Chlorpromazine Increases the Expression of Polysialic Acid (PolySia) in Human Neuroblastoma Cells and Mouse Prefrontal Cortex

Abstract

The neural cell adhesion molecule (NCAM) is modified by polysialic acid (polySia or PSA) in embryonic brains. In adult brains, polySia modification of NCAM is only observed in restricted areas where neural plasticity, remodeling of neural connections, or neural generation is ongoing although the amount of NCAM remains unchanged. Impairments of the polySia-expression and several single nucleotide polymorphisms (SNPs) of the polysialyltransferase (polyST) ST8SIA2 gene are reported to be associated with schizophrenia and bipolar disorder. Chlorpromazine (CPZ) is well-known as an agent for treating schizophrenia, and our hypothesis is that CPZ may affect the polySia expression or the gene expression of polySTs or NCAM. To test this hypothesis, we analyzed the effects of CPZ on the expression of polySia-NCAM on human neuroblastoma cell line, IMR-32 cells, by immunochemical and chemical methods. Interestingly, the cell surface expression of polySia, especially those with lower chain lengths, was significantly increased on the CPZ-treated cells, while mRNAs for polySTs and NCAM, and the amounts of total polySia-NCAM remained unchanged. The addition of brefeldin A, an inhibitor of endocytosis, suppressed the CPZ-induced cell surface polySia expression. In addition, polySia-NCAM was also observed in the vesicle compartment inside the cell. All these data suggest that the level of cell surface expression of polySia in IMR-32 is highly regulated and that CPZ changes the rate of the recycling of polySia-NCAM, leading to the up-regulation of polySia-NCAM on the cell surface. We also analyzed the effect of CPZ on polySia-expression in various brain regions in adult mice and found that CPZ only influenced the total amounts of polySia-NCAM in prefrontal cortex. These results suggest a brain-region-specific effect of CPZ on the expression of total polySia in mouse brain. Collectively, anti-schizophrenia agent CPZ consistently up-regulates the expression polySia at both cellular and animal levels.

Recent advances in the analysis of polysialic acid from complex biological systems

“…PolySia with DP > 90 has been visualised by DMB labelling/HAPEC-FD from as little as 200 ng derivatised colominic acid has been reported . This approach has been widely-used for studying polySia from cells and tissues with detectable DP ranging from 18 to 60 (Inoue & Inoue, 2001a, 2001bPoongodi et al, 2002;Yabe, Sato, Matsuda, & Kitajima, 2003;Simon et al, 2013;Nishimura et al, 2014;Kaese et al, 2015;Abe et al, 2017). However, the main challenge of mild acid hydrolysis coupled with HPAEC-FD is the potential partial hydrolysis of long-chain polySia during the hydrolysis reaction and DMB labelling, as noted by Inoue (Lin et al, 1999;Inoue et al, 2001).…”

“…Therefore, relative quantitation of polySia of a specific DP can be performed by comparing fluorescence peak areas between calibration standards and biological samples. Besides the advantage of providing quantitative analysis of polySia of specific DP, the HPAEC-FD approach can also determine quantitative differences in polySia DP distribution (Poongodi et al, 2002;Nishimura et al, 2014;Abe et al, 2017). A DNAPac-PA100 column combined with a fluorescence detector was used to investigate the relative quantitative changes in expression of polySia chain lengths with time (24-84 h) in IMR-32 (human neuroblastoma) and PC-12 (rat pheochromocytoma) cancer cells (Poongodi et al 2002).…”

“…This approach was also used to compare the expression levels of polySia containing more than 6 Sia units from embryonic and adult pig brains, and found the relative quantitative result was in good agreement with that obtained from an ELISA study utilising anti-polySia antibodies (Nishimura et al, 2014). Furthermore, changes in cellular expression levels of polySia (DP > 6) in response to drug treatment have been described (Abe et al, 2017). However, HPAEC-FD has relatively poor sensitivity in the measurement of longer chain polySia, probably due to the de-polymerisation during fluorescent derivatisation (as described in Section 3).…”

“…However, HPAEC-FD has relatively poor sensitivity in the measurement of longer chain polySia, probably due to the de-polymerisation during fluorescent derivatisation (as described in Section 3). Another disadvantage is that derivatisation of polySia requires lengthy reaction times (up to 24 h), albeit at low temperature (4 °C), to minimise the partial hydrolysis of long-chain polySia, as discussed earlier (Poongodi et al, 2002;Nishimura et al, 2014;Abe et al, 2017).…”

“…Western blotting is a widely-used technique for evaluating relative expression levels of polysialylated-proteins in biological samples (von Der Ohe et al, 2002;Korja et al, 2009;Valentiner, Muhlenhoff, Lehmann, Hildebrandt, & Schumacher, 2011;Nishimura et al, 2014;Westphal, Kleene, Lutz, Theis, & Schachner, 2016;Abe et al, 2017;Chen, Ren, Zhang, Troy, & Wang, 2017;Klobucar et al, 2017). Similarly to ELISA, Western blotting also recognises polySia proteins via immunodetection.…”