Vladimir Grubišić

Ca(2+) sources for the exocytotic release of glutamate from astrocytes

Astrocytes can exocytotically release the gliotransmitter glutamate from vesicular compartments. Increased cytosolic Ca(2+) concentration is necessary and sufficient for this process. The predominant source of Ca(2+) for exocytosis in astrocytes resides within the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate and ryanodine receptors of the ER provide a conduit for the release of Ca(2+) to the cytosol. The ER store is (re)filled by the store-specific Ca(2+)-ATPase. Ultimately, the depleted ER is replenished by Ca(2+) which enters from the extracellular space to the cytosol via store-operated Ca(2+) entry; the TRPC1 protein has been implicated in this part of the astrocytic exocytotic process. Voltage-gated Ca(2+) channels and plasma membrane Na(+)/Ca(2+) exchangers are additional means for cytosolic Ca(2+) entry. Cytosolic Ca(2+) levels can be modulated by mitochondria, which can take up cytosolic Ca(2+) via the Ca(2+) uniporter and release Ca(2+) into cytosol via the mitochondrial Na(+)/Ca(2+) exchanger, as well as by the formation of the mitochondrial permeability transition pore. The interplay between various Ca(2+) sources generates cytosolic Ca(2+) dynamics that can drive Ca(2+)-dependent exocytotic release of glutamate from astrocytes. An understanding of this process in vivo will reveal some of the astrocytic functions in health and disease of the brain. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Ca2+ Responses in Enteric Glia Are Mediated by Connexin-43 Hemichannels and Modulate Colonic Transit in Mice

BACKGROUND & AIMS In the enteric nervous system, neurotransmitters initiate changes in calcium (Ca(2+) responses) in glia, but it is not clear how this process affects intestinal function. We investigated whether Ca(2+)-mediated responses in enteric glia are required to maintain gastrointestinal function. METHODS We used in situ Ca(2+) imaging to monitor glial Ca(2+) responses, which were manipulated with pharmacologic agents or via glia-specific disruption of the gene encoding connexin-43 (Cx43) (hGFAP::CreER(T2+/-)/Cx43(f/f) mice). Gastrointestinal function was assessed based on pellet output, total gut transit, colonic bead expulsion, and muscle tension recordings. Proteins were localized and quantified by immunohistochemistry, immunoblot, and reverse transcription polymerase chain reaction analyses. RESULTS Ca(2+) responses in enteric glia of mice were mediated by Cx43 hemichannels. Cx43 immunoreactivity was confined to enteric glia within the myenteric plexus of the mouse colon; the Cx43 inhibitors carbenoxolone and 43Gap26 inhibited the ability of enteric glia to propagate Ca(2+) responses. In vivo attenuation of Ca(2+) responses in the enteric glial network slowed gut transit overall and delayed colonic transit--these changes are also observed during normal aging. Altered motility with increasing age was associated with reduced glial Ca(2+)-mediated responses and changes in glial expression of Cx43 messenger RNA and protein. CONCLUSIONS Ca(2+)-mediated responses in enteric glia regulate gastrointestinal function in mice. Altered intercellular signaling between enteric glia and neurons might contribute to motility disorders.

Single-vesicle architecture of synaptobrevin2 in astrocytes

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 glial cells in the brain. We tag Sb2 protein at C- and N termini with a pair of fluorophores, which allows us to determine the Sb2 length and geometry. To estimate total number of Sb2 proteins per vesicle and the quantity necessary for the formation of fusion pores, we treat cells with ATP to stimulate Ca2+-dependent exocytosis, increase intracellular alkalinity to enhance the fluorescence presentation of yellow-shifted pHluorin (YpH), appended to the vesicle lumen domain of Sb2, and perform photobleaching of YpH fluorophores. Fluorescence intensity analysis reveals that the total number of endogenous Sb2 units or molecules per vesicle is ≤25.

Enteric glia: the most alimentary of all glia.

Glia (from Greek γλοία meaning ‘glue’) pertains to non‐neuronal cells in the central (CNS) and peripheral nervous system (PNS) that nourish neurons and maintain homeostasis. In addition, glia are now increasingly appreciated as active regulators of numerous physiological processes initially considered exclusively under neuronal regulation. For instance, enteric glia, a collection of glial cells residing within the walls of the intestinal tract, regulate intestinal motility, a well‐characterized reflex controlled by enteric neurons. Enteric glia also interact with various non‐neuronal cell types in the gut wall such as enterocytes, enteroendocrine and immune cells and are therefore emerging as important local regulators of diverse gut functions. The intricate molecular mechanisms that govern glia‐mediated regulation are beginning to be discovered, but much remains unknown about the functions of enteric glia in health and disease. Here we present a current view of the enteric glia and their regulatory roles in gastrointestinal (GI) (patho)physiology; from GI motility and epithelial barrier function to enteric neuroinflammation.

