INTRODUCTION
Tetracyclines are a family of antibiotics that inhibit bacterial protein synthesis by binding to the small ribosomal subunit and blocking the binding of aminoacyltRNAs to the ribosome A site [1]. Although tetracycline effects as antibiotics are rather old, there has been a renewing interest in these molecules, as it has been reported that, in addition to their antibacterial activity, they induce important pharmacological effects which may have clinical applications to various disease states. In recent years, preclinical studies have suggested that tetracyclines have neuroprotective properties that are worth reusing for neurodegenerative diseases [2-4].
In particular, doxycycline (DOX), an antibiotic belonging to the tetracycline family, has not only been reported to exhibit neuroprotective activity in Parkinson’s disease (PD) animal models like 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine, attenuating the loss of dopaminergic neurons in thesubstantia nigra pars compacta and striatal nerve terminals [5,6], but it also remodels toxic α-synucein oligomers into a non-toxic, non-seeding form [7], interferes with tau amyloid aggregation [8] and decreases the expression of the main markers of inflammation in microglial cultures treated with LPS [9], indicating that its underlying mechanism is the ability of DOX to reduce neuroinflammation [2,10].
A new family of chemically modified tetracyclines (CMTs) has been structurally rearranged to eliminate their antimicrobial activities while retaining their complementary mechanisms, such as inhibition of inflammation, proteolysis, angiogenesis, among others [11-14]. To date, more than eight CMTs are available [15]. Because CMTs might represent a safer treatment compared to antimicrobial treatment with tetracyclines, since their administration in laboratory animals does not produce tetracycline resistant microorganisms in the oral and intestinal flora [16], some of them, as CMT-1, CMT3 and CMT-8 have been tested in pre-clinical and clinical applications, including cancer trials and neurological disease studies [10,17,18].
Chemically modified tetracycline 3 (CMT-3,6-demethyl-6-deoxy-4-de[dimethylamino]-tetracycline) might be a promising drug for oxidative stress-related disorders, as neurodegenerative diseases, due to the advantage of being able to be used in chronic treatments without generating serious side effects. CMT-3, also called Incyclinide or COL-3 is produced by deletion of the dimethylamino group from carbon 4 in the A ring of tetracyclines, which eliminates the antibiotic activity without affecting other important properties of the molecule [19] (Figure 1). COL-3 is the only CMT tested in clinical trials in cancer patients [20,21], and its pleiotropic properties offer impressive therapeutic potential to reduce the excessive degradation of connective tissue during various pathological processes, including inflammatory conditions [22-25]. COL-3 molecule is highly lipophilic, so it can also cross the blood-brain barrier and thus act within the brain [14,26]. COL-3 mechanism of action in the central nervous system includes anti-apoptotic effects, anti-protein aggregation, removal of ROS, inhibition of matrix metalloproteinase, and protection against mitochondrial dysfunctions, among others [2,17]. COL-3 has been also proposed as an anti-inflammatory molecule, as it can inhibit lipopolysaccharide (LPS)-induced microglial activation and cytokine expression in the brain [26]; however, the mechanism through which this molecule reduces neuroinflammation is yet to be described.
In the present study, we used COL-3 to further explore the mechanisms underlying its anti-inflammatory action and compare its effects with a reference tetracycline that presents antibacterial activity, DOX. To this aim, we used culture of microglial cells isolated from postnatal mouse brains and an activation paradigm using the classical inflammogen LPS. Since the aggregation process that affects α-synuclein (αS) synaptic protein may represent a potential trigger for the activation of microglial cells, which is involved in the pathogenesis of synucleinopathies including PD [27-29], we also used the aggregated version of αS (αSa) as an inflammogen. We observed that COL-3 exerts robust anti-inflammatory effects on microglial cells by inhibiting ROS signaling events and glucose-dependent synthesis of NADPH, a cofactor required for activation of NADPH oxidase and the generation of ROS. Surprisingly, the anti-inflammatory action of COL-3 was significantly higher than that evoked by DOX, as lower concentrations of COL-3 were sufficient to induce the same anti-inflammatory effect that a higher concentration of DOX.