Multiple sclerosis drug FTY-720 toxicity is mediated by the heterotypic fusion of organelles in neuroendocrine cells

FTY-720 (Fingolimod) was one of the first compounds authorized for the treatment of multiple sclerosis. Among its other activities, this sphingosine analogue enhances exocytosis in neuroendocrine chromaffin cells, altering the quantal release of catecholamines. Surprisingly, the size of chromaffin granules is reduced within few minutes of treatment, a process that is paralleled by the homotypic fusion of granules and their heterotypic fusion with mitochondria, as witnessed by dynamic confocal and TIRF microscopy. Electron microscopy studies support these observations, revealing the fusion of several vesicles with individual mitochondria to form large, round mixed organelles. This cross-fusion is SNARE-dependent, being partially prevented by the expression of an inactive form of SNAP-25. Fused mitochondria exhibit an altered redox potential, which dramatically enhances cell death. Therefore, the cross-fusion of intracellular organelles appears to be a new mechanism to be borne in mind when considering the effect of FTY-720 on the survival of neuroendocrine cells

Superior efficacy of calcipotriene and betamethasone dipropionate aerosol foam versus ointment in patients with psoriasis vulgaris – A randomized phase II study

Background: An aerosol foam formulation of fixed combination calcipotriene 0.005% (as hydrate; Cal) plus betamethasone dipropionate 0.064% (BD) was developed to improve psoriasis treatment. Objectives: To compare the efficacy and safety of Cal/BD aerosol foam with Cal/BD ointment after 4 weeks. Methods: In this Phase II, multicenter, investigator-blind, 4-week trial, adult patients with psoriasis vulgaris were randomized to Cal/BD aerosol foam, Cal/BD ointment, aerosol foam vehicle or ointment vehicle (3:3:1:1). The primary efficacy endpoint was the proportion of patients at week 4 who achieved treatment success (clear or almost clear with at least a two-step improvement) according to the physician’s global assessment of disease severity. Results: In total, 376 patients were randomized. At week 4, significantly more patients using Cal/BD aerosol foam achieved treatment success (54.6% versus 43.0% [ointment]; p = 0.025); mean modified (excluding the head, which was not treated) psoriasis area and severity index score was significantly different between Cal/BD aerosol foam and Cal/BD ointment (mean difference –0.6; p = 0.005). Rapid, continuous itch relief occurred with both active treatments. One adverse drug reaction was reported with Cal/BD aerosol foam (application site itch). Conclusions: Cal/BD aerosol foam demonstrates significantly greater efficacy and similar tolerability compared with Cal/BD ointment for psoriasis treatment

Sodium sensing in neurons with a dendrimer-based nanoprobe.

Ion imaging is a powerful methodology to assess fundamental biological processes in live cells. The limited efficiency of some ion-sensing probes and their fast leakage from cells are important restrictions to this approach. In this study, we present a novel strategy based on the use of dendrimer nanoparticles to obtain better intracellular retention of fluorescent probes and perform prolonged fluorescence imaging of intracellular ion dynamics. A new sodium-sensitive nanoprobe was generated by encapsulating a sodium dye in a PAMAM dendrimer nanocontainer. This nanoprobe is very stable and has high sodium sensitivity and selectivity. When loaded in neurons in live brain tissue, it homogenously fills the entire cell volume, including small processes, and stays for long durations, with no detectable alterations of cell functional properties. We demonstrate the suitability of this new sodium nanosensor for monitoring physiological sodium responses such as those occurring during neuronal activity

State-Dependent cAMP Binding to Functioning HCN Channels Studied by Patch-Clamp Fluorometry

One major goal of ion channel research is to delineate the molecular events from the detection of the stimuli to the movement of channel gates. For ligand-gated channels, it is challenging to separate ligand binding from channel gating. Here we studied the cyclic adenosine monophosphate (cAMP)-dependent gating in hyperpolarization-activated cAMP-regulated (HCN) channel by simultaneously recording channel opening and ligand binding, using the patch-clamp fluorometry technique with a unique fluorescent cAMP analog that fluoresces strongly in the hydrophobic binding pocket and exerts regulatory effects on HCN channels similar to those imposed by cAMP. Corresponding to voltage-dependent channel activation, we observed a robust, close-to-threefold increase in ligand binding, which was more pronounced at subsaturating ligand concentrations than higher concentrations. This observation supported the cyclic allosteric models and indicated that protein allostery can be implemented through differentiating ligand binding affinities between resting and active states. The kinetics of ligand binding largely matched channel activation. However, during channel deactivation, ligand unbinding was slower than channel closing, suggesting a delayed response to membrane potential by the ligand binding machinery. Our results provide what we believe to be new insights into the cAMP-dependent gating in HCN channel and the interpretation of protein allostery for general ligand-gated channels and receptors

Structural Basis for the cAMP-dependent Gating in the Human HCN4 Channel*

Hyperpolarization-activated cAMP-regulated (HCN) channels play important physiological roles in both cardiovascular and central nervous systems. Among the four HCN isoforms, HCN2 and HCN4 show high expression levels in the human heart, with HCN4 being the major cardiac isoform. The previously published crystal structure of the mouse HCN2 (mHCN2) C-terminal fragment, including the C-linker and the cyclic-nucleotide binding domain (CNBD), has provided many insights into cAMP-dependent gating in HCN channels. However, structures of other mammalian HCN channel isoforms have been lacking. Here we used a combination of approaches including structural biology, biochemistry, and electrophysiology to study cAMP-dependent gating in HCN4 channel. First we solved the crystal structure of the C-terminal fragment of human HCN4 (hHCN4) channel at 2.4 Å. Overall we observed a high similarity between mHCN2 and hHCN4 crystal structures. Functional comparison between two isoforms revealed that compared with mHCN2, the hHCN4 protein exhibited marked different contributions to channel function, such as a ∼3-fold reduction in the response to cAMP. Guided by structural differences in the loop region between β4 and β5 strands, we identified residues that could partially account for the differences in response to cAMP between mHCN2 and hHCN4 proteins. Moreover, upon cAMP binding, the hHCN4 C-terminal protein exerts a much prolonged effect in channel deactivation that could have significant physiological contributions