Fig 1.
Phenserine mitigates oxidative stress and glutamate excitotoxicity in cultured neuronal cells.
(A) Human SH-SY5Y cells and rat primary cortical neurons (B and C) were treated with and without phenserine and challenged with either oxidative stress (H2O2) or glutamate excitotoxicity. Cell viability was quantified by MTS (A and C) or LDH (B) assay at 24 h. * and # designate comparisons with control cells and either H2O2 or glutamate challenged cells, respectively (*p<0.05, **p<0.01, ***p<0.001 (comparison with Control cells); #p<0.05, ##p<0.01, (comparison with challenged cells), N≥4 per group.
Fig 2.
Phenserine mitigates mTBI-induced impairments in visual and spatial memory.
(A) mTBI mice demonstrate a deficit in visual memory compared with control (**p<0.01). Phenserine administration, at both doses utilized in this study significantly ameliorated behavioral deficits (##p<0.01). One-way ANOVA revealed a significant effect between groups [F(5,98) = 7.770, p = 0.000]. Fisher’s LSD post hoc analysis revealed that the preference index of the “mTBI” group was significantly lower than all other groups (**p<0.01). (B) mTBI mice demonstrate a significant deficit in spatial memory compared with control (**p<0.01). Phenserine administration significantly ameliorated this damage (##p<0.01 for 2.5mg/kg, and #p<0.05 for 5mg/kg). One-way ANOVA revealed a significant effect between groups [F(5,105) = 6.190, p = 0.000]. Fisher’s LSD post hoc analysis revealed that the preference index of the “mTBI” group was significantly lower than all other groups, other than “Phenserine only” 5 mg/kg (*p<0.05, **p<0.01).
Fig 3.
Phenserine augments endogenous antioxidant homeostatic mechanisms relevant to mTBI.
Quantification within the ipisilateral hippocampus at 5 and 14 days following mTBI or a sham procedure with and without phenserine (PHEN) treatment: (A) levels of thiobarbituric acid reactive substances (TBARS), (B) superoxide dismutase (SOD) activity, (C) glutathione peroxidase (GPx) activity, and (D) SOD1 (E) SOD2 and (F) GPx1 protein levels (each normalized to total protein, as measured by Ponceau S). Values were significantly different from either control (sham) or phenserine in the absence of mTBI: *p<0.05, **p<0.01, ***p<0.001. Values were significantly different between the untreated and phenserine-treated mTBI challenged groups: #p<0.05, ##p<0.01. N = 6 per group for day 5, and N = 4–6 per group for day 14 evaluation.
Fig 4.
Significantly regulated genes, Gene Ontologies and Canonical Pathways observed in hippocampal tissues from mTBI, mTBI/PHEN and PHEN animals are presented.
Red arrows indicate up regulated genes, Green arrows indicate down regulated genes. A: Genes: by 14 days after injury, 50 and 129 genes were commonly up and down regulated, respectively, in the three treatment groups. The largest numbers of genes exclusively regulated were observed in the mTBI+PHEN treated animals, 332 genes and 528 genes were exclusively up and down regulated, respectively. Due to the large numbers of genes the identities and Z-ratios of the genes are provided in S1 Table. B: Gene ontologies: ontologies derived from altered genes in hippocampal tissues indicate that 20 and 22 gene ontologies were commonly up and down regulated, respectively, in the three treatment groups. Several CNS or neuronal gene ontology listings are described in the results section; however, all ontology identities and Z-scores are provided in S2 Table. C: Pathways: molecular pathways derived from altered genes in hippocampal tissues indicate no pathways were commonly regulated in the three treatment groups. Several pathways were CNS- or neuronal-related. These pathways are described in the results section. A list of pathways with corresponding Z-scores is provided in S3 Table.
Fig 5.
CNS—neuronal canonical and non-canonical pathways observed to be regulated by mTBI compared to sham, and the effects of post-injury treatment with phenserine on mTBI-regulated pathways are presented.
R* refers to Reactome. Most pathways were observed to be down regulated by mTBI, with only two that were up regulated. Two pathways were associated with neurodegenerative disease: ‘Blalock Alzheimer’s Disease Up’ and ‘Biocarta Parkin Pathway’. While post-injury treatment of animals with phenserine had no effect on the majority of pathways, one Alzheimer’s disease-related pathway was further regulated by this drug. Drug treatment reversed the effects of mTBI on the Alzheimer’s disease pathway.
Fig 6.
Partial array validation for the genes Fos, Arc and Tmem66 illustrate similarities between the fold changes in the gene transcripts measured by q-RT-PCR and array methods.
The fold change measurements are indicated in the figure. The mTBI-induced changes for gene transcript levels of Fos were significantly elevated compared to sham levels, p = 0.01387. The mTBI-induced changes in transcript levels for Arc and Tmem66 were not significantly different from the sham values (p = 0.1780 and p = 0.1506, respectively); however the fold changes were similar. Values are expressed as mean±SEM of n observations. Sham n = 5 and mTBI n = 5.