3 and Kalirin protein levels. Moreover, dendritic spine density in epileptic rats was evaluated using Golgi staining. RESULTS: Modulation of EphB3 functionality influenced acute seizure severity, latency duration, and frequency of spontaneous recurrent seizures. Golgi staining disclosed an EphB3-driven alteration in dendritic spine density within the hippocampus of epileptic rats, underscoring its pivotal role in the reconfiguration of hippocampal neural circuits. Furthermore, our data propose Kalirin as a prospective downstream mediator of the EphB3 receptor. CONCLUSIONS: Our findings elucidate that EphB3 impacts the action potential dynamics in isolated rat hippocampal slices and alters dendritic spine density in the inner molecular layer of epileptic rat hippocampi, likely through Kalirin-mediated pathways. This hints at EphB3's significant role in shaping excitatory circuit loops and recurrent seizure activity via Kalirin.

An epilepsy-associated KV1.2 charge-transfer-center mutation impairs KV1.2 and KV1.4 trafficking.

We report on a heterozygous KCNA2 variant in a child with epilepsy. KCNA2 encodes KV1.2 subunits, which form homotetrameric potassium channels and participate in heterotetrameric channel complexes with other KV1-family subunits, regulating neuronal excitability. The mutation causes substitution F233S at the KV1.2 charge transfer center of the voltage-sensing domain. Immunocytochemical trafficking assays showed that KV1.2(F233S) subunits are trafficking deficient and reduce the surface expression of wild-type KV1.2 and KV1.4: a dominant-negative phenotype extending beyond KCNA2, likely profoundly perturbing electrical signaling. Yet some KV1.2(F233S) trafficking was rescued by wild-type KV1.2 and KV1.4 subunits, likely in permissible heterotetrameric stoichiometries: electrophysiological studies utilizing applied transcriptomics and concatemer constructs support that up to one or two KV1.2(F233S) subunits can participate in trafficking-capable heterotetramers with wild-type KV1.2 or KV1.4, respectively, and that both early and late events along the biosynthesis and secretion pathway impair trafficking. These studies suggested that F233S causes a depolarizing shift of ∼48 mV on KV1.2 voltage dependence. Optical tracking of the KV1.2(F233S) voltage-sensing domain (rescued by wild-type KV1.2 or KV1.4) revealed that it operates with modestly perturbed voltage dependence and retains pore coupling, evidenced by off-charge immobilization. The equivalent mutation in the Shaker K+ channel (F290S) was reported to modestly affect trafficking and strongly affect function: an ∼80-mV depolarizing shift, disrupted voltage sensor activation and pore coupling. Our work exposes the multigenic, molecular etiology of a variant associated with epilepsy and reveals that charge-transfer-center disruption has different effects in KV1.2 and Shaker, the archetypes for potassium channel structure and function.

Retinal Dysfunction in a Mouse Model of HCN1 Genetic Epilepsy.

Pathogenic variants in HCN1 are associated with a range of epilepsy syndromes including a developmental and epileptic encephalopathy. The recurrent de novo HCN1 pathogenic variant (M305L) results in a cation leak, allowing the flux of excitatory ions at potentials where the wild-type channels are closed. The Hcn1M294L mouse recapitulates patient seizure and behavioral phenotypes. As HCN1 channels are highly expressed in rod and cone photoreceptor inner segments, where they shape the light response, mutated channels are likely to impact visual function. Electroretinogram (ERG) recordings from male and female mice Hcn1M294L mice revealed a significant decrease in the photoreceptor sensitivity to light, as well as attenuated bipolar cell (P2) and retinal ganglion cell responses. Hcn1M294L mice also showed attenuated ERG responses to flickering lights. ERG abnormalities are consistent with the response recorded from a single female human subject. There was no impact of the variant on the structure or expression of the Hcn1 protein in the retina. In silico modeling of photoreceptors revealed that the mutated HCN1 channel dramatically reduced light-induced hyperpolarization, resulting in more Ca2+ flux during the response when compared with the wild-type situation. We propose that the light-induced change in glutamate release from photoreceptors during a stimulus will be diminished, significantly blunting the dynamic range of this response. Our data highlight the importance of HCN1 channels to retinal function and suggest that patients with HCN1 pathogenic variants are likely to have a dramatically reduced sensitivity to light and a limited ability to process temporal information.SIGNIFICANCE STATEMENT Pathogenic variants in HCN1 are emerging as an important cause of catastrophic epilepsy. HCN1 channels are ubiquitously expressed throughout the body, including the retina. Electroretinogram recordings from a mouse model of HCN1 genetic epilepsy showed a marked decrease in the photoreceptor sensitivity to light and a reduced ability to respond to high rates of light flicker. No morphologic deficits were noted. Simulation data suggest that the mutated HCN1 channel blunts light-induced hyperpolarization and consequently limits the dynamic range of this response. Our results provide insights into the role HCN1 channels play in retinal function as well as highlighting the need to consider retinal dysfunction in disease caused by HCN1 variants. The characteristic changes in the electroretinogram open the possibility of using this tool as a biomarker for this HCN1 epilepsy variant and to facilitate development of treatments.

