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.

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.

Semaphorin 3A signaling through neuropilin-1 is an early trigger for distal axonopathy in the SOD1G93A mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a motor neuron disease characterized by progressive distal axonopathy that precedes actual motor neuron death. Triggers for neuromuscular junction degeneration remain to be determined, but the axon repulsion factor semaphorin 3A (Sema3A), which is derived from terminal Schwann cells, is a plausible candidate. This study examines the hypothesis that Sema3A signaling through its motor neuron neuropilin-1 (NRP1) receptor triggers distal axonopathy and muscle denervation in the SOD1 mouse model of ALS. Neuropilin-1 was found to be expressed in axonal terminals at the mouse neuromuscular junction in vivo and in NSC-34 motor neuron-like cells in vitro. In differentiated NSC-34 cells, an anti-NRP1 antibody that selectively blocks Sema3A binding to NRP1 prevented Sema3A-induced growth cone collapse. Furthermore, intraperitoneal injections of anti-NRP1 antibody administered twice weekly from age 40 days significantly delayed and even temporarily reversed motor functional decline while prolonging the life span of SOD1 mice. Histologic evaluation at 90 and 125 days revealed that anti-NRP1 antibody reduced neuromuscular junction denervation and attenuated pathologic alterations in ventral roots at late-stage disease. These data suggest that peripheral NRP1 signaling is involved in the pathobiology of this ALS model and that antagonizing Sema3A/NRP1 binding or downstream signals could have implications for the treatment of ALS.

A derivative of the brain metabolite lanthionine ketimine improves cognition and diminishes pathology in the 3 × Tg-AD mouse model of Alzheimer disease.

Lanthionine ketimine ([LK] 3,4-dihydro-2H-1,4-thiazine-3,5-dicarboxylic acid) is the archetype for a family of naturally occurring brain sulfur amino acid metabolites, the physiologic function of which is unknown. Lanthionine ketimine and its synthetic derivatives have recently demonstrated neurotrophic, neuroprotective, and antineuroinflammatory properties in vitro through a proposed mechanism involving the microtubule-associated protein collapsin response mediator protein 2. Therefore, studies were undertaken to test the effects of a bioavailable LK ester in the 3 × Tg-AD mouse model of Alzheimer disease. Lanthionine ketimine ester treatment substantially diminished cognitive decline and brain amyloid-ß (Aß) peptide deposition and phospho-Tau accumulation in 3 × Tg-AD mice and also reduced the density of Iba1-positive microglia. Furthermore, LK ester treatment altered collapsin response mediator protein 2 phosphorylation. These findings suggest that LK may not be a metabolic waste but rather a purposeful neurochemical, the synthetic derivatives of which constitute a new class of experimental therapeutics for Alzheimer disease and related entities.

Collapsin response mediator protein-2: an emerging pathologic feature and therapeutic target for neurodisease indications.

Collapsin response mediator protein-2 (DPYSL2 or CRMP2) is a multifunctional adaptor protein within the central nervous system. In the developing brain or cell cultures, CRMP2 performs structural and regulatory functions related to cytoskeletal dynamics, vesicle trafficking and synaptic physiology whereas CRMP2 functions in adult brain are still being elucidated. CRMP2 has been associated with several neuropathologic or psychiatric conditions including Alzheimer's disease (AD) and schizophrenia, either at the level of genetic polymorphisms; protein expression; post-translational modifications; or protein/protein interactions. In AD, CRMP2 is phosphorylated by glycogen synthase kinase-3ß (GSK3ß) and cyclin dependent protein kinase-5 (CDK5), the same kinases that act on tau protein in generating neurofibrillary tangles (NFTs). Phosphorylated CRMP2 collects in NFTs in association with the synaptic structure-regulating SRA1/WAVE1 (specifically Rac1-associated protein-1/WASP family verprolin-homologous protein-1) complex. This phenomenon could plausibly contribute to deficits in neural and synaptic structure that have been well documented in AD. This review discusses the essential biology of CRMP2 in the context of nascent data implicating CRMP2 perturbations as either a correlate of, or plausible contributor to, diverse neuropathologies. A discussion is made of recent findings that the atypical antidepressant tianeptine increases CRMP2 expression, whereas other, neuroactive small molecules including the epilepsy drug lacosamide and the natural brain metabolite lanthionine ketimine appear to bind CRMP2 directly with concomitant affects on neural structure. These findings constitute proofs-of-concept that pharmacological manipulation of CRMP2 is possible and hence, may offer new opportunities for therapy development against certain neurological diseases.

Emerging biological importance of central nervous system lanthionines.

