Effects of Prolonged GRP78 Haploinsufficiency on Organ Homeostasis, Behavior, Cancer and Chemotoxic Resistance in Aged Mice

Abstract

GRP78, a multifunctional protein with potent cytoprotective properties, is an emerging therapeutic target to combat cancer development, progression and drug resistance. The biological consequences of prolonged reduction in expression of this essential chaperone which so far has been studied primarily in young mice, was investigated in older mice, as older individuals are likely to be important recipients of anti-GRP78 therapy. We followed cohorts of Grp78^+/+ and Grp78^+/- male and female mice up to 2 years of age in three different genetic backgrounds and characterized them with respect to body weight, organ integrity, behavioral and memory performance, cancer, inflammation and chemotoxic response. Our results reveal that body weight, organ development and integrity were not impaired in aged Grp78^+/- mice. No significant effect on cancer incidence and inflammation was observed in aging mice. Interestingly, our studies detected some subtle differential trends between the WT and Grp78^+/- mice in some test parameters dependent on gender and genetic background. Our studies provide the first evidence that GRP78 haploinsufficiency for up to 2 years of age has no major deleterious effect in rodents of different genetic background, supporting the merit of anti-GRP78 drugs in treatment of cancer and other diseases affecting the elderly.

Figure 1. Sustained reduction of GRP78 protein level in organs of aged Grp78⁺/⁻ mice.

(A) GRP78 protein levels in pancreas, liver, spleen, white adipose tissue, skeletal muscle and brain were examined by Western blotting in 7 month old Grp78^+/+ and ^+/− mice of C57BL/6*129sv genetic background (n ≥ 3 for each genotype). Top panel: representative blots. Bottom panel: levels normalized against α-actin (muscle) or β-actin (other tissues) indicated as mean ± SEM. *unadjusted P < 0.05, **unadjusted P < 0.01. (B) Protein levels of ER chaperones GRP78, GRP94, calnexin (CNX), and calreticulin (CRT) were examined by Western blotting in liver of 7 month old Grp78^+/+ and ^+/− mice of C57BL/6*129sv genetic background (n ≥ 3 for each genotype). Left panel: representative blots. Right panel: levels normalized against β-actin indicated as mean ± SEM. **unadjusted P < 0.01. (C) Protein levels of ER chaperones GRP78 and GRP94 from lung of 22 month old Grp78^+/+ and ^+/− mice of C57BL/6 genetic background (n = 3 for each genotype). Top panel: representative blots. Bottom panel: levels averaged for each genotype and normalized against β-actin plotted as mean ± SEM. *unadjusted P < 0.05.

Figure 2. Aged C57BL/6 Grp78⁺/⁻ mice exhibited normal body weight, organ size and morphology.

(A) 23–25 month old C57BL/6 mice were sacrificed and body/organ weights were measured and plotted as mean ± SEM. Male: Grp78^+/+ (n = 13); Grp78^+/− (n = 18); female: Grp78^+/+ (n = 11); Grp78^+/− (n = 11). *unadjusted P < 0.05, ***unadjusted P < 0.001. (B). Representative histological sections of lung and renal cortex of the indicated genotypes. Both organs show normal tissue architecture. Lungs of GRP78^+/+ and GRP78^+/− mice show unremarkable alveolar spaces (a) of normal sizes and lined by alveolar septae of similar and normal thickness. Bronchioles (b) are lined by respiratory epithelium. The kidneys show glomeruli (arrows) with normal cellularity and unremarkable renal tubules. Magnification bars: 100 microns.

Figure 3. Similar body weight and organ size in aged Grp78⁺/⁻ mice of 129sv and C57BL/6*129sv genetic backgrounds.

23–25 month old male (A) 129sv mice or (B) C57BL/6*129sv mice were sacrificed and body/organ weights were measured and plotted as mean ± SEM. (A) Grp78^+/+ (n = 3); Grp78^+/− (n = 9). (B) Grp78^+/+ (n = 5); Grp78^+/− (n = 6).

Figure 4. Normal circulating levels of glucose and IGF-I in aged Grp78⁺/⁻ mice.

(A) Serum glucose levels in 23-25 month old mice of the indicated genotypes, genders, and genetic backgrounds. C57BL/6 [male: Grp78^+/+ (n = 13); Grp78^+/− (n = 18); female: Grp78^+/+ (n = 11)]; Grp78^+/− (n = 11). (B) 129sv: [male: Grp78^+/+ (n = 3); Grp78^+/− (n = 9); female: Grp78^+/+ (n = 7)] or (C) C57BL/6*129sv mice [male: Grp78^+/+ (n = 5); Grp78^+/− (n = 6)].

Figure 5. Number of circulating hematopoietic cells in aged Grp78⁺/⁻ mice.

Complete blood cell counts were obtained for 23–25 month old (A) C57BL/6 mice and (B) 129sv mice. White blood cells (WBC), lymphocytes (Lymph), monocytes (Mon), platelets (PLT), red blood cells (RBC) and hemoglobin (HGB) levels are shown. (A) Male Grp78^+/+ (n = 9), male Grp78^+/− (n = 10), female Grp78^+/+ (n = 5) and female Grp78^+/− (n = 7). (B) Male Grp78^+/+ (n = 2), and Grp78^+/− (n = 6). Bars: mean ± SEM.

Figure 6. Normal retinal morphology in 2-year-old Grp78⁺/⁻ mice.

Light micrograph of retinal sections from 2-year-old C57BL/6 Grp78^+/+ mice (A) and Grp78^+/− mice (B). RPE, retinal pigmented epithelium; OS and IS, photoreceptor outer segment and inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar = 40 μm. (C) Outer nuclear layer thickness was measured at 20 different positions along the inferior (I) and superior (S) axis along the central meridian for control (Grp78^+/+, N = 3) and Grp78^+/− (N = 4) mice. The plot shows average thickness ± SD.

Figure 7. Behavioral performance of aged Grp78⁺/⁻ mice.

(A) Best Rotarod performance score in 19–22 month old female and male C57BL/6 Grp78^+/+ and Grp78^+/− mice. N = 12-13/group. Rotarod learning performance over the 6 trial period is shown for each gender and genotype. (B) Best Rotarod performance score in 23 month old male C57BL/6*129sv Grp78^+/+ and Grp78^+/− mice. N = 5/group. Rotarod learning performance over the 6 trial period is shown for each genotype. (C) Spontaneous alternation behavior (SAB) and number of arm entries in the Y-maze for C57BL/6*129sv Grp78^+/+ and Grp78^+/− male mice. N = 4-6/group. (D) Recognition index at 23 month for C57BL/6*129sv Grp78^+/+ and Grp78^+/− male mice in the novel object recognition task. Exploration time of the old vs. old (trial 1) or novel object (trial 2). N = 4-6/group. Each data point represents mean ± SEM.

Figure 8. Representative examples of neoplastic tumors in aged Grp78⁺/⁺ and Grp78+/− mice.

(A) Section of spleen from a Grp78^+/+ mouse showing extensive infiltration by a lymphoma outlined by the dashed lines; the inset shows a magnified representative section of the tumor, illustrating a monoclonal population of cells with pleomorphic nuclei and a high mitotic rate. (B) Section of liver from a Grp78^+/− mouse showing an adenoma outlined by the dashed line. Sections of lung from Grp78^+/+ (C) and Grp78^+/− (D) mice. In each case the pulmonary parenchyma is infiltrated by an adenocarcinoma, which is extensive in (C) and microscopic in (D). Both tumors show papillary architecture. Tumors in panels A and D are from C57BL/6 mice while those in panels B and C are from C57BL/6*129sv mice. Magnification bars: 50 microns in A, 100 microns in (B–D).

Figure 9. Chemotoxic stress response of aged Grp78⁺/⁻ mice.

(A) male and (B) female 20–22 month old C57BL/6 Grp78^+/+ and Grp78^+/− mice received 8 mg/kg intravenous inoculations of Doxorubicin (DXR) at the time points indicated by dashed lines. Chemotoxicity-induced weight loss and survival are presented for each gender and genotype. N = 6-7/group.