Neurosteroid modulation of GABAA receptor function by independent action at multiple specific binding sites

: Neurosteroids are endogenous modulators of GABAA receptors that mediate anxiety, pain, mood and arousal. The 3-hydroxyl epimers, allopregnanolone (3α-OH) and epi-allopregnanolone (3β-OH) are both prevalent in mammalian brain and produce opposite effects on GABAA receptor function, acting as positive and negative allosteric modulators respectively. This Perspective provides a model to explain the actions of 3α-OH and 3β-OH neurosteroids. The model is based on evidence that the neurosteroid epimers bind to an overlapping subset of specific sites on GABAA receptors, with their net functional effect on channel gating being the sum of their independent effects at each site.

The BMP antagonist Gremlin1 contributes to the development of cortical excitatory neurons, motor balance and fear responses

Bone morphogenetic protein (BMP) signaling is required for early forebrain development and cortical formation. How the endogenous modulators of BMP signaling regulate the structural and functional maturation of the developing brain remains unclear. Here we show that expression of the BMP antagonist, Grem1, marks committed layer Ⅴ and Ⅵ glutamatergic neurons in the embryonic mouse brain. Lineage tracing of Grem1-expressing cells in the embryonic brain was examined by administration of tamoxifen to pregnant Grem1creERT; Rosa26LSLTdtomato mice at 13.5 days post coitum (dpc), followed by collection of embryos later in gestation. In addition, at 14.5 dpc, bulk mRNA seq analysis of differentially expressed transcripts between FACS sorted Grem1 positive and negative cells was performed. We also generated Emx1-cre mediated Grem1 conditional knockout mice (Emx1-Cre;Grem1flox/flox) in which the Grem1 gene was deleted specifically in the dorsal telencephalon. Grem1Emx1cKO animals had reduced cortical thickness, especially layers Ⅴ and Ⅵ and impaired motor balance and fear sensitivity compared to littermate controls. This study has revealed new roles for Grem1 in the structural and functional maturation of the developing cortex.

Arginine methylation: the promise of a ‘silver bullet’ for brain tumours?

Despite intense research efforts, our pharmaceutical repertoire against high-grade brain tumours has not been able to increase patient survival for a decade and life expectancy remains at less than 16 months after diagnosis, on average. Inhibitors of protein arginine methyltransferases (PRMTs) have been developed and investigated over the past 15 years and have now entered oncology clinical trials, including for brain tumours. This review collates recent advances in the understanding of the role of PRMTs and arginine methylation in brain tumours. We provide an up-to-date literature review on the mechanisms for PRMT regulation. These include endogenous modulators such as alternative splicing, miRNA, post-translational modifications and PRMT–protein interactions, and synthetic inhibitors. We discuss the relevance of PRMTs in brain tumours with a particular focus on PRMT1, -2, -5 and -8. Finally, we include a future perspective where we discuss possible routes for further research on arginine methylation and on the use of PRMT inhibitors in the context of brain tumours.

Sirt6 Deacetylase: A Potential Key Regulator in the Prevention of Obesity, Diabetes and Neurodegenerative Disease

Sirtuins, NAD + dependent proteins belonging to class III histone deacetylases, are involved in regulating numerous cellular processes including cellular stress, insulin resistance, inflammation, mitochondrial biogenesis, chromatin silencing, cell cycle regulation, transcription, and apoptosis. Of the seven mammalian sirtuins present in humans, Sirt6 is an essential nuclear sirtuin. Until recently, Sirt6 was thought to regulate chromatin silencing, but new research indicates its role in aging, diabetes, cardiovascular disease, lipid metabolism, neurodegenerative diseases, and cancer. Various murine models demonstrate that Sirt6 activation is beneficial in alleviating many disease conditions and increasing lifespan, showing that Sirt6 is a critical therapeutic target in the treatment of various disease conditions in humans. Sirt6 also regulates the pathogenesis of multiple diseases by acting on histone proteins and non-histone proteins. Endogenous and non-endogenous modulators regulate both activation and inhibition of Sirt6. Few Sirt6 specific non-endogenous modulators have been identified. Hence the identification of Sirt6 specific modulators may have potential therapeutic roles in the diseases described above. In this review, we describe the development of Sirt6, the role it plays in the human condition, the functional role and therapeutic importance in disease processes, and specific modulators and molecular mechanism of Sirt6 in the regulation of metabolic homeostasis, cardiovascular disease, aging, and neurodegenerative disease.

