Segnalazione dopaminergica negli astrociti striatali: un focus sulla comunicazione astrocita-neurone

Astrocytes have emerged as critical regulators of central nervous system (CNS) function, engaging in complex communication with neurons and other glial cells. Recent evidence shows that several astrocytic functions are modulated by neuronal activity, primarily triggered by neurotransmitter release. Astrocytes express a comprehensive range of receptors, including all five types of dopamine receptors, allowing them to sense and respond to this neurotransmitter. Notably, dopamine has been shown to influence astrocytic activities, including their secretory function. However, the mechanisms through which dopamine-mediated astrocyte-derived signals affect neurons and synaptic function remain poorly understood. This PhD project aimed to investigate the physiological role of dopamine signaling via dopamine receptor 1 (D1R), activated under sustained neuronal activity, in shaping the astrocyte secretome and influencing astrocyte-to-neuron communication. On this ground, we characterized primary astrocyte cultures for dopamine receptor expression and their ability to respond to dopamine using quantitative PCR (qPCR) and western blotting (WB). Results showed that striatal astrocytes express higher levels of D1R compared to cortical and midbrain ones, making them a focus for further analysis. Astrocyte-conditioned medium (ACM) was produced by treating striatal astrocytes with the D1R agonist SKF-38393, and its effects on neuronal growth and synaptic development were evaluated. Morphological analysis of neurons cultured in ACM revealed that astrocytic D1R activation promotes enhanced dendritic complexity and growth cone formation, with increased levels of Drebrin E, a marker of axonal and dendritic development. Furthermore, synaptic analysis using immunofluorescence (IF) and WB showed that acute treatment with ACM from D1R-activated astrocytes significantly increased the number of synapses, suggesting a role for astrocytic D1R signaling in promoting synaptic matching and maturation. To uncover the molecular basis underlying the observed effects, a proteomic analysis of the astrocyte secretome was performed. Results revealed that D1R activation alters the composition of astrocyte-secreted proteins, enriching for factors involved in neuronal growth and synaptic modulation. A bioinformatic analysis allowed to identify a pool of promising candidates expressed by astrocytes and capable of acting as extracellular signaling molecules. Concurrently, we isolated D1R-expressing astrocytes via fluorescent-activated cell sorting (FACS), which represent approximately 10% of the initial cell preparation. Following stimulation of this homogeneous cell population, D1R-induced astrocyte-secreted proteins should accumulate in the ACM and simplifying the hit detection and validation. We are now planning to utilize specific antibodies and inhibitors to block the target proteins in ACM or their target site of binding on neurons to validate their roles as mediators of astrocyte-neuron communication. Overall, these findings highlight the critical role of astrocytic D1R signaling in regulating neuronal and synaptic development, providing new insights into astrocyte-neuron crosstalk. Given the involvement of dopamine in both movement and cognitive disorders, this PhD project opens avenues for exploring astrocyte dysfunctions in neurological diseases, particularly those related to dysregulation of dopamine signaling.