Dendrobium nobile Lindl alkaloid, a novel autophagy inducer, protects against axonal degeneration induced by Aβ25-35 in hippocampus neurons in vitro

Abstract

Aims: Axonal degeneration is a pathological symbol in the early stage of Alzheimer's disease (AD), which can be triggered by amyloid-β (Aβ) peptide deposition. Growing evidence indicates that deficit of autophagy eventually leads to the axonal degeneration. Our previous studies have shown that Dendrobium nobile Lindl alkaloid (DNLA) had protective effect on neuron impairment in vivo and in vitro; however, the underlying mechanisms is still unclear.

Methods: We exposed cultured hippocampus neurons to Aβ_25-35 to investigate the effect of DNLA in vitro. Axonal degeneration was evaluated by immunofluorescence staining and MTT assay. Neurons overexpressing GFP-LC3B were used to measure the formation of autophagosome. Autophagosome-lysosome fusion, the lysosomal pH, and cathepsin activity were assessed to reflect autophagy process. Proteins of interest were analyzed by Western blot.

Results: DNLA pretreatment significantly inhibited axonal degeneration induced by Aβ_25-35 peptide in vitro. Further studies revealed DNLA treatment increased autophagic flux through promoting formation and degradation of autophagosome in hippocampus neurons. Moreover, enhancement of autophagic flux was responsible for the protective effects of DNLA on axonal degeneration.

Conclusions: DNLA prevents Aβ_25-35 -induced axonal degeneration via activation of autophagy process and could be a novel therapeutic target.

Keywords: Alzheimer's disease; Dendrobium nobile Lindl alkaloid; amyloid-β; autophagy; axonal degeneration.

Figure 1

DNLA protect hippocampus neurons axonal degeneration from Aβ_25‐35 cytotoxicity. Hippocampus neurons were pretreated with 10⁻⁷ mol/L of Aβ_23‐35 for 2 hours and then exposed to DNLA (3.5, 35, 350 ng/mL) for 24, 48, and 72 hours, respectively, for immunofluorescence staining, MTT assay, and Western blot analysis. (A) Representative images of neurons suffering from axonal degeneration. (B) The quantification of axonal degeneration namely degeneration index in (A). Raw data were obtained from five random visual fields from three independent wells. (C) Cell viability was measured by MTT assay. (D) The protein levels of PSD95 and SYN were analyzed by Western blot. (E) The statistical bar graph of quantification analysis of protein bands in (D). Compared with nontreatment group ^▲ P<.05, compared with alone Aβ_23‐35 treatment group *P<.05. Scale bar: 50 μm

Figure 2

DNLA increases autophagosome formation and promotes autophagosome‐lysosome fusion in hippocampus primary neurons of rats. Scale bar: 10 μm. (A) and (C) Representative fluorescence images of autophagosome in hippocampus neurons overexpressing GFP or GFP‐LC3B. In (A), neurons were treated with DNLA or DMSO for 24 hours. Red arrows indicate autophagosome marked by GFP‐LC3B. In (C), hippocampus neurons were treated with DNLA (350 ng/mL) or DMSO alone or in the presence of 3MA (10⁻³ mol/L) or HCQ (5×10⁻⁵ mol/L) for 24 hours. (B) and (D) Bar graph of result of GFP‐LC3B puncta quantification in (A) and (C), respectively. (E) Representative fluorescence images of autophagosome‐lysosome fusion. Neurons overexpressing GFP‐LC3B were treated with DNLA (350 ng/mL) or HCQ (5×10⁻⁵ mol/L) for 24 hours or maintained in EBSS medium for 4 hours. Autophagosomes were shown by green fluorescence signal, and lysosome was shown by red fluorescence signals. Merged images display colocalization of green and red fluorescence signal. Enlarged images are cut from the merge panels (white borders). (F) Quantification of Pearson's correlation coefficient as a statistic for quantifying colocalization. More than 50 cells were counted in every group. Compared with DMSO group *P<.05

Figure 3

DNLA induces autophagic flux in hippocampus neurons of rats in a dose‐ and time‐dependent manner. (A) Endogenous LC3B II, SQSTM1/p62, Beclin1, and ATG5 were analyzed by Western blot in neurons treated with DNLA for 24 hours. (B) and (C) Statistical bar graph of quantification of protein expression in (A). (D) Representative bands of endogenous LC3B II, SQSTM1/p62, Beclin1, and ATG5 analyzed by Western blot. (E) and (F) Statistical bar graph of quantification of proteins expression in (D). Raw data were obtained from at least five independent samples. * indicates a significant difference from controls, P<.05

Figure 4

DNLA enhances lysosomal cathepsin activity via acidifying lysosome compartment to promote degradation of lysosome pathway. (A) Representative pictures of neurons stained by AO. (B) Quantification of red/green intensity ratio of (A), raw data originated from at least three independent visual fields from three independent wells. (C) Endogenous CTSB and CTSD protein levels were surveyed by Western blot in neurons treated with DNLA for 24 hours. Pro‐CTSB and pro‐CTSD, precursor form of CTSB and CTSD; mature‐CTSB and mature‐CTSD, mature form of CTSB and CTSD. (D) Statistical bar graph of quantification of proteins expression in (C). (E) Enzymatic activity of CTSB and CTSD. (F) Representative pictures of neurons marked by DQ Red BSA. (G) Statistical bar graph of fluorescence intensity of DQ Red BSA in (F), data were showed as the mean±SD of at least five independent visual fields from three independent wells. (D) Colocalization of green fluorescence signal of GFP‐LC3B and red fluorescence signal of DQ Red BSA. Scale bar: 10 μm. * indicates a significant statistical difference from DMSO treatment group,*P<.05

Figure 5

Autophagic flux was enhanced by DNLA treatment in neurons of axonal degeneration induced by Aβ_23‐35 peptide. (A) LC3B, SQSTM1/p62, Beclin1, and ATG5 were analyzed by Western blot in neurons treated with DNLA for 24 hours. (B) and (C) The statistical bar graph of quantification analysis of protein bands in (A). Data from at least five independent samples were used for statistical analysis, compared with nontreatment group ^▲ P<.05, compared with alone Aβ_23‐35 treatment group *P<.05

Figure 6

BAFA inhibits protective effect of DNLA on axonal degeneration induced by Aβ_25‐35 peptide. (A) Representative images of neurons of axonal degeneration. Cells were visualized by immunofluorescence assay as description in Figure 4. (B) The statistical bar graph of degeneration index was presented. (C) The statistical bar graph of cell viability decided by MTT assay. (D‐F) and (G‐I) Quantification analysis of protein levels of PSD95, SYN, LC3B, and SQSTM1/p62 by Western blot. All data were presented as the mean±SD of 5 samples. Scale bar: 50 μm