Perlecan, CollagenXVIII, and Agrin Expression in Normo‐, Hypo‐, and Aganglionic Segments in Hirschsprung's Disease

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Expression patterns of CXCR4 in different colon tissue segments of patients with Hirschsprung's disease

Expression pattern and localization of alpha-synuclein in the human enteric nervous system

Cellular uptake of several viruses and polybasic macromolecules requires the expression of cell-surface heparan sulfate proteoglycan (HSPG) through as yet ill defined mechanisms. We unexpectedly found that among several cell-surface-binding single chain variable fragment (scFv) anti-HS antibody (alphaHS) clones, only one, AO4B08, efficiently translocated macromolecular cargo to intracellular vesicles through induction of HSPG endocytosis. Interestingly, AO4B08-induced PG internalization was strictly dependent on HS 2-O-sulfation and appeared independent of intact N-sulfation. AO4B08 and human immunodeficiency virus (HIV)-Tat, i.e. a well known cell-penetrating peptide, were shown to compete for the internalizing PG population. To obtain a more detailed characterization of this pathway, we have developed a procedure for the isolation of endocytic vesicles by conjugating AO4B08 with superparamagnetic nanoparticles. [(35)S]sulfate-labeled HSPG was found to accumulate in isolated, AO4B08-containing vesicles, providing the first biochemical evidence for intact HSPG co-internalization with its ligand. Further analysis revealed the existence of both syndecan, i.e. a transmembrane HSPG, and glycosyl-phosphatidyl-inositol-anchored glypican in purified vesicles. Importantly, internalized syndecan and glypican were found to co-localize in AO4B08-containing vesicles. Our data establish HSPGs as true internalizing receptors of macromolecular cargo and indicate that the sorting of cell-surface HSPG to endocytic vesicles is determined by a specific HS epitope that can be carried by both syndecan and glypican core protein.

Heparan sulfate proteoglycans (HSPG) are involved in the binding and uptake of apolipoprotein (apo) E-enriched remnant lipoproteins by cultured cells in vitro. To define the role of hepatic HSPG in remnant lipoprotein clearance in vivo, heparinase (30 units) was infused intravenously into mice to hydrolyze the liver HSPG and determine the effect of HSPG hydrolysis on remnant clearance by the liver. Liver HSPG were prelabeled by peritoneal injection of [35S]Na2SO4. Injection of heparinase decreased the amount of 35S-labeled liver HSPG by approximately 20-40% within 10-15 min. Heparinase infusion significantly inhibited the clearance of chylomicrons, chylomicron remnants, chylomicron remnants + apoE, rabbit beta-very low density lipoproteins (beta-VLDL), and beta-VLDL + apoE. Compared with saline injection in control mice, heparinase injection retarded the plasma clearance of the remnants by 1.5- to 2-fold and decreased liver uptake by 1.3- to 1.6-fold. Confocal fluorescence microscopy of thick slices of liver from mice injected with 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine-labeled beta-VLDL + apoE revealed markedly less intense fluorescence from hepatocytes in heparinase-treated animals compared with those in saline-treated control animals. Intravenous heparinase infusion did not inhibit the clearance of mouse low density lipoproteins (LDL), a ligand for the LDL receptor, and did not affect the clearance of alpha 2-macroglobulin, a ligand for the LDL receptor-related protein. The results suggest an important role of the liver HSPG in remnant clearance in vivo.

Segmental distribution of colonic neuropeptides in Hirschsprung's disease

The plasma clearance of intestinally derived remnant lipoproteins by the liver is a process that likely involves three steps. Our model suggests that the initial rapid clearance by the liver begins with sequestration of the remnants within the space of Disse, where apolipoprotein E secreted by hepatocytes enhances remnant binding and uptake. Heparan sulfate proteoglycans (HSPG), which are also abundant in the space of Disse, mediate this enhanced binding. Next, the remnants undergo further processing in the space of Disse by hepatic and lipoprotein lipases, which may also serve as ligands mediating remnant uptake. The final step, endocytosis by hepatocytes, appears to be mediated, at least in part, by the low density lipoprotein (LDL) receptor and by the LDL receptor-related protein (LRP). Cell-surface HSPG play a critical role in remnant uptake, not only in the important initial sequestration or capture step in the space of Disse, but also as an essential or integral component of the HSPG-LRP pathway. In addition, HSPG appear to function alone as a receptor and display unique handling properties for specific isoforms of apolipoprotein E.

