Late-Onset of Spinal Neurodegeneration in Knock-In Mice Expressing a Mutant BiP

<div><p>Most human neurodegenerative diseases are sporadic, and appear later in life. While the underlying mechanisms of the progression of those diseases are still unclear, investigations into the familial forms of comparable diseases suggest that endoplasmic reticulum (ER) stress is involved in the pathogenesis. Binding immunoglobulin protein (BiP) is an ER chaperone that is central to ER function. We produced knock-in mice expressing a mutant BiP that lacked the retrieval sequence in order to evaluate the effect of a functional defect in an ER chaperone in multi-cellular organisms. Here we report that heterozygous mutant BiP mice revealed motor disabilities in aging. We found a degeneration of some motoneurons in the spinal cord accompanied by accumulations of ubiquitinated proteins. The defect in retrieval of BiP by the KDEL receptor leads to impaired activities in quality control and autophagy, suggesting that functional defects in the ER chaperones may contribute to the late onset of neurodegenerative diseases.</p></div

Motoneurons at the anterior horn of spinal cords of aged mutant BiP mice suffer from ER stress.

<p>(A) Motoneurons stained by an anti-choline acetyltransferase antibody (red) at the anterior horn in the spinal cord of both a 6 month-old wild type (+/+, 6 m) and a 6 month-old mutant BiP mouse (Bm/+, 6 m) express ER chaperones as well (green). Scale bars, 20 um. (B) The immunoreactivity with an anti-choline acetyltransferase antibody at the anterior horn is reduced in the aged 29 month-old mutant spinal cord (Bm/+, 29 m). Scale bars, 20 um. (C) Large cells at the anterior horn of the aged 29 month-old mutant spinal cord (Bm/+, 29 m) express ER chaperones as well as CHOP. Scale bars, 10 um. The nuclei were stained with Hoechst 33258 (blue, A and C).</p

The expressions of chaperones in the mutant BiP mice.

<p>The heterozygous mutant BiP mice and the litter mate wild type mice were anesthetized by pentobarbital, and the brains and spinal cords were removed. They were subjected to Western blot analysis with an anti-KDEL mouse mAb for BiP and GRP94, an anti-HA mouse mAb for mutant BiP, an anti-CHOP rabbit antiserum, and an anti-SOD1 rabbit antiserum.</p

Aggregations were obvious in the mutant BiP MEF.

<p>The aggregations by transient expressions of SOD1-GFP were evaluated by immunofluorescence microscopy with labeling by using a rabbit anti-Derlin1 antibody for the ER staining (red) and SOD1-GFP (green) in wild type (+/+) and the homozygous mutant (Bm/Bm) MEF with Hoechst 33342 for nuclear staining. Scale bars, 10 um. Aggregations of SOD1were observed in the mutant BiP MEF as well as in the wild type MEF treated with a proteasome inhibitor, ALLN (10 ug/ml), at 37°C for 12 h.</p

Some motoneurons in the spinal cord revealed a degeneration accompanied by accumulations of ubiquitinated proteins.

<p>(A) TUNEL staining revealed some apoptotic cells at the anterior horn in the spinal cord of a 29 month-old mutant BiP mouse (Bm/+, 29 m). Scale bars, 10 um. (B) The immunoreactivity with an anti-GFAP antibody at the anterior horn is increased in a 17 month-old mutant spinal cord (Bm/+, 17 m). Scale bars, 20 um. GFAP positive cells are counted (five fields in each mouse, GFAP positive cells/the number of nucleus). +/+, 16 m; 33/199, 35/174, 34/192, 40/181, 42/207, Bm/+, 17 m; 42/132, 50/140, 59/147, 39/154, 58/143 +/+, 6 m; 5/143, 4/131, 4/141, 0/95, 0/107. The ratio of GFAP positive cells is significantly higher in the mutant BiP spinal cord (Bm/+, 17 m) compared to that in the wild type (+/+, 16 m) by t test (p value is 0.0009). (C) The aggregations were stained by an anti-ubiquitin antibody in the large cells at the anterior horn of the 29 month-old mutant spinal cord (Bm/+, 29 m, arrowheads). Scale bars, 10 um.</p

Downregulation of RAS inhibits growth and enhances cytotoxicity of doxorubicin and bortezomib in <i>RAS</i>-mutated MM cell lines.

