Saša Trkov

Astrocytic Vesicle Mobility in Health and Disease

Astrocytes are no longer considered subservient to neurons, and are, instead, now understood to play an active role in brain signaling. The intercellular communication of astrocytes with neurons and other non-neuronal cells involves the exchange of molecules by exocytotic and endocytotic processes through the trafficking of intracellular vesicles. Recent studies of single vesicle mobility in astrocytes have prompted new views of how astrocytes contribute to information processing in nervous tissue. Here, we review the trafficking of several types of membrane-bound vesicles that are specifically involved in the processes of (i) intercellular communication by gliotransmitters (glutamate, adenosine 5′-triphosphate, atrial natriuretic peptide), (ii) plasma membrane exchange of transporters and receptors (EAAT2, MHC-II), and (iii) the involvement of vesicle mobility carrying aquaporins (AQP4) in water homeostasis. The properties of vesicle traffic in astrocytes are discussed in respect to networking with neighboring cells in physiologic and pathologic conditions, such as amyotrophic lateral sclerosis, multiple sclerosis, and states in which astrocytes contribute to neuroinflammatory conditions.

Fingolimod—A sphingosine-like molecule inhibits vesicle mobility and secretion in astrocytes

In the brain, astrocytes signal to the neighboring cells by the release of chemical messengers (gliotransmitters) via regulated exocytosis. Recent studies uncovered a potential role of signaling lipids in modulation of exocytosis. Hence, we investigated whether sphingosine and the structural analog fingolimod/FTY720, a recently introduced therapeutic for multiple sclerosis, affect (i) intracellular vesicle mobility and (ii) vesicle cargo discharge from cultured rat astrocytes. Distinct types of vesicles, peptidergic, glutamatergic, and endosomes/lysosomes, were fluorescently prelabeled by cell transfection with plasmids encoding atrial natriuretic peptide tagged with mutant green fluorescent protein and vesicular glutamate transporter tagged with enhanced green fluorescent protein or by LysoTracker staining, respectively. The confocal and total internal reflection fluorescence microscopies were used to monitor vesicle mobility in the cytoplasm and near the basal plasma membrane, respectively. Sphingosine and FTY720, but not the membrane impermeable lipid analogs, dose‐dependently attenuated vesicle mobility in the subcellular regions studied, and significantly inhibited stimulated exocytotic peptide and glutamate release. We conclude that in astrocytes, cell permeable sphingosine‐like lipids affect regulated exocytosis by attenuating vesicle mobility, thereby preventing effective vesicle access/interaction with the plasma membrane docking/release sites.

Expression of familial Alzheimer disease presenilin 1 gene attenuates vesicle traffic and reduces peptide secretion in cultured astrocytes devoid of pathologic tissue environment

In the brain, astrocytes provide metabolic and trophic support to neurones. Failure in executing astroglial homeostatic functions may contribute to the initiation and propagation of diseases, including Alzheimer disease (AD), characterized by a progressive loss of neurones over years. Here, we examined whether astrocytes from a mice model of AD isolated in the presymptomatic phase of the disease exhibit alterations in vesicle traffic, vesicular peptide release and purinergic calcium signaling. In cultured astrocytes isolated from a newborn wild‐type (wt) and 3xTg‐AD mouse, secretory vesicles and acidic endosomes/lysosomes were labeled by transfection with plasmid encoding atrial natriuretic peptide tagged with mutant green fluorescent protein (ANP.emd) and by LysoTracker, respectively. The intracellular Ca2+ concentration ([Ca2+]i) was monitored with Fluo‐2 and visualized by confocal microscopy. In comparison with controls, spontaneous mobility of ANP‐ and LysoTracker‐labeled vesicles was diminished in 3xTg‐AD astrocytes; the track length (TL), maximal displacement (MD) and directionality index (DI) were all reduced in peptidergic vesicles and in endosomes/lysosomes (P < 0.001), as was the ATP‐evoked attenuation of vesicle mobility. Similar impairment of peptidergic vesicle trafficking was observed in wt rat astrocytes transfected to express mutated presenilin 1 (PS1M146V). The ATP‐evoked ANP discharge from single vesicles was less efficient in 3xTg‐AD and PS1M146V‐expressing astrocytes than in respective wt controls (P < 0.05). Purinergic stimulation evoked biphasic and oscillatory [Ca2+]i responses; the latter were less frequent (P < 0.001) in 3xTg‐AD astrocytes. Expression of PS1M146V in astrocytes impairs vesicle dynamics and reduces evoked secretion of the signaling molecule ANP; both may contribute to the development of AD. GLIA 2016;64:317–329

Amyotrophic lateral sclerosis immunoglobulins G enhance the mobility of Lysotracker-labelled vesicles in cultured rat astrocytes

Aim:  We examined the effect of purified immunoglobulins G (IgG) from patients with amyotrophic lateral sclerosis (ALS) on the mobility and exocytotic release from Lysotracker‐stained vesicles in cultured rat astrocytes.

