International Journal of Applied Research in Natural Products Vol. 7 (4), pp. 39-48. Directory of Open Access Journals ©2008-2014. IJARNP-HS Publication
Original Research Eugenia jambolana Lam. Increases lifespan and ameliorates experimentally induced neurodegeneration in C. elegans Maria de Fátima Bezerra1, Brandon York Jamison2, Yuki Gomada2, Katia Cristina Borges2, Roberta Targino Pinto Correia#1, Dhiraj Anil Vattem*2 1 Laboratory of Food Bioactive Compounds and Dairy Technology, Chemical Engineering Department, Federal University of Rio Grande do Norte, Campus Lagoa Nova, Natal - RN, Brazil. 59075-180. 2 Nutrition Biomedicine and Biotechnology, Texas State University. San Marcos, TX, USA, 78666.
Summary. Type-2 diabetes mellitus (T2DM), dyslipidemia (DL) and inflammation (IF) are associated with reduced lifespan (LS) and increased risk of neurodegenerative diseases (NDG). Dysregulation in insulin/insulin-like growth factor-1 (IGF-1) (IIS) signaling, forkhead box O transcription factor (FOXO) and Silent Information Regulators or Sirtuins (SIRT) may be responsible. We investigated the effect of spray dried Jambolan (Eugenia jambolana Lam.) fruit in Caenorhabditis elegans model for lifespan, amyloid 1-42 (A1-42) aggregation induced paralysis and MPP+ (1-methyl-4-phenylpyridinium) induced neurodegeneration. Effect on modulating critical genes involved signaling pathways important in IIS, LS and NDG were also studied in C. elegans. Results show suggest statistically significant increase in lifespan (9-22.7%) coupled with a delay in A1-42 induced paralysis (11.5%) and MPP+ induced paralysis (38-43%). Gene expression studies indicated a significant upregulation in expression of C. elegans homologs of foxo, sirt1, dopamine D1 receptor and suggested a non-FOXO mediated mechanism of action. Industrial relevance. Jambolan is a bioactive-rich tropical fruit with high colorant potential. Despite this fact, its perishability has hampered its market and industrial use beyond the countries where it is cultivated. Considering that drying is a popular technique able to extend fruits shelf life and concentrate their natural bioactive compounds, this research investigates the health relevance of spray dried jambolan. Here we addressed the potential of dried Jambolan fruit to extend lifespan and inhibit the progression of experimentally induced neurodegeneration using the C. elegans model. We demonstrated that this convenient fruit product was able to increase the lifespan of C. elegans. The jambolan extracts also influenced some critical genes of signaling pathways relevant to metabolic diseases, aging and neurodegeneration. Based on our results, some insight about the mechanism of action that would lead to the observed anti-aging and anti-neurodegenerative effects is discussed. The data shown here reveals fresh data concerning the health-relevant attributes of spray dried Jambolan fruit and provides the scientific rationale for a better exploitation and valorization of this tropical fruit. Keywords. Caenorhabditis elegans; Eugenia jambolana; Lifespan; Neurodegeneration; Insulin signaling; sirtuin
INTRODUCTION Eugenia jambolana Lam., is a tropical evergreen tree native to the Indian subcontinent and produces an intensely dark purple fruit with sweet to astringent taste (Sant’Ana et al., 2011) The fruit known as Jamun or black plum, is cherished for its health promoting effects in Ayurveda and other forms of traditional medicine(Ayyanar & Subash-Babu, 2012). During colonial times, this plant was introduced to Central and South American countries, especially Brazil, where it is known as Jambolan and is cultivated widely. High perishability and lack of optimized postharvest processing technologies has restricted the popularity of this fruit outside the countries where it is grown (Bennett et al., 2011). We have recently shown that dehydration, especially spray drying and lyophilization can be applied to increase shelf-life without compromising the nutritive and organoleptic properties of Jambolan and other tropical fruit pulps and residues (Correia, Borges, Medeiros, & Genovese, 2012; Fujita, Borges, Correia, Franco, & Genovese, 2013) While traditional medicine has described several beneficial properties associated with the consumption of Jambolan, research in rodent models has provided scientific evidence for managing diabetes, dyslipidemia and inflammation (Baliga, Fernandes, Thilakchand, D’souza, & Rao, 2013; Srivastava & Chandra, 2013). These therapeutic properties have been associated with the presence of diverse group of secondary metabolites, especially, phenolic acids, flavonoids, anthocyanains and terpenes that are abundant in Jambolan pulp and skin (Sant’Ana et al., 2011; Srivastava & Chandra, 2013). However, information on the potential mechanism of health promotion by Jambolan and their constituent active metabolites is limited and not well understood. Emerging research has indicated that metabolic aberrations resulting from insulin resistance, type 2 diabetes mellitus (T2DM) and dyslipedimia are associated with accelerated aging, cognitive decline and neurodegeneration (Cholerton, Baker, & Craft, 2011; Cole & Frautschy, 2007). Dysregulation of metabolic signaling especially of insulin/insulin-like growth factor-1 (IGF-1) ___________________ *#Corresponding Author(s). [email protected]
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(IIS) has been shown to attenuate a robust stress response mediated via forkhead box O transcription factor or FOXO and Silent Information Regulators or Sirtuins (SIRT) to decrease longevity(Gan & Mucke, 2008). Additionally, insulin resistance, dyslipidemia and inflammation have been linked to abnormal amyloid peptide processing and hyperphosphorylation of tau proteins to initiate aggregation into plaques and fibrillary tangles and cause protein toxicity in neurons (Cohen & Dillin, 2008; Donmez, 2012). Moreoever, metabolic syndrome has also been shown to reduce protesomal and autophagic protein clearance (degradation), induce metabolic dysfunction and increase oxidative stress. Together, these factors cause abnormal neuronal function, accelerate neuronal cell death to initiate cognitive decline and neurodegeneration , such as Alzheimer’s disease (AD) and Parkinson’s disease (PD)(Lionaki, Markaki, & Tavernarakis, 2013). Therefore, therapeutic agents that can reduce insulin resistance, manage dyslipidemia and control inflammation via the activation of FOXO and SIRT mediated signaling can increase lifespan and delay the onset of and progression of neurodeneration(Herskovits & Guarente, 2013). Since Jambolan has been shown to have anti-diabetic, hypolipidemic and anti-inflammatory effects we hypothesized that it may also have anti-aging and anti-neurodegenerative properties. Therefore, the objective of this study was to investigate the potential of Jambolan fruit extract to extend lifespan and abate the progression of experimentally induced neurodegeneration in the nematode Caenorhabditis elegans. The effect of Jambolan extracts on critical genes of IIS and SIRT signaling pathways relevant to metabolic diseases, aging and neurodeneration was also investigated in vivo in C. elegans.