<p>(A) INA6 cells were transiently transfected with non-targeting or <i>NRAS</i> siRNA, and RPMI-8226 and RPMI-8226 DOX40 cells were transiently transfected with non-targeting or <i>KRAS</i> siRNA. The cell number and viability 48 h later were assessed with trypan blue exclusion. Whole-cell lysates were subjected to western blotting to confirm the downregulation of NRAS and KRAS expression using NRAS, KRAS, HRAS, and β-actin Abs. Data are the mean ± SD of triplicate wells. (B) INA6 and NCI-H929 cells were transiently transfected with non-targeting or <i>NRAS</i> siRNA, and RPMI-8226 and RPMI-8226 DOX40 cells were transiently transfected with non-targeting or <i>KRAS</i> siRNA. After 48 h, whole-cell lysates were subjected to western blotting using p27, cyclin D1, NRAS and β-actin Abs. (C) INA6 cells were transiently transfected with non-targeting or <i>NRAS</i> siRNA and then treated with or without bortezomib (5 nM) for 48 h. RPMI-8226 cells were transiently transfected with non-targeting or <i>KRAS</i> siRNA and then treated with or without doxorubicin (0.1 μM) for 48 h. In each case, cell viability was assessed with the MTT assay of triplicate cultures and expressed as the percentage of the untreated control. Data are the mean ± SD. (D) RPMI-8226 DOX40 cells were transiently transfected with non-targeting or <i>KRAS</i> siRNA and then treated with or without doxorubicin (1 μM) for 24 h. Cell viability was assessed with the MTT assay of triplicate cultures and expressed as the percentage of the untreated control. Data are the mean ± SD.</p

TAS-116 effects on cell viability and RAS-RAF-MEK-ERK signaling in <i>RAS</i>-mutated MM cell lines.

<p>(A) NCI-H929, INA6, MM.1S, and RPMI-8226 MM cell lines were cultured with TAS-116 (0–5 μM) for 24, 48, or 72 h. In each case, cell viability was assessed with the MTT assay of triplicate cultures and expressed as the percentage of the untreated control. Data are the mean ± SD. (B) NCI-H929 and RPMI-8226 cells were treated with the indicated doses of TAS-116 for 24 h. Whole-cell lysates were subjected to western blotting using p-B-Raf, B-Raf, p-C-Raf, C-Raf, p-MEK1/2, MEK1/2, p-ERK, ERK, p-Akt (S473), Akt, PARP, and β-actin Abs. FL, full-length; CF, cleaved form.</p

Combination of TAS-116 plus tipifarnib, dabrafenib, or AZD6244 blocks the growth stimulatory effect of the bone marrow microenvironment.

<p>(A) NCI-H929, INA6, MM.1S, and RPMI-8226 cells were treated with TAS-116 (1 μM) either alone or in combination with tipifarnib (NCI-H929: 2 μM, INA6: 0.5 μM, MM.1S: 2 μM, RPMI-8226: 2 μM), dabrafenib (NCI-H929: 10 μM, INA6: 2 μM, MM.1S: 10 μM, RPMI-8226: 10 μM), or AZD6244 (NCI-H929: 10 μM, INA6: 2 μM, MM.1S: 20 μM, RPMI-8226: 20 μM) for 48 h. Apoptotic cells were analyzed with flow cytometry using annexin V/PI staining. Each treatment was tested in triplicate wells, and apoptosis was assessed as the percentage of annexin V-positive cells. TAS, TAS-116; Tipi, tipifarnib; Dabra, dabrafenib; AZD, AZD6244. (B) MM.1S and NCI-H929 cells were cultured with TAS-116 (2 μM), AZD6244 (20 μM), or TAS-116 plus AZD6244 for 24 h in the presence or absence of BMSC supernatant. Whole-cell lysates were subjected to western blotting using PARP, caspase 3, and β-actin Abs. FL, full-length; CF, cleaved form; SN, supernatant. (C) MM.1S cells were cultured with TAS-116 (1 μM), AZD6244 (20 μM), or TAS-116 plus AZD6244; and NCI-H929 cells were cultured with TAS-116 (1 μM), AZD6244 (10 μM), or TAS-116 plus AZD6244 for 48 h in the presence or absence of BMSC supernatant. Apoptotic cells were analyzed with flow cytometry using annexin V/PI staining. Each treatment was tested in triplicate wells, and apoptosis was assessed as the percentage of annexin V-positive cells. TAS, TAS-116; AZD, AZD6244; SN, supernatant (*: <i>P</i> < 0.05; **: <i>P</i> < 0.01).</p

