The Purinergic P2X7 Receptor as a Target for Adjunctive Treatment for Drug-Refractory Epilepsy.

Epilepsy is one of the most common neurological diseases worldwide. Anti-seizure medications (ASMs) with anticonvulsants remain the mainstay of epilepsy treatment. Currently used ASMs are, however, ineffective to suppress seizures in about one third of all patients. Moreover, ASMs show no significant impact on the pathogenic mechanisms involved in epilepsy development or disease progression and may cause serious side-effects, highlighting the need for the identification of new drug targets for a more causal therapy. Compelling evidence has demonstrated a role for purinergic signalling, including the nucleotide adenosine 5'-triphosphate (ATP) during the generation of seizures and epilepsy. Consequently, drugs targeting specific ATP-gated purinergic receptors have been suggested as promising treatment options for epilepsy including the cationic P2X7 receptor (P27XR). P2X7R protein levels have been shown to be increased in the brain of experimental models of epilepsy and in the resected brain tissue of patients with epilepsy. Animal studies have provided evidence that P2X7R blocking can reduce the severity of acute seizures and the epileptic phenotype. The current review will provide a brief summary of recent key findings on P2X7R signalling during seizures and epilepsy focusing on the potential clinical use of treatments based on the P2X7R as an adjunctive therapeutic strategy for drug-refractory seizures and epilepsy.

Opposing effects of the purinergic P2X7 receptor on seizures in neurons and microglia in male mice.

BACKGROUND: The purinergic ATP-gated P2X7 receptor (P2X7R) is increasingly recognized to contribute to pathological neuroinflammation and brain hyperexcitability. P2X7R expression has been shown to be increased in the brain, including both microglia and neurons, in experimental models of epilepsy and patients. To date, the cell type-specific downstream effects of P2X7Rs during seizures remain, however, incompletely understood. METHODS: Effects of P2X7R signaling on seizures and epilepsy were analyzed in induced seizure models using male mice including the kainic acid model of status epilepticus and pentylenetetrazole model and in male and female mice in a genetic model of Dravet syndrome. RNA sequencing was used to analyze P2X7R downstream signaling during seizures. To investigate the cell type-specific role of the P2X7R during seizures and epilepsy, we generated mice lacking exon 2 of the P2rx7 gene in either microglia (P2rx7:Cx3cr1-Cre) or neurons (P2rx7:Thy-1-Cre). To investigate the protective potential of overexpressing P2X7R in GABAergic interneurons, P2X7Rs were overexpressed using adeno-associated virus transduction under the mDlx promoter. RESULTS: RNA sequencing of hippocampal tissue from wild-type and P2X7R knock-out mice identified both glial and neuronal genes, in particular genes involved in GABAergic signaling, under the control of the P2X7R following seizures. Mice with deleted P2rx7 in microglia displayed less severe acute seizures and developed a milder form of epilepsy, and microglia displayed an anti-inflammatory molecular profile. In contrast, mice lacking P2rx7 in neurons showed a more severe seizure phenotype when compared to epileptic wild-type mice. Analysis of single-cell expression data revealed that human P2RX7 expression is elevated in the hippocampus of patients with temporal lobe epilepsy in excitatory and inhibitory neurons. Functional studies determined that GABAergic interneurons display increased responses to P2X7R activation in experimental epilepsy. Finally, we show that viral transduction of P2X7R in GABAergic interneurons protects against evoked and spontaneous seizures in experimental temporal lobe epilepsy and in mice lacking Scn1a, a model of Dravet syndrome. CONCLUSIONS: Our results suggest a dual and opposing action of P2X7R in epilepsy and suggest P2X7R overexpression in GABAergic interneurons as a novel therapeutic strategy for acquired and, possibly, genetic forms of epilepsy.

Post-translational regulation of the mTORC1 pathway: A switch that regulates metabolism-related gene expression.

The mechanistic target of rapamycin complex 1 (mTORC1) is a kinase complex that plays a crucial role in coordinating cell growth in response to various signals, including amino acids, growth factors, oxygen, and ATP. Activation of mTORC1 promotes cell growth and anabolism, while its suppression leads to catabolism and inhibition of cell growth, enabling cells to withstand nutrient scarcity and stress. Dysregulation of mTORC1 activity is associated with numerous diseases, such as cancer, metabolic disorders, and neurodegenerative conditions. This review focuses on how post-translational modifications, particularly phosphorylation and ubiquitination, modulate mTORC1 signaling pathway and their consequential implications for pathogenesis. Understanding the impact of phosphorylation and ubiquitination on the mTORC1 signaling pathway provides valuable insights into the regulation of cellular growth and potential therapeutic targets for related diseases.

