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Astrocyte-specific NRCAM deficiency promotes GABAergic synapse pruning to drive central sensitization in bone cancer
Zuoxia Zhang1, Yunfan Hou1, Yanting Mao1
1Department of Anesthesiology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, Jiangsu, China.
Background:
Astrocytic activation is critically involved in the development and maintenance of bone cancer pain (BCP). Recent studies have shown that astrocytes participate in synaptic remodeling through synaptic phagocytosis, whereas neuronal cell adhesion molecule (NRCAM) restricts the extension of perisynaptic astrocytic processes and exerts an anti-phagocytic effect. However, whether astrocyte-mediated synaptic phagocytosis contributes to BCP remains to be explored. Hence, this study investigated the role of astrocytic NRCAM deficiency in synaptic remodeling and central sensitization in BCP.
Methods:
A mouse model of BCP was established by intrafemoral inoculation of fibrosarcoma cells. Pain-related behaviors were evaluated by spontaneous pain assessment and mechanical withdrawal threshold testing. Bone destruction and tumor infiltration were examined by hematoxylin and eosin staining. Synaptic alterations and astrocytic activation were assessed by western blotting and immunofluorescence. Astrocyte-mediated synaptic engulfment was analyzed using immunofluorescence, three-dimensional reconstruction, magnetic-activated cell sorting, and Golgi-Cox staining. Moreover, fluorocitrate was used to inhibit the astrocytic activation. Astrocytic NRCAM was overexpressed via adeno-associated virus (AAV)-mediated gene delivery.
Results:
Fibrosarcoma cell inoculation induced persistent spontaneous pain and mechanical hyperalgesia in C3H/HeN mice, accompanied by trabecular bone destruction and tumor infiltration. In the spinal cord, expression of the excitatory synaptic markers VGLUT1 and PSD95 was increased, whereas expression of the inhibitory synaptic markers VGAT and Gephyrin was decreased, together with marked astrocytic activation. Multiple complementary assays consistently showed that astrocytes engulfed both excitatory and inhibitory synapses, with a clear preference for GABAergic synapses. Pharmacological inhibition of astrocytic activation with fluorocitrate alleviated pain behaviors and improved synaptic remodeling. NRCAM expression was significantly downregulated in tumor-bearing mice. Restoration of astrocytic NRCAM expression by AAV markedly reduced astrocyte-mediated engulfment towards GABAergic synapses, had minimal effect on excitatory synapses, and significantly attenuated BCP.
Conclusions:
Reactive astrocytes preferentially phagocytose GABAergic synapses in BCP, thereby contributing to synaptic imbalance and central sensitization. This process is associated with downregulation of astrocytic NRCAM. Restoring astrocytic NRCAM alleviates BCP by suppressing excessive astrocyte-mediated phagocytosis of GABAergic synapses. These findings identify astrocytic NRCAM-dependent synaptic phagocytosis as an unrecognized mechanism underlying BCP and as a potential therapeutic target.
