pubmed.ncbi.nlm.nih.gov

Transgenic mice expressing the Nmnat1 protein manifest robust delay in axonal degeneration in vivo - PubMed

  • ️Thu Jan 01 2009

Transgenic mice expressing the Nmnat1 protein manifest robust delay in axonal degeneration in vivo

Yo Sasaki et al. J Neurosci. 2009.

Abstract

Axonal degeneration is a key component of a variety of neurological diseases. Studies using wld(s) mutant mice have demonstrated that delaying axonal degeneration slows disease course and prolongs survival in neurodegenerative disease models. The Wld(s) protein is normally localized to the nucleus, and contains the N terminus of ubiquitination factor Ube4b fused to full-length Nmnat1, an NAD biosynthetic enzyme. While Nmnat enzymatic activity is necessary for Wld(s)-mediated axonal protection, several important questions remain including whether the Ube4b component of Wld(s) also plays a role, and in which cellular compartment (nucleus vs cytosol) the axonal protective effects of Nmnat activity are mediated. While Nmnat alone is clearly sufficient to delay axonal degeneration in cultured neurons, we sought to determine whether it was also sufficient to promote axonal protection in vivo. Using cytNmnat1, an engineered mutant of Nmnat1 localized only to the cytoplasm and axon, that provides more potent axonal protection than that afforded by Wld(s) or Nmnat1, we generated transgenic mice using the prion protein promoter (PrP). The sciatic nerve of these cytNmnat1 transgenic mice was transected, and microscopic analysis of the distal nerve segment 7 d later revealed no evidence of axonal loss or myelin debris, indicating that Nmnat alone, without any other Wld(s) sequences, is all that is required to delay axonal degeneration in vivo. These results highlight the importance of understanding the mechanism of Nmnat-mediated axonal protection for the development of new treatment strategies for neurological disorders.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.

Cytoplasmic Nmnat1 (cytNmnat1) provides more robust axonal protection than Wlds protein or wild-type Nmnat1. A, Representative images of EGFP fluorescence from the DRG cell bodies and phase-contrast images of axons at 0 and 72 h after transection are shown. DRG neurons were infected with relatively low amounts of adenovirus (106 CFU per well) expressing the indicated proteins. Only cytNmnat1-expressing neurons have intact axons 3 d after axotomy. B, The axonal degeneration indices (±SD) were plotted against time after axotomy. The number indicated to the right of each plot is the relative expression level of each protein calculated from EGFP fluorescent images (see Materials and Methods). Note that axons expressing cytNmnat1 were protected at lower expression levels than those expressing Nmnat1 or Wlds. C–E, To determine a wider dose–response curve, DRG neurons were infected with the indicated amount of adenovirus (103 to 107 CFU per well) and analyzed as above. The relative transgene expression was plotted against the axonal degeneration index 3 d after axotomy. DRG cultures from three independent litters of mice (17–19 wells total) were analyzed for each protein. Axons from Nmnat1- and Wlds-expressing DRG neurons remained intact at relative transgene expression levels above 16 a.u. and 1.6 a.u., whereas axons from cytNmnat1-expressing DRG neurons showed no axonal degeneration until transgene expression levels dropped below 0.3 a.u. Importantly, at expression levels between 0.3 and 1 a.u., axons from cytNmnat1-expressing neurons were significantly more intact than those of Nmnat1-, or Wlds-expressing DRGs (p < 0.01 by ANOVA and Tukey's post hoc test). Note that DI > 0.2 corresponds to visibly degenerated axons. Scale bar, 100 μm.

Figure 2.
Figure 2.

cytNmnat1 is present in axons. DRG neurons were infected with lentivirus expressing 6xHis epitope-tagged Nmnat1 or cytNmnat1. The infected neurons were stained with anti-6xHis antibody. Nmnat1 was predominantly expressed in the nucleus and cytNmnat1 is expressed diffusely in the cytosol (top row). cytNmnat1 was detected in axons at high levels, whereas no Nmnat1 was detected within the axon (middle row). Phase-contrast pictures corresponding to the axonal area stained with anti-6xHis antibody are shown (bottom row). Scale bar, 25 μm.

Figure 3.
Figure 3.

Nmnat enzymatic activity is increased in Prp-cytNmnat1 Tg mouse brain. A, cytNmnat1 expression levels in lysates prepared from the 6-week-old brains of two independent PrP-cytNmnat1 Tg mice of all four (A–D) lines and wild-type (wt) mice were analyzed by Western blotting using anti-6xHis tag antibody. Transgene expression was observed in all four lines, with the lowest level in line A and the highest level in lines C and D. B, Nmnat enzymatic activity was measured in 6-week-old brain lysates prepared from each of the PrP-cytNmnat1 Tg lines, wlds, and wild-type mice. Each of the four Tg lines showed significant increases in Nmnat enzymatic activity that was well correlated with the protein expression levels observed in A. Three mice of each genotype were analyzed and the data are displayed as mean ± SD.

Figure 4.
Figure 4.