Lanthionine (Lan), the thioether analog of cystine, is a natural but nonproteogenic amino acid thought to form naturally in mammals through promiscuous reactivity of the transsulfuration enzyme cystathionine-beta-synthase (CbetaS). Lanthionine exists at appreciable concentrations in mammalian brain, where it undergoes aminotransferase conversion to yield an unusual cyclic thioether, lanthionine ketimine (LK; 2H-1,4-thiazine-5,6-dihydro-3,5-dicarboxylic acid). Recently, LK was discovered to possess neuroprotective, neuritigenic and anti-inflammatory activities. Moreover, both LK and the ubiquitous redox regulator glutathione (gamma-glutamyl-cysteine-glycine) bind to mammalian lanthionine synthetase-like protein-1 (LanCL1) protein which, along with its homolog LanCL2, has been associated with important physiological processes including signal transduction and insulin sensitization. These findings begin to suggest that Lan and its downstream metabolites may be physiologically important substances rather than mere metabolic waste. This review summarizes the current state of knowledge about lanthionyl metabolites with emphasis on their possible relationships to LanCL1/2 proteins and glutathione. The potential significance of lanthionines in paracrine signaling is discussed with reference to opportunities for utilizing bioavailable pro-drug derivatives of these compounds as novel pharmacophores.

Exposure of the amygdala to elevated levels of corticosterone alters colonic motility in response to acute psychological stress.

The amygdala is important for integrating the emotional, endocrine and autonomic responses to stress. Exposure of the amygdala to elevated levels of corticosterone (CORT) induces anxiety-like behavior and a hypersensitive colon in rodents; however, effects on colonic transit are unknown. Micropellets releasing CORT alone or combined with a selective glucocorticoid (GR) or mineralocorticoid (MR) receptor antagonist were implanted bilaterally at the dorsal boundary of the central amygdala in male rats. Inactive cholesterol implants served as controls. Seven days later, rats received water avoidance stress (WAS) for 1 h and the fecal pellet output was measured. Colorectal transit was also evaluated following the stressor by recording the time for expulsion of a glass bead placed into the colorectum. Plasma CORT levels were evaluated before WAS, after 60 min of WAS and 90 min post-WAS. Exposure of the amygdala to elevated CORT did not alter daily fecal pellet production or the number of fecal pellets released in response to WAS. However, following WAS, rats with CORT implants on the amygdala showed a delay in colorectal transit compared to cholesterol-implanted controls. Plasma CORT measurements showed that basal and WAS-induced increases in plasma CORT were similar in all groups but a prolonged increase in plasma CORT was observed 90 after cessation of WAS in rats with CORT implants. The post-WAS changes in colonic motility and plasma CORT were prevented by antagonism of GR or MR in the amygdala, suggesting their importance in driving stress-associated changes in colonic motility.

Efficacy of ipamorelin, a novel ghrelin mimetic, in a rodent model of postoperative ileus.

Ghrelin and ghrelin mimetics stimulate appetite and enhance gastric motility. The present study investigates whether ipamorelin, a selective growth hormone secretagogue and agonist of the ghrelin receptor, would accelerate gastrointestinal transit and ameliorate the symptoms in a rodent model of postoperative ileus (POI). Fasted male rats were subjected to laparotomy and intestinal manipulation. At the end of surgery, a dye marker was infused in the proximal colon to evaluate postsurgical colonic transit time, which was the time to the first bowel movement. In addition, fecal pellet output, food intake, and body weight were monitored regularly for 48 h. Ipamorelin (0.01-1 mg/kg), growth hormone-releasing peptide (GHRP)-6 (20 microg/kg), or vehicle (saline) were administered via intravenous bolus infusion after a single dosing or a 2-day repetitive dosing regimen (four doses a day at 3-h intervals). Compared with the vehicle, a single dose of ipamorelin (1 mg/kg) or GHRP-6 (20 microg/kg) decreased the time to the first bowel movement but had no effect on cumulative fecal output, food intake, or body weight gain measured 48 h after the surgery. In contrast, repetitive dosing of ipamorelin (0.1 or 1 mg/kg) significantly increased the cumulative fecal pellet output, food intake, and body weight gain. The results suggest that postsurgical intravenous infusions of ipamorelin may ameliorate the symptoms in patients with POI.

Effect of TZP-201, a novel motilin receptor antagonist, in the colon of the musk shrew (Suncus murinus).

