Parkinson’s disease (PD) is the second most common progressive neurodegenerative disorder after Alzheimer’s disease (AD) and as such represents a serious social burden. It is characterized by selective loss of dopaminergic neurons from substantia nigra pars compacta
(SNPC) and their projections to the striatum [1
]. Impairment of motor function and memory are the primary clinical characteristics of PD observed both in patients and experimental animal models.
Several lines of independent data convergently indicate that dysregulated oxidative responses play a major role in disease onset, maintenance, and progression [4
]. One of the major sources of reactive oxygen species (ROS) in SNPC may originate from changed DA metabolism [7
]. Studies in patients with PD showed increased levels of oxidized lipids, proteins, and DNA, as well as decreased levels of reduced glutathione (GSH) [8
] in SNPC. Consequences of OS are intracellular accumulation and extracellular release of proteins with abnormal conformation, which then penetrate into adjacent neurons, thus stimulating the neurodegenerative progression [9
Current therapy for the disease is symptomatic and fails to influence the course of the disease and its progression [10
Hence, as PD remains largely an unmet medical need, obtaining a better understanding of the pathogenic mechanisms operating in PD development, including the dysregulated OS responses strongly warrant discovery of novel pathogenic-tailored approaches for the treatment of the disease. One potential approach to the discovery of new therapeutic opportunities relies on the development of medications based on naturally occurring substances.
Along this line of research, we presently examine three natural bio-antioxidants (AOs), namely the plant monoterpene myrtenal (Myrt), the polyphenol ellagic acid (EA), and the natural dithiol alpha-lipoic acid (LA). They all are strong bio-antioxidants, which have shown promising preclinical and clinical effects in several pathological conditions (for structures of compounds, see Figure 1
LA and EA derivatives and some supplements representing a rich source of both have been tested on humans, and have improved some symptoms that also occur in PD. The administration of LA in combination with omega-3 fatty acids slowed cognitive and functional decline in a 12 month trial in AD patients [14
]. Another study showed significant improvement of total antioxidant capacity in LA-treated multiple sclerosis patients in comparison to non-treated ones after 12 weeks [15
]. EA, on the other hand, restored cognitive performance related to mild age-related declines in overweight participants aged around 50 years after 12 weeks EA treatment course [16
]. Supplementation with EA-rich foods and beverages as pomegranate juice has been shown to improve the antioxidant status in aged healthy humans (aged over 60 years) after four weeks of treatment [17
To our knowledge, none of these substances has been applied in clinical trials on PD patients to date, and there are only scarce in vivo or in vitro data for ameliorating disease effects for some of them.
]chromene-5-10-dione) is the substance mainly contributing to the various and notorious beneficial effects of Punica granatum
L. (pomegranate) extracts. EA is also widely available in most berries and nuts [19
Of particular interest for this study is the reported ability of EA to lower the rotenone-induced generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in PC12 cells [22
] as well as its ability to exhibit a neuroprotective effect against oxidative damage in diabetic rats by suppressing oxidative stress [23
]. From cognitive and behavioral points of view, EA prevents scopolamine- and diazepam-induced cognitive impairments [24
] and may be of potential benefit in brain stroke by alleviating the oxidative stress characteristic for this condition [26
]. Data also supports EA’s brain-protective properties against 6-OHDA-induced neuroinflammation in rats as well as cognitive and long-term potentiation deficits following traumatic brain injury [27
EA alleviates oxidative damage through several mechanisms: (1) activation of antioxidant response using the Nrf2 (nuclear erythroid factor 2) [28
]; (2) inhibition of cyclooxygenase 2 (COX-2) [30
] and cytokines regulated through NF-kB (nuclear factor-kappa (B) [31
]; (3) modulation of cell survival and/or apoptosis [33