MATERIALS AND METHODS Jambolan powder production. Jambolan (Eugenia jambolana Lam.) fruits harvested from Northeastern Brazil and authenticated at Federal University of Rio Grande do Norte (Natal, Brazil). The fruits were screened for uniformity and only fruits with no visible defects were selected. After washing in running water, the fruits were processed into fruit pulp by using a pilot depulping machine (model Compacta, ITAMETAL, Itabuna, Brazil) and they were kept frozen. For the spray drying process, the jambolan pulp was mixed with a solution of arabic gum 10%. The mixtures were atomized using a spray dryer [model LM MSD 1.0 (Labmaq do Brasil LTDA, Ribeirão Preto, Brazil).] at 110 oC with a 1.3-mm diameter atomizing nozzle at an airflow and 45 L/min and a feed flow of 0.53 mL/min. Extraction procedures and preparation of samples. The low and high molecular weight fractions of Jambolan powder were prepared using a method developed previously(Huerta, Mihalik, Becket, Maitin, & Vattem, 2010) Extraction of low molecular weight fraction (EJ-L). Five grams of Jambolan powder was suspended in 100 ml of water in an Erlenmeyer flask at 60ºC with stir bar and allowed to mix for 30 minutes at 250 rpm. The samples were then centrifuged at 4000 rpm at 10oC for 15 min. The supernatant was filtered under vacuum using a Buchner funnel equipped with Whatman No. 1 filter paper. The filtrate was mixed with acetone in 1:1 (v/v) ratio and again centrifuged to precipitate soluble gums and fibers in the extract. The acetone in the supernantant was evaporated in a Rotavapor® (Model: RII, Buchi, Switzerland) at 60oC for 5 min under vacuum. The acetone free solution was filter-sterilized (Corning, North Bend, OH) and labeled as EJ-L. Extraction of high molecular weight fraction (EJ-H). The residue from the filter paper (item 2.2.1) was scraped with a spatula into an Erlenmeyer flask and combined with the sediment from centrifugation (from previous step above). To this 66.6 ml of 4N NaOH was added, and the mixture was stirred for 30 minutes at 250 rpm on a magnetic stirrer. The mixture was then centrifuged and vacuum filtered as described above. The pH of the filtrate was adjusted immediately to 7.0 and filter-sterilized into a sterile bottle labeled as EJ-H. Phenolic characterization and standardization. Since the biological functionality of food ingredients has been predominantly associated with the polyphenolic secondary metabolites, prior to our C. elegans studies, EJ-L and EJ-H fractions were assayed for total phenolic content by Folin-Ciocalteu spectrophotometric assay described elsewhere(Correia et al., 2012). The total phenolic content of the two fractions was expressed as microgram of gallic acid equivalents per mg dry weight of the jambolan powder [g (GAE)/mg]. To compare the effectiveness of both fractions they were standardized to a total phenolic content of 260 ±35 [g (GAE)/mg]. The concentration of both EJ-L and EJ-H in all C. elegans experiment was optimized to 1.0 % (v/v) in NGM after several iterations. We determined from our initial screening experiments (data not shown) at this concentration, extracts did not interfere with the normal development of C. elegans and yielded results that within the sensitivity of various in vivo C. elegans assays. C. elegans strains and maintenance. Wild type and transgenic C. elegans strains carrying green fluorescent protein (gfp)- or heat shock protein (hsp) promoter fusions of different genes relevant were obtained from the Caenorhabditis Genetics Center (CGC) [University of Minnesota (Minneapolis, MN)]. (Table 1). Worms were grown and maintained at 20oC (Except CL4176 which was maintained at 16oC) on 60 mm culture plates with Nematode Growth Medium (NGM) (1.7% agar, 0.3% NaCl, 0.25% Peptone, 1mM CaCl, 1mM MgSO4, 5mg/L Cholesterol, 2.5 mM KPO4) at 16-20oC (Brenner, 1974). Media was poured aseptically into culture plates (10 ml for 60 mm) using a peristaltic pump and allowed to solidify for 36 hours. NGM culture plates were then inoculated with 50 μl of Escherichia coli OP50 [CGC, University of Minnesota (Minneapolis, MN)] overnight cultures and incubated for 8 hours at 37oC. Strains of C. elegans were maintained by picking 2-3 young adult worms onto freshly inoculated NGM plates every 4-7 days. Age synchronization. Prior to the beginning of the experiment C. elegans were age synchronized. Ten worms at L4 stage (F0) were transferred to a single NGM plates and incubated at 20oC until they progressed to adulthood and layed eggs. Adults were immediately removed from the plates and the eggs were allowed to hatch (F1) and grow to L4 at 20oC. L4 worms of F1 generation were again transferred to fresh NGM plates and allowed to mature into gravid adults and lay eggs at 20oC. Adults were quickly removed from the plates were returned to 20oC incubator to facilitate egg hatching. L1 worms (F2) generation were collected from the plate by washing with S-basal buffer (0.59% NaCl, 5% 1M KPO4, 5 mg/ml Cholesterol in ethanol) (Ching & Hsu, 2011;
Eugenia jambolana increases lifespan and ameliorates neurodegeneration in C. elegans