Alterations of calcium homoeostasis in cultured rat astrocytes evoked by bioactive sphingolipids

In the brain, alterations in sphingolipid metabolism contribute to several neurological disorders; however, their effect on astrocytes is largely unknown. Here, we identified bioactive sphingolipids that affect intracellular free calcium concentration ([Ca2+]i), mobility of peptidergic secretory vesicles, signalling pathways involved in alterations of calcium homoeostasis and explored the relationship between the stimulus‐evoked increase in [Ca2+]i and attenuation of vesicle mobility.

Immunoglobulins G from patients with sporadic amyotrophic lateral sclerosis affects cytosolic Ca2+ homeostasis in cultured rat astrocytes.

Astrocytes are considered essential in the etiopathogenesis of amyotrophic lateral sclerosis (ALS). We have demonstrated previously that immunoglobulins G (IgG) isolated from patients with ALS enhance the mobility of acidic vesicles in cultured astrocytes in a Ca(2+)-dependent manner. Here we directly examined the impact of purified sporadic ALS IgG on cytosolic [Ca(2+)] ([Ca(2+)]i) in astrocytes. Confocal time-lapse images were acquired and fluorescence of a non-ratiometric Ca(2+) indicator was recorded before and after the application of IgG. ALS IgG (0.1 mg/ml) from 7 patients evoked transient increases in [Ca(2+)]i in ~50% of tested astrocytes. The probability of observing a response was independent of extracellular Ca(2+). The peak increase in [Ca(2+)]i developed ~3 times faster and the time integral of evoked transients was ~2-fold larger; the peak amplitude itself was not affected by extracellular Ca(2+). Application of pharmacological inhibitors revealed that activation of inositol-1,4,5-triphosphate receptors is necessary and sufficient to initiate transients in [Ca(2+)]i; the Ca(2+) influx through store-operated calcium entry prolongs the transient increase in [Ca(2+)]i. Thus, ALS IgG acutely affect [Ca(2+)]i by mobilizing both, intra- and extracellular Ca(2+) into the cytosol of cultured astrocytes.

Neural regulation of acetylcholinesterase‐associated collagen Q in rat skeletal muscles

Acetylcholinesterase‐associated collagen Q is expressed also outside of neuromuscular junctions in the slow soleus muscle, but not in fast muscles. We examined the nerve dependence of muscle collagen Q expression and mechanisms responsible for these differences. Denervation decreased extrajunctional collagen Q mRNA levels in the soleus muscles and junctional levels in fast sternomastoid muscles to about one third. Cross‐innervation of denervated soleus muscles by a fast muscle nerve, or electrical stimulation by ‘fast’ impulse pattern, reduced their extrajunctional collagen Q mRNA levels by 70–80%. In contrast, stimulation of fast muscles by ‘slow’ impulse pattern had no effect on collagen Q expression. Calcineurin inhibitors tacrolimus and cyclosporin A decreased collagen Q mRNA levels in the soleus muscles to about 35%, but did not affect collagen Q expression in denervated soleus muscles or the junctional expression in fast muscles. Therefore, high extrajunctional expression of collagen Q in the soleus muscle is maintained by its tonic nerve‐induced activation pattern via the activated Ca2+‐calcineurin signaling pathway. The extrajunctional collagen Q expression in fast muscles is independent of muscle activation pattern and seems irreversibly suppressed. The junctional expression of collagen Q in fast muscles is partly nerve‐dependent, but does not encompass the Ca2+‐calcineurin signaling pathway.

Pathophysiology of Vesicle Dynamics in Astrocytes

In astrocytes, as in other eukaryotic cells, vesicles have key cellular functions including constitutive housekeeping of the plasma membrane structure and cell-to-cell communication. On the one hand, vesicle traffic is associated with cell surface morphology exhibiting distinct glial microdomains. These determine the signaling potential and metabolic support for neighboring cells. On the other hand, vesicles are used in astrocytes for the release of vesicle-laden chemical messengers. This chapter addresses the properties of membrane-bound vesicles that store gliotransmitters (glutamate, adenosine 5’-triphosphate, peptides), other recycling vesicles, and endocytotic vesicles that are involved in the traffic of plasma membrane receptors such as the class II major histocompatibility molecules and membrane transporters (aquaporin 4 and excitatory amino acid transporter 2). Vesicle dynamics are also considered in view of diseases such as amyotrophic lateral sclerosis, multiple sclerosis, autistic disorders, trauma, edema, and states in which astrocytes contribute to neuroinflammation. In multiple sclerosis, for example, fingolimod, a recently introduced drug, apparently also affects vesicle traffic and gliotransmitter release from astrocytes, indicating that these processes may well be used as a target for the development of new therapies.