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Methods for monitoring endoplasmic reticulum stress and the unfolded protein response.

Samali A, Fitzgerald U, Deegan S, Gupta S - Int J Cell Biol (2010)

Bottom Line: Research into ER stress and the UPR continues to grow at a rapid rate as many new investigators are entering the field.There are also many researchers not working directly on ER stress, but who wish to determine whether this response is activated in the system they are studying: thus, it is important to list a standard set of criteria for monitoring UPR in different model systems.We would like to emphasize that no individual assay is guaranteed to be the most appropriate one in every situation and strongly recommend the use of multiple assays to verify UPR activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, National University of Ireland, Galway, Galway, Ireland.

ABSTRACT
The endoplasmic reticulum (ER) is the site of folding of membrane and secreted proteins in the cell. Physiological or pathological processes that disturb protein folding in the endoplasmic reticulum cause ER stress and activate a set of signaling pathways termed the Unfolded Protein Response (UPR). The UPR can promote cellular repair and sustained survival by reducing the load of unfolded proteins through upregulation of chaperones and global attenuation of protein synthesis. Research into ER stress and the UPR continues to grow at a rapid rate as many new investigators are entering the field. There are also many researchers not working directly on ER stress, but who wish to determine whether this response is activated in the system they are studying: thus, it is important to list a standard set of criteria for monitoring UPR in different model systems. Here, we discuss approaches that can be used by researchers to plan and interpret experiments aimed at evaluating whether the UPR and related processes are activated. We would like to emphasize that no individual assay is guaranteed to be the most appropriate one in every situation and strongly recommend the use of multiple assays to verify UPR activation.

No MeSH data available.


Related in: MedlinePlus

Detection of protein levels of UPR target genes. (a) Immunohistochemical detection of CHOP, GRP78, and XBP1 in Multiple Sclerosis patient postmortem brain tissue. Representative images showing upregulation of CHOP (ii) at the edge (LE) of a chronic active lesion, in comparison to (i) NAWM. GRP78 expression was downregulated in the center of a chronic active lesion (iii) when compared to the edge (iv) of actively demyelinating lesions. Sample images illustrate the variety of morphologically distinct cell types that express CHOP or GRP78 including macrophages (Mø), astrocytes (a), and oligodendrocytes (o). Increased expression of XBP1 was found at the edge of a chronic active lesion (vi), when compared to normal-appearing white matter (v). XBP1 immunostaining is also apparent in a large number of oligodendrocytes (o). All immunoperoxidase-stained cells were detected using the chromogen, DAB (brown), and counterstained with hematoxylin for nuclei (blue). Scale bars = 250 μm. Letter codes are as follows: NAWM = normal-appearing white matter; LC = lesion center; LE = lesion edge. Red astrices indicate location of lesion within brain sections analyzed. (b) PC12 cells were treated with 0.25 μM of Tg for 0, 2, 4, 6, and 8 hours. Whole cell lysates were analyzed by Western Blot for GRP78, CHOP, spliced XBP1, phospho-eIF-2α, and total- eIF-2α. β-Actin was used to determine equal loading of samples.
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fig2: Detection of protein levels of UPR target genes. (a) Immunohistochemical detection of CHOP, GRP78, and XBP1 in Multiple Sclerosis patient postmortem brain tissue. Representative images showing upregulation of CHOP (ii) at the edge (LE) of a chronic active lesion, in comparison to (i) NAWM. GRP78 expression was downregulated in the center of a chronic active lesion (iii) when compared to the edge (iv) of actively demyelinating lesions. Sample images illustrate the variety of morphologically distinct cell types that express CHOP or GRP78 including macrophages (Mø), astrocytes (a), and oligodendrocytes (o). Increased expression of XBP1 was found at the edge of a chronic active lesion (vi), when compared to normal-appearing white matter (v). XBP1 immunostaining is also apparent in a large number of oligodendrocytes (o). All immunoperoxidase-stained cells were detected using the chromogen, DAB (brown), and counterstained with hematoxylin for nuclei (blue). Scale bars = 250 μm. Letter codes are as follows: NAWM = normal-appearing white matter; LC = lesion center; LE = lesion edge. Red astrices indicate location of lesion within brain sections analyzed. (b) PC12 cells were treated with 0.25 μM of Tg for 0, 2, 4, 6, and 8 hours. Whole cell lysates were analyzed by Western Blot for GRP78, CHOP, spliced XBP1, phospho-eIF-2α, and total- eIF-2α. β-Actin was used to determine equal loading of samples.

