A combined microRNA and proteome profiling to investigate the effect of ZnO nanoparticles on neuronal cells
Authors: Smriti Singh Yadav
Sanjeev Kumar Yadav
Saumya Mishra
Sanjay Yadav
Devendra Parmar
Ankur Kumar Srivastava
Journal: Nanotoxicology (2020)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: MicroRNA; ZnO nanoparticles; cellular bioenergetics; neuronal differentiation; proteomics.
Affiliations: Developmental Toxicology Laboratory, Systems Toxicology, and Health Risk Assessment Group, CSIR- Indian Institute of Toxicology Research (CSIR-IITR), Vishvigyan Bhawan, Lucknow, Uttar Pradesh, India., Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India., All India Institute of Medical Sciences (AIIMS), Raebareli, Uttar Pradesh, India.

Abstract:  Zinc oxide nanoparticles (ZnO NPs) are one of the most broadly used engineered nanomaterials. The toxicity potential of ZnO NPs has been explored in several studies; however, its neurotoxicity, especially its molecular mechanism, has not been studied in depth. In this study, we have used a cellular model of neuronal differentiation (nerve growth factor differentiated PC12 cells) to compare the effect of ZnO NPs exposure on neuronal (differentiated or mature neurons) and non-neuronal (undifferentiated) cells. Our studies have shown that the noncytotoxic concentration of ZnO NPs causes neurite shortening and degeneration in differentiated PC12 cells. Brain-specific microRNA (miRNA) array and liquid chromatography with tandem mass spectrometry (LC-MS/MS) are used to carry out profiling of miRNAs and proteins in PC12 cells exposed with ZnO NPs. Exposure of ZnO NPs produced significant deregulation of a higher number of miRNAs (15) and proteins (267) in neuronal cells in comparison to miRNAs (8) and proteins (207) of non-neuronal cells (8). In silico pathway analysis of miRNAs and proteins deregulated in ZnO NPs exposed differentiated PC12 cells have shown pathways leading to neurodegenerative diseases and mitochondrial dysfunctions are primarily targeted pathways. Further, a bioenergetics study carried out using Seahorse XFp metabolic flux analyzer has confirmed the involvement of mitochondrial dysfunctions in ZnO NPs exposed differentiated PC12 cells. In conclusion, differentiated PC12 cells (neuronal) were found more vulnerable than undifferentiated (non-neuronal PC12 cells) toward the exposure of ZnO NPs and deregulation of miRNAs and mitochondrial dysfunctions play a significant role in its toxicity. 

Retracted: No

PubPeer comments:
 ![file] The whole paper is based on the idea of different responses of undifferentiated and differentiated PC-12 cells to ZnO nanoparticles toxicity, but no non-cytotoxic dose study was performed for delicate u201cNeuron likeu201d differentiated PC12 cells. The same dose used for differentiated PC-12 harshly damaged the cells which can be clearly seen in flat line in the XFp analyzer graph (fig 7 a2). Can the authors explain the u201cflat curveu201d in seahorse experiment for differentiated PC-12 cells? , 
 #1 ![file] Very good observation, I want to add that, in XFp analyzer graphs ( Figure : 7) comparison should have been between undifferentiated vs differentiated based on the hypothesis. If you pay close attention to values on Y-axis ( indicated by same color arrows) there is no significant difference between ZnO nano-particles treated undifferentiated and differentiated cells. , 
 ![file] In figure 7 (d2) differentiated PC-12 cells are showing a negative ATP production, how ATP production can go in negative range in a functional  neuron like cells, where supposedly ~75% cells are alive after ZnO  non-cytotoxic dose treatment? XFp analyzer data also raises question about the combined miRNA and proteomic profiling data for differentiated PC-12 cells, as same dose was used for those experiments too. , 
 **Answer to Comments of Asteroporpa Muricatopatella:** Cell viability assay was carried out only to identify the non-cytotoxic dose for further studies. Our study involved a comparison of the effect of ZnO NPs on undifferentiated and differentiated PC12 cells, so we have taken the same dose for both groups. So non-cytotoxic concentrations were calculated from undifferentiated cells only. We have taken same concentration (non-cytotoxic concentration 8 u00b5g ml) for undifferentiated and differentiated PC12 cells. Same question was asked by one of the reviewer and we have provided the above reply and he was satisfied. When we carried out different mitochondrial function assays in both undifferentiated and differentiated PC12 cells at concentration of 8 u00b5g ml, we have found that differentiated cells are more sensitive towards exposure of ZnO NPs and same has been discussed in our manuscript and is one of the findings of our work. If you compare values of y axis in both graphs, it can be seen that control differentiated cells have very high OCR at 0 minute (~ 400), however in undifferentiated control cells it is ~ 80. Lower OCR values observed in ZnO exposed differentiated PC12 cells are the effect of ZnO NP exposure, which was the only difference between two groups. Line is looking flatter due to high OCR values of control differentiated cells. OCR values has dropped substantially after the addition of Rotenone in ZnO NP exposed differentiated cells. **Answer to Comments of Crematogaster Limata: ** Control differentiated cells show around 10 times more OCR values (~300) in basal respiration than control undifferentiated cells (~30), how effect of any chemical can be compared between undifferentiated and differentiated. Ankur K. Srivastav (First Author) , 
 #4 1- Isn t it obvious that differentiated cells which is characterized by cell cycle arrest will respond to a toxic insult differently than a constantly proliferating undifferentiated cells? what was preventing the authors to identify different non-cytotoxic doses for both cell types!! Is there any guideline that non-cytotoxic dose should be always same for all the different cell types cell lines ? 2- Value may be higher on Y-axis, but OCR of ZnO treated undifferentiated cells are ~50% at 0 minute compared to control and 20% in ZnO treated differentiated cells compared to control. which clearly indicate the  Neuron like differentiated PC-12 are highly damaged by ZnO treatment. , 
 **Crematogaster Limata s** comment also indicates the drastically low basal respiration value in ZnO NPs treated differentiated PC-12 compared to control is not due to different response but due to cellular damage by potentially toxic dose of ZnO nanoparticles. Percentage difference of basal respiration in undifferentiated cell vs control is ~40% (figure 7b1) and ~5% only in ZnO NPs treated differentiated PC-12 cells vs control (figure 7 b 2). ,