Distribution, interacting partners, and function of polysialic acid in the brainstem and spinal cord of adult rodents

Theoretical thesis.Bibliography: pages 191-231.Chapter 1: Introduction -- Chapter 2: Polysialic acid distribution within the spinal cord, brainstem, and trigeminal ganglion -- Chapter 3: Identification of polysialic acid interacting partners in the dorsal horn of rat and mouse and the trigeminal nucleus of rat -- Chapter 4: Viral vector induced expression of polysialic acid in the rostral ventrolateral medulla: expression pattern and cardiorespiratory effects -- Chapter 5: Synthesis and future directions.Glycans, a major class of biomolecules, are expressed on every cells surface and play an important role in maintaining health with their perturbation causing disease. Polysialic acid (polySia) is a large cell surface glycan widely express ed in the developing brain with an essential role in brain development. Mice deficient in polysialyltransferases ST8Sia II and ST8Sia IV, enzymes required for polySiasynthesis, show severe developmental deficits in their nervous system with mostly dying wit hin a month. In adult brain, however, polySia exhibits a discrete expression pattern which in the higher brain regions is often associated with plasticity. The expression and function of polySia in the brainstem and spinal cord is poorly understood. We investigated the distribution and cellular localization of polySia in the adult rat brainstem, spinal cord, and trigeminal ganglion resulting in the identification of novel polySia positive regions including the spinal trigeminal nucleus caudalis (Sp5C) and the intermediolateral cell column (IML). PolySia was associated with neurons and fine astrocytic processes, confirmed by ultrastructural analyses. Within the superficial laminae of the dorsal horn, some association of polySia with inhibitory neurons was found. The sugar also coated some neurons, satellite glial cells, and fibres in the trigeminal ganglia, which provides input to Sp5C. Comparing the expression pattern of polySia in rats and mice showed mostly common patterns of labelling although areas such as the spinal cord dorsal horn differed between species. Moreover, comparison of the pattern of polySia immunolabe l ling using the two most common antibodies, mAb 735 and mAb 5324, demonstrated similar patterns of expression. Next, in order to understand the function of polySia in the spinal cord and the biological processes it regulates, binding partners of polySia in the dorsal horn were investigated using co - immunoprecipitation (IP) followed by label - free liquid chromatography tandem mass spectrometry. Thirteen potential protein partners were identified, with more than half associated with signalling. Five protein s (receptor expression - enhancing protein 5, guanine nucleotide - binding protein G(o) subunit alpha, sodium/potassium - transporting ATPase subunits alpha - 2 and alpha - 3, and clathrin heavy chain) were further vali dated using co - IP/reverse co - IP followed by western blotting and confocal microscopy. The interaction with validated candidates was also demonstrated in the Sp5C and IML of adult rats as well as in the dorsal horn of adult mice, indicating that the interactions were neither region nor species specific.Recently our laboratory found that polySia is required for the normal transmission of information through the nucleus of solitary tract (NTS) . Information from the NTS is conveyed to the rostral ventrolateral medulla (RVLM), which generates vasomotor tone and integrates a range of cardiorespiratory reflexes. We used a viral vector that drives the expression of polysialyltransferase ST8SiaIV in the RVLM where polySia expression is normally low. We determined the consequences of increasing polySia expression in basal cardiorespiratory and reflex function. Induced expression of polySia was demonstrated but did not change baseline cardiorespiratory function or alter four reflexes tested (Bezold - Jarisch reflex, responses to hypercapnia, hypoxia, and acute intermittent hypoxia). The lower plateau of sympathetic baroreceptor reflex curve was elevated, possibly due to increased non - barosensitive sympathe tic activity; however, total activity was unchanged. Due to substantial variability it remains unclear whether polySia did not alter neurotransmission in the RVLM or its expression, particularly in cardiorespiratory neurons was not enough to alter its function. These data greatly expand the lists of polySia positive regions and potential binding partners in the brainstem and spinal cord that will help in delineating the function and mechanisms of action, particularly with respect to signal l ing, of polySia in these regions.1 online resource (xii, 232 pages

Polysialic acid in the rat brainstem and thoracolumbar spinal cord: distribution, cellular location, and comparison with mouse

Polysialic acid (polySia), a homopolymer of α2,8-linked glycans, is a posttranslational modification on a few glycoproteins, most commonly in the brain, on the neural cell adhesion molecule. Most research in the adult central nervous system has focused on its expression in higher brain regions, where its distribution coincides with regions known to exhibit high levels of synaptic plasticity. In contrast, scant attention has been paid to the expression of polySia in the hindbrain. The main aims of the study were to examine the distribution of polySia immunoreactivity in the brainstem and thoracolumbar spinal cord, to compare the distribution of polySia revealed by two commercial antibodies commonly used for its investigation, and to compare labeling in the rat and mouse. We present a comprehensive atlas of polySia immunoreactivity: we report that polySia labeling is particularly dense in the dorsal tegmentum, medial vestibular nuclei and lateral parabrachial nucleus, and in brainstem regions associated with autonomic function, including the dorsal vagal complex, A5, rostral ventral medulla, A1, and midline raphe, as well as sympathetic preganglionic neurons in the spinal cord and central targets of primary sensory afferents (nucleus of the solitary tract, spinal trigeminal nucleus, and dorsal horn [DH]). Ultrastructural examination showed labeling was present predominantly on the plasma membrane/within the extracellular space/in or on astrocytes. Labeling throughout the brainstem and spinal cord were very similar for the two antibodies and was eliminated by the polySia-specific sialidase, Endo-NF. Similar patterns of distribution were found in rat and mouse brainstem with differences evident in DH

