A cell-penetrating ester of the neural metabolite lanthionine ketimine stimulates autophagy through the mTORC1 pathway: Evidence for a mechanism of action with pharmacological implications for neurodegenerative pathologies

Abstract

Autophagy is a fundamental cellular recycling process vulnerable to compromise in neurodegeneration. We now report that a cell-penetrating neurotrophic and neuroprotective derivative of the central nervous system (CNS) metabolite, lanthionine ketimine (LK), stimulates autophagy in RG2 glioma and SH-SY5Y neuroblastoma cells at concentrations within or below pharmacological levels reported in previous mouse studies. Autophagy stimulation was evidenced by increased lipidation of microtubule-associated protein 1 light chain 3 (LC3) both in the absence and presence of bafilomycin-A1 which discriminates between effects on autophagic flux versus blockage of autophagy clearance. LKE treatment caused changes in protein level or phosphorylation state of multiple autophagy pathway proteins including mTOR; p70S6 kinase; unc-51-like-kinase-1 (ULK1); beclin-1 and LC3 in a manner essentially identical to effects observed after rapamycin treatment. The LKE site of action was near mTOR because neither LKE nor the mTOR inhibitor rapamycin affected tuberous sclerosis complex (TSC) phosphorylation status upstream from mTOR. Confocal immunofluorescence imaging revealed that LKE specifically decreased mTOR (but not TSC2) colocalization with LAMP2(+) lysosomes in RG2 cells, a necessary event for mTORC1-mediated autophagy suppression, whereas rapamycin had no effect. Suppression of the LK-binding adaptor protein CRMP2 (collapsin response mediator protein-2) by means of shRNA resulted in diminished autophagy flux, suggesting that the LKE action on mTOR localization may occur through a novel mechanism involving CRMP2-mediated intracellular trafficking. These findings clarify the mechanism-of-action for LKE in preclinical models of CNS disease, while suggesting possible roles for natural lanthionine metabolites in regulating CNS autophagy.

Keywords: Autophagy; CRMP2; DPYSL2; Lanthionine ketimine; mTOR complex (mTORC).

Figure 1. LKE stimulates autophagy in SH-SY5Y neuroblastoma and RG2 glioma cells

A: SH-SY5Y cells were treated for 4d then lysed and blotted for LC3. B: The ratio of LC3-II/actin was expressed as % of control for six independent experiments; *P<0.05. C,D: Confirmation that the LC3-II increase was cause by increased autophagy flux rather than blockage of late-stage autophagy clearance steps was accomplished by co-treating cells 4h with 10 µM LKE in the presence or absence of bafilomycin A-1. Persistence of the LKE-induced LC3-II increase in the presence of bafilomycin was taken to indicate that the LKE effect was due to autophagy stimulation rather than clearance inhibition.

Figure 2. LKE stimulation of autophagy is due to inhibition of mTOR pathway signaling and maps to a site of action near the mTORC1

RG2 glioma cells were treated 4h or 24h with 50 nM rapamycin or 10 µM LKE, lysed and blotted with antibodies against the indicated proteins and phospho-epitopes. A: The current autophagy regulation model, inspired by but substantially modified from Ravikumar et al. (2010). B: Action of LKE and rapamycin on the archetypical mTORC1 target, p70S6 kinase; C: Pathway analysis to map site of action for LKE relative to rapamycin. Note that the first common site of action for LKE and rapamycin is at mTOR phosphorylation on residues 2448 (autophosphorylation site) and 2481 (S6K feedback phosphorylation site) but no effects on TSC1/2 occur that would be consistent with a pro-autophagic activity. The stimulation of AKT(p403) by rapamycin is known to occur through disinibition of mTORC2 after mTORC1 inhibition (Guertin et al., 2006).

Figure 3. LKE effect on ULK1 is a sensitive indicator of autophagy activation

A: RG2 glioma cells were treated 24 h with the indicated concentration of LKE, lysed and probed for total ULK1 or ULK1 phosphorylated on the mTOR target residue 757. B: The ratio of ULK1/ULK1(p757) band densities was plotted as a function of LKE concentration.

Figure 4. LKE, but not rapamycin, specifically decreases mTOR localization at lysosomes

Confocal images of RG2 glioma cells double-immunofluorescently labeled for mTOR (green) and the lysosomal marker LAMP2 (red). Co-localization of the two markers (yellow) indicates positioning of mTOR at lysosomal sites. LKE (10 µM, overnight) significantly reduced the mTOR/LAMP2 co-localization but did not affect TSC2 localization relative to lysosomes. Rapamycin (50 nM, overnight) had no significant effect on either mTOR or TSC2 localization. Data represent analyses of 120 images from 12 slides of 4 experiments per treatment are presented in graph.

Figure 5. Partial knock-down of the LK binding, cargo protein CRMP2 slows autophagy flux in SH-SY5Y neuroblastoma cells

A: Relative to vector control infected cells, SH-SY5Y cells stably transfected with shRNA against CRMP2 expressed 50 ± 10% (P<0.01) as much CRMP2 protein, and LC3-II was relatively more elevated in the basal condition. B: CRMP2-knockdown (CRMP2-KD) cells had more LC3-II in the basal state than vector control cells, but displayed proportionally less response to bafilomycin-A, suggesting the increased LC3-II was likely due to impaired autophagy clearance or generally slower autophagy flux. C: LKE increased LC3-II in CRMP2-KD cells both with and without bafilomycin co-treatment, indicating increased autophagy flux induced by the drug even in the presence of diminished CRMP2. D: ULK1 protein was decreased in CRMP2-KD cells but increased by LKE treatment in both vector control and CRMP2-KD cells.