Assessing neuroprotective effects of diroximel fumarate and siponimod via modulation of pacemaker channels in an experimental model of remyelination.

Cuprizone (CPZ)-induced alterations in axonal myelination are associated with a period of neuronal hyperexcitability and increased activity of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels in the thalamocortical (TC) system. Substances used for the treatment of multiple sclerosis (MS) have been shown to normalize neuronal excitability in CPZ-treated mice. Therefore, we aimed to examine the effects of diroximel fumarate (DRF) and the sphingosine 1-phospate receptor (S1PR) modulator siponimod on action potential firing and the inward current (Ih) carried by HCN ion channels in naive conditions and during different stages of de- and remyelination. Here, DRF application reduced Ih current density in ex vivo patch clamp recordings from TC neurons of the ventrobasal thalamic complex (VB), thereby counteracting the increase of Ih during early remyelination. Siponimod reduced Ih in VB neurons under control conditions but had no effect in neurons of the auditory cortex (AU). Furthermore, siponimod increased and decreased AP firing properties of neurons in VB and AU, respectively. Computational modeling revealed that both DRF and siponimod influenced thalamic bursting during early remyelination by delaying the onset and decreasing the interburst frequency. Thus, substances used in MS treatment normalize excitability in the TC system by influencing AP firing and Ih.

Optimized synthesis and pharmacological evaluation of HCN channel inhibitor EC18.

HCN4 channels are considered to be a promising target for cardiac pathologies, epilepsy, and multiple sclerosis. However, there are no subtype-selective HCN channel blockers available, and only a few compounds are reported to display subtype preferences, one of which is EC18 (cis-1). Herein, we report the optimized synthetic route for the preparation of EC18 and its evaluation in three different pharmacological models, allowing us to assess its activity on cardiac function, thalamocortical neurons, and immune cells.

Effects of Axonal Demyelination, Inflammatory Cytokines and Divalent Cation Chelators on Thalamic HCN Channels and Oscillatory Bursting.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system that is characterized by the progressive loss of oligodendrocytes and myelin and is associated with thalamic dysfunction. Cuprizone (CPZ)-induced general demyelination in rodents is a valuable model for studying different aspects of MS pathology. CPZ feeding is associated with the altered distribution and expression of different ion channels along neuronal somata and axons. However, it is largely unknown whether the copper chelator CPZ directly influences ion channels. Therefore, we assessed the effects of different divalent cations (copper; zinc) and trace metal chelators (EDTA; Tricine; the water-soluble derivative of CPZ, BiMPi) on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that are major mediators of thalamic function and pathology. In addition, alterations of HCN channels induced by CPZ treatment and MS-related proinflammatory cytokines (IL-1ß; IL-6; INF-α; INF-ß) were characterized in C57Bl/6J mice. Thus, the hyperpolarization-activated inward current (Ih) was recorded in thalamocortical (TC) neurons and heterologous expression systems (mHCN2 expressing HEK cells; hHCN4 expressing oocytes). A number of electrophysiological characteristics of Ih (potential of half-maximal activation (V0.5); current density; activation kinetics) were unchanged following the extracellular application of trace metals and divalent cation chelators to native neurons, cell cultures or oocytes. Mice were fed a diet containing 0.2% CPZ for 35 days, resulting in general demyelination in the brain. Withdrawal of CPZ from the diet resulted in rapid remyelination, the effects of which were assessed at three time points after stopping CPZ feeding (Day1, Day7, Day25). In TC neurons, Ih was decreased on Day1 and Day25 and revealed a transient increased availability on Day7. In addition, we challenged naive TC neurons with INF-α and IL-1ß. It was found that Ih parameters were differentially altered by the application of the two cytokines to thalamic cells, while IL-1ß increased the availability of HCN channels (depolarized V0.5; increased current density) and the excitability of TC neurons (depolarized resting membrane potential (RMP); increased the number of action potentials (APs); produced a larger voltage sag; promoted higher input resistance; increased the number of burst spikes; hyperpolarized the AP threshold), INF-α mediated contrary effects. The effect of cytokine modulation on thalamic bursting was further assessed in horizontal slices and a computational model of slow thalamic oscillations. Here, IL-1ß and INF-α increased and reduced oscillatory bursting, respectively. We conclude that HCN channels are not directly modulated by trace metals and divalent cation chelators but are subject to modulation by different MS-related cytokines.

Piperazine squaric acid diamides, a novel class of allosteric P2X7 receptor antagonists.

