According to a 2015 United Nations report, the number of people over 60 is expected to double in the next 35 years to almost 2.1 billion people. As our population shifts to a larger proportion of elderly individuals, neurodegenerative disorders including amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and Alzheimer’s disease (AD) will continue to become more prevalent. Because most neurodegenerative diseases are primarily sporadic in nature, an effective method of treatment would be to prevent or slow the dying of neurons in affected brain regions. Inflammation, misfolded proteins, mitochondrial dysfunction, oxidative stress, nitrosative stress, apoptosis, and excitotoxicity are all considered major factors underlying the pathology of various neurodegenerative diseases [1
]. Thus, therapeutic agents that target several of these disease mechanisms may be the most viable treatment options.
Since ancient times, plants have been used as natural treatments for acute and chronic disorders. Over the last few decades, natural compounds possessing medicinal benefits (i.e., nutraceuticals) have been proposed as promising treatment options for many diseases due to their intrinsic antioxidant abilities in scavenging reactive oxygen species (ROS) and reactive nitrogen species (RNS) [3
]. The activities of many of these compounds, such as rosmarinic acid and carnosic acid, have been extensively investigated using in vitro and in vivo models [5
]. Manufactured or commercially synthesized drugs sometimes display more activity than natural compounds. However, natural compounds often produce long term benefits and generally elicit fewer side effects [6
]. Many nutraceuticals are classified as (poly)phenolic antioxidant compounds, like phenolic acids, flavonoids, tannins, and lignans [7
The rosemary shrub (Rosmrinus officinalis
) is a member of the mint family (Lamiaceae
) found originally in the Mediterranean region, and now found abundantly throughout the world. Commonly used as an herbal spice in food, rosemary has long been used as an alternative therapy for different illnesses including headaches, inflammatory diseases, and stomach problems [9
]. Rosemary has a significant intrinsic antioxidant activity due to its molecular components, such as rosmarinic acid and carnosic acid, and both compounds have demonstrated neuroprotective effects in different neurodegenerative diseases. The main constituent, rosmarinic acid (Figure 1
A), is a naturally occurring hydroxylated polyphenol molecule found in several plant families, including Lamiaceae
, and Blechnaceae
]. It has been shown to have antioxidant, anti-inflammatory, anti-viral, anti-mutagenic, and anti-angiogenic capabilities [9
]. Moreover, rosmarinic acid appears to be neuroprotective and has been demonstrated to induce neuroprotection from reactive glial cells in in vitro models of AD, spinal cord injury, and PD by inhibiting nitric oxide (NO) production [18
]. Rosmarinic acid protected against memory deficits in ischemic mice and alleviated neurological symptoms in the G93A mutant hSOD1 mouse model of ALS [21
Carnosic acid (Figure 1
B), a polyphenolic compound also found in the rosemary herb, possesses many of the biological activities discussed above. The robust antioxidant activity of carnosic acid may be related to its ability to up-regulate endogenous free radical scavenging enzymes via activation of the Nrf2 transcriptional pathway [24
]. In several in vitro models, carnosic acid has been shown to have protective effects against neurotoxicity by promoting pro-survival pathways including modulation of apoptosis, autophagy, and regulation of the parkin pathway [26
]. Carnosic acid showed a neuroprotective effect both in vitro and in vivo in a mouse model of neurotoxicity and in a rat model of PD [28
The purpose of this study was to directly compare the neuroprotective effects of rosmarinic acid and carnosic acid against cell death induced by nitrosative stress, excitotoxicity, and caspase activation in an in vitro model of primary rat cerebellar granule neuron (CGN) cultures. Apoptosis was measured morphologically by Hoechst staining to visualize nuclear size and morphology, in addition to bright field imaging or immunocytochemistry to visualize neuronal processes and the microtubule network, respectively. To further investigate the pro-survival pathway that carnosic acid activates to block caspase-dependent cell death, we used several different inhibitors, including wortmannin to inhibit PI3K, PD98059 to block MAPK (MEK1/2), and Akt inhibitor XIII to inhibit Akt-dependent pro-survival signaling. Our data demonstrate that rosmarinic acid and carnosic acid show overlapping and unique neuroprotective effects against diverse stressors that cause neuronal cell death. The unique neuroprotective effects of these two polyphenols has direct implications for designing future preclinical studies to evaluate the therapeutic potential of these compounds in neurodegenerative diseases.
