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

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 improves the survival rate of PDAPP AD mice.

<p>Kaplan-Meier survival curve of PDAPP mice with normal (PDAPP, N = 51), hyperactive (PDAPP <i>Becn1</i>^FA/FA, N = 34), or deficient (PDAPP <i>Bcl2</i>^AAA, N = 33) autophagy monitored over time for 9 months. Statistical significance was analyzed by the log-rank test.</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

<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

<i>Becn1</i>^FA/FA mutation ameliorates cerebral Aβ accumulation, memory deficits and mortality of Alzheimer’s mouse models.

<p><b>(A-B)</b> Dot-blot assays and quantification of soluble <b>(A)</b> and insoluble <b>(B)</b> Aβ42 levels in homogenated brain samples of 6-month old 5XFAD <i>Becn1</i>^+/+ and 5XFAD <i>Becn1</i>^FA/FA mice, immunostained with anti-Aβ42 antibody. Total protein loading was labeled by Ponceau S. Triplicate experiments from 6 mice in each group were shown. ***, P<0.001, t test. <b>(C)</b> ELISA analyses of total (soluble and insoluble) Aβ42 levels in the cortex of 6-month old 5XFAD <i>Becn1</i>^+/+ and 5XFAD <i>Becn1</i>^FA/FA mice. N = 7. <b>(D)</b> Representative images (upper) and quantification (lower) of amyloid plaques stained by Thioflavin S in brain (cortex and hippocampus) of 5XFAD <i>Becn1</i>^+/+ and 5XFAD <i>Becn1</i>^FA/FA mice. Magnification of the enclosed regions is shown on the right. N = 6–8. Scale bar: 500 μm. ***, P<0.001, t test. <b>(E)</b> Dot-blot assays and quantification of total Aβ42 levels in homogenated brain samples of 6-month old 5XFAD <i>Becn1</i>^FA/FA mice treated with the autophagy inhibitor SBI-0206965 or vehicle once per day for 7 days. Total protein loading was labeled by Ponceau S. N = 5. **, P<0.01, t test. <b>(F)</b> Morris water maze test of 6-month old WT, 5XFAD <i>Becn1</i>^+/+ and 5XFAD <i>Becn1</i>^FA/FA mice. Escape latency and total distance traveled in visible platform test and hidden platform test are shown. N = 11–20. Results represent mean ± s.e.m. Two-way repeated measures ANOVA.</p

The <i>Becn1</i> F121A mutation inhibits the BECN1-BCL2 interaction in vivo.

<p><b>(A)</b> Schematic representation of the strategy for hyperactive autophagy via the <i>Becn1</i>^F121A knockin allele. F121A blocks binding of BECN1 with its inhibitor BCL2, which leads to upregulated BECN1 function and constitutively high autophagy. <b>(B)</b> Co-immunoprecipitation of BECN1 by BCL2 in skeletal muscle and brain tissues from wild-type (WT) and <i>Becn1</i>^F121A mice. Less BECN1^F121A is immunoprecipitated by BCL2 than WT BECN1, quantified by the BECN1/BCL2 ratio in the IP samples from 3 independent experiments. FA/FA, <i>Becn1</i>^F121A homozygous knock-in mice. ***, P<0.001, t test.</p

ML246 and voluntary exercise decrease cerebral amyloid plaques and ameliorate memory deficits in 5XFAD AD mice.

<p><b>(A, B)</b> Representative images (upper) and quantification (lower) of dot-blot assays on soluble <b>(A)</b> and insoluble <b>(B)</b> Aβ42 levels in brain samples of 6-month old 5XFAD and 5XFAD <i>Becn1</i>^+/- KO mice after 4 months of voluntary running, immunostained with anti-Aβ42 antibody. Total protein loading was labeled by Ponceau S. Triplicate experiments from 4–5 mice in each group were shown. <b>(C)</b> Representative images (left) and quantification (right) of amyloid deposits stained by Thioflavin S in brain of 6-month old 5XFAD mice, and 5XFAD mice subject to 5 weeks of ML246 treatment or 4 months of voluntary exercise. Scale bar: 500 μm. Results represent mean ± s.e.m. N = 6–8. *, P<0.05; **, P<0.01, t test. <b>(D)</b> Morris water maze test of 6-month old WT, 5XFAD, and 5XFAD mice after 5 weeks of ML246 treatment or 4 months of voluntary running. Escape latency and total distance traveled in visible platform test and hidden platform test are shown. N = 10–11. Results represent mean ± s.e.m. Two-way repeated measures ANOVA.</p

An autophagy-inducing compound ML246 reduces amyloid load in an autophagy-dependent manner in vitro and in vivo.

<p><b>(A)</b> Chemical structure of ML246. <b>(B)</b> Dot-blot assays (left) and quantification (right) of secreted Aβ42 levels in conditioned media of HEK293 cells stably expressing APP treated with vehicle (DMSO) or ML246 for 24 h, immunostained with anti-Aβ42 antibody. Cells were transfected with non-targeting control (NC) or <i>ATG7</i> siRNA 24 h prior to ML246 treatment. Results are quantified from 4 independent experiments. <b>(C)</b> Representative images (left) and quantification (right) of TUNEL signals (red) in WT primary cortical neurons treated with conditioned media from (<b>B</b>) for 24 h. Nuclei were stained with DAPI. Scale bar, 100 μm. N = 10 fields (each field containing 20–30 neurons). <b>(D, E)</b> Representative images (upper) and quantification (lower) of dot-blot assays on soluble <b>(D)</b> and insoluble <b>(E)</b> Aβ42 levels in brain samples of 6-month old 5XFAD and 5XFAD <i>Becn1</i>^+/- KO mice after 5 weeks of ML246 treatment, immunostained with anti-Aβ42 antibody. Total protein loading was labeled by Ponceau S. Triplicate experiments from 4–5 mice in each group were shown. Results represent mean ± s.e.m. NS, not significant; *, P<0.05; **, P<0.01; ***, P<0.001, t test.</p