Marko Kreft

University of Ljubljana · Biotechnical Faculty

Astrocytes, a highly heterogeneous neuroglial cell type, execute a myriad of homeostatic roles in the central nervous system. Although these cells are not firing action potential, they are excitable as demonstrated by an increase in the concentration of cytosolic second messengers, including free calcium concentration ([Ca2+]i) and cyclic adenosine...

A single sub-anesthetic dose of ketamine evokes rapid and long-lasting beneficial effects in patients with a major depressive disorder. However, the mechanisms underlying this effect are unknown. It has been proposed that astrocyte dysregulation of extracellular K+ concentration ([K+]o) alters neuronal excitability, thus contributing to depression....

Gaussian noise is an important stimulus for the study of biological systems, especially sensory and neural systems. Since these systems are inherently nonlinear, the properties of the noise strongly influence the outcome of the analysis. Therefore, it is crucial to use a well-defined and controlled noise stimulus. In this paper, we first use the ex...

Regulated exocytosis consists of the fusion between vesicles and the plasma membranes, leading to the formation of a narrow fusion pore through which secretions exit the vesicle lumen into the extracellular space. An increase in the cytosolic concentration of free Ca²⁺ ([Ca²⁺]i) is considered the stimulus of this process. However, whether this mech...

The aim of this study was to quantify the effectiveness of intradermal application of autologous fibroblasts on lean tissue structures. The histological sections of the skin were analysed and evaluated for the expansion potential of autologous fibroblasts in the control skin patch area and the nearby pre-treated skin patch into which we had injecte...

Gaussian noise is one of the principal inputs used for studying biological systems, especially sensory and neuronal systems. Because of the inherent nonlinearities of these systems, the properties of the noise strongly influence the outcome of the analysis. We show for the case of an insect filiform sensillum, a simple mechanoreceptor with a single...

G-protein-coupled receptors (GPCRs) represent a family with over 800 members in humans, and one-third of these are targets for approved drugs. A large number of GPCRs have unknown physiologic roles. Here, we investigated GPR27, an orphan GPCR belonging to the family of super conserved receptor expressed in the brain, with unknown functions. Cytosol...

Interactive mechanical forces between pairs of individual SNARE proteins synaptobrevin 2 (Sb2) and syntaxin 1A (Sx1A) may be sufficient to mediate vesicle docking. This notion, based on force spectroscopy single molecule measurements probing recombinant Sx1A an Sb2 in silico, questioned a predominant view of docking via the ternary SNARE complex fo...

In some lysosomal storage diseases (LSD) cholesterol accumulates in vesicles. Whether increased vesicle cholesterol affects vesicle fusion with the plasmalemma, where the fusion pore, a channel between the vesicle lumen and the extracellular space, is formed, is unknown. Super-resolution microscopy revealed that after stimulation of exocytosis, pit...

Astroglial aerobic glycolysis, a process during which d-glucose is converted to l-lactate, a brain fuel and signal, is regulated by the plasmalemmal receptors, including adrenergic receptors (ARs) and purinergic receptors (PRs), modulating intracellular Ca²⁺ and cAMP signals. However, the extent to which the two signals regulate astroglial aerobic...

During cognitive efforts mediated by local neuronal networks, approximately 20% of additional energy is required; this is mediated by chemical messengers such as noradrenaline (NA). NA targets astroglial aerobic glycolysis, the hallmark of which is the end product l‐lactate, a fuel for neurons. Biochemical studies have revealed that astrocytes exhi...

OBJECTIVES GDI1 gene encodes for αGDI, a protein controlling the cycling of small GTPases, reputed to orchestrate vesicle trafficking. Mutations in human GDI1 are responsible for intellectual disability (ID). In mice with ablated Gdi1, a model of ID, impaired working and associative short-term memory was recorded. This cognitive phenotype worsens i...

Eukaryotic vesicles fuse with the plasmalemma to form the fusion pore, previously considered to be unstable with widening of the pore diameter. Recent studies established that the pore diameter is stable, reflecting balanced forces of widening and closure. Proteins are considered key regulators of the fusion pore, whereas the role of membrane lipid...

Nasal drug formulations can be effective for local delivery of therapeutic drugs to the sinonasal mucosa or for systemic drug delivery by absorption directly into the bloodstream. The growing field of potential nasal therapies includes nasal vaccination and even treatment of neurodegenerative diseases. However, it is important that nasal drug formu...