Rosmarinic acid and carnosic acid have many bioactivities, including neuroprotective, antioxidant, and anti-inflammatory effects, that may make them viable therapeutic options to diminish the underlying pathophysiology of various neurodegenerative diseases. In the current study, we compared the neuroprotective effects of rosmarinic acid and carnosic acid against insults modeling nitrosative stress, excitotoxicity, and caspase-dependent apoptosis in cultured rat CGNs, a well-established in vitro model to examine neuronal cell death [34
]. Moreover, we studied diverse mechanisms that may underlie the neuroprotective effects of carnosic acid against intrinsic apoptosis.
Our data show that rosmarinic acid and carnosic acid protect CGNs from an in vitro model of nitrosative stress induced by SNP. This compound is a NO donor which can promote the formation of toxic RNS [35
]. The formation of RNS causes extensive cellular damage resulting in cell death [35
]. The neuroprotective effects of rosmarinic acid against nitrosative stress in vivo likely involves its capacity to downregulate NF-ĸB and inhibit NO production from activated glial cells [36
]. However, the effects observed in vitro in CGNs are likely due to rosmarinic acid directly scavenging NO. Moreover, both rosmarinic acid and carnosic acid share a structural motif; a catechol moiety that acts as a hydrogen donor to free radicals and uses oxygen as an electron acceptor [40
]. Indeed, we have shown that polyphenols containing catechol moieties demonstrate robust neuroprotective effects against NO-induced toxicity [41
]. Our results are also consistent with a previous study in which rosmarinic acid inhibited NO production in RAW2647 mouse macrophages [43
]. In a similar manner, carnosic acid has been shown to inhibit NO production induced by LPS in microglial cells [44
]. Thus, both rosmarinic acid and carnosic acid appear to mitigate nitrosative stress via two mechanisms. First, these compounds blunt NO production from macrophages and microglia. Second, they act as direct scavengers of RNS, including NO.
Excitotoxicity has been shown to play a role in these diseases and therefore, we tested these agents to determine their neuroprotective effects against glutamate/glycine-induced toxicity in CGNs [45
]. To replicate excitotoxicity in vitro, CGNs were exposed to glutamate and its co-agonist glycine, to cause excitotoxicity by causing an influx of calcium. The molecules bind to ionotropic glutamate receptors, allowing calcium to enter, facilitating depolarization of the cell. One consequence of excitotoxicity is neuronal nitric oxide synthase (nNOS) activation and increase in NO, leading to nitrosative and oxidative stress [35
]. We show that rosmarinic acid displays a neuroprotective effect against glutamate/glycine-induced excitotoxicity. This result has been substantiated in another study performed in human neuroblastoma cells [46
]. Conversely, carnosic acid exacerbated the excitotoxic cell death in CGN cultures. The opposing effects of these compounds on excitotoxic cell death are striking and are not readily explained. Additional studies are necessary to define the mechanisms underlying this result.
In our study, we found that carnosic acid exacerbated the effects of excitotoxicity in CGNs. Other studies have shown it to antagonize intracellular calcium mobilization in leukocytes, and interestingly, it provided anti-excitotoxic effects in vivo and in vitro in HT-22 hippocampal cells [25
]. The contrasting findings between our study and these prior reports may be due to differences in how the excitotoxic cell death is executed within the distinct cell types being tested. Regardless of these effects, carnosic acid did significantly protect CGNs against 5K apoptotic conditions, whereas rosmarinic acid did not.
