The inhibitory effects of inhibitors targeted MAPK/ERK pathway can down-regulate the SLP-2 expression.

<p><b>Aa, Da:</b> Both KYSE510 cells (A) and EC9706 (D) were pretreated with two different ERK1/2 inhibitors (PD98059, 1 µM and U0126, 1 µM) for 3 hours, and then treated with PMA (100 nM) for 15 minutes to analyze the level of phosphorylated ERK1/2 and total ERK1/2. And then around 24 hours to detect SLP-2 expression. <b>Ab, Db:</b> The graph represents densitometry of the results of 3 independent experiments (mean±SD). <b>Ba, Ea:</b> Both KYSE510 cells (B) and EC9706 cells (E) were pretreated with two different AKT inhibitors (LY294002, 1 µM and MK-2206, 1 µM) for 3 hours, and then treated with PMA (100 nM) for 15 minutes to analyze the level of phosphorylated AKT and total AKT. Then, the rest of procedure of detection is similar. <b>Bb, Eb:</b> The graph represents densitometry of the results of 3 independent experiments (mean±SD). <b>Ca, Fa, Ga:</b> Both KYSE510 cells (C) and EC9706 cells (F, G) were pretreated with two different ERK1/2 inhibitors and two AKT inhibitors for 3 hours, and then treated with EGF (10 nM) for 15 minutes to analyze the level of phosphorylated ERK1/2, AKT and total ERK1/2, AKT. And then around 24 hours to detect SLP-2 expression. <b>Cb, Fb, Gb:</b> The graph represents densitometry of the results of 3 independent experiments (mean±SD).</p

Expression patterns of SLP-2 protein in invasive ESCC.

<p><b>Aa:</b> Overexpressions of SLP-2 in ESCC tissue than that in normal margin were detected by Western blot. Also, higher expression level of SLP-2 can be detected in invasive front tissues compared with that in tumor central tissues. ß-actin was used as a loading control. <b>Ab:</b> The graph represents densitometry of the results of 3 independent experiments (mean±SD). *, Statistically different, compared with the SLP-2 expression in normal margin (<i>P</i><0.05). <b>B:</b> The distribution patterns of SLP-2 and cytoskeleton actin protein were observed by immunofluorescence staining. SLP-2 and actin protein were marked with green and red fluorescence, respectively. Merge image showed that ESCC cells infiltrated esophageal smooth muscles and SLP-2 protein expressed mainly at the invasive front. <b>Ca–g:</b> Predominant positive staining of SLP-2 at the invasive margin of cancer nests, which were indicated with black arrows. <b>Ch–i:</b> A single invasive cell, which was marked with black arrows showed SLP-2 staining (IHC staining, original magnification ×200).</p

The MAPK pathway and AKT pathway activation might probably be correlated with SLP-2 up-regulation.

<p><b>A, D:</b> In both KYSE510 cells (A) and EC9706 cells (D), PMA can activate MAPK/ERK, MAPK/JNK, MAPK/p38, and AKT pathway, induced p-ERK1/2, p-JNK, p-p38, and p-AKT high-expression. Whereas total ERK1/2, JNK, p38, and AKT expression remained unchanged. Also, followed by MAPK/AKT pathway activation, SLP-2 expression was raised obviously, which was identified by Western blot analysis. <b>Ba, b, Ea, b:</b> Similarly, in both KYSE510 cells (B) and EC9706 cells (E), EGF can up-regulate SLP-2 expression in both concentration and time dependent manners. <b>Ca, b, Fa, b:</b> KYSE510 cells (C), EC9706 cells (F). The graph represents densitometry of the results of 3 independent experiments (mean±SD). *, Statistically different, compared with the control cells (<i>P</i><0.05).</p

SLP-2 regulates invasion of ESCC cell lines in vitro by down-regulating MMP-2 transcriptionally.

<p><b>Aa, Ba:</b> Human ESCC KYSE510 cells (A) and EC9706 cells (B) were transfected with either SLP-2 siRNA or control siRNA. After 48 h to 72 h, SLP-2 expressions were inhibited obviously, which were detected by Western blot. ß-actin was used as a loading control. <b>Ab, Bb:</b> The graph represents densitometry of the results of 3 independent experiments. *, Statistically different (<i>P</i><0.05). <b>Ac, Bc:</b> The cells, which were transfected with either SLP-2 siRNA or control siRNA for 72 hours were transferred to Transwell chambers. After 20 hours, the cells were checked for invasion. *, Significant differences were observed between control and SLP-2 siRNA groups (<i>P</i><0.05). <b>C, D:</b> KYSE510 cells (C) and EC9706 cells (D) were transfected with siRNA for control or SLP-2 for 72 hours, respectively. RT-PCR (Ca, Da) and Western blot assay (Cb, Db) were carried out. ß-actin was used as a loading control. The graph represents densitometry of the results of 3 independent experiments. *, Statistically different, compared with the control cells (<i>P</i><0.05).</p

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

Reversible Bond/Cation-Coupled Electron Transfer on Phenylenediamine-Based Rhodamine B and Its Application on Electrochromism

A biomimetic system on reversible bond-coupled electron transfer (BCET) has been proposed and investigated in a switchable Rh–N molecule with redox active subunits. We discover that energy barrier of C–N bond breaking is reduced dramatically to less than 1/7 (from 40.4 to 5.5 kcal/mol), and 1/3 of the oxidation potential is simultaneously lowered (from 0.67 to 0.43 V) with the oxidation of Rh–N. The concept, cation-coupled electron transfer (CCET), is highly recommended by analyzing existing proton coupled electron transfer (PCET) and metal coupled electron transfer (MCET) along with aforementioned BCET, which have same characteristic of transferring positive charges, such as proton, metal ion, and organic cation. Molecular switch can be controlled directly by electricity through BCET process. Solid electrochromic device was fabricated with extremely high coloration efficiency (720 cm²/C), great reversibility (no degradation for 600 cycles), and quick respond time (30 ms)

