Co-culture of Glutamatergic Neurons and Pediatric High-Grade Glioma Cells Into Microfluidic Devices to Assess Electrical Interactions.

Pediatric high-grade gliomas (pHGG) represent childhood and adolescent brain cancers that carry a rapid dismal prognosis. Since there is a need to overcome the resistance to current treatments and find a new way of cure, modeling the disease as close as possible in an in vitro setting to test new drugs and therapeutic procedures is highly demanding. Studying their fundamental pathobiological processes, including glutamatergic neuron hyperexcitability, will be a real advance in understanding interactions between the environmental brain and pHGG cells. Therefore, to recreate neurons/pHGG cell interactions, this work shows the development of a functional in vitro model co-culturing human-induced Pluripotent Stem (hiPS)-derived cortical glutamatergic neurons pHGG cells into compartmentalized microfluidic devices and a process to record their electrophysiological modifications. The first step was to differentiate and characterize human glutamatergic neurons. Secondly, the cells were cultured in microfluidic devices with pHGG derived cell lines. Brain microenvironment and neuronal activity were then included in this model to analyze the electrical impact of pHGG cells on these micro-environmental neurons. Electrophysiological recordings are coupled using multielectrode arrays (MEA) to these microfluidic devices to mimic physiological conditions and to record the electrical activity of the entire neural network. A significant increase in neuron excitability was underlined in the presence of tumor cells.

Cytotoxic CD8+ T lymphocytes expressing ALS-causing SOD1 mutant selectively trigger death of spinal motoneurons.

Adaptive immune response is part of the dynamic changes that accompany motoneuron loss in amyotrophic lateral sclerosis (ALS). CD4+ T cells that regulate a protective immunity during the neurodegenerative process have received the most attention. CD8+ T cells are also observed in the spinal cord of patients and ALS mice although their contribution to the disease still remains elusive. Here, we found that activated CD8+ T lymphocytes infiltrate the central nervous system (CNS) of a mouse model of ALS at the symptomatic stage. Selective ablation of CD8+ T cells in mice expressing the ALS-associated superoxide dismutase-1 (SOD1)G93A mutant decreased spinal motoneuron loss. Using motoneuron-CD8+ T cell coculture systems, we found that mutant SOD1-expressing CD8+ T lymphocytes selectively kill motoneurons. This cytotoxicity activity requires the recognition of the peptide-MHC-I complex (where MHC-I represents major histocompatibility complex class I). Measurement of interaction strength by atomic force microscopy-based single-cell force spectroscopy demonstrated a specific MHC-I-dependent interaction between motoneuron and SOD1G93A CD8+ T cells. Activated mutant SOD1 CD8+ T cells produce interferon-γ, which elicits the expression of the MHC-I complex in motoneurons and exerts their cytotoxic function through Fas and granzyme pathways. In addition, analysis of the clonal diversity of CD8+ T cells in the periphery and CNS of ALS mice identified an antigen-restricted repertoire of their T cell receptor in the CNS. Our results suggest that self-directed immune response takes place during the course of the disease, contributing to the selective elimination of a subset of motoneurons in ALS.