• Alzheimer's disease
• long term potentiation
• memory deterioration
• neurodegeneration
• synaptic impairment

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by declining memory and cognitive function. The hallmark neuropathological features include extracellular amyloid-β (Aβ) plaques and intracellular neurofibrillary tangles (NFTs), both of which contribute to neuronal loss, synaptic dysfunctions, and widespread neurodegeneration. Despite significant advancements in understanding the pathological roles of Aβ and tau protein, the mechanisms underlying their interdependent contribution to synaptic dysfunction in Alzheimer’s disease remain unclear. Moreover, neuroinflammation is a pivotal feature of AD pathogenesis, with microglial cells, the primary driver of this process, which have been implicated in Aβ and tau pathology. In particular, in AD microglial cells activation contributes to Aβ clearance, as microglia efficiently uptake soluble Aβ via micropinocytosis and insoluble fibrils through phagocytosis. However, prolonged activation, potentially resulting from uptake system failure, leads to neurotoxic responses. This highlights microglia as a pivotal connection between protein accumulation and synaptic dysfunction. The entorhinal cortex (EC) is a key brain region within the medial temporal lobe involved in memory, navigation, and spatial representation. The EC represents one of the first brain regions affected in AD and is highly susceptible to neurodegeneration. Core neuropathological markers of AD, including Aβ accumulation and NFTs, initially develop within the EC during mild AD and progressively extend to the hippocampus and other cortical structures as the disease advances. Moreover, the EC-hippocampal circuit has been suggested as a primary route for the spread of synaptic impairments and neurodegenerative processes in AD. However, the precise mechanisms underlying the spread of synaptic dysfunction remain elusive, and it is yet to be determined whether early EC vulnerability actively contributes to EC-hippocampal circuits neurodegeneration. In recent years, EVs have emerged as a pivotal factor for transcellular signaling in the brain. In particular, given their ability to transport molecular cargo, EVs are now recognized as potential carriers of pathogenic proteins, highlighting their role in disease propagation. Moreover, different studies have shown that microglia-derived EVs could play a role in the spreading of synaptic dysfunction in AD. The aim of this thesis was to investigate whether microglia-derived extracellular vesicles carrying tau aggregates (tau-EVs) can lead to electrophysiological and behavioral deficits. In order to do this, we used primary microglia cell cultures that were exposed to a pathologic concentration of oligomeric wild-type form of Tau (200 nM). Then, samples enriched in Tau-EVs or control-EVs (ctrl-EVs), the latter derived from microglia not exposed to oligomeric wt Tau, were isolated by differential centrifugation. Western Blot analysis have been performed in order to detect the presence of oligomeric wt Tau within microglia-derived EVs. We confirmed the ability of microglia to internalize administered Tau in vitro, as suggested by different studies. Moreover, we found that microglia-derived EVs internalize exogenous oligomeric wt Tau when it is administered in primary microglial cells culture. Having established that microglia-derived EVs efficiently internalize oligomeric wild-type Tau, we next explored their potential role in triggering and spreading synaptic impairments throughout the entorhinal-hippocampal network. Synaptic dysfunction within the entorhinal cortex-hippocampal circuitry is a hallmark of early AD pathology. The EC is one of the first regions to be affected, with long term potentiation (LTP) deficits emerging before amyloid plaques form. This dysfunction propagates trans-synaptically to the hippocampus, contributing to widespread neural network abnormalities and cognitive decline. In vitro electrophysiological recordings, particularly extracellular field potential recordings, provide a powerful tool for studying synaptic dysfunction in AD model. This technique allows to measure synaptic responses in acute brain slices, with the aim of assess synaptic transmission, plasticity, and network activity in real time. Here, we performed in vitro electrophysiological recording to investigate whether Tau-EVs contribute to the propagation of synaptic dysfunction within the entorhinal-hippocampal circuit. Using EC-hippocampal slices, we assessed LTP—a synaptic plasticity mechanism underlying learning and memory— in both the EC and its primary target, the hippocampal dentate gyrus, at 1 h and 24 h following stereotaxic injection of Tau-EVs or ctrl-EVs into the EC of adult mice. One hour after stereotaxic injection of Tau-EVs, LTP was completely abolished in EC layer II, while synaptic plasticity at the PP–DG synapse remained intact. However, by 24 hours post-injection, LTP was significantly impaired at the level of the DG, indicating a delayed propagation of synaptic dysfunction along the EC-DG pathway. These findings suggest that Tau-EVs facilitate a progressive spread of synaptic deficits across functionally connected brain regions. These results further support the idea that pathogenic proteins in association with EVs can propagate synaptic dysfunction along the EC-hippocampal circuit. Interestingly, the electrophysiological results suggest the existence of a well-defined temporal pattern of neuronal dysfunction, with an ordered involvement of distinct circuits within the hippocampal formation as synaptic impairments advance. This structured progression may correspond to the gradual deterioration of memory functions. In the last years, accumulating research suggests that the EC plays a critical role in episodic associative memory formation, with its medial and lateral subdivisions believed to process and integrate spatial and non-spatial contextual cues, facilitating memory encoding and retrieval. In order to establish whether the spread of tau-EVs-dependent synaptic dysfunctions is associated with a progressive memory impairment, we used specific memory tasks. In particular, we employed the novel object recognition test (ORT) and the novel object place/context recognition test (OPCRT), which respectively are used to investigate the non-associative hippocampal dependent memory and the lateral-EC-dependent associative memory. In agreement with our hypothesis, the behavioral assessments revealed that 1h after the injection of tau-EVs, mice are not capable of discriminating between familiar and novel OPC association, but their performance in the ORT is comparable to controls. When mice where tested at 24 h after the injection with tau-EVs, a memory deficit was also detected using the hippocampal dependent task (ORT). In conclusion, these findings further support the hypothesis that EVs can play a crucial role in the spreading of the deleterious effect of tau within the medial temporal lobe and may represent a new neurobiological mechanism for the progression of AD neurodegeneration.