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The intracellular accumulation of polymeric neuroserpin explains the severity of the dementia FENIB.

Miranda E, MacLeod I, Davies MJ, Pérez J, Römisch K, Crowther DC, Lomas DA - Hum. Mol. Genet. (2008)

Bottom Line: Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is an autosomal dominant dementia that is characterized by the retention of polymers of neuroserpin as inclusions within the endoplasmic reticulum (ER) of neurons.We show a direct correlation between the severity of the disease-causing mutation and the accumulation of neuroserpin polymers in cell and fly models of the disease.Moreover, mutant neuroserpin causes locomotor deficits in the fly allowing us to demonstrate a direct link between polymer accumulation and neuronal toxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK. em285@cam.ac.uk

ABSTRACT
Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is an autosomal dominant dementia that is characterized by the retention of polymers of neuroserpin as inclusions within the endoplasmic reticulum (ER) of neurons. We have developed monoclonal antibodies that detect polymerized neuroserpin and have used COS-7 cells, stably transfected PC12 cell lines and transgenic Drosophila melanogaster to characterize the cellular handling of all four mutant forms of neuroserpin that cause FENIB. We show a direct correlation between the severity of the disease-causing mutation and the accumulation of neuroserpin polymers in cell and fly models of the disease. Moreover, mutant neuroserpin causes locomotor deficits in the fly allowing us to demonstrate a direct link between polymer accumulation and neuronal toxicity.

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Related in: MedlinePlus

Trafficking of wild-type and mutant neuroserpin in stably transfected PC12 cell lines. (A) Lysates from PC12-Tet-On, PC12-Wt, PC12-S52R and PC12-G392E cells cultured with (on) or without (off) 10 µg/ml doxycycline to induce neuroserpin expression were analysed by SDS and non-denaturing PAGE followed by western-blot analysis with an anti-neuroserpin polyclonal antibody. The membrane from the SDS–PAGE was also analysed with an anti-GAPDH antibody as a loading control. (B) Neuroserpin from lysed PC12-Tet-On, PC12-Wt, PC12-S52R and PC12-G392E cells induced for 4 days was immunoprecipitated with the 1A10 anti-neuroserpin monoclonal antibody and treated (+) or not (−) with endoglycosidase H (eH) and analysed by SDS–PAGE and western blot analysis. S52R and G392E neuroserpin were sensitive to endoglycosidase H (arrow) whereas wild-type neuroserpin was resistant (arrowhead). (C) Immuno-co-localization of wild-type and G392E neuroserpin with resident proteins of the secretory pathway. PC12-wildtype and PC12-G392E cells were differentiated to a neuronal phenotype by plating in collagen and treating with NGF (150 ng/ml) for 7 days, and then induced to express neuroserpin with 10 µg/ml doxycycline for 3 days. Cells were co-stained with either a polyclonal anti-neuroserpin antibody or one of the anti-neuroserpin monoclonal antibodies (1A10 for total neuroserpin or 7C6 for neuroserpin polymers) and antibodies against calreticulin (ER), GM130 (Golgi), ERGIC-53/p58 (ERGIC) or chromogranin A (trafficked through the regulated secretory pathway). The colour corresponding to each antibody (red or green) is shown above the figure and only the merged images are presented. Yellow represents areas of overlapping red and green. The nucleus appears blue due to DNA staining with DAPI. Scale bar: 10 µm. (D) PC12-Wt, PC12-S52R and PC12-G392E cells were induced to express neuroserpin for 3 days with 10 µg/ml doxycycline and then incubated for 15 min with control or release buffer containing 5 or 55 mm KCl, respectively, to assess regulated secretion from dense core secretory granules. Neuroserpin was analysed in cell lysates and buffer solutions by SDS–PAGE and western blot and quantified by sandwich ELISA. The graph shows the averages for three independent experiments analysed by ELISA, expressed as percentages ± SEM. The amount of neuroserpin secreted from each cell line when treated with release buffer was statistically different when analysed by one-way ANOVA (P = 0.0003) followed by a post-test for linear trend (R2 = 0.93, P = 0.0001).
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DDN041F4: Trafficking of wild-type and mutant neuroserpin in stably transfected PC12 cell lines. (A) Lysates from PC12-Tet-On, PC12-Wt, PC12-S52R and PC12-G392E cells cultured with (on) or without (off) 10 µg/ml doxycycline to induce neuroserpin expression were analysed by SDS and non-denaturing PAGE followed by western-blot analysis with an anti-neuroserpin polyclonal antibody. The membrane from the SDS–PAGE was also analysed with an anti-GAPDH antibody as a loading control. (B) Neuroserpin from lysed PC12-Tet-On, PC12-Wt, PC12-S52R and PC12-G392E cells induced for 4 days was immunoprecipitated with the 1A10 anti-neuroserpin monoclonal antibody and treated (+) or not (−) with endoglycosidase H (eH) and analysed by SDS–PAGE and western blot analysis. S52R and G392E neuroserpin were sensitive to endoglycosidase H (arrow) whereas wild-type neuroserpin was resistant (arrowhead). (C) Immuno-co-localization of wild-type and G392E neuroserpin with resident proteins of the secretory pathway. PC12-wildtype and PC12-G392E cells were differentiated to a neuronal phenotype by plating in collagen and treating with NGF (150 ng/ml) for 7 days, and then induced to express neuroserpin with 10 µg/ml doxycycline for 3 days. Cells were co-stained with either a polyclonal anti-neuroserpin antibody or one of the anti-neuroserpin monoclonal antibodies (1A10 for total neuroserpin or 7C6 for neuroserpin polymers) and antibodies against calreticulin (ER), GM130 (Golgi), ERGIC-53/p58 (ERGIC) or chromogranin A (trafficked through the regulated secretory pathway). The colour corresponding to each antibody (red or green) is shown above the figure and only the merged images are presented. Yellow represents areas of overlapping red and green. The nucleus appears blue due to DNA staining with DAPI. Scale bar: 10 µm. (D) PC12-Wt, PC12-S52R and PC12-G392E cells were induced to express neuroserpin for 3 days with 10 µg/ml doxycycline and then incubated for 15 min with control or release buffer containing 5 or 55 mm KCl, respectively, to assess regulated secretion from dense core secretory granules. Neuroserpin was analysed in cell lysates and buffer solutions by SDS–PAGE and western blot and quantified by sandwich ELISA. The graph shows the averages for three independent experiments analysed by ELISA, expressed as percentages ± SEM. The amount of neuroserpin secreted from each cell line when treated with release buffer was statistically different when analysed by one-way ANOVA (P = 0.0003) followed by a post-test for linear trend (R2 = 0.93, P = 0.0001).

