br Materials and methods br Results br Discussion

Materials and methods
Results
Discussion The present study shows that 1) overexpression of wild-type or mutant α-SYN interferes with forskolin-induced DBH up-regulation, 2) α-SYN enters into the nucleus and is able to bind to CRE region, compete for CREB, and attenuate CRE-mediated transcription of DBH, and 3) A53T Tg mice exhibit abnormal regulation of DBH and TH expression in response to the IMO stress, and show higher anxiety like behaviors in the normal condition and abnormal behaviors following the IMO stress than normal mice. Adaptation to psychological or physiological stressful circumstances requires integration of behavioral, neuroendocrine, and autonomic responses to maintain homeostasis. The LC is regarded as a part of the central ‘stress circuitry’, which is robustly activated after stressful stimuli in experimental animals (Itoi and Sugimoto, 2010). Because there are close relationship between the central noradrenergic system and anxiety states/or depression (Itoi and Sugimoto, 2010), abnormal regulation of noradrenergic activity under stressful condition might play a causative role in the development of anxiety/or depression. Gene expression of NE synthesizing enzymes, TH and DBH, is triggered by various types of stress. In the present study, IMO resulted in elevated TH and DBH immunoreactive proteins in the LC of normal mice (Fig. 5). Immunohistochemistry results suggest that TH and DBH protein levels in WT mice after the IMO stress increased compared with non-stressed levels. In contrast, A53T Tg mice exhibited the maladaptive response to the repeated stress. TH protein expression did not significantly regulated by IMO stress. Furthermore, in A 804598 to the WT mice, the IMO stress did influence DBH protein levels in LC of the A53T Tg mice. Behavior results strongly further suggest that the A53T Tg mice exhibit significantly heightened anxiety-like behaviors (increased innate fear) than their littermate control mice in non-stress condition. Interestingly, the A53T Tg mice exhibit abnormal anxiety behaviors following IMO stress exposure compared with their behaviors in non-stress condition whereas the littermate control mice showed more anxiety behaviors after IMO stress exposure indicating that WT mice sensitively respond to the acute stress to cope with the unexpected stress/threat but A53T Tg mice exhibit impaired and maladaptive responses probably due to the down regulation of DBH. There are large amount of evidences demonstrating the pathogenesis of DArgic neurodegeneraion in PD (Olanow and Tatton, 1999), however, the molecular mechanisms leading to the anxious/depressive symptoms in PD are obscure. Our data from previous (Kim et al., 2011) and present studies using NE-producing SK-N-BE(2) cells show that CRE-mediated up-regulation of TH and DBH is significantly suppressed by overexpression of α-SYN, especially A53T (Fig. 2). In addition, stress-induced up-regulation of TH and DBH is attenuated in A53T Tg mice (Fig. 5). Although we did not evaluate in the present study by which mechanism is mainly involved in the up-regulation of TH and DBH expression under IMO, many evidences have shown that stress activates cAMP pathway in the LC (Melia et al., 1992). It has been also reported that TH and DBH have CRE region in their promoter (Kim et al., 1994) and cAMP/PKA-dependent CREB signaling plays a crucial role in the regulation of TH and DBH gene expressions (Kim et al., 1994, Lamouroux et al., 1993, Swanson et al., 1997). Our reports demonstrate for the first time that mutant α-SYN contributes to the abnormal regulation of stress response-related TH and DBH expression in NE-producing cells, and impaired and maladaptive responses to stress. The crucial role of CRE-mediated adaptive response to stress and emotional behavior is supported by the findings that anxiogenic-like response is increased in CREB-deficient mice (Valverde et al., 2004); under stressful situation, CREB-deficiency caused a reduction in locomotor activity and an increase in the percentage of the time spent in the closed sector, suggesting an anxiogenic-like response. It is corroborated by our results showing that A53T Tg mice exhibit significantly heightened anxiety-like behaviors than control mice in no stress condition (Fig. 6). More interestingly, abnormal stress response is observed in A53T Tg mice (Fig. 6). These observations demonstrate that changes in a transcription factor and gene expression of target proteins could produce diverse modification in emotional behavior, and α-SYN mutation could play a crucial role in the generation of non-motor symptom in PD patients.