Article Figures & Data
Figures
- Figure 1.
LLLT exerts neuroprotection against Aβ toxicity. A, LLLT (2 J/cm2) protects SH-SY5Y cells against Aβ25–35 (25 μm) neurotoxicity using the CCK-8 assay after being irradiated with LLLT for 12 h, 24 h, or 48 h. B, Primary hippocampal neurons or SH-SY5Y cells were exposed to Aβ25–35 followed by irradiation with LLLT at 0.5 J/cm2, 1 J/cm2, 2 J/cm2, or 4 J/cm2, respectively. Cell viability was assessed by the CCK-8 assay after 24 h. C, LLLT (2 J/cm2) protects primary hippocampal neurons against Aβ1–42 (25 μm) neurotoxicity using the CCK-8 assay after irradiation with LLLT. D–G, Primary hippocampal neurons treated with Aβ25–35 and/or LLLT or Aβ1–42 and/or LLLT were double stained with apoptosis markers Annexin V/PI using flow cytometric analysis. H–I, Apoptosis in SH-SY5Y cells was analyzed in H and I. J, Representative immunofluorescent images of 5 DIV hippocampal neurons under indicated treatments with MAP2 antibody to visualize dendrite (green). Staining with Rhodamine-labeled phalloidin to visualize F-actin (red). Scale bar, 10 μm. K, Effects of these treatments on the number of primary dendrites per neurons. For each group, >25 neurons were measured. L, Effects of these treatments on average dendritic length per neuron. For each group, >25 neurons were measured. M, Representative photomicrographs of FITC-phalloidin labeling in hippocampal neurons derived from APP/PS1 mice embryo on 10 DIV under the treatment with or without LLLT. Scale bar, 10 μm. N, Quantification of primary dendrite numbers per neurons under indicated treatments. For each group, >25 neurons were measured. All the data in these figures are presented as mean ± SEM four individual experiments. *p < 0.05 versus control group; #p < 0.05 versus indicated group.
- Figure 2.
LLLT upregulates BDNF expression in cultured neurons derived from APP/PS1 transgenic mice and in Aβ-treated hippocampal neurons. A, Western blot analysis of hippocampal lysates from APP/PS1 transgenic mice and age-matched WT littermate mice at 6 months of age. B, Western blot analysis of BDNF expression in Aβ25–35-and Aβ1–42-treated primary hippocampal neurons. C, Representative Western blot assay for detecting the time-dependent effect of 2 J/cm2 LLLT on BDNF expression in SH-SY5Y cells. D, Representative Western blot assay for detecting the dose-dependent effect of LLLT on BDNF expression after 24 h. E, Western blot was performed to detect the BDNF, nerve growth factor (NGF), and NT-3 expression after Aβ25–35 treatment with or without LLLT in primary hippocampal neurons. F–G, BDNF expression was detected by Western blot (F) and ELISA (G) in hippocampal neurons derived from WT mice embryos or APP/PS1 embryos at 14 DIV with or without LLLT. H, Representative immunofluorescent images of 5 DIV hippocampal neurons under indicated treatments with MAP2 antibody to visualize dendrite (green). Staining with Rhodamine-labeled phalloidin to visualize F-actin (red). Scale bar, 10 μm. I, Effects of these treatments on the number of primary dendrites per neurons. For each group, >25 neurons were measured. J, Effects of these treatments on average dendritic length per neuron. For each group, >25 neurons were measured. All the data in these figures are presented as mean ± SEM four individual experiments. *p < 0.05 versus control group; #p < 0.05 versus indicated group.
- Figure 3.
LLLT-induced activation of CREB enhances BDNF transcription upon Aβ treatment. A, Act D inhibits LLLT-induced increase in BDNF mRNA expression in Aβ25–35-treated SH-S5Y cells. B, SH-SY5Y cells were transfected with CREB siRNA. Western blot analysis of BDNF expression after Aβ25–35 treatment with or without LLLT. C, Western bolt analysis of BDNF expression in SH-SY5Y cells under indicated treatments. The concentration of MTA is 200 nm. D, Western blot analysis of CREB phosphorylation at Ser133 in primary hippocampal neurons treated with Aβ25–35 and/or LLLT. E, Representative immunofluorescent images of p-CREB (Ser 133) (green) in SH-SY5Y cells under the indicated treatments. Staining with propidium iodide (PI) to visualize nucleus. F, ChIP assay was done to examine DNA-binding activity of CREB to BDNF promoter in SH-SY5Y cells and primary hippocampal neurons with different treatments. All the data in these figures are presented as mean ± SEM four individual experiments. *p < 0.05 versus control group; #p < 0.05 versus indicated group.