Autophagy activation by novel inducers prevents BECN2-mediated drug tolerance to cannabinoids

<p>Cannabinoids and related drugs generate profound behavioral effects (such as analgesic effects) through activating CNR1 (cannabinoid receptor 1 [brain]). However, repeated cannabinoid administration triggers lysosomal degradation of the receptor and rapid development of drug tolerance, limiting the medical use of marijuana in chronic diseases. The pathogenic mechanisms of cannabinoid tolerance are not fully understood, and little is known about its prevention. Here we show that a protein involved in macroautophagy/autophagy (a conserved lysosomal degradation pathway), BECN2 (beclin 2), mediates cannabinoid tolerance by preventing CNR1 recycling and resensitization after prolonged agonist exposure, and deletion of Becn2 rescues CNR1 activity in mouse brain and conveys resistance to analgesic tolerance to chronic cannabinoids. To target BECN2 therapeutically, we established a competitive recruitment model of BECN2 and identified novel synthetic, natural or physiological stimuli of autophagy that sequester BECN2 from its binding with GPRASP1, a receptor protein for CNR1 degradation. Co-administration of these autophagy inducers effectively restores the level and signaling of brain CNR1 and protects mice from developing tolerance to repeated cannabinoid usage. Overall, our findings demonstrate the functional link among autophagy, receptor signaling and animal behavior regulated by psychoactive drugs, and develop a new strategy to prevent tolerance and improve medical efficacy of cannabinoids by modulating the BECN2 interactome and autophagy activity.</p

A <i>Becn1</i> mutation mediates hyperactive autophagic sequestration of amyloid oligomers and improved cognition in Alzheimer's disease

<div><p>Impairment of the autophagy pathway has been observed during the pathogenesis of Alzheimer’s disease (AD), a neurodegenerative disorder characterized by abnormal deposition of extracellular and intracellular amyloid β (Aβ) peptides. Yet the role of autophagy in Aβ production and AD progression is complex. To study whether increased basal autophagy plays a beneficial role in Aβ clearance and cognitive improvement, we developed a novel genetic model to hyperactivate autophagy in vivo. We found that knock-in of a point mutation F121A in the essential autophagy gene Beclin 1/<i>Becn1</i> in mice significantly reduces the interaction of BECN1 with its inhibitor BCL2, and thus leads to constitutively active autophagy even under non-autophagy-inducing conditions in multiple tissues, including brain. <i>Becn1</i>^F121A-mediated autophagy hyperactivation significantly decreases amyloid accumulation, prevents cognitive decline, and restores survival in AD mouse models. Using an immunoisolation method, we found biochemically that Aβ oligomers are autophagic substrates and sequestered inside autophagosomes in the brain of autophagy-hyperactive AD mice. In addition to genetic activation of autophagy by <i>Becn1</i> gain-of-function, we also found that ML246, a small-molecule autophagy inducer, as well as voluntary exercise, a physiological autophagy inducer, exert similar <i>Becn1</i>-dependent protective effects on Aβ removal and memory in AD mice. Taken together, these results demonstrate that genetically disrupting BECN1-BCL2 binding hyperactivates autophagy in vivo, which sequestrates amyloid oligomers and prevents AD progression. The study establishes new approaches to activate autophagy in the brain, and reveals the important function of <i>Becn1</i>-mediated autophagy hyperactivation in the prevention of AD.</p></div

<i>Becn1</i>^F121A leads to autophagy hyperactivation in vivo.

<p><b>(A, B)</b> Representative images (left panel) and quantification (right panel) of GFP-LC3 puncta (autophagosomes) in skeletal muscle <b>(A)</b> and brain <b>(B)</b> of GFP-LC3 <i>Becn1</i>^+/+ and GFP-LC3 <i>Becn1</i>^FA/FA mice at non-autophagy-inducing conditions (fed and rested), after 90-min exercise, or after 48 hours of starvation. Scale bar: 25 μm. Results represent mean ± s.e.m. N = 5. *, P<0.05, **, P<0.01, t test. <b>(C)</b> Western blot analysis (left panel) and quantification (right panel) of LC3 and p62 levels in skeletal muscle from <i>Becn1</i>^+/+ and <i>Becn1</i>^FA/FA mice injected with one dose of PBS or 50 mg/kg lysosomal inhibitor chloroquine. The autophagy flux is measured by the difference in the p62 and LC3 levels between mice injected with PBS and with chloroquine. Results represent mean ± s.e.m. N = 3. **, P<0.01, two-way ANOVA for comparison of magnitude of changes between different groups in mice of different genotypes.</p

Autophagosomal sequestration of Aβ42 in brain of autophagy-hyperactive mice.

<p>(Left) Scheme of immunoisolation of autophagosomes from brain of 12-week old 5XFAD <i>Becn1</i>^FA/FA mice expressing GFP-LC3. Briefly, post-nucleus extracts of the brain lysates was obtained by centrifugation at a low speed of 1,000 xg. Autophagosomes were enriched by centrifugation at a high speed of 20,000 xg, and pulled down by an anti-GFP antibody using magnetic beads. (Right) Western blot detection of Aβ42 fibrillar and oligomeric species inside autophagosomes immunoprecipitated by GFP antibody as in the scheme. A known autophagy cargo p62 serves as a positive control, and a cytosolic enzyme GAPDH is a negative control.</p