Abstract

Bipolar-disorder’s pathophysiology and the mechanism by which medications exert their beneficial effect is yet unknown, but others’ and our data implicate patients’ brain mitochondrial-dysfunction and its amendment by mood-stabilizers. We recently designed a novel mouse bipolar-disorder-like model using chronic administration of a low-dose of the oxidative-phosphorylation complex I inhibitor, rotenone. Four and eight weeks rotenone treatment induced manic- and depressive-like behavior, respectively, accompanied by mood-related neurochemical changes. Here we aimed to investigate whether each of the autophagy-enhancers lithium (a mood-stabilizer), trehalose and resveratrol and/or each of the reactive oxygen species (ROS)-scavengers, resveratrol and N-acetylcystein and/or the combinations lithium+resveratrol or trehalose+N-acetylcystein, can ameliorate behavioral and neurochemical consequences of neuronal mild mitochondrial-dysfunction. We observed that lithium, trehalose and N-acetylcystein reversed rotenone-induced manic-like behavior as well as deviations in protein levels of mitochondrial complexes and the autophagy marker LC3-II. This raises the possibility that mild mitochondrial-dysfunction accompanied by impaired autophagy and a very mild increase in ROS levels are related to predisposition to manic-like behavior. On the other hand, although, as expected, most of the drugs tested eliminated the eight weeks rotenone-induced increase in protein levels of all hippocampal mitochondrial complexes, only lithium ubiquitously ameliorated the depressive-like behaviors. We cautiously deduce that aberrant autophagy and/or elevated ROS levels are not involved in predisposition to the depressive phase of bipolar-like behavior. Rather, that amending the depressive–like characteristics requires different mitochondria-related interventions. The latter might be antagonizing N-methyl-D-aspartate receptors (NMDARs), thus protecting from disruption of mitochondrial calcium homeostasis and its detrimental consequences. In conclusion, our findings suggest that by-and-large, among the autophagy-enhancers and ROS-scavengers tested, lithium is the most effective in counteracting rotenone-induced changes. Trehalose and N-acetylcystein may also be effective in attenuating manic-like behavior.

Introduction

The pathophysiology of bipolar-disorder (BD) is not yet unraveled, but substantial evidence points at the involvement of mitochondrial-dysfunction [1,2,3]. In support of the latter, mood-stabilizers, the predominant treatment for BD, although discovered by serendipity and, as of now, with mechanism of action poorly understood, were shown to enhance mitochondrial-function and protect from oxidative-stress [4, 5], resulting in neuroprotection. Building on the involvement of mitochondrial-dysfunction in the etiology and treatment of BD we have recently designed a novel mouse model of BD-like behavior and neurochemistry [6] achieved by chronic treatment with a never used before low-dose rotenone, a mitochondrial complex-I (CoI) inhibitor, which induces mild mitochondrial-dysfunction.

Mitochondrial-dysfunction, sometimes caused by oxidative-stress and excess of reactive oxygen species (ROS), consequently leads to cell damage [7]. A rescue process which, most of the time, protects cells under these circumstances is autophagy/mitophagy [7,8,9,10]. Hence, here we reiterated our in vivo BD-like model and examined whether autophagy-enhancers and/or ROS-scavengers, comprised of either GRAS (generally regarded as safe) compounds or of approved mood-stabilizers, can fully, or at least partially, counteract rotenone-induced mitochondrial-dysfunction. We used drugs which we recently demonstrated to alleviate some rotenone-induced mild mitochondrial changes in SH-SY5Y cells [11].

The drugs included I. Autophagy-enhancers: (i) lithium salts (Li), the prototype mood-stabilizer used to treat BD patients [12,13,14]. In bipolar patients it induces anti-manic, anti-suicidal and prophylactic effects. In addition, it is used as adjunctive treatment to antidepressants in major depression [15, 16]. Several key cell signaling-related enzymes were found to be lithium-inhibitable, resulting in enhanced autophagy, elevated neurotrophic factors release, antioxidant effects, neuroinflammation and cytokine release attenuation and mitochondrial energy metabolism enhancement [17,18,19,20]. The involvement of these processes in a variety of neuropsychiatric disorders beyond BD raised, in the last decade, the interest in exploring Li’s potential neuroprotective property not only in psychiatric disorders but also in neurodegenerative and neurodevelopmental ones [17, 21,22,23,24,25,26].

