URIDINE PROMOTES NEURITE OUTGROWTH IN NEUROBLASTOMA CELLS
J. Spathies, G. C. Tucker* and B. P. Nathan
Department of Biological Sciences, Eastern Illinois University, 600 Lincoln Avenue, Charleston, IL 61920, USA
*Corresponding Author’s email: gctucker@eiu.edu
ABSTRACT
Neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease are the main causes of age-related dementia. These diseases can be due to neuronal cell death and/or impairment of neuronal growth and connections. Giant oyster mushroom (GOM), Pleurotus giganteus, is used as a nootropic to improve cognitive function. GOM can also be used to prevent the onset of dementia. The underlying mechanism behind the medicinal property of GOM is unclear. Previous studies have shown that GOM has a high concentration of uridine. In this study, we examined the effects of uridine on neurite outgrowth in the Neuro-2a (N2a) neuroblastoma cell line. We also examined the effects of various concentrations of uridine on neurite outgrowth in N2a cells. When exposed to uridine, N2a cells produced significantly longer neurite extensions (p≤0.001) and exhibited a significant increase in neurite-bearing cells (p≤0.001). The peak neurite promoting effect of uridine was at 100 πM. Our results suggest uridine promotes neurite outgrowth in N2a cells (p≤0.001). Future studies are required to identify the mechanism(s) behind therapeutic potential of uridine on neurodegenerative diseases.
Keywords: Uridine, giant oyster mushroom, Pleurotus giganteus, neurite outgrowth, herbal medicine, Neuro2a, nerve regeneration, Alzheimer’s disease, Parkinson’s disease.
INTRODUCTION
Neurodegenerative diseases have major social and economic burdens on the world’s increasing population. The number of people living with dementia-related illnesses, such as Alzheimer’s disease (AD)and Parkinson’s disease (PD), is continuing to increase at a rapid rate (Hebert et al., 2003). A large portion of the geriatric population will experience some form of dementia-related illnesses, increasing the economic burden of heathcare. With the central nervous system’s (CNS) limited ability to repair itself after injury or progressive disease-related damage, there is currently no treatment for recovering lost CNS function. Most therapeutic medicine found to prevent neurodegenerative disease cannot cross the blood-brain barrier (Allen et al., 2013; Daneman et al., 2015). Given there is no treatment or cure for neurodegenerative diseases, like AD and PD, many people are turning to a more holistic approach.
One approach to this issue is to address age-related diseases and to act towards their prevention through dietary supplements and functional foods. An increasing amount of research has recently focused on functional foods and their bioactive constituents in our everyday diet (Khan and Tania 2012). Mushrooms have become known for their health benefits beyond providing nutrients. With an entire kingdom containing unique secondary metabolites, some can be used as an unlimited source of new pharmaceutical products (Wasser, 2002). A community-based study in Singapore focused on men and women,60 years old and older, who consumed two portions of mushrooms per week. These individuals had a 43 % reduced risk of developing mild cognitive impairment (MCI), a brain condition that may lead to more serious diseases such as AD (Feng et al. 2019).
Culinary-medicinal mushrooms have been used for their bioactive, secondary metabolites to reduce amyloid-induced neurotoxicity, neurite outgrowth stimulation, nerve growth factor synthesis, neuroprotective function, antioxidant, and anti-inflammatory effects (Phan. et al. 2015). Vitamin D-enriched white button mushrooms, Agaricus bisporus (J.E.Lange) Imbach, have been shown to improve the memory in a mouse model for AD (Bennett et al. 2013). Mushrooms have even been found to produce compounds that promote neurite outgrowth. As an example, erinacines and hericenones, isolated from the mushroom Hericium erinaceus Persoon, commonly called lion’s mane, for their neuroprotective properties (Wong et al. 2012). Since these brain-improving compounds can only be isolated from the mushrooms that produce them, an increasing amount of research is needed to identify the mechanism behind their medicinal properties.
