1. Introduction
Neurodegenerative diseases comprise a condition in which nerve cells from brain and spinal cord are lost leading to either functional loss (ataxia) or sensory dysfunction (dementia). Mitochondrial dysfunctions, excitotoxicity, and, finally, apoptosis have been reported as pathological causes for aging and neurodegenerative diseases such as Parkinson’s disease (PD), Alzheimer’s disease (AD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS) [
1]. Although any disease mentioned above is characterized by its own molecular mechanism and clinical onset, common pathways can be identified as: 1) oxidative stress and free radicals formation; 2) metal dyshomeostasis; 3) protein misfolding and aggregation; and 4) mitochondrial dysfunctions [
2]. Oxidative stress plays a key role in neurodegenerative diseases: it arises due to an imbalance between pro-oxidant/antioxidant homeostasis that further takes part in generation of ROS and free radicals potentially toxic for neuronal cells [
3]. Furthermore, in all forms of ROS generation molecular oxygen needs to be activated by a range of metallo-enzymes, thus facilitating ROS generation upon interaction of redox metals with O
2 using various catalytic pathways. Since free radicals are toxic to cells, under normal circumstances, cells have an efficient regulating system for O
2 and metal ion interaction leading to free radicals and ROS generation [
4]. In fact, during the Fenton reaction, hydroxyl radicals are produced from hydrogen peroxide in the presence of a metal in a low oxidation state:
Fenton chemistry may occur in neurons of the nervous tissue where levels of both hydrogen peroxide and cerebral biometals—such as Fe
3+; Cu
2+; and Zn
2+—are found. Several studies have indicated that cerebral biometal dyshomeostasis and oxidative stress are intimately associated [
5]. An
in vitro AD model has shown that both Aβ-40 and Aβ-42 deposits are formed after incubation of immobilized β-amyloid oligomers with Cu
2+, Zn
2+, or Fe
3+. In these conditions, Fe
3+ promoted the deposition of fibrillar amyloid plaques, while Cu
2+ and Zn
2+ only induced the formation of amorphous aggregates [
5]. In an
in vitro PD model, it has been found that Fe
3+ enhanced intracellular aggregation of α-synuclein and led to the formation of advanced glycation end products. The accumulation of these factors strongly contributed to the progression of the neurodegenerative process [
6].
Increasingly medicinal chemistry approaches are currently under study to discover new drugs able to remove excess of specific metals [
7,
8] and to prevent or block the oxidative process that characterizes PD and AD [
9,
10,
11,
12]. Taking into account that drugs with two or more useful biological activities for the same pathology may represent an important pharmacological advance, we are currently interested in multifunctional drugs that combine potent antioxidant, chelant, and neuroprotective properties in a single molecule for the treatment of PD and AD [
13,
14,
15,
16]. For this purpose, to design a novel class of compounds with a multimodal mechanism of action, we selected the hydroxyquinoline (HQ) scaffold as a privileged structure since it is a clinically relevant bioactive metal chelator. Recently, 8-hydroxyquinoline (8-HQ,
1) derivatives have found application in PD and AD drug discovery [
17] since 8-HQ: 1) is able to cross the blood–brain barrier (BBB) [
18]; 2) is a strong iron chelator with antioxidant property [
19,
20,
21]; and 3) is able to protect against the precipitation of β-amyloid plaques in presence of Cu
2+, Fe
3+, Zn
2+—compared to clioquinol—due to its ability to chelate these metals [
22].
The aim of this work was to combine the antioxidant and neuroprotective properties of (R)-alpha-lipoic acid (LA,
3) and the chelant activities of 8-HQ (
1) [
23] to obtain a novel multi-target ligand, LA-HQ-LA (
5) with multifunctional neuroprotective profile. LA-HQ-LA was obtained by linking via two ester bonds the 8-HQ derivative (5-hydroxymethyl-8-hydroxyquinoline,
2) to LA, thus increasing the lipophilicity of this molecule. LA-HQ-LA can cross plasma membranes and release HQ and two molecules of LA, thus triggering a significant decrease in oxidative stress from human SH-SY5Y neuroblastoma cells. In addition, due to the different chemical nature of the ester bonds, the derivative
5 could gradually provide a continuative and time-controlled release of LA—an elevator of GSH levels that are lower in some cerebral areas of patients affected by neurodegenerative diseases [
24]—and HQ directly to specific groups of neurons characterized by cellular stress and metals accumulation.
2. Results and Discussion
Starting from 8-HQ (
1), the required starting material 5-hydroxymethyl-8-hydroxyquinoline (
2) was obtained in good yield, using a known procedure [
25]. The new multi-target ligand LA-HQ-LA (
5) was synthesized by direct condensation of 5-hydroxymethyl-8-hydroxyquinoline (
2) and LA-NHS (
4), previously prepared as reported by Nefkens
et al. [
26] (
Scheme 1). The chemical structure of LA-HQ-LA was confirmed by
1H-,
13C-NMR, IR, and MS spectra data.
Scheme 1.
Synthesis of LA-HQ-LA (5).