Modulatory Roles of ATP and Adenosine in Cholinergic Neuromuscular Transmission

A review of the data on the modulatory action of adenosine 5’-triphosphate (ATP), the main co-transmitter with acetylcholine, and adenosine, the final ATP metabolite in the synaptic cleft, on neuromuscular transmission is presented. The effects of these endogenous modulators on pre- and post-synaptic processes are discussed. The contribution of purines to the processes of quantal and non-quantal secretion of acetylcholine into the synaptic cleft, as well as the influence of the postsynaptic effects of ATP and adenosine on the functioning of cholinergic receptors, are evaluated. As usual, the P2-receptor-mediated influence is minimal under physiological conditions, but it becomes very important in some pathophysiological situations such as hypothermia, stress, or ischemia. There are some data demonstrating the same in neuromuscular transmission. It is suggested that the role of endogenous purines is primarily to provide a safety factor for the efficiency of cholinergic neuromuscular transmission.

Mapping of CaM, S100A1 and PIP2-Binding Epitopes in the Intracellular N- and C-Termini of TRPM4

Molecular determinants of the binding of various endogenous modulators to transient receptor potential (TRP) channels are crucial for the understanding of necessary cellular pathways, as well as new paths for rational drug designs. The aim of this study was to characterise interactions between the TRP cation channel subfamily melastatin member 4 (TRPM4) and endogenous intracellular modulators—calcium-binding proteins (calmodulin (CaM) and S100A1) and phosphatidylinositol 4, 5-bisphosphate (PIP2). We have found binding epitopes at the N- and C-termini of TRPM4 shared by CaM, S100A1 and PIP2. The binding affinities of short peptides representing the binding epitopes of N- and C-termini were measured by means of fluorescence anisotropy (FA). The importance of representative basic amino acids and their combinations from both peptides for the binding of endogenous TRPM4 modulators was proved using point alanine-scanning mutagenesis. In silico protein–protein docking of both peptides to CaM and S100A1 and extensive molecular dynamics (MD) simulations enabled the description of key stabilising interactions at the atomic level. Recently solved cryo-Electron Microscopy (EM) structures made it possible to put our findings into the context of the entire TRPM4 channel and to deduce how the binding of these endogenous modulators could allosterically affect the gating of TRPM4. Moreover, both identified binding epitopes seem to be ideally positioned to mediate the involvement of TRPM4 in higher-order hetero-multimeric complexes with important physiological functions.

Arachidonoylcholine and Other Unsaturated Long-Chain Acylcholines Are Endogenous Modulators of the Acetylcholine Signaling System

Cholines acylated with unsaturated fatty acids are a recently discovered family of endogenous lipids. However, the data on the biological activity of acylcholines remain very limited. We hypothesized that acylcholines containing residues of arachidonic (AA-CHOL), oleic (Ol-CHOL), linoleic (Ln-CHOL), and docosahexaenoic (DHA-CHOL) acids act as modulators of the acetylcholine signaling system. In the radioligand binding assay, acylcholines showed inhibition in the micromolar range of both α7 neuronal nAChR overexpressed in GH4C1 cells and muscle type nAChR from Torpedo californica, as well as Lymnaea stagnalis acetylcholine binding protein. Functional response was checked in two cell lines endogenously expressing α7 nAChR. In SH-SY5Y cells, these compounds did not induce Ca2+ rise, but inhibited the acetylcholine-evoked Ca2+ rise with IC50 9 to 12 μM. In the A549 lung cancer cells, where α7 nAChR activation stimulates proliferation, Ol-CHOL, Ln-CHOL, and AA-CHOL dose-dependently decreased cell viability by up to 45%. AA-CHOL inhibited human erythrocyte acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BChE) by a mixed type mechanism with Ki = 16.7 ± 1.5 μM and αKi = 51.4 ± 4.1 μM for AChE and Ki = 70.5 ± 6.3 μM and αKi = 214 ± 17 μM for BChE, being a weak substrate of the last enzyme only, agrees with molecular docking results. Thus, long-chain unsaturated acylcholines could be viewed as endogenous modulators of the acetylcholine signaling system.