Identification of natural products with neuronal and metabolic benefits through autophagy induction

<p>Autophagy is a housekeeping lysosomal degradation pathway important for cellular survival, homeostasis and function. Various disease models have shown that upregulation of autophagy may be beneficial to combat disease pathogenesis. However, despite several recently reported small-molecule screens for synthetic autophagy inducers, natural chemicals of diverse structures and functions have not been included in the synthetic libraries, and characterization of their roles in autophagy has been lacking. To discover novel autophagy-regulating compounds and study their therapeutic mechanisms, we used analytic chemistry approaches to isolate natural phytochemicals from a reservoir of medicinal plants used in traditional remedies. From this pilot plant metabolite library, we identified several novel autophagy-inducing phytochemicals, including Rg2. Rg2 is a steroid glycoside chemical that activates autophagy in an AMPK-ULK1-dependent and MTOR-independent manner. Induction of autophagy by Rg2 enhances the clearance of protein aggregates in a cell-based model, improves cognitive behaviors in a mouse model of Alzheimer disease, and prevents high-fat diet-induced insulin resistance. Thus, we discovered a series of autophagy-inducing phytochemicals from medicinal plants, and found that one of the compounds Rg2 mediates metabolic and neurotrophic effects dependent on activation of the autophagy pathway. These findings may help explain how medicinal plants exert the therapeutic functions against metabolic diseases.</p

The Two Endocytic Pathways Mediated by the Carbohydrate Recognition Domain and Regulated by the Collagen-like Domain of Galectin-3 in Vascular Endothelial Cells

<div><p>Galectin-3 plays an important role in endothelial morphogenesis and angiogenesis. We investigated the endocytosis of galectin-3 in human vascular endothelial cells and showed that galectin-3 could associate with and internalized into the cells in a carbohydrate-dependent manner. Our work also revealed that galectin-3 was transported to the early/recycling endosomes and then partitioned into two routes – recycling back to the plasma membrane or targeting to the late endosomes/lysosomes. Various N- and C-terminal truncated forms of galectin-3 were constructed and compared with the full-length protein. These comparisons showed that the carbohydrate-recognition domain of galectin-3 was required for galectin-3 binding and endocytosis. The N-terminal half of the protein, which comprises the N-terminal leader domain and the collagen-like internal repeating domain, could not mediate binding and endocytosis alone. The collagen-like domain, although it was largely irrelevant to galectin-3 trafficking to the early/recycling endosomes, was required for targeting galectin-3 to the late endosomes/lysosomes. In contrast, the leader domain was irrelevant to both binding and intracellular trafficking. The data presented in this study correlate well with different cellular behaviors induced by the full-length and the truncated galectin-3 and provide an alternative way of understanding its angiogenic mechanisms.</p> </div

The exocytosis of Gal-3.

<p>A: HUVECs were incubated in SFM with 15 µg/ml Gal-3 for 30 min at 37°C and then washed five times at 4°C. The cells were placed in fresh SFM and then incubated at 37°C or 4°C for a total of 120 min with fresh media changes every 30 min. The media collected from each time point were precipitated by TCA and analyzed by western blotting. B: The HUVECs were incubated with 15 µg/ml Gal-3 for 30 min at 4°C and then washed five times at 4°C. The cells were then placed in fresh SFM and incubated at 37°C for 0, 20, 40, 60, or 120 min. Both the cells and the media were analyzed with western blotting.</p

The endocytosis of Gal-3.

<p>The HUVECs were incubated with biotinylated Gal-3 for 30 min and then either permeabilized or not before examination by IF analysis with Rhodamine- streptavidin. Images from a fluorescent microscope. Scale bar, 10 µm.</p

Primers.

<p>Primers.</p

The two endocytic pathways of Gal-3 observed in HUVECs are also in HMEC-1 cells.

<p>A: Fluorescent images showing the kinetics of Gal-3 endocytosis. DTAF-Gal-3 was incubated with HMEC-1 for 10 min, 30 min, or 120 min at 37°C and then processed for IF analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052430#pone-0052430-g004" target="_blank">Figure 4</a>. The images in this figure were obtained with a fluorescence microscope. Scale bar, 10 µm. B:Western blot showing the exocytosis of Gal-3. The HMEC-1 cells were incubated with or without (w/o) Gal-3 at 4°C for 30 min. After extensive washing, the cells were either immediately lysed (0 min) or incubated in fresh SFM at 37°C for 60 min. After incubation, both the medium and the cells were analyzed with western blotting.</p

The roles of Gal-3 N-terminal domains in endocytosis and exocytosis.

<p>A: The association of various Gal-3 proteins with HUVEC at different temperatures. The cells were incubated with 15 µg/ml Gal-3, G13-250, G69-250 or G111-250 for 60 min at 4°C, 20°C, or 37°C. The amount of protein added in the medium is shown in the top panel (medium). Cell associated proteins were analyzed by western blotting with polyclonal antibodies against Gal-3 and actin. B: The exocytosis of various Gal-3 proteins by the cells. The cells were incubated with or without (w/o) Gal-3, G13-250, G69-250 and G111-250 at 4°C for 30 min. After extensive washing, the cells were either lysed immediately (0 min) or incubated in fresh SFM at 37°C for 60 min. After incubation, both the media and the cells were analyzed with western blotting.</p

Preparation of the full-length and truncated Gal-3.

<p>A: Schematics of the full-length and truncated Gal-3. B: Purity was confirmed by 12% SDS-PAGE. Gal-3 refers to the full-length protein, composed of 250 amino acid residues; G13-250, G69-250 and G111-250 refer to the N-terminal truncated forms of Gal-3, containing the amino acids from 13 to 250, 69 to 250, and 111 to 250 respectively; GST-Gal-3 refers to the full-length Gal-3 fused to the GST tag; GST-G1-108 refers to the N-terminal 108 amino acids fused to the GST tag.</p

Schematic of the endocytic pathways of Gal-3 in endothelial cells.

<p>(i) Gal-3 bound with the glycol-ligand on the plasma membrane (PM); (ii) Gal-3 was endocytosed and transported to the early/recycling endosomes (EE/RE); (iii) A fraction of the endocytic Gal-3 was recycled to the PM and then released into the medium; (vi) A fraction of the endocytic Gal-3 was transported to late endosome/lysosomes (LE/lysosomes).</p

The association of Gal-3 with HUVECs.

<p>A: Cell association is concentration dependent. The cells were incubated for 30 min in SFM containing 0, 0.1, 0.2, 0.5, 1, 2, 5 or 15 µg/ml Gal-3. B: Cell association is time dependent. The cells were incubated in SFM containing either 2 or 15 µg/ml Gal-3 for 5, 30, 60, 120, or 240 min. C: Cell association is lectin specific. The cells were incubated for 30 min in SFM containing 15 µg/ml Gal-3 in the presence or absence of 50 mM lactose or sucrose. After incubation, the cells were extensively washed with cold SFM. Western blotting analysis was performed with anti-Gal-3 and anti-actin antibodies. Arrowhead, degraded form of Gal-3.</p