Mentions: We recommend determining the protein levels of established UPR target genes whose induction has been reported to occur during relevant conditions of ER stress. Activation of the UPR has been found in various pathological states of the brain including ischemia and degenerative diseases. Increased phosphorylation of PERK has been shown after cerebral Ischemia and reperfusion by immunohistochemical analysis [32]. Several postmortem studies of primary human Alzheimer's disease brain tissues show evidence of ER stress in the form of enhanced ER chaperone expression and immunohistochemical reactivity for specific markers of the UPR [33, 34]. Recently we found increased expression of GRP78, CHOP, and XBP1 in acute, active, and chronic multiple sclerosis (MS) lesions by immunohistochemical and dual-immunofluorescent analyses [35]. Figure 2(a) shows immunohistochemical staining of fixed frozen paraffin-embedded (FFPE) brain tissue sections from MS patient which showed upregulation of CHOP, GRP78, and XBP1. Specific antibodies used are detailed in Table 1. FFPE tissue was used in preference to frozen blocks as in our hands it yielded higher quality staining with lower background and fewer staining artifacts. Following deparaffinization, all sections were incubated for 10 minutes at room temperature in 3% hydrogen peroxide in methanol (Sigma-Aldrich, Dublin), to block endogenous peroxidases. For CHOP and GRP78 staining, antigen retrieval was achieved by incubating sections in 0.01 M Tris-EDTA pH 9 (Sigma-Aldrich, Dublin) for 2 minutes in a Tefal pressure cooker at full steam. To retrieve antigen before XBP1 staining, tissue was placed in 0.01 M citrate pH 9 (Sigma-Aldrich, Dublin) before microwaving it for 20 minutes in a 700 watt Sanyo microwave. Bound CHOP, GRP78, or XBP1 antibody was detected following incubation for 30 minutes at room temperature in peroxidase-labeled EnVision anti-mouse or anti-rabbit antibody (Dako, Ely, UK) with 3,3′- diaminobenzidine (DAB) as a chromogen (Dako, Ely, UK).


Methods for monitoring endoplasmic reticulum stress and the unfolded protein response.

Samali A, Fitzgerald U, Deegan S, Gupta S - Int J Cell Biol (2010)

Detection of protein levels of UPR target genes. (a) Immunohistochemical detection of CHOP, GRP78, and XBP1 in Multiple Sclerosis patient postmortem brain tissue. Representative images showing upregulation of CHOP (ii) at the edge (LE) of a chronic active lesion, in comparison to (i) NAWM. GRP78 expression was downregulated in the center of a chronic active lesion (iii) when compared to the edge (iv) of actively demyelinating lesions. Sample images illustrate the variety of morphologically distinct cell types that express CHOP or GRP78 including macrophages (Mø), astrocytes (a), and oligodendrocytes (o). Increased expression of XBP1 was found at the edge of a chronic active lesion (vi), when compared to normal-appearing white matter (v). XBP1 immunostaining is also apparent in a large number of oligodendrocytes (o). All immunoperoxidase-stained cells were detected using the chromogen, DAB (brown), and counterstained with hematoxylin for nuclei (blue). Scale bars = 250 μm. Letter codes are as follows: NAWM = normal-appearing white matter; LC = lesion center; LE = lesion edge. Red astrices indicate location of lesion within brain sections analyzed. (b) PC12 cells were treated with 0.25 μM of Tg for 0, 2, 4, 6, and 8 hours. Whole cell lysates were analyzed by Western Blot for GRP78, CHOP, spliced XBP1, phospho-eIF-2α, and total- eIF-2α. β-Actin was used to determine equal loading of samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2821749&req=5

