The Isolated and Combined Effects of Folic Acid and Synthetic Bioactive Compounds against Aβ(25-35)-Induced Toxicity in Human Microglial Cells

Folic acid plays an important role in neuronal development. A series of newly synthesized bioactive compounds (NSCs) was reported to exhibit immunoactive and neuroprotective functions. The isolated and combined effects of folic acid and NSCs against β-amyloid (Aβ)-induced cytotoxicity are poorly understood. These effects were tested using human microglia cells (C13NJ) subjected to Aβ(25-35) challenge. According to an MTT assay, treatment of C13NJ cells with Aβ(25-35) at 10~100 μM for 48 h induced 18%~43% cellular death in a dose-dependent manner (p < 0.05). Aβ(25-35) treatment at 25 μM induced nitrite oxide (NO) release, elevated superoxide production, and reduced the distribution of cells in the S phase. Preincubation of C13NJ with 100 μM folic acid protected against Aβ(25-35)-induced cell death, which coincided with a reduction in NO release by folic acid supplements. NSC47 at a level of 50 μM protected against Aβ(25-35)-induced cell death and reduced Aβ-promoted superoxide production (p < 0.05). Folic acid in combination with NSC47 at their cytoprotective doses did not synergistically ameliorate Aβ(25-35)-associated NO release, superoxide production, or cell cycle arrest. Taken together, folic acid or NSC treatment alone, but not the combined regimen, protected against Aβ(25-35)-induced cell death, which may partially, if not completely, be mediated by free radical-scavenging effects.


Introduction
Although the etiology of Alzheimer's disease (AD) is not clearly understood, the neurodegenerative process is thought to involve extracellular plaques of β-amyloid (Aβ) and neurofibrillar tangles of tau proteins which accumulate in brain tissues [1,2]. Elevated oxidative stress is associated with the deposition of Aβ peptide, which induces neurotoxicity [3]. Various neuronal populations are differentially susceptible to oxidative damage associated with Aβ peptide challenge. Brain microglial cells, the principal immune cells in the central nervous system (CNS) which protect against microorganism invasion, are vulnerable to Aβ-induced toxicity [4]. At an early stage of Aβ-protein deposition, microglial cells are activated to release toxic immunocytokines such as nitrite oxide (NO) and products of superoxide which exert neurotoxicity [5,6]. Microglial cultures were found to clear up AD's Aβ peptides [7]. The Aβ-induced oxidative immunotoxicity by glial activation and/or by promoting cell death was proposed to be involved in the neurodegenerative process.
Folate serves as both a carbon donor and acceptor in the de novo synthesis of thymidylate and amino acid interconversion, which are critical for cell proliferation and neuronal development [8].
Deprivation of folate in human neuroblastoma cells induces elevated oxidative stress, alters cytosolic calcium, and induces apoptotic death [9]. Cultivation of hippocampal cells in folate-deficient medium promotes cell death [10]. Folate supplementation, on the other hand, is associated with improving memory deficits among cognitively impaired subjects. Higher folate intake is correlated with lower risks of AD [11,12]. Folic acid (pteroylmonoglutamic acid) is the molecular form used to fortify foods and dietary supplements. It was proposed that folic acid supplements can scavenge peroxyl radicals, azide radicals, and hydroxyl radicals in an in vitro radical reaction model system [13]. Growing evidence suggests a potential role of folic acid in in vivo and in vitro antioxidant actions [14,15]. Those studies raised the possibility that folic acid supplementation might be effective in protecting against Aβ-induced toxicity through its antioxidant action, although a direct link has not been established.
A series of chemical compounds (NSCs) with a piperidine structure was newly synthesized according to the molecular characteristics of numerous bioactive compounds [16,17]. The bioactive functions of NSCs were first demonstrated by Chou et al.; NSCs enhanced cellular proliferation of peripheral blood mononuclear cells and elevated interleukin (IL)-2 and interferon (IFN)-γ production [18]. Another study showed that treatment of synapse neurons with NSCs ameliorated glutamate release [19]. Among NSCs, NSC42 has the skeleton of an indolizidine and NSC43 has the skeleton of a quinolizidine. It is well known that indolizidines and quinolizidines are important frameworks of many natural products, and have many interesting biological activities [20]. NSC47 belongs to a lesscommon framework of pyridoazepines, but some pyridoazepines were found to have very strong insecticidal activities [21], and were studied as potential dopamine D1 receptors [22] and nicotinic acetylcholinergic receptor ligands [23]. Synthesis methods for structures like NSC42, -43, and -47 were previously reported by our lab [24,25]. Although a few functional properties of NSC42, -43, and -47 were reported, it remains unclear if such bioactive NSCs possess any antioxidant activity to protect against Aβ-induced cytotoxicity. In light of accumulating evidence of the plausible roles of folic acid supplementation and NSCs in neuronal protection, we hypothesized that folic acid and NSCs, alone or in a combined regimen, can protect microglial cells against Aβ-induced oxidative cytotoxicity. A human microglial cell line (C13NJ) was used as the experimental model to test this hypothesis. The 11-amino acid fragment of the Aβ peptide, Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) , located in the hydrophobic domain at the Cterminal end of Aβ  , was shown to mimic some of the pathological processes in the AD brain [26]. Markers of cell death, elevated oxidative stress including NO release and superoxide synthesis, and cell cycle arrest were measured to explore the working mechanisms. (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)

