Sac domain-containing proteins belong to a newly identified family of phosphoinositide phosphatases （the PIPPase family）. Despite well-characterized enzymatic activity, the biological functions of this mammalian Sac...Sac domain-containing proteins belong to a newly identified family of phosphoinositide phosphatases （the PIPPase family）. Despite well-characterized enzymatic activity, the biological functions of this mammalian Sac domain PIPPase family remain largely unknown. We identified a novel Sac domain-containing protein, rat Sac3 （rSac3）, which is widely expressed in various tissues and localized to the endoplasmic reticulum, Golgi complex and recycling endosomes, rSac3 displays PIPPase activity with PI（3）P, PI（4）P and PI（3,5）P2 as substrates in vitro, and a mutation in the catalytic core of the Sac domain abolishes its enzymatic activity. The expression of rSac3 is upregulated during nerve growth factor （NGF）-stimulated PC 12 cell neuronal differentiation, and overexpression of this protein promotes neurite outgrowth in PC 12 cells. Conversely, inhibition ofrSac3 expression by antisense oligonucleotides reduces neurite outgrowth of NGF- stimulated PC 12 cells, and the active site mutation of rSac3 eliminates its neurite-outgrowth-promoting activity. These results indicate that rSac3 promotes neurite outgrowth in differentiating neurons through its PIPPase activity, suggesting that Sac domain PIPPase proteins may participate in forward membrane trafficking from the endoplasmic reticulum and Golgi complex to the plasma membrane, and may function as regulators of this crucial process of neuronal cell growth and differentiation.展开更多
Objective Tyro-3 and Axl receptors are expressed in brain in a region-specific manner and their bioactivities in the central nervous system remain still elusive.The aim of the present study was to investigate their fu...Objective Tyro-3 and Axl receptors are expressed in brain in a region-specific manner and their bioactivities in the central nervous system remain still elusive.The aim of the present study was to investigate their functions in neuronal differentiation.Methods PC12 cells overexpressing Tyro-3 or Axl were established by transfection with full-length CMV-Tyro3-eCFP or CMV-Axl-eGFP plasmid,respectively.CMV-eGFP plasmid served as a control vector.After that,the fluorescence intensity and distributions of green fluorescent protein （GFP） and cyan fluorescent protein （CFP） in the cells with or without nerve growth factor （NGF） treatment were real-time monitored.Results Expressions of Tyro-3 and Axl receptors were under the regulation of NGF and associated with neuronal differentiation.This was not observed in CMV-eGFP-transfected PC12 cells.Besides,confocal microscopy revealed that NGF affected intracellular localization of full-length Axl-eGFP and Tyro-3eCFP in PC12 cells.Moreover,the development of outgrowth of differentiated PC12 cells under stimulation of NGF was promoted by overexpression of Tyro-3 or Axl.Conclusion Expressions of Tyro-3 and Axl receptors are under the regulation of NGF and are involved in NGF-induced neuronal differentiation of PC12 cells.展开更多
Objective To study the apoptotic effects of 1 methyl 4 phenyl 1,2,3,6 tetrahydropyridine (MPTP) on the nigral dopaminergic neurons of mice and 1 methyl 4 phenylpyridium ion (MPP +) on pheochromocytoma (P...Objective To study the apoptotic effects of 1 methyl 4 phenyl 1,2,3,6 tetrahydropyridine (MPTP) on the nigral dopaminergic neurons of mice and 1 methyl 4 phenylpyridium ion (MPP +) on pheochromocytoma (PC12) cells, as well as the antagonism of Eldepryl against MPTP's apoptotic effect Methods Three groups of C 57 BL mice were treated with MPTP, Eldepryl plus MPTP and normal saline, respectively, for 7 days before performing TUNEL (terminal deoxyneucleotidyl transferase mediated dUTP x nick end labeling) and FACS (fluorescence activated cell sorting) analyses of neuronal apoptosis in the substantia nigra The same tests were employed in cell culture to examine apoptosis in PC12 cells treated with MPP +, MPTP or PBS Results Intraperitoneal administration of MPTP 30?mg/kg could induce nigral apoptosis, and oral use of Eldepryl prior to MPTP treatment could completely prevent the nigral apoptosis caused by MPTP MPP +, an intermediate metabolite of MPTP, could lead to the apoptosis of PC12 cells, whereas MPTP itself had no such effect on PC12 cells Conclusions The experiment indicated that the neurotoxin, MPTP, might cause the death of nigral neurons through a mechanism of apoptosis and this effect might be mediated by its bioactive intermediate metabolite MPP + Eldepryl could protect the neurotoxicity from MPTP展开更多
基金We thank Dr Bin Zhang （University of Michigan, USA） for providing the MCFD2 antibodies. This work was supported in part by the Life Science Special Fund of the Chinese Academy of Sciences for Human Genome Research （KJ95T-06 and KSCX1-Y02 to BML, NHJ and MLJ）, the National Natural Science Foundation of China （30225023 and 30430240 to BML and 90208011, 30300174, 30470856, 30421005 and 30623003 to NHJ）, the National Key Basic Research and Development Program of China （2006CB500807 to BML and 2002CB713802, 2005CB522704 and 2006CB943902 to NHJ）, the National High-Tech Research and Development Program of China （2006AA02ZI99 to BML and 2006AA02Z186 to NHJ）, the Shanghai Key Project of Basic Science Research （04DZ14005 to BML and 04DZ14005, 04DZ05608, 06DJI4001 and 06DZ22032 to NHJ）, the Council of the Shanghai Municipal for Science and Technology （05814578 to NHJ）, and the US National Institutes of Health （DA013471 and DA020555 to LY）.