Glycogen synthase kinase 3 (GSK-3) plays an important role in metabolic regulation in skeletal muscles, and both insulin and adrenaline stimulate GKS-3 phosphorylation. The aim of the present study was to study the effect of insulin and adrenaline on GSK-3 localisation in skeletal muscles.We characterized subcellular localization of (GSK-3) signal...

In obesity, the skeletal muscle capillary network regresses and the insulin-mediated capillary recruitment is impaired. However, it has been shown that in the early stage of advanced obesity, an increased functional vascular response can partially compensate for other mechanisms of insulin resistance. The present study aimed to investigate the chan...

The ventral compound eye of many insects contains polarization-sensitive photoreceptors, but little is known about how they are integrated into visual functions. In female horseflies, polarized reflections from animal fur are a key stimulus for host detection. To understand how polarization vision is mediated by the ventral compound eye, we investi...

The original version of this article unfortunately contained a mistake in Author name. In Pia Pužar Dominkuš, "Pužar" should be classified as Familyname.

As part of the blood-brain-barrier, astrocytes are ideally positioned between cerebral vasculature and neuronal synapses to mediate nutrient uptake from the systemic circulation. In addition, astrocytes have a robust enzymatic capacity of glycolysis, glycogenesis and lipid metabolism, managing nutrient support in the brain parenchyma for neuronal c...

Once infected by HIV-1, microglia abundantly produce accessory protein Nef that enhances virus production and infectivity, but little is known about its intracellular compartmentalization, trafficking mode(s), and release from microglia. Here, we transfected immortalized human microglia with a plasmid encoding Nef tagged with green fluorescent prot...

Besides being a neuronal fuel, L-lactate is also a signal in the brain. Whether extracellular L-lactate affects brain metabolism, in particular astrocytes, abundant neuroglial cells, which produce L-lactate in aerobic glycolysis, is unclear. Recent studies suggested that astrocytes express low levels of the L-lactate GPR81 receptor (EC50 ≈ 5 mM) th...

PKH lipophilic dyes are highly fluorescent and stain membranes by intercalating their aliphatic portion into the exposed lipid bilayer. They have established use in labeling and tracking of cells in vivo and in vitro. Despite wide use of PKH-labeled extracellular vesicles (EVs) in cell targeting and functional studies, nonEV-associated fluorescent...

Microvillar photoreceptors are intrinsically capable of detecting the orientation of e-vector of linearly polarized light. They provide most invertebrates with an additional sensory channel to detect important features of their visual environment. However, polarization sensitivity (PS) of photoreceptors may lead to the detection of polarization-ind...

Aim: Alzheimer disease (AD) is largely considered a neuron-derived insult, but also involves failure of astroglia. A recent study indicated that mutated presenilin 1 (PS1M146V), a putative endoplasmic reticulum (ER) Ca2+ channel with decreased Ca2+ conductance, impairs the traffic of astroglial peptidergic vesicles. Whether other pathogenically re...

A number of recent studies revealed that astrocytes are a target of the locus coeruleus (LC)-nerve terminals that supply noradrenaline (NA) throughout the central nervous system (CNS). LC activation triggers a global CNS excitation, associated with arousal, which affects many processes including metabolism and memory formation. Although electricall...

Capillary network characteristics are invaluable for diagnostics of muscle diseases. Biopsy material is limited in size and mostly not accessible for intensive research. Therefore, especially in human tissue, studies are performed on autopsy material. To approach the problem whether it is reliable to deduce hypotheses from autopsy material to expla...

Uroplakins (UPs) play an essential role in maintaining an effective urothelial permeability barrier at the level of superficial urothelial cell (UC) layer. Although the organization of UPs in the apical plasma membrane (PM) of UCs is well known, their transport in UCs is only partially understood. Here, we dissected trafficking of UPs and its diffe...

Dipeptidyl peptidase 4 (DPP4) is a transmembrane glycoprotein involved in protein degradation. Due to its action on incretins, which increase insulin secretion, DPP4 is considered a therapeutic target for type 2 diabetes. Here we have studied the role of single and combined effects of hypoxia and inactivity on the expression of DPP4 in human adipos...

