THE JANUS FACE OF BILIRUBIN: FROM NEONATAL TOXICITY TO THE POTENTIAL PROTECTION IN NEURODEGENERATIVE DISEASES

TASK 1: Bilirubin-induced neonatal neurotoxicity Unconjugated bilirubin is a metabolite of heme catabolism, and it exhibits Janus face for owning neurotoxicity and neuroprotective effects. It can induce neurotoxicity in neonates (kernicterus). It has been already used in animal models as well as in clinics in other diseases with relevant results. As a model of kernicterus, we used the Gunn rat. We observed the full protection given by Curc by preventing cerebellar hypoplasia by counteracting inflammation, redox imbalance, and glutamate neurotoxicity. The results suggest the potential of Curc in the clinical scenario, especially in the settings where phototherapy and exchange transfusion are not feasible to perform. TASK 2: Bilirubin protection in neurodegenerative diseases The second face of bilirubin studied in our project is its neuroprotective potential. Despite it having long been considered a cytotoxic pigment, last decades UCB has emerged with its plethora of therapeutic potential. Mildly elevated bilirubin level (as in the Gilbert syndrome population), is protective against an array of extra CNS diseases as well as in laboratory models of multiple sclerosis and glioma. Based on this background, we aimed to assess UCB therapeutic potential in PD. To the goal, we used the ex vivo PD model which is able to mimic the stages of the human disease up to the diagnosis phase. We demonstrated that 0.5uM and 1uM UCB fully protect DOPAn loss observed in the PD model. Importantly, we demonstrated that, among the pro-inflammatory markers altered in PD and modulated by UCB, Tnf-alpha is the determinant of DOPAn loss and UCB protection. Despite its milestone as a potential single-therapy candidate for PD in our aforementioned experiment, the use of UCB as a therapeutic agent suffers from the challenge of reaching a so-tuned UCB concentration in the substantia nigra of PD subjects. In collaboration with the University of Turin that priory developed NBs able to carry therapeutic agents, we started screening different formulations of nanobubbles (glycol chitosan [GC]; GC-deferoxamine [GC-DFO], and para-magnetic NBs, GC-DFO-iron [GC-DFO-Fe] and for their safety and efficacy in protecting from DOPAn loss in the model of PD. GC and GC-DFO are safe to healthy slices (not challenged with Rot) at dilutions higher than 1:192. After loading different formulations of NBs with UCB, the restoration of DOPAn reach up to 80% under GC-UCB and GC-DFO-UCB treatment. While the administration of UCB-loaded NBs at diagnosis to stop the progression of DOPAn loss is rational, it will neither revert nor prevent the already occurred damage, as well as a repeated delivery of NBs to the CNS is not a suitable possibility. As we noticed in Task 1 that curcumin (Curc) protects the Gunn rat brain against the toxicity of very high UCB levels also by normalizing TNF- alpha, and considering that Curc may be considered as a nutraceutical – lifestyle – prophylactic - chronic approach to Parkinson, we have begun to explore its effect in our ex vivo PD model. Among the range concentrations of Curc tested, 5uM of the spice fully protected from rotenone-induced DOPAn loss, and the protection was lost at higher concentrations. Preliminary data seems to confirm that Curc also acts through Tnf-alpha normalization.