Myoglobin mutant with enhanced nitrite reductase activity regulates intracellular oxidative stress in human breast cancer cells.

Heme proteins play vital roles in regulating the reactive oxygen/nitrogen species (ROS/RNS) levels in cells. In this study, we overexpressed human wild-type (WT) myoglobin (Mb) and its double mutant, F43H/H64A Mb with enhanced nitrite reductase (NIR) activity, in the typical representative triple-negative breast cancer cell, MDA-MB-231 cells. The results showed that the overexpression of F43H/H64A Mb increased the level of nitric oxide (NO) and the degree of oxidative stress, and then activated Akt/MAPK mediated apoptotic cascade, whereas WT Mb showed the opposite effect. This study indicates that Mb plays an important role in maintaining the balance of the cellular redox system and could thus be a valuable target for cancer therapy.

Large-conductance calcium-activated potassium channels mediate lipopolysaccharide-induced activation of murine microglia.

Large-conductance calcium-activated potassium (BK) channels are ubiquitously expressed in most cell types where they regulate many cellular, organ, and organismal functions. Although BK currents have been recorded specifically in activated murine and human microglia, it is not yet clear whether and how the function of this channel is related to microglia activation. Here, using patch-clamping, Griess reaction, ELISA, immunocytochemistry, and immunoblotting approaches, we show that specific inhibition of the BK channel with paxilline (10 µm) or siRNA-mediated knockdown of its expression significantly suppresses lipopolysaccharide (LPS)-induced (100 ng/ml) BV-2 and primary mouse microglial cell activation. We found that membrane BK current is activated by LPS at a very early stage through Toll-like receptor 4 (TLR4), leading to nuclear translocation of NF-κB and to production of inflammatory cytokines. Furthermore, we noted that BK channels are also expressed intracellularly, and their nuclear expression significantly increases in late stages of LPS-mediated microglia activation, possibly contributing to production of nitric oxide, tumor necrosis factor-α, and interleukin-6. Of note, a specific TLR4 inhibitor suppressed BK channel expression, whereas an NF-κB inhibitor did not. Taken together, our findings indicate that BK channels participate in both the early and the late stages of LPS-stimulated murine microglia activation involving both membrane-associated and nuclear BK channels.

KCTD: A new gene family involved in neurodevelopmental and neuropsychiatric disorders.

The underlying molecular basis for neurodevelopmental or neuropsychiatric disorders is not known. In contrast, mechanistic understanding of other brain disorders including neurodegeneration has advanced considerably. Yet, these do not approach the knowledge accrued for many cancers with precision therapeutics acting on well-characterized targets. Although the identification of genes responsible for neurodevelopmental and neuropsychiatric disorders remains a major obstacle, the few causally associated genes are ripe for discovery by focusing efforts to dissect their mechanisms. Here, we make a case for delving into mechanisms of the poorly characterized human KCTD gene family. Varying levels of evidence support their roles in neurocognitive disorders (KCTD3), neurodevelopmental disease (KCTD7), bipolar disorder (KCTD12), autism and schizophrenia (KCTD13), movement disorders (KCTD17), cancer (KCTD11), and obesity (KCTD15). Collective knowledge about these genes adds enhanced value, and critical insights into potential disease mechanisms have come from unexpected sources. Translation of basic research on the KCTD-related yeast protein Whi2 has revealed roles in nutrient signaling to mTORC1 (KCTD11) and an autophagy-lysosome pathway affecting mitochondria (KCTD7). Recent biochemical and structure-based studies (KCTD12, KCTD13, KCTD16) reveal mechanisms of regulating membrane channel activities through modulation of distinct GTPases. We explore how these seemingly varied functions may be disease related.

Whi2 is a conserved negative regulator of TORC1 in response to low amino acids.