]; and (4) potentiation of biological antioxidants and antioxidant enzymes activity [34
)-5-(1,2-dithiolan-3-yl)pentanoic acid) is a naturally occurring substance, a dithiol that plays fundamental role in mitochondrial metabolism as a coenzyme for pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. It is also a substrate for the NADPH-dependent enzyme glutathione reductase. According to present understanding, LA is most likely synthesized in human and animal mitochondria [35
]. It is not considered to be a vitamin but is able to affect the levels of some vitamins in the organism [36
]. Its protective in vivo and in vitro activities against a range of pathophysiological conditions have been reported [37
], including protection against MPP+
-induced toxicity in neuronal cells [38
LA has been widely used in clinical setting as a supplementary treatment for different conditions associated with increased oxidative stress. Several studies have shown that LA exerts protective effects in in vivo and in vitro experimental models of neurodegenerative diseases, including Alzheimer’s disease (AD), macular degeneration, and PD [39
]. Administration in moderate doses has produced no evidence of serious side effects [38
)-6,6-dimethylbicyclo[3.1.1]hept-2-ene-2-carboxaldehyde) is a bicyclic monoterpenoid established in many plant essential oils, including Cuminum cyminum
] and Lavandula
]. Myrt demonstrates multiple biopharmacological activities in different experimental conditions. Accordingly, Myrt influences apoptotic and pro-apoptotic signaling pathways, stabilizes intrinsic antioxidant protection, suppresses TNF-α [51
] expression, inhibits tumor growth, regulates the activity of number of lysosomal and mitochondrial enzymes, and influences the processes of gluconeogenesis in tumor cells [51
]. This monoterpenoid is relatively poorly studied in the field of neuroscience. Our previous research demonstrated preventive effects of Myrt on Alzheimer’s type dementia and amnesia in ICR mice and Wistar rats, thus re-confirming the role of OS mechanisms in AD pathogenesis [40
]. So far, there is no data in the literature about effects of Myrt on PD progression.
These multiple lines of evidence propelled us to investigate influence of these three natural AOs, namely EA, LA and Myrt, in a head-to-head comparison on the development of the neurodegenerative process of experimental PD induced by intrastriatal administration of 6-hydroxydopamine (6-OHDA).
Whilst the etiology of PD remains unclear so far, numerous reports suggest a connection between OS and the pathogenesis of the disease [78
]. One of the concepts of PD pathogenesis focuses on the formation of ROS and the role of oxidative stress leading to damage of SNPC neurons. This pathogenetic hypothesis is supported both by preclinical studies in animal models of PD [80
] and extensive postmortem studies in PD patients, where it has been observed to have caused impaired mitochondrial function, alterations in antioxidant protective systems (most notably superoxide dismutase and reduced glutathione), extensive oxidative damage to lipids, proteins, and DNA [81
This converging evidence indicating a key role of OS in PD propel the search for appropriate antioxidants to neutralize oxidative processes. However, treatment with common antioxidants such as vitamin C, vitamin E, and coenzyme Q10 exhibited poor effects in disease delay. For this reason, the search for either synthetic or other natural substances with rich biological properties and complex multi-target mechanisms continue for effective therapy or the prevention of PD [82
The antioxidants EA and LA have been investigated as a new therapeutic alternative for neurodegenerative disorders such as AD and PD, as well as for depression, ischemia, and other disorders [84
Our previous studies also established the significant preventive effect of EA and LA as well as of Myrt on experimental scopolamine (Sco) induced dementia from types of Alzheimer’s disease in rodents. We have proved that neuroprotective, neuromodulatory, and improving learning and memory effects, accompanied by strong AO activity allow multi-target treatment of the neurodegenerative process [25
The 6-OHDA hemilesioned PD rat model that we have presently used is a well-recognized in vivo tool to investigate mechanism and therapeutic strategies for PD treatment.
