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.

Mild mitochondrial metabolic deficits by α-ketoglutarate dehydrogenase inhibition cause prominent changes in intracellular autophagic signaling: Potential role in the pathobiology of Alzheimer's disease

“…Annexin V is one member of the family of calcium and phospholipid binding proteins. Early ellipsometry studies that focused on phosphatidylserine demonstrate that adsorption proteins is calcium-dependent and is completely reversible upon calcium depletion (Andree et al, 1990; Chu et al, 2013; Hara et al, 2006; Harris-White et al, 2015; Yang et al, 2009; Vives-Bauza et al, 2010; Zhang et al, 2009). Treatments that alter membrane phospholipids and/or their charge will alter Annexin V binding (Andree et al, 1990).…”

“…Migration of some specific cytosolic proteins is a central part of autophagy that is particularly targeted towards the mitochondria (mitophagy), which in turn influences and promotes the selective survival of the existing healthy mitochondria within the cells (Banerjee et al, 2015; Lemasters et al, 2005). For example: (a) parkin migration to the mitochondria is a common early step in depolarization-induced mitophagy (Vives-Bauza et al, 2010); (b) microtubule-associated protein 1A/1B-light chain 3 (LC3) is a soluble protein which is distributed ubiquitously in mammalian tissues and cultured cells, and upon autophagic activation the cytoplasmic LC3 (LC3I) protein is conjugated to phosphatidylethanolamine to form the LC3-phosphatidylethanolamine conjugate (LC3II) which, in turn, is recruited to autophagosomal membranes (Harris-White et al, 2015); (c) the movement of cardiolipin (CL) from the inner mitochondrial membrane (IMM) to the outer mitochondrial membrane (OMM) recruits LC3 and induces mitophagy (Chu et al, 2013). Depolarization and increased fission of the mitochondria make the organelles more fragmented and favor their selection for mitophagy.…”

H2S- and NO-Signaling Pathways in Alzheimer's Amyloid Vasculopathy: Synergism or Antagonism?

“…Brain transsulfuration pathway involving cysteine, homocysteine, and cystathionine generates H 2 S and the atypical amino acid lanthionine (Hensley and Denton, 2015 ). The latter regulates brain cells autophagy (Harris-White et al, 2015 ), neuritis outgrowth (Hubbard et al, 2013 ), and is currently discussed as a candidate for correction of neurological alterations seen in neurodegeneration (Hensley and Denton, 2015 ).…”

Analysis of lanthionine ketimine ethyl ester in mouse serum, whole blood and tissues using ultrahigh‐pressure liquid chromatography/tandem mass spectrometry

“…LK has anti‐neuroinflammatory and neurotrophic activities, and the synthetic ethyl ester of LK (LKE, Figure ) has been reported to be more permeable to the blood–brain barrier than the acid form, a property that has made this compound a candidate for studies that focus on neurodegenerative diseases and processes including Alzheimer's, ischemia, amyotropic lateral sclerosis, multiple sclerosis and Batten's disease . Additionally, LKE stimulates autophagy in RG2 glioma and SH‐SY5Y neuroblastoma cells, and has anti‐apoptotic properties in both in vitro and in vivo models …”