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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),... more
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 Ca(2+) concentration ([Ca(2+) ]i ) was monitored with Fluo-2 and visualized by confocal microscopy. In comparison with controls, spontaneous mobility of ANP- and LysoTracker-labeled ...
Research Interests: Glia and Neurosciences
Fundamentally, all brain disorders can be broadly defined as the homeostatic failure of this organ. As the brain is composed of many different cells types, including but not limited to neurons and glia, it is only logical that all the... more
Fundamentally, all brain disorders can be broadly defined as the homeostatic failure of this organ. As the brain is composed of many different cells types, including but not limited to neurons and glia, it is only logical that all the cell types/constituents could play a role in health and disease. Yet, for a long time the sole conceptualization of brain pathology was focused on the well-being of neurons. Here, we challenge this neuron-centric view and present neuroglia as a key element in neuropathology, a process that has a toll on astrocytes, which undergo complex morpho-functional changes that can in turn affect the course of the disorder. Such changes can be grossly identified as reactivity, atrophy with loss of function and pathological remodeling. We outline the pathogenic potential of astrocytes in variety of disorders, ranging from neurotrauma, infection, toxic damage, stroke, epilepsy, neurodevelopmental, neurodegenerative and psychiatric disorders, Alexander disease to neoplastic changes seen in gliomas. We hope that in near future we would witness glial-based translational medicine with generation of deliverables for the containment and cure of disorders. We point out that such as a task will require a holistic and multi-disciplinary approach that will take in consideration the concerted operation of all the cell types in the brain.
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Astrocytes play an important housekeeping role in the central nervous system. Additionally, as secretory cells, they actively participate in cell-to-cell communication, which can be mediated by membrane-bound vesicles. The gliosignaling... more
Astrocytes play an important housekeeping role in the central nervous system. Additionally, as secretory cells, they actively participate in cell-to-cell communication, which can be mediated by membrane-bound vesicles. The gliosignaling molecules stored in these vesicles are discharged into the extracellular space after the vesicle membrane fuses with the plasma membrane. This process is termed exocytosis, regulated by SNARE proteins, and triggered by elevations in cytosolic calcium levels, which are necessary and sufficient for exocytosis in astrocytes. For astrocytic exocytosis, calcium is sourced from the intracellular endoplasmic reticulum store, although its entry from the extracellular space contributes to cytosolic calcium dynamics in astrocytes. Here, we discuss calcium management in astrocytic exocytosis and the properties of the membrane-bound vesicles that store gliosignaling molecules, including the vesicle fusion machinery and kinetics of vesicle content discharge. In a...
Research Interests: Glia and Neurosciences
We previously used the expression of various combinations and configurations of MyoD and E12, two basic helix-loop-helix transcription factors (TF), to produce populations of myotubes assuming distinct morphology, myofibrillar development... more
We previously used the expression of various combinations and configurations of MyoD and E12, two basic helix-loop-helix transcription factors (TF), to produce populations of myotubes assuming distinct morphology, myofibrillar development and Ca2+ dynamics, from mammalian C2C12 myoblasts in non-differentiation growth conditions. Here, we assessed the synthetically generated myotubes in terms of energetics, otherwise necessary to sustain their mechanical output as bio-actuators. We found that the myotubes exhibit changed expression of key regulators for the uptake and utilization of two major cellular fuels, glucose and lactate. Furthermore, while lactate transport was uniformly slowed in all the populations of myotubes, glucose uptake and utilization were modified by particular TF configuration. Our approach allows the production of a class of biomaterials with predetermined energetics that could be applied in biorobotics, where fuel of choice could be used, and also in reparative m...
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Astrocytes provide a principal pathway for glutamate uptake in the mammalian brain, a task accomplished by the powerful action of excitatory amino acid transporters (EAAT) 1 and 2. These transporters are synthesized within the endoplasmic... more
Astrocytes provide a principal pathway for glutamate uptake in the mammalian brain, a task accomplished by the powerful action of excitatory amino acid transporters (EAAT) 1 and 2. These transporters are synthesized within the endoplasmic reticulum and are then trafficked to the plasma membrane. The characteristics of their intracellular traffic within astrocytes have not been investigated. We monitored the trafficking of secretory vesicles laden with the recombinant fluorescent protein chimera of EAAT2 in cultured astrocytes. Such vesicles appeared as fluorescent puncta, and their trafficking parameters were obtained using original algorithms, which we describe here in detail. We determined the maximal displacement, average instantaneous speed, and trajectory angle of individual puncta/vesicles, with angles near 0° indicating radial movement directly away from or toward the nucleus and angles near 90° indicating tangential movement. Analysis of these trafficking parameters demonstr...
