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Uridine from Pleurotus giganteus and Its Neurite Outgrowth Stimulatory Effects with Underlying Mechanism.

Phan CW, David P, Wong KH, Naidu M, Sabaratnam V - PLoS ONE (2015)

Bottom Line: An edible mushroom, Pleurotus giganteus was found to stimulate neurite outgrowth in vitro but the chemical constituents and the underlying mechanism is yet to be elucidated.Uridine (100 μM) was found to increase the percentage of neurite-bearing cells of differentiating neuroblastoma (N2a) cells by 43.1 ± 0.5%, which was 1.8-fold higher than NGF (50 ng/mL)-treated cells.MEK/ERK and PI3K-Akt-mTOR further induced phosphorylation of cAMP-response element binding protein (CREB) and expression of growth associated protein 43 (GAP43); all of which promoted neurite outgrowth of N2a cells.

View Article: PubMed Central - PubMed

Affiliation: Mushroom Research Centre, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.

ABSTRACT
Neurodegenerative diseases are linked to neuronal cell death and impairment of neurite outgrowth. An edible mushroom, Pleurotus giganteus was found to stimulate neurite outgrowth in vitro but the chemical constituents and the underlying mechanism is yet to be elucidated. The chemical constituents of P. giganteus (linoleic acid, oleic acid, cinnamic acid, caffeic acid, p-coumaric acid, succinic acid, benzoic acid, and uridine) were tested for neurite outgrowth activity. Uridine (100 μM) was found to increase the percentage of neurite-bearing cells of differentiating neuroblastoma (N2a) cells by 43.1 ± 0.5%, which was 1.8-fold higher than NGF (50 ng/mL)-treated cells. Uridine which was present in P. giganteus (1.80 ± 0.03 g/100g mushroom extract) increased the phosphorylation of extracellular-signal regulated kinases (ERKs) and protein kinase B (Akt). Further, phosphorylation of the mammalian target of rapamycin (mTOR) was also increased. MEK/ERK and PI3K-Akt-mTOR further induced phosphorylation of cAMP-response element binding protein (CREB) and expression of growth associated protein 43 (GAP43); all of which promoted neurite outgrowth of N2a cells. This study demonstrated that P. giganteus may enhance neurite outgrowth and one of the key bioactive molecules responsible for neurite outgrowth is uridine.

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Enhancement of uridine-induced neurite outgrowth is (a) ERK-, (b) Akt-, (c) MEK- & mTOR-, and (d) CREB-dependent as evidenced by the expression of phospho-ERK1/2 (p44/p42), Akt (Thr308), MEK (Ser217/221) & mTOR (Ser2448), and CREB (Ser133) as detected using specific antibodies.U0126 and PD98059 are the inhibitors for ERK. LY294002 is the inhibitor for PI3K/Akt. Data are expressed as mean ± S.D. for n = 3. Different alphabets represent significant differences between samples (p < 0.05). *p < 0.05 significantly lower relative to group without inhibitor treatment. #p < 0.05 significantly higher relative to NGF.
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pone.0143004.g006: Enhancement of uridine-induced neurite outgrowth is (a) ERK-, (b) Akt-, (c) MEK- & mTOR-, and (d) CREB-dependent as evidenced by the expression of phospho-ERK1/2 (p44/p42), Akt (Thr308), MEK (Ser217/221) & mTOR (Ser2448), and CREB (Ser133) as detected using specific antibodies.U0126 and PD98059 are the inhibitors for ERK. LY294002 is the inhibitor for PI3K/Akt. Data are expressed as mean ± S.D. for n = 3. Different alphabets represent significant differences between samples (p < 0.05). *p < 0.05 significantly lower relative to group without inhibitor treatment. #p < 0.05 significantly higher relative to NGF.

Mentions: Since the uridine-induced neurite outgrowth activity was found to be mediated by P2Y, MAPK/ERK1/2, and PI3K/Akt pathways, the ability of uridine to activate the specific protein kinases responsible for the pathways was tested. The phosphorylation of the downstream target proteins was measured. P-ERK activity (Fig 6A) was significantly (p < 0.05) enhanced by uridine when compared to the NGF-treated cells. The presence of the aqueous and ethanol extracts also triggered significantly (p < 0.05) higher phospho-p44/42 (Thr202/Tyr204) levels when compared to cells with medium only. Besides, uridine resulted in the highest expression of p-ERK (Fig 6A). As expected, ERK phosphorylation was significantly (p < 0.05) suppressed in the presence of inhibitors U0126 and PD98059. N2a cells treated with uridine also exhibited a significantly higher (p < 0.05) Akt (Thr308) phosphorylation when compared to the NGF control (Fig 6B). Similarly, AKT inhibitor LY294002-treated cells added with mushroom extracts, uridine, or NGF did not result in phosphorylation of Akt. Considering the above results showing that uridine significantly (p < 0.05) enhanced the expression of p-ERK and p-Akt, it will be crucial to evaluate whether uridine could also increase p-MEK and p-mTOR. Treatment of N2a cells with uridine caused a significant (p < 0.05) and sustained increase of phosphorylation of MEK and mTOR in dose dependent manner (Fig 6C). The highest phosphorylation levels of MEK and mTOR (2.65±0.2 and 3.33±0.2, respectively) were observed with 500 μM uridine. CREB is critical for activating the transcription of genes controlled by the cAMP-response element, and many of these genes may be involved in neuronal outgrowth and plasticity. Therefore, the phosphorylation of CREB was investigated. As shown in Fig 6D, treatment of N2a cells with 500 μM of uridine caused approximately 2.8 fold increase of CREB phosphorylation when compared with the vehicle group.


