Abstract
The generation of human neural tissue-like 3D structures holds great promise for disease modeling, drug discovery and regenerative medicine strategies. Promoting the establishment of complex cell-cell interactions, 3D culture systems enable the development of human cell-based models with increased physiological relevance, over monolayer cultures. Here, we demonstrate the establishment of neuronal and astrocytic metabolic signatures and shuttles in a human 3D neural cell model, namely the glutamine-glutamate-GABA shuttle. This was indicated by labeling of neuronal GABA following incubation with the glia-specific substrate [2-13C]acetate, which decreased by methionine sulfoximine-induced inhibition of the glial enzyme glutamine synthetase. Cell metabolic specialization was further demonstrated by higher pyruvate carboxylase-derived labeling in glutamine than in glutamate, indicating its activity in astrocytes and not in neurons. Exposure to the neurotoxin acrylamide resulted in intracellular accumulation of glutamate and decreased GABA synthesis. These results suggest an acrylamide-induced impairment of neuronal synaptic vesicle trafficking and imbalanced glutamine-glutamate-GABA cycle, due to loss of cell-cell contacts at synaptic sites. This work demonstrates, for the first time to our knowledge, that neural differentiation of human cells in a 3D setting recapitulates neuronal-astrocytic metabolic interactions, highlighting the relevance of these models for toxicology and better understanding the crosstalk between human neural cells.
Highlights
There has been an increasing demand for a paradigm shift from animal cells towards robust human cell models of high physiological relevance[1]
Ntera2/clone D1 (NT2) aggregates differentiation towards the neural lineage can be achieved upon retinoic acid (RA) induction and subsequent maturation (Fig. 2A), resulting in highly viable (Fig. 2B) differentiated neurospheres composed mainly of βIII-tubulin-positive neurons and glial fibrillary acidic protein (GFAP)-positive astrocytes (Fig. 2C), as previously reported by our group[15]
The amounts of [2-13C]glutamine were higher relative to [2-13C]glutamate, while the labeling in C3 was similar for both. This reflects the different contributions of the anaplerotic and oxidative pathways for the synthesis of these two metabolites. These contributions can be expressed as the ratio pyruvate carboxylase (PC)/pyruvate dehydrogenase (PDH), which can be estimated by dividing the difference between C2 and C3 by C4 ((C2-C3)/C4)[48]
Summary
There has been an increasing demand for a paradigm shift from animal cells towards robust human cell models of high physiological relevance[1]. NT2 neural differentiation has been shown to recapitulate developmental processes observed during in vivo neurogenesis[12], generating neuronal cells able to establish functional synapses and elicit action potentials following depolarization[13,14] Given these properties, the NT2 cell line has been reported to be a promising human cell source for toxicological applications[15,16,17,18]. Other important neural metabolic hallmarks, including the glutamine-glutamate-GABA cycle between neurons and astrocytes[32] have not been previously reported for these stem-cell derived co-cultures cultures. An example of this metabolic specialization is the fact that glucose is taken up mostly by neurons[36], while astrocytes have been shown to metabolize acetate[37,38]
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