Studio in vivo di nuovi inibitori dell'inflammasoma NLRP3 in un modello murino di malattia di Parkinson

Inflammasomes are a multiprotein complex involved in the inflammatory response of human immune system to defend itself from different stimuli. The thesis work is focused on considering NLRP3 (NLR family pyrin domain containing 3) inflammasome as a possible target to be used to treat Parkinson’s disease. Two new molecules (INF245 and INF246), synthesized by the SynBioMed research group of the University of Turin, were tested in a mouse model of Parkinson’s disease induced by the neurotoxin 6-hydroxydopamine (6-OHDA), in the laboratory of prof. Cristovao, at the University of Beira Interior in Portugal. On the basis of the characterization reported in a previous thesis, compound INF246 was administered intraperitoneally, whereas its acetylated derivative INF245 was intra-brain injected, using a minipump system implanted in mice brains. Mice treated with INF245 or INF246 have been compared with both a positive control group of animals that were not injected with 6-OHDA, and a negative control group, which received only the intracranial injection of 6-OHDA. To complete the study, a group of mice were treated intraperitoneally with the reference compound MCC950 (i.e. a well known inhibitor of the NLRP3 inflammasome). After 7 days treatment, mice brains were collected and cut in slices by a cryostat machine. They were treated following an immunohistochemistry protocol, where diaminobenzidine (DAB) was used as a chromogen. DAB, reacting with horseradish peroxidase (HRP) conjugated to the antibody that targets tyrosine hydroxylase (TH), produces a brown precipitate, thus allowing the count of TH positive neurons as an index of dopaminergic neurons vitality. TH+ neurons on each brain slice was counted and the results show that INF245 was able to reduce the dopaminergic neuron loss caused by 6-OHDA as well as MCC950, while INF246 did not significantly reduce TH+ neurons loss, although it was possible to observe a positive trend with INF246 treatment. In conclusion, the NLRP3 inflammasome inhibition can be considered a new promising strategy to reduce the neuronal loss, one of the 3 characteristic hallmarks of Parkinson’s disease.