The neurotoxin 6-OHDA is a structural analogue of dopamine and noradrenaline and possesses high affinity for several catecholamine-ergic plasma membrane transporters (DAT and NAT respectively), which recognize and uptake 6-OHDA, which is a highly active dopaminergic neurotoxin that disrupts the catecholamine transport system and induces neurotoxicity by selective generation of OS and neuroinflammation in basal ganglia [88
]. In addition, 6-OHDA is toxic both at a peripheral and central level. However, since the neurotoxin is incapable of crossing the blood-brain barrier, its toxicity in the CNS is achieved only when directly injected into the brain by stereotaxic surgery. The classical method of intracerebral infusion 6-OHDA involves a massive destruction of nigrostriatal dopaminergic neurons and is largely used to investigate motor and biochemical dysfunctions in Parkinson’s disease [89
]. The neurotoxin causes selective and massive loss of dopaminergic neurons via generation of oxidative stress [91
], mitochondrial dysfunction [95
], and neuroinflammation with microglial activation [96
The so-induced model mimics PD-like motor deficits relatively reliably and can partly recapitulate the progression of the PD pathology [97
In this study, several PD-like behaviors were comprehensively evaluated using the model, including: the apomorphine-induced rotational behavior, which is related to the degree of nigrostriatal DA loss and is considered to be the “gold standard” in the hemilesioned rat model [97
]; the rotarod test which is often used to appraise the balance skills and motor co-ordination in the PD rats [98
]; and the step through test often used to evaluate learning and memory performance in PD animals. The PD model establishment worked clearly well, as the behavioral performance of the PD rats was impaired compared to in SO rats. In particular, 6-OHDA produced an increase in the number of contralateral rotations induced by apomorphine. The latter is a DA receptor agonist that at low doses causes contralateral turning by stimulating both supersensitive D1 and D2 receptors preferentially on the denervated side [99
]. The neurotoxin treatment also increased the number of falls/min in rotarod test and decreased STL in a step-trough test.
Our experimental data showed that the three tested substances decreased significantly the number of contralateral rotation in apomorphine test with the best effect being observed 3 weeks post lesion for EA and decreased the number of falls/min in rotarod test with best effect for Myrt, 3 weeks after lesion. This result confirmed the reported improvement in motor coordination effect of EA and decreased the contralateral rotation effect of LA in 6-OHDA-induced neurotoxicity on a rat model of hemi-parkinsonism [87
]. To the best of our knowledge, no studies have so far been conducted with Myrt on PD models. The data from our behavioral test showed that EA, LA, and Myrt had comparable neuroprotective effects against 6-OHDA induced neural oxidative damage.
In addition, particularly interesting results were obtained from the step through test, where alteration in learning and memory performance was evaluated by the change in STL time. In our experiment, we found that all three tested substances exhibited very good memory-enhancing effects, with the best result again for Myrt at three weeks post lesion. It is well known that learning and memory processes are primarily dependent on the cholinergic neurons in the hippocampus, cerebral cortex, and some parts of the new striatum [101
]. The ability of EA to regulate cognitive function through inhibition of the acetylcholinesterase (AChE) activity (IC50 = 13.79 μg/mL) and through acetylcholine upregulation was already reported [102
]. A memory-enhancing effect and increased acetylcholine (ACh) level in the brain of dement animals after Myrt treatment (in combination with LA) was indicated for the first time in our data that was reported previously [57
]. The AChE inhibitory effect of Myrt was only established in vitro in 2011 [59
Damage and loss of dopaminergic neurons in the SNPC lead to decreased DA levels in the neostriatum, which underlies the symptoms of Parkinson’s disease [103
]. Therefore, we detected DA level in the brain in this study, and as expected, the results showed that the impaired behavior were due to the depletion of DA in the striatum in PD- rats in comparison to SO group. The three tested compounds, Myrt, EA, and LA, restored DA brain levels in the ipsilateral and contralateral side, but showed hemisphere lateralization. The strongest (more than 10-fold vs. the 6-OHDA group) was the effect of Myrt and this effect was more pronounced in the contralateral side of the brain. The effects of EA on DA were weaker than that of LA and for both, the observed effects were stronger in ipsilateral (lesioned) side of the brain. The ability of LA to restore DA levels was reported in 6-OHDA- and MPTP-induced models of PD [104
]. In our previous studies with Sco-induced model of dementia, we have established that Myrt significantly increases DA levels in the brain [87
]. The ability of the tested AOs to increase dopamine levels in the brain after the neurotoxin action (6-OHDA) provides additional strong in vivo proof-of-concept of their neuroprotective properties. In addition to pure neuronal preservation, our results hinted at a possible neuromodulatory effect, which was especially evident for Myrt.