Scheme 1.
Synthesis of LA-HQ-LA (5).
The neuroprotective and antioxidant capacities of LA-HQ-LA against oxidative stress were assayed by using the human SH-SY5Y neuroblastoma cell line, which is a reliable model for studying the neurotoxic effect of agents such as H
2O
2, 6-OHDA, and frequently used for elucidating the mechanisms of neurodegenerative diseases [
27].
First of all, to define the suitable concentration range, the effects on cell proliferation of LA, HQ, and LA-HQ-LA were determined by colorimetric MTT assay (
Figure 1). Thus, we performed dose-response experiments (with compound concentrations of 1, 10, and 100 μM) to verify if, 24 h after the treatment, the compounds added to the cells had any effect on the cell proliferative capacity. The compound concentrations of 1 and 10 μM did not show significant differences compared to the control, while at 100 μΜ, an antiproliferative activity was observed (
Figure 1,
Figure 2 and
Figure 3). In particular, at 100 μM, LA still retained a proliferative activity, while LA-HQ-LA resulted in an antiproliferative activity, since there are no visible proliferating cells (
Figure 2). Based on the results obtained, the utilized compound concentration in all the experiments reported in this study was 1 μM.
Figure 1.
Dose-response effects of LA, HQ, and LA-HQ-LA in undifferentiated SH-SY5Y human neuroblastoma cellsa.
Figure 1.
Dose-response effects of LA, HQ, and LA-HQ-LA in undifferentiated SH-SY5Y human neuroblastoma cellsa.
Figure 2.
Morphological analysis of undifferentiated SH-SY5Y human neuroblastoma cellsin the presence of LA, HQ, and LA-HQ-LA.a
Figure 2.
Morphological analysis of undifferentiated SH-SY5Y human neuroblastoma cellsin the presence of LA, HQ, and LA-HQ-LA.a
Because the sensitivity of cells to toxic agents may be dependent on the state of their neuronal differentiation, we used undifferentiated and RA-differentiated SH-SY5Y cells to study the neuroprotective effect of our compounds following H
2O
2 insult [
28,
29]. Thus, undifferentiated SH-SY5Y cells were pre-treated with LA, HQ, and LA-HQ-LA for 24 h and then exposed to H
2O
2 (25-150-300 μM) for other 24 h (cell viability was detected using MTT assay as shown in
Figure 3). Both compounds (LA-HQ-LA and LA) showed a significant protective effect against H
2O
2 at 25 and 150 μM H
2O
2. Moreover, the neuroprotective effect of LA-HQ-LA was appreciably higher in respect to LA and the control at 300 μM. In
Figure 3, we did not insert the results obtained for HQ since, as evidenced by the morphological evaluation (
Figure 4), at 1 μM and in presence of 25 μM of H
2O
2 it was demonstrated as toxic for the undifferentiated SH-SY5Y cells. This deleterious effect on SH-SY5Y cells could be due to a synergic action between HQ and H
2O
2. HQ, being a metal chelator of iron, copper, and zinc (transition metals that react easily with ROS), avoids the Haber-Weiss reaction between the metal and the superoxide anion; in these oxidative stress conditions, superoxide anion is overproduced, thereby damaging cells. On the other hand, H
2O
2 directly produces a high quantity of ROS further damaging the cells. These two combined actions determine a deleterious synergic effect on cells.
Figure 3.
Neuroprotective effects of LA and LA-HQ-LA in undifferentiated and H2O2-lesioned SH-SY5Y human neuroblastoma cells.a
Figure 3.
Neuroprotective effects of LA and LA-HQ-LA in undifferentiated and H2O2-lesioned SH-SY5Y human neuroblastoma cells.a
Many evidences indicated that the neuronal differentiation of SH-SY5Y neuroblastoma cells seems appropriate for studying neurotoxicity since the proliferation rate is limited and cells morphologically resemble the neuronal phenotype [
30]. Thus, we differentiated our cells with RA to increase the cholinergic properties of SH-SY5Y cell line and we lesioned them with increasing concentrations (25–150–300 μM) of H
2O
2 (
Figure 5). Results confirmed a significant neuroprotective effect of LA-HQ-LA, at all doses of H
2O
2, against LA and control. These results confirmed several previous reports [
28,
31,
32] which showed that the differentiation of SH-SY5Y cells by RA enhanced their resistance to the action of neurotoxic agents. In fact, comparing
Figure 3 and
Figure 5, indicating cellular viability, we observed that at high concentrations of H
2O
2 (300 μ M), the optical density O.D. values were reduced more in the undifferentiated than differentiated SH-SY5Y cells. Thus, the undifferentiated cells were much more vulnerable to H
2O
2 than RA-treated ones.
Figure 4.
Morphological analysis of undifferentiated and H2O2-lesioned SH-SY5Y human neuroblastoma cells in the presence of LA, LA-HQ-LA, and HQ.a
Figure 4.
Morphological analysis of undifferentiated and H2O2-lesioned SH-SY5Y human neuroblastoma cells in the presence of LA, LA-HQ-LA, and HQ.a
Figure 5.
