| Summary: | 碩士 === 國立臺灣大學 === 分子與細胞生物學研究所 === 105 === It has been reported that the protein level of muscle-specific NogoA is abnormally up-regulated in ALS patients. Since the animal model that is able to express NogoA in mature stage is less developed, we generated a zebrafish transgenic line Tg(pZα: TetON-Rtn4a l), which enables to conditionally expresses Rtn4a l in muscle after doxycycline induction. Results demonstrated that denervation at neuromuscular junction was observed after induction for one week; severe necrosis in myofibers was observed at the third week; and the weight of adult fish was greatly decreased at the fourth week. These dynamic phenotypes occurring in this transgenic line suggest that muscle-specific overexpression of Rtn4a1 in zebrafish model may exhibit similar symptoms that were suffered in human ALS disease.
The molecular mechanism underlying how over-expression of NogoA causes the ALS-like disease in zebrafish remains unclear. Previously we found that the Protein P was decreased in the condition medium (CM) obtained from culturing muscle cell line Sol8 which was over-expressed NogoA (Sol8-NogoA). In this continuous study, we demonstrated that the neurite outgrowth of neuron could also be retarded if neuron cell line NSC34 was cultured in Sol8-NogoA CM. When Protein P was directly added into Sol8-NogoA CM, the neurite outgrowth retardation occurred in NSC34 cells could be rescued. In contrast, when antiserum specifically against Protein P was added into Sol8-NogoA CM, the neurite outgrowth retardation of NSC34 remained intact, suggesting that Protein P is an essential factor for NSC 34 cells to grow neurites. Additionally, we proved that exogenous Protein P caused the decrease of the protein levels of p-Limk1 S323 and p-Cofiliin S3 in NSC34 cells; but that of p-Limk1 T508, which is the downstream of NogoA through Nogo 66 domain, remained unchanged, suggesting that the signal pathway affected by Protein P is independent of knowning Nogo 66 domain receptor (NgR1)-related pathways, including p-Limk1 T508. Furthermore, we constructed two mutated forms of Limk1 such as Limk1 T508V and S323A and transfected them to NSC34 cells. Results showed that the reduction of p-Cofilin S3 induced by Protein P was not altered by adding Limk1 T508V. Instead, the reduction of p-Cofilin S3 induced by Protein P was abolished by adding Limk1 S323A, suggesting that the normal function of endogenous Limk1 S323 in NSC34 was competed out by the mutated Limk1 S323A. We also found that addition of Nogo 66 caused the increase of ROCK2, p-Limk1 T508, p-Limk1 S323 and p-Cofilin S3. However, when Protein P was added, the levels p-Limk1 S323 and p-Cofilin S3 were decreased, suggesting that increasing p-Cofilin S3 induced by Nogo66 was ablated by Protein P through decreasing p-Limk1 S323. Meanwhile, when NAP2, a NgR1 blocking peptide, was added, the all levels of ROCK2, p-Limk1 S323, p-Limk1 T508 and p-Cofilin S3 were decreased due to no impacts of Nogo 66. Interestingly, when Protein P was added in the presence of NgR1, the levels of ROCK2 and p-Limk1 T508 did not change, suggesting that Protein P causes specifically the decrease of p-Cofilin S3 through p-Limk1 S323, which is independent of ROCK2/p-Limk1 T508 axis.
Lastly, using Western blot analysis, we demonstrated that the expression levels of neuron differentiated markers MAP2 and GAP43 and the neuron functional markers Syn1 and ChAT were decreased in NSC34 cells treated with Sol8-NogoA CM. However, if Protein P was added in the Sol8-NogoA CM, the expression levels of MAP2, GAP43, Syn1 and ChAT were all increased. The Syn1 signal was also detected in the growth cone of NSC34 using immunostaining. demonstrated that NSC34 treated with exogenous P protein exhibited more Syn1 signal at growth cone. The line of above evidence, we concluded that when NogoA was over-expressed in muscle led to decrease the secretion of Protein P, causing to reduce the ability of Protein P to decrease p-Cofilin S3 through p-Limk1 S323, resulting in inhibiting the neurite outgrowth of NSC34 cells.
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