: Drosophila Wingless (Wg) is the homologue of the vertebrate Wnt-1 that is implicated in breast cancer. I have investigated the role of heparan sulfate proteoglycans (HSPGs) in Wg signaling. HSPGs are cell surface macromolecules that consist of a protein core to which heparan sulfate (HS) glycosaminoglycan (GAG) chains are attached. My genetic studies in Drosophila have uncovered critical functions of HSPGs in Wg signaling. First, I have shown that in the absence Sulfateless (Sfl), an essential enzyme involved in the biosynthsis of HS GAGs, Wg signaling is defective, suggesting that HSPGs play key role(s) in Wg signaling. I further demonstrated that Division abnormally delayed (Dally), a Drosophila HSPG of Glypican-type, is involved in Wg signaling. Genetic interaction experiments are consistent with a model in which Dally acts as a co-receptor for Wg. Finally, I have found that Dally-like protein (Dlp), a 2nd member of Drosophila Glypican play a key role in the regulation of the extracellular distribution of Wg protein. These findings provide new insights into our understanding of the extracellular regulation of Wnt signaling and will help for therapeutic interventions by the development of specific drugs for the prevention and treatment of breast cancer.

Immunocytochemical characterization of supporting cells in the enteric nervous system in Hirschsprung's disease

Hirschsprung's disease is a congenital abnormality of the enteric nervous system (ENS) presenting severe constipation soon after birth due to the lack of ganglion cells in the distal gut. Surgery for Hirschsprung's disease requires an intraoperative histopathological diagnosis to assess the extent of aganglionosis. Confocal laser endomicroscopy (CLE) is a novel endoscopic technique allowing real-time, in vivo analysis of cellular details during ongoing endoscopy. In this study, we evaluated the possibility of a new application of CLE to provide real-time observations of the ENS in patients with Hirschsprung's disease. In this preclinical feasibility study, we assessed the visualization of the ENS by CLE using surgically resected intestines. The subjects were nine patients who underwent pull-through surgery for Hirschsprung's disease between September 2014 and March 2016. The colon specimens were stained with 0.1% cresyl violet and evaluated using CLE. We compared the CLE findings with those of the histopathological examination. The ENS was clearly visualized as a ladder-like structure in the ganglionic segment but was not observed in the aganglionic segment. Of the 69 samples, corresponding positive and negative results for both CLE and the histopathology were obtained in 61 (88%). In addition, CLE was able to visualize unique, wavy structures comprising thick nerve bundles characteristic of the aganglionic/transition zone in Hirschsprung's disease. As a novel tool for visualizing the human ENS, CLE has the potential to revolutionize how pediatric surgeons identify the level of ganglionosis during surgery for Hirschsprung's disease and may be a superior alternative to intraoperative histopathological diagnosis.

An immunocytochemical study was performed to assess the role of beta-1 integrins in the pathogenesis of Hirschsprung's disease. Fresh tissue samples from both aganglionic and ganglionic segments of five patients who were undergoing surgery for Hirschsprung's disease were obtained. Samples were rapidly frozen in liquid nitrogen. Sections were cut and stained using anti alpha-1, 2, 3, 4, 5, 6 and beta-1 monoclonals according to the indirect immunoperoxidase method. The evaluation did not reveal any significant change of pattern in the distribution of beta-1 integrins in the non-neural elements of both aganglionic and ganglionic segments of colon, such as the epithelium, muscularis mucosa, muscularis externa, connective tissues and blood vessels. Nerve fibres in both aganglionic and ganglionic segments strongly expressed the alpha-6 chain of very late activation antigen which led to their increase in the aganglionic segment. In addition to revealing the increase, alpha-6 monoclonals also had in situ positive control due to their presence in non-neural elements. Hence, immunostaining of the suction biopsies with anti alpha-6 monoclonals may be employed as a new and simple method in the diagnosis of Hirschsprung's disease. On the other hand, beta-1 integrins do not seem to play a role in the defective migration of ganglion cells occurring in Hirschsprung's disease.