Yeast WHI2 was originally identified in a genetic screen for regulators of cell cycle arrest and later suggested to function in general stress responses. However, the function of Whi2 is unknown. Whi2 has predicted structure and sequence similarity to human KCTD family proteins, which have been implicated in several cancers and are causally associated with neurological disorders but are largely uncharacterized. The identification of conserved functions between these yeast and human proteins may provide insight into disease mechanisms. We report that yeast WHI2 is a new negative regulator of TORC1 required to suppress TORC1 activity and cell growth specifically in response to low amino acids. In contrast to current opinion, WHI2 is dispensable for TORC1 inhibition in low glucose. The only widely conserved mechanism that actively suppresses both yeast and mammalian TORC1 specifically in response to low amino acids is the conserved SEACIT/GATOR1 complex that inactivates the TORC1-activating RAG-like GTPases. Unexpectedly, Whi2 acts independently and simultaneously with these established GATOR1-like Npr2-Npr3-Iml1 and RAG-like Gtr1-Gtr2 complexes, and also acts independently of the PKA pathway. Instead, Whi2 inhibits TORC1 activity through its binding partners, protein phosphatases Psr1 and Psr2, which were previously thought to only regulate amino acid levels downstream of TORC1. Furthermore, the ability to suppress TORC1 is conserved in the SKP1/BTB/POZ domain-containing, Whi2-like human protein KCTD11 but not other KCTD family members tested.

Cell death in genome evolution.

Inappropriate survival of abnormal cells underlies tumorigenesis. Most discoveries about programmed cell death have come from studying model organisms. Revisiting the experimental contexts that inspired these discoveries helps explain confounding biases that inevitably accompany such discoveries. Amending early biases has added a newcomer to the collection of cell death models. Analysis of gene-dependent death in yeast revealed the surprising influence of single gene mutations on subsequent eukaryotic genome evolution. Similar events may influence the selection for mutations during early tumorigenesis. The possibility that any early random mutation might drive the selection for a cancer driver mutation is conceivable but difficult to demonstrate. This was tested in yeast, revealing that mutation of almost any gene appears to specify the selection for a new second mutation. Some human tumors contain pairs of mutant genes homologous to co-occurring mutant genes in yeast. Here we consider how yeast again provide novel insights into tumorigenesis.

Genome evolution in yeast reveals connections between rare mutations in human cancers.

Cancer cells are riddled with mutations. Less than one percent of these are thought to be mutations that drive cancer phenotypes. However, a recent study conducted on the yeast knockout collections by Teng et al. [Mol. Cell (2013) 52: 485-494] provides hard evidence that single gene deletions/mutations in most non-essential genes can drive the selection for cancer-like mutations.

Genome-wide consequences of deleting any single gene.

Loss or duplication of chromosome segments can lead to further genomic changes associated with cancer. However, it is not known whether only a select subset of genes is responsible for driving further changes. To determine whether perturbation of any given gene in a genome suffices to drive subsequent genetic changes, we analyzed the yeast knockout collection for secondary mutations of functional consequence. Unlike wild-type, most gene knockout strains were found to have one additional mutant gene affecting nutrient responses and/or heat-stress-induced cell death. Moreover, independent knockouts of the same gene often evolved mutations in the same secondary gene. Genome sequencing identified acquired mutations in several human tumor suppressor homologs. Thus, mutation of any single gene may cause a genomic imbalance, with consequences sufficient to drive adaptive genetic changes. This complicates genetic analyses but is a logical consequence of losing a functional unit originally acquired under pressure during evolution.

Important roles of the conserved linker-KKS in human neuronal growth inhibitory factor.

Metallothinein-3 (MT3), also named neuronal growth inhibitory factor (GIF), is attractive by its distinct neuronal growth inhibitory activity, which is not shared by other MT isoforms. The polypeptide chain of GIF is folded into two individual domains, which are connected by a highly conserved linker, KKS. In order to figure out the significance of the conserved segment, we constructed several mutants of human GIF (hGIF), including the K31/32A mutant, the K31/32E mutant and the KKS-SP mutant by site-directed mutagenesis. pH titration and DTNB reaction exhibited that all the three mutations made the beta-domain lower in stability and looser. More significantly, change of KKS to SP also altered the general backbone conformation and metal-thiolate cluster geometry. Notably, bioassay results showed that the bioactivity of the K31/32A mutant and the K31/32E mutant decreased obviously, while the KKS-SP mutant lost inhibitory activity completely. Based on these results, we proposed that the KKS linker was a crucial factor in modulating the stability and the solvent accessibility of the Cd(3)S(9) cluster in the beta-domain through domain-domain interactions, thus was indispensable to the biological activity of hGIF.