fig2: Detection of protein levels of UPR target genes. (a) Immunohistochemical detection of CHOP, GRP78, and XBP1 in Multiple Sclerosis patient postmortem brain tissue. Representative images showing upregulation of CHOP (ii) at the edge (LE) of a chronic active lesion, in comparison to (i) NAWM. GRP78 expression was downregulated in the center of a chronic active lesion (iii) when compared to the edge (iv) of actively demyelinating lesions. Sample images illustrate the variety of morphologically distinct cell types that express CHOP or GRP78 including macrophages (Mø), astrocytes (a), and oligodendrocytes (o). Increased expression of XBP1 was found at the edge of a chronic active lesion (vi), when compared to normal-appearing white matter (v). XBP1 immunostaining is also apparent in a large number of oligodendrocytes (o). All immunoperoxidase-stained cells were detected using the chromogen, DAB (brown), and counterstained with hematoxylin for nuclei (blue). Scale bars = 250 μm. Letter codes are as follows: NAWM = normal-appearing white matter; LC = lesion center; LE = lesion edge. Red astrices indicate location of lesion within brain sections analyzed. (b) PC12 cells were treated with 0.25 μM of Tg for 0, 2, 4, 6, and 8 hours. Whole cell lysates were analyzed by Western Blot for GRP78, CHOP, spliced XBP1, phospho-eIF-2α, and total- eIF-2α. β-Actin was used to determine equal loading of samples.
Mentions: We recommend determining the protein levels of established UPR target genes whose induction has been reported to occur during relevant conditions of ER stress. Activation of the UPR has been found in various pathological states of the brain including ischemia and degenerative diseases. Increased phosphorylation of PERK has been shown after cerebral Ischemia and reperfusion by immunohistochemical analysis [32]. Several postmortem studies of primary human Alzheimer's disease brain tissues show evidence of ER stress in the form of enhanced ER chaperone expression and immunohistochemical reactivity for specific markers of the UPR [33, 34]. Recently we found increased expression of GRP78, CHOP, and XBP1 in acute, active, and chronic multiple sclerosis (MS) lesions by immunohistochemical and dual-immunofluorescent analyses [35]. Figure 2(a) shows immunohistochemical staining of fixed frozen paraffin-embedded (FFPE) brain tissue sections from MS patient which showed upregulation of CHOP, GRP78, and XBP1. Specific antibodies used are detailed in Table 1. FFPE tissue was used in preference to frozen blocks as in our hands it yielded higher quality staining with lower background and fewer staining artifacts. Following deparaffinization, all sections were incubated for 10 minutes at room temperature in 3% hydrogen peroxide in methanol (Sigma-Aldrich, Dublin), to block endogenous peroxidases. For CHOP and GRP78 staining, antigen retrieval was achieved by incubating sections in 0.01 M Tris-EDTA pH 9 (Sigma-Aldrich, Dublin) for 2 minutes in a Tefal pressure cooker at full steam. To retrieve antigen before XBP1 staining, tissue was placed in 0.01 M citrate pH 9 (Sigma-Aldrich, Dublin) before microwaving it for 20 minutes in a 700 watt Sanyo microwave. Bound CHOP, GRP78, or XBP1 antibody was detected following incubation for 30 minutes at room temperature in peroxidase-labeled EnVision anti-mouse or anti-rabbit antibody (Dako, Ely, UK) with 3,3′- diaminobenzidine (DAB) as a chromogen (Dako, Ely, UK).

Bottom Line: Research into ER stress and the UPR continues to grow at a rapid rate as many new investigators are entering the field.There are also many researchers not working directly on ER stress, but who wish to determine whether this response is activated in the system they are studying: thus, it is important to list a standard set of criteria for monitoring UPR in different model systems.We would like to emphasize that no individual assay is guaranteed to be the most appropriate one in every situation and strongly recommend the use of multiple assays to verify UPR activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, National University of Ireland, Galway, Galway, Ireland.

ABSTRACT
The endoplasmic reticulum (ER) is the site of folding of membrane and secreted proteins in the cell. Physiological or pathological processes that disturb protein folding in the endoplasmic reticulum cause ER stress and activate a set of signaling pathways termed the Unfolded Protein Response (UPR). The UPR can promote cellular repair and sustained survival by reducing the load of unfolded proteins through upregulation of chaperones and global attenuation of protein synthesis. Research into ER stress and the UPR continues to grow at a rapid rate as many new investigators are entering the field. There are also many researchers not working directly on ER stress, but who wish to determine whether this response is activated in the system they are studying: thus, it is important to list a standard set of criteria for monitoring UPR in different model systems. Here, we discuss approaches that can be used by researchers to plan and interpret experiments aimed at evaluating whether the UPR and related processes are activated. We would like to emphasize that no individual assay is guaranteed to be the most appropriate one in every situation and strongly recommend the use of multiple assays to verify UPR activation.

No MeSH data available.


Related in: MedlinePlus