Nanopore sensing of botulinum toxin type B by discriminating an enzymatically cleaved Peptide from a synaptic protein synaptobrevin 2 derivative.

Botulinum neurotoxins (BoNTs) are the most lethal toxin known to human. Biodefense requires early and rapid detection of BoNTs. Traditionally, BoNTs can be detected by looking for signs of botulism in mice that receive an injection of human material, serum or stool. While the living animal assay remains the most sensitive approach, it is costly, slow and associated with legal and ethical constrains. Various biochemical, optical and mechanical methods have been developed for BoNTs detection with improved speed, but with lesser sensitivity. Here, we report a novel nanopore-based BoNT type B (BoNT-B) sensor that monitors the toxin’s enzymatic activity on its substrate, a recombinant synaptic protein synaptobrevin 2 derivative. By analyzing the modulation of the pore current caused by the specific BoNT-B-digested peptide as a marker, the presence of BoNT-B at a subnanomolar concentration was identified within minutes. The nanopore detector would fill the niche for a much needed rapid and highly sensitive detection of neurotoxins, and provide an excellent system to explore biophysical mechanisms for biopolymer transportation.

Pitt-Hopkins Mouse Model has Altered Particular Gastrointestinal Transits In Vivo.

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 similar between the groups, indicating normal gross growth of PTHS mice and their overall physiological GI motility and intestinal secretion/absorption. There were no significant differences in gut length and gross appearance pointing out that PTHS mice have normal gut in gross anatomical terms. However, the assessment of gut transit indicates that, while whole‐gut transit velocity was similar between the groups, the upper GI and distal colon transit velocities were significantly reduced in the PTHS mice. This is the first evidence of specific gut related problems in the PTHS mice. Our study also validates the TCF4 functional knockout mice as an animal model to study PTHS‐associated GI disturbances. Autism Res 2015, 8: 629–633.

Enteric glial activity regulates secretomotor function in the mouse colon but does not acutely affect gut permeability

The role of enteric glial cell activity in the acute regulation of epithelial barrier and secretomotor functions of the intestines under physiological conditions is not clear. We used transgenic mice to modify glial activity and found that enteric glia significantly contribute to the neurogenic ion transport while glial activity does not appear to play a major role in the acute regulation of barrier function. The selective activation of glial activity evoked electrogenic ion transport primarily through neural pathways and was sufficient to drive electrogenic ion transport to an extent equal to the direct activation of neurogenic ion transport. These findings provide novel insight into the cellular mechanisms that control fluid transport homeostasis in the intestine and might provide novel therapeutic avenues for functional diarrheal diseases.

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

Abstract 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.

Enteric glia regulate gut motility in health and disease

The enteric nervous system, often referred to as the second brain, is the largest assembly of neurons and glia outside the central nervous system. The enteric nervous system resides within the wall of the digestive tract and regulates local gut reflexes involved in gastrointestinal motility and fluid transport; these functions can be accomplished in the absence of the extrinsic innervation from the central nervous system. It is neurons and their circuitry within the enteric nervous system that govern the gut reflexes. However, it is becoming clear that enteric glial cells are also actively involved in this process through the bidirectional signaling with neurons and other cells in the gut wall. We synthesize the recently discovered modulatory roles of enteric gliotransmission in gut motility and provide our perspective for future lines of research.

The second brain in autism spectrum disorder: could connexin 43 expressed in enteric glial cells play a role?

Autism spectrum disorder (ASD) is an umbrella term for a heterogeneous group of developmental disorders that present with persistent deficits in social communication/interaction and repetitive/restricted patterns of behavior, interests, or activities that cannot be better explained by intellectual disability or global developmental delay (DSM-5 APA, 2013). The recent estimate for ASD in 8-year old children in the United States is one in 68 (Baio and Autism and Developmental Disabilities Monitoring Network Surveillance Year 2010 Principal Investigators, 2014). Although a genetic cause can be identified in 20–25% cases (reviewed in Miles, 2011), the etiology of ASD still remains largely unclear. Several possible factors have been investigated to explain the etiology of ASD, i.e., immune dysregulation and inflammation, oxidative stress, mitochondrial dysfunction, and environmental factors (Rossignol and Frye, 2012).