In the present study we confirmed the relation between neuronal degeneration in 6-OHDA model of PD and OS as a main pathogenic factor in disease progression [4
]. Oxidation of 6-OHDA by molecular oxygen or monoamine oxidase underlies its neurotoxicity in the brain and leads to production of intracellular H2
which can be transformed into highly reactive hydroxyl radicals, a reduction in glutathione (GSH) and SOD activity, an increase in LPO and production of superoxide free radicals causing cell damage [106
]. A decrease of GSH in SNPC is the earliest known indicator of oxidative stress in pre-symptomatic PD, which precedes decreases in dopamine levels [81
Our results established increased LPO and reduced glutathione levels in PD- brain and this alteration was available both in the ipsilateral and contralateral sides. In agreement with this post-mortem analyses and further strengthening the preclinical relevance of the 6-OHDA model of PD presently used, we also observed a significant decrease in activities of main AO enzymes (SOD, CAT and GPx) in PD rats in comparison to the SO group (Figure 7
). Along with this, the administration of the three tested bio-AOs, Myrt, LA, and EA for five consecutive days demonstrated that:
The three bio- AOs decreased LPO levels, ipsilateral in the brain and the strength of the effect diminished in the following sequence EA = Myrt > LA;
LA and Myrt increased GSH brain levels. The effect of the dithiol compound was better and contralaterally located. The monoterpenoid Myrt enhanced GSH both ipsilateral and contralateral effects in the same way.
Altogether, it could be suggested that the use of Myrt, LA, and especially EA, alone or in combination may contribute to alleviate the progression of PD.
All tested substances increased CAT activity. Increased CAT activity by EA and Myrt was more pronounced ipsilaterally and by LA — contralaterally. The most powerful effect in this setting was that of Myrt. In our experimental conditions, we did not establish significantly increased SOD activity to be caused by EA, as reported by Sarkaki et al. [86
]. This difference may be due to different experimental condition. We applied EA before lesion, while Sarkaki et al.—after lesion [86
]. It can be concluded that all three tested compounds enhanced the cerebral antioxidant defense with different strength and hemispheric specificity, but all led to a reduction of oxidative stress.
While on the one hand we confirmed previous data on neuroprotective action exhibited by EA and LA against 6-OHDA-induced neural oxidative damage [86
], on the other we demonstrate the powerful pharmacological effects of Myrt towards experimental PD here for the first time.
We also established statistically some common antioxidant mechanisms of PD recovery in rats in two major brain areas [4
]. There is a direct relationship between levels of oxidative stress and function of the DA neurons in PD animals. A direct correlation between DA system recovery and improvement in motor performance by AO in rats was established. Decrease of oxidative stress and particularly lipid peroxidation was directly associated with improved behavioral performance in AO treated animals. The respective data are presented in Table 1
In order to distinguish general from specific mechanisms of the neuroprotective action of the three natural antioxidants used, we performed a cluster analysis where we used the principal component scores derived from a set of biochemical data (DA levels together with OS parameters) and behavioral parameters (motor coordination and memory) (Figure 5
). Cluster analysis correctly grouped and isolated the data points from all different treatments (SO, 6-OHDA, EA, LA, Myrt). It correctly distinguishes the 6-OHDA (black dots) from other treatments. This approach provided quantitatively supports two conclusions:
Significant PD recovery by the three AOs—there is clear evidence for neuroprotective AO effects and differentiation of the treated groups from PD rats.
There are close but yet distinguishable mechanisms of neuroprotection for each of three AOs: LA, EA, and Myrt.
Observed specific PD—the preventive effect of EA, LA, and Myrt was related to their diverse rich biological activity. The improving memory effect of EA probably is specific and was due to its active urolithins metabolites, which can easily penetrate the blood–brain barrier [107
]. The recovering memory effect of LA in a mouse model of AD type dementia [40
] is related to direct or indirect AO effect of LA [58
] and to neuromodulatory activity of Myrt [26
On the basis of the present experiment, we presumed that under the similar neurodegenerative effects of the three AOs lay down different mechanisms related to their powerful biological activity. This could explain why in spite of their different chemical structure and specific biologic activities, the three compounds could effectively prevent the neurodegenerative process in PD animals using their diverse multi-target capacities.
In this regard, future studies with combined use of Myrt, LA and EA in different combinations may prove useful to evaluate whether these compound specific-effects and their hemispheric specificity may be synergistically complemented by their combined use. If Myrt, EA, and LA exert synergistic effects in vivo, lower doses of each compound may afford an even stronger anti-Parkinsonian effect than when they are applied individually.