Association of cystic fibrosis transmembrane conductance regulator with epithelial sodium channel subunits carrying Liddle's syndrome mutations.

The association of the cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) in the pathophysiology of cystic fibrosis (CF) is controversial. Previously, we demonstrated a close physical association between wild-type (WT) CFTR and WT ENaC. We have also shown that the F508del CFTR fails to associate with ENaC unless the mutant protein is rescued pharmacologically or by low temperature. In this study, we present the evidence for a direct physical association between WT CFTR and ENaC subunits carrying Liddle's syndrome mutations. We show that all three ENaC subunits bearing Liddle's syndrome mutations (both point mutations and the complete truncation of the carboxy terminus), could be coimmunoprecipitated with WT CFTR. The biochemical studies were complemented by fluorescence lifetime imaging microscopy (FLIM), a distance-dependent approach that monitors protein-protein interactions between fluorescently labeled molecules. Our measurements revealed significantly increased fluorescence resonance energy transfer between CFTR and all tested ENaC combinations as compared with controls (ECFP and EYFP cotransfected cells). Our findings are consistent with the notion that CFTR and ENaC are within reach of each other even in the setting of Liddle's syndrome mutations, suggestive of a direct intermolecular interaction between these two proteins.

Astroglial Regulation of Magnocellular Neuroendocrine Cell Activities in the Supraoptic Nucleus.

Studies on the interactions between astrocytes and neurons in the hypothalamo-neurohypophysial system have significantly facilitated our understanding of the regulation of neural activities. This has been exemplified in the interactions between astrocytes and magnocellular neuroendocrine cells (MNCs) in the supraoptic nucleus (SON), specifically during osmotic stimulation and lactation. In response to changes in neurochemical environment in the SON, astrocytic morphology and functions change significantly, which further modulates MNC activity and the secretion of vasopressin and oxytocin. In osmotic regulation, short-term dehydration or water overload causes transient retraction or expansion of astrocytic processes, which increases or decreases the activity of SON neurons, respectively. Prolonged osmotic stimulation causes adaptive change in astrocytic plasticity in the SON, which allows osmosensory neurons to reserve osmosensitivity at new levels. During lactation, changes in neurochemical environment cause retraction of astrocytic processes around oxytocin neurons, which increases MNC's ability to secrete oxytocin. During suckling by a baby/pup, astrocytic processes in the mother/dams exhibit alternative retraction and expansion around oxytocin neurons, which mirrors intermittently synchronized activation of oxytocin neurons and the post-excitation inhibition, respectively. The morphological and functional plasticities of astrocytes depend on a series of cellular events involving glial fibrillary acidic protein, aquaporin 4, volume regulated anion channels, transporters and other astrocytic functional molecules. This review further explores mechanisms underlying astroglial regulation of the neuroendocrine neuronal activities in acute processes based on the knowledge from studies on the SON.

Two Metabolic Fuels, Glucose and Lactate, Differentially Modulate Exocytotic Glutamate Release from Cultured Astrocytes.

Astrocytes have a prominent role in metabolic homeostasis of the brain and can signal to adjacent neurons by releasing glutamate via a process of regulated exocytosis. Astrocytes synthesize glutamate de novo owing to the pyruvate entry to the citric/tricarboxylic acid cycle via pyruvate carboxylase, an astrocyte specific enzyme. Pyruvate can be sourced from two metabolic fuels, glucose and lactate. Thus, we investigated the role of these energy/carbon sources in exocytotic glutamate release from astrocytes. Purified astrocyte cultures were acutely incubated (1 h) in glucose and/or lactate-containing media. Astrocytes were mechanically stimulated, a procedure known to increase intracellular Ca2+ levels and cause exocytotic glutamate release, the dynamics of which were monitored using single cell fluorescence microscopy. Our data indicate that glucose, either taken-up from the extracellular space or mobilized from the intracellular glycogen storage, sustained glutamate release, while the availability of lactate significantly reduced the release of glutamate from astrocytes. Based on further pharmacological manipulation during imaging along with tandem mass spectrometry (proteomics) analysis, lactate alone, but not in the hybrid fuel, caused metabolic changes consistent with an increased synthesis of fatty acids. Proteomics analysis further unveiled complex changes in protein profiles, which were condition-dependent and generally included changes in levels of cytoskeletal proteins, proteins of secretory organelle/vesicle traffic and recycling at the plasma membrane in aglycemic, lactate or hybrid-fueled astrocytes. These findings support the notion that the availability of energy sources and metabolic milieu play a significant role in gliotransmission.

