p150glued缺失对神经元中Rab蛋白表达水平的影响
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摘要
目的探讨家族性运动神经元病致病基因DCTN1编码的p150glued蛋白缺失对于原代培养的皮质神经元细胞活性及Rab蛋白表达的影响。方法以野生型(WT)和p150glued缺失(p150glued KO)新生小鼠为细胞来源,体外培养皮质神经元7d和14d后,分别利用MTT法和Western blot法检测皮质神经元存活率以及Rab蛋白家族成分表达水平。结果与WT小鼠皮质神经元相比,p150glued KO小鼠皮质神经元在体外培养7d和14d时,细胞活力无明显差异,14d时Rab4、Rab5、Rab9、Rab11、Rab24蛋白表达水平未发生显著变化,而Rab7蛋白表达水平显著降低(P<0.001)。结论 p150glued缺失对原代皮质神经元存活以及调控囊泡运输的Rab蛋白家族中Rab4、Rab5、Rab9、Rab11、Rab24的表达水平无明显影响,但特异性减弱晚期内体标志物Rab7蛋白的表达,可阻碍胞内溶酶体降解通路。
Objective To investigate the effects of motor neuron disease(MND) related DCTN1 gene encoded p150glued protein deficiency on cell viability and expression of Rab proteins in cultured mouse cortical neurons. Method The cortical neurons from wild type(WT) and p150glued knock out(p150glued KO) newborn mice were cultured in vitro for 7 to 14 days. The cell viability and the expressions of Rab proteins were detected with MTT assay and Western blot, respectively. Results Compared to WT mice, cortical neurons from p150glued KO mice showed comparable cell viability on 7 and 14 days in vitro. On 14 days in vitro, cortical neurons from p150glued KO mice showed decreased level of Rab7 only(P<0.001). Conclusion p150glued deficiency specifically reduces the expression of vesicular transport related protein Rab7 in cultured cortical neurons.
Objective To investigate the effects of motor neuron disease(MND) related DCTN1 gene encoded p150glued protein deficiency on cell viability and expression of Rab proteins in cultured mouse cortical neurons. Method The cortical neurons from wild type(WT) and p150glued knock out(p150glued KO) newborn mice were cultured in vitro for 7 to 14 days. The cell viability and the expressions of Rab proteins were detected with MTT assay and Western blot, respectively. Results Compared to WT mice, cortical neurons from p150glued KO mice showed comparable cell viability on 7 and 14 days in vitro. On 14 days in vitro, cortical neurons from p150glued KO mice showed decreased level of Rab7 only(P<0.001). Conclusion p150glued deficiency specifically reduces the expression of vesicular transport related protein Rab7 in cultured cortical neurons.
引文
[1]Langemeyer L,Frohlich F,Ungermann C.Rab GTPase function in endosome and lysosome biogenesis[J].Trends Cell Biol,2018,pii:S0962-8924(18)30108-30109.
[2]Yu J,Lai C,Shim H,et al.Genetic ablation of dynactin p150(Glued)in postnatal neurons causes preferential degeneration of spinal motor neurons in aged mice[J].Mol Neurodegener,2018,13(1):10.
[3]Parisiadou L,Yu J,Sgobio C,et al.LRRK2 regulates synaptogenesis and dopamine receptor activation through modulation of PKA activity[J].Nat Neurosci,2014,17(3):367-376.
[4]Yu J,Sun M,Chen Z,et al.Magnesium modulates amyloid-beta protein precursor trafficking and processing[J].J Alzheimers Dis,2010,20(4):1091-1106.
[5]Cho HJ,Yu J,Xie C,et al.Leucine-rich repeat kinase 2 regulates Sec16A at ER exit sites to allow ER-Golgi export[J].EMBO J,2014,33(20):2314-2331.
[6]Saxena S,Bucci C,Weis J,et al.The small GTPase Rab7 controls the endosomal trafficking and neuritogenic signaling of the nerve growth factor receptor TrkA[J].J Neurosci,2005,25(47):10930-10940.
[7]Deinhardt K,Salinas S,Verastegui C,et al.Rab5 and Rab7 control endocytic sorting along the axonal retrograde transport pathway[J].Neuron,2006,52(2):293-305.