Mushrooms of the genus Pleurotus are widely consumed worldwide, for their flavor and high nutritional value (Khan and Tania 2012). Pleurotus giganteus (Berk.) Karunarathna and Hyde, commonly known as giant oyster mushroom (GOM), is used for culinary purposes. The consumption of this mushroom goes as far back as the indigenous people of Peninsular Malaysia (Lee et al., 2009). Many in vitro studies of GOM have evaluated its anticancer, antioxidative, antifungal, hepatoprotective, and neurite outgrowth capabilities (Karunarathna et al. 2012; Phan et al. 2013, 2015; Baskaran et al. 2017). The GOM extracts exhibited neurite outgrowth in rat pheochromocytoma (PC12) and N2a cells (Phan et al. 2012, 2013). The main bioactive constituent of GOM is believed to be uridine.
Uridine is an RNA nucleotide and has been identified in several mushroom species (Yang et al., 2012). Specifically, it was recognized as one of the main bioactive compounds in the medicinal mushroom species, Cordyceps militaris (L.) Link, containing 45.4 mg/kg extract of uridine (Das et al., 2010). Uridine is also present in breast milk (Thorell et al., 1996) and, as a nucleotide; it is reported to have important physiological roles in breastfeeding infants (Leach et al., 1995). Uridine contributes to brain phosphatidylcholine synthesis via the Kennedy pathway (Cansev, 2016; Pooler et al., 2005). Its uptake into the brain and through the blood-brain barrier is initiated by specific nucleotide transporters. The rate at which uptake occurs is a major factor determining phosphatide synthesis. With uridine being a precursor of phosphatidylcholine, a membrane constituent, its presence in GOM may give us a better understanding of its medicinal properties. Previous studies have shown GOM extract increases neuronal outgrowth with uridine being the active ingredient (Phan et al., 2015).
In the present study, we examined the effects of uridine on neurite outgrowth in N2a neuroblastoma cell line. We found that treatment of N2a cultures with uridine significantly increased neurite length, combined length of neurites per cell, as well as the percent of neurite-bearing cells.
MATERIALS AND METHODS
Mouse neuroblastoma cells (N2a) were obtained from American Type Culture Collection (Manassas, VA). The N2a cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), with L-glutamine, penicillin-streptomycin (Thermo-Fisher Scientific) and fetal bovine serum (FBS) (Atlanta Biologicals). Uridine (Sigma Chemicals) was dissolved in ethanol due to its poor solubility in water. Three different concentrations of uridine were studied: 50 πM, 100 πM, and 200 πM. All the cultures were maintained at 37 °C and 6.5% CO2 in a humidified incubator. The cells were subcultured at 2-day intervals (Nathan and Tucker 2022).
The N2a cells were plated in tissue culture plates at an initial concentration of 50,000 cells per plate containing DMEM medium with 1X L-glutamine, 1X penicillin-streptomycin, 10 mM of glucose, and 10% of FBS for 48 hours. To examine the effects of uridine on neurite outgrowth, the medium was replaced with FBS-free medium containing 1X L-glutamine, 1X penicillin-streptomycin, 10 mM of glucose and either 50 πM, 100 πM or 200 πM of uridine in ethanol along with an ethanol-alone counterpart for each concentration. The uridine concentration tested in our study was based on previously reported uridine concentration of 1.6-1.8% (g/100g) in Giant Oyster Mushroom extract, which corresponds to 1.5 mM of uridine (Phan et al., 2015). This study also showed that 100 mM elicited the maximum neurite outgrowth in NGF-treated neuronal cultures. The cells were photographed using an Amscope MU 1400-CK digital camera attached to a phase-contrast microscope. Our methods follow those used in a previous study of plant derived compounds (Asiatic and Madecassic acids) on cultured neurite growth (Nathan and Tucker 2019).
