NMDAR inhibition-independent antidepressant actions of ketamine metabolites

Major depressive disorder afflicts ~16 percent of the world population at some point in their lives. Despite a number of available monoaminergic-based antidepressants, most patients require many weeks, if not months, to respond to these treatments, and many patients never attain sustained remission of their symptoms. The non-competitive glutamatergic N-methyl-D-aspartate receptor (NMDAR) antagonist, (R,S)-ketamine (ketamine), exerts rapid and sustained antidepressant effects following a single dose in depressed patients. Here we show that the metabolism of ketamine to (2S,6S;2R,6R)-hydroxynorketamine (HNK) is essential for its antidepressant effects, and that the (2R,6R)-HNK enantiomer exerts behavioural, electroencephalographic, electrophysiological and cellular antidepressant actions in vivo. Notably, we demonstrate that these antidepressant actions are NMDAR inhibition-independent but they involve early and sustained α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor activation. We also establish that (2R,6R)-HNK lacks ketamine-related side-effects. Our results indicate a novel mechanism underlying ketamine’s unique antidepressant properties, which involves the required activity of a distinct metabolite and is independent of NMDAR inhibition. These findings have relevance for the development of next generation, rapid-acting antidepressants

Resveratrol and Its Metabolites Bind to PPARs

Resveratrol, a modulator of several signaling proteins, can exert off-target effects involving PPAR transcription factor. However, evidence for the direct interaction between this polyphenol and PPARs is lacking. Here, we addressed the hypothesis that resveratrol and its metabolites control aspects of PPAR transcriptional activity through direct interaction with PPARs. Bioaffinity chromatographic studies with the immobilized ligand-binding domains (LBDs) of PPARγ and PPARα and isothermal titration calorimetry (ITC) allowed the determination of the binding affinities of resveratrol, resveratrol 3-O-glucuronide, resveratrol 4-O-glucuronide and resveratrol 3-O-sulfate to both PPAR-LBDs. Interaction of resveratrol, resveratrol 3-O-glucuronide and resveratrol 4-O-glucuronide with PPARγ-LBD occurred with binding affinities of 1.4, 1.1 and 0.8 μM, respectively, while only resveratrol bound to the PPARα-LBD with a binding affinity of 2.7 μM. Subsequently, X-ray crystallographic studies were carried out to characterize resveratrol binding to the PPARγ-LBD at the molecular level. The electron density map from the crystal structure of the complex between PPARγ-LBD and resveratrol revealed the presence of one molecule of resveratrol bound into the LBD of PPARγ, with the ligand occupying a position close to that of other know PPARγ ligands. Transactivation assays were also performed in HepG2 cells and the results showed that resveratrol was not a PPAR agonist but, instead was able to displace rosiglitazone from PPARγ and Wy-14,643 from PPARα with IC50 values of 27.4 ± 1.8 μM and 31.7 ± 2.5 μM, respectively. We propose that resveratrol acts as a PPAR antagonist through its direct interaction with PPARγ and PPARα

Differential sensitivity of prefrontal cortex and hippocampus to alcohol-induced toxicity.

The prefrontal cortex (PFC) is a brain region responsible for executive functions including working memory, impulse control and decision making. The loss of these functions may ultimately lead to addiction. Using histological analysis combined with stereological technique, we demonstrated that the PFC is more vulnerable to chronic alcohol-induced oxidative stress and neuronal cell death than the hippocampus. This increased vulnerability is evidenced by elevated oxidative stress-induced DNA damage and enhanced expression of apoptotic markers in PFC neurons. We also found that one-carbon metabolism (OCM) impairment plays a significant role in alcohol toxicity to the PFC seen from the difference in the effects of acute and chronic alcohol exposure on DNA repair and from exaggeration of the damaging effects upon additional OCM impairment in mice deficient in a key OCM enzyme, methylenetetrahydrofolate reductase (MTHFR). Given that damage to the PFC leads to loss of executive function and addiction, our study may shed light on the mechanism of alcohol addiction

Identification of CB1/CB2 Ligands from <i>Zanthoxylum bungeanum</i>

In order to study cannabinoid receptor ligands, a novel plate-based assay was developed previously to measure internalization of CB1/CB2 receptors by determining the change in the intracellular levels of the radiolabeled agonists. This plate-based assay was also used for screening against complex matrices, specifically, in the present study screening for CB1/CB2 receptor activity of extracts for several species of the plant genus <i>Zanthoxylum</i>. The objective of this screen was to identify novel antagonists of the CB1 receptor, which simultaneously displayed agonist activity against the CB2 receptor, since compounds matching this criterion could be potential candidates for the treatment of type-1 diabetes. As a result, two <i>Z. bungeanum</i> extracts were deemed active, leading to the identification of eight compounds, of which compound <b>7</b> [(2<i>E</i>,4<i>E</i>,8<i>E</i>,10<i>E</i>,12<i>E</i>)-<i>N</i>-isobutyl-2,4,8,10,12-tetradecapentaenamide, γ-sanshool] was obtained as a promising lead compound

Templates for qPCR-based BER activity assay.

<p>Whole cell extracts were isolated from brain tissue and exposed to a template DNA containing a single nucleotide lesion (A) and control template (B). BER activity was calculated by comparing the ΔΔCt values (Ct is the number of cycles required for the fluorescent signal to cross the threshold) of the repaired and control templates.</p

OCM impairment is involved in ethanol-induced oxidative DNA damage and neuronal apoptosis effects in the PFC.

<p>The brain sections (PFC) of WT and <i>Mthfr+/−</i> mice exposed for 3 weeks or 4 days (acute) to the Lieber-DeCarli liquid diet with- or without ethanol (5%) were triple-labeled with NeuN (purple), TUNEL (green) and cleaved caspase-3 (red). Hoechst 33342 (blue) was used to identify all cell nuclei. Fluorescence was visualized by confocal microscopy. Scale bar = 20 µm. Note increased number of TUNEL/caspase-3-positive neurons in PFC of <i>Mthfr+/−</i> mice chronically exposed to ethanol, compared with corresponding WT mice (arrows). Also, note a higher density of TUNEL/caspase-3-positive neurons in PFC of chronically, compared with acutely exposed to ethanol WT mice (arrows).</p

PFC is more vulnerable to ethanol-induced neuronal apoptosis than hippocampus.

<p>Brain sections double-labeled with TUNEL and MAP-2 in the PFC and hippocampus (Hip) and quantified by stereological counting; Values are means ± SEM; *p<0.01. Neither the number of MAP-2-positive cells (neurons) nor volumes of the brain structures were affected by chronic 3-week ethanol exposure. Note significantly higher density of TUNEL- positive neurons in PFC than in hippocampus of ethanol-exposed mice.</p

PFC is more vulnerable to ethanol-induced oxidative stress than hippocampus.

<p>(A) oxo-8dG expression in neurons (neuronal marker MAP-2) was quantified in PFC and hippocampus (Hip) by stereological counting. Neither the number of neurons nor volumes of the brain structures were affected. Values are mean ± SEM; *p<0.01. (B) Oxo-8dG expression in neurons normalized to corresponding controls (Δ). Note significantly higher density of oxo-8dG -labeled neurons and Δ in PFC, compared with hippocampus of ethanol-exposed mice. (C) DNA repair activity in response to oxidative DNA damage (oxo-8dG) assessed by qPCR in whole cell extracts obtained from PFC and hippocampus (Hip) of control mice and mice exposed to acute or chronic ethanol. Values are means ± SEM; *p<0.05, **p<0.01. Note the response to oxidative DNA damage by PFC lysate is significantly stronger than those in the hippocampus.</p