Regulated exocytosis can be split into a sequence of steps ending with the formation and the dilation of a fusion pore, a neck-like connection between the vesicle and the plasma membrane. Each of these steps is precisely controlled to achieve the optimal spatial and temporal profile of the release of signalling molecules. At the level of the fusion...

Background: Dynamin is a multidomain GTPase exhibiting mechanochemical and catalytic properties involved in vesicle scission from the plasmalemma during endocytosis. New evidence indicates that dynamin is also involved in exocytotic release of catecholamines, suggesting the existence of a dynamin-regulated structure that couples endo- to exocytosi...

X-linked non-syndromic intellectual disability (XLID) is a common mental disorder recognized by cognitive and behavioral deficits. Mutations in the brain-specific αGDI, shown to alter a subset of RAB GTPases redistribution in cells, are linked to XLID, likely via changes in vesicle traffic in neurons. Here, we show directly that isolated XLID mice...

In the past, vesicle content release was thought to occur immediately and completely after triggering of exocytosis. However, vesicles may merge with the plasma membrane to form an Ångstrom diameter fusion pore that prevents the exit of secretions from the vesicle lumen. The advantage of such a narrow pore is to minimize the delay between the trigg...

Astrocytes are excitable neural cells that contribute to brain information processing via bidirectional communication with neurons. This involves the release of gliosignaling molecules that affect synapses patterning and activity. Mechanisms mediating the release of these molecules likely consist of non-vesicular and vesicular-based mechanisms. It...

We are reviewing and commenting highlights of the research published in Image Analysis and Stereology journal (IAS), volume 35, where 16 original research papers on image analysis, computer vision, modelling, and other approaches were published. We have reported on the precision of curve length estimation in the plane. Further, a focus was on a rob...

Uporaba dvo-fotonskega konfokalnega mikroskopa za raziskovanje encimske aktivnosti ektomikoriznih gliv in analiza slik s programom ImageJ Simbioza med ektomikoriznimi glivami in gozdnimi drevesi prispeva k rasti dreves in delovanju ekosistema. Ektomikorizne glive so razvile visoko diferencirane strukture, ki omogočajo različne načine privzemanja hr...

In the fluorescence microscopy of biological specimens the structures in cells and tissues usually need to be labelled with various fluorescent markers. The three main groups of fluorescent markers are small organic fluorochromes, fluorescent proteins and quantum dots. Fluorescent markers differ according to photophysical properties and binding spe...

Key support for vesicle-based release of gliotransmitters comes from studies of transgenic mice with astrocyte-specific expression of a dominant-negative domain of synaptobrevin 2 protein (dnSNARE). To determine how this peptide affects exocytosis, we used super-resolution stimulated emission depletion microscopy and structured illumination microsc...

Ketamine is an anesthetic that exhibits analgesic, psychotomimetic, and rapid antidepressant effects that are of particular neuropharmacological interest. Recent studies revealed astrocytic Ca ²⁺ signaling and regulated exocytosis as ketamine‐targeted processes. Thus high‐resolution cell‐attached membrane capacitance measurements were performed to...

Buckwheat ( Fagopyrum esculentum Moench, F. tataricum Gaertner) groats and flour have been established globally as nutritional foods because of their high levels of proteins, polyphenols and minerals. In some regions, buckwheat herb is used as a functional food. In the present study, reports of in vitro studies, preclinical and clinical trials deal...

Edema in the central nervous system can rapidly result in life-threatening complications. Vasogenic edema is clinically manageable, but there is no established medical treatment for cytotoxic edema, which affects astrocytes and is a primary trigger of acute post-traumatic neuronal death. To test the hypothesis that adrenergic receptor agonists, inc...

Regulated exocytosis is a multistage process involving a merger between the vesicle and the plasma membrane, leading to the formation of a fusion pore, a channel, through which secretions are released from the vesicle to the cell exterior. A stimulus may influence the pore by either dilating it completely (full‐fusion exocytosis) or mediating a rev...

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 iso...

Dipeptidyl peptidase 4 (DPP4), a transmembrane protein, has been identified in human adipose tissue and is considered to be associated with obesity-related type 2 diabetes. Since adipose tissue is relatively hypoxic in obese participants, we investigated the expression of DPP4 in human preadipocytes (hPA) and adipocytes in hypoxia, during different...

Most of the human brain mass is occupied by the neocortex, which consists of neurons and non-neurons. The latter cells include astrocytes, a heterogeneous glial cell type. While astrocytes have been considered as neuronsubservient entities for almost a century, it is now becoming evident that they are essential in providing homeostatic support to n...