Vayndorf, Lee, & Liu, 2013) into a sterile 15 ml centrifuge tube. Worms from a minimum of 5 plates were pooled into a single centrifuge tube and centrifuged at 8000 rpm for 10 mins at 10oC. The supernatant was carefully aspirated and the worms were washed again in by S-basal buffer, centrifuged and aspirated to leave approximately 1 ml of S-basal in the centrifuge tube. The tube was gently agitated to disperse the worms and 20 μl was pipetted onto a slide and the number of worms was counted under a stereo microscope. The concentration of the worms was adjusted to be 10-15 worms by diluting with S-complete liquid media (97.7% S-basal, 1% potassium citrate, 1% trace metals, 0.3% CaCl2, 0.3% MgSO4). A 100 mg/ml suspension of E. coli OP50 was prepared by centrifuging 100 ml of an overnight E. coli OP50 culture in LB at 3500 rpm. Spent LB was aspirated and pellet was washed several times by resuspension and centrifugation in sterile distilled water. The weight of the resultant pellet was determined and adjusted to 100 mg/ml using S-complete medium. E. coli OP50 was added to the vial of age synchronized worms to yield a final concentration of 5 mg/ml and used immediately to set up experiments. Table 1. List of C. elegans strains included are the C. elegans gene names with their human homolog. Strain N2 GR1352 BC10837 LG326 BC14613 CF1553
Transgene Wild Type xrIs87[daf16alpha::GFP::DAF16B + rol-6(su1006)]. sEx10837[rCesB0334 .8::GFP + pCeh361]. geIs7 [skn-1b::GFP]. sEx14613[rCesW08G 11.4::GFP + pCeh361]. muIs84[pAD76(sod3::GFP)].
Wormbase Gene ID* N/A
Forkhead Box O (FOXO)
[rCesB0334.8::GFP + pCeh361]; Age-1::gfp
Phoshoinositide 3-kinase (PI3K)
Nuclear factor-erythroid-2 related factor-2 (Nrf2) transcription factor
[W08G11.4::gfp] transcriptional fusion. Driven by pptr-1
Protein Phosphatase 2A regulatory subunit B56
sod-3::GFP (driven by sod-3)
Iron/Manganese superoxide dismutase
Catalase (ctl-1, ctl-2 and ctl-3)
WBGene00000830 WBGene00000831 WBGene00013220
Human Amyloid β1-42
Dopamine receptor 1
wuIs151[ctl-1 + ctl-2 [ctl-1 + ctl-2 + ctl-3 + myo+ ctl-3 + myo2::GFP] 2::GFP] leEx3295 [sir2.1::mCherry + [sir-2.1::mCherry + UL3295 R11A8.5::GFP + rolR11A8.5::GFP + rol-6(su1006)] 6(su1006)]. smg-1(cc546) I; dvIs27 X. dvIs27[pAF29(myoABeta42 CL4176 3/A-Beta 1-42/let UTR) + pRF4(rol6(su1006))] pha-1(e2123) III; F15A8.5::GFP = dopOH2411 otEx233. 1prom1::GFP. *Information obtained from www.wormbase.org. GA800
Life span assay in microtiter plates. The lifespan of C. elegans N2 strain was examined as previously described (Solis & Petrascheck, 2011). To a synchronous culture of L1 C. elegans worms (described above) in S-complete medium (10-15 worms /20 l) containing 5 mg/ml of E. coli OP50, penicillin-streptomycin mixture was added to a concentration of 1% (v/v). 120 μl of the worm suspension was transferred to each well of a 96 well plate, placed on a microtiter plate shaker for 2 minutes and incubated for 45 hours at 20°C until the animals reach the L4 stage. 30 μl of a 0.6 mM Fluorodeoxyuridine (FUDR) stock solution was added to each well to sterilize the animals. 8 to 12 hours after the adding FUDR, EJ-L and EJ-H fractions were added at 1% (v/v) and sealed with Glad Press‘n’Seal ® (Glad, Okland, CA) and incubated at 20oC. Five l of the E. coli OP50 (100 mg/ml) was added to each well to prevent starvation. Worms were scored every other day by counting the number of alive (swimming morphology) animals under stereo microscope to determine lifespan and survival rates. Three replicates per experiment with n >300 worms were used per treatment. Alzheimer’s Disease assay in transgenic C. elegans. Transgenic C. elegans (CL4176) containing a heat-sensitive mutation developed to express human amyloid β1-42 in the muscle tissue was maintained on NGM plates at 16oC (Dostal, Roberts, & Link, 2010). Prior to the beginning of the experiment C. elegans were age synchronized (at 16 oC) as described above. L1 worms from F2 generation were transferred to control or treatment plates and allowed to mature to gravid adult stage to lay eggs. Control plates did not contain any treatment whereas treatment plates contained EJ-L or EJ-H fractions of Jambolan at 1 % (v/v). Fractions were added to the NGM media just prior to pouring. NGM culture plates were then inoculated with 50 μl of Escherichia coli (E. coli) OP50 overnight cultures and incubated for 24 hours at 23oC (Sutphin & Kaeberlein, 2009). For treatment plates, E. coli OP50 was mixed with jambolan fractions at 1 % (v/v) before spreading. To standardize the food supply, the plates were then incubated under UV light in a Stratagene UV Stratalinker 2400 (La Jolla, CA) at maximum dose for 5 min to arrest growth of the E. coli OP50. Upon development of the eggs to L3 worm stage, the plates were upshifted to 25°C for induction of amyloid β1-42 expression. Mobility scoring was conducted beginning 20 h after temperature up-shift and continued in 2 hour increments until all C. elegans are paralyzed. For statistical purposes five replicates per experiment with a minimum of