We use an Atomic Force Microscope based single molecule measurements to evaluate the activation free energy in the interaction of SNARE proteins syntaxin 1A, SNAP25B and synaptobrevin 2 which regulate intracellular fusion of vesicles with... more
We use an Atomic Force Microscope based single molecule measurements to evaluate the activation free energy in the interaction of SNARE proteins syntaxin 1A, SNAP25B and synaptobrevin 2 which regulate intracellular fusion of vesicles with target membranes. The dissociation rate of the binary syntaxin-synaptobrevin and the ternary syntaxin-SNAP25B-synaptobrevin complex was measured from the rupture force distribution as a function of the rate of applied force. The temperature dependence of the spontaneous dissociation rate was used to obtain the activation energy to the transition state of 19.8 +/- 3.5 kcal/mol = 33 +/- 6 k(B)T and 25.7 +/- 3.0 kcal/mol = 43 +/- 5 k(B)T for the binary and ternary complex, respectively. They are consistent with those measured previously for the ternary complex in lipid membranes and are of order expected for bilayer fusion and pore formation. The DeltaG was 12.4-16.6 kcal/mol = 21-28 k(B)T and 13.8-18.0 kcal/mol = 23-30 k(B)T for the binary and ternar...
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Carbon nanotubes, owing to their electrical, chemical, mechanical, and thermal properties, are one of the most promising nanomaterials for the electronics, computer, and aerospace industries. More recently, these unique materials are... more
Carbon nanotubes, owing to their electrical, chemical, mechanical, and thermal properties, are one of the most promising nanomaterials for the electronics, computer, and aerospace industries. More recently, these unique materials are finding their niche in neuroscience. Here, we discuss the use of carbon nanotubes as scaffolds for neuronal growth. The chemical properties of carbon nanotubes can be systematically varied by attaching different functional groups. Such functionalized carbon nanotubes can be used to control the outgrowth and branching pattern of neuronal processes. We also discuss electrical interactions between neurons and carbon nanotubes. The electrical properties of nanotubes can provide a mechanism to monitor or stimulate neurons through the scaffold itself. The ease of which carbon nanotubes can be patterned makes them attractive for studying the organization of neural networks and has the potential to develop new devices for neural prosthesis. We note that additio...
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Pitt-Hopkins syndrome (PTHS) is a neurodevelopmental disorder, classified as an autism spectrum disorder that is caused by the haploinsufficiency of Transcription Factor 4 (TCF4). The most common non-neurological symptoms in PTHS patients... more
Pitt-Hopkins syndrome (PTHS) is a neurodevelopmental disorder, classified as an autism spectrum disorder that is caused by the haploinsufficiency of Transcription Factor 4 (TCF4). The most common non-neurological symptoms in PTHS patients are gastrointestinal (GI) disturbances, mainly gastroesophageal reflux and severe constipation (in about 30 and 75% of PTHS patients, respectively). We hypothesized that the recently recognized mouse model of PTHS will exhibit problems with their gut function. We conducted series of in vivo tests on 15- to 19- week old male mice, heterozygous for the TCF4 functional deletion, mimicking the TCF4 haploinsufficiency in PTHS patients, and their wild type littermates. Data collection and initial analysis were performed blindly, that is, the genotyping key was received after the mean values were calculated for each individual animal, and then mean/median of each group was subsequently calculated. Body weight, fecal pellet output, and fluid content were s...
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Neurotransmitters released at synapses activate neighboring astrocytes, which in turn, modulate neuronal activity by the release of diverse neuroactive substances that include classical neurotransmitters such as glutamate, GABA or ATP.... more
Neurotransmitters released at synapses activate neighboring astrocytes, which in turn, modulate neuronal activity by the release of diverse neuroactive substances that include classical neurotransmitters such as glutamate, GABA or ATP. Neuroactive substances are released from astrocytes through several distinct molecular mechanisms, for example, by diffusion through membrane channels, by translocation by plasmalemmal transporters or by vesicular exocytosis. Vesicular release regulated by a stimulus-mediated increase in cytosolic calcium involves SNARE-dependent merger of vesicle membrane with the plasmalemma. Up to 25 molecules of synaptobrevin 2, a SNARE complex protein, appear clustered at a single astroglial vesicle. These proteins determine the properties of the fusion pore, an aqueous channel that forms a pathway for the discharge of the vesicular content into the extracellular space. Several types of vesicles storing amino acids, peptides and ATP are present in astrocytes. In the present essay we shall overview multiple aspects of vesicular architecture and their dynamics in astroglial cells.
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A clear consensus concerning the mechanisms of intracellular secretory vesicle trafficking in astrocytes is lacking in the physiological literature. A good characterization of vesicle trafficking that may assist researchers in achieving... more
A clear consensus concerning the mechanisms of intracellular secretory vesicle trafficking in astrocytes is lacking in the physiological literature. A good characterization of vesicle trafficking that may assist researchers in achieving that goal is the trajectory angle, defined as the angle between the trajectory of a vesicle and a line radial to the cell's nucleus. In this study, we provide a precise definition of the trajectory angle, describe and compare two methods for its calculation in terms of measureable trafficking parameters, and give recommendations for the appropriate use of each method. We investigated the trafficking of vesicles containing excitatory amino acid transporter 2 (EAAT2) fluorescently tagged with enhanced green fluorescent protein (EGFP) to quantify and validate the precision of each method. The motion of fluorescent puncta--taken to represent vesicles containing EAAT2-EGFP--was found to be typical of secretory vesicle trafficking. An exact method for calculating the trajectory angle of these puncta produced no error but required large computation time. An approximate method reduced the requisite computation time but produced an error depending on the inverse of the ratio of the punctum's initial distance from the nucleus centroid to its maximal displacement. Fitting this dependence to a power function allowed us to establish an exclusion distance from the centroid, beyond which the approximate method is less likely to produce an error above an acceptable 5%. We recommend that the exact method be used to calculate the trajectory angle for puncta closer to the nucleus centroid than this exclusion distance.