Effects of folic acid or/and NSC47 on NO production of Aβ (25-35) -treated C13NJ cells
To understand if the protective effects of folic acid and NSC47 against Aβ-induced cytotoxicity are associated with oxidative modification, cellular release of nitric oxide (NO) with the various treatments was measured ( Figure 3). When treated with Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) for 48 h, C13JN cells released a significantly higher level of NO than the controls. Folic acid supplementation at a cytoprotective level (100 μM) significantly reduced NO production of Aβ-treated C13NJ cells (p < 0.05). NSC47 at a cytoprotective level (50 μM) reduced NO release of Aβ-treated cells, yet it did not reach statistical significance. When FA and NSC47 were combined at a cytoprotective level, synergistic effects of the combined regimen against NO release of Aβ-treated C13NJ cells were not observed.

Effects of folic acid or/and NSC47 on superoxide production of Aβ (25-35) -treated C13NJ cells
Oxidative stress was assessed by measuring differential levels of superoxide production among the experimental groups ( Figure 4). Previous studies revealed that preincubation of cells with folate supplements for 24~72 h or with NSC47 for 3~24 h produced similar protective effects against Aβinduced cytotoxicity and NO release (data not shown). A time period of 24 h was selected to study the protective mechanisms of the two molecules against Aβ-induced superoxide production. Preincubation of Aβ-treated C13NJ cells with folic acid at a cytoprotective level did not affect superoxide production by Aβ-treated cells. NSC47 at a cytoprotective level significantly reduced superoxide levels of Aβtreated cells. Combining folic acid with NSC47 did not significantly diminish superoxide levels of Aβtreated C13NJ cells.  C13NJ cells were preincubated with FA, NSC47, and their combination at cytoprotective doses for 24 h followed by Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) treatment for 48 h. Superoxide production was analyzed as described in the Experimental section. Data are expressed as a percentage of the control. Values are presented as the mean ± SD, n = 3 independent experiments. * p < 0.05, the Aβ-treated (+Aβ) group compared to the control. # p < 0.05, the FA-NSCs-, and combined-treated groups compared to the Aβ-treated control (+Aβ) group. (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)