TESOT: a teaching modality targeting the learning obstacles in global medical education.

In higher education, it is a great challenge for instructors to teach international medical students (IMSs) efficiently. These students usually have different learning obstacles and learning style preferences from domestic students. Thus it is necessary to use teaching modalities targeting the specific characteristics of IMSs. Accordingly, we have developed a teaching modality composed of classical teacher-centered approach (TCA), enriched with components of student-centered approach (SCA) and online interactions targeting the learning characteristics of IMSs, which we defined as TESOT (an acronym made of the underlined words' initials). Aside from the online interactions that provide both answers to questions raised by students and guidance throughout a course, this modality contains additional in-classroom components (i.e., pre-lecture quiz, student-led summary, and post-lecture quiz). The effectiveness of this modality was tested in the nervous system module of the Physiology course for IMSs. The final exam scores in the nervous system module in the year taught with TESOT were higher than those earned by students taught with a classical TCA modality in preceding 2 yr. The improvement of teaching effectiveness is attributable to increasing communication, bridging course contexts, and meeting diverse learning style preferences. These results indicate that TESOT as an effective teaching modality is useful for enhancing efficiency of teaching IMSs.

Astrocytes as secretory cells of the central nervous system: idiosyncrasies of vesicular secretion.

Astrocytes are housekeepers of the central nervous system (CNS) and are important for CNS development, homeostasis and defence. They communicate with neurones and other glial cells through the release of signalling molecules. Astrocytes secrete a wide array of classic neurotransmitters, neuromodulators and hormones, as well as metabolic, trophic and plastic factors, all of which contribute to the gliocrine system. The release of neuroactive substances from astrocytes occurs through several distinct pathways that include diffusion through plasmalemmal channels, translocation by multiple transporters and regulated exocytosis. As in other eukaryotic cells, exocytotic secretion from astrocytes involves divergent secretory organelles (synaptic-like microvesicles, dense-core vesicles, lysosomes, exosomes and ectosomes), which differ in size, origin, cargo, membrane composition, dynamics and functions. In this review, we summarize the features and functions of secretory organelles in astrocytes. We focus on the biogenesis and trafficking of secretory organelles and on the regulation of the exocytotic secretory system in the context of healthy and diseased astrocytes.

Evolution of Neuroglia.

As the nervous system evolved from the diffused to centralised form, the neurones were joined by the appearance of the supportive cells, the neuroglia. Arguably, these non-neuronal cells evolve into a more diversified cell family than the neurones are. The first ancestral neuroglia appeared in flatworms being mesenchymal in origin. In the nematode C. elegans proto-astrocytes/supportive glia of ectodermal origin emerged, albeit the ensheathment of axons by glial cells occurred later in prawns. The multilayered myelin occurred by convergent evolution of oligodendrocytes and Schwann cells in vertebrates above the jawless fishes. Nutritive partitioning of the brain from the rest of the body appeared in insects when the hemolymph-brain barrier, a predecessor of the blood-brain barrier was formed. The defensive cellular mechanism required specialisation of bona fide immune cells, microglia, a process that occurred in the nervous system of leeches, bivalves, snails, insects and above. In ascending phylogeny, new type of glial cells, such as scaffolding radial glia, appeared and as the bran sizes enlarged, the glia to neurone ratio increased. Humans possess some unique glial cells not seen in other animals.

The Concept of Neuroglia.

Neuroglia represent a diverse population of non-neuronal cells in the nervous systems, be that peripheral, central, enteric or autonomic nervous system. Arguably, these cells represent about half of the volume of the human brain. This volumetric ratio, and by extension glia to neurone ratio, not only widely differ depending on the size of the animal species brain and its positioning on the phylogenetic tree, but also vary between the regions of an individual brain. Neuroglia derived from a dual origin (ectoderm and mesodermal) and in an assorted morphology, yet their functional traits can be mainly classified into being keepers of homeostasis (water, ions, neurotransmitters, metabolites, fuels, etc.) and defenders (e.g., against microbial organisms, etc.) of the nervous system. As these capabilities go awry, neuroglia ultimately define their fundamental role in most, if not, all neuropathologies. This concept presented in this chapter serves as a general introduction into the world of neuroglia and subsequent topics covered by this book.