OBJECTIVES: Motilin is the main gut peptide that stimulates propulsive motility in the upper gastrointestinal (GI) tract. Motilin receptors exist in the colon but little is known about their functional role, and species-dependent differences present a major obstacle to understanding the physiological significance and potential therapeutic implications of motilin receptors in the colon. Our study aimed to define whether a motilin receptor is functionally expressed in the colon of the Asian musk (or house) shrew (Suncus murinus) and to investigate the effect of a novel motilin receptor antagonist, TZP-201. METHODS: GI tissue (gastric antrum, small intestine and colon) was isolated from male shrews and the effects of a motilin receptor agonist [Nle13]motilin and the antagonist TZP-201 on contractile activity and mucosal electrogenic transport of water and electrolytes were investigated in vitro. KEY FINDINGS: [Nle13]motilin induced a moderate increase in spontaneous contractility in the stomach and no significant changes in the small intestine; a marked increase in contractility was found in the colon. Motilin-induced contractions in the colon were abolished by tetrodotoxin or atropine, and dose-dependently inhibited by 0.01-10 muM TZP-201. Neither [Nle13]motilin nor TZP-201 had any effect on basal mucosal transport. CONCLUSIONS: Shrew colon expresses a functional motilin receptor that induces contractile activity by the activation of enteric cholinergic neurons. TZP-201 inhibited motilin-induced colonic contractions. Motilin antagonists may represent a new approach for the treatment of GI motility disorders characterised by hypercontractility.

Effect of the ghrelin receptor agonist TZP-101 on colonic transit in a rat model of postoperative ileus.

Ghrelin, the natural ligand of the growth hormone secretagogue receptor (ghrelin receptor), is an orexigenic gut hormone with prokinetic action in the upper gastrointestinal tract. Previously we have shown in a rodent model of postoperative ileus that the synthetic ghrelin receptor agonist TZP-101 prevents the delay in gastric emptying and improves small intestinal transit. The goal of the present study was to investigate whether TZP-101 affects colonic transit and food intake in rats with postoperative ileus. Fasted rats were treated with morphine and subjected to laparotomy under isoflurane anesthesia. Following surgery the animals were placed in clean home cages and fecal pellet output and food intake were monitored for 48 h. TZP-101 or vehicle were administered as 3 i.v. bolus infusions at 0 h, 2 h and 4 h post-surgery. TZP-101 (0.03-1 mg/kg) dose-dependently decreased the time to first bowel movement and increased fecal pellet output measured at 12 h and 24 h post-surgery compared to the vehicle. The administration of TZP-101 was not associated with a significant alteration in food intake. In conclusion, this study provides the first experimental evidence that a novel ghrelin receptor agonist improves large bowel function in rats with postoperative ileus, suggesting that TZP-101 may be useful in the clinic to accelerate upper gastrointestinal transit and to shorten the time to the first bowel movement following surgery.

Mineralocorticoid and glucocorticoid receptors in the amygdala regulate distinct responses to colorectal distension.

Previously we found that exposure of the amygdala to elevated levels of corticosterone (CORT) induces anxiety-like behavior coupled to colonic hypersensitivity to distension, however, the specific corticoid receptor mediating the CORT responses remains controversial. In this study we investigated, through the use of selective antagonists, the relative role of amygdaloid mineralocorticoid (MR) versus glucocorticoid receptors (GR) in CORT-mediated spinal and cardiovascular pseudoaffective reflex responses to colorectal distension (CRD). Micropellets containing, CORT and a selective MR antagonist (spironolactone) or GR antagonist (mifepristone) were implanted stereotaxically onto the dorsal margin of the amygdala in rats. On day 7 post-implantation in response to graded CRD we measured: (i) changes in the electrical activity of dorsal horn neurons in the L6-S1 spinal cord and (ii) the cardiovascular depressor responses. Exposure of the amygdala to CORT-releasing micropellets increased the proportion of spinal neurons showing high-threshold and/or long-lasting responses and potentiated the magnitude of excitation. Elevated levels of amygdala CORT also increased the magnitude of the cardiovascular depressor response to CRD. MR but not GR antagonism prevented the increase in spinal cord neuronal excitation, whereas either the MR or GR antagonist decreased the magnitude of the depressor cardiovascular response to CRD. We conclude that MR in the amygdala trigger descending pathways facilitating viscero-nociceptive processing in the spinal cord, whereas MR and GR have a non-redundant role in CORT-induced potentiation of the autonomic pseudoaffective responses to colorectal stimuli.

Application of ghrelin to gastrointestinal diseases.

This review outlines recent findings related to the therapeutic potential of ghrelin agonists or antagonists for the treatment of gastrointestinal (GI) motility disorders and the regulation of the hypothalamic control of feeding. The ability of ghrelin to promote GI motility in patients with postoperative ileus or gastroparesis, and to restore the normal feeding pattern and energy balance in the elderly and in cancer patients with impaired appetite are highlighted. A critical analysis of the possible mechanism of action and the potential benefits of using synthetic ghrelin agonists is based on data from preclinical research and recent clinical studies.

