3D human stem-cell-derived neuronal spheroids for in vitro neurotoxicity testing of methylglyoxal, highly reactive glycolysis byproduct and potent glycating agent.

Human-derived three-dimensional (3D) in vitro models are advanced human cell-based model for their complexity, relevance and application in toxicity testing. Intracellular accumulation of methylglyoxal (MGO), the most potent glycating agent in humans, mainly generated as a by-product of glycolysis, is associated with age-related diseases including neurodegenerative disorders. In our study, 3D human stem-cell-derived neuronal spheroids were set up and applied to evaluate cytotoxic effects after short-term (5 to 48 h) treatments with different MGO concentrations, including low levels, taking into consideration several biochemical endpoints. In MGO-treated neurospheroids, reduced cell growth proliferation and decreased cell viability occurred early from 5-10 µM, and their compactness diminished starting from 100 µM, apparently without affecting spheroid size. MGO markedly caused loss of the neuronal markers MAP-2 and NSE from 10-50 µM, decreased the detoxifying Glo1 enzyme from 50 µM, and activated NF-kB by nuclear translocation. The cytochemical evaluation of the 3D sections showed the presence of necrotic cells with loss of nuclei. Apoptotic cells were observed from 50 µM MGO after 48 h, and from 100 µM after 24 h. MGO (50-10 µM) also induced modifications of the cell-cell and cell-ECM interactions. These effects worsened at the higher concentrations (300-500 µM). In 3D neuronal spheroids, MGO tested concentrations comparable to human samples levels measured in MGO-associated diseases, altered neuronal key signalling endpoints relevant for the pathogenesis of neurodegenerative diseases and aging. The findings also demonstrated that the use of 3D neuronal spheroids of human origin can be useful in a strategy in vitro for testing MGO and other dicarbonyls evaluation.

Human Astrocyte Spheroids as Suitable In Vitro Screening Model to Evaluate Synthetic Cannabinoid MAM2201-Induced Effects on CNS.

There is growing concern about the consumption of synthetic cannabinoids (SCs), one of the largest groups of new psychoactive substances, its consequence on human health (general population and workers), and the continuous placing of new SCs on the market. Although drug-induced alterations in neuronal function remain an essential component for theories of drug addiction, accumulating evidence indicates the important role of activated astrocytes, whose essential and pleiotropic role in brain physiology and pathology is well recognized. The study aims to clarify the mechanisms of neurotoxicity induced by one of the most potent SCs, named MAM-2201 (a naphthoyl-indole derivative), by applying a novel three-dimensional (3D) cell culture model, mimicking the physiological and biochemical properties of brain tissues better than traditional two-dimensional in vitro systems. Specifically, human astrocyte spheroids, generated from the D384 astrocyte cell line, were treated with different MAM-2201 concentrations (1-30 µM) and exposure times (24-48 h). MAM-2201 affected, in a concentration- and time-dependent manner, the cell growth and viability, size and morphological structure, E-cadherin and extracellular matrix, CB1-receptors, glial fibrillary acidic protein, and caspase-3/7 activity. The findings demonstrate MAM-2201-induced cytotoxicity to astrocyte spheroids, and support the use of this human 3D cell-based model as species-specific in vitro tool suitable for the evaluation of neurotoxicity induced by other SCs.