# PC12 and THP-1 Cell Lines as Neuronal and Microglia Model in Neurobiological Research
## 3 Results and Discussion
### PC12 Cells and Amyloid \(\beta\) (25-35)  
PC12 cells induced to differentiation and treated with amyloid \(\beta\) (25-35) presented a decrease in mitochondrial metabolic activity in a dose-dependent manner (Figure 2A). Moreover, the differences in MTT assay results between differentiated and undifferentiated ones are statistically significant in the entire studied concentration range--activity was 17.9-35.2% lower in differentiated cells. As observed in numerous previous studies, A\(\beta\) presents a pronounced affinity towards the mitochondrial membrane and neurons are physiologically characterized by the presence of many mitochondria in each cell, especially in branching neurites that can be observed only in differentiated PC12 cells [23]. It is currently believed that amyloid \(\beta\) might be a member of the antimicrobial peptides family. It is assumed that A\(\beta\) possesses the ability to self-aggregate in the presence of bacteria or fungi and bind to their particles leading to the neutralization of such harmful agents. Unfortunately, due to mitochondria's origin (theory of endosymbiosis), these organelle share some similarities in membrane composition and properties with microbes, making them especially prone to amyloid toxicity [8; 9].  
Figure 2: Effect of A\(\beta_{25-35}\) on undifferentiated and differentiated PC12 cells: (**A**) metabolic activity (MTT assay); (**B**) cytotoxicity (LDH assay); (**C**) level of reactive oxygen species (DCF-DA assay); (**D**) double-stranded DNA damage (FHA assay); Control—cell culture incubated without A\(\beta_{25-35}\);* \(p<0.05\)—significant difference compared to control; # \(p<0.05\)—significant difference between undifferentiated and differentiated PC12 cells.  
A concentration dependence was also observed in the LDH assay (Figure 2B). In the concentrations between 0.1 and 5.0 uM, slightly higher cytotoxicity measured as LDH release was noted in the culture of differentiated PC12 cells compared to undifferentiated (by 3.7-16.4% but without the statistical significance of difference).  
Literature data show that amyloid \(\beta\) is pro-oxidative in both in vitro studies on neuron-like cells and in Alzheimer's disease patients. Our study found a pro-oxidative effect of A\(\beta_{25\text{-}35}\) at concentrations of 1 and 5 uM, regardless of whether the cell cultures were differentiated (Figure 2C). In our previous paper, however, we showed that A\(\beta_{25\text{-}35}\) exhibits antioxidant activity at low physiological concentrations. Results of the DCF-DA assay in this study at 0.001 and 0.1 uM A\(\beta_{25\text{-}35}\) concentrations in both cell types and 0.1 uM A\(\beta_{25\text{-}35}\) in undifferentiated PC12 cells confirm this observation. Different antioxidant and pro-oxidative responses depending on cell cultures' differentiation may result from different phenotypes and cell activity. Undifferentiated cells are in constant proliferation, so their metabolism is faster, and in this case, higher ROS production is possible.  
A\(\beta_{25\text{-}35}\) caused a significant increase in the amount of DNA damage, at concentrations of 1 and 5 uM A\(\beta_{25\text{-}35}\) in both types of cells, and in differentiated PC12 cells also at the concentration of 0.1 uM A\(\beta_{25\text{-}35}\) (Figure 2D). The genotoxicity of A\(\beta_{25\text{-}35}\) in concentrations of 0.1-5 uM caused more than twice as much damage (from 71.8% to 116.4%) in differentiated cells than in undifferentiated cultures. The difference was most profoundly demonstrated at the highest A\(\beta_{25\text{-}35}\) concentration (5.0 uM). Exemplary micrographs of the nuclear halo taken in the FHA assay are shown in Figure 3.