Fig 1.
Differentiation of SH-SY5Y cells using laminin and IGF-1.
SH-SY5Y cells were plated on laminin and grown for 24 hours in DMEM/F12 supplement and 10% FBS. To induce differentiation, FBS was removed and 50 nm IGF-1 was added; cells were allowed to grow for 72 hours. (A) Differential interference contrast (DIC) image of the undifferentiated controls. Red arrows marked the neurites in undifferentiated cells that were characteristic for neuroblast-like cells. (B) DIC image of the differentiated cells. The increase in neurite length upon differentiation was marked out by the green arrow heads. (C and D) To quantify the change in the length of the neurites, two days into the differentiation protocol, cells were transfected with GFP cDNA and imaged on day 3 using fluorescence microscopy. Transfecting with GFP highlighted the neurites among the confluent cell layers, allowing for easy quantification. (C) An overview of the undifferentiated controls. Red arrows marked out the neurite of each GFP transfected cells. (D) Differentiated cells displayed long neurites as outlined by green arrow heads. (E) The increase in the length of the neurites upon differentiation was statistically significant (unpaired t-test, **** indicates P-val < 0.0001).
Table 1.
Number of mRNAs altered by differentiation and Bacopa treatment.
Fig 2.
Results of the gene ontology analysis by Partek Genomics Suite.
The bar graphs indicate the percentage of transcripts that belong to Biological Processes (blue), Cellular Components (red) and Molecular Functions (green) that were most affected by Bacopa treatment.
Table 2.
Summary of neuronal genes whose mRNAs were altered by Bacopa treatment of SH-SY5Y cells.
Table 3.
Genes regulated by Bacopa identified by gene-level analysis of the RNA-Seq data.
Fig 3.
Absolute values of Fold Change (absFC) caused by Bacopa treatment.
RT-PCR was performed on undifferentiated cells and differentiated cells which were treated with vehicle (DMSO) or with Bacopa for either 4 h (blue) or 24 h (orange). The gray area indicated an absFC value smaller than 1. Genes marked with * were results from the treatment on undifferentiated cells. (1) NPTN_A and (2) NPTN_A were results generated from 2 sets of primers priming for NPTN transcript A.
Table 4.
Results from GSEA (Pre-ranked).
Fig 4.
Enrichment Plot, MA Plot and enriched gene list for the ‘oxidative stress response’ pathway.
(A) Enrichment Plot for the designated pathway. The graph represented the incremental change in the enrichment score for this pathway, when queried along the ranked list of genes during gene-set enrichment analysis (GSEA). Maximal enrichment score was observed at 0.45. The relative ranks of the genes belonging to this pathway were indicated by the bars under the graph. Lines clustered to the left (marked in red) demonstrated strong enrichment of 39 highly ranked genes for this pathway that were upregulated by Bacopa. (B) The 39 genes (leading up to the maximal enrichment score) that contributed positively to the core enrichment of the ‘oxidative stress response’ pathway in GSEA. (C) Mean-Average (MA) plot analysis of the Oxidative Stress Response pathway. The MA plot compares the distribution of differential gene expression as a function of the magnitude of expression signals. The Y-axis plotted the log ratio of treatment (Bacopa) vs. Control (DMSO) and X-axis recorded the log of the average FPKM score between the two groups. The distribution of log ratios for all genes queried by RNA sequencing were shown in light gray circles. Genes belonging to the Oxidative Stress Response pathway (238 genes) were shown in orange and red circles. The 39 genes contributing to significant enrichment of this pathway (by GSEA) were shown in red whereas the remaining pathway genes were shown in orange.
Table 5.
Ingenuity Pathway Analysis: Over-represented biological functions.
Table 6.
Ingenuity Pathway Analysis: Upstream regulators.
Fig 5.
H2O2 toxicity and protection by Bacopa.
(A) Using a high-content screening (HCS) microscopy platform (MetaXpress, Molecular Devices) we measured the number of live SH-SY5Y cells after a 24-hour treatment with H2O2 at the concentrations indicated. The number of live cells per field of view was normalized to the control value (B) H2O2 survival curve fitted with the logistics equation: The IC50 (concentration that kills 50% of the cells) was 105 μM. (C) Neuroprotection assay for Bacopa. The bar graphs represented live cell counts per field of view (means ± SEM) for (i) control cultures (Ctrl), (ii) cells treated with 100 μM peroxide for 24 hours, and cells treated with the same amount of H2O2 supplemented with a (iii) high and (iv) low concentration of Bacopa (Bac). The high concentration Bacopa showed significant protection, while the low concentration failed to protect against peroxide toxicity. * P-val < 0.05, *** P-val < 0.001, one-way ANOVA, Dunnett post-test.
Table 7.
Summary of SLC-mediated transport functions up-regulated by Bacopa.
Fig 6.
Pathways identified by IPA connects Bacopa to memory, neuroprotection and AD.
The light-blue box summarizes the effect of Bacopa on the three transcription factors identified by the Ingenuity Pathway Analysis (IPA). Bacopa activates ATF4 and NRF2, while it inhibits the function of FOXO3. The color of boxes and arrows indicates the effects of Bacopa: green indicates increase/activate, orange indicates decrease/inhibit. Blue boxed are three biological endpoints: learning & memory, neuroprotection, and Alzheimer’s disease (AD). Explanation of the numbered connections: (1) ATF4 and NRF2 functionally interact with each other [40–43]. (2) ATF4 is implicated in memory [59–62]. (3) NRF is implicated in memory [63–67]. (4) Translation initiation factor eIF2-alpha stimulates translation of ATF4 [68–71]. (5) eIF2-alpha is implicated in memory formation [72, 73]. (6) ATF4 has been implicated in Alzheimer’s disease (AD) [74–78]. (7) NRF2 has also been linked to AD [63, 65, 67, 79]. (8) NRF2 plays a critical role in neuroprotection [80–83]. (9) FOXO3 mediates oxidative stress-induced neuronal cell death [84–87]. Inhibition of FOXO3 by Bacopa could explain its neuroprotective effect.