Developmental Change of Sialidase Neu4 Expression in Murine Brain and Its Involvement in the Regulation of Neuronal Cell Differentiation

Abstract

Sialidase Neu4 is reported to be dominantly expressed in the mouse brain, but its functional significance is not fully understood. We previously demonstrated that sialidase Neu3, also rich in mouse brain, is up-regulated during neuronal differentiation with involvement in acceleration of neurite formation. To elucidate physiological functions of Neu4, as well as Neu3, we determined expression during mouse brain development by quantitative RT-PCR. Expression was relatively low in the embryonic stage and then rapidly increased at 3-14 days after birth, whereas Neu3 demonstrated high levels in the embryonic stage and down-regulation after birth. Murine Neu4 was found to possess two isoforms differing in expression levels, developmental pattern, and enzymatic character. Distinct from the human isoforms, the murine forms, to a different extent, both catalyzed the removal of sialic acid from gangliosides as well as glycoproteins, and one isoform seemed to act on polysialylated NCAM efficiently, despite the low activity toward ordinary substrates. In situ hybridization demonstrated Neu4 mRNA to be present mainly in the hippocampus in which NCAM is rich and decreases after birth. During retinoic acid-induced differentiation, Neu4 expression was down-regulated in Neuro2a cells. Overexpression of Neu4 resulted in suppression of neurite formation, and its knockdown showed the acceleration. Thin layer chromatography of the glycolipids from Neu4-transfected cells showed ganglioside compositions to be only slightly affected, although lectin blot analysis revealed increased binding to Ricinus communis agglutinin (RCA) lectin of a ϳ95-kDa glycoprotein, which decreased with cell differentiation. These results suggest that mouse Neu4 plays an important regulatory role in neurite formation, possibly through desialylation of glycoproteins.

Sialidase NEU4 Hydrolyzes Polysialic Acids of Neural Cell Adhesion Molecules and Negatively Regulates Neurite Formation by Hippocampal Neurons

“…Quantitative Reverse Transcription-PCR Analysis-The mRNA levels of the four endogenous mouse sialidases and two polysialyltransferases in hippocampal neuron were evaluated by quantitative RT-PCR (real-time PCR, Light Cycler, Roche Diagnostics) as described previously (25). Total RNA was isolated, and first-strand cDNA was synthesized by reverse transcription and used as a template.…”

“…Sialidase Activity-For sialidase enzymatic studies, HEK293T cells were transiently transfected with the respective expression plasmids for murine sialidase genes Neu2 (21), Neu3 (21), and two splicing isoforms Neu4a and Neu4b (25) and for rat Neu1 gene (27), previously prepared. For NEU1 activity, the protective protein gene (pp11) was sometimes co-transfected (28).…”

“…Of the four types, NEU3 preferentially hydrolyzes gangliosides other than GM1 and GM2, and is up-regulated during neuronal differentiation with involvement in acceleration of neurite formation (21)(22)(23)(24). In contrast, NEU4 possessing two isoforms, both catalyzing removal of sialic acid from oligosaccharides and glycoproteins as well as gangliosides, is likely to behave during neural differentiation in a way opposite to NEU3 (25). In the mouse, NEU4 is expressed predominantly in the brain (25,26).…”

“…We previously demonstrated that the levels are relatively low in the embryonic stage, rapidly increasing at 3-14 days after birth, while NEU3 shows high levels in embryonic stages and post-partum downregulation. In the adult mouse brain, in situ hybridization exhibited NEU4 to be mainly present in the hippocampus, in which polySia-NCAM is rich and decreases after birth (25). In the present study, building on our previous findings, we investigated whether murine sialidases, especially NEU4, catalyze the degradation of polySia, and found that NEU4 efficiently hydrolyzes polySia-NCAM and negatively regulates neurite outgrowth of hippocampal neurons.…”

“…In contrast, NEU4 possessing two isoforms, both catalyzing removal of sialic acid from oligosaccharides and glycoproteins as well as gangliosides, is likely to behave during neural differentiation in a way opposite to NEU3 (25). In the mouse, NEU4 is expressed predominantly in the brain (25,26). We previously demonstrated that the levels are relatively low in the embryonic stage, rapidly increasing at 3-14 days after birth, while NEU3 shows high levels in embryonic stages and post-partum downregulation.…”