文摘Sac domain-containing proteins belong to a newly identified family of phosphoinositide phosphatases （the PIPPase family）. Despite well-characterized enzymatic activity, the biological functions of this mammalian Sac domain PIPPase family remain largely unknown. We identified a novel Sac domain-containing protein, rat Sac3 （rSac3）, which is widely expressed in various tissues and localized to the endoplasmic reticulum, Golgi complex and recycling endosomes, rSac3 displays PIPPase activity with PI（3）P, PI（4）P and PI（3,5）P2 as substrates in vitro, and a mutation in the catalytic core of the Sac domain abolishes its enzymatic activity. The expression of rSac3 is upregulated during nerve growth factor （NGF）-stimulated PC 12 cell neuronal differentiation, and overexpression of this protein promotes neurite outgrowth in PC 12 cells. Conversely, inhibition ofrSac3 expression by antisense oligonucleotides reduces neurite outgrowth of NGF- stimulated PC 12 cells, and the active site mutation of rSac3 eliminates its neurite-outgrowth-promoting activity. These results indicate that rSac3 promotes neurite outgrowth in differentiating neurons through its PIPPase activity, suggesting that Sac domain PIPPase proteins may participate in forward membrane trafficking from the endoplasmic reticulum and Golgi complex to the plasma membrane, and may function as regulators of this crucial process of neuronal cell growth and differentiation.
基金supported by the National Basic Research Development Program of China(No.2006CB500700)the National Natural Science Foundation of China(No. 30900421/c090201)
文摘Objective Tyro-3 and Axl receptors are expressed in brain in a region-specific manner and their bioactivities in the central nervous system remain still elusive.The aim of the present study was to investigate their functions in neuronal differentiation.Methods PC12 cells overexpressing Tyro-3 or Axl were established by transfection with full-length CMV-Tyro3-eCFP or CMV-Axl-eGFP plasmid,respectively.CMV-eGFP plasmid served as a control vector.After that,the fluorescence intensity and distributions of green fluorescent protein （GFP） and cyan fluorescent protein （CFP） in the cells with or without nerve growth factor （NGF） treatment were real-time monitored.Results Expressions of Tyro-3 and Axl receptors were under the regulation of NGF and associated with neuronal differentiation.This was not observed in CMV-eGFP-transfected PC12 cells.Besides,confocal microscopy revealed that NGF affected intracellular localization of full-length Axl-eGFP and Tyro-3eCFP in PC12 cells.Moreover,the development of outgrowth of differentiated PC12 cells under stimulation of NGF was promoted by overexpression of Tyro-3 or Axl.Conclusion Expressions of Tyro-3 and Axl receptors are under the regulation of NGF and are involved in NGF-induced neuronal differentiation of PC12 cells.
文摘Objective To study the apoptotic effects of 1 methyl 4 phenyl 1,2,3,6 tetrahydropyridine (MPTP) on the nigral dopaminergic neurons of mice and 1 methyl 4 phenylpyridium ion (MPP +) on pheochromocytoma (PC12) cells, as well as the antagonism of Eldepryl against MPTP's apoptotic effect Methods Three groups of C 57 BL mice were treated with MPTP, Eldepryl plus MPTP and normal saline, respectively, for 7 days before performing TUNEL (terminal deoxyneucleotidyl transferase mediated dUTP x nick end labeling) and FACS (fluorescence activated cell sorting) analyses of neuronal apoptosis in the substantia nigra The same tests were employed in cell culture to examine apoptosis in PC12 cells treated with MPP +, MPTP or PBS Results Intraperitoneal administration of MPTP 30?mg/kg could induce nigral apoptosis, and oral use of Eldepryl prior to MPTP treatment could completely prevent the nigral apoptosis caused by MPTP MPP +, an intermediate metabolite of MPTP, could lead to the apoptosis of PC12 cells, whereas MPTP itself had no such effect on PC12 cells Conclusions The experiment indicated that the neurotoxin, MPTP, might cause the death of nigral neurons through a mechanism of apoptosis and this effect might be mediated by its bioactive intermediate metabolite MPP + Eldepryl could protect the neurotoxicity from MPTP
