Methylone is a rapid-acting neuroplastogen with less off-target activity than MDMA

Warner-Schmidt and colleagues situate their study in the context of limited, slow-acting pharmacotherapies for post-traumatic stress disorder (PTSD) and the recent clinical interest in MDMA-assisted psychotherapy. Earlier work shows that methylone, a structural analogue of MDMA, has rapid, robust and durable antidepressant- and anxiolytic-like effects in preclinical models and encouraging open-label case series in humans, but the molecular mechanisms that might underlie these effects are not well defined. The authors note key practical differences between methylone and MDMA reported clinically and preclinically, including the absence of hallucinogenic effects for methylone, tolerability, and a potentially reduced requirement for extensive concomitant psychotherapy and compatibility with selective serotonin reuptake inhibitors (SSRIs).

Multiple complementary experimental approaches were used to compare methylone and MDMA. Male Sprague Dawley rats (~200 g) were used for in vitro binding, uptake and release assays (performed at Gifford Bioscience, UK) and for in-life RNA sequencing (RNA-seq) and immunohistochemistry (performed at WuXi Apptec, USA). For radioligand binding, rat brain membranes were prepared and competitive binding performed with [3H] citalopram, [3H] nisoxetine and [3H] WIN35428 to estimate inhibition constants (K i ). Uptake inhibition assays used synaptosomes loaded with radiolabeled 5-HT, DA or NE to determine IC50 values, and release assays used superfusion of preloaded synaptosomes to calculate drug-evoked release and EC50 values. Standard non-linear curve-fitting routines (Prism) were used to derive pharmacological parameters. Off-target GPCR activity was assessed using the GPCRmax high-throughput screen at Eurofins DiscoverX, testing agonist and antagonist activity across a library of 168 G-protein coupled receptors. In silico docking was performed with MOE 2022.02 using the Amber10 force field to examine potential ligand fits to experimentally determined receptor structures, with ligand conformer generation and docking-score evaluation described in the methods. For transcriptomics, rats received a single intraperitoneal (IP) dose of methylone (10 mg/kg), MDMA (10 mg/kg) or vehicle and brains were harvested 8 hours later. Frontal cortex and amygdala were dissected, total RNA extracted (Qiagen RNeasy), mRNA-enriched libraries prepared (NEBNext PolyA selection) and sequenced on an Illumina platform with 2 × 150 bp paired-end reads. Reads were trimmed, aligned to the ENSEMBL reference genome using STAR, and unique exon read counts obtained with featureCounts. Differential expression and functional enrichment were performed; pathway analysis used Metascape on gene lists selected by a reported statistical cutoff (extracted text gives the cutoff as “0.32 ≥ log2FC < -0.32, padj ≤0.1”, which is unclear in sign convention). Immunohistochemistry was conducted on rats treated with the same single doses and sacrificed 24 h later; brain sections were stained for myelin basic protein (MBP) and imaged at 20×. For non-RNA-seq experiments, statistics used t-tests or one-way ANOVA with Fisher’s LSD post-hoc testing and p < 0.05 as the threshold for significance.