-treated C13NJ cells
As oxidative stress may signal modulation of the cell cycle of dividing cells, we further investigated the effect of folic acid or/and NSC47 on the cell cycle of Aβ-treated C13NJ cells. Figure 5 shows that Aβ challenge significantly reduced the percentages of cells in the S phase (the phase of DNA synthesis), suggesting that Aβ-induced cytotoxicity of C13NJ cells may be partially mediated by affecting DNA synthesis. Preincubation of Aβ-induced C13NJ cells with NSC47, but not with folic acid or the combined regimen, restored the proportions of S-phase cells back to the value of control cells. C13NJ cells were preincubated with folic acid supplements, NSC47, and their combination at a cytoprotective dose for 24 h followed by Aβ (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) treatment for 48 h. Cell cycle distributions were analyzed as described in the Experimental section. Values are the mean ± SD, n = 3 independent experiments. * p < 0.05, compared to the control.
We found that folic acid supplementation was effective in protecting human microglial cells against Aβ-induced cell death and NO release. Mechanisms through which folate might impact suppressed production and/or increased degradation of NO are not known. NO is synthesized by a specific enzyme, NO synthase (NOS), the activity of which increases in cerebral microvessels during AD development [30]. It was reported that folic acid treatment of SOD1 G93A transgenic mice suppressed activation of microglia and inhibited the expression of inducible (i)NOS in the spinal cord [31]. Alternatively, increased degradation of NO by superoxide, rather than impaired formation of NO, is predominantly observed in early atherosclerosis [32,33]. Given the fact that folic acid supplementation did not affect superoxide production of microglial cells upon Aβ challenge, the NO-superoxide interaction cycle might not contribute to the effects of folic acid in NO reduction. Determining whether the protective effect of folic acid against NO release of Aβ-treated microglia is linked to altered expression of iNOS requires further studies.
Preincubation of microglial cells with NSC47 protected against Aβ-induced cell death, superoxide generation, and disruption of cell cycle progress. The data suggested that NSC47 may act as an antioxidant to scavenge superoxides. The mechanisms by which NSC47 scavenges superoxide of Aβtreated microglial cells are not clear. Intracellular superoxide levels can be elevated by several enzyme systems, including xanthine oxidase, uncoupled NOS, complexes I and III of the mitochondrial electron transport system, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. NADPH oxidases are a family of protein complexes believed to be responsible for the localized and limited production of superoxide radicals [34,35]. Alternatively, Aβ-associated elevated superoxide generation may result from impaired antioxidant defense and/or superoxide generation elicited by mitochondrial dysfunction [36]. Mitochondrial dysfunction including impaired respiration and depolarization of the mitochondrial membrane potential may induce a vicious cycle of superoxide production and release of apoptotic signals which promote cellular death [37]. Determining whether NSC47 can block the activity of NADPH oxidase and/or modulate mitochondrial function to scavenge Aβ-induced superoxide warrants further studies.
We found that folic acid in combination with NSC47 at their individual cytoprotective doses was unable to act synergistically to scavenge NO or superoxide levels of Aβ-treated microglial cells. The reasons are unclear at the present time. We speculated that folic acid and NSC47, when added together to the culture medium, may interact or compete with each other for cellular uptake. In that case, the combined molecules might not achieve their individual dose effectiveness inside cells to scavenge free radicals. The fact that folic acid and NSC47 have similar bicyclic ring structures [21,24,25] suggests that interactions between these two bioactive molecules for cellular uptake may be possible. On the other hand, the current cytoprotective dose of folic acid supplement (100 μM) was not effective in intracellular superoxide scavenging, which may provide an alternative explanation of why the combined molecules did not have a synergistic effect on superoxide levels of Aβ-treated microglial cells. It was reported that suppression of superoxide production was folic acid-dependent with the most effective dose at the high level of 1,500 μM in a macrophage reference model [38]. This concentration is 15 times greater than the concentration used in the present study. A high dose of folic acid supplementation was required to resume mitochondrial respiratory function in Cu 2+ -stimulated primary hepatocytes [39]. Further studies are warranted to investigate the effective dose of folic acid alone or in combination with NSC47 to completely scavenge Aβ-induced radicals in microglial cells. (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)

NO release assay
C13NJ cells (5 × 10 5 cells) were cultured with FA or/and NSC at designated concentrations and time periods, then treated with 25 μM Aβ 25-35 for 48 h. Cultured medium at 80 μL was mixed with 200 μL/well Griess reagent (1% sulfanilamide and 0.1% N-1-naphthylethylene diamide dihydrochloride and 2.5% H 3 PO 4 ) for 5 min. Nitrite concentrations were determined at 540 nm on an ELISA reader, and then calculated with a standard curve of sodium nitrite.

Intracellular reactive oxygen species (ROS) levels
Intracellular ROS, particular superoxide production, were assayed using the fluorescent dye, hydroethidine (HE) [40]. HE oxidation is particularly sensitive to superoxide anions. Thirty minutes before Aβ treatment was terminated, HE (5 mmol/L) was added to cultured cells. At each indicated time point, cells were harvested, and the fluorescence intensity of intracellular hydroethidine (with emission at 585 nm) was monitored on a Coulter EPICS XL-MCL flow cytometer (Miami, FL, USA).

Cell cycle analysis
After 48 h of Aβ treatment, control cells and cells in each group were harvested, fixed in ice-cold 100% ethanol, and treated with ribonuclease A (500 mg/L) and 0.5% Triton at 37 °C for 60 min. Cells were then stained with propidium iodide (PI; 50 mg/L for DNA staining) for 20 min. After centrifugation, the pellet was resuspended in PBS with the same concentration of PI for 10 min. Cellular DNA in 10,000 cells was analyzed in a Coulter EPICS XL-MCL flow cytometer. The percentage of cells in each phase of the cell cycle was determined by the WinMDI 2.8 program.

Statistical analysis
All data are presented as the mean ± SD. One-way analysis of variance (ANOVA) and Duncan's test (SPSS software) or Student's t-test were used for comparisons among groups. A significant difference was indicated when the p value was < 0.05.