The P2X7 receptor (P2X7R) stands out among the purinergic receptors due to its strong involvement in the regulation of tumor growth and metastasis formation as well as in innate immune responses and afferent signal transmission. Numerous studies have pointed out the beneficial effects of P2X7R antagonism for the treatment of a variety of cancer types, inflammatory diseases, and chronic pain. Herein we describe the development of novel P2X7R antagonists, incorporating piperazine squaric diamides as a central element. Besides improving the antagonists' potency from pIC50 values of 5.7-7.6, ADME properties (logD7.4 value, plasma protein binding, in vitro metabolic stability) of the generated compounds were investigated and optimized to provide novel P2X7R antagonists with drug-like properties. Furthermore, docking studies revealed the antagonists binding to the allosteric binding pocket in two distinct binding poses, depending on the substitution of the central piperazine moiety.

Impact of Diverse Ion Channels on Regulatory T Cell Functions.

The population of regulatory T cells (Tregs) is critical for immunological self-tolerance and homeostasis. Proper ion regulation contributes to Treg lineage identity, regulation, and effector function. Identified ion channels include Ca2+ release-activated Ca2+, transient receptor potential, P2X, volume-regulated anion and K+ channels Kv1.3 and KCa3.1. Ion channel modulation represents a promising therapeutic approach for the treatment of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. This review summarizes studies with gene-targeted mice and pharmacological modulators affecting Treg number and function. Furthermore, participation of ion channels is illustrated and the power of future research possibilities is discussed.

Loss of Kat2a enhances transcriptional noise and depletes acute myeloid leukemia stem-like cells.

Acute Myeloid Leukemia (AML) is an aggressive hematological malignancy with abnormal progenitor self-renewal and defective white blood cell differentiation. Its pathogenesis comprises subversion of transcriptional regulation, through mutation and by hijacking normal chromatin regulation. Kat2a is a histone acetyltransferase central to promoter activity, that we recently associated with stability of pluripotency networks, and identified as a genetic vulnerability in AML. Through combined chromatin profiling and single-cell transcriptomics of a conditional knockout mouse, we demonstrate that Kat2a contributes to leukemia propagation through preservation of leukemia stem-like cells. Kat2a loss impacts transcription factor binding and reduces transcriptional burst frequency in a subset of gene promoters, generating enhanced variability of transcript levels. Destabilization of target programs shifts leukemia cell fate out of self-renewal into differentiation. We propose that control of transcriptional variability is central to leukemia stem-like cell propagation, and establish a paradigm exploitable in different tumors and distinct stages of cancer evolution.

Less than 30% of patients with acute myeloid leukaemia ­ an aggressive cancer of the white blood cells ­ survive five years post-diagnosis. This disease disrupts the maturation of white blood cells, resulting in the accumulation of immature cells that multiply and survive but are incapable of completing their maturation process. Amongst these, a group of cancer cells known as leukemic stem cells is responsible for continually replenishing the leukaemia, thus perpetuating its growth. Cancers develop when cells in the body acquire changes or mutations to their genetic makeup. The mutations that lead to acute myeloid leukaemia often affect the activity of genes known as epigenetic regulators. These genes regulate which proteins and other molecules cells make by controlling the way in which cells 'read' their genetic instructions. The epigenetic regulator Kat2a is thought to 'tune' the frequency at which cells read their genetic instructions. This tuning mechanism decreases random fluctuations in the execution of the instructions cells receive to make proteins and other molecules. In turn, this helps to ensure that individual cells of the same type behave in a similar way, for example by keeping leukaemia cells in an immature state. Here, Domingues, Kulkarni et al. investigated whether interfering with Kat2a can make acute myeloid leukaemia less aggressive by allowing the immature white blood cells to mature. Domingues, Kulkarni et al. genetically engineered mice to remove Kat2a from blood cells on demand and then inserted a mutation that causes acute myeloid leukaemia. The experiments showed that the loss of Kat2a delayed the development of leukaemia in the mice and progressively depleted leukaemia stem cells, causing the disease to become less aggressive. The results also showed that loss of Kat2a caused more fluctuations in how the white blood cells read their genetic code, which resulted in more variability in the molecules they produced and increased the tendency of the cells to mature. These findings establish that loss of Kat2a causes leukaemia stem cells to mature and stop multiplying by untuning the frequency at which the cells read their genetic instructions. In the future, it may be possible to develop drugs that target human KAT2A to treat acute myeloid leukaemia.