(ii) Trehalose, a natural disaccharide comprised of two glucose molecules, is an autophagy modulator suggested as a treatment for neurodegenerative diseases in which autophagy has been shown to play a role [27,28,29]. In animal models trehalose was found to stimulate autophagy through the adenosine monophosphate–activated protein kinase (AMPK) [30], however, its role in autophagy is still controversial [31]. It functions as a stabilizer of proteins and is able to protect protein structural integrity. As previously reported by our lab, trehalose affected mouse brain autophagy markers in a manner indicative of enhanced autophagy, augmented manic-like behavior induced by amphetamine and attenuated immobility in the forced-swim test, the two latter effects being compatible with an anti-depressant-like effect [32]. Trehalose was also found to prevent neurodegeneration through decreasing damaged cell components [31].

II. A ROS-scavenger: NAC (N-Acetyl-L-cysteine) is a precursor of intracellular cysteine and of reduced glutathione (GSH) and is, thus, an inhibitor of ROS production, probably through conversion of NAC into hydrogen sulfide [33,34,35]. NAC improved neuronal deficits and nulled rotenone’s effect [36], increased midbrain dopaminergic neurons’ survival after rotenone treatment [37] and reduced methamphetamine-induced hyperthermia without affecting mice locomotor activity [38]. Clinical studies found a beneficial effect of NAC on the dopaminergic system in Parkinson’s disease patients, along with increased dopamine-transporter (DAT) binding in the caudate and the putamen [37, 39].

III. An autophagy-enhancer and a ROS-scavenger: resveratrol (RES, 3,5,4′-trihydroxy-trans-stilbene), a Sirt1 stimulator belonging to class-III histone deacetylases [40]. Resveratrol plays a role in autophagy regulation through mTOR inhibition [41, 42] and is also a ROS-scavenger [42,43,44]. As such, it inhibits excessive ROS production and lipid peroxidation, elevates mitochondrial membrane potential and inhibits cytochrome C release from the inner mitochondrial membrane, thus ameliorating aberrant mitochondrial distribution [45, 46]. Together, all resveratrol’s effects culminate, in the central nervous system (CNS), as follows. Reduced neurodegeneration in murine cerebral cortex and enhanced memory recovery following exposure to fluoride [46], as well as improved cognition, learning and memory in rats with vascular dementia and in mice models of Alzheimer’s disease [47].

Since we hypothesized that 1. Autophagy-enhancers and/or ROS-scavengers can fully, or at least partially, counteract/alleviate consequences of brain mild mitochondrial- dysfunction; 2. Combined treatment with an autophagy-enhancer and a ROS-scavenger is superior than each of them by its own in counteracting/alleviating consequences of brain mild mitochondrial-dysfunction, we also studied the effects of two pairs of drugs, lithium+resveratrol and trehalose+NAC on rotenone-induced mild mitochondrial-dysfunction as reflected in behavioral and neurochemical characteristics.

Methods

Animals

Male, five or eight weeks old mice (Envigo, Israel) were used. Animals were maintained on a 12 h/12 h light/dark cycle (lights on 7:00 a.m. to 7:00 p.m.) with constant temperature at 23 ± 1 ^oC and ad-libitum access to food and water. Tests were performed during the light phase of the cycle between 9:00 am and 7:00 pm. Mice were allowed to acclimatize to the new environment for one week before treatment initiation. Animals were weighed once every third day and their general well-being assessed by fur examination and general appearance. No randomization method to allocate mice to a specific group/treatment was used. Blindness of the experimenter was kept during all experiments by the fact that the Ethovision software (see below) is independent of the experimenter. All experimental procedures followed the Israeli guidelines for treatment and care of experimental animals and were approved by the Ben-Gurion University animal experimentation ethics committee (Authorization Numbers: IL-50-07-2015 & IL-40-06-2019).

Drugs’ delivery

Rotenone, dissolved in saline supplemented with 0.5% DMSO, was injected subcutaneously (s.c.), 0.75 mg/kg once daily for four or eight weeks. 

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https://www.nature.com/articles/s41380-023-01955-x


FYI:  Dr. Carmen-Silva Sergiou