Mentions: Neuroserpin is constitutively secreted in COS-7 cells. However, in glandular and neural tissues, neuroserpin is trafficked through the regulated secretory pathway, stored in dense core vesicles and secreted upon stimulation (22–24). We therefore developed stably transfected PC12 cell lines that conditionally expressed wild-type neuroserpin (PC12-Wt) or mutants that cause moderate (S52R; PC12-S52R) and severe (G392E; PC12-G392E) forms of FENIB (Fig. 4A). Neuroserpin was detected by SDS–PAGE and western-blot analysis in lysates of the three cell lines following induction with doxycycline (Fig. 4A, top panel, lanes On). The steady-state level of neuroserpin was lower for cells expressing the mutant proteins. This must be due to increased ER-associated degradation of S52R and G392E neuroserpin, as the three cell lines expressed similar levels of protein when assessed by the pulse-chase analysis (results not shown) and both mutant proteins accumulated within the cells upon treatment with the proteasomal inhibitors lactacystin and MG132 (data not shown). Non-denaturing PAGE and western-blot analysis of cell lysate from PC12-Wt cells showed a single band characteristic of monomeric wild-type neuroserpin (Fig. 4A, bottom panel, lane On/Wt, arrow), while PC12-S52R and PC12-G392E cell lysates showed high molecular mass ladders typical of polymers (Fig. 4A, bottom panel, lanes On/S52R and On/G392E, curly bracket). A weak monomeric band was also apparent in lysates from the PC12-S52R cells (Fig. 4A, bottom panel, lane On/S52R, arrow).