Cortical Parvalbumin-Positive Interneuron Development and Function Are Altered in the APC Conditional Knockout Mouse Model of Infantile and Epileptic Spasms Syndrome.

Infantile and epileptic spasms syndrome (IESS) is a childhood epilepsy syndrome characterized by infantile or late-onset spasms, abnormal neonatal EEG, and epilepsy. Few treatments exist for IESS, clinical outcomes are poor, and the molecular and circuit-level etiologies of IESS are not well understood. Multiple human IESS risk genes are linked to Wnt/ß-catenin signaling, a pathway that controls developmental transcriptional programs and promotes glutamatergic excitation via ß-catenin's role as a synaptic scaffold. We previously showed that deleting adenomatous polyposis coli (APC), a component of the ß-catenin destruction complex, in excitatory neurons (APC cKO mice, APCfl/fl x CaMKIIαCre) increased ß-catenin levels in developing glutamatergic neurons and led to infantile behavioral spasms, abnormal neonatal EEG, and adult epilepsy. Here, we tested the hypothesis that the development of GABAergic interneurons (INs) is disrupted in APC cKO male and female mice. IN dysfunction is implicated in human IESS, is a feature of other rodent models of IESS, and may contribute to the manifestation of spasms and seizures. We found that parvalbumin-positive INs (PV+ INs), an important source of cortical inhibition, were decreased in number, underwent disproportionate developmental apoptosis, and had altered dendrite morphology at P9, the peak of behavioral spasms. PV+ INs received excessive excitatory input, and their intrinsic ability to fire action potentials was reduced at all time points examined (P9, P14, P60). Subsequently, GABAergic transmission onto pyramidal neurons was uniquely altered in the somatosensory cortex of APC cKO mice at all ages, with both decreased IPSC input at P14 and enhanced IPSC input at P9 and P60. These results indicate that inhibitory circuit dysfunction occurs in APC cKOs and, along with known changes in excitation, may contribute to IESS-related phenotypes.SIGNIFICANCE STATEMENT Infantile and epileptic spasms syndrome (IESS) is a devastating epilepsy with limited treatment options and poor clinical outcomes. The molecular, cellular, and circuit disruptions that cause infantile spasms and seizures are largely unknown, but inhibitory GABAergic interneuron dysfunction has been implicated in rodent models of IESS and may contribute to human IESS. Here, we use a rodent model of IESS, the APC cKO mouse, in which ß-catenin signaling is increased in excitatory neurons. This results in altered parvalbumin-positive GABAergic interneuron development and GABAergic synaptic dysfunction throughout life, showing that pathology arising in excitatory neurons can initiate long-term interneuron dysfunction. Our findings further implicate GABAergic dysfunction in IESS, even when pathology is initiated in other neuronal types.

The effect of the HMGB1/RAGE/TLR4/NF-κB signalling pathway in patients with idiopathic epilepsy and its relationship with toxoplasmosis.

This study aims to investigate the relationship between toxoplasmosis and this pathway, which may be effective in the formation of epilepsy by acting through the HMGB1/RAGE/TLR4/NF-κB signalling pathway in patients with idiopathic epilepsy. In the study, four different experimental groups were formed by selecting Toxoplasma gondii IgG positive and negative patients with idiopathic epilepsy and healthy controls. Experimental groups were as follows: Group 1: Epilepsy+/Toxo- (E+, T-) (n = 10), Group 2: Epilepsy-/Toxo- (E-, T-) (n = 10), Group 3: Epilepsy-/Toxo+ (E-, T+) (n = 10), Group 4: Epilepsy+/Toxo+ (E+, T+) (n = 10). HMGB1, RAGE, TLR4, TLR1, TLR2, TLR3, IRAK1, IRAK2, IKBKB, IKBKG, BCL3, IL1ß, IL10, 1 L8 and TNFα mRNA expression levels in the HMGB/RAGE/TLR4/NF-κB signalling pathway were determined by quantitative simultaneous PCR (qRT-PCR) after collecting blood samples from all patients in the groups. Statistical analysis was performed by one-way ANOVA followed by LSD post-hoc tests, and p < 0.05 was considered to denote statistical significance. The gene expression levels of HMGB1, TLR4, IL10, IL1B, IL8, and TLR2 were significantly higher in the G1 group than in the other groups (p < 0.05). In the G3 group, RAGE and BCL3 gene expression levels were significantly higher than in the other groups (p < 0.05). In the G4 group, however, IRAK2, IKBKB, and IKBKG gene expression levels were significantly higher than in the other groups (p < 0.05). HMGB1, TLR4, IRAK2, IKBKB, IL10, IL1B, IL1B, and IL8 in this signalling pathway are highly expressed in epilepsy patients in G1 and seizures occur with the stimulation of excitatory mechanisms by acting through this pathway. The signalling pathway in epilepsy may be activated by HMGB1, TLR4, and TLR2, which are considered to increase the level of proinflammatory cytokines. In T. gondii, this pathway is activated by RAGE and BCL3.