TAS-116 induces synergistic cytotoxicity with dabrafenib in the BRAF-mutated U266 MM cell line.

<p>U266 cells were cultured with TAS-116 (0–5 μM) (A) or dabrafenib (0–5 μM) (B) for 24, 48, or 72 h. Cell viability was assessed with the MTT assay of triplicate cultures and expressed as the percentage of the untreated control. Data are the mean ± SD. (C) U266 cells were treated with the indicated concentrations of TAS-116, dabrafenib, or TAS-116 plus dabrafenib for 48 h, and then the viability was analyzed with the MTT assay. Isobologram analysis shows the synergistic cytotoxic effect of TAS-116 and dabrafenib. The graph (left) is derived from the values given in the table (right). The numbers 1–4 in the graph correspond to the combinations shown in the table. CI values < 1.0 indicate synergism; CI = 1.0 indicates an additive effect; and CI > 1.0 indicates antagonism. (D) U266 cells were treated with TAS-116 (0.5 μM), dabrafenib (1 μM), or TAS-116 plus dabrafenib for 48 h. Apoptotic cells were analyzed with flow cytometry using annexin V/PI staining. Each treatment was tested in triplicate wells, and apoptosis was assessed as the percentage of annexin V-positive cells. (E) U266 cells were treated with TAS-116 (1 μM), dabrafenib (2 μM), or TAS-116 plus dabrafenib for 24 h. Whole-cell lysates were subjected to western blotting using p-B-Raf, B-Raf, p-C-Raf, C-Raf, p-MEK1/2, MEK1/2, p-ERK, ERK, p-Akt (S473), Akt, PARP, caspase 3, and β-actin Abs. FL, full-length; CF, cleaved form.</p

RAS pathway inhibitors induce cytotoxicity and apoptosis in <i>RAS</i>-mutated MM cell lines.

<p>(A) NCI-H929, INA6, MM.1S, and RPMI-8226 MM cell lines were cultured with tipifarnib (0–20 μM), dabrafenib (0–20 μM), or AZD6244 (0–20 μM) for 72 h. In each case, cell viability was assessed with the MTT assay of triplicate cultures and expressed as the percentage of the untreated control. Data are the mean ± SD. (B) NCI-H929 and RPMI-8226 cells were treated with TAS-116 (3 μM), tipifarnib (10 μM), dabrafenib (20 μM), or AZD6244 (20 μM) for 24 h. Whole-cell lysates were subjected to western blotting using NRAS, KRAS, p-B-Raf, B-Raf, p-C-Raf, C-Raf, p-MEK1/2, MEK1/2, p-ERK, ERK, p-Akt (S473), Akt, PARP, and β-actin Abs. FL, full-length; CF, cleaved form. (C) NCI-H929, INA6, MM.1S, and RPMI-8226 cells were treated with tipifarnib (0–20 μM), dabrafenib (0–20 μM), or AZD6244 (0–20 μM) for 48 h. Apoptotic cells were analyzed with flow cytometry using annexin V/PI staining. Each treatment was tested in triplicate wells, and apoptosis was assessed as the percentage of annexin V-positive cells.</p