expression level also increased in the SCN, but decreased in the PFC and OB. The decrease was also confirmed via chemical detection method. To understand the mechanism, we analyzed the mRNA expression prior and subsequent to the TS and found that the expression of St8sia4 and NCAM in SCN increased consequent to TS, which was consistent with the increased polySia expression level in SCN. However, in the OB and PFC, there were no alterations in the expression levels of polySia-associated genes; such as St8sia2, St8sia4, and NCAM. In addition, the NCAM protein expression level was unaltered. Thus, we sought to ascertain the possibility of corticosterone as a key regulator as the increased serum level of corticosterone is directly proportional to the acute stress induced by TS. Consequent to injection of corticosterone into the vein, the serum corticosterone level increased; however, the polySia expression level remained unaltered. The same result was obtained using the cell line IMR-32. Then we considered the possibility of the exercise because mice move vigorously during TS. Mice were placed into a ball for 3 min (movement time in TS), and the serum corticosterone level was measured prior to the test. This setup did not increase the corticosterone level, indicating that it did not induce acute stress in the mice. We further analyzed the polySia expression level and found that it was unaltered, indicating that the fear condition, not the movement nor corticosterone, changed the polySia in brains during the TS. In order to elucidate the underlying mechanism, we hypothesized that sialidase secreted from microglia cells was involved in this decrease because using a microglial cell line, we demonstrated that microglia secreted sialidase on the exosome after LPS-activation7). Therefore, to confirm this in vivo, we injected DANA, a sialidase inhibitor, into the mice prior to TS. The subsequent analysis of polySia expression level in the OB and PFC revealed that the expected decrease in the acute stress-induced polySia expression level was inhibited, clearly indicating that sialidase contributed to the decrease in the acute stress-induced polySia expression level. In addition, we elucidated the contribution of microglia in this mechanism. We used minocycline, a reagent for microglia inhibition. Consequent to the injection with minocycline, the expected decrease in the acute stress-induced polySia expression level was only inhibited in the OB but not in the PFC. Lastly, we elucidated the contribution of astrocyte in the decrease of acute stress-induced polySia expression level in the PFC. We used gabapentin, a reagent for astrocyte inhibition. Following injection, the expected decrease in the acute stress-induced polySia expression level was inhibited. Therefore, the decrease in the acute stress-induced polySia expression level was due to the sialidase activity, and the origins of the enzyme were microglia and astrocytes in the OB and PFC, respectively (Figure 2). These results demonstrate that negative environmental factor, such as acute stress, altered the polySia expression level in specific brain regions4). Progress after grant periodWe analyzed the polySia expression level using DISC1 mutant mice (L100P), which are considered to express similar phenotypes as that of patients with schizophrenia, impaired working memory, and PPI. Interestingly, DISC1 mice exhibited decreased polySia expression level in the SCN, indicating that this specific brain region might be a target site when considering the schizophrenia pathology8). For polySia detection, we compared several types of polySia-detection methods using brain samples, and subsequently compared the polySia expression level with that of the vertebrate brains9). Evolution and pathology are two sides of a coin. Further analysis and elucidation of polySia is required for the deep understanding of the biological significance of the existence of polySia in nature, which is presently ongoing.Future perspectiveOur research clearly demonstrates that polySia is expressed in an appropriate time (T), at appropriate cell types, and of an appropriate brain region (P), and that the expression of polySia can thus be defined by the T and P parameters (Figure 3). We determined the T and P parameters at the normal state. Once normal, polySia structures were impaired by genetic (G) and/Figure 2. Demonstration of polySia change after acute stress In the olfactory bulb, microglia cells are activated by acute stress to secrete sialidases, which consequently bind to polySia chains and cleaves. In the prefrontal cortex, astrocyte cells are activated by acute stress to secrete sialidases, which consequently bind to polySia chains and cleaves. The mechanisms were demonstrated using the sialidase inhibitor (DANA), microglia inhibitor (minocycline, Mino), and astrocyte inhibitor (gabapentin, GBP).61
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