The effect of NAMPT deletion in projection neurons on the function and structure of neuromuscular junction (NMJ) in mice

Abstract

Nicotinamide adenine dinucleotide (NAD +) plays a critical role in energy metabolism and bioenergetic homeostasis. Most NAD + in mammalian cells is synthesized via the NAD + salvage pathway, where nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme, converting nicotinamide into nicotinamide mononucleotide (NMN). Using a Thy1-Nampt −/− projection neuron conditional knockout (cKO) mouse, we studied the impact of NAMPT on synaptic vesicle cycling in the neuromuscular junction (NMJ), end-plate structure of NMJs and muscle contractility of semitendinosus muscles. Loss of NAMPT impaired synaptic vesicle endocytosis/exocytosis in the NMJs. The cKO mice also had motor endplates with significantly reduced area and thickness. When the cKO mice were treated with NMN, vesicle endocytosis/exocytosis was improved and endplate morphology was restored. Electrical stimulation induced muscle contraction was significantly impacted in the cKO mice in a frequency dependent manner. The cKO mice were unresponsive to high frequency stimulation (100 Hz), while the NMN-treated cKO mice responded similarly to the control mice. Transmission electron microscopy (TEM) revealed sarcomere misalignment and changes to mitochondrial morphology in the cKO mice, with NMN treatment restoring sarcomere alignment but not mitochondrial morphology. This study demonstrates that neuronal NAMPT is important for pre-/ post-synaptic NMJ function, and maintaining skeletal muscular function and structure. Nicotinamide adenine dinucleotide (NAD +) is found in all cells of the human body and is an important cofactor or co-substrate, used in numerous enzymatic processes including glycolysis, the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, DNA repair, and protein deacetylation 1. NAD + levels may be important for many aspects of health and aging, including cellular metabolism, sarcopenia, and neurodegeneration 2. NAD + can be synthesized through multiple enzymatic pathways. One is a de novo pathway that begins with the amino acid tryptophan, while other pathways utilize different metabolites capable of being converted into NAD +. In mammalian cells, the majority of NAD + is produced from metabolites entering the NAD + salvage pathway 3. The rate limiting enzyme of the salvage pathway is nicotinamide phosphoribosyltransferase (NAMPT), which condenses nicotinamide (NAM) and 5-phosphoribosyl pyrophosphate (PRPP) into nicotinamide mononucleotide (NMN). NMN is subsequently synthesized into NAD + by nicotinamide mononucleotide adenylyltransferases (NMNATs) 4. NAD + levels decline with age and in different diseases. However, administration of NