Innovative Neurotoxicology Models: From Cell Cultures to CRISPR/Cas9

Neurotoxicology, as a branch of toxicology, has experienced steady growth in the recent past in the form of models and techniques to assess the impacts of environmental toxins on neural functioning. The previously used approaches embraced the usage of animal models, and, although they are useful, they are associated with certain ethical issues and cannot accurately reflect the human organism’s response. This has prompted researchers to come up with in vitro culture models and some of the current best technologies, like CRISPR/Cas9. These progressions improve not only the knowledge of how neurotoxins work but also provide the right direction for proper and ethical use in studying their application.

The Rise of In Vitro Models in Neurotoxicology

Among all the in vitro models used in neurotoxicology studies, the SH-SY5Y cell line is rather popular. These human neuroblastoma cells are best suited for differentiation into neuron-like cells; thus, they are a good model to study neurotoxicity. On the one hand, differentiated SH-SY5Y cells have a higher ATP level and plasma membrane activity; on the other hand, they do not have significant cell strength against oxidative stress, a frequent phenomenon in toxic contexts of neural cells.

Research has shown that SH-SY5Y cells are suitable for working out the influence of various environmental chemicals. For instance, chlorpyrifos and cypermethrin, labeled as insecticides, caused apoptosis and oxidative stress in these cells. This conforms with the rest of the world’s view that environmental pollutants interfere with the normal functioning of cells and set off the neurodegenerative process.

In addition, the SH-SY5Y model has been used in the discovery of neuroprotective agents against neurotoxicity. Such natural compounds as Moringa oleifera extracts were effective in reducing the level of oxidative stress and apoptosis induced by DEHP in SH-SY5Y cells.

Advanced High-Throughput Screening Techniques

High-throughput screening is a novel approach that has transformed neurotoxicology because it helps scientists determine how thousands of chemicals and drugs impact neural cells. For instance, Tox21 10K compound library has been used in the identification of inhibitors of acetylcholinesterase using cells as well as enzyme activity-based assays. The present approach not only lays the basis for discovering new neurotoxic liabilities but is also beneficial in determining their modes of action using molecular docking and time-dependent inhibition studies.

HTS has also helped in finding new chemical entities with neuroprotective characteristics. For instance, screening methodology was used to discover new acetylcholinesterase inhibitors, including cilostazol and chelerythrine chloride, in the past. These compounds can therefore be recommended as potential therapeutic agents for diseases characterized by neurotoxicity.

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The Impact of CRISPR/Cas9 in Neurotoxicology

The CRISPR/Cas9 approach has added a new cutting-edge tool for neurotoxicology studies by providing methods that allow for the introduction of specific vs. genetic changes. This tool helps scientists get rid of certain genes and then analyze the phenotypic consequences; thus, learning the molecular genetics of neurotoxicity can be accomplished by employing this tool.

One of the most important areas of using CRISPR/Cas9 in neurotoxicology can be discussed as genome-wide screening. Loadman et al. (2013) have used CRISPR/Cas9 to knock off genes in SH-SY5Y cells to study pathways that are associated with the cellular response to chronic dieldrine treatment. This pesticide, being an environmental carcinogen, has been associated with neurodegenerative diseases, including Parkinson’s. CER_warned Key_housekeepers, using CRISPR/Cas9 screening, found that several components of the ubiquitin-proteasome system, as well as the mTOR pathway, were, therefore, pinpointed as organizing dieldrin toxicity. This underlines the value of CRISPR/Cas9 for identifying new targets for the treatment of diseases based on a tumor’s genetically defined features.

CRISPR/Cas9 has been applied to the assessment of gene function in modulating toxicant-induced NE injury through the deletion of certain genes. For example, disruption of the gene for HDAC3 was reported to abolish neuronal cell death promoted by Aroclor 1254, a mixture of polychlorinated biphenyl. Moreover, it strengthens the hypothesis of an essential role of epigenetic mechanisms in neurotoxicity and prospects for further investigation of neuroprotective approaches.

The Role of Autophagy in Neurotoxicity

The cellular degradation process has been recognized more and more as being involved in the neurotoxicity process. These analyses conducted together with SH-SY5Y cells depict that autophagy is a double-edged sword and plays either a protective or deleterious role depending on the circumstances. For instance, TOCP causes autophagy in SH-SY5Y cells; this is a protective response at first. But if it progresses, autophagy can result in cell death and thus exhibit a fine line between being a protective and pathogenic process in neurotoxicity.

Studies have also shown that improving autophagy can reduce the impact of some neurotoxic compounds. Some experimental evidence also showed that phagocytosis, a precondition of phagocytosis, can reduce the toxicity of some particular neurotoxic compounds. For example, it has been demonstrated that isoflavones increase the levels of autophagy and thus prevent the apoptosis of SH-SY5Y cells exposed to atrazine. This protective effect is through the BEX2 protein signaling that points to the possible treatment option through BEX kinases against neurotoxicity.

Oxidative Stress and its Implications

The generation of reactive oxygen species seems to be involved in the toxic action of many chemicals affecting the nervous system. This video depicts SH-SY5Y cells, which have earlier been used to model oxidative stress elicited by different environmental pollutants. For instance, BPA exposure offers a proliferation of nitric oxide levels and oxidative damage in SH-SY5Y cells. In doing so, this oxidative stress can lead to apoptosis of the cellular structures and neurodegenerative processes.

It has been considered that antioxidants can have a protective effect on oxidative stress-induced neurotoxicity. Flavonol quercin has been identified to attenuate apoptosis induced by dieldrin and associated with ER stress in dopaminergic neuronal cells. Such findings indicate pro-oxidative stress as a potential mechanism that could be targeted to reduce neuronal toxicity.

In Vitro Models Beyond SH-SY5Y

Although SH-SY5Y cells are widely used for neurotoxicity studies, other in vitro models have shown usefulness as well. Primary neuronal cultures, neuronal and glial co-cultures, and organotypic cultures are more complex and closer to in vivo conditions to study neurotoxicity. The ability of these models to simulate other cell types in the brain means that they are more likely to reflect the way different cells communicate, thus giving a more versatile picture of how neurotoxic agents are likely to impact neural connections.

For example, primary dopaminergic neuron cultures have been adopted in investigating the impacts of the perfluorinated compound PFOS on dopamine transmission. These studies showed that PFOS can change the density and activity of dopamine transporters, which gives an understanding of how environmental pollutants affect Parkinson’s disease.

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Directions for Future Research and Recommendations for Practice

The sustained advancement of novel neurotoxicology models hints at the chances of improving neuroscience’s knowledge about the impact of enviable toxicants on neural integrity. As these models advance, they can completely supplement classical animal models, which are in tune with the 3Rs principles in animal use.

Further, in vitro, neurotoxicology is expected to undergo a radical change with the developments of organ-on-a-chip and 3D bioprinting technologies. They can develop many new technologies that will be closer to the actual physiological structure and functional organization of the human brain. Together with these approaches, genetic techniques, for instance, CRISPR/Cas9, will help investigators examine molecular aspects of neurotoxicity in great detail.

Thus, neurotoxicology is currently in the process of transitioning to a new level due to the use of progressive in vitro models and genetic approaches. These innovations not only enhance the understanding of the processes that take place during neurotoxic effects but also create the basis for the further development of therapeutic approaches against neurodegenerative diseases. In the future development of research, these models should be further optimised and aligned with human physical characteristics as much as possible to achieve a greater beneficial effect on people’s health.

References

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