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>75 worms were used. Failure to respond to touch (prodding with a worm pick) and, absence of pharyngeal pumping was used to score paralyzed/dead worms. Kaplan-Meier method was used to compare the mobility curves. The time in hours post temperature at which 50% of worms were completely paralyzed/dead (PT50 ) was calculated from the survival curves. MPP+ induced Parkinson’s Disease assay in C. elegans. Forty microliters of age synchronized L3 worms in S-Complete (around 17-23 worms) containing 5 mg/ml of E. coli OP50 was mixed with 10μl of MPP+ (1.13 mg/ml in DH20) and 1 % (v/v) EJ-L or EJ-H fractions of Jambolan (Control had distilled water) and transferred to each well of a sterile 96 well plate. A minimum of eight such wells were prepared for each treatment and the plates were sealed with Glad Press‘n’Seal ® (Glad, Okland, CA) and incubated at 20oC. Mobility scoring was conducted every 12 h and the total number of paralyzed worms were recorded after 48 h. For statistical purposes eight replicates per experiment with a minimum of > 100 worms were used. Failure to respond to touch (prodding with a worm pick) and, absence of pharyngeal pumping was used to score paralyzed/dead worms (Braungart, Gerlach, Riederer, Baumeister, & Hoener, 2004). In vivo gene expression studies in transgenic C. elegans. . Fifty l of age synchronized L1 worms in S-Complete (around 1723worms) containing 5 mg/ml of E. coli OP50 and 1 % (v/v) EJ-L or EJ-H fractions of Jambolan (Control had distilled water) was transferred to each well of a sterile 96 well plate. A minimum of eight such wells were prepared for each treatment and the plates were sealed with Glad Press‘n’Seal ® (Glad, Okland, CA) and incubated at 20 oC. When the worms matured to L4 stage they were transferred a to a 35 mm culture plate containing a solidified 2ml layer of 1% Phytagel (Sigma-Aldrich, St. Louis, MO). Worms were immobilized by adding 5-10 l of 25mM sodium azide solution in Magnesium buffer and used for direct in vivo fluorescence imaging of GFP using the Nikon SMZ1500 fluorescence microscope with Ri1 CCD camera (Nikon, Japan). Eight replicates with a minimum of > 120 worms were used per treatment. L4 worms were randomly selected and relative fluorescence, with respect to control, was quantified from the corrected total fluorescence using the National Institute of Health’s ImageJ software (Iser & Wolkow, 2007) Statistical Analyses. Results were expressed as means ± standard error. For the lifespan assays, Kaplan-Meier method was used to compare the lifespan survival curves and the survival differences were tested for significance (p 100; * = p < 0.05 (ANOVA)]
Effect of Jambolan on in vivo expression of genes relevant to IIS, aging and neurodegeneration in C. elegans.We evaluated the effect of Jambolan fractions on modulation of multiple genes implicated in development of neuro/degenerative and aging related diseases in transgenic strains of C. elegans (Table 1). Relative to control, the expression of the fork-head transcription factor daf-16 (Figure 4A) increased significantly by 117% in response to treatment with EJ-L. Similarly, in C. elegans treated with EJ-H, the expression of daf-16 was significantly upregulated by 112.5% relative to control (Figure 4A). Among EJ-L and EJ-H, the expression of daf-16 was higher with EJ-L than with EJ-H (Figure 4A). However, the expression of
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mitochondrial superoxide dismutase sod-3 (Figure 4B) and catalases [ctl-1, ctl-2, ctl-3, (Figure 4C)], which are under positive transcriptional regulation of daf-16 did not change in response to treatment with the two Jambolan fractions. Relative to control, the expression of Nuclear factor-erythroid-2 related factor-2 (Nrf2) transcription factor or skn-1 that regulates the expression of several antioxidant response genes did not change in response to treatment with either EJ-L or EJ-H (Figure 4D). However, the expression of sirtuin (SIRT1) or sir2.1 gene (Figure 4E) in C. elegans was significantly upregulated in response to treatment with both EJ-L and EJ-H (Figure 4E). The expression of two genes in the insulin/insulin like growth factors
Figure 4. Relative change (% of Control) in expression of (A) daf-16 (B) sod-3 (C) ctl-1+ctl-2+ctl-3 (D) skn-1 (E) sir2.1 (F) age-1 (G) pptr1(H) dop-1 in transgenic C. elegans fed CONT: [NGM]; EJ-L: [NGM + 1% (v/v) EJ-L]; and EJ-H: [NGM + 1% (v/v) EJ-H] medium. [E. coli OP50 (5mg/ml) ; n > 100; *(vs. CONT); #(EJ-L vs. EL-H) = p < 0.05 (ANOVA)]
signaling (IIS) pathway Phoshoinositide 3-kinase (PI3K) or age-1 (Figure 4F) in C. elegans and Protein Phosphatase 2A or pptr1 (Figure 4G) also remained unchanged relative to control in response to treatment with EJ-L and EJ-H. The expression of the
Eugenia jambolana increases lifespan and ameliorates neurodegeneration in C. elegans
dopamine receptor gene dop-1 was significantly unpregulated in the C. elegans neurons in response to treatment with EJ-L and EJ-H relative to control (Figure 4H).
