NAXE deficiency: A neurometabolic disorder of NAD(P)HX repair amenable for metabolic correction

Abstract

The NAD(P)HX repair system is a metabolite damage repair mechanism responsible for restoration of NADH and NADPH after their inactivation by hydration. Deficiency in either of its two enzymes, NAD(P)HX dehydratase (NAXD) or NAD(P)HX epimerase (NAXE), causes a fatal neurometabolic disorder characterized by decompensations precipitated by inflammatory stress. Clinical findings include rapidly progressive muscle weakness, ataxia, ophthalmoplegia, and motor and cognitive regression, while neuroimaging abnormalities are subtle or nonspecific, making a clinical diagnosis challenging. During stress, nonenzymatic conversion of NAD(P)H to NAD(P)HX increases, and in the absence of repair, NAD(P)H is depleted, and NAD(P)HX accumulates, leading to decompensation; however, the contribution of each to the metabolic derangement is not established. Herein, we summarize the clinical knowledge of NAXE deficiency from 30 cases and lessons learned about disease pathogenesis from cell cultures and model organisms and describe a metabolomics signature obtained by untargeted metabolomics analysis in one case at the time of crisis and after initiation of treatment. Overall, biochemical findings support a model of acute depletion of NAD⁺, signs of mitochondrial dysfunction, and altered lipidomics. These findings are further substantiated by untargeted metabolomics six months post-crisis showing that niacin supplementation reverses primary metabolomic abnormalities concurrent with improved clinical status.

Keywords: Fever induced encephalopathy; Mitochondrial dysfunction; NAD(+); NAD(P)HX epimerase; NAXE; Neurometabolic disorders; PEBEL1; Pellagra.

Fig. 1.

The generation of NAD(P)HX and its repair by NAXD and NAXE. The formation of NAD(P)HX, a hydrated form of NAD(P)H, occurs either nonenzymatically from NAD(P)H or from side reactions of other enzymes (e.g., glyceraldehyde-3-phosphate dehydrogenase, GAPDH), and is accelerated by acidic or hyperthermic conditions. NAD(P)HX can inhibit cellular dehydrogenases (red arrows) and can further cyclize into a toxic metabolite that is thought to inhibit the mitochondrial respiratory chain [13]. The repair of NAD(P)HX is performed by its cofactor repair system, comprised of NAXD and NAXE (green arrows).

Fig. 2.

Plasma untargeted metabolomics during an acute crisis and 6 months post-niacin therapy. A. Alterations in analytes within the NAD⁺ catabolic pathways. In red, taken at time of crisis, there is absence of N¹-methylnicotinamide (MNA) and adenosine-diphosphoribose (ADPR), and low N¹-methyl-2-pyridone-5-carboxamide (2PY) (Z-score − 7.5). NAD⁺ is metabolized to nicotinamide (NAM) and ADPR by several intracellular enzymes; the former is further methylated by nicotinamide-N-methyltransferase (NNMT) into MNA, which is oxidized to 2PY (see Fig. 3 for pathway). Significantly low levels of these analytes strongly indicate low level of available NAD⁺. Levels of NAD⁺/NADH in biofluids are not accurately analyzed in clinical metabolomics of biofluids. NAM itself is only modestly reduced (Z-score − 1.5) during crisis (red), see discussion above. Relative analyte levels 6 months post-crisis (green) show repletion while on niacin supplementation. B-D: Heat map visualization of altered metabolite Z-scores (+1.5>Z-score < −1.5) (B) amino acids and their metabolites; (C) lysophospholipids, phosphatidylcholines, phosphatidylethanolamines, plasmalogens; and, (D) fatty acids and acylcarnitines are shown. Red shades represent higher Z-scores, blue – lower Z-scores. The Z-scores illustrate whether a given metabolite is significantly altered relative to standardized references (see Methods in the Supplementary File). In general, a Z-score greater than +1.75 or lower than −1.75 is considered significantly different from the control. B. Alterations in amino acids during crisis and relative normalization after 6 months of niacin therapy. Isovalerylglycine (Z-score + 2.6), 2-hydroxy-3-methylvalerate (Z-score + 2.4), isobutyrylglycine (Z-score + 2.2), and arginine (Z-score + 2.0), elevated at time of crisis, are associated with mitochondrial dysfunction and normalized post-treatment. In addition, elevation of asparagine (Z-score + 2) and reduction of hydroxyasparagine (Z score − 2.7) may indicate asparaginyl hydroxylase inhibition, which can be seen during hypoxic stress [51-53]. Elevated 2-pyrrolidinone (Z-score + 2.4) and guanidinobutanoate (Z-score + 2.4) can indicate inhibition of succinic semialdehyde dehydrogenase (SSADH), which utilizes NAD(P)⁺ as a substrate (the more pathognomonic metabolite, γ-hydroxybutyrate, is not detected in our untargeted assay). All these derivatives were normalized with long-term niacin supplementation. C. and D. Heat maps of lipid alterations show accumulation of several 18-carbon diacylglycerols and 16- and 18-carbon phosphatidylcholines, and depletion of lysophospholipids and several medium chain-fatty acids and acylcarnitines, with a reversion of these perturbations post-crisis. See text for further discussion.

Fig. 3.

Selected pathways of NAD⁺ metabolism. NAD⁺ is generated and consumed by multiple cellular pathways. Left, in black shading, the repair system restores spontaneously generated NAD(P)HX back to NAD(P)H. NAD⁺ is utilized by several enzymes, including members of the sirtuin family (SIRT), poly-ADP-ribose polymerase (PARP), and cyclic ADP ribose hydrolase (CD38). The product of SIRT and PARP, nicotinamide (NAM), can be recycled back to NAD⁺ by the salvage pathway (green shading, top right) or is further converted into MNA by NNMT and the co-factor SAM. MNA is then oxidized by aldehyde oxidase (AOX1) into 2PY. This degradation is highlighted by the red shading, lower right. The de novo (kynurenine) NAD⁺ biosynthesis pathway is indicated in the bottom left. Abbreviations: NMN = nicotinamide mononucleotide; NAMPT = nicotinamide phosphoribosyltransferase; NMNAT = nicotinamide mononucleotide adenylyl transferases. TRP = tryptophan; Quin = quinolinic acid; NAM = nicotinamide. ADPR = adenosine-5′-diphosphoribose; MNA = N¹-methylnicotinamide; SAM = S-adenosylmethionine; AOX1 = aldehyde oxidase; 2PY = N¹-methyl-2-pyridone-5-carboxamide.