In regulated exocytosis vesicular and plasma membranes merge to form a fusion pore in response to stimulation. The nonselective cation HCN channels are involved in the regulation of unitary exocytotic events by at least two mechanisms. They can affect SNARE-dependent exocytotic activity indirectly, via the modulation of free intracellular calcium;...

Astrocytes contain glycogen, an energy buffer, which can bridge local short-term energy requirements in the brain. Glycogen levels reflect a dynamic equilibrium between glycogen synthesis and glycogenolysis. Many factors that include hormones and neuropeptides, such as insulin and insulin-like growth factor 1 (IGF-1) likely modulate glycogen stores...

Hormone and neurotransmitter release from vesicles is mediated by regulated exocytosis, where an aqueous channel-like structure, termed a fusion pore, is formed. It was recently shown that second messenger cAMP modulates the fusion pore, but the detailed mechanisms remain elusive. In this study, we asked whether the hyperpolarization-activated cycl...

Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence res...

It is becoming clear that astrocytes, the most abundant type of glial cells in the mammalian brain, share many properties with neurons. One such property involves vesicles, which play a key role in cell-to-cell signaling. On the one hand, vesicles determine the signaling potential by delivering various receptors and transporters to the plasma membr...

Background During development of recombinant monoclonal antibodies in Chinese hamster ovary (CHO) cells, C-terminal amidated species are observed. C-terminal amidation is catalysed by peptidylglycine α-amidating monooxygenase (PAM), an enzyme known to be expressed in CHO cells. The significant variations between clones during clone selection, and t...

Exocytic transmitter release is regulated by the SNARE complex, which contains a vesicular protein, synaptobrevin2 (Sb2). However, Sb2 vesicular arrangement is unclear. Here we use super-resolution fluorescence microscopy to study the prevalence and distribution of endogenous and exogenous Sb2 in single vesicles of astrocytes, the most abundant gli...

AimIn the brain, alterations in sphingolipid metabolism contribute to several neurologic 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, signaling pathways involved in alterations...

The morphology of astrocytes, likely regulated by cAMP, determines the structural association between astrocytes and the synapse, consequently modulating synaptic function. β-Adrenergic receptors (β-AR), which increase cytosolic cAMP concentration ([cAMP]i ), may affect cell morphology. However, the real-time dynamics of β-AR-mediated cAMP signalin...

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 metabol...

Astrocytes, the most abundant type of glial cells, play a number of important functions in information processing in the brain. They ensheath synapses and modulate synaptic transmission by many mechanisms which include the exocytotic release of gliotransmitters into the extracellular space. However, the anatomy and nature of exocytotic vesicle inte...

How cholesterol, a key membrane constituent, affects membrane surface area dynamics in secretory cells is unclear. Using methyl-beta-cyclodextrin (MbetaCD) to deplete cholesterol, we imaged melanotrophs from male Wistar rats in real-time and monitored membrane capacitance (C(m)), fluctuations of which reflect exocytosis and endocytosis. Treatment w...

Neuroendocrine cells contain small and large vesicles, but the functional significance of vesicle diameter is unclear. We studied unitary exocytic events of prolactin-containing vesicles in lactotrophs by monitoring discrete steps in membrane capacitance. In the presence of sphingosine, which recruits VAMP2 for SNARE complex formation, the frequenc...

Aquaporin 4 (AQP4) is the predominant water channel in the brain, expressed mainly in astrocytes and involved in water transport in physiologic and pathologic conditions. Besides the classical isoforms M1 (a) and M23 (c), additional ones may be present at the plasma membrane, such as the recently described AQP4b, d, e, and f. Water permeability reg...

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....

In order to understand exocytosis and endocytosis, it is necessary to study these processes directly. An elegant way to do this is by measuring plasma membrane capacitance (Cm), a parameter proportional to cell surface area, the fluctuations of which are due to fusion and fission of secretory and other vesicles. Here we describe protocols that enab...

Regulated exocytosis mediates the release of hormones and transmitters. The last step of this process is represented by the merger between the vesicle and the plasma membranes, and the formation of a fusion pore. Once formed, the initially stable and narrow fusion pore may reversibly widen (transient exocytosis) or fully open (full-fusion exocytosi...