DISCUSSION Lifespan in C. elegans, like in all animals is controlled by a multitude of etiopathological factors, but the role of insulin/insulinlike growth factor-1 (IGF-1) signaling (IIS) has been most extensively studied. The components of the IIS signaling pathway in C. elegans are highly conserved and demonstrate a striking homology to mammalian (including human) systems(Landis & Murphy, 2010). The IIS signaling cascade is activated by the binding of a ligand to the IGF-1 receptor (daf-2) and results in the phosphorylation (and activation) of Phosphatidylinositide 3-kinase or PI3Kinase (age-1) to produce PIP3. Formation of this glycolipid, facilitates the recruitment and phosphorylation mediated activation of an important regulatory serine/threoninespecific protein kinase known as protein kinases B (PKB/AKT)(Landis & Murphy, 2010). Among the many targets of PKB, it phosphorylates forkhead box O transcription factor or FOXO (Daf-16) and facilitates its cytoplasmic retention. PKB also phosphorylates and inactivates the transcription factor Nuclear factor (erythroid-derived 2)-like 2 or NRF-2(An et al., 2005). It is known that un-phosphorylated Daf-16 dissociates from the 14-3-3 protein in the cytoplasm and undergoes nuclear translocation to induce the expression itself and of several genes involved in nutrient, oxidant and stress response including, the iron/manganese superoxide dismutase (SOD-3) in mitochondria and cytoplasmic catalases (CTL-1; CTL-2; CTL-3)(Wen, Gao, & Qin, 2013). An active SKN-1 factor on the other hand transloctaes into the nucleus and binds to the antioxidant responsive element (ARE) to activate the expression of the putative phase-II detoxification and glutathione biosynthetic enzymes(An et al., 2005). The IIS pathway is inactivated via dephosphorylation of AGE-1 and PKB by Phosphatase and tensin homolog or PTEN (DAF-18) and Protein Phosphatase 2A Regulatory subunit (PPTR-1) respectively(Landis & Murphy, 2010). Inactivation of IIS either due to dietary restriction (DR) or by gene silencing has been linked to increase in longevity via activation of DAF-16 mediated gene expression(Berdichevsky, Viswanathan, Horvitz, & Guarente, 2006). In our studies with Jambolan extracts, dietary supplementation with low (EJ-L) and high (EJ-H) molecular weight fractions of Jambolan the lifespan of C. elegans increased by 22.7% and 9% respectively. We also observed that the expression of AGE-1 and PPTR-1 was not significantly different in worms feeding on Jambolan extracts than in control, suggesting that the IIS pathway was neither inhibited nor operating at a lower flux. Surprisingly, the expression of DAF-16 increased by 12-17% when the worms diet was supplemented with 1% (v/v) of EJ-L and EJ-H. However, the expression of SOD-3 and CTL (CTL-1; CTL-2; CTL-3) under transcription regulation of DAF-16 remained unchanged after Jambolan treatment, suggesting an upregulated bur transcriptionally inactive DAF-16. In addition to DAF-16, other pathways are also known to modulate DAF-16 expression, independent of IIS (Wang, Bohmann, & Jasper, 2005). A recent study exploring the lifespan promoting effect of Reishi mushroom in C. elegans showed that increased expression of DAF-16 and enhanced lifespan via activation of Toll-interleukin 1 receptor intracellular domain (TIR-1) pathway mediated by a mitogen activated protein kinases (MAPK) and independent of IIS (Chuang, Chiou, Huang, Yang, & Wong, 2009). Silent Information Regulators or Sirtuins (SIRT) are important histone deacetylases that are activated by dietary and environmental stresses to regulate several biological process, including stress response and lifespan(Bamps, Wirtz, Savory, Lake, & Hope, 2009; Calabrese et al., 2012). Moreover, like DAF-16, organisms with increased expression of Sir2.1 have longer lifespan(Bamps et al., 2009). In our studies, treatment with both the Jambolan fractions resulted in a significant upregulation of Sir2.1 expression by ~ 11.5 % relative to control. Several studies have shown that SIRT1 (Sir2.1) is necessary for anti-aging and antioxidant response upon DAF-16 activation. Inside the nucleus Sir2.1 form a transcriptionally active complex in the nucleus with DAF-16 and facilitates the recruitment of RNA polymerase to the promoter region to activate gene expression (Bamps et al., 2009; Calabrese et al., 2012). However, recent studies have shown that nuclear translocation of DAF-16 upon disassociation from 14-3-3 protein alone is not sufficient to form a transcriptionally active complex with Sir2.1(Y. Wang et al., 2006). It is now known that that upon disassociation, even 14-3-3 proteins translocates into the nucleus, where it facilitates the interaction of DAF-16 with Sir2.1, however, this interaction is only favored when both DAF-16 and the 14-3-3 proteins are covalently modified by stress induced acetylation in the cytoplasm prior to nuclear translocation (Berdichevsky et al., 2006). Absence of stress induced acetylation, results in an unstable DAF-16/14-3-3-/Sir2.1 complex and promotes nuclear eviction of DAF-16(Y. Wang et al., 2006). In our study, the relative constant expression of SKN-1 and CTL (CTL-1; CTL-2; CTL-3), both induced by oxidative stress indicate a relatively normoxic environment, and may explain the absence of transcriptional induction of genes under DAF-16 regulation notwithstanding upregulation in expression of both DAF-16 and Sir2.1. These results are similar to the observations made in a similar study investigating the anti-aging role of polydatin (a natural resveratrol glycoside) in C. elegans, where an increased DAF-16 expression was observed without any increase in SOD-3 and CTL (CTL-1; CTL-2; CTL-3) (Wen et al., 2013). Taken together, these results may suggest an alternative mechanism of anti-aging properties of Jambolan and other natural products independent of DAF-16. In response to dietary supplementation with either EJ-L or EJ-H fractions of Jambolan the heat-shock induced aggregation of A1-42 peptide and resultant paralysis was significantly delayed in C. elegans model. Mitochondrial dysfunction, oxidative stress and activation of apoptotic signaling due to intra- and extracellular accumulation of misfolded or damaged proteins has been linked to the etiopathology of aging and neurodegenerative diseases(Cohen & Dillin, 2008; Radak, Zhao, Goto, & Koltai, 2011). Several studies have unsuccessfully investigated the role of antioxidant therapy aging processes and nerudegenerative diseases, thereby questioning the role of oxidative stress(Shukla, Mishra, & Pant, 2011). The transgenic model of C. elegans for Alzhimers disease used in our study, specifically measures the intracellular aggregation of the A1-42 peptide in muscle tissues and permits the investigation of non-free radical contributors, primarily proteotoxicity in aging and neurodegeration. Since accumulation of
