Distribution, interacting partners, and function of polysialic acid in the brainstem and spinal cord of adult rodents
thesisposted on 2022-03-28, 14:00 authored by Shila Shahbazian
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.