We measured three parameters of neuronal growth: percent neurite-bearing cells, longest neurite length, and average combined length (Nathan and Tucker 2022). Neurite length was measured in at least 50 cells using ImageJ © software. The percentage of neurite-bearing cells is the number of neurons with a neurite extension measuring at least 30 πm divided by the total number of cells in a field and then multiplied by 100%. For measuring the longest neurite and combined length of neurites, photos were taken from 12 different quadrants evenly distributed throughout the plate. Given that 50 photos were taken per plate and three trials were performed, a total of 150 neurons were measured for each treatment condition (Nathan and Tucker 2022).
This experiment consisted of three separate trials with different N2a cultures and reagents each time. All experimental data was expressed as mean ± standard error. Statistical differences between groups were calculated by one-way analysis of variance (ANOVA).
RESULTS AND DISCUSSION
Uridine has been recognized as one of the main bioactive compounds in the medicinal mushroom Pleurotus giganteus (GOM). We examined uridine’s effects to promote neuronal outgrowth in N2a cells. The N2a neuroblastoma cell line is derived from mouse C1300 tumor and differentiated into neuron-like cells (Radio et al., 2008). The cells contain an intermediate filament, neurofilament 200, used as a structural axonal protein (Posmantur et al., 2009). Other studies have shown that neurofilament 200 were expressed in N2a cells after treatment with GOM extract (Phan et al., 2013). We used the N2a cell line as an in vitro model for this study to determine the effect of uridine on neurite outgrowth.
Incubation of N2a cells with ethanol alone (vehicle) had no effect on the percentage of neurite-bearing cells as compared to cell grown in medium alone (Figure 1). Treatment of N2a cells with uridine significantly (p≤0.001) increased the percentage of neurite bearing cells as compared to cells grown in vehicle alone (figure 2A). Uridine at a concentration of 100 πM stimulated the highest percentage of neurite-bearing cells, almost double the amount seen in the vehicle control. The increase in uridine concentration from 100 πM to 200 πM caused a slight decrease in the percent of neurite-bearing cells. Previous studies have shown GOM has a uridine concentration of 1.5 πM, and can increase neural growth in a dose-dependent manner, up to 100 πM (Baskaran et al. 2017). These findings support our results that 100 πM is the ideal concentration of uridine for optimal neural growth.
N2a cells treated with uridine significantly increased (p≤0.001) neurite length as compared to cells incubated with vehicle alone (Figure2B). In addition, the combined length of all neurite extensions showed a significant increase (p≤0.001) when treated with uridine as compared to the vehicle alone (Figure2C). These results suggest uridine promotes neurite outgrowth in N2a cells. Previous studies have shown the rate at which brain neurons form new dendritic spines depend upon three limiting compounds: uridine, DHA, and choline. All three compounds are precursors of the phosphatides in neural membranes. Uridine supplements can increase brain phosphatide levels. Moreover, uridine can be an agonist for P2Y2 receptors stimulating the production of synaptic proteins (Wurtman et al., 2010). Another study suggests when uridine binds to P2Y receptors, it stimulates the MEK/ERK and PI3K/AKt/mTOR pathways, which in turn increases neuronal growth (Phan et al., 2015).
The results from our study demonstrate that treatment of N2a cells with uridine increased the percentage of neurite-bearing cells, neurite extension, and combined length of neurites per cell. The optimum uridine concentration on neurite outgrowth was 100 πM. Future in vivo studies are desirable, to examine the beneficial effects of uridine in animal models and further elucidate its therapeutic potential in neurodegenerative diseases.

Medium 1

Ethanol

Uridine
Figure 1–Representative phase contrast micrographs of N2a cells incubated in medium alone (A), medium containing ethanol (vehicle, B) and 100 πM of uridine (C). Scale bar = 20 µm.




Figure 2 - Incubation of N2a cells with uridine significantly (*p≤0.001) increased the percentage of neurite bearing cells (A), neurite length (B), and combined length of neurite (C) as compared to cells incubated with ethanol alone (vehicle). Data are mean +/- SE from three different experiments.
List of abbreviations: GOM, Pleurotus giganteus; N2a, Neuro2a murine neuroblastoma; CNS, Central nervous system; and MCI, mild cognitive impairment.