Recent Advances of Functional Proteomics in Gastrointestinal Cancers- a Path towards the Identification of Candidate Diagnostic, Prognostic, and Therapeutic Molecular Biomarkers

Gastrointestinal (GI) cancer remains one of the common causes of morbidity and mortality. A high number of cases are diagnosed at an advanced stage, leading to a poor survival rate. This is primarily attributed to the lack of reliable diagnostic biomarkers and limited treatment options. Therefore, more sensitive, specific biomarkers and curative treatments are desirable. Functional proteomics as a research area in the proteomic field aims to elucidate the biological function of unknown proteins and unravel the cellular mechanisms at the molecular level. Phosphoproteomic and glycoproteomic studies have emerged as two efficient functional proteomics approaches used to identify diagnostic biomarkers, therapeutic targets, the molecular basis of disease and mechanisms underlying drug resistance in GI cancers. In this review, we present an overview on how functional proteomics may contribute to the understanding of GI cancers, namely colorectal, gastric, hepatocellular carcinoma and pancreatic cancers. Moreover, we have summarized recent methodological developments in phosphoproteomics and glycoproteomics for GI cancer studies

Manipulating root water supply elicits major shifts in the shoot proteome

Substantial reductions in yield caused by drought stress can occur when parts of the root system experience water deficit even though other parts have sufficient access to soil water. To identify proteins associated to drought signaling, rice (Oryza sativa L. cv. IR64.) plants were transplanted into plastic pots with an internal wall dividing each pot into two equal compartments, allowing for equal distribution of soil and the root system between these compartments. The following treatments were applied: either both compartments were watered daily ("wet" roots), or water was withheld from both compartments ("dry" roots), or water was withheld from only one of the two compartments in each pot ("wet" and "dry" roots). The substantial differences in physiological parameters of different growth conditions were accompanied by differential changes in protein abundances. Label-free quantitative shotgun proteomics have resulted in identification of 1383 reproducible proteins across all three conditions. Differentially expressed proteins were categorized within 17 functional groups. The patterns observed were interesting in that in some categories such as protein metabolism and oxidation-reduction, substantial numbers of proteins were most abundant when leaves were receiving signals from "wet" and "dry" roots. In yet other categories such as transport, several key transporters were surprisingly abundant in leaves supported by partially or completely droughted root systems, especially plasma membrane and vacuolar transporters. Stress-related proteins behaved very consistently by increasing in droughted plants but notably some proteins were most abundant when roots of the same plant were growing in both wet and dry soils. Changes in carbohydrate-processing proteins were consistent with the passive accumulation of soluble sugars in shoots under drought, with hydrolysis of sucrose and starch synthesis both enhanced. These results suggest that drought signals are complex interactions and not simply the additive effect of water supply to the roots.10 page(s

Manipulating Root Water Supply Elicits Major Shifts in the Shoot Proteome

Substantial reductions in yield caused by drought stress can occur when parts of the root system experience water deficit even though other parts have sufficient access to soil water. To identify proteins associated to drought signaling, rice (<i>Oryza sativa</i> L. cv. IR64.) plants were transplanted into plastic pots with an internal wall dividing each pot into two equal compartments, allowing for equal distribution of soil and the root system between these compartments. The following treatments were applied: either both compartments were watered daily (“wet” roots), or water was withheld from both compartments (“dry” roots), or water was withheld from only one of the two compartments in each pot (“wet” and “dry” roots). The substantial differences in physiological parameters of different growth conditions were accompanied by differential changes in protein abundances. Label-free quantitative shotgun proteomics have resulted in identification of 1383 reproducible proteins across all three conditions. Differentially expressed proteins were categorized within 17 functional groups. The patterns observed were interesting in that in some categories such as protein metabolism and oxidation–reduction, substantial numbers of proteins were most abundant when leaves were receiving signals from “wet” and “dry” roots. In yet other categories such as transport, several key transporters were surprisingly abundant in leaves supported by partially or completely droughted root systems, especially plasma membrane and vacuolar transporters. Stress-related proteins behaved very consistently by increasing in droughted plants but notably some proteins were most abundant when roots of the same plant were growing in both wet and dry soils. Changes in carbohydrate-processing proteins were consistent with the passive accumulation of soluble sugars in shoots under drought, with hydrolysis of sucrose and starch synthesis both enhanced. These results suggest that drought signals are complex interactions and not simply the additive effect of water supply to the roots