Neuronal expression of the cytNmnat1 transgene. Immunofluorescence with the anti-6xHis tag antibody was used to examine the expression of the cytNmnat1 transgene in 6-week-old PrP-cytNmnat1(D) Tg brain (A, B, C, E) and DRG (F). Wild-type brain is shown in D. The highest cytNmnat1 expression was detected in the hippocampus (A). Higher magnification revealed cytNmnat1 expression in the cytosol and neuronal projections in the CA1 pyramidal cells, cortical neurons, and neurons in the dentate gyrus (B, C, E; arrows indicating representative neuronal projections). High levels of cytNmnat1 were also detected in DRG neurons (F). Scale bars: (in D) A, D, 250 μm; (in F) B, C, E, F, 50 μm.

Figure 5.
Figure 5.

Delayed axonal degeneration in cultured DRG neurons from PrP-cytNmnat1(A) Tg mice. A, Axons from wild-type and PrP-cytNmnat1(A) Tg DRG neurons were transected after 14 d in culture. Degeneration indices were calculated 0, 1, 2, and 3 d later. Wild-type axons were completely degenerated after 1 d whereas transgenic axons were intact even 3 d after injury (n = 10). B, DRG neurons from PrP-cytNmnat1(A) Tg mice were cultured for 14 d, then treated with 4 μ

m

vincristine. Degeneration indices were calculated at 2 and 3 d after vincristine addition. Wild-type axons were completely degenerated after 2 d whereas transgenic axons were intact 3 d after vincristine treatment (n = 10). *Significant difference (p < 0.001, Student's t test) between neurons from PrP-cytNmnat1 and wild type of the indicated time.

Figure 6.
Figure 6.

Axonal degeneration after sciatic nerve transection is delayed in PrP-cytNmnat1 Tg mice. A, Neurofilament (165 kDa component) levels remaining in the distal sciatic nerve segment 7 d after transection were measured by Western blot using anti-neurofilament antibody to examine axonal loss. Neurofilament levels in transected distal segment (i) of PrP-cytNmnat1(D) Tg mice were equivalent to those in the uninjured control (c) nerve. No neurofilament remained in the wild-type distal segment after injury. Transected distal segment from wlds mice also contained neurofilament. B, Plastic thin sections (4 μm) of distal sciatic nerve taken from PrP-Nmnat1(A–D) Tg, wlds, or wild-type mice 7 d after transection were stained with toluidine blue. Axons and the myelin structures of PrP-cytNmnat1 Tg and wlds mice were well preserved, whereas the wild-type nerve structure was completely disorganized with massive axonal loss and large amounts of myelin debris. C, Electron microscopic evaluation of these nerves revealed that the wild-type axonal structure was completely degenerated 7 d after axotomy (middle) compared with the uninjured nerve control (top). In contrast, the distal nerve from Prp-cytNmnat1(D) was well preserved (bottom). Higher magnification clearly showed an intact nerve structure including microtubules and mitochondria inside the axon in the transected transgenic nerve. Scale bars: B, 50 μm; C, left column, 10 μm; C, right column, 2 μm.

Figure 7.
Figure 7.

Axonal and synaptic integrity are both protected in PrP-cytNmnat1(D) Tg mice after axotomy. The CMAP amplitude is shown as a percentage of the initial value (0 h) after stimulation distal to site of sciatic transaction in wild-type, PrP-cytNmnat1(D) Tg, and wlds mice. At 10 weeks of age, wild-type mice had no response 2 d after axotomy, whereas PrP-cytNmnat1 and wlds mice retained ∼80% of initial CMAP amplitude. This response declined to ∼10% in both PrP-cytNmnat1 Tg and wlds mice at 7 d after axotomy. At 17 weeks, PrP-cytNmnat1 Tg mice had an ∼90% decline in the CMAP response 7 d after axotomy, similar to that observed in 10-week-old Tg mice. * and ** indicate significant difference (p < 0.001 for * and p < 0.05 for **, Student's t test) between relative CMAP at indicated time and 0 h. *** indicates no significant difference (p > 0.1, Student's t test) between 10-week-old and 17-week-old PrP-cytNmnat1 of CMAP at indicated time.

Similar articles

Cited by

References

    1. Araki T, Sasaki Y, Milbrandt J. Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration. Science. 2004;305:1010–1013. - PubMed
    1. Avery MA, Sheehan AE, Kerr KS, Wang J, Freeman MR. Wld S requires Nmnat1 enzymatic activity and N16-VCP interactions to suppress Wallerian degeneration. J Cell Biol. 2009;184:501–513. - PMC - PubMed
    1. Baloh RH, Strickland A, Ryu E, Le N, Fahrner T, Yang M, Nagarajan R, Milbrandt J. Congenital hypomyelinating neuropathy with lethal conduction failure in mice carrying the Egr2 I268N mutation. J Neurosci. 2009;29:2312–2321. - PMC - PubMed
    1. Beirowski B, Babetto E, Gilley J, Mazzola F, Conforti L, Janeckova L, Magni G, Ribchester RR, Coleman MP. Non-nuclear Wld(S) determines its neuroprotective efficacy for axons and synapses in vivo. J Neurosci. 2009;29:653–668. - PMC - PubMed
    1. Berger F, Lau C, Dahlmann M, Ziegler M. Subcellular compartmentation and differential catalytic properties of the three human nicotinamide mononucleotide adenylyltransferase isoforms. J Biol Chem. 2005;280:36334–36341. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources