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1.
FIGURE 8.

FIGURE 8. From: Diabetes-induced Central Neuritic Dystrophy and Cognitive Deficits Are Associated with the Formation of Oligomeric Reticulon-3 via Oxidative Stress.

High glucose induced the formation of protein carbonyls and RTN3 aggregates in SH-SY5Y cells. Images representing the detection of protein carbonyls (A) and RTN3 (C) by Western blotting of the extracts from SH-SY5Y cells after 1 h of treatment with 300 μm H2O2, 500 μm H2O2, 25 mm glucose, 300 μm H2O2 + 25 mm glucose, 500 μm H2O2 + 25 mm glucose or 0.5% DMSO. Statistical data of B and D indicated that H2O2 and/or 25 mm glucose treatment induced significant increases in the amount of protein carbonyls (DNP) and HW-RTN3 and a decrease in monomeric RTN3 in SH-SY5Y cells. All values are reported as the means ± S.E. (n = 3–5 per group). * and # represent p < 0.05 versus the control and DMSO, respectively.

Bei Zhao, et al. J Biol Chem. 2013 May 31;288(22):15590-15599.
2.
FIGURE 2.

FIGURE 2. From: Diabetes-induced Central Neuritic Dystrophy and Cognitive Deficits Are Associated with the Formation of Oligomeric Reticulon-3 via Oxidative Stress.

Diabetic rats showed impaired performance in water maze tests. A, in non-visible trials, diabetic rats took longer to find the platform on training days 4 and 5 compared with control (Ctrl) rats. B, in visible platform trials, rats showed similar motor function in the control and diabetic groups. C and D, in probe trials, typical swimming patterns were recorded on training day 6 in both the control and diabetic rats (C), and the results showed that diabetic rats spent significantly less time in the target quadrant (D). All values are reported as the means ± S.E. (n = 8 and 9 for control and diabetic group, respectively). * represents p < 0.05 compared with the controls.

Bei Zhao, et al. J Biol Chem. 2013 May 31;288(22):15590-15599.
3.
FIGURE 5.

FIGURE 5. From: Diabetes-induced Central Neuritic Dystrophy and Cognitive Deficits Are Associated with the Formation of Oligomeric Reticulon-3 via Oxidative Stress.

Diabetes induced Aβ generation via increased BACE1 activity in rat brain. A, Western blots revealed bands for full-length APP (FL-APP), APP-C99, BACE1-cleaved APP product, and APP-C83, an α-secretase cleaved product, in the cortex of control (Ctrl) and diabetic (DM) rats. Ratios of densitometry over actin were calculated and are represented graphically for FL-APP (B), APP-C99 (C), and APP-C83 (D) in the cortex. E, photos show Aβ immunopositive stained (Aβ+) cells in the cortex of control and diabetic rat brains. Bar = 100 μm. Diabetes significantly increased the number of Aβ+ cells in cortical neurons (F) and the amounts of Aβ40 and 42 in cortical tissues (G). All values are reported as the means ± S.E. (n = 5 per group). * represents p < 0.05 compared with the controls.

Bei Zhao, et al. J Biol Chem. 2013 May 31;288(22):15590-15599.
4.
FIGURE 1.

FIGURE 1. From: Diabetes-induced Central Neuritic Dystrophy and Cognitive Deficits Are Associated with the Formation of Oligomeric Reticulon-3 via Oxidative Stress.

Changes of body weight, biochemical parameters, and insulin sensitivity in diabetic rats. Body weight, plasma glucose (PGL), plasma total cholesterol (PTC), and plasma triglyceride (PTG), brain weight, and insulin sensitivity were determined in diabetic rats. Compared with control (Ctrl, n = 10) rats, DM (n = 15) rats showed time-dependent increases in body weight within 3 months (A) and increased in glucose, cholesterol, and triglyceride (C) but showed no changes in brain weight (B). An intravenous insulin glucose tolerance test was performed to calculate the k value based on the glucose disappearance rate within 10 min by the average slope. These results demonstrated that the k value decreased in DM rats (D, n = 5/group), indicating reduced insulin sensitivity in the diabetic rats. Data are shown as the mean ± S.E. * represents p < 0.05 versus the controls.

Bei Zhao, et al. J Biol Chem. 2013 May 31;288(22):15590-15599.
5.
FIGURE 3.

FIGURE 3. From: Diabetes-induced Central Neuritic Dystrophy and Cognitive Deficits Are Associated with the Formation of Oligomeric Reticulon-3 via Oxidative Stress.

Diabetes induced changes in RTN3 protein expression in rat brains. A, tissue extracts from cortex in control and diabetic brains were analyzed by Western blotting with antibody R458 to the RTN3 C terminus. Bands near 24 kDa and above 175 kDa, respectively, demonstrated the presence of RTN3 and HW-RTN3 proteins (n = 4 in each group). B, HW-RTN3 proteins were increased, whereas monomeric RTN3 proteins were reduced in the cortex of diabetic rats compared with controls. C and D, RTN3 mRNA was detected by RT-PCR analysis (C) and showed no significant changes in the cortex (D) between control and diabetic brains (n = 3 in each group). Data are represented as the mean ± S.E. * represents p < 0.05 compared with the controls. E–H, shown is immunofluorescence staining of RTN3 in the parietal cerebral cortex of control (E and F) and diabetic rats (G and H). Photographs F and H are magnified images corresponding to the squares labeled with f and h in photos E and F, respectively.

Bei Zhao, et al. J Biol Chem. 2013 May 31;288(22):15590-15599.
6.
FIGURE 6.

FIGURE 6. From: Diabetes-induced Central Neuritic Dystrophy and Cognitive Deficits Are Associated with the Formation of Oligomeric Reticulon-3 via Oxidative Stress.

Diabetes triggered the formation of RTN3 oligomer aggregates and neuritic dystrophy in rat brain. Double fluorescent immunostaining of RTN3 with NeuN (A and B), SMI32 (C and D), or oligomer (E and F) in the parietal cerebral cortex of control (A, C, and E) and diabetic rats (B, D, and F). The bar = 100 μm in photos A–D, and the bar = 50 μm in photos (E and F). The images show that RTN3-positive (RTN3+) staining was evenly distributed in neuronal cell bodies in control rats and co-localized with the neuronal marker, NeuN (A). RTN3+ neurites in diabetic rats did not stain with NeuN (B) and SMI32 (D) but were co-stained by oligomer (F). G, tissue extracts (200 μg of protein) from the cortex were first precipitated (IP) with oligomer antibody (1 μl) and then detected on Western blots (IB) with RTN3 antibody. An arrowhead in G indicates IgG light chain (ILC). The results showed that HW-RTN3 was present in diabetic rat brains (input) and could be precipitated by anti-oligomer antibody in diabetic brains tissues (DM) but not in control tissues (Ctrl).

Bei Zhao, et al. J Biol Chem. 2013 May 31;288(22):15590-15599.
7.
FIGURE 4.

FIGURE 4. From: Diabetes-induced Central Neuritic Dystrophy and Cognitive Deficits Are Associated with the Formation of Oligomeric Reticulon-3 via Oxidative Stress.

Diabetic rats showed reduced interaction of RTN3 with BACE1 in rat brain. A, BACE1-immunostained cells were present in the pyramidal neurons of the parietal cerebral cortex in control (Ctrl) and diabetic (DM) rats. Bar = 100 μm. B and C, tissue lysates extracted from cortex were used to examine BACE1 levels by Western blotting. The results show that the levels of BACE1 expression in the cortex were not significantly different between control and diabetic rats. D and E, enriched total tissue extract from control and diabetic rat brains (200 μg) were immunoprecipitated (IP) with a limited amount of anti-BACE1 antibody (1 μl) and then examined by Western blotting with anti-RTN3 (R458) antibody. The results show that BACE1-RTN3-binding proteins, but not BACE1 protein, were reduced in diabetic cortex. F and G, in the reversed immunoprecipitation assay, tissue lysates (200 μg) were immunoprecipitated with R458 antibody (1 μl) and examined by BACE1 antibody. The results show that BACE1-RTN3 binding proteins, but not RTN3 protein, were reduced in the diabetic cortex. IHC in D and F, IgG heavy Chain. Data are shown graphically as the mean ± S.E. (n = 4 per group). * represents p < 0.05 compared with the controls.

Bei Zhao, et al. J Biol Chem. 2013 May 31;288(22):15590-15599.
8.
FIGURE 7.

FIGURE 7. From: Diabetes-induced Central Neuritic Dystrophy and Cognitive Deficits Are Associated with the Formation of Oligomeric Reticulon-3 via Oxidative Stress.

Diabetes induced an increase in oxidative DNA and protein damage in rat brain. A, immunostaining for 8-oxo-dG, a marker of oxidative DNA damage, revealed positive cell staining (8-oxo-dG+) in both control (Ctrl) and diabetic (DM) brains. Bar = 100 μm. B and C, rat brain extracts were derivatized with 10 mm DNPH in 2 n HCl and detected with an antibody against DNP on Western blots to identify protein carbonyls. Western blotting (IB; B) shows a statistically significant increase in the number and density of protein carbonyls in DM brain (C). D and E, extracts from rat brains were immunoprecipitated (IP) with DNP antibody after DNPH derivatization and then probed with anti-RTN3 antibody to detect RTN3 carbonyls (first and second lanes). The results showed that the mono-RTN3 band could be detected in diabetic brain tissues after immunoprecipitation with DNP antibody, indicating that diabetes induced the formation of RTN3 carbonyls in brain. RTN3 carbonyls were significantly increased in diabetic brains compared with controls. Treatment of extracts from diabetic brains with vehicle or 2 n HCl revealed that HW-RTN3 bands could be detected in the control sample (third lane) but not in the acid-treated sample (fourth lane), suggesting that HW-RTN3 aggregates were unstable in acidic conditions and dissociated to mono-RTN3. All values are reported as the means ± S.E. (n = 6 per group). * represents p < 0.05 compared with the controls.

Bei Zhao, et al. J Biol Chem. 2013 May 31;288(22):15590-15599.

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