| Summary: | 碩士 === 高雄醫學大學 === 醫學系生理學科碩士班 === 103 === Abstract
Spinal muscular atrophy (SMA) is a genetic neurodegenerative disease. survival of motor neuron (SMN) protein has been found in cell nucleus as well as the neurite and growth cone of developing motor neurons. Nonetheless, it is still not clarified that why SMN deficiency causes neurodegeneration. In addition, accumulating evidence have shown that extracellular purines such as adenosine triphosphate (ATP) and adenosine diphosphate (ADP) act as neurotransmitters to modulate nerve synapse. Purines binds to the purinergic receptor (P2 receptor) such as P2Y1 and P2Y2 on the cell membrane of neurons, leading to an increase in intracellular calcium concentration ([Ca2+]i) and downstream signal transduction. It is proposed that in SMA, purinergic signaling pathway is compromised, leading to disruption in neuronal differentiation. We hypothesize that the disruption of SMN in motor neurons may cause disruption in purinergic signaling, contributing to neurodegeneration.We used NSC-34 cell line, a hybrid cell produced by the fusion of neuroblastoma and motor neuron, as the cell model. The neuronal differentiation of NSC-34 cells was stimulated by fetal bovine serum (FBS) and horse serum (HS). Inducible RNAi-stable NSC-34 cells were treated with doxycycline for 5 days to knock down SMN gene. The vector without an insert was also stably transfected into NSC-34 cells. Normal NSC34 cells only treated with doxycycline were used as control group. Intracellular Ca2+ homeostasis was recorded by Ca2+ imaging. Western blotting and immunofluorescence cytochemistry was used to examine SMN, neuronal marker protein α-tubulin, MAP2, synaptophysin, P2Y1 and P2Y2 receptors, total and phosphorylated IP3 receptor.
We found that in SMN gene knocked down cells the neuronal differentiation was diminished and P2Y receptor-mediated calcium signaling was imparied. After knockdown of SMN, the protein expression of P2Y1 and P2Y2 purinergic receptor isoforms were decreased, and phosphorylated (i.e. inactive) IP3 receptor was increased, probably causing a disruption in purinergic signaling. In addition, stimulation by extracellular ATP increased the protein expression of SMN, while P2Y receptor antagonist RB2 blocked the stimulatory effect of ATP, and decreased the protein expression of SMN, MAP2 and synaptophysin.
Our findings show that SMN protein is associated with purinergic signaling pathway and calcium homeostasis. Extracellular ATP enhanced SMN protein expression and motor neuron development. Interactions between SMN protein and purinergic signaling may play important roles in the pathogenesis of SMA.
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