Métodos de estudos da neuropatia da hanseníase: avaliando o efeito da colest-4-en-3-ona no comportamento neuronal in vitro

Abstract

Leprosy is an infectious disease caused by Mycobacterium leprae (M. leprae) that mainly affects the skin and peripheral nerves, leading to severe degeneration of nervous tissue. While much has been studied about the impact of the bacillus on the pathophysiology of the nerve and its interaction with Schwann cells, considered as the main host cells, the interaction M. Leprae-neurons, as well as the effect of this pathogen metabolites on neuronal cells are still unclear. A potential metabolite is cholest-4-en-3- one (cholestenone), an oxycholesterol related to cytotoxicity and neurodegeneration, synthesized by M. leprae. Thus, the present study aimed to investigate the impact of cholestenone on neuronal behavior and viability, as well as on the bacteria’s internalization capacity in neurons. To that end, mice dorsal root ganglia (DRG) neurons were cultured and stimulated with cholestenone, and evaluated by using optical microscopy for morphology, degeneration and cell viability. Furthermore, human neurons from the SK-N-AS (SK) lineage were cultured and exposed to dead M. leprae and cholestenone, and subsequently analyzed with flow cytometry and microscopy for bacillus internalization and cell viability. Our results reveal cholestenone as a potential compound with a detrimental effect on neuronal morphology, altering the length and complexity of neurites by around 30%. Moreover, preliminary data suggest the possibility of the compound acting as a degenerative inducer. Finally, we show, for the first time, the ability of dead M. leprae to be internalized into human neurons, paving new perspectives on the contribution of neuron–bacteria interactions to the course of the disease. Taken together, these results point to a new path for the study of neural leprosy pathogenesis, providing new insights and models on the contribution of the bacillus and its pathogenic elements to the neuropathology caused during its infection.