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damaged and misfolded proteins is an important cellular signal for senescence and apoptosis, protein quantity control is maintained by activating ubiquitin, chaperone and heat-shock protein mediated recognition and delivery of old, damaged and misfolded proteins to proteosomal and autophagic processes for lysosomal and extra-lysosomal protein turnover(Wilson, Zou, Mattson, Pallauf, & Rimbach, 2013). Dysregulation and decreased efficiency of proteosomal and autophagy processes has been linked to reduced protein clearance, senescence and neurodegeneration(Dostal et al., 2010). Activation of protein quality control processes, especially autophagy is therefore an important strategy for delaying aging and neurodegenerative processes(Wilson et al., 2013). Several studies have shown that activation of Sir2.1 expression by dietary restriction and polyphenols (resveratrol) activate autophagy signal cascades in a DAF-16 independent manner to confer resistance towards cellular protein toxicity to delay aging and neurodegenerative processes(Wilson et al., 2013). Increased lifespan, delayed A1-42 aggregation induced paralysis coupled with increased expression of Sir2.1 in C. elegans upon dietary supplementation with Jambolan observed in our studies could potentially be due to activation of the above mentioned autophagy processes. Similar studies done with caffeine and ginkgo biloba have suggested other non-antioxidant mediated mechanism for reducing A1-42 aggregation induced proteotoxicity(Dostal et al., 2010; Wu et al., 2006). These alternative mechanisms of actions attributed to the presence of bioactive compounds similar to curcumin(Wu et al., 2006), with a spatial configuration facilitating an increased thermodynamically stable interaction with amyloidogenic proteins to prevent their oligomerization (and thus aggregation) may partially explain the effects we observed with Jambolan extracts(Wu et al., 2006). MPP+ (1-methyl-4-phenylpyridinium) is the active metabolite of the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6tetrahydropyridine) produced by the monoamineoxidas-B (MAO-B) in the glial cells(Chakraborty, Bornhorst, Nguyen, & Aschner, 2013). Once formed, MPP+ shows an enhanced affinity towards the dopamine (DA) transporter (DAT), where it is taken up by the pre-synaptic dopmaninergic cell during the DA reuptake process. Once internalized, MPP+ inhibits the mitochondrial complex-I protein of the electron transport chain and causes oxidative stress, ATP depletion and mitochondrial dysfunction and dopaminergic neuronal cell death for Parkinson’s disease (PD) like symptoms (Chakraborty et al., 2013). The MPP+ induced neurotoxicity is a popular system to study PD in vertebrate and invertebrate models, especially C. elegans(Braungart et al., 2004). In our study, treatment with Jambolan fraction resulted in a 38-43% reduction in MPP+ induced paralysis. Moreover, the expression of the dopamine D1 receptor (dop-1) was upregulated by 37% and 67% in response to dietary supplementation with EJ-L and EJ-H fractions of Jambolan. Dop-1 is a post-synaptic DA receptor in C. elegans that is a homologous to the human D1 and is under the transcriptional regulation of multiple neutrophic factors(Do, Kerr, & Kuzhikandathil, 2007). Dop-1 is the most abundant form of DA receptor and is a transmembrane G-protein coupled receptor (GPCR), which upon ligand binding results in activation of adenylate cyclase to produce cyclic AMP (cAMP) and triggers a signaling cascade mediated by protein kinase-A (PKA), phospholipase-C (PLC) and calcium eventually resulting in activation of several MAPK that contribute to synaptic plasticity and neuroprotection (Beaulieu & Gainetdinov, 2011; Nishi, Kuroiwa, & Shuto, 2011). Therefore, activation of dop-1 function, is an active target for therapeutic management of PD. Increased dop-1 expression has recently been shown to confer protection against hydrogen peroxide induced neuronal cell death via activation of extracellular signal-regulated kinase (ERK), p38 dependent MAPK signaling resulting in upregulation of several anti-apoptotic factors (Li, Li, Fan, Zheng, & Ma, 2012)and may potentially explain the potential neuroprotective effects of Jambolan in our study.
CONCLUSIONS Our results suggest that dietary supplementation with Jambolan significantly increased lifespan in C. elegans. Additionally, in worms feeding on Jambola extracts the A1-42 aggregation induced paralysis was abrogated. Results also suggest that increased in longevity and reduced protein toxicity in C. elegans was potentially due to IIS and DAF-16 independent mechanism activated by Sir2.1. We also observed significant attenuation of MPP+ induced oxidative dopaminergic neurotoxicity in C. elegans model of PD, potentially via the activation of dop-1 mediated neuroprotective effects. In our previous experiments we have shown that low and high molecular fractions of natural products had distinct biological functionality (Huerta et al., 2010; Huerta, Mihalik, Maitin, Crixell, & Vattem, 2007). This was attributed to the extraction free phenolics, phenolic acids and flavonoids in low molecular weight fractions and release of insoluble polymerized phenolics, tannins, lignins and lignans from proteins and carbohydrates during high molecular fractionation (Huerta et al., 2010). Interestingly, in this study, both EJ-L and EJ-H fractions performed similarly in different assays, with the EJ-L being marginally better than EJ-H. Further studies on elucidating the potential molecular mechanism of action using advanced genetic tools and characterizing the potential bioactive principals are imperative to establish the potential health promoting effects of Jambolan are currently underway.
