Joachim Kirsch

Proinflammatory cytokines increase glial fibrillary acidic protein expression in enteric glia

Background: Enteric glia protect the integrity of the gut, as loss of enteric glial fibrillary acidic protein (GFAP) positive (+) glia leads to a haemorrhagic jejunoileitis. Crohn’s disease (CD) and necrotising enterocolitis (NEC) show pathological changes in enteric glia. Therefore, factors controlling GFAP+ enteric glia are of great interest. The aim of the present study was to characterise enteric glia and determine the effect of interleukin 1β (IL-1β), interleukin 4 (IL-4), tumour necrosis factor α (TNF-α), and lipopolysaccharides (LPS) on cultured enteric glia. Methods: Dissected rat colon and cultured enteric glia cells were double labelled with anti-GFAP and anti-S-100 antibodies. For regulatory studies, enteric glia cells were treated with cytokines and LPS. Proliferation was assayed using bromodeoxyuridine (BrdU) and mitosis of enteric glia was blocked by demecolcine. Results: We were able to distinguish GFAP negative (−) from GFAP+ glia subtypes in situ and in primary cultures. Incubation of cells with IL-1β, TNF-α, and LPS led to a significant increase in GFAP+ enteric glia while IL-4 had no effect on GFAP expression. After incubation with IL-1β, total intracellular GFAP of enteric glia cells was increased. Upregulation of GFAP+ enteric glia could also be observed after stimulation with IL-1β on blocking mitosis. BrdU uptake in stimulated enteric glia showed no increased proliferation rate. Conclusions: Two different types of enteric glia based on GFAP expression exist in the gut. Proinflammatory cytokines and LPS cause a dramatic increase in GFAP+ enteric glia. This suggests that cytokines play an important role in controlling GFAP+ enteric glia which might in turn be involved in modulating the integrity of the bowel during inflammation.

Proinflammatory cytokines induce neurotrophic factor expression in enteric glia: a key to the regulation of epithelial apoptosis in Crohn's disease.

OBJECTIVESnImbalanced apoptosis of enterocytes is likely to be 1 of the mechanisms underlying Crohns disease (CD). Apoptosis of enterocytes is regulated by glial-derived neurotrophic factor (GDNF), which is increased in CD. The cellular source of GDNF during gut inflammation is unclear. The aim of the study was to identify the source of GDNF in CD during gut inflammation.nnnMATERIALS AND METHODSnGlial fibrillary acidic protein (GFAP), GDNF, and smooth muscle actin (SMA) was detected in the gut from patients with CD by immunohistochemistry. Cultured enteric glia cells (EGC) were labeled with anti-GFAP, anti-GDNF, and antibodies and a Golgi marker (anti-58K antibodies) after blocking Golgi export with monensin. Cultured EGCs were treated with interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, and lipopolysaccharides. Secretion of neurotrophic factors was detected by enzyme-linked immunosorbent assay.nnnRESULTSnMucosal GFAP-positive EGCs are increased in the colon of patients with CD. This type of glia but not subepithelial myofibroblasts expresses significant amounts of GDNF. In vitro GDNF is continuously secreted from cultured EGCs. The neurotrophic factor secretion could be stimulated by IL-1beta, tumor necrosis factor-alpha, and lipopolysaccharides in a time- and dose-dependent manner. The increased GDNF secretion by EGCs sustained for>12 hours after withdrawal of the proinflammatory cytokines.nnnCONCLUSIONSnA mucosal GFAP expressing EGC population is dramatically increased in CD. This population is a major cellular source of the upregulated GDNF in the inflamed gut. Therefore, mucosal EGC may play a key role in protecting the gut epithelium and may contribute to reestablish the integrity of the injured epithelium.

Interaction of the C-terminal region of the rat serotonin transporter with MacMARCKS modulates 5-HT uptake regulation by protein kinase C.

The serotonin transporter (SERT) mediates the re-uptake of released serotonin into presynaptic nerve terminals. Its activity is regulated by different mechanisms including protein kinase C (PKC) triggered internalization. Here, we used yeast 2-hybrid screening and cotransfection into 293 cells to identify a homologue of the myristoylated alanine-rich C kinase substrate (MARCKS), MacMARCKS, as a C-terminally interacting protein of SERT. Upon cotransfection with SERT, MacMARCKS caused a reduction in the maximal rate of [(3)H]serotonin uptake and reduced its down-regulation elicited by activation of PKC. Our data are consistent with MARCKS proteins regulating the plasma membrane dynamics of neurotransmitter transporters.

Assembly of signaling machinery at the postsynaptic membrane.

The postsynaptic membrane and the subsynaptic cell compartment are specialized for inter- and intracellular signaling. Recent work has focused on the role of synaptic activity in regulating the surface distribution of neurotransmitter receptors. In addition, several components of secondary signaling pathways involved in the long-term regulation of synaptic efficacy have been identified.