Acknowledgements: We thank the Department of Biological Sciences, Eastern Illinois University, for providing facilities and funding for the completion of this study.
Statement of Authors Contributions: The project was conceived by BP and JS. All authors contributed to research, writing, editing, and creating figures for the manuscript.
REFERENCES
- Allen, S. J., J.J. Watson, D.K. Shoemark, N.U. Barua and N.K. Patel. (2013). GDNF, NGF and BDNF as therapeutic options for neurodegeneration. Pharmacology and Therapeutics 138: 155–175. https://doi.org/10.1016/j.pharmthera.2013.01.004
- Baskaran, A., K.H. Chua, V. Sabaratnam, M.R. Ram and U. R. Kuppusamy. (2017). Pleurotus giganteus, the giant oyster mushroom inhibits NO production in LPS/H2O2 stimulated RAW 264.7 cells via STAT 3 and COX-2 pathways. BMC Complementary and Alternative Medicine 17: 40. DOI: 1186/s12906-016-1546-6
- Bennett, L., C. Kersaitis, S.L. Macaulay, G. Münch, G. Niedermayer, J. Nigro and M. Bird. (2013). Vitamin D2-Enriched Button Mushroom (Agaricus bisporus) Improves Memory in Both Wild Type and APPswe/PS1dE9 Transgenic Mice. PLOS ONE 76362. DOI: 1371/journal.pone.0076362
- Cansev, M. (2006). Uridine and cytidine in the brain: their transport and utilization. Brain Research Reviews 52: 389–397. DOI: 1016/j.brainresrev.2006.05.001
- Daneman, R. and A. Prat. (2015). The Blood–Brain Barrier. Cold Spring Harbor Perspectives in Biology 7: 1-14. https://doi.org/10.1101/cshperspect.a020412
- Das, S. K., M. Masuda, A. Sakurai. and M. Sakakibara (2010). Medicinal uses of the mushroom Cordyceps militaris: Current state and prospects. Fitoterapia 81: 961–968. DOI: 1016/j.fitote.2010.07.010
- Feng, L., I. Cheah, M.M. Ng, J. Li, S. Chan, S. Lim, R. Mahendran, E. Kua and B. Halliwell. (2019). The Association between Mushroom Consumption and Mild Cognitive Impairment: A Community-Based Cross-Sectional Study in Singapore. Journal of Alzheimer’s Disease 68: 197--203. DOI: 3233/JAD-180959
- Hebert, L.E., P.A. Scherr, J.L. Bienias, D.A. Bennett and D.A. Evans. (2003). Alzheimer Disease in the US Population: Prevalence Estimates Using the 2000 Census. Archives of Neurology 60(8): 1119–1122. DOI: 1001/archneur.60.8.1119
- Karunarathna, S.C., Z. L. Yang, O. Raspé, T.W. Ko Ko, E.C. Vellinga, R.-L. Zhao andK.D. Hyde, (2012). Lentinus giganteus revisited: New collections from Sri Lanka and Thailand. Mycotaxon 118: 57-71. DOI:5248/118.57
- Khan, M. A. and M. Tania. (2012). Nutritional and Medicinal Importance of Pleurotus Mushrooms: An Overview. Food Reviews International 28: 313–329. https://doi.org/10.1080/87559129.2011.637267
- Leach, J. L., J. H. Baxter, B. E. Molitor, M. B. Ramstack and M.L. Masor. (1995). Total potentially available nucleosides of human milk by stage of lactation. American Journal of Clinical Nutrition 61: 1224–1230. DOI: 1093/ajcn/61.6.1224
- Lee, S. S., Y. S. Chang and M. N. R. Noraswati. (2009). Utilization of macrofungi by some indigenous communities for food and medicine in Peninsular Malaysia. Forest Ecology and Management 257: 2062–2065. DOI:1016/j.foreco.2008.09.044
- Nathan, M. and G. C. Tucker (2019). Asiatic acid and Madecassic acid promote neurite outgrowth in Neuro-2a Cells. Transactions of the Illinois State Academy of Science 112: 1-4.
