Metal Ion Regulation in the Central Nervous System and in Glutamatergic Synapses: Role of the Cellular Prion Protein

Despite many efforts, the molecular mechanisms underlying the pathophysiology of neurodegenerative disorders have not been fully understood. Results published in literature highlight that different neurodegenerative diseases share common features: protein aggregation in neuronal tissue; oxidation of neuronal tissue mediated by redox-active metal ions interaction with a target protein; and functional demise. So, unveiling the physiological function of protein that aggregate in neurodegenerating tissues, as well as their interplay with metal ions, becomes a prominent issue, in order to understand the etiological trigger and to define a possible therapeutic strategy. Metal ions are essential elements for cellular processes, but at the same they are potentially dangerous since they can give rise to Fenton reaction and oxidative/nitrosative stress. So, their homeostasis is strictly regulated in each district of the organism, but in particular in the brain. The brain, having the highest metabolic rate and depending predominantly on oxidative metabolism for its energy, has developed fine mechanisms to compartmentalize, distribute, uptake and excrete the different ionic species. Alterations in one of these mechanisms can lead to great neuronal damages, and maybe neurodegenerative disorders. This work has been focused on the cellular prion protein (PrPC), whose conformational isoform, the scrapie prion protein (PrPSc) is the causative agent of prion disordes and whose function has not been clearly defined, yet. Metal ions are a common denominator to all the cellular pathways in which PrPC seems to be actively involved. In particular, metal ions homeostasis maintainance, neuroprotection in excitotoxic condition and ionotropic receptor modulation have been studied. In the first part of the project, PrPC role in metal ion homeostais maintainance has been investigated. To this aim, copper, manganese, zinc and iron content, as well as metal binding proteins expression have been measured in a PrP knockout murine model, compared to wild-type. The results describe the global rearrangement occurring in the expression of metal binding proteins to maintain trace metals homeostasis, trying to compensate PrPC absence. At the same time, a pronounced decrease in Ceruloplasmin ferroxidase activity has been detected in PrP null mouse serum, pointing out a global impairment in copper metabolism in PrPC absence. In the second part of the project, the importance of the interaction between PrPC and copper ions in excitotoxic conditions and in synapses functionality has been studied. It has been published that PrP null mice show higher levels of neuronal cell death in stressful conditions and when subjected to toxic treatment with glutamate receptor agonists. Moreover, these mice show altered kinetics of N-methyl-D-aspartate (NMDA) receptor current. These alterations appears to be due to an inhibitory regulation that PrPC exerts on NMDA receptors via copper ions, lacking in PrP null hippocampi. First, the enhanced suceptibility to excitotoxicity of PrP knockout mice has been verified and characterised in organotypic hippocampal cultures upon treatment with NMDA. Higher neuronal cell death levels have been detected in all the investigated hippocampal regions. To identify which cellular regulatory mechanism is alterd in PrPC absence, the expression of the proteins mainly involved in excitotoxicity has been compared between PrP knockout and wild-type hippocampi. Among other minor differences, a different modulation of calcium transporters expression has been identified in PrP knockout hippocampi and brains. This global alteration appears to be necessary to maintain calcium homeostasis, since calcium content measurements did not reveal any strong difference between PrP null and wild-type samples. NMDA receptors can be S-nitrosylated on extracellular cysteines and this reaction is always inhibitory. S-nytrosilation requires an electron acceptor to occur, for this reason copper ions are often involved in these kind of reactions. Moreover, copper ions are known to modulate NMDA receptor activity, but the precise mechanism has not been described, yet. Since PrPC is known to support the S-nitrosylation of other membrane proteins, the S-nitrosylation levels of NMDA receptor subunits GluN1 and GluN2A have been measured in PrP knockout hippocampi from adult mice and compared to wild-type ones. Results show that the S-nitrosylated fractions of both GluN1 and GluN2A are reduced in PrP absence. So, this reveals that PrPC modulates NMDA receptor activity providing the copper ions necessary to support their inhibitory S-nitrosylation reaction. Through this mechanism, PrPC contributes to inhibit NMDA receptor currents, as well as to protect neurons in excitotoxic conditions.