NAD and axon degeneration: from the Wlds gene to neurochemistry

Abstract

Neurodegenerative diseases have become a global issue due to the aging population. These disorders affect a vast patient population and represent a huge area of unmet therapeutic need. Axon degeneration is a common pathological character of those neurodegenerative diseases. It results in the loss of communication between neurons. Two decades ago, the Wallerian degeneration slow (Wlds) mouse strain was identified, in which the degeneration of transected axons is delayed. The phenotype is attributed to the overexpression of a chimeric protein Wlds which contains a short fragment of the ubiquitin assembly protein UFD2 and the full-length nicotinamide adenine dinucleotide (NAD) synthetic enzyme Nicotinamide mononucleotide adenylyl-transferase-1 (Nmnat-1). However, the underlying molecular mechanism remains largely unknown. Recently, it's reported by independent researchers that the full length coding sequence of mouse Nmnat-1 could mimic the axonal protective effect of the Wlds gene when overexpressed in primary neural cultures. Together with a significant number of subsequential reports, this finding highlighted the substantial role of nicotinamide adenine dinucleotide (NAD) in the process of axon degeneration. Here we reviewed the history of axon degeneration research from a neurochemical standpoint and discuss the potential involvement of NAD synthesis, NAD consumption and NAD-dependent proteins and small molecules in axon degeneration.

Figure 1

The components of the Wlds chimeric gene. The Wlds gene contains the N-terminal 70-amino acid short fragment of the ubiquitin assembly protein UFD2 and the full-length nicotinamide adenine dinucleotide (NAD) synthetic enzyme Nicotinamide mononucleotide adenylyl-transferase-1 (Nmnat-1).

Figure 2

The mammalian NAD synthesis pathways. In mammalian cells, NAD could be synthesized from three different precursors, such as from tryptophan (the de novo synthesis), as while as from nicotinamide or nicotinic acid (the salvage synthesis). Nmnat-1 is a indispensable enzyme controlling the last step of both de novo and salvage NAD biosynthesis pathways. Nicotinamide represents the main source of NAD in most cells in mammals, including neurons, and it is also the product of NAD⁺ hydrolysis catalyzed by NAD⁺ consuming proteins.

Figure 3

The method used to measure axonal NAD⁺ level. Axons from 12 culture DRG explants were washed with PBS and extracted with perchloric acid. The precipitated protein was separated by centrifuge. The amount of the precipitated protein was measured by the modified BCA protein assay. The protein-free extract was neutralized and applied to HPLC. The axonal NAD⁺ level is normalized against the protein contents in the same sample and expressed as nmol/mg.

Figure 4

NAD⁺ is essential for ATP-synthesizing redox reactions. Both cytosolic glycolysis and mitochondrial oxidative phosphorylation required the participation of NAD⁺. Cytosolic NAD⁺ is required in the glycolytic pathway for the conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate. Thus, upon cytosolic NAD⁺ depletion, glucose can no longer be converted to the pyruvate needed to fuel the oxidative phosphorylation in mitochondria.

Figure 5

The interaction between NAD⁺ and ATP during Wallerian degeneration. (A) Increase of the AMP/ATP ration indicates low cellular energy level, and would induce the activation of AMPKK, which would further catalyze the phosphorylation of AMPK. (B) NAD⁺ is required for ATP generation, while ATP is one substrate of NAD synthesis from nicotinamide. (C) the proposed molecular pathway participated in axon degeneration.

Figure 6

The three families of NAD⁺ consuming proteins. All the reactions catalyzed by these NAD consuming proteins use NAD⁺ as a donor of ADPR, while generating nicotinamide (NAm) as a side product. Nicotinamide has the product inhibition effect on the enzymatic activities of most of the NAD⁺ consuming proteins, probably by competing for the NAD⁺ binding pocket.

Figure 7

PBEF inhibitor FK866 induces neuronal atrophy in cultured hippocampal neurons (A and B) The effects of FK866 on cellular NAD and ATP levels. Mature hippocampal neurons (DIV = 22) were treated with FK866 at different concentration, and collected at indicated time-points post-KF866 treatment for HPLC analysis. The NAD and ATP levels were expressed as percentages of the control groups without treatment. (C) Representative confocal images showing that FK866 treatment induces decreases of dendritic spines and dendritic branching in hippocampal neurons. DIV 25 neurons were treated with FK866 (10 nM) alone, or FK866 (10 nM) together with NAD (5 mM) for three days. To visualized individual neurons, the same cultures were transfected with enhanced GFP expressing plasmids by the calcium phosphate transfection method. Hippocampal neurons were cultured at a high density (about 1,000 neurons per mm²). Transfection efficiency is less than 0.1%. The top panel shows the representative higher magnification views of dendritic segments (Scale bar: 5 µm). The bottom panel is the representative images of whole the entire neuron showing dendritic branching patterns (Scale bar: 50 µm).

Figure 8

Age-related NAD decrease in mouse tissues (A–D) Hippocampus (A), cerebellum (B), cortex (C) or liver (D) tissues from mice of indicated age were subjected to HPLC analysis. NAD levels were normalized against the total weight of the tissue and expressed as nmol/mg. Quantification was performed on results from duplicate experiments from at least three animals at each age. Statistical analysis was done by student t-test. **p < 0.01, ***p < 0.001.