NMNAT2基因敲除HEK293细胞

Axon degeneration is a central pathological feature of many neurodegenerative diseases. Sterile alpha and Toll/interleukin-1 receptor motif-containing 1 (SARM1) is a nicotinamide adenine dinucleotide (NAD)-cleaving enzyme whose activation triggers axon destruction. Loss of the biosynthetic enzyme NMNAT2, which converts nicotinamide mononucleotide (NMN) to NAD, activates SARM1 via an unknown mechanism. Using structural, biochemical, biophysical, and cellular assays, we demonstrate that SARM1 is activated by an increase in the ratio of NMN to NAD and show that both metabolites compete for binding to the auto-inhibitory N-terminal armadillo repeat (ARM) domain of SARM1. We report structures of the SARM1 ARM domain bound to NMN and of the homo-octameric SARM1 complex in the absence of ligands. We show that NMN influences the structure of SARM1 and demonstrate via mutagenesis that NMN binding is required for injury-induced SARM1 activation and axon destruction. Hence, SARM1 is a metabolic sensor responding to an increased NMN/NAD ratio by cleaving residual NAD, thereby inducing feedforward metabolic catastrophe and axonal demise.

NMNAT2 is an essential but labile protein required for axon integrity. It is rapidly degraded after nerve injury, promoting axon degeneration. However, the mechanisms regulating NMNAT2 ubiquitination and turnover in neurons remain unclear. In this study, we identify the F-box protein FBXO21 as an NMNAT2-binding protein, and its deficiency confers axonal protection via increasing NMNAT2 abundance. FBXO21 recruits SKP1, CUL1, and RBX1 to form an SCFFBXO21 complex, which promotes NMNAT2 ubiquitination in vivo and in vitro. SCFFBXO21 ubiquitinates NMNAT2 at K155 within an isoform-specific targeting and interaction domain of the family of NMNATs, which underlies the unique labile nature of NMNAT2. The ubiquitination-deficient NMNAT2-K155R exhibits substantially reduced protein turnover and enhanced axon-protective capacity. Finally, in Fbxo21 knockout mice, NMNAT2 levels are markedly increased and the survival of injured sciatic nerves is significantly prolonged. Collectively, our findings reveal a crucial role of FBXO21 in axon degeneration, highlighting the SCFFBXO21 complex as a potential target for modulating NMNAT2-dependent axon survival.

SARM1 is an inducible NAD hydrolase that is the central executioner of pathological axon loss. Recently, we elucidated the molecular mechanism of SARM1 activation, demonstrating that SARM1 is a metabolic sensor regulated by the levels of NAD and its precursor, nicotinamide mononucleotide (NMN), via their competitive binding to an allosteric site within the SARM1 N-terminal ARM domain. In healthy neurons with abundant NAD, binding of NAD blocks access of NMN to this allosteric site. However, with injury or disease the levels of the NAD biosynthetic enzyme NMNAT2 drop, increasing the NMN/ NAD ratio and thereby promoting NMN binding to the SARM1 allosteric site, which in turn induces a conformational change activating the SARM1 NAD hydrolase. Hence, NAD metabolites both regulate the activation of SARM1 and, in turn, are regulated by the SARM1 NAD hydrolase. This dual upstream and downstream role for NAD metabolites in SARM1 function has hindered mechanistic understanding of axoprotective mechanisms that manipulate the NAD metabolome. Here we reevaluate two methods that potently block axon degeneration via modulation of NAD related metabolites, 1) the administration of the NMN biosynthesis inhibitor FK866 in conjunction with the NAD precursor nicotinic acid riboside (NaR) and 2) the neuronal expression of the bacterial enzyme NMN deamidase. We find that these approaches not only lead to a decrease in the levels of the SARM1 activator NMN, but also an increase in the levels of the NAD precursor nicotinic acid mononucleotide (NaMN). We show that NaMN inhibits SARM1 activation, and demonstrate that this NaMN-mediated inhibition is important for the long-term axon protection induced by these treatments. Analysis of the NaMN-ARM domain co-crystal structure shows that NaMN competes with NMN for binding to the SARM1 allosteric site and promotes the open, autoinhibited configuration of SARM1 ARM domain. Together, these results demonstrate that the SARM1 allosteric pocket can bind a diverse set of metabolites including NMN, NAD, and NaMN to monitor cellular NAD homeostasis and regulate SARM1 NAD hydrolase activity. The relative promiscuity of the allosteric site may enable the development of potent pharmacological inhibitors of SARM1 activation for the treatment of neurodegenerative disorders.