Metabolomic and transcriptional profiling reveals bioenergetic stress and activation of cell death and inflammatory pathways in vivo after neuronal deletion of NAMPT

Abstract

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the NAD+ salvage pathway. Our previous study demonstrated that deletion of NAMPT gene in projection neurons using Thy1-NAMPT−/− conditional knockout (cKO) mice causes neuronal degeneration, muscle atrophy, neuromuscular junction abnormalities, paralysis and eventually death. Here we conducted a combined metabolomic and transcriptional profiling study in vivo in an attempt to further investigate the mechanism of neuronal degeneration at metabolite and mRNA levels after NAMPT deletion. Here using steady-state metabolomics, we demonstrate that deletion of NAMPT causes a significant decrease of NAD+ metabolome and bioenergetics, a buildup of metabolic intermediates upstream of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in glycolysis, and an increase of oxidative stress. RNA-seq shows that NAMPT deletion leads to the increase of mRNA levels of enzymes in NAD metabolism, in particular PARP family of NAD+ consumption enzymes, as well as glycolytic genes Glut1, Hk2 and PFBFK3 before GAPDH. GO, KEGG and GSEA analyses show the activations of apoptosis, inflammation and immune responsive pathways and the inhibition of neuronal/synaptic function in the cKO mice. The current study suggests that increased oxidative stress, apoptosis and neuroinflammation contribute to neurodegeneration and mouse death as a direct consequence of bioenergetic stress after NAMPT deletion.

Nuclear NAD+-biosynthetic enzyme NMNAT1 facilitates survival of developing retinal neurons

“…The existence of the aforementioned metabolomics studies on tissues deficient in NAMPT, which catalyzes the NAD + synthesis step immediately prior to the NMNAT1-catalyzed step and exhibits dynamic subcellular localization (Svoboda et al, 2019), permits identification of potential NMNAT1-specific metabolic changes. Impaired glycolytic flux appears to be a more general feature of tissue NAD + depletion, as studies reporting NAMPT inhibition or deletion in projection neurons and skeletal muscle myotubes report accumulation of glycolytic metabolites upstream of GAPDH (Oakey et al, 2019;Lundt et al, 2021). Furthermore, utilizing stable isotope tracers, Lundt et al present evidence that glycolysis blockage reverses glycolytic flux in NAMPT-inhibited myotubes, an effect which we believe may explain decreased levels of G3P and DHAP in our model.…”

“…Several recent studies examine levels of select metabolites in mature NMNAT1-deficient retinas (Sasaki et al, 2020a), or more broadly examine tissue metabolomes after perturbation of the NMN-synthesizing enzyme NAMPT (Lin et al, 2016; Oakey et al, 2019; Lundt et al, 2021). Our metabolomics results approximate that NMNAT1 synthesizes ∼40% of the total retinal NAD + pool, which is generally consistent with a previous model (Sasaki et al, 2020a).…”

Nuclear NAD+-biosynthetic enzyme NMNAT1 facilitates development and early survival of retinal neurons

“…Several recent studies examine levels of select metabolites in mature NMNAT1-deficient retinas ( Sasaki et al, 2020b ), or more broadly examine tissue metabolomes after perturbation of the NMN-synthesizing enzyme NAMPT ( Lin et al, 2016 ; Oakey et al, 2018 ; Lundt et al, 2021 ), but to date there have been no comprehensive metabolomic studies on NMNAT1-deficient tissues. Our metabolomics results approximate that NMNAT1 synthesizes ~40% of the total retinal NAD + pool, which is generally consistent with a previous model ( Sasaki et al, 2020b ).…”

“…Several recent studies examine levels of select metabolites in mature NMNAT1-deficient retinas (Sasaki et al, 2020b), or more broadly examine tissue metabolomes after perturbation of the NMN-synthesizing enzyme NAMPT (Lin et al, 2016;Oakey et al, 2019;Lundt et al, 2021), but to date there have been no comprehensive metabolomic studies on NMNAT1-deficient tissues.…”

Adipokines as Immune Cell Modulators in Multiple Sclerosis

“…In line with these intracellular effects of NAMPT inhibition, neuron-specific deletion of intracellular NAMPT causes neuronal degeneration and led to muscle atrophy, paralysis, and eventually death in mice. Metabolomics of the cortical tissue of these mice revealed that NAMPT deletion activates apoptotic, inflammatory, and immune-responsive pathways and inhibits pathways involved in neuronal/synaptic function [ 156 ]. This supports the differential role of intracellular NAMPT as a key enzyme in cell metabolism and extracellular NAMPT as a signaling, cytokine-like molecule to activate inflammatory pathways.…”