Uridine from Pleurotus giganteus and Its Neurite Outgrowth Stimulatory Effects with Underlying Mechanism.

Phan CW, David P, Wong KH, Naidu M, Sabaratnam V - PLoS ONE (2015)

Enhancement of uridine-induced neurite outgrowth is (a) ERK-, (b) Akt-, (c) MEK- & mTOR-, and (d) CREB-dependent as evidenced by the expression of phospho-ERK1/2 (p44/p42), Akt (Thr308), MEK (Ser217/221) & mTOR (Ser2448), and CREB (Ser133) as detected using specific antibodies.U0126 and PD98059 are the inhibitors for ERK. LY294002 is the inhibitor for PI3K/Akt. Data are expressed as mean ± S.D. for n = 3. Different alphabets represent significant differences between samples (p < 0.05). *p < 0.05 significantly lower relative to group without inhibitor treatment. #p < 0.05 significantly higher relative to NGF.
© Copyright Policy
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pone.0143004.g006: Enhancement of uridine-induced neurite outgrowth is (a) ERK-, (b) Akt-, (c) MEK- & mTOR-, and (d) CREB-dependent as evidenced by the expression of phospho-ERK1/2 (p44/p42), Akt (Thr308), MEK (Ser217/221) & mTOR (Ser2448), and CREB (Ser133) as detected using specific antibodies.U0126 and PD98059 are the inhibitors for ERK. LY294002 is the inhibitor for PI3K/Akt. Data are expressed as mean ± S.D. for n = 3. Different alphabets represent significant differences between samples (p < 0.05). *p < 0.05 significantly lower relative to group without inhibitor treatment. #p < 0.05 significantly higher relative to NGF.
Mentions: Since the uridine-induced neurite outgrowth activity was found to be mediated by P2Y, MAPK/ERK1/2, and PI3K/Akt pathways, the ability of uridine to activate the specific protein kinases responsible for the pathways was tested. The phosphorylation of the downstream target proteins was measured. P-ERK activity (Fig 6A) was significantly (p < 0.05) enhanced by uridine when compared to the NGF-treated cells. The presence of the aqueous and ethanol extracts also triggered significantly (p < 0.05) higher phospho-p44/42 (Thr202/Tyr204) levels when compared to cells with medium only. Besides, uridine resulted in the highest expression of p-ERK (Fig 6A). As expected, ERK phosphorylation was significantly (p < 0.05) suppressed in the presence of inhibitors U0126 and PD98059. N2a cells treated with uridine also exhibited a significantly higher (p < 0.05) Akt (Thr308) phosphorylation when compared to the NGF control (Fig 6B). Similarly, AKT inhibitor LY294002-treated cells added with mushroom extracts, uridine, or NGF did not result in phosphorylation of Akt. Considering the above results showing that uridine significantly (p < 0.05) enhanced the expression of p-ERK and p-Akt, it will be crucial to evaluate whether uridine could also increase p-MEK and p-mTOR. Treatment of N2a cells with uridine caused a significant (p < 0.05) and sustained increase of phosphorylation of MEK and mTOR in dose dependent manner (Fig 6C). The highest phosphorylation levels of MEK and mTOR (2.65±0.2 and 3.33±0.2, respectively) were observed with 500 μM uridine. CREB is critical for activating the transcription of genes controlled by the cAMP-response element, and many of these genes may be involved in neuronal outgrowth and plasticity. Therefore, the phosphorylation of CREB was investigated. As shown in Fig 6D, treatment of N2a cells with 500 μM of uridine caused approximately 2.8 fold increase of CREB phosphorylation when compared with the vehicle group.

Bottom Line: An edible mushroom, Pleurotus giganteus was found to stimulate neurite outgrowth in vitro but the chemical constituents and the underlying mechanism is yet to be elucidated.Uridine (100 μM) was found to increase the percentage of neurite-bearing cells of differentiating neuroblastoma (N2a) cells by 43.1 ± 0.5%, which was 1.8-fold higher than NGF (50 ng/mL)-treated cells.MEK/ERK and PI3K-Akt-mTOR further induced phosphorylation of cAMP-response element binding protein (CREB) and expression of growth associated protein 43 (GAP43); all of which promoted neurite outgrowth of N2a cells.

View Article: PubMed Central - PubMed

Affiliation: Mushroom Research Centre, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.

ABSTRACT
Neurodegenerative diseases are linked to neuronal cell death and impairment of neurite outgrowth. An edible mushroom, Pleurotus giganteus was found to stimulate neurite outgrowth in vitro but the chemical constituents and the underlying mechanism is yet to be elucidated. The chemical constituents of P. giganteus (linoleic acid, oleic acid, cinnamic acid, caffeic acid, p-coumaric acid, succinic acid, benzoic acid, and uridine) were tested for neurite outgrowth activity. Uridine (100 μM) was found to increase the percentage of neurite-bearing cells of differentiating neuroblastoma (N2a) cells by 43.1 ± 0.5%, which was 1.8-fold higher than NGF (50 ng/mL)-treated cells. Uridine which was present in P. giganteus (1.80 ± 0.03 g/100g mushroom extract) increased the phosphorylation of extracellular-signal regulated kinases (ERKs) and protein kinase B (Akt). Further, phosphorylation of the mammalian target of rapamycin (mTOR) was also increased. MEK/ERK and PI3K-Akt-mTOR further induced phosphorylation of cAMP-response element binding protein (CREB) and expression of growth associated protein 43 (GAP43); all of which promoted neurite outgrowth of N2a cells. This study demonstrated that P. giganteus may enhance neurite outgrowth and one of the key bioactive molecules responsible for neurite outgrowth is uridine.

Show MeSH
Related in: MedlinePlus