[8]Munch C,Sedlmeier R,Meyer T,et al.Point mutations of the p150subunit of dynactin(DCTN1)gene in ALS[J].Neurology,2004,63(4):724-726.
[9]Laird FM,Farah MH,Ackerley S,et al.Motor neuron disease occurring in a mutant dynactin mouse model is characterized by defects in vesicular trafficking[J].J Neurosci,2008,28(9):1997-2005.
[10]Ikenaka K,Kawai K,Katsuno M,et al.dnc-1/dynactin 1 knockdown disrupts transport of autophagosomes and induces motor neuron degeneration[J].PLoS One,2013,8(2):e54511.
[11]Jiang YM,Yamamoto M,Tanaka F,et al.Gene expressions specifically detected in motor neurons(dynactin 1,early growth response 3,acetyl-CoA transporter,death receptor 5,and cyclin C)differentially correlate to pathologic markers in sporadic amyotrophic lateral sclerosis[J].J Neuropathol Exp Neurol,2007,66(7):617-627.
[12]Bhuin T,Roy JK.Rab proteins:the key regulators of intracellular vesicle transport[J].Exp Cell Res,2014,328(1):1-19.
[13]Veleri S,Punnakkal P,Dunbar GL,et al.Molecular insights into the roles of rab proteins in intracellular dynamics and neurodegenerative diseases[J].Neuromolecular Med,2018,20(1):18-36.
[14]Hegedus K,Takats S,Boda A,et al.The Ccz1-Mon1-Rab7 module and Rab5 control distinct steps of autophagy[J].Mol Biol Cell,2016,27(20):3132-3142.
[15]Grosshans BL,Ortiz D,Novick P.Rabs and their effectors:achieving specificity in membrane traffic[J].Proc Natl Acad Sci U S A,2006,103(32):11821-11827.
[16]Bucci C,Alifano P,Cogli L.The role of rab proteins in neuronal cells and in the trafficking of neurotrophin receptors[J].Membranes(Basel),2014,4(4):642-677.
[17]Amaya C,Militello RD,Calligaris SD,et al.Rab24 interacts with the Rab7/Rab interacting lysosomal protein complex to regulate endosomal degradation[J].Traffic,2016,17(11):1181-1196.
[18]Munafo DB,Colombo MI.Induction of autophagy causes dramatic changes in the subcellular distribution of GFP-Rab24[J].Traffic,2002,3(7):472-482.
[19]Hyttinen JM,Niittykoski M,Salminen A,et al.Maturation of autophagosomes and endosomes:a key role for Rab7[J].Biochim Biophys Acta,2013,1833(3):503-510.
[20]Guerra F,Bucci C.Multiple roles of the small GTPase Rab7[J].Cells,2016,5(3)pii:E34.
[21]Vanlandingham PA,Ceresa BP.Rab7 regulates late endocytic trafficking downstream of multivesicular body biogenesis and cargo sequestration[J].J Biol Chem,2009,284(18):12110-12124.
[22]Bucci C,Thomsen P,Nicoziani P,et al.Rab7:a key to lysosome biogenesis[J].Mol Biol Cell,2000,11(2):467-480.
[23]De Luca M,Cogli L,Progida C,et al.RILP regulates vacuolar ATPase through interaction with the V1G1 subunit[J].J Cell Sci,2015,128(14):2565.
[24]Maxson ME,Grinstein S.The vacuolar-type H(+)-ATPase at a glance-more than a proton pump[J].J Cell Sci,2014,127(Pt23):4987-4993.
[2]Yu J,Lai C,Shim H,et al.Genetic ablation of dynactin p150(Glued)in postnatal neurons causes preferential degeneration of spinal motor neurons in aged mice[J].Mol Neurodegener,2018,13(1):10.
[3]Parisiadou L,Yu J,Sgobio C,et al.LRRK2 regulates synaptogenesis and dopamine receptor activation through modulation of PKA activity[J].Nat Neurosci,2014,17(3):367-376.
[4]Yu J,Sun M,Chen Z,et al.Magnesium modulates amyloid-beta protein precursor trafficking and processing[J].J Alzheimers Dis,2010,20(4):1091-1106.
