Addition of polysialic acid, a homopolymer of α-2,8-linked sialic acid, is a unique posttranslational modification primarily of the neural cell adhesion molecule (NCAM). Polysialylated NCAM (PSA-NCAM) is widely expressed on neural cells in the brain during embryonic and neonatal stages, and expression is significantly reduced in adult stages (3
). In adults, however, PSA-NCAM persists in areas of active neurogenesis and synapse formation, such as the subventricular zone (SVZ), migration pathways to the olfactory bulb, and the hippocampus (6
). Polysialic acid is synthesized by two polysialyltransferases, ST8SiaII (also called STX) and ST8SiaIV (also called PST), which are expressed in a spatiotemporally regulated manner in the central nervous system (3
). Due to its polyanionic nature and extended helical structure, polysialic acid can block both cis
-interactions between NCAM molecules on the same cell and trans
-interactions between adjacent cells. Polysialic acid also influences interaction of NCAM with other molecules, such as heparan sulfate proteoglycans and brain-derived neurotrophic factor (BDNF) (17
). These observations suggest that polysialic acid modifications on NCAM affect molecular interactions involved in neural development and potentially synaptic plasticity.
Studies of NCAM-deficient mice (NCAM−/−
) or enzymatic removal of polysialic acid by endoneuraminidase show that PSA-NCAM plays diverse roles in promoting neurogenesis, neuronal pathfinding, defasciculation, and synapse formation (3
). For example, PSA-NCAM functions in cell-cell interactions necessary for chain migration of neural precursors from the SVZ to the olfactory bulb (10
). However, NCAM−/−
mice do not exhibit clear defects in other types of cell migration required for cerebrum and cerebellum formation in vivo. Interestingly, ST8SiaII and ST8SiaIV single mouse mutants exhibit a normal rostral migratory stream (RMS) forming the olfactory bulb (4
), indicating that olfactory interneuron precursors from the SVZ can use polysialic acid synthesized by either polysialyltransferase. Inactivation of either ST8SiaII or ST8SiaIV differentially impairs learning and memory and/or fear-associated conditioning associated with hippocampal function (4
), suggesting that ST8SiaII and ST8SiaIV play distinct roles in neuronal plasticity. Studies also show that single knockout mice do not lose all polysialic acid expression, since ST8SiaII and ST8SiaIV expression likely overlaps (4
). Therefore, to determine the role of polysialic acid in vivo, it was necessary to inactivate both ST8SiaII and ST8SiaIV.
Recently, Weinhold et al. reported that ST8SiaII and ST8SiaIV double knockout mice exhibit postnatal lethality and morphological brain anomalies, such as hydrocephalus, reduction in size of the internal capsule, and malformation of the anterior commissure and corticospinal tract (46
). These phenotypes were not observed in mice deficient in NCAM, ST8SiaII, or ST8SiaIV alone. Interestingly, many of these phenotypes were rescued in mice lacking NCAM, ST8SiaII, and ST8SiaIV (46
), indicating that NCAM protein in polysialic acid-deficient mice is responsible for the severe phenotypes seen in ST8SiaII/ST8SiaIV double knockout mice. However, it is still unclear how polysialic acid deficiency impairs neural cell function. It is also important to determine whether specific deficiencies are NCAM dependent or polysialic acid dependent. Several transcription factors, growth factors, and cell adhesion molecules are required for neural cell migration and differentiation, and mutations in the genes encoding these proteins are associated with brain malformations observed in human neurological disorders (7
). It is important then to determine if loss of polysialic acid affects function of these molecules and corresponding diseases.
Here we have undertaken cellular and molecular analyses of ST8SiaII and ST8SiaIV double mutant mice, which completely lack polysialic acid. We employed immunohistochemistry to detect specific cell types, such as glial cells, pyramidal cells, and dividing and migrating neural precursors, in both wild-type (WT) and double knockout mice. We identified a new role for polysialic acid in migration of both neurons and glial cells during cortex formation. Many neural cells lacking polysialic acid underwent apoptosis, which was rarely detected in mice deficient in NCAM, ST8SiaII, and ST8SiaIV alone. In vitro differentiation assays using neurosphere culture indicated that polysialic acid rather than NCAM regulates differentiation of glial precursors. Reverse transcription-PCR (RT-PCR) experiments analyzing genes associated with neural cell migration and differentiation showed decreased transcription of Pax6, which is required for cell migration in the cortex, and increased expression of glial fibrillary acidic protein (GFAP) in polysialic acid-deficient mice. These studies collectively show that polysialic acid plays critical roles in regulating cell migration, neural cell differentiation, and establishment of the glial cell lineage.