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Accumulation of alpha-synuclein (α-syn) is a key pathological hallmark of synucleinopathies and has been shown to negatively impact neuronal function and activity. α-syn is an important factor contributing to astrocyte overactivation, though the effect of astrocyte overactivation on neurons remains unclear. Single-cell RNA sequencing data of mouse brain frontal cortex and midbrain from Hua-Syn (A53T) and wild type mice were utilized from the GEO database. Enrichment analysis, protein-protein interaction networks, and cell-cell interaction networks all indicated enhanced communication between astrocytes and neurons, along with the involvement of TNF and inflammation-related signaling pathways. In vitro experiments were performed to further explore the mechanism of neurotoxicity in astrocyte-neuron crosstalk. Astrocytes were treated by α-syn, neuronal TNFR1 receptors were antagonized by R-7050, and the cells were co-cultured after 24 h treatment. ELISA results revealed that cytokines such as TNF-α and IL-6 were significantly upregulated in astrocytes following the endocytosis of α-syn. Immunofluorescence (IF) showed neuronal dendritic reduction, axon elongation and increased co-localisation of TNFR1 receptor expression. Western blot showed up-regulation of PKR, P-eIF2α and ATF4 protein expression. Conversely, after antagonizing neuronal TNFR1 receptors with the R-7050 chemical inhibitor, neuronal synaptic structure was significantly restored and the expression of PKR, P-eIF2α and ATF4 was down-regulated. In summary, TNF-α acts as a signaling molecule mediating the up-regulated astrocyte-neuron crosstalk, providing new insights into the pathogenesis of α-syn-related neurological disorders., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)