Gliocrine System: Astroglia as Secretory Cells of the CNS.

Astrocytes are secretory cells, actively participating in cell-to-cell communication in the central nervous system (CNS). They sense signaling molecules in the extracellular space, around the nearby synapses and also those released at much farther locations in the CNS, by their cell surface receptors, get excited to then release their own signaling molecules. This contributes to the brain information processing, based on diffusion within the extracellular space around the synapses and on convection when locales relatively far away from the release sites are involved. These functions resemble secretion from endocrine cells, therefore astrocytes were termed to be a part of the gliocrine system in 2015. An important mechanism, by which astrocytes release signaling molecules is the merger of the vesicle membrane with the plasmalemma, i.e., exocytosis. Signaling molecules stored in astroglial secretory vesicles can be discharged into the extracellular space after the vesicle membrane fuses with the plasma membrane. This leads to a fusion pore formation, a channel that must widen to allow the exit of the Vesiclal cargo. Upon complete vesicle membrane fusion, this process also integrates other proteins, such as receptors, transporters and channels into the plasma membrane, determining astroglial surface signaling landscape. Vesiclal cargo, together with the whole vesicle can also exit astrocytes by the fusion of multivesicular bodies with the plasma membrane (exosomes) or by budding of vesicles (ectosomes) from the plasma membrane into the extracellular space. These astroglia-derived extracellular vesicles can later interact with various target cells. Here, the characteristics of four types of astroglial secretory vesicles: synaptic-like microvesicles, dense-core vesicles, secretory lysosomes, and extracellular vesicles, are discussed. Then machinery for vesicle-based exocytosis, second messenger regulation and the kinetics of exocytotic vesicle content discharge or release of extracellular vesicles are considered. In comparison to rapidly responsive, electrically excitable neurons, the receptor-mediated cytosolic excitability-mediated astroglial exocytotic vesicle-based transmitter release is a relatively slow process.

Physiology of Astroglia.

Astrocytes are principal cells responsible for maintaining the brain homeostasis. Additionally, these glial cells are also involved in homocellular (astrocyte-astrocyte) and heterocellular (astrocyte-other cell types) signalling and metabolism. These astroglial functions require an expression of the assortment of molecules, be that transporters or pumps, to maintain ion concentration gradients across the plasmalemma and the membrane of the endoplasmic reticulum. Astrocytes sense and balance their neurochemical environment via variety of transmitter receptors and transporters. As they are electrically non-excitable, astrocytes display intracellular calcium and sodium fluctuations, which are not only used for operative signalling but can also affect metabolism. In this chapter we discuss the molecules that achieve ionic gradients and underlie astrocyte signalling.

General Pathophysiology of Astroglia.

Astroglial cells are involved in most if not in all pathologies of the brain. These cells can change the morpho-functional properties in response to pathology or innate changes of these cells can lead to pathologies. Overall pathological changes in astroglia are complex and diverse and often vary with different disease stages. We classify astrogliopathologies into reactive astrogliosis, astrodegeneration with astroglial atrophy and loss of function, and pathological remodelling of astrocytes. Such changes can occur in neurological, neurodevelopmental, metabolic and psychiatric disorders as well as in infection and toxic insults. Mutation in astrocyte-specific genes leads to specific pathologies, such as Alexander disease, which is a leukodystrophy. We discuss changes in astroglia in the pathological context and identify some molecular entities underlying pathology. These entities within astroglia may repent targets for novel therapeutic intervention in the management of brain pathologies.

Neuroglia in Ageing.

Ageing reduces the functional capacity of all organs, so does that of the nervous system; the latter is evident in the reduction of cognitive abilities, learning and memory. While the exact mechanisms of ageing of the nervous system remain elusive, it is without doubt that morpho-functional changes in a variety of neuroglial cells contribute to this process. The age-dependent changes in neuroglia are characterised by a progressive loss of function. This reduces glial ability to homeostatically nurture, protect and regenerate the nervous tissue. Such neuroglial paralysis also facilitates neurodegenerative processes. Ageing of neuroglia is variable and can be affected by environmental factors and comorbidities.

Astroglia in Alzheimer's Disease.

Alzheimer's disease is the most common cause of dementia. Cellular changes in the brains of the patients suffering from Alzheimer's disease occur well in advance of the clinical symptoms. At the cellular level, the most dramatic is a demise of neurones. As astroglial cells carry out homeostatic functions of the brain, it is certain that these cells are at least in part a cause of Alzheimer's disease. Historically, Alois Alzheimer himself has recognised this at the dawn of the disease description. However, the role of astroglia in this disease has been understudied. In this chapter, we summarise the various aspects of glial contribution to this disease and outline the potential of using these cells in prevention (exercise and environmental enrichment) and intervention of this devastating disease.

Homer1 Scaffold Proteins Govern Ca2+ Dynamics in Normal and Reactive Astrocytes.

In astrocytes, the intracellular calcium (Ca2+) signaling mediated by activation of metabotropic glutamate receptor 5 (mGlu5) is crucially involved in the modulation of many aspects of brain physiology, including gliotransmission. Here, we find that the mGlu5-mediated Ca2+ signaling leading to release of glutamate is governed by mGlu5 interaction with Homer1 scaffolding proteins. We show that the long splice variants Homer1b/c are expressed in astrocytic processes, where they cluster with mGlu5 at sites displaying intense local Ca2+ activity. We show that the structural and functional significance of the Homer1b/c-mGlu5 interaction is to relocate endoplasmic reticulum (ER) to the proximity of the plasma membrane and to optimize Ca2+ signaling and glutamate release. We also show that in reactive astrocytes the short dominant-negative splice variant Homer1a is upregulated. Homer1a, by precluding the mGlu5-ER interaction decreases the intensity of Ca2+ signaling thus limiting the intensity and the duration of glutamate release by astrocytes. Hindering upregulation of Homer1a with a local injection of short interfering RNA in vivo restores mGlu5-mediated Ca2+ signaling and glutamate release and sensitizes astrocytes to apoptosis. We propose that Homer1a may represent one of the cellular mechanisms by which inflammatory astrocytic reactions are beneficial for limiting brain injury.

Two modes of enteric gliotransmission differentially affect gut physiology.

Enteric glia (EG) in the enteric nervous system can modulate neuronally regulated gut functions. Using molecular genetics, we assessed the effects that molecular entities expressed in EG and otherwise mediating two distinct mechanisms of gliotransmitter release, connexin 43 (Cx43) hemichannel vs. Ca2+ -dependent exocytosis, have on gut function. The expression of mutated Cx43G138R (which favors hemichannel, as opposed to gap-junctional activity) in EG increased gut motility in vivo, while a knock-down of Cx43 in EG resulted in the reduction of gut motility. However, inhibition of Ca2+ -dependent exocytosis in EG did not affect gut motility in vivo; rather, it increased the fecal pellet fluid content. Hampering either Cx43 expression or Ca2+ -dependent exocytosis in EG had an effect on colonic migrating motor complexes, mainly decreasing frequency and velocity of contractions ex vivo. Thus, EG can differentially modulate gut reflexes using the above two distinct mechanisms of gliotransmission.

Astroglial calcium signalling in Alzheimer's disease.

Neuroglial contribution to Alzheimer's disease (AD) is pathologically relevant and highly heterogeneous. Reactive astrogliosis and activation of microglia contribute to neuroinflammation, whereas astroglial and oligodendroglial atrophy affect synaptic transmission and underlie the overall disruption of the central nervous system (CNS) connectome. Astroglial function is tightly integrated with the intracellular ionic signalling mediated by complex dynamics of cytosolic concentrations of free Ca2+ and Na+. Astroglial ionic signalling is mediated by plasmalemmal ion channels, mainly associated with ionotropic receptors, pumps and solute carrier transporters, and by intracellular organelles comprised of the endoplasmic reticulum and mitochondria. The relative contribution of these molecular cascades/organelles can be plastically remodelled in development and under environmental stress. In AD astroglial Ca2+ signalling undergoes substantial reorganisation due to an abnormal regulation of expression of Ca2+ handling molecular cascades.