Tau is normally a highly soluble phosphoprotein found predominantly in neurons. Six different isoforms of tau are expressed in the adult human CNS. Under pathological conditions, phosphorylated tau aggregates are a defining feature of neurodegenerative disorders called tauopathies. Recent findings have suggested a potential role of the gut-brain axis in CNS homeostasis, and therefore we set out to examine the isoform profile and phosphorylation state of tau in the enteric nervous system (ENS) under physiological conditions and in tauopathies. Surgical specimens of human colon from controls, Parkinson’s disease (PD) and progressive supranuclear palsy (PSP) patients were analyzed by Western Blot and immunohistochemistry using a panel of anti-tau antibodies. We found that adult human ENS primarily expresses two tau isoforms, localized in the cell bodies and neuronal processes. We did not observe any difference in the enteric tau isoform profile and phosphorylation state between PSP, PD and control subjects. The htau mouse model of tauopathy also expressed two main isoforms of human tau in the ENS, and there were no apparent differences in ENS tau localization or phosphorylation between wild-type and htau mice. Tau in both human and mouse ENS was found to be phosphorylated but poorly susceptible to dephosphorylation with lambda phosphatase. To investigate ENS tau phosphorylation further, primary cultures from rat enteric neurons, which express four isoforms of tau, were pharmacologically manipulated to show that ENS tau phosphorylation state can be regulated, at least in vitro. Our study is the first to characterize tau in the rodent and human ENS. As a whole, our findings provide a basis to unravel the functions of tau in the ENS and to further investigate the possibility of pathological changes in enteric neuropathies and tauopathies.

Objective To detect the expressions of neurexin and neuroligin in the human and ro-dent enteric nervous system (ENS) and intestine tissues of the patients with Hischsprung's disease (HD). Methods Longitudinal muscle with adherent myenteric plexus (LMMP) were dissected from the intestines of mice,rats,guinea pigs and humans. The myenteric plexus was identified by staining neuron markers of neurexin-1 and Hu,prepsynaptic markers of neurexin-1 and synaptophysin and postsynaptic markers of neuroligin-1 and PSD95. The expressions of the Neurexin and Neurelogin in 20 HD cases were detected by immunohistochemistry. Results Neurexin-1,neuroligin-1-4 and Hu were detected in the some cells of the myenteric plexus of both rodents and humans. The expressions of Neurexin-1 and synaptophysin,and Neuroligin-1 and PSD95 were co-localized in the same tissues. The expressions of neurexin and neuroligin were prominent in the ganglionic colonic segment of HD,they were decreased in the transitional segments,and negative in the aganglionic colonic segment. In the same HD tissue,no difference was found between neurexin and neuroligin expression. Conclusions Neurexin and Neuroligin are expressed in the ENS of both rodents and humans. Dysregulated ex-pressions of Neurexin and Neuroligin may play important roles in the pathogenesis of HD. Key words: Enteric nervous system; Synapses; Hirschsprung disease

Glycosaminoglycans: use in treatment of diabetic nephropathy.

Leukaemia inhibitory factor (LIF) is a neuropoietic cytokine, which promotes the development of enteric neurons in vitro, particularly when administered together with neurotrophin-3 (NT-3). The purpose of this study was to map the LIF immunoreactivity in the human enteric nervous system in foetuses, children, adults, and in patients with Hirschsprung's disease. Normal bowel specimens were obtained at postmortem examination of 13 foetuses, at 13-31 weeks of gestation, and at surgery in five children and two adults. Bowel resected in seven patients with Hirschsprung's disease was also investigated. Immunohistochemical analysis was performed on material fixed in formalin and embedded in paraffin. The specimens were exposed to antibodies raised against LIF. The ABC-complex method was used to visualise binding of antibodies to the corresponding antigen. LIF immunoreactivity was disclosed in the myenteric and submucous ganglion cells at 13-31 weeks of gestation, in childhood cases, and adults. LIF-immunoreactive ganglion cells were absent in aganglionic bowel, where the ganglia in the intermuscular layer were replaced by hypertrophic nerve bundles. These morphological findings indicate that LIF may play a role in the development of the enteric nervous system.