WldS but not Nmnat1 protects dopaminergic neurites from MPP+ neurotoxicity

Abstract

Background: The WldS mouse mutant ("Wallerian degeneration-slow") delays axonal degeneration in a variety of disorders including in vivo models of Parkinson's disease. The mechanisms underlying WldS -mediated axonal protection are unclear, although many studies have attributed WldS neuroprotection to the NAD+-synthesizing Nmnat1 portion of the fusion protein. Here, we used dissociated dopaminergic cultures to test the hypothesis that catalytically active Nmnat1 protects dopaminergic neurons from toxin-mediated axonal injury.

Results: Using mutant mice and lentiviral transduction of dopaminergic neurons, the present findings demonstrate that WldS but not Nmnat1, Nmnat3, or cytoplasmically-targeted Nmnat1 protects dopamine axons from the parkinsonian mimetic N-methyl-4-phenylpyridinium (MPP+). Moreover, NAD+ synthesis is not required since enzymatically-inactive WldS still protects. In addition, NAD+ by itself is axonally protective and together with WldS is additive in the MPP+ model.

Conclusions: Our data suggest that NAD+ and WldS act through separate and possibly parallel mechanisms to protect dopamine axons. As MPP+ is thought to impair mitochondrial function, these results suggest that WldS might be involved in preserving mitochondrial health or maintaining cellular metabolism.

Figure 1

Wld^s protects dopaminergic neurons from MPP⁺ toxicity. (A) Dissociated dopaminergic cultures from both WT and Wld^s mice were treated with 2 μm MPP⁺ for 48 hours, and processed for TH immunoreactivity. (B) Quantification of TH+ cell bodies and (C) TH+ neurites was done using unbiased stereology. Data are normalized to control cultures and denote the mean ± SEM of representative determinations made in three separate cultures. *p < 0.01; **p < 0.001.

Figure 2

Cytoplasmic Wld^s protects dopaminergic neurons from MPP⁺ toxicity. (A) Dissociated dopaminergic cultures from both WT and cyto Wld^s mice were co-stained with TH and Wld^s antibodies to confirm the subcellular localization of Wld^s. (B) Cultures were treated with 2 μm MPP⁺ for 48 hours prior to fixing and staining. (C) Quantification of TH+ cell bodies and (D) TH+ neurites shows that cytoplasmic Wld^S protected both cell bodies and neurites against MPP⁺. Data are normalized to control cultures and denote the mean ± SEM of representative determinations made in three separate cultures. *p < 0.05.

Figure 3

Nmnat by itself does not protect dopaminergic neurons from MPP⁺ toxicity. (A) Diagram of the lentiviral constructs used to transduce WT dissociated dopaminergic neurons. (B) Western blot of cell lysates from transduced primary midbrain cultures illustrates that all the transduced transgenes exhibit similar levels of expression. (C) Similar transduction efficiencies of the different lentiviruses were confirmed by quantifying the number of TH+ and GFP+ cells following transduction of dopaminergic cultures. (D) Quantification of TH+ cell bodies and (E) TH+ neurites show that only Wld^S-transduced cultures protected neurites against MPP⁺. Data are normalized to control cultures and denote the mean ± SEM of representative determinations made in three separate cultures. *p < 0.001.

Figure 4

Wld^s and cytoplasmic Nmnat1 protect DRG axons from vincristine toxicity. (A) DRG cultures from E14 mice transduced with GFP, Wld^s, cyto Nmnat1, or inactive Wld^s were processed for acetylated tubulin immunoreactivity 24 hours after vincristine treatment. Inserts in bottom middle panels show 40× images of DRG cell bodies transduced with Wld^s and cytoplasmic Nmnat1, respectively, to illustrate no overt nuclear enrichment of Nmnat1. (B) Quantification of neurites shows that both Wld^s and cyto Nmnat1 protects DRG neurites from vincristine toxicity. Data are normalized to control cultures and denote the mean ± SEM of representative determinations made in three separate cultures. *p < 0.05.

Figure 5

Inactive Wld^s also protects dopaminergic neurons from MPP⁺ toxicity. (A) Dissociated dopaminergic neurons transduced with GFP or inactive Wld^s were treated and processed as described. (B) Quantification of TH+ cell bodies and (C) TH+ neurites. Data are normalized to control cultures and denote the mean ± SEM of representative determinations made in three separate cultures. *p < 0.05.

Figure 6

NAD⁺ protects dopaminergic cells and neurites from MPP⁺ toxicity. (A) NAD⁺ biosynthetic pathway [75]. (B) Dissociated WT dopaminergic cultures were pretreated with NAD⁺, Nmn, or Namn 24 hours before addition of 2 μm MPP⁺. Quantification of TH+ cell bodies and (C) TH+ neurites show that NAD⁺ and Nmn, but not Namn, protected cells and neurites from MPP⁺. Data are normalized to control cultures and denote the mean ± SEM of representative determinations made in three separate cultures. *p < 0.05, **p,0.01, ***p < 0.001.

Figure 7

NAD⁺ does not protect dopaminergic neurons through the Sirt1 pathway. (A) Dissociated midbrain cultures from both WT and Sirt1 KO mice were pretreated with NAD⁺ 24 hours before addition of 2 μm MPP⁺. (B) Quantification of TH+ cell bodies and (C) TH+ neurites show that NAD⁺ protects cells and neurites from MPP⁺ in both WT and Sirt1 KO cultures. Data are normalized to control cultures and denote the mean ± SEM of representative determinations made in three separate cultures. *p < 0.05.

Figure 8

The protective effect of NAD⁺ and Wld^S are additive. (A) Dissociated midbrain cultures from both WT and Wld^S mice were pretreated with NAD⁺ 24 hours before addition of 2 μm MPP⁺. (B) Quantification of TH+ cell bodies and (C) TH+ neurites show that NAD⁺ pretreatment was more effective in protecting Wld^S neurites from MPP⁺ versus untreated Wld^S cultures. (D) NAD⁺ dose response curve showing that the protection seen with 1 mM NAD⁺ is maximal. Addition of 10 mM NAD⁺ before MPP⁺ treatment induced 50% cell death in dopaminergic neurons (data not shown). Data are normalized to control cultures and denote the mean ± SEM of representative determinations made in three separate cultures. *p < 0.05; **p < 0.001.