NAD synthase NMNAT acts as a chaperone to protect against neurodegeneration

Abstract

Neurodegeneration can be triggered by genetic or environmental factors. Although the precise cause is often unknown, many neurodegenerative diseases share common features such as protein aggregation and age dependence. Recent studies in Drosophila have uncovered protective effects of NAD synthase nicotinamide mononucleotide adenylyltransferase (NMNAT) against activity-induced neurodegeneration and injury-induced axonal degeneration. Here we show that NMNAT overexpression can also protect against spinocerebellar ataxia 1 (SCA1)-induced neurodegeneration, suggesting a general neuroprotective function of NMNAT. It protects against neurodegeneration partly through a proteasome-mediated pathway in a manner similar to heat-shock protein 70 (Hsp70). NMNAT displays chaperone function both in biochemical assays and cultured cells, and it shares significant structural similarity with known chaperones. Furthermore, it is upregulated in the brain upon overexpression of poly-glutamine expanded protein and recruited with the chaperone Hsp70 into protein aggregates. Our results implicate NMNAT as a stress-response protein that acts as a chaperone for neuronal maintenance and protection. Our studies provide an entry point for understanding how normal neurons maintain activity, and offer clues for the common mechanisms underlying different neurodegenerative conditions.

Figure 1. NMNAT suppresses toxicity induced by ataxin-1

a–h, Ommatidial morphology of cross section (a–d) and horizontal section (e–h) of 15-day-old control fly (a, e), and flies overexpressing dAtx-1 (b–d, f–h). Overexpression of wild-type NMNAT (c, g) or enzyme-inactive NMNAT-WR (d, h) restores the rhabdomere degeneration phenotype. i–p, Eye exterior morphology and ommatidial morphology of two-day-old control fly (i, m), and flies overexpressing hAtx-1[82Q] (j–l, n–p). Overexpression of hAtx-1[82Q] leads to rough eye (j) and reduced rhabdomere size (n) phenotypes that are partly suppressed by co-expression of NMNAT (k, o) or NMNAT-WR (l, p).

Figure 2. NMNAT acts as a chaperone in cultured cells

a–c, Cos7 cells are co-transfected with hAtx-1[82Q]-GFP (green) and NMNAT (red). TOTO3 staining (blue) marks nuclei. Scale bar in a for a–c, 20 μm. d, Quantification of GFP fluorescence. Aggregates are defined as objects at least 0.4 μm² and 3,600 intensity units. Error bars, s.e.m. *P < 0.05, **P < 0.01, ***P < 0.005 (analysis of variance post-hoc test). e–h, Western analysis (e, f) and quantification (g, h) of detergent-insoluble fractions of cells transfected with hAtx-1[82Q]-GFP (e) or hAtx-1[2Q]-GFP (f) and vector, NMNAT or Hsp70, and treated with either DMSO or 10 μM MG132. The level of insoluble hAtx-1[82Q] (g) or hAtx-1[2Q] (h) in the DMSO-treated cells is equal to 1 (fold). Error bars, s.e.m.; n = 4.

Figure 3. Endogenous and overexpressed NMNAT proteins are recruited with Hsp70 into the hAtx-1[82Q] aggregates

a–e, Seven-day-old adult fly brains were stained for NMNAT (green; f–j), hAtx-1[82Q] (red; k–o) and Hsp70 (blue; p–t). Higher magnifications of boxed areas are shown in u–y. Endogenous NMNAT is primarily nuclear (f). hAtx-1[82Q] forms nuclear aggregates (l). Endogenous NMNAT is upregulated and recruited to the aggregates (g). NMNAT is distributed to the cytoplasm when singly overexpressed (h), but is recruited to ataxin aggregates when co-expressed with hAtx-1[82Q] (i, n). Enzymatically inactive NMNAT-WR is also recruited into ataxin aggregates when co-overexpressed with hAtx-1[82Q] (j, o). Hsp70 is upregulated and recruited into the aggregates (q, s, t).

Figure 4. In vivo and in vitro chaperone activity assays

a, b, Luciferase activity was measured without heat shock as controls (blue columns), after 15 min heat shock at 45 °C (red columns) or after 3 h recovery at room temperature (yellow columns). The luciferase activities relative to the controls are displayed. Error bars, s.e.m. *P <0.05, **P <0.01; (analysis of variance post-hoc test); n = 4, triplicate sampling. c, Protein aggregation and its inhibition by chaperones. d–h, Aggregation of citrate synthase is prevented by addition of Hsp70 (e, g), NMNAT (f, g) or human hsNMNAT3 (g) but not by lysozyme (d). g, The relative aggregation rate is calculated by the change in absorbance per minute and the aggregation rate of CS alone is set to 1. (h) The percentage of remaining aggregation is calculated at the saturation point (8000 s). i, Insulin aggregation is inhibited by Hsp70, NMNAT, NMNAT-WR or hsNMNAT3 but not BSA or lysozyme.