To further characterize the protective effects of carnosic acid against intrinsic apoptosis, three different inhibitors were used to test the involvement of specific cell survival pathways. Inhibition of the PI3K pro-survival pathway with wortmannin blocked the protective effects of carnosic acid seen in 5K apoptotic medium. This demonstrated that the neuroprotective effects of this compound are dependent upon activation of a PI3K signaling pathway. Downstream of PI3K, two major pro-survival pathways can be activated, the Akt pathway and the Ras/MAPK pathway. When we inhibited Akt downstream of PI3K, carnosic acid still significantly protected CGNs against 5K-induced apoptosis suggesting that neuroprotection is not through Akt activation. Next, we examined the Ras/MAPK pathway by inhibiting MEK1/2 with PD98059. As with the Akt inhibitor, carnosic acid protected against 5K-induced apoptosis even in the presences of PD98059. This result shows that carnosic acid protects against intrinsic apoptosis independently of the Ras/MAPK pro-survival pathway.
We have tested two major pro-survival cell signaling pathways activated downstream of PI3K (Akt and Ras/MAPK) and shown that neither was essential to the protective effects of carnosic acid against intrinsic apoptosis, indicating that carnosic acid must protect CGNs through another PI3K-dependent pathway. One PI3K-dependent mechanism not tested, but potentially neuroprotective, is activation of the Nrf2 pathway. Previous studies reported that pretreating IMR-32 cells with wortmannin decreases Nrf2 nuclear translocation [48
]. Furthermore, Satoh et al. have reported that carnosic acid activates Nrf2 in vivo and in vitro by activating the transcriptional antioxidant response element (ARE) modulating the cellular redox state [24
]. Thus, the PI3K-dependent neuroprotection by carnosic acid against intrinsic apoptosis observed in CGNs may involve activation of the Nrf2 pathway. Another possibility is a PI3K-dependent activation of Rac GTPase and subsequent activation of p21-activated kinase (PAK), which we have previously shown to have pro-survival effects in CGNs [49
]. Future studies will be necessary to examine if carnosic acid activates Rac/PAK pro-survival signaling in CGNs via PI3K and if this pathway is ultimately responsible for the protective effects of this polyphenol against intrinsic apoptosis.
In summary, rosmarinic acid and carnosic acid displayed both shared and unique neuroprotective effects when evaluated in cultured CGNs. Both compounds provided significant neuroprotection against the NO donor SNP, likely due to their shared catechol structural motif. On the other hand, only rosmarinic acid protected CGNs from excitotoxicity while carnosic acid uniquely protected CGNs from caspase-dependent apoptosis. Both of these compounds have been shown to have impressive antioxidant and neuroprotective effects and these effects nicely complement one another. Therefore, future preclinical studies should explore not only the individual therapeutic actions of these compounds, but also the potential complementary effects of these rosemary polyphenols on neurodegenerative disease progression.
4. Materials and Methods
4.1. CGN Culture
Cerebellar granule neurons (CGNs) were isolated as previously described [52
] from seven-day-old Sprague Dawley rat pups. Cells were plated on poly-l
-lysine coated six-well plates (35 mm-diameter), with a density of approximately 2 × 106
cells/well in Basal Medium Eagle’s supplemented with 25 mM potassium chloride, 2 mM l
-glutamate, 10% fetal bovine serum, and 2 mM penicillin-streptomycin (100 U/mL/100 µg/mL). Cytosine arabinoside (10 µM) was added to the culture medium 24 h after plating to inhibit the growth of non-neuronal cells. Cultures were ~95% pure for granule neurons. The resulting CGNs were then incubated at 37 °C in 10% CO2
for six to seven days in culture prior to experimentation. All animal manipulations were performed in accordance with a protocol approved by the University of Denver Institutional Animal Care and Use Committee.
Sodium nitroprusside (SNP) and PD98059 were obtained from Calbiochem (San Diego, CA, USA). Carnosic acid ((4aR,10aS)-5,6-Dihydroxy-1,1-dimethyl-7-propan-2-yl-2,3,4,9,10,10 a-hexahydrophenanthrene-4a-carboxylic acid) and rosmarinic acid ((R)-O-(3,4-dihydroxy- cinnamoyl)-3-(3,4-dihydroxyphenyl) lactic acid, 3,4-Dihydroxycinnamic acid (R)-1-carboxy-2-(3,4-dihydroxyphenyl) ethyl ester) were obtained from AG Scientific (San Diego, CA, USA). Glutamic acid was purchased from MP biomedical (Santa Ana, CA, USA). Glycine, Hoechst, wortmannin, and monoclonal antibody to β-tubulin were obtained from Sigma Aldrich (St. Louis, MO, USA). Akt Inhibitor XIII was purchased from Calbiochem (San Diego, CA, USA).