Astroglial Vesicular Trafficking in Neurodegenerative Diseases.

The neocortex represents one of the largest estates of the human brain. This structure comprises ~30-40 billions of neurones and even more of non-neuronal cells. Astrocytes, highly heterogeneous homoeostatic glial cells, are fundamental for housekeeping of the brain and contribute to information processing in neuronal networks. Gray matter astrocytes tightly enwrap synapses, contact blood vessels and, naturally, are also in contact with the extracellular space, where convection of fluid takes place. Thus astrocytes receive signals from several distinct extracellular domains and can get excited by numerous mechanisms, which regulate cytosolic concentration of second messengers, such as Ca2+ and cAMP. Excited astrocytes often secrete diverse substances (generally referred to as gliosignalling molecules) that include classical neurotransmitters such as glutamate and ATP or neuromodulators such as D-serine or neuropeptides. Astrocytic secretion occurs through several mechanisms: by diffusion through membrane channels, by translocation via plasmalemmal transporters or by vesicular exocytosis. Vesicular release of gliosignalling molecules appears fundamentally similar to that operating in neurones, since it depends on the SNARE proteins-dependent merger of the vesicle membrane with the plasmalemma. However, the coupling between the stimulus and astroglial vesicular secretion is at least one order of magnitude slower than that in neurones. Here we review mechanisms of astrocytic excitability and the molecular, anatomical and physiological properties of vesicular apparatus mediating the release of gliosignalling molecules in health and in the neurodegenerative pathology.

Glutamate and ATP at the Interface Between Signaling and Metabolism in Astroglia: Examples from Pathology.

Glutamate is the main excitatory transmitter in the brain, while ATP represents the most important energy currency in any living cell. Yet, these chemicals play an important role in both processes, enabling them with dual-acting functions in metabolic and intercellular signaling pathways. Glutamate can fuel ATP production, while ATP can act as a transmitter in intercellular signaling. We discuss the interface between glutamate and ATP in signaling and metabolism of astrocytes. Not only do glutamate and ATP cross each other's paths in physiology of the brain, but they also do so in its pathology. We present the fabric of this process in (patho)physiology through the discussion of synthesis and metabolism of ATP and glutamate in astrocytes as well as by providing a general description of astroglial receptors for these molecules along with the downstream signaling pathways that may be activated. It is astroglial receptors for these dual-acting molecules that could hold a key for medical intervention in pathological conditions. We focus on two examples disclosing the role of activation of astroglial ATP and glutamate receptors in pathology of two kinds of brain tissue, gray matter and white matter, respectively. Interventions at the interface of metabolism and signaling show promise for translational medicine.

Spatio-temporal characteristics of metabotropic glutamate receptor 5 traffic at or near the plasma membrane in astrocytes.

Astrocytes can sense extracellular glutamate and respond to it by elevating their intracellular Ca(2+) levels via the activation of G-protein coupled receptors, such as metabotropic glutamate receptor 5 (mGluR5), which, during early postnatal development, is the primary receptor responsible for glutamatergic signaling in astrocytes. However, the detailed spatio-temporal characteristics of mGluR5 traffic at or near the plasma membrane of astrocytes are not well understood. To address this issue, we expressed recombinant fluorescent protein chimera of mGluR5 and used total internal reflection fluorescence microscopy on rat visual cortical astrocytes in culture. We used astrocytes lacking major processes, otherwise posing as a diffusion barrier, to infer into the general dynamics of this receptor. We found that plasmalemmal mGluR5 clusters in distinct areas, the size, and initial spatio-temporal level of occupancy of which dictated mGluR5 trafficking characteristics upon glutamate stimulation. These findings will be valuable in the interpretation of point-to-point information transfer and volume transmission between astrocytes and neurons, as well as that of paracrine signaling within astrocytic networks.

Loose excitation-secretion coupling in astrocytes.

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 astrocytes, the delay between the increase in cytosolic calcium activity and the discharge of secretions from the vesicular lumen is orders of magnitude longer than that in neurons. This relatively loose excitation-secretion coupling is likely tailored to the participation of astrocytes in modulating neural network processing.