Neuronal small extracellular vesicles carrying miR-181c-5p contribute to the pathogenesis of epilepsy by regulating the protein kinase C-δ/glutamate transporter-1 axis in astrocytes.

Information exchange between neurons and astrocytes mediated by extracellular vesicles (EVs) is known to play a key role in the pathogenesis of central nervous system diseases. A key driver of epilepsy is the dysregulation of intersynaptic excitatory neurotransmitters mediated by astrocytes. Thus, we investigated the potential association between neuronal EV microRNAs (miRNAs) and astrocyte glutamate uptake ability in epilepsy. Here, we showed that astrocytes were able to engulf epileptogenic neuronal EVs, inducing a significant increase in the glutamate concentration in the extracellular fluid of astrocytes, which was linked to a decrease in glutamate transporter-1 (GLT-1) protein expression. Using sequencing and gene ontology (GO) functional analysis, miR-181c-5p was found to be the most significantly upregulated miRNA in epileptogenic neuronal EVs and was linked to glutamate metabolism. Moreover, we found that neuronal EV-derived miR-181c-5p interacted with protein kinase C-delta (PKCδ), downregulated PKCδ and GLT-1 protein expression and increased glutamate concentrations in astrocytes both in vitro and in vivo. Our findings demonstrated that epileptogenic neuronal EVs carrying miR-181c-5p decrease the glutamate uptake ability of astrocytes, thus promoting susceptibility to epilepsy.

Sirtuin 3 regulates astrocyte activation by reducing Notch1 signaling after status epilepticus.

Sirtuin3 (Sirt3) is a nicotinamide adenine dinucleotide enzyme that contributes to aging, cancer, and neurodegenerative diseases. Recent studies have reported that Sirt3 exerts anti-inflammatory effects in several neuropathophysiological disorders. As epilepsy is a common neurological disease, in the present study, we investigated the role of Sirt3 in astrocyte activation and inflammatory processes after epileptic seizures. We found the elevated expression of Sirt3 within reactive astrocytes as well as in the surrounding cells in the hippocampus of patients with temporal lobe epilepsy and a mouse model of pilocarpine-induced status epilepticus (SE). The upregulation of Sirt3 by treatment with adjudin, a potential Sirt3 activator, alleviated SE-induced astrocyte activation; whereas, Sirt3 deficiency exacerbated astrocyte activation in the hippocampus after SE. In addition, our results showed that Sirt3 upregulation attenuated the activation of Notch1 signaling, nuclear factor kappa B (NF-κB) activity, and the production of interleukin-1ß (IL1ß) in the hippocampus after SE. By contrast, Sirt3 deficiency enhanced the activity of Notch1/NF-κB signaling and the production of IL1ß. These findings suggest that Sirt3 regulates astrocyte activation by affecting the Notch1/NF-κB signaling pathway, which contributes to the inflammatory response after SE. Therefore, therapies targeting Sirt3 may be a worthy direction for limiting inflammatory responses following epileptic brain injury.

Milestone review: GABA, from chemistry, conformations, ionotropic receptors, modulators, epilepsy, flavonoids, and stress to neuro-nutraceuticals.

Arising out of a PhD project more than 50 years ago to synthesise analogues of the neurotransmitter GABA, a series of new chemical entities were found to have selective actions on ionotropic GABA receptors. Several of these neurochemicals are now commercially available. A new subtype of these receptors was discovered that could be a target for the treatment of myopia, the facilitation of learning and memory, and the improvement of post-stroke motor recovery. The development of these new chemical entities over many years demonstrates the importance of neurochemicals with which to investigate selective aspects of GABA receptors and illustrates the significance of collaboration between chemists and biologists in neurochemistry. Vital were the improvements in synthetic organic chemistry and the use of functional human receptors expressed in oocytes. Current interest in ionotropic GABA receptors includes the clinical development of subtype-specific agents and the role of gain-of-function receptor variants in epilepsy. Dietary flavonoids were found to cross the blood-brain barrier to influence brain function. Natural and synthetic flavonoids had a range of effects on GABA receptors, ranging from positive, silent, and negative allosteric modulators, to even second-order modulation of first-order modulators. Flavonoids have been called "a new family of benzodiazepines." Like benzodiazepines, flavonoids reduce stress. Stress produces changes in GABA receptors in the brain that may be because of changes in endogenous modulators, such as neurosteroids and corticosteroids. GABA also occurs naturally in the diet leading to studies of the effects of oral GABA on brain function. This finding has resulted in studies of GABA and related neurochemicals as neuro-nutraceuticals. GABA systems in the gut microbiome are essential to such studies. The actions of oral GABA and of GABA-enriched beverages and foodstuffs are now an area of considerable scientific and commercial interest. GABA is a deceptively simple chemical that can take up many shapes, which may underlie its complex functions. The need for new chemical entities with selective actions for further studies highlights the need for continuing collaboration between chemists and biologists.