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Human model neurons as predictive in vitro test system for developmental neurotoxicity

Project Summary:

The developing human nervous system is more sensitive to exposure with environmental chemicals   than the adult. To properly assess the risk of chemicals for human health, data on developmental neurotoxicity (DNT) are necessary. Current DNT testing guidelines (OECD 2007; USEPA 1998) propose in vivo investigations in animal models, such as rats. These tests require more than a thousand female animals to be killed for each chemical in question which is problematic both in terms of animal welfare and costs and time consumption. Thus, the existing guidelines for DNT testing will require amend­ments to address the expected future regulatory demands for DNT of the thousands of chemicals for which there are few to no DNT data yet. Therefore, according to the “3R principle” (reduction, replacement, and refinement), alternative testing strategies are needed which address the problem of animal welfare by refining and reducing animal experiments and the problem of costs

In this project we explore a human cell-based test system to assess DNT potential of chemicals at an early stage of brain development. A human neural pre­cursor cell line is tested for suitability for semi-automated high-throughput DNT screening. We establish assays for detecting disturbances in two basic pro­cesses of brain development in 96-well scale: neuronal dif­ferentiation and migration using the human Ntera2 (NT2) cell line. We assess the effects of test compounds with well-established DNT potential in comparison with compounds without specific DNT potential. We find that human NT2 cell cultures treated with the morphogen, retinoic acid, imitate neuronal differentiation, and migra­tion in vitro. The developmental neurotoxicants methyl­mercury chloride, sodium arsenite, sodium valproate, and methylazoxymethanol significantly reduce the expression of the neuronal marker β-tubulin type III and decrease the migration distance in developing NT2 cells. Both endpoints, differentiation and migration, can be read out directly in a standard fluorescence plate reader, enabling high-throughput screening. Currently, we are exploring the DNT potential of pesticide compounds in the assays. We conclude that NT2 cell tests are suited as components of a human cell-based modular in vitro DNT test system.

Because the development of therapeutic treatments for brain damage requires drug testing not only on neurons of experimental animals, but also on human neurons, we are also incorporating other endpoints in our test system, such as neurite initiation, extension, and network formation. This offers the possibility to analyze the effects of drugs as positive regulators of neural regeneration. For example, using the NT2 neurons, we find that the pain reliever Ibuprofen decreases RhoA (Ras homolog gene family, member A GTPase) activation and promotes neurite growth. Inhibition of the downstream effector Rho kinase by the drugs Y-27632 and Fasudil results in a strong increase in neurite outgrowth. The results are encouraging for using assays based on NT2 model neurons as ethical and efficient method in preclinical drug development.

 

Funding:

German Ministry of Education and Research (0313925D/0315522D) and German Research Foundation (BI 262/16-2).

 

Project Lead:

Prof. Dr. Gerd Bicker (gerd.bickertiho-hannover.de; 0511-856-7765)

PD Dr. Michael Stern (michael.stern@tiho-hannover.de; 0511-856-7767)

Institute of Physiology and Cell Biology, University of Veterinary Medicine Hannover, Germany

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