ACKNOWLEDGMENTS MFB was supported by financial support and scholarship from CAPES Foundation (Process: BEX 14647 / 12-5; Ministry of Education of Brazil, Brasília, Brazil). DAV was supported by a grant from the Vrdür Foundation. Special thanks also to R. Corey DeLeon, Christen Lester, Leanna Mcmillin and Deanna Townsend for their assistance with the C. elegans models
Eugenia jambolana increases lifespan and ameliorates neurodegeneration in C. elegans
REFERENCES An, J. H., Vranas, K., Lucke, M., Inoue, H., Hisamoto, N., Matsumoto, K., & Blackwell, T. K. (2005). Regulation of the Caenorhabditis elegans oxidative stress defense protein SKN-1 by glycogen synthase kinase-3. Proceedings of the National Academy of Sciences of the United States of America, 102(45), 16275–16280. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1283458&tool=pmcentrez&rendertype=abstract Ayyanar, M., & Subash-Babu, P. (2012). Syzygium cumini (L.) Skeels: A review of its phytochemical constituents and traditional uses. Asian Pacific Journal of Tropical Biomedicine, 2(3), 240–6. doi:10.1016/S2221-1691(12)60050-1 Baliga, M. S., Fernandes, S., Thilakchand, K. R., D’souza, P., & Rao, S. (2013). Scientific validation of the antidiabetic effects of Syzygium jambolanum DC (black plum), a traditional medicinal plant of India. Journal of Alternative and Complementary Medicine (New York, N.Y.), 19(3), 191–7. doi:10.1089/acm.2011.0752 Bamps, S., Wirtz, J., Savory, F. R., Lake, D., & Hope, I. A. (2009). The Caenorhabditis elegans sirtuin gene, sir-2.1, is widely expressed and induced upon caloric restriction. Mechanisms of Ageing and Development, 130(11), 762–770. Retrieved from http://www.sciencedirect.com/science/article/pii/S0047637409001468 Beaulieu, J.-M., & Gainetdinov, R. R. (2011). The physiology, signaling, and pharmacology of dopamine receptors. Pharmacological Reviews, 63(1), 182–217. doi:10.1124/pr.110.002642 Bennett, L. E., Jegasothy, H., Konczak, I., Frank, D., Sudharmarajan, S., & Clingeleffer, P. R. (2011). Total polyphenolics and anti-oxidant properties of selected dried fruits and relationships to drying conditions. Journal of Functional Foods, 3(2), 115–124. Retrieved from http://www.sciencedirect.com/science/article/pii/S1756464611000314 Berdichevsky, A., Viswanathan, M., Horvitz, H. R., & Guarente, L. (2006). C. elegans SIR-2.1 Interacts with 14-3-3 Proteins to Activate DAF-16 and Extend Life Span. Cell, 125(6), 1165–1177. Retrieved from http://www.sciencedirect.com/science/article/pii/S0092867406006180 Braungart, E., Gerlach, M., Riederer, P., Baumeister, R., & Hoener, M. C. (2004). Caenorhabditis elegans MPP + Model of Parkinson ’ s Disease for High-Throughput Drug Screenings. Neurodegenerative Diseases, 175–183. Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics, 77(1), 71–94. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1213120&tool=pmcentrez&rendertype=abstract Calabrese, V., Cornelius, C., Leso, V., Trovato-Salinaro, a, Ventimiglia, B., Cavallaro, M., … Castellino, P. (2012). Oxidative stress, glutathione status, sirtuin and cellular stress response in type 2 diabetes. Biochimica et Biophysica Acta, 1822(5), 729–36. doi:10.1016/j.bbadis.2011.12.003 Chakraborty, S., Bornhorst, J., Nguyen, T. T., & Aschner, M. (2013). Oxidative Stress Mechanisms Underlying Parkinson’s Disease-Associated Neurodegeneration in C. elegans. International Journal of Molecular Sciences, 14(11), 23103–28. doi:10.3390/ijms141123103 Ching, T.-T., & Hsu, A.-L. (2011). Solid plate-based dietary restriction in Caenorhabditis elegans. Journal of Visualized Experiments : JoVE, (51), e2701. doi:10.3791/2701 Cholerton, B., Baker, L. D., & Craft, S. (2011). Insulin resistance and pathological brain ageing. Diabetic Medicine : A Journal of the British Diabetic Association, 28(12), 1463–75. doi:10.1111/j.1464-5491.2011.03464.x Chuang, M.-H., Chiou, S.-H., Huang, C.-H., Yang, W.-B., & Wong, C.-H. (2009). The lifespan-promoting effect of acetic acid and Reishi polysaccharide. Bioorganic & Medicinal Chemistry, 17(22), 7831–7840. Retrieved from http://www.sciencedirect.com/science/article/pii/S096808960900844X Cohen, E., & Dillin, A. (2008). The insulin paradox: aging, proteotoxicity and neurodegeneration. Nature Reviews. Neuroscience, 9(10), 759–67. doi:10.1038/nrn2474 Cole, G. M., & Frautschy, S. a. (2007). The role of insulin and neurotrophic factor signaling in brain aging and Alzheimer’s Disease. Experimental Gerontology, 42(1-2), 10–21. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/17049785 Correia, R. T. P., Borges, K. C., Medeiros, M. F., & Genovese, M. I. (2012). Bioactive compounds and phenolic-linked functionality of powdered tropical fruit residues. Food Science and Technology International = Ciencia Y Tecnología de Los Alimentos Internacional, 18(6), 539–47. doi:10.1177/1082013211433077 Do, T., Kerr, B., & Kuzhikandathil, E. V. (2007). Brain-derived neurotrophic factor regulates the expression of D1 dopamine receptors. Journal of Neurochemistry, 100(2), 416–28. doi:10.1111/j.1471-4159.2006.04249.x Donmez, G. (2012). The neurobiology of sirtuins and their role in neurodegeneration. Trends in Pharmacological Sciences, 33(9), 494–501. doi:10.1016/j.tips.2012.05.007 Dostal, V., Roberts, C. M., & Link, C. D. (2010). Genetic mechanisms