Prokinetic effects of a new ghrelin receptor agonist TZP-101 in a rat model of postoperative ileus.

Postoperative ileus (POI) is a major cause of postoperative complications and prolonged hospitalization. Ghrelin, which is the endogenous ligand for the growth hormone secretagogue receptor, has been found to stimulate gastric motility and accelerate gastric emptying. The present study investigates whether TZP-101 (0.03-1 mg/kg i.v.), a synthetic ghrelin-receptor agonist, could improve gastrointestinal transit in rats with POI. Since the main factors for the development of POI are the surgical manipulation and the gastrointestinal effects of opioid-receptor agonists used for pain management, the effect of TZP-101 was investigated in rats subjected to surgery, to morphine treatment (3 mg/kg s.c.), or to a combination of both. The results showed that TZP-101 is equally effective against the delayed gastrointestinal transit induced by surgery, by morphine, or by the combination of both interventions. The prokinetic action of TZP-101 was more pronounced in the stomach compared to the small intestine.

Effects of TAK-637 on NK(1) receptor-mediated mechanisms regulating colonic secretion.

This study investigates the effect of a selective NK(1) receptor antagonist TAK-637 on enteric mechanisms involved in regulation of epithelial secretion in the colon. Mucosal sheets isolated from guinea-pig colon were placed in modified Ussing chambers and the net active transport of electrolytes was measured as short-circuit current (Isc). GR-73632, a selective NK(1) receptor agonist, induced an increase in basal Isc, which was inhibited by TAK-637 (IC(50) of 21 nM). The increase in Isc induced by GR-73632 was significantly attenuated by tetrodotoxin (TTX, 1 microM), indicating that TAK-637 inhibits neuronal NK(1) receptors. Moreover, TAK-637 reduced the TTX-resistant component of the response to GR-73632 suggesting that NK(1) receptors expressed by epithelial cells are inhibited by TAK-637. In separate experiments, TAK-637 partially inhibited the submaximal Isc induced by electrical field stimulation (EFS, 0.5 ms, 15 Hz) of enteric nerves or by activation of primary afferent fibers using capsaicin (50 microM). TAK-637 had no significant effect on the basal Isc or on responses induced by neurokinin A (NKA), senktide, or forskolin. The results imply that inhibition of peripheral NK(1) receptors may reduce autonomic epithelial secretion in response to activation of autonomic secretomotor pathways, while having no significant effect on basal epithelial transport.

Oral treatment with recombinant human interleukin-11 improves mucosal transport in the colon of human leukocyte antigen-B27 transgenic rats.

Recombinant human interleukin (IL)-11 is a pleiotropic cytokine with anti-inflammatory activity. The objective of the study was to investigate whether oral treatment with rhIL-11 improves colonic epithelial dysfunction in the human leukocyte antigen (HLA)-B27 transgenic rat model of spontaneous chronic inflammation. Experiments were performed using adult male HLAB27 rats, whereas healthy nontransgenic F344 rats served as controls. Enteric-coated rhIL-11 multi-particles (equivalent to 500 microg/kg rhIL11) or placebo (formulation lacking rhIL-11) were administrated orally on alternate days for 2 weeks to HLA-B27 or F344 rats. Stool character was observed daily during the treatment period. Animals were euthanized at the end of treatment and colonic inflammation was evaluated be measuring tissue myeloperoxidase (MPO) activity. Epithelial transport in isolated colonic mucosal sheets was studied in modified Ussing chambers. Oral treatment of HLA-B27 rats with rhIL-11 reduced MPO activity in the colon and suppressed the clinical signs of diarrhea. The electrophysiological characteristics of mucosal transport were improved in the HLA-B27 rats treated with rhIL-11 compared with placebo. After rhIL-11 treatment the basal transepithelial resistance and the estimated paracellular resistance were significantly increased, neurally mediated secretory responses to electrical field stimulation were improved, and cholinoceptor sensitivity was normalized. Treatment with rhIL-11 had no significant effect on basal short circuit current and the maximal secretory response to carbachol or substance P. Our data demonstrate that oral rhIL-11 therapy is associated with suppression of mucosal inflammation and a concomitant improvement of epithelial resistance and neurally mediated secretion in a model of chronic HLA-B27 colitis.

Seirogan (wood creosote) inhibits stress-induced ion secretion in rat intestinal epithelium.