Enteric nervous plasticity and development: dependence on neurotrophic factors

The enteric nervous system in the mammalian gut is histologically and to some extent functionally similar to the central nervous system. Thus, structural and functional similarities between these systems are evident. As shown for the central nervous system, differentiation of neural crest-derived precursor cells of the enteric nervous system also depends essentially on different neurotrophic factors. Moreover, recent studies have revealed that these trophic factors also play a critical role throughout life by regulating neurotransmitter and neuropeptide synthesis, and by influencing neuronal morphology and synaptic functions. Consequently, our understanding of these complex interactions of the enteric nervous system and neurotrophic factors requires synergistic efforts from neurophysiology, biochemistry, and pharmacology in order to understand the complex phenomena of enteric nervous development and plasticity in the gut. Knowledge of these mechanisms might help to develop strategies for therapy of neuronal abnormalities, which cause different gastrointestinal diseases.

PAT1a Modulates Intracellular Transport and Processing of Amyloid Precursor Protein (APP), APLP1, and APLP2

Understanding the intracellular transport of the β-amyloid precursor protein (APP) is a major key to elucidate the regulation of APP processing and thus β-amyloid peptide generation in Alzheimer disease pathogenesis. APP and its two paralogues, APLP1 and APLP2 (APLPs), are processed in a very similar manner by the same protease activities. A putative candidate involved in APP transport is protein interacting with APP tail 1 (PAT1), which was reported to interact with the APP intracellular domain. We show that PAT1a, which is 99.0% identical to PAT1, binds to APP, APLP1, and APLP2 in vivo and describe their co-localization in trans-Golgi network vesicles or endosomes in primary neurons. We further demonstrate a direct interaction of PAT1a with the basolateral sorting signal of APP/APLPs. Moreover, we provide evidence for a direct role of PAT1a in APP/APLP transport as overexpression or RNA interference-mediated knockdown of PAT1a modulates APP/APLPs levels at the cell surface. Finally, we show that PAT1a promotes APP/APLPs processing, resulting in increased secretion ofβ-amyloid peptide. Taken together, our data establish PAT1a as a functional link between APP/APLPs transport and their processing.

Raver2, a new member of the hnRNP family

Raver2 was identified as a novel member of the hnRNP family based on sequence homology within three RNA recognition motifs and its general domain organization reminiscent of the previously described raver1 protein. Like raver1, raver2 contains two putative nuclear localization signals and a potential nuclear export sequence, and also displays nucleo‐cytoplasmic shuttling in a heterokaryon assay. In glia cells and neurons, raver2 localizes to the nucleus. Moreover, the protein interacts with polypyrimidine tract binding protein (PTB) suggesting that it may participate in PTB‐mediated nuclear functions. In contrast to ubiquitously expressed raver1, raver2 exerts a distinct spatio‐temporal expression pattern during embryogenesis and is essentially restricted to brain, lung, and kidney in the adult mouse.

Gut inflammation modulated by the enteric nervous system and neurotrophic factors

The gastrointestinal tract is characterized by an extensive and elaborate intrinsic nervous system, i.e., the enteric nervous system (ENS) (1). It extends from the oesophagus to the sphincter ani internus and includes the nervous elements within the walls of the gallbladder, the cystic duct, the common bile duct and the pancreas. Because of the high number of neurons (10) and the aforementioned structural and functional characteristics rex8f ecting certain parallels with the central nervous system, the ENS is called the brain of the gut (2). Interestingly, most of the enteric neurons are not directly innervated by pre-ganglionic input from the brain or spinal cord. The ENS can be regarded as a fully independent part of the autonomous nervous system, which includes the sympathetic and parasympathetic systems. Located in close proximity to intestinal effector systems, the ENS controls motility (3, 4), exocrine, endocrine and paracrine secretion (5), and is involved in the regulation of microcirculation, absorption (6) and gut sensation (7, 8). The ENS also seems to have an effect on immune and inx8f ammatory processes of the gut (9). This modulating function during inx8f ammatory processes appears to be mediated in parts by neurotrophic factors.

Mover is a novel vertebrate‐specific presynaptic protein with differential distribution at subsets of CNS synapses

Presynaptic nerve terminals contain scaffolding proteins that orchestrate neurotransmitter release at active zones. Here we describe mover, a yet unknown non‐transmembrane protein that is targeted to presynaptic terminals when overexpressed in cultured neurons. Confocal immunomicroscopy revealed that mover colocalizes with presynaptic markers in the calyx of Held. In the hippocampus, mover localizes to mossy fibre terminals, but is absent from inhibitory nerve terminals. By contrast, mover localizes to inhibitory terminals throughout the cerebellar cortex. Our results suggest that mover may act in concert with generally expressed scaffolding proteins in distinct sets of presynaptic terminals.

Glutamate Receptor Subunit Expression in Primary Enteric Glia Cultures

Excitotoxicity, which is mediated via glutamate receptors, is also a phenomenon of the enteric nervous system. Whether enteric glial cells (EGCs), which resemble astrocytes of the central nervous system, express glutamate receptors and hence are involved in gut excitotoxicity is not yet known. To investigate glutamate receptor subunit expression in EGCs, primary EGC cultures of the myenteric plexus were analyzed by real-time PCR and Western blotting. These studies indeed showed that in EGC cultures, mRNA of the glutamate receptor subunits NR1, NR2A/B, GluR1, GluR3, and GluR5 and the protein bands of the glutamate receptor subunits NR2A/B, GluR1, GluR3, and GluR5 could be detected. Thus, in the enteric nervous system, glutamate receptor subunits are also expressed by EGCs, indicating that these cells might be involved in gut excitotoxicity.