Nicotinamide mononucleotide adenylyl transferase-mediated axonal protection requires enzymatic activity but not increased levels of neuronal nicotinamide adenine dinucleotide

Abstract

Axonal degeneration is a hallmark of many neurological disorders. Studies in animal models of neurodegenerative diseases indicate that axonal degeneration is an early event in the disease process, and delaying this process can lead to decreased progression of the disease and survival extension. Overexpression of the Wallerian degeneration slow (Wld(s)) protein can delay axonal degeneration initiated via axotomy, chemotherapeutic agents, or genetic mutations. The Wld(s) protein consists of the N-terminal portion of the ubiquitination factor Ube4b fused to the nicotinamide adenine dinucleotide (NAD(+)) biosynthetic enzyme nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1). We previously showed that the Nmnat1 portion of this fusion protein was the critical moiety for Wld(s)-mediated axonal protection. Here, we describe the development of an automated quantitative assay for assessing axonal degeneration. This method successfully showed that Nmnat1 enzymatic activity is important for axonal protection as mutants with reduced enzymatic activity lacked axon protective activity. We also found that Nmnat enzymes with diverse sequences and structures from various species, including Drosophila melanogaster, Saccharomyces cerevisiae, and archaebacterium Methanocaldococcus jannaschii, which encodes a protein with no homology to eukaryotic Nmnat enzymes, all mediate robust axonal protection after axotomy. Besides the importance of Nmnat enzymatic activity, we did not observe changes in the steady-state NAD(+) level, and we found that inhibition of nicotinamide phosphoribosyltransferase (Nampt), which synthesizes substrate for Nmnat in mammalian cells, did not affect the protective activity of Nmnat1. These results provide the possibility of a role for new Nmnat enzymatic activity in axonal protection in addition to NAD(+) synthesis.

Figure 1.

Quantitative axonal degeneration assay. A, Representative pictures of original images, binarized images, and fragmented axons from intact and degenerated DRG axons. B, DRG neurons were cultured for 14 d, and the axons were then transected. Representative images of the same fields of axons at the indicated times after axotomy are shown. The time after axotomy is shown above, and the degeneration index is shown below, each image. C, Images from the same field after axotomy were analyzed by two different methods. The bar graph shows the percentage of remaining axons ± SD calculated with the visual rating system used by Araki et al. (2004). The degeneration index calculated from these images was plotted against time after axotomy and displayed as a line graph. The extent of axonal degeneration detected by these two methods is well correlated.

Figure 2.

Nmnat enzymatic activity is critical for axonal protection. A, Representative images of axons of DRG neurons infected with lentivirus expressing EGFP, Nmnat1, Nmnat1(H24A), or Nmnat1(W170A) are shown. Axons from Nmnat1-expressing DRG neurons were protected after axotomy, whereas axons from Nmnat1(H24A)- or Nmnat1(W170A)-expressing neurons degenerated similarly to those expressing EGFP. B, The quantification of axonal degeneration in DRG neurons expressing EGFP, Nmnat1, Nmnat1(H24A), or Nmnat1(W170A). Twelve fields were evaluated for each condition, and each experiment was repeated three times. The degeneration index value ± SD at 24, 48, and 72 h is displayed. Only Nmnat1 shows significant axonal protection (p < 0.001, n = 3).

Figure 3.

Nmnat axonal protective activity is conserved throughout evolution. A, The Nmnat enzymatic activities from HEK293T cell lysates expressing EGFP, Nmnat1, dNmnat, yNmnat, or mjNmnat demonstrated that each enzyme had similar NAD⁺ synthetic activity. Error bars represent SD. B, The rate of axonal degeneration after injury was quantified in DRG neurons expressing EGFP, mouse Nmnat1, dNmnat, yNmnat, or mjNmnat. Twelve fields were evaluated for each condition, and each experiment was repeated three times. The degeneration index value ± SD at 24, 48, and 72 h is displayed. All Nmnat enzymes, from mammalian to Archaea, delayed axonal degeneration of injured DRG neurons in a comparable manner. Nmnat1-, dNmnat-, yNmnat-, or mjNmnat- versus EGFP-expressing neurons, p < 0.001 (n = 3).

Figure 4.

Axonal degeneration proceeds normally in Cd38^−/−Parp1^−/− mice despite high NAD⁺ levels. A, In vitro axonal degeneration assays were performed using DRG neurons from mice with the indicated genotypes infected with lentivirus expressing cytNmnat1 or EGFP. The DI was calculated as outlined in Materials and Methods. Sixteen fields were evaluated for each condition, and each experiment was repeated three times. The degeneration index value ± SD at 0, 24, 48, and 72 h is displayed. *Significant difference (p < 0.001) between neurons expressing cytNmnat1 and those expressing EGFP of the indicated genotypes. B, Sciatic nerves in mice of the indicated genotypes were transected, and the distal segments were harvested 7 d later. Transverse sections of the distal segments stained with toluidine blue are displayed. While axons from wld^s mice are preserved, degenerating axon profiles are abundant in nerves from Cd38^−/−Parp1^−/− and wild-type (wt) mice.

Figure 5.

The Nampt inhibitor FK866 reduces neuronal NAD⁺ levels but does not affect Nmnat-mediated axonal protection. A, FK866 (1 μm) treatment of DRG neurons expressing either EGFP or cytNmnat1 reduced NAD⁺ levels by ∼65% within 24 h. Error bars represent SD. B, Axonal degeneration assays were performed using neurons expressing EGFP or cytNmnat1 that were treated with FK866 or DMSO (control). The degeneration indices were calculated. Twelve fields were evaluated for each condition, and each experiment was repeated three times. The degeneration index value ± SD at 24, 48, and 72 h is displayed. *Significant difference (p < 0.001) between neurons expressing cytNmnat1 and those expressing EGFP of the indicated genotypes.

Figure 6.

Neurons expressing Nampt siRNA have low levels of NAD⁺, but Nmnat-mediated axonal protection is intact. A, DRG neurons were infected with lentiviruses expressing EGFP or cytNmnat1 as well as siRNAs targeting Nampt or luciferase. After 8 d, NAD⁺ levels were measured using HPLC. Both Nampt siRNAs effectively reduced NAD⁺ levels. Overexpression of cytNmnat1 had no effect on NAD⁺ levels. Values are from three independent experiments; *p < 0.001 versus luciferase siRNA-expressing neurons. Error bars represent SD. B, Axonal degeneration assays were performed using neurons infected with lentiviruses expressing the indicated siRNA and either cytNmnat1 or EGFP, and degeneration indices were calculated. Sixteen fields were evaluated for each condition, and each experiment was repeated three times. The degeneration index value ± SD at 0, 24, 48, and 72 h is displayed. *Significant difference (p < 0.001) between cytNmnat1- and EGFP-expressing neurons expressing either luciferase or Nampt siRNA. Note that Nampt siRNA had no effect on Nmnat-mediated axonal protection.