[5]Cho HJ,Yu J,Xie C,et al.Leucine-rich repeat kinase 2 regulates Sec16A at ER exit sites to allow ER-Golgi export[J].EMBO J,2014,33(20):2314-2331.
[6]Saxena S,Bucci C,Weis J,et al.The small GTPase Rab7 controls the endosomal trafficking and neuritogenic signaling of the nerve growth factor receptor TrkA[J].J Neurosci,2005,25(47):10930-10940.
[7]Deinhardt K,Salinas S,Verastegui C,et al.Rab5 and Rab7 control endocytic sorting along the axonal retrograde transport pathway[J].Neuron,2006,52(2):293-305.
[8]Munch C,Sedlmeier R,Meyer T,et al.Point mutations of the p150subunit of dynactin(DCTN1)gene in ALS[J].Neurology,2004,63(4):724-726.
[9]Laird FM,Farah MH,Ackerley S,et al.Motor neuron disease occurring in a mutant dynactin mouse model is characterized by defects in vesicular trafficking[J].J Neurosci,2008,28(9):1997-2005.
[10]Ikenaka K,Kawai K,Katsuno M,et al.dnc-1/dynactin 1 knockdown disrupts transport of autophagosomes and induces motor neuron degeneration[J].PLoS One,2013,8(2):e54511.
[11]Jiang YM,Yamamoto M,Tanaka F,et al.Gene expressions specifically detected in motor neurons(dynactin 1,early growth response 3,acetyl-CoA transporter,death receptor 5,and cyclin C)differentially correlate to pathologic markers in sporadic amyotrophic lateral sclerosis[J].J Neuropathol Exp Neurol,2007,66(7):617-627.
[12]Bhuin T,Roy JK.Rab proteins:the key regulators of intracellular vesicle transport[J].Exp Cell Res,2014,328(1):1-19.
[13]Veleri S,Punnakkal P,Dunbar GL,et al.Molecular insights into the roles of rab proteins in intracellular dynamics and neurodegenerative diseases[J].Neuromolecular Med,2018,20(1):18-36.
[14]Hegedus K,Takats S,Boda A,et al.The Ccz1-Mon1-Rab7 module and Rab5 control distinct steps of autophagy[J].Mol Biol Cell,2016,27(20):3132-3142.
[15]Grosshans BL,Ortiz D,Novick P.Rabs and their effectors:achieving specificity in membrane traffic[J].Proc Natl Acad Sci U S A,2006,103(32):11821-11827.
[16]Bucci C,Alifano P,Cogli L.The role of rab proteins in neuronal cells and in the trafficking of neurotrophin receptors[J].Membranes(Basel),2014,4(4):642-677.
[17]Amaya C,Militello RD,Calligaris SD,et al.Rab24 interacts with the Rab7/Rab interacting lysosomal protein complex to regulate endosomal degradation[J].Traffic,2016,17(11):1181-1196.
[18]Munafo DB,Colombo MI.Induction of autophagy causes dramatic changes in the subcellular distribution of GFP-Rab24[J].Traffic,2002,3(7):472-482.
[19]Hyttinen JM,Niittykoski M,Salminen A,et al.Maturation of autophagosomes and endosomes:a key role for Rab7[J].Biochim Biophys Acta,2013,1833(3):503-510.
[20]Guerra F,Bucci C.Multiple roles of the small GTPase Rab7[J].Cells,2016,5(3)pii:E34.
[21]Vanlandingham PA,Ceresa BP.Rab7 regulates late endocytic trafficking downstream of multivesicular body biogenesis and cargo sequestration[J].J Biol Chem,2009,284(18):12110-12124.
[22]Bucci C,Thomsen P,Nicoziani P,et al.Rab7:a key to lysosome biogenesis[J].Mol Biol Cell,2000,11(2):467-480.
[23]De Luca M,Cogli L,Progida C,et al.RILP regulates vacuolar ATPase through interaction with the V1G1 subunit[J].J Cell Sci,2015,128(14):2565.
[24]Maxson ME,Grinstein S.The vacuolar-type H(+)-ATPase at a glance-more than a proton pump[J].J Cell Sci,2014,127(Pt23):4987-4993.