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.