NT-020 treatment reduces inflammation and augments Nrf-2 and Wnt signaling in aged rats

We measured the expression of 84 cytokine and chemokine genes in the hippocampus of young and old (3–5 and 22–25, respectively) rats using the RT-PCR cytokine array from Qiagen PARN-150Z. The aged samples exhibited increased expression of inflammatory genes TNF and IL-1β (Table 1). IL24 is a modulator of immune response associated with the anti-inflammatory alternative activations state of microglia. It was decreased threefold in our data set (Table 1) [27]. These results support previous studies that reported high levels of cytokine and chemokine expression in the serum of aged animals. These results provide a picture of the differential expression of cytokines and chemokines in the aged hippocampus.

In a previous study of NT-020, aged rats fed an NT-020 diet for 3 weeks had significantly lower numbers of OX-6 (MHC II) positive microglia [28]. Present results confirm that this decreased number of activated microglia is paralleled by a decrease in cytokine and chemokine expression (Table 2).We again analyzed the expression of 84 cytokines and chemokines in the hippocampus of aged rats fed either a control NIH diet or an NT-020 diet. IL24, IL4, and IL10 are cytokines with anti-inflammatory or immune-modulating effects whose expression was increased in the NT-020 diet group (Table 2). Further, expression of CX3CL1 (fractalkine), LIF, and GDNF was also observed (Tables 2 and 4). Expression of these markers is indicative of microglia in an M2 or alternative activation state which supports tissue repair and stem cell proliferation [27, 29]. Expression of the pro-inflammatory cytokine genes IL-1α and IL18 (Table 2) were decreased as was expression of chemokines CCL19 and CCL2 known to be involved in the trafficking of T cells to the CNS [30]. We also performed an ELISA assay to measure levels of both TNF-α and IL-1β. Protein levels were significantly increased in the old hippocampus (Fig. 1). NT-020 treatment decreased protein levels of both TNF-α and IL-1β in 22-month-old rats (Fig. 1). These results suggest that dietary supplementation with NT-020 increases expression of immune-modulatory molecules within the hippocampus. Interestingly as in a previous study [28] with NT-020, this was also accompanied by an improvement in performance on a spatial learning task, in this case, the radial arm water maze in the aged rats (Additional file 1: Figure S1), but not in young rats (Additional file 2: Figure S2).

In an attempt to identify possible regulators upstream of our target molecules, we performed upstream and downstream analysis in the bioinformatics program Ingenuity Pathway Analysis (Fig. 2). The upstream analysis predicts that inhibition of RELA, the p65 subunit of NF-κB, could explain the treatment effect which is consistent with previous reports that EGCG was capable of inhibiting NF-κB in T cells in a model of MS [31]. In addition, there is a predicted increase in the activity of corticosterone (CORT) (Fig. 2b). Low levels and short durations of corticosteroids are known to have anti-inflammatory effects; however, here, it is unlikely that supplementation triggered release of these compounds. Instead, we propose that in the primed state of the aged hippocampus, there may be a blunted response to corticosterone signaling which is restored after supplementation. A recent study examined the potential role of excess glucocorticoids leading to brain aging and AD; a novel finding was observed when comparing the glucocorticoid transcriptome with the aging transcriptome. Although the prevailing hypothesis relates to overactivation of corticosteroids with age, these authors observed that many genes including the inflammatory genes that are upregulated with age are downregulated by cortisol treatments suggesting a downregulation of glucocorticoid responses with age in some cells that could be responsible for the increased inflammatory response with age [32]. Further studies are needed to determine if these upstream regulators are altered in the aged rats with NT-020 treatment. Downstream analysis of bio-functions gives an interpretation of the gene expression data. This analysis is consistent with a decrease in the synthesis of nitric oxide, a major source of oxidative stress in the CNS, the influx of granulocytes, and the TH1 immune response (Fig. 2c). Notably, the genes at the center of these predictions include the anti-inflammatory genes IL4 and IL10. Taking a systematic view of the data has demonstrated that the anti-inflammatory effects of dietary supplementation are broad and involve modulating the activity of multiple cell types in the niche.

