Complement system as glutamate modulator and synaptic organizer in EAE animal model.

In recent years, there has been a growing interest in the interaction between the central nervous system (CNS) and the immune system. This has led to the finding that the CNS is an immune-privileged environment. A principal component of the innate immune system is the complement system, which is essential for the primary defense of the human body against pathogens. The complement system is frequently described as a "double-edged sword" in the context of the CNS. On one hand, it facilitates the physiological maturation of neural networks and plays a crucial role in promoting synaptic pruning during brain development. On the other hand, a dysregulation of this system can lead to excessive synaptic loss, which could contribute to the progression of neurodegenerative diseases. Additionally, research conducted over the past two decades has revealed a "non-canonical" role for the complement system in modulating neurotransmission at chemical synapses. This thesis aims to examine the function of the complement system in pathological conditions, in both aberrant synaptic elimination and glutamate modulation, as promoter of neurodegenerative processes. In particular, the study employed an experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis (MS). To evaluate the first aim regarding the role of the complement system as a synaptic modulator of neurotransmission, three specific objectives were pursued: first, to assess complement-induced glutamate release from both synapses and astrocytes in healthy mice, second to evaluate complement system alterations and glutamate release in EAE mice at various disease stages, and third to explore the role of excitatory amino acid transporters in these processes. To elucidate the second aim regarding the involvement of excessive synaptic pruning in neurodegeneration, I focused on developing appropriate methods to highlight microglial phagocytosis of cortical nerve terminals (synaptosomes). Next, the objective was to quantify pruning differences between EAE and healthy mice, and to explore the impact of complement proteins (C3) on synaptic vulnerability. In the initial phase of the study, an investigation was conducted into structural synapse impairments within the cortex and hippocampus of EAE mice. It was observed that while inflammatory markers GFAP and CD11b were elevated, only the cortical region exhibited changes in the postsynaptic marker PSD95, that resulted overexpressed. This may be a compensatory mechanism for reduced glutamate release efficiency. In addition to astrocytosis and microgliosis an overexpression in the protein density of C1q and C3 complement components was detected in EAE mice. Building on these findings, I analysed the role of the complement system in modulating glutamate transmission. The results showed that complement-induced glutamate release varies depending on the particle type, specifically whether it is synaptosomes or gliosomes. Astrocytic gliosomes exhibit higher release activity, which is mediated by glutamate transporters working in reverse mode. In EAE mice, this modulation becomes imbalanced, with reduced release in synaptosomes and increased release in gliosomes. This imbalance, supported by proteomic changes in glutamate transporters EAAT1 and EAAT2, suggests that complement activity can be involved in a maladaptation of the astrocyte-neuron communication, indicating a potential excitotoxic imbalance. The second phase of the study examines complement-mediated synaptic pruning, employing new techniques to measure microglial phagocytosis of synaptosomes. The results demonstrate that EAE synaptosomes have an increase susceptibility to pruning, which may be driven by the interaction between C3 and its receptor, resulting in the phagocytosis of synapses by microglia. These findings collectively highlight the complex role of the complement system in synaptic dysfunction, providing insights into potential therapeutic targets for MS and other neurodegenerative diseases.