Neuroprotective effects of LA and LA-HQ-LA in RA-differentiated and H2O2-lesioned SH-SY5Y human neuroblastoma cells.a
Figure 5.
Neuroprotective effects of LA and LA-HQ-LA in RA-differentiated and H2O2-lesioned SH-SY5Y human neuroblastoma cells.a
Figure 6.
Antioxidant capabilities of LA and LA-HQ-LA in RA-differentiated and H2O2-lesioned SH-SY5Y human neuroblastoma cells a.
Figure 6.
Antioxidant capabilities of LA and LA-HQ-LA in RA-differentiated and H2O2-lesioned SH-SY5Y human neuroblastoma cells a.
In the above reported experiments, exposing SH-SY5Y neuroblastoma cells to increasing concentrations of H
2O
2, we induced reactive oxygen species (ROS) generation. An overproduction of ROS and a lower antioxidant capability of the cells result in oxidative stress that characterizes several neurodegenerative pathologies. To accurately measure ROS and the cell capability to counteract this insult, we used cell permeable fluorescent and chemiluminescent probes. 2′-7′-Dichlorodihydrofluorescein diacetate (H
2DCF-DA) is one of the most widely used techniques for directly measuring the redox state of a cell [
33]. Our results showed that, at t
0-t
5 and at all the concentrations of H
2O
2, LA-HQ-LA and LA exerted a powerful antioxidant effect returning ROS levels similar to the control (Fig. 6). These data confirmed that our compound possesses good antioxidant and neuroprotective capabilities.
Comparing
Figure 3,
Figure 5, and
Figure 6 we can observe that LA-HQ-LA showed a long-lasting neuroprotective activity respect to LA only in long-term experiments (Fig. 3, 5) probably due to a time-controlled release of its components.
Figure 7.
Neuroprotective effects of LA, HQ, and LA-HQ-LA in undifferentiated and 6-OHDA-lesioned SH-SY5Y human neuroblastoma cellsa.
Figure 7.
Neuroprotective effects of LA, HQ, and LA-HQ-LA in undifferentiated and 6-OHDA-lesioned SH-SY5Y human neuroblastoma cellsa.
To further investigate the neuroprotective role in PD, it was necessary to differentiate SH-SY5Y neuroblastoma cells toward the DAergic phenotype using RA/PMA [
34,
35]. We used the neurotoxin 6-OHDA as a toxic agent to lesionate the cells, as it is commonly used as a dopaminergic degeneration model for both
in vitro and
in vivo studies [
36]. Like DA, 6-OHDA is quickly oxidized to form a variety of free radical species, and also induces ROS-dependent apoptosis in dopaminergic cells. In this second toxicity model, the neuroprotective effect of LA, HQ, and LA-HQ-LA against oxidative stress was evaluated by using the neurotoxin 6-OHDA in both the undifferentiated and RA/PMA-differentiated cells. The undifferentiated SH-SY5Y cells were treated with LA, HQ, and LA-HQ-LA for 1 h and then exposed to increasing concentrations of 6-OHDA (25-50-75-150 μM). After further 24 h of incubation, the cultures were assessed for viability by MTT assay (
Figure 7). LA-HQ-LA was the most neuroprotective compound, among the ones investigated, mostly at the strongly neurotoxic 6-OHDA concentration of 150 μM. In RA/PMA-differentiated cells (
Figure 8), LA-HQ-LA showed a strong neuroprotective effect at all the used concentrations of 6-OHDA. Also in these experiments, we observed a major susceptibility to toxic insult of undifferentiated respect to differentiated cells (
Figure 7 and
Figure 8).
Figure 8.
Neuroprotective effect of LA, HQ, and LA-HQ-LA in RA/PMA-differentiated and 6-OHDA-lesioned SH-SY5Y human neuroblastoma cells.a
Figure 8.
Neuroprotective effect of LA, HQ, and LA-HQ-LA in RA/PMA-differentiated and 6-OHDA-lesioned SH-SY5Y human neuroblastoma cells.a
Our data showed that LA-HQ-LA has a significant neuroprotective effect against both H
2O
2 and 6-OHDA, higher than LA and HQ, up to 150 μM. However, LA-HQ-LA showed a different behavior in the response (mainly with 6-OHDA) in differentiated cells compared to the undifferentiated ones. This difference probably depends on the variable sensitivity of cells against neurotoxic agents. In fact, RA/PMA-differentiated cells exhibit 6-fold higher levels of dopamine D
2 and D
3 receptors in respect to undifferentiated or RA-differentiated cells [
37]. Furthermore, it is also reasonable that LA-HQ-LA showed the greatest effect against neurotoxic agents, because LA itself is a neuroprotective agent for dopaminergic neurons [
38]. The obtained results confirm that LA-HQ-LA is a valid molecule, potentially more effective in respect to LA and HQ. In particular, LA-HQ-LA—at low concentrations (1 μM)—might be a good therapeutic choice in diseases such as PD, where oxidative stress caused by oxidants plays a key role.