The intracellular accumulation of polymeric neuroserpin explains the severity of the dementia FENIB.

Miranda E, MacLeod I, Davies MJ, Pérez J, Römisch K, Crowther DC, Lomas DA - Hum. Mol. Genet. (2008)

Trafficking of wild-type and mutant neuroserpin in stably transfected PC12 cell lines. (A) Lysates from PC12-Tet-On, PC12-Wt, PC12-S52R and PC12-G392E cells cultured with (on) or without (off) 10 µg/ml doxycycline to induce neuroserpin expression were analysed by SDS and non-denaturing PAGE followed by western-blot analysis with an anti-neuroserpin polyclonal antibody. The membrane from the SDS–PAGE was also analysed with an anti-GAPDH antibody as a loading control. (B) Neuroserpin from lysed PC12-Tet-On, PC12-Wt, PC12-S52R and PC12-G392E cells induced for 4 days was immunoprecipitated with the 1A10 anti-neuroserpin monoclonal antibody and treated (+) or not (−) with endoglycosidase H (eH) and analysed by SDS–PAGE and western blot analysis. S52R and G392E neuroserpin were sensitive to endoglycosidase H (arrow) whereas wild-type neuroserpin was resistant (arrowhead). (C) Immuno-co-localization of wild-type and G392E neuroserpin with resident proteins of the secretory pathway. PC12-wildtype and PC12-G392E cells were differentiated to a neuronal phenotype by plating in collagen and treating with NGF (150 ng/ml) for 7 days, and then induced to express neuroserpin with 10 µg/ml doxycycline for 3 days. Cells were co-stained with either a polyclonal anti-neuroserpin antibody or one of the anti-neuroserpin monoclonal antibodies (1A10 for total neuroserpin or 7C6 for neuroserpin polymers) and antibodies against calreticulin (ER), GM130 (Golgi), ERGIC-53/p58 (ERGIC) or chromogranin A (trafficked through the regulated secretory pathway). The colour corresponding to each antibody (red or green) is shown above the figure and only the merged images are presented. Yellow represents areas of overlapping red and green. The nucleus appears blue due to DNA staining with DAPI. Scale bar: 10 µm. (D) PC12-Wt, PC12-S52R and PC12-G392E cells were induced to express neuroserpin for 3 days with 10 µg/ml doxycycline and then incubated for 15 min with control or release buffer containing 5 or 55 mm KCl, respectively, to assess regulated secretion from dense core secretory granules. Neuroserpin was analysed in cell lysates and buffer solutions by SDS–PAGE and western blot and quantified by sandwich ELISA. The graph shows the averages for three independent experiments analysed by ELISA, expressed as percentages ± SEM. The amount of neuroserpin secreted from each cell line when treated with release buffer was statistically different when analysed by one-way ANOVA (P = 0.0003) followed by a post-test for linear trend (R2 = 0.93, P = 0.0001).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2387220&req=5