4.3. Treatment Protocols
Treatment with glutamate/glycine and SNP:CGNs were co-treated with either carnosic acid or rosmarinic acid and the stressors SNP (100 μM) or glutamate/glycine (100 μM/10 μM), for 24 h before fixation and Hoechst staining for quantification of cell death. For all experiments, an untreated control and an SNP- or glutamate/glycine-only control was used to compare cell death and neuroprotection. Prior to treatment, the plating medium was removed and replaced with serum free medium containing 25 mM potassium chloride to prevent any potential protective effects of the serum.
4.3.1. Treatment with 5K Apoptotic Medium
CGNs were co-treated with either carnosic acid or rosmarinic acid and 5K apoptotic medium. Prior to treatment, the plating medium was aspirated, and the cells were washed once with 5K apoptotic medium to remove any leftover serum. 5K apoptotic medium contains Basal Medium Eagle’s supplemented with 5 mM potassium chloride, 2 mM l-glutamate, and 2 mM penicillin-streptomycin (100 U/mL/100 µg/mL). After the first wash with 5K, the medium was removed and replaced with 1 mL of 5K apoptotic medium, which was left in each well. Wells in which the plating medium was never replaced, and wells containing 5K apoptotic medium alone were used as controls. In the remaining experimental wells, the appropriate concentration of rosmarinic acid or carnosic acid was added and the wells were left in an incubator at 3 °C for 24 h before fixation and staining with Hoechst for quantification of apoptosis.
4.3.2. Protocol for Treatment with Inhibitors
CGNs were co-treated with varying concentrations of carnosic acid, and then either 5K apoptotic medium alone or containing 10 µM PD98059, 100 nM wortmannin, or 10 μM AKT Inhibitor XIII. Wells in which the plating medium was never replaced, and wells containing 5K apoptotic medium alone were used as controls. Wells containing 5K apoptotic medium plus the wortmannin, PD98059, and AKT Inhibitor XIII without carnosic acid were also included as controls. Cells were treated and then left in an incubator for 24 h before fixation, Hoechst staining, and quantification of apoptosis.
4.4. Fixation, Hoechst Staining, and Immunocytochemistry
Following treatment, CGNs were washed once with phosphate buffered saline (PBS; pH = 7.4) and fixed for one hour at room temperature in 4% paraformaldehyde. Cells were then washed again with PBS and stained with Hoechst at a concentration of 10 µg/mL. After washing with PBS, the cells were imaged using a Zeiss Axiovert-200M epi-fluorescence microscope. Five images were taken per well to assess apoptosis, with either duplicate or triplicate wells per experiment. Cells were counted and scored as either living or apoptotic based on nuclear morphology; CGNs having condensed or fragmented nuclei were counted as apoptotic. In some cases, the microtubule network was visualized by staining with an antibody to β-tubulin and a fluorescein isothiocyanate (FITC)-conjugated secondary antibody.
4.5. MTT Cell Viability Assay
As an alternative means of assessing cell death, some experiments were evaluated using an MTT viability assay. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a tetrazolium dye which is reduced by NAD(P)H-dependent cellular oxidoreductase enzymes, primarily within the mitochondria of viable cells, to yield an insoluble formazan derivative which can be solubilized and assayed colorimetrically as an indicator of cell viability. MTT data presented were obtained from four wells per treatment.
4.6. Data Analysis
Each experiment was performed using either duplicate or triplicate wells for each treatment, with each experiment being performed four independent times. Data are presented as the means ± SEM of the total number of experiments. Data was analyzed using a one-way ANOVA with a post hoc Tukey’s test. A p value of <0.05 was considered statistically significant.