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.

Emerging evidence for the modulation of exocytosis by signalling lipids.

Membrane fusion is a key event in exocytosis of neurotransmitters and hormones stored in intracellular vesicles. In this process, soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins are essential components of the exocytotic molecular machinery, while lipids have been seen traditionally as structural elements. However, the so-called signalling lipids, such as sphingosine and arachidonic acid, interact with SNAREs and directly modulate the frequency and mode of fusion events.

Sphingomimetic multiple sclerosis drug FTY720 activates vesicular synaptobrevin and augments neuroendocrine secretion.

Neurotransmission and secretion of hormones involve a sequence of protein/lipid interactions with lipid turnover impacting on vesicle trafficking and ultimately fusion of secretory vesicles with the plasma membrane. We previously demonstrated that sphingosine, a sphingolipid metabolite, promotes formation of the SNARE complex required for membrane fusion and also increases the rate of exocytosis in isolated nerve terminals, neuromuscular junctions, neuroendocrine cells and in hippocampal neurons. Recently a fungi-derived sphingosine homologue, FTY720, has been approved for treatment of multiple sclerosis.

The position of mitochondria and ER in relation to that of the secretory sites in chromaffin cells.

Knowledge of the distribution of mitochondria and endoplasmic reticulum (ER) in relation to the position of exocytotic sites is relevant to understanding the influence of these organelles in tuning Ca(2+) signals and secretion. Confocal images of probes tagged to mitochondria and the F-actin cytoskeleton revealed the existence of two populations of mitochondria, one that was cortical and one that was perinuclear. This mitochondrial distribution was also confirmed by using electron microscopy.

Lipid metabolites enhance secretion acting on SNARE microdomains and altering the extent and kinetics of single release events in bovine adrenal chromaffin cells.

Lipid molecules such as arachidonic acid (AA) and sphingolipid metabolites have been implicated in modulation of neuronal and endocrine secretion. Here we compare the effects of these lipids on secretion from cultured bovine chromaffin cells. First, we demonstrate that exogenous sphingosine and AA interact with the secretory apparatus as confirmed by FRET experiments.

Cortical F-actin affects the localization and dynamics of SNAP-25 membrane clusters in chromaffin cells.

It has been proposed recently that the F-actin cytoskeleton organizes the relative disposition of the SNARE proteins and calcium channels that form part of the secretory machinery in chromaffin cells, a neurosecretory model. To test this idea, we used confocal microscopy do determine if DsRed-SNAP-25 microdomains, which define the final sites of exocytosis along with syntaxin-1, preferentially remain in contact with F-actin cortical structures labelled by lifeact-EGFP. A quantitative analysis showed that in cells over-expressing these constructs there is a preferential colocalization, rather than a random distribution of SNAP-25 patches.

F-actin-myosin II inhibitors affect chromaffin granule plasma membrane distance and fusion kinetics by retraction of the cytoskeletal cortex.

Chromaffin cell catecholamines are released when specialized secretory vesicles undergo exocytotic membrane fusion. Evidence indicates that vesicle supply and fusion are controlled by the activity of the cortical F-actin-myosin II network. To study in detail cell cortex and vesicle interactions, we use fluorescent labeling with GFP-lifeact and acidotropic dyes in confocal and evanescent wave microscopy.

The F-actin cortex in chromaffin granule dynamics and fusion: a minireview.

Chromaffin granules are restrained in a dense cortical cytoskeleton before releasing their complex mix of active substances in response to cell stimulation. In recent years, the complex organization and dynamics of the chromaffin cell cortex has been unveiled through its analysis with a range of techniques to visualize this structure, including confocal fluorescence, transmitted light, and evanescent field microscopy. Accordingly, it has become apparent that the cortex is a dense F-actin mesh that contains open polygonal spaces through which vesicles can access the submembrane space.

Association of SNAREs and calcium channels with the borders of cytoskeletal cages organizes the secretory machinery in chromaffin cells.

In chromaffin cells, SNARE proteins, forming the basic exocytotic machinery are present in membrane clusters of 500-600 nm in diameter. These microdomains containing both SNAP-25 and syntaxin-1 are dynamic and the expression of altered forms of SNAREs modifies not only their motion but also the mobility of the associated granules. It is also clear that SNARE microdomain location defines the place for individual vesicle fusion and that the alteration of cluster dynamics affects the fusion process itself.

Metabolic activity of cerebellar and basal ganglia-thalamic neurons is reduced in parkinsonism.

We have examined whether degeneration of nigrostriatal dopaminergic neurons causes dysfunction of both the basal ganglia-thalamic and cerebello-thalamic pathways. Changes in the activity of thalamic neurons receiving input from the basal ganglia or the cerebellum were examined in two models of Parkinson's disease, 6-hydroxydopamine (6-OHDA)-lesioned rats and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys. Metabolic activity of the neurons was evaluated at the cellular level by quantitative in situ hybridization, using the expression of messenger RNA for subunit I of cytochrome oxidase (COI), encoded by the mitochondrial genome, as the marker.