DDN041F4: Trafficking of wild-type and mutant neuroserpin in stably transfected PC12 cell lines. (A) Lysates from PC12-Tet-On, PC12-Wt, PC12-S52R and PC12-G392E cells cultured with (on) or without (off) 10 µg/ml doxycycline to induce neuroserpin expression were analysed by SDS and non-denaturing PAGE followed by western-blot analysis with an anti-neuroserpin polyclonal antibody. The membrane from the SDS–PAGE was also analysed with an anti-GAPDH antibody as a loading control. (B) Neuroserpin from lysed PC12-Tet-On, PC12-Wt, PC12-S52R and PC12-G392E cells induced for 4 days was immunoprecipitated with the 1A10 anti-neuroserpin monoclonal antibody and treated (+) or not (−) with endoglycosidase H (eH) and analysed by SDS–PAGE and western blot analysis. S52R and G392E neuroserpin were sensitive to endoglycosidase H (arrow) whereas wild-type neuroserpin was resistant (arrowhead). (C) Immuno-co-localization of wild-type and G392E neuroserpin with resident proteins of the secretory pathway. PC12-wildtype and PC12-G392E cells were differentiated to a neuronal phenotype by plating in collagen and treating with NGF (150 ng/ml) for 7 days, and then induced to express neuroserpin with 10 µg/ml doxycycline for 3 days. Cells were co-stained with either a polyclonal anti-neuroserpin antibody or one of the anti-neuroserpin monoclonal antibodies (1A10 for total neuroserpin or 7C6 for neuroserpin polymers) and antibodies against calreticulin (ER), GM130 (Golgi), ERGIC-53/p58 (ERGIC) or chromogranin A (trafficked through the regulated secretory pathway). The colour corresponding to each antibody (red or green) is shown above the figure and only the merged images are presented. Yellow represents areas of overlapping red and green. The nucleus appears blue due to DNA staining with DAPI. Scale bar: 10 µm. (D) PC12-Wt, PC12-S52R and PC12-G392E cells were induced to express neuroserpin for 3 days with 10 µg/ml doxycycline and then incubated for 15 min with control or release buffer containing 5 or 55 mm KCl, respectively, to assess regulated secretion from dense core secretory granules. Neuroserpin was analysed in cell lysates and buffer solutions by SDS–PAGE and western blot and quantified by sandwich ELISA. The graph shows the averages for three independent experiments analysed by ELISA, expressed as percentages ± SEM. The amount of neuroserpin secreted from each cell line when treated with release buffer was statistically different when analysed by one-way ANOVA (P = 0.0003) followed by a post-test for linear trend (R2 = 0.93, P = 0.0001).
Mentions: Neuroserpin is constitutively secreted in COS-7 cells. However, in glandular and neural tissues, neuroserpin is trafficked through the regulated secretory pathway, stored in dense core vesicles and secreted upon stimulation (22–24). We therefore developed stably transfected PC12 cell lines that conditionally expressed wild-type neuroserpin (PC12-Wt) or mutants that cause moderate (S52R; PC12-S52R) and severe (G392E; PC12-G392E) forms of FENIB (Fig. 4A). Neuroserpin was detected by SDS–PAGE and western-blot analysis in lysates of the three cell lines following induction with doxycycline (Fig. 4A, top panel, lanes On). The steady-state level of neuroserpin was lower for cells expressing the mutant proteins. This must be due to increased ER-associated degradation of S52R and G392E neuroserpin, as the three cell lines expressed similar levels of protein when assessed by the pulse-chase analysis (results not shown) and both mutant proteins accumulated within the cells upon treatment with the proteasomal inhibitors lactacystin and MG132 (data not shown). Non-denaturing PAGE and western-blot analysis of cell lysate from PC12-Wt cells showed a single band characteristic of monomeric wild-type neuroserpin (Fig. 4A, bottom panel, lane On/Wt, arrow), while PC12-S52R and PC12-G392E cell lysates showed high molecular mass ladders typical of polymers (Fig. 4A, bottom panel, lanes On/S52R and On/G392E, curly bracket). A weak monomeric band was also apparent in lysates from the PC12-S52R cells (Fig. 4A, bottom panel, lane On/S52R, arrow).

Bottom Line: Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is an autosomal dominant dementia that is characterized by the retention of polymers of neuroserpin as inclusions within the endoplasmic reticulum (ER) of neurons.We show a direct correlation between the severity of the disease-causing mutation and the accumulation of neuroserpin polymers in cell and fly models of the disease.Moreover, mutant neuroserpin causes locomotor deficits in the fly allowing us to demonstrate a direct link between polymer accumulation and neuronal toxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK. em285@cam.ac.uk

ABSTRACT
Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is an autosomal dominant dementia that is characterized by the retention of polymers of neuroserpin as inclusions within the endoplasmic reticulum (ER) of neurons. We have developed monoclonal antibodies that detect polymerized neuroserpin and have used COS-7 cells, stably transfected PC12 cell lines and transgenic Drosophila melanogaster to characterize the cellular handling of all four mutant forms of neuroserpin that cause FENIB. We show a direct correlation between the severity of the disease-causing mutation and the accumulation of neuroserpin polymers in cell and fly models of the disease. Moreover, mutant neuroserpin causes locomotor deficits in the fly allowing us to demonstrate a direct link between polymer accumulation and neuronal toxicity.

Show MeSH
Related in: MedlinePlus