Pharmacology at monoamine transporters showed that methylone and MDMA are both uptake inhibitors and releasers of serotonin (5-HT), norepinephrine (NE) and dopamine (DA), but with differing affinities. Competitive binding yielded K i values at SERT, NET and DAT of 4.15, 1.15 and 5.73 μM for methylone versus 2.62, 0.79 and 5.11 μM for MDMA, respectively. In synaptosomal uptake assays methylone inhibited 5-HT uptake more potently than MDMA (IC50 = 0.43 μM vs 1.8 μM), while NE and DA uptake inhibition were comparable between the drugs (NE IC50 = 0.13 vs 0.12 μM; DA IC50 = 2.3 vs 2.5 μM). Release assays indicated that methylone evoked less DA release than MDMA while producing similar 5-HT and NE release; reported EC50 values were 5-HT: 0.62 vs 0.16 mM, DA: 4.79 vs 1.42 mM, and NE: 0.33 vs 0.49 mM. A statistical comparison for DA effects reported drug: F(1,4) = 9.932, p < 0.05 and concentration: F(2,9) = 28.66, p < 0.0001. The GPCR screen across 168 receptors found no significant agonist or antagonist activity for methylone at 1 or 10 μM; the largest signal (5HT5A at 13.9%) was well below the assay's 30% activity threshold. In contrast, MDMA (10 μM) approached the agonist cutoff for 5HT2A (27.5%) and 5HT2C (28.0%) and showed notable antagonist activity at 5HT2C (52.2%). These differences were statistically significant in the reported comparisons (e.g., 5HT2A agonism: F(3,8) = 354.8, p < 0.0001). Docking simulations agreed with the screening results: MDMA fitted the 5HT2A and 5HT2C binding pockets, whereas methylone—whose structure differs by a ketone substituent—could not be accommodated without steric clashes, consistent with lack of observed activity. RNA-seq of amygdala and frontal cortex 8 h after a single 10 mg/kg IP dose revealed region- and drug-specific transcriptional responses. In the amygdala, MDMA regulated substantially more genes than methylone; nearly all genes changed by methylone were also changed by MDMA, but MDMA altered an additional 1,313 genes. Functional enrichment of MDMA-specific changes included G alpha (q) and GPCR signalling, receptor tyrosine kinase signalling, protein folding, orexin receptor pathway and cytokine signalling. Both drugs downregulated a set of myelin-associated genes in the amygdala: nearly 20% of downregulated transcripts were myelin-related. Immunohistochemistry confirmed a reduction in myelin basic protein (MBP) in the basolateral (F(2,13) = 4.417, p < 0.05) and central nuclei (F(2,13) = 4.062, p < 0.05) of the amygdala after drug treatment, whereas MBP in cortex was unchanged (F(2,13) = 3.776, not significant). The most strongly downregulated gene after methylone in the amygdala was Ankk1, a locus linked to dopamine synthesis and PTSD susceptibility. In the frontal cortex, MDMA again regulated more genes (~825) than methylone (~480), with 154 genes commonly regulated. Methylone specifically upregulated pathways tied to synaptic plasticity, cytogenesis, survival and neurotrophin signalling, including BDNF signalling. Among the top methylone-upregulated transcripts were Vgf, Camk1g, Selenom, Nfil3, Psrc1 and Nptx2—genes implicated by the authors in various aspects of synaptic and neurotrophic plasticity. The authors also report that methylone produced a rapid and robust induction of BDNF (reported as ~60% increase versus controls in the discussion), and that MDMA regulated related neuroplasticity genes but additionally modulated stress-response and protein-folding pathways that were not seen with methylone. The authors note greater inter-animal variability in the methylone gene-expression effects compared with MDMA.

Warner-Schmidt and colleagues interpret the convergent pharmacology and transcriptomics as evidence that methylone is a rapid-acting neuroplastogen with a narrower off-target profile than MDMA. Both drugs act as monoamine uptake inhibitors and releasers, but methylone released less dopamine than MDMA, which the authors suggest may reduce tendencies for impulsivity, heightened emotional sensitivity or addictive behaviours that are linked to dopaminergic transmission. Differences in GPCR activity and docking—MDMA showing activity at 5HT2A/2C and methylone showing none—are offered as molecular explanations for clinical observations that methylone is non-hallucinogenic and better tolerated in some patients. A major finding highlighted by the authors is the rapid downregulation of myelin-related genes in the amygdala after either drug and the corroborating reduction in MBP immunoreactivity. They propose that rapid myelin plasticity may render amygdala circuits more malleable, potentially facilitating the disruption of pathological fear memories relevant to PTSD. In the frontal cortex, rapid upregulation of neuroplasticity and neurotrophin-related genes—most notably BDNF and several synaptic plasticity regulators—provides a candidate mechanism for the fast antidepressant- and anxiolytic-like behavioural effects previously reported for methylone. The authors acknowledge several uncertainties. A discrepancy with prior reports about the rank order of monoamine release (their study: NE > 5HT > DA; prior studies: NE > DA > 5HT) may reflect different experimental systems (rat synaptosomes here versus transfected HEK cells elsewhere). They also note that methylone-induced gene-expression changes were more variable between individual animals than MDMA-induced changes, which could reflect biological heterogeneity or differences in neurotransmitter responses. Additional pathways regulated by MDMA only—heat shock proteins, orexin signalling and cytokine pathways—may underlie some of MDMA's distinct acute effects such as changes in energy, appetite and immune stress, and could relate to abuse liability; the authors caution that the roles of these pathways require further study. Finally, they recommend future experiments to examine structural and functional neuroplasticity and circuit-level effects to test the mechanistic hypotheses generated by the transcriptomic and immunohistochemical results.