Figure 5

The Oxidoreductase Activity of PDI Is Protective against ER Stress and Inhibition of ER-Golgi Transport Induced by Mutant TDP-43

(A) Detection of nuclear immunoreactivity to XBP-1 in cells expressing mCherry-tagged TDP-43. Cells expressing mCherry (row 1), TDP-WT (row 2), or mutant TDP-Q331K (row 3), co-expressing PDI-WT or PDI-QUAD, or treated with BMC (rows 4, 5, 6), arrows representing XBP-1 activation.

(B) The proportion of cells expressing nuclear XBP-1 decreased when PDI-WT was co-expressed or treated with BMC (∗p < 0.05), unlike PDI-QUAD.

(C) Immunofluorescence detection of nuclear immunoreactivity to XBP-1 in EGFP-tagged TDP-43 cells. Cells expressing TDP-WT (row 1), or TDP-M337V (row 2), co-expressing PDI-WT or PDI-QUAD, or treatment with BMC (rows 3, 4, 5), arrows represent XBP-1 activation.

(D) The proportion of cells expressing nuclear XBP-1 decreased when PDI-WT was co-expressed, or BMC was administered to TDP-M337V cells (∗∗p < 0.01). More cells with nuclear XBP-1 were found in populations expressing PDI-QUAD compared with PDI-WT, and PDI-QUAD compared with BMC treatment (∗p < 0.05).

(E) Detection of nuclear immunoreactivity to CHOP in EGFP-tagged TDP-43 cells. Cells expressing pEGFP (row 1), TDP-WT (row 2), TDP-M337V (row 3), co-expressing PDI-WT or PDI-QUAD, or treated with BMC (rows 4, 5, 6), arrows represent CHOP activation.

(F) The proportion of cells expressing nuclear CHOP was decreased when PDI-WT was co-expressed or BMC was administered to TDP-M337V cells (∗∗p < 0.01). There was a significant difference between TDP-M337V cells co-expressing PDI-WT and PDI-QUAD (∗∗p < 0.01), and TDP-M337V cells co-expressing PDI-QUAD and treated with BMC (∗∗p < 0.01).

(G) PDI's oxidoreductase activity rescues inhibition of ER-Golgi transport induced by mutant TDP-43. Quantification of the degree of co-localization of VSVG^ts045 with the ER and Golgi compartments using Mander's coefficient following immunocytochemistry for calnexin and GM130. Data are presented as mean ± SEM, n = 20. A significant difference was observed (∗p < 0.05) in the co-localization between VSVG^ts045 and the ER (calnexin) between cells expressing TDP-Q331K with empty vector and those expressing PDI-WT, and also with (∗p < 0.05) BMC-treatment . A significant difference was also observed (∗p < 0.05) in co-localization between VSVG^ts045 and the Golgi (GM130) between cells expressing TDP-Q331K and PDI-WT and BMC-treated cells.

Scale bars: 10 μm in (A) and (C), 4 μm in (E).