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Research Interests: EM, Signal Integrity, Neurochemistry, Soc, Signal Transduction, and 18 moreGlutamate, Neuropeptides, Humans, Animals, Astrocyte, Glial Cell, Central Nervous System, Astrocytes, NMDA, Amino Acids, Exocytosis, Amino Acid Profile, Neurosciences, Plasma Membrane, TIRF, Nucleotides, Adenosine Triphosphate, and Cell Membrane
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ABSTRACT The atomic force microscope (AFM) is capable of imaging surfaces at very high resolution. The AFM has been used to image living glial cells in culture. Typical images reveal the three‐dimensional shape of the cell and often... more
ABSTRACT The atomic force microscope (AFM) is capable of imaging surfaces at very high resolution. The AFM has been used to image living glial cells in culture. Typical images reveal the three‐dimensional shape of the cell and often internal cellular structures are visible. In this report, it is shown that by increasing the imaging force, cells can be removed from the surface on which they are grown. Although the forces involved in this process are complex, it is possible to compare relative adhesion of different types of living cells to a particular substrate.
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J. Neurochem. (2012) 121, 4–27.J. Neurochem. (2012) 121, 4–27.AbstractNeuroglial cells define brain homeostasis and mount defense against pathological insults. Astroglia regulate neurogenesis and development of brain circuits. In the... more
J. Neurochem. (2012) 121, 4–27.J. Neurochem. (2012) 121, 4–27.AbstractNeuroglial cells define brain homeostasis and mount defense against pathological insults. Astroglia regulate neurogenesis and development of brain circuits. In the adult brain, astrocytes enter into intimate dynamic relationship with neurons, especially at synaptic sites where they functionally form the tripartite synapse. At these sites, astrocytes regulate ion and neurotransmitter homeostasis, metabolically support neurons and monitor synaptic activity; one of the readouts of the latter manifests in astrocytic intracellular Ca2+ signals. This form of astrocytic excitability can lead to release of chemical transmitters via Ca2+-dependent exocytosis. Once in the extracellular space, gliotransmitters can modulate synaptic plasticity and cause changes in behavior. Besides these physiological tasks, astrocytes are fundamental for progression and outcome of neurological diseases. In Alzheimer’s disease, for example, astrocytes may contribute to the etiology of this disorder. Highly lethal glial-derived tumors use signaling trickery to coerce normal brain cells to assist tumor invasiveness. This review not only sheds new light on the brain operation in health and disease, but also points to many unknowns.Neuroglial cells define brain homeostasis and mount defense against pathological insults. Astroglia regulate neurogenesis and development of brain circuits. In the adult brain, astrocytes enter into intimate dynamic relationship with neurons, especially at synaptic sites where they functionally form the tripartite synapse. At these sites, astrocytes regulate ion and neurotransmitter homeostasis, metabolically support neurons and monitor synaptic activity; one of the readouts of the latter manifests in astrocytic intracellular Ca2+ signals. This form of astrocytic excitability can lead to release of chemical transmitters via Ca2+-dependent exocytosis. Once in the extracellular space, gliotransmitters can modulate synaptic plasticity and cause changes in behavior. Besides these physiological tasks, astrocytes are fundamental for progression and outcome of neurological diseases. In Alzheimer’s disease, for example, astrocytes may contribute to the etiology of this disorder. Highly lethal glial-derived tumors use signaling trickery to coerce normal brain cells to assist tumor invasiveness. This review not only sheds new light on the brain operation in health and disease, but also points to many unknowns.
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Research Interests: Calcium, Animals, Astrocytes, Rats, Exocytosis, and 5 moreWistar Rats, Glia, Neurosciences, Glutamic Acid, and Sphingosine
Astrocytes can release a variety of transmitters, including glutamate and ATP, in response to stimuli that induce increases in intracellular Ca(2+) levels. This release occurs via a regulated, exocytotic pathway. As evidence of this,... more
Astrocytes can release a variety of transmitters, including glutamate and ATP, in response to stimuli that induce increases in intracellular Ca(2+) levels. This release occurs via a regulated, exocytotic pathway. As evidence of this, astrocytes express protein components of the vesicular secretory apparatus, including synaptobrevin 2, syntaxin, and SNAP-23. Additionally, astrocytes possess vesicular organelles, the essential morphological elements required for regulated Ca(2+)-dependent transmitter release. The location of specific exocytotic sites on these cells, however, remains to be unequivocally determined.