ATP extracellulare e neuroinfiammazione nella patogenesi della malattia di Alzheimer

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive memory loss and two histopathological hallmarks: amyloid-beta (Aβ) plaques and neurofibrillary tangles. Extracellular ATP (eATP) acts as a key danger signal in inflammation, increasing from nanomolar to micromolar levels in inflamed tissue. At these high concentrations, it activates the P2X7 receptor (P2X7R), which triggers NLRP3 inflammasome activation and the release of the pro-inflammatory cytokines IL-1β and IL-18. eATP levels are regulated by ectoenzymes that sequentially convert it into the anti-inflammatory mediator adenosine. Besides its role in neuroinflammation, it has been proposed that the balance between eATP and its metabolites, modulated by microglia, influences neuronal excitability. Upon neuroinflammatory conditions, microglia are actively involved through the release of cytokines, complement proteins, and ROS in response to pathological stimuli. My PhD project provides a longitudinal characterization of the AD neuroinflammatory state and I investigated the role of the eATP/P2X7R axis in neuroinflammation and neuronal circuit dysfunction as a potential therapeutic target. Employing the PS2APP (B6152H) mouse model (carrying both PS2-N141I and APPSwe mutations), I analyzed three different disease stages: 2 months (pre-Aβ plaques), 6 months (onset of Aβ plaque deposition), and 9 months (overt cognitive decline). To assess the contribution of P2X7R to AD-associated neuroinflammation and to test whether its deletion could rescue AD phenotypes, AD mice were crossed with P2X7R knockout mice (AD-P2X7RKO). Using in vivo bioluminescent imaging, I measured for the first time eATP levels in the AD brain. I found elevated eATP at 6 and 9 months, consistent with disease progression. Surprisingly, increased eATP was already detectable at 2 months, well before pathological and cognitive changes, and was normalized in AD-P2X7RKO mice. I found that this accumulation did not depend on the degradative pathway, as CD39, CD73, and ADA levels remained unchanged. At all three ages, eATP rise is associated with a neuroinflammatory state, in terms of NLRP3 inflammasome activation and pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) increase, normalized as well by P2X7R deletion. Recent data obtained in our laboratory showed that cortical microglial reactivity is also present at 2 months, leading to P2X7R-dependent aberrant synaptic pruning. I carried out Ca²⁺ imaging experiments in acute slices from somato-sensory cortex (SSCx) and I demonstrated that these early changes are also in concomitance with neuronal hyperexcitability in AD mice, with a higher percentage of active and hyperactive neurons and more sustained Ca2+ responses upon electrical stimulation. AD-P2X7RKO mice showed reduced neuronal hyperactivity, indicating that P2X7R contributes to early neuronal dysfunction and neuroinflammation. From a translational point of view, I explored an RNA-based gene therapy approach to acutely silence P2X7R. While widespread P2X7R downregulation across the brain resulted in systemic side effects, local intracortical injections into the SSCx achieved effective silencing without adverse consequences. Preliminary data suggest that this approach could rescue neuronal hyperactivity in AD mice, indicating that P2X7R modulation may help restore cortical network function. In summary, AD mouse brains display early eATP accumulation, accompanied by neuroinflammation and neuronal hyperactivity, which occur prior to Aβ plaque deposition and are reversed by P2X7R deletion. These findings highlight P2X7R as a promising therapeutic target in the early stages of AD, with the potential to slow or even prevent disease progression.