Elucidation of neuronal and glial cell phenotypes and functions utilizing mass spectrometry techniques

Abstract

Microglia and brain macrophages are increasingly evidenced as key regulators in CNS development, homeostasis and pathology. A better understanding of the cellular dynamics and mechanisms that regulate microglial homeostasis and function will provide the means to manipulate these cells for therapeutic purposes. In the first section of the study, the cellular complexity of CNS myeloid compartment were unraveled using an adoptive transfer experiment with gene-modified bone marrow cells, bone marrow transplantation in a mouse model of facial nerve axotomy or high-throughput techniques such as single-cell RNA sequencing (scRNA-Seq). These findings demonstrated that, followed CNS conditioning, bone marrow-derived cells are recruited to the CNS including the retina, preferentially to the lesioned sites of the CNS. These infiltrating bone marrow-derived macrophages stably integrated into the CNS myeloid cell compartment of the lesioned brain. Similarly, the results obtained from scRNA-Seq revealed spatial and temporal microglial heterogeneity in both mouse and human brain. In diseased brain, the composition of microglial sub-populations was altered, and their microglial signatures could be rapidly changed during neurodegeneration (such as facial nerve axotomy) and/or neuroinflammation (such as multiple sclerosis). In the second part of the study, microglial heterogeneity was investigated in human post-mortem brain tissue and fresh brain biopsies at the single-cell protein level. Again, these results highlight the cellular complexity of the CNS myeloid compartment including the microglia subpopulations described in the first section, which complemented the transcriptomic signatures revealed by scRNA-Seq. Moreover, the findings translated mouse microglial phenotypes to the human system, emphasizing the translational potential of the methodology for further investigation in clinical applications. Besides single-cell phenotypic and functional characterization by mass cytometry, functions and metabolomics of CNS cells can be assessed using mass spectrometry. For this approach, the human neuroblastoma cell line SH-SY5Y was used for the establishment of the methodology. The study demonstrated that the SHSY5Y cell line was capable of synthesizing targeted metabolites (in this case “morphine”). Briefly, SH-SY5Y cells were cultured in the presence of 13C-, 2H- or 18O-labelled precursors of the morphine biosynthetic pathway. SH-SY5Y cells de novo incorporate the stable isotope-labelledprecursors into endogenously synthesized morphine. The finding unequivocally proved the capacity of de novo morphine synthesis of human neuroblastoma cells. This established methodology can be applied for future metabolomic study of CNS cells including microglial cells.