Gap junction intercellular communications regulates activation of SARM1 and protects against axonal degeneration

Sterile alpha and Toll/interleukin-1 receptor motif containing 1 (SARM1), a nicotinamide adenine dinucleotide (NAD)-utilizing enzyme, mediates axon degeneration (AxD) in various neurodegenerative diseases. It is activated by nicotinamide mononucleotide (NMN) to produce a calcium messenger, cyclic ADP-ribose (cADPR). This activity is blocked by elevated NAD level. Here, we verified this metabolic regulation in somatic HEK-293T cells by overexpressing NMN-adenyltransferase to elevate cellular NAD, which resulted not only in inhibition of their own SARM1 from producing cADPR but, surprisingly, also in the 5-10 neighboring wildtype cells in mixed cultures via connexin (Cx)-43. Direct visualization of gap junction intercellular communication (GJIC) was achieved by incubating cells with a permeant probe, PC11, which is converted by SARM1 into PAD11, a fluorescent NAD analog capable of traversing GJs. Extending the findings to dorsal root ganglion neurons, we further showed that CZ-48, a permeant NMN analog, or axotomy, activated SARM1 and the produced PAD11 was transferred to contacting axons via GJIC. The gap junction involved was identified as Cx36 instead. This neuronal GJIC was demonstrated to be functional, enabling healthy neurons to protect adjacent axotomized axons from degeneration. Inhibition of GJIC in mice by AAV-PHP.eB-mediated knockdown of Cx36 in brain induced neuroinflammation, which in turn activated SARM1 and resulted in axon degeneration as well as behavioral deficits. Our results demonstrate a novel intercellular regulation mechanism of SARM1 and reveal a protective role of healthy tissue against AxD induced by injury or neuroinflammation.