- Phan, C.-W., P. David, M. Naidu, K.-H. Wong, and V. Sabaratnam. (2013). Neurite outgrowth stimulatory effects of culinary-medicinal mushrooms and their toxicity assessment using differentiating Neuro-2a and embryonic fibroblast BALB/3T3. BMC Complementary and Alternative Medicine 13: 261. DOI: 1186/1472-6882-13-261
- Phan, C.-W., P. David, M. Naidu, K.-H. Wong, and V. Sabaratnam. (2015a). Therapeutic potential of culinary-medicinal mushrooms for the management of neurodegenerative diseases: Diversity, metabolite, and mechanism. Critical Reviews in Biotechnology 35: 355–368. DOI: 3109/07388551.2014.887649
- Phan, C.-W., P. David, M. Naidu, K.-H. Wong, and V. Sabaratnam. (2015b). Uridine from Pleurotus giganteus and Its Neurite Outgrowth Stimulatory Effects with Underlying Mechanism. PLOS ONE: 0143004. DOI: 1371/journal.pone.0143004
- Phan, C.-W., G.-S. Lee, I. G. Macreadie, S. N. A. Malek, D. Pamela and V. Sabaratnam. (2013). Lipid Constituents of the Edible Mushroom, Pleurotus giganteus Demonstrate Anti-Candida Activity. Natural Product Communications 8(12):1763-5.
- Phan, C.-W., W.-L. Wong, P. David, M. Naidu and V. Sabaratnam. (2012). Pleurotus giganteus (Berk.) Karunarathna and K.D. Hyde: Nutritional value and in vitro neurite outgrowth activity in rat pheochromocytoma cells. BMC Complementary and Alternative Medicine 12: 1054. DOI: 1186/1472-6882-12-102
- Pooler, A. M., D. H. Guez, R. Benedictus and R. J. Wurtman. (2005). Uridine enhances neurite outgrowth in nerve growth factor-differentiated PC12 [corrected]. Neuroscience 134: 207–214. DOI: 1016/j.neuroscience.2005.03.050
- Posmantur, R., R.L. Hayes, C.E. Dixon and W.C. Taft. (2009). Neurofilament 68 and Neurofilament 200 Protein Levels Decrease After Traumatic Brain Injury. Journal of Neurotrauma 11:533--545. DOI: 1089/neu.1994.11.533
- Radio, N. M. and W. R. Mundy. (2008). Developmental neurotoxicity testing in vitro: Models for assessing chemical effects on neurite outgrowth. NeuroToxicology 29: 361–376. DOI: 1016/j.neuro.2008.02.011
- Thorell, L., L. B. Sjöberg and O. Hernell. (1996). Nucleotides in human milk: Sources and metabolism by the newborn infant. Pediatric Research 40: 845–852. DOI: 1203/00006450-199612000-00012
- Wasser, S. P. (2002). Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides. Applied Microbiology and Biotechnology 60: 258–274. https://doi.org/10.1007/s00253-002-1076-7
- Wong, K.-H., M. Naidu, R. P. David, R. Bakar and V. Sabaratnam. (2012). Neuroregenerative potential of lion’s mane mushroom, Hericium erinaceus in the treatment of peripheral nerve injury. International Journal of Medicinal Mushrooms 14: 427–446. DOI: 1615/intjmedmushr.v14.i5.10
- Wurtman, R. J., M. Cansev, T. Sakamoto and I. Ulus. (2010). Nutritional modifiers of aging brain function: Use of uridine and other phosphatide precursors to increase formation of brain synapses. Nutrition Reviews 68: 88–101. DOI: 1111/j.1753-4887.2010.00344.x
- Yang, F., R. Lv, Y.-I. Zhang and Z. Xia. (2012). Comparison study on nucleosides and nucleotides in edible mushroom species by capillary zone electrophoresis. Analytical Methods 4: 546–549. DOI:1039/C2AY05793J
|