Polyphenolic compounds can activate adaptive stress response pathways in the cell, most notably Nrf2-ARE [9]. Because activation of Nrf2 is capable of attenuating the inflammatory response, we investigated NT-020’s ability to drive Nrf2 activation in vivo [26]. Immunohistochemistry was used to examine cellular localization of Nrf-2 and one of the enzymes regulated by Nrf-2, heme oxygenase 1 in the dentate gyrus of the hippocampus. Doublecortin (DCX), IBA-1, NeuN, and GFAP were used to label immature neurons, microglia, neurons, and astrocytes, respectively. Using confocal microscopy, we randomly counted 100 cells per animal and then determined if expression of Nrf-2 was observed in the nucleus (Fig. 3). The percent of cells with nuclear co-localization of Nrf-2 and HO-1 was significantly increased in all four cell types in the rats that received the NT-020 diet (Fig. 4). This increase occurred with treatment in both young and old rats suggesting that these effects are independent of basal inflammation status. This supports that at least some of the polyphenolic activity in vivo comes from an activation of antioxidant response element pathways across most cell types in the CNS. Interestingly, the role of Nrf-2 in microglial priming has become delineated in many studies including a recent study suggesting that Nrf2 is important for phagocytosis [33]. In addition, Nrf-2 is a regulator of CX3CL1 actions, one of the chemokines upregulated by NT-020 treatment reported in Table 2 [34]. Similar results on Nrf-2 and HO-1 were observed in the subventricular zone (SVZ), the other neurogenic-rich niche in the CNS (Additional file 3: Figure S3).

Wnt ligand binding to its receptor triggers an accumulation of intra-cellular β-catenin that correlates with overall Wnt activity [35]. To gain better insight into the activity level of the Wnt pathway, we stained hippocampus sections as above for Nrf2 for nuclear co-localization with β-catenin. The aged group had reduced percent of DCX positive cells with nuclear co-labeled β-catenin although it only approached significance (Fig. 4). However, compared to control, rats on the NT-020 diet have a higher percent of nuclear co-localization of β-catenin in all cell types. This supports the hypothesis that the NT-020 diet increases overall activity of the Wnt pathway in aged animals. Similar results are observed in the SVZ (Additional file 3: Figure S3). To examine this further, we analyzed gene expression of Wnt target genes.

Analysis of Wnt signaling revealed significant changes in genes responsible for regulating both the cell cycle and cell fate decisions (Table 2). The causes of age-related decline in neurogenesis in the hippocampus are still not fully clear, but several studies examining Wnt ligands suggest that the decreased signaling particularly the decrease in the Wnt3a ligand is at least partly responsible for the decline [17]. Wnt target genes associated with cell cycles Cyclin D1, C-Myc, and AHR were decreased by 1.6-, 2.1-, and 2.12-fold, respectively (Table 3). Growth factors GDNF and IGF2 expression were reduced by 2.7- and 3.6-fold, respectively. Extracellular antagonists block ligand access to the FZD receptors preventing signal transduction. We observed increases in Wnt signaling antagonists SFRP2 and TLE1 which were increased by 1.51- and 1.58-fold, respectively (Table 3). Also, DKK1 was also increased but only approached significance. In addition to extracellular antagonists, several co-repressors exist in the nucleus to antagonize TCF/LEF signaling. TLE1 or Groucho is one such repressor that binds TCF and prevents its transcriptional activity in the absence of β-catenin. These results suggest that antagonism of Wnt signaling is a significant factor affecting Wnt signal transduction in the aged brain. Taken together, these alterations paint the picture of a cell environment with a decrease in extracellular growth and survival signals which correlates with the observed effects of aging on neurogenesis.

Gene expression from NT-020-treated rats revealed a complex pattern of gene regulation that may represent a novel mechanism by which these compounds affect neurogenesis (Table 4). Growth factors GDNF and FGF4 levels were increased, both of which support proliferation and survival of young neural progenitors. An increase in NANOG supports the notion that more pluripotent cells are present in treated animals. Paradoxically, expression of the Wnt3a gene was reduced in the supplemented group while nuclear co-localization of β-catenin was increased. One possible explanation is that restoration of Wnt signal transduction by reduction of antagonists SFRP2, TLE1, and WISP1 triggered a negative feedback mechanism. Expression of transcription factor DLK1, a transmembrane member of the Notch family, was increased after supplementation. Studies on DLK1 indicate that it promotes neurogenesis through its modulation of Notch and BMP pathways [36]. Overall, the gene expression changes are consistent with increased WNT downstream signaling. We postulate that this is a result of reduced expression of negative regulators. These results put forward several possible mechanisms by which NT-020 rescues neurogenesis in the aged niche, and further analysis is necessary to understand what role these changes play in attenuation of the aged phenotype.