of coffee extract protection in a Caenorhabditis elegans model of βamyloid peptide toxicity. Genetics, 186(3), 857–866. doi:10.1534/genetics.110.120436 Fujita, A., Borges, K., Correia, R., Franco, B. D. G. de M., & Genovese, M. I. (2013). Impact of spouted bed drying on bioactive compounds, antimicrobial and antioxidant activities of commercial frozen pulp of camu-camu (Myrciaria dubia Mc. Vaugh). Food Research International, 54(1), 495–500. Retrieved from http://www.sciencedirect.com/science/article/pii/S0963996913003918 Gan, L., & Mucke, L. (2008). Paths of convergence: sirtuins in aging and neurodegeneration. Neuron, 58(1), 10–4. doi:10.1016/j.neuron.2008.03.015 Herskovits, A. Z., & Guarente, L. (2013). Sirtuin deacetylases in neurodegenerative diseases of aging. Cell Research, 23(6), 746–58. doi:10.1038/cr.2013.70 Huerta, V., Mihalik, K., Becket, K., Maitin, V., & Vattem, D. (2010). Anti-diabetic and Anti-energy Harvesting Properties of Common Traditional Herbs, Spices and Medicinal Plants from India. Journal of Natural Remedies, 10(2), 123–135. Retrieved from http://www.jnronline.com/index.php/jnr/article/view/28939 Huerta, V., Mihalik, K., Maitin, V., Crixell, S. H., & Vattem, D. A. (2007). Effect of Central/South American medicinal plants on energy harvesting ability of the mammalian GI tract. J. Med. Plants Res, 1(2), 38–49. Iser, W. B., & Wolkow, C. a. (2007). DAF-2/insulin-like signaling in C. elegans modifies effects of dietary restriction and nutrient stress on aging, stress and growth. PloS One, 2(11), e1240. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2080776&tool=pmcentrez&rendertype=abstract Landis, J. N., & Murphy, C. T. (2010). Integration of diverse inputs in the regulation of Caenorhabditis elegans DAF-16/FOXO. Developmental Dynamics : An Official Publication of the American Association of Anatomists, 239(5), 1405–12. doi:10.1002/dvdy.22244
Bezerra et al.
Li, G.-Y., Li, T., Fan, B., Zheng, Y.-C., & Ma, T.-H. (2012). The D1 dopamine receptor agonist, SKF83959, attenuates hydrogen peroxideinduced injury in RGC-5 cells involving the extracellular signal-regulated kinase/p38 pathways. Molecular Vision, 18, 2882–2895. Retrieved from http://www.molvis.org/molvis/v18/a294/ Lionaki, E., Markaki, M., & Tavernarakis, N. (2013). Autophagy and ageing: Insights from invertebrate model organisms. Ageing Research Reviews, 12(1), 413–28. doi:10.1016/j.arr.2012.05.001 Nishi, A., Kuroiwa, M., & Shuto, T. (2011). Mechanisms for the modulation of dopamine d(1) receptor signaling in striatal neurons. Frontiers in Neuroanatomy, 5(July), 43. doi:10.3389/fnana.2011.00043 Radak, Z., Zhao, Z., Goto, S., & Koltai, E. (2011). Age-associated neurodegeneration and oxidative damage to lipids, proteins and DNA. Mol Aspects Med, 32(4-6), 305–315. doi:10.1016/j.mam.2011.10.010 Sant’Ana, A. de S., Baliga, M. S., Bhat, H. P., Baliga, B. R. V., Wilson, R., & Palatty, P. L. (2011). Phytochemistry, traditional uses and pharmacology of Eugenia jambolana Lam. (black plum): A review. Food Research International, 44(7), 1776–1789. Retrieved from http://www.sciencedirect.com/science/article/pii/S0963996911000949 Shukla, V., Mishra, S. K., & Pant, H. C. (2011). Oxidative stress in neurodegeneration. Advances in Pharmacological Sciences, 2011, 572634. doi:10.1155/2011/572634 Srivastava, S., & Chandra, D. (2013). Pharmacological potentials of Syzygium cumini: a review. Journal of the Science of Food and Agriculture, 93(9), 2084–93. doi:10.1002/jsfa.6111 Sutphin, G. L., & Kaeberlein, M. (2009). Measuring Caenorhabditis elegans life span on solid media. Journal of Visualized Experiments : JoVE, (27), 1–7. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2794294&tool=pmcentrez&rendertype=abstract Vayndorf, E. M., Lee, S. S., & Liu, R. H. (2013). Whole apple extracts increase lifespan, healthspan and resistance to stress in Caenorhabditis elegans. Journal of Functional Foods, 5(3), 1236–1243. doi:10.1016/j.jff.2013.04.006 Wang, M., Bohmann, D., & Jasper, H. (2005). JNK extends life span and limits growth by antagonizing cellular and organism-wide responses to insulin signaling. Cell, 121(1), 115–125. doi:10.1016/j.cell.2005.02.030 Wang, Y., Oh, S. W., Deplancke, B., Luo, J., Walhout, A. J. M., & Tissenbaum, H. A. (2006). C. elegans 14-3-3 proteins regulate life span and interact with SIR-2.1 and DAF-16/FOXO. Mechanisms of Ageing and Development, 127(9), 741–747. Retrieved from http://www.sciencedirect.com/science/article/pii/S0047637406001448 Wen, H., Gao, X., & Qin, J. (2013). Probing the anti-aging role of polydatin in Caenorhabditis elegans on a chip. Integrative Biology : Quantitative Biosciences from Nano to Macro, 6(1), 35–43. doi:10.1039/c3ib40191j Wilson, M., Zou, S., Mattson, M. P., Pallauf, K., & Rimbach, G. (2013). Autophagy, polyphenols and healthy ageing. Ageing Research Reviews, 12(1), 237–252. Retrieved from http://www.sciencedirect.com/science/article/pii/S1568163712000529 Wu, Y., Wu, Z., Butko, P., Christen, Y., Lambert, M. P., Klein, W. L., … Luo, Y. (2006). Amyloid-beta-induced pathological behaviors are suppressed by Ginkgo biloba extract EGb 761 and ginkgolides in transgenic Caenorhabditis elegans. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 26(50), 13102–13113. doi:10.1523/JNEUROSCI.3448-06.2006