The tricyclic antidepressant clomipramine inhibits neuronal autophagic flux

Abstract

Antidepressants are commonly prescribed psychotropic substances for the symptomatic treatment of mood disorders. Their primary mechanism of action is the modulation of neurotransmission and the consequent accumulation of monoamines, such as serotonin and noradrenaline. However, antidepressants have additional molecular targets that, through multiple signaling cascades, may ultimately alter essential cellular processes. In this regard, it was previously demonstrated that clomipramine, a widely used FDA-approved tricyclic antidepressant, interferes with the autophagic flux and severely compromises the viability of tumorigenic cells upon cytotoxic stress. Consistent with this line of evidence, we report here that clomipramine undermines autophagosome formation and cargo degradation in primary dissociated neurons. A similar pattern was observed in the frontal cortex and liver of treated mice, as well as in the nematode Caenorhabditis elegans exposed to clomipramine. Together, our findings indicate that clomipramine may negatively regulate the autophagic flux in various tissues, with potential metabolic and functional implications for the homeostatic maintenance of differentiated cells.

Figure 1

Clomipramine and fluoxetine treatments reduce autophagy flux in cortical neurons. (A,B) Primary cortical neurons were treated with (A) clomipramine or (B) fluoxetine at concentrations of 1 and 5 μM for 12, 24 and 48 h. Densitometric analysis of LC3-II (left) and p62 (right) is reported. β-Actin was used as loading control. Bars represent mean ± S.E.M. Each group results from 6 independent neuronal cultures. (C) Cortical neurons were incubated with clomipramine (5 μM) or fluoxetine (5 μM) for 12 h, while they were exposed to 20 nM Baf A1 for only 3 h. Densitometric analysis of LC3-II represents mean ± S.E.M of 4 independent neuronal cultures (⁺p = 0.0867, *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 2

Autophagic flux is decreased by clomipramine treatment in mouse tissues. LC3-II and p62 levels in the (A,B) liver and (C,D) frontal cortex of mice treated with (A–C) clomipramine (Clo) or (B–D) fluoxetine (Fluo), compared to untreated animals (Ctrl). Data are relative to ex vivo tissues incubated with (+) and without (−) NH₄Cl and leupeptin (inhib). β-Actin was used as a loading control. For the densitometric analysis, bars represent mean ± S.E.M. of 7 mice for each group (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 3

Clomipramine and fluoxetine modulate C. elegans autophagy in a different manner. (A) Quantification of GFP::LGG-1 positive puncta at the confocal microscope (n animals = 60, ****p < 0.0001). Nematodes overexpressing lgg-1p::gfp::lgg-1 were exposed to clomipramine and fluoxetine for 24 h. Data information = ₁₀Clo and ₁₀Fluo = 10 μg/ml in NGM agar; ₅₀Clo and ₅₀Fluo = 50 μg/ml in NGM agar; C = control (equivalent volume of DMSO as vehicle). (B,C) Quantification of Q40::YFP containing puncta in nematodes upon treatment with 10 μg/ml clomipramine or fluoxetine. (B) L3 larvae expressing unc-54p::Q40::YFP transgene were exposed to antidepressants for (B) 24 h (n animals = 40, ***p < 0.0001), while (C) young adults were grown for 24 h and 96 h on drug-containing NGM agar plates (n animals = 40, *p < 0.05 **p < 0.01). (D) Percentage of paralyzed nematodes overexpressing human β-amyloid peptide. Animals were treated for 7 consecutive days with 10 μg/ml clomipramine or fluoxetine (n animals = 150, **p < 0.01). Percentage of paralyzed animals was determined every 24 h.