- Figure 4.
LLLT upregulates BDNF expression and CREB Ser 133 phosphorylation in Aβ25–35-treated cells via ERK activation. A, Representative Western blot assay of CREB Ser133 phosphorylation stimulated with Aβ25–35 and/or LLLT in the presence of Gö6983 (20 μm), API-2 (2 μm), and PD98059 (1 μm) in SH-SY5Y cells. B, Representative Western blot assay of the CREB Ser133 phosphorylation stimulated with Aβ25–35 and/or LLLT in the presence of PD98059 (1 μm) and H89 (10 μm) in primary hippocampal neurons. C, Primary hippocampal neurons were treated with Aβ25–35 and/or LLLT, or pretreated with PD98059. Western blot analysis was performed to detect the levels of BDNF, total-ERK, p-ERK, total-CREB, and p-CREB. D, Representative Western blot assay for detecting the levels of ERK phosphorylation after indicated treatments in cytoplasm and nuclear lysates, respectively. E, Representative Western blot assay of the p-ERK, total-ERK, GTP-Ras, and total-Ras stimulated with Aβ25–35 and/or LLLT in the presence of PD98059 (1 μm) and FTS (20 μm) in primary hippocampal neurons. F, Representative Western blot assay of the GTP-Ras and total-Ras pretreated with Ca2+-free medium, or xestospongin B (xpB, 40 μm). All the data in these figures are presented as mean ± SEM four individual experiments. *p < 0.05 and **p < 0.05 versus control group; #p < 0.05 versus indicated group.
- Figure 5.
Altered dendritic morphology after LLLT in Aβ25–35-treated hippocampal neurons at 5 DIV via ERK/CREB pathway. A, Representative images of 5 DIV hippocampal neurons with indicated treatments. Scale bar, 10 μm. B, E, Quantification of primary dendrites numbers per neurons under indicated treatments. For each group, >25 neurons were measured. C, F, Quantification of average dendritic length per neurons under indicated treatments. For each group, >25 neurons were measured. D, Hippocampal neurons were transfected with CREB siRNA at 3 DIV and treated with Aβ25–35 and LLLT at 4 DIV. Representative immunofluorescent images with MAP2 antibody are shown at 5 DIV. Scale bar, 10 μm. All the data in these figures are presented as mean ± SEM four individual experiments. *p < 0.05 versus control group; #p < 0.05 versus indicated group.
- Figure 6.
Dendritic cytoskeletal protein contributes to LLLT-promoted dendrite growth. A, LLLT induced Rac1 activity in Aβ25–35-treated hippocampal neurons, as indicated by an increase in GTP-bound Rac1. B, Quantitative analysis of the levels of GTP-bound Rac1 after different treatments. All the data in these figures are presented as mean ± SEM four individual experiments. *p < 0.05 versus control group; #p < 0.05 versus indicated group.
- Figure 7.
LLLT rescues dendritic spine loss in neurons derived from APP/PS1 transgenic mice. A, Representative photomicrographs of FITC-phalloidin labeling in hippocampal neurons derived from APP/PS1 mice on 14 DIV under the treatment with or without LLLT. Scale bar, 10 μm. B, Quantification of spine density under indicated treatments. For each group, we measured >25 dendrites from 13 to 15 neurons. C, Representative Western blot analysis was performed to detect the levels of PSD-95 in Aβ25–35 and/or LLLT-treated hippocampal neurons. D, Quantitative analysis of the levels of PSD-95 after indicated treatments. All the data in these figures are presented as mean ± SEM four individual experiments. *p < 0.05 versus control group; #p < 0.05 versus indicated group.