Acute stress in often associated with abnormalities in gastrointestinal function, including enhanced secretion of water and electrolytes that leads to diarrhea. The goal of our study was to investigate whether Seirogan inhibits stress-induced intestinal secretion in Wistar-Kyoto rats. Electrogenic ion secretion was measured in modified Ussing chambers as an increase in basal short-circuit current (Isc) across isolated rat jejunal or colonic mucosal sheets. Mucosal preparations from rats exposed to cold restraint stress showed a significant increase in basal Isc compared to controls. The cumulative addition of Seirogan to the Ussing chamber caused a concentration-dependent reduction of the stress-induced increase of basal Isc to levels resembling nonstressed controls. In a separate experiment, Seirogan (15 mg/kg) administered by oral gavage inhibited stress-induced secretion and normalized basal Isc in the jejunum and colon. The results suggest that Seirogan may be an effective therapy for patients with stress-associated diarrhea.

NK1 receptor-mediated mechanisms regulate colonic hypersensitivity in the guinea pig.

Neurokinin-1 (NK(1)) receptors activated by substance P (SP) are involved in the processing of nociceptive information and are a potential target for therapy of visceral pain. We have evaluated the role of NK(1) receptors using a selective antagonist of NK(1) receptors in two animal models of colorectal hypersensitivity. The behavioral response to colorectal distension was assessed in freely moving guinea pigs by recording visceromotor reflex contractions of the abdominal musculature. Colonic hypersensitivity was induced by intracolonic administration of a chemical irritant (0.6% of acetic acid), or by acute partial restraint stress. Sensitization was characterized by an exaggerated visceromotor response to a low level of colorectal distension (10 mm Hg). In both models of colonic hypersensitivity, oral administration of TAK-637 (0.1-10 mg/kg) normalized visceromotor responses. The intracerebroventricular (10 microg/kg) or intrathecal (10 microg/kg) administration of TAK-637 inhibited colonic hypersensitivity, suggesting an interaction with central NK(1) receptors. In contrast, TAK-637 had no effect on visceromotor responses to colorectal distension at 40 mm Hg in guinea pigs with normosensitive (nonsensitized) colons. In conclusion, central NK(1) receptors play a significant role in colonic hypersensitivity induced by visceral afferent nerve sensitization from gastrointestinal origin or acute psychosomatic stress, but not in the perception of colorectal distension in animals with normosensitive colons.

Peripheral activity of a new NK1 receptor antagonist TAK-637 in the gastrointestinal tract.

Pathways controlling gastrointestinal function involve the activation of neurokinin NK1 receptors by substance P (SP) under normal and pathological conditions. Our aim was to pharmacologically characterize the effect of a nonpeptide NK1 receptor antagonist TAK-637 [(aR,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,11-tetrahydro-9-methyl-5-(4-methylphenyl)-7H-[1,4]diazocino[2,1-g] [1,7]naphthyridine-6,13-dione] and determine key mechanisms of TAK-637 action in the gastrointestinal tract. Experiments were performed using intestinal preparations isolated from the guinea pig. The selective agonists of NK1 receptors, [Sar9,Met(O2)11]-SP and GR 73632 [H2N-(CH2)4-CO-Phe-Phe-Pro-NMe-Leu-Met-NH2], induced contractions in colonic longitudinal muscle pretreated with atropine. TAK-637 (1-100 nM) caused a rightward shift of the concentration-response curves showing nanomolar affinity against [Sar9,Met(O2)11]-SP (Kb = 4.7 nM) and GR 73632 (K(b) = 1.8 nM). This antagonist effect remained unchanged by tetrodotoxin. Furthermore, neither the contractions of colonic circular muscle induced by selective activation of NK2 receptors by GR 64349 (Lys-Asp-Ser-Phe-Val-Gly-R-gamma-lactam-Leu-Met-NH2) nor the responses of taenia coli induced by the selective NK3 receptor agonist senktide were affected by TAK-637 (100 nM). Studies of electrically induced neurogenic contractions showed that TAK-637 had no effect on cholinergic responses to single-pulse (0.5 ms) stimulation or stimulation with increasing frequency (1-16 Hz, 0.5 ms, 5-s train duration). In contrast, TAK-637 significantly reduced nonadrenergic, noncholinergic contractions of colonic longitudinal muscle evoked at frequencies of 8 to 16 Hz and prevented the development of capsaicin-induced contractions in isolated segments of terminal ileum. Our results indicate that TAK-637 is a selective antagonist of smooth muscle NK(1) receptors that activate intestinal muscle contraction. Additionally TAK-637 inhibits neuronal NK1 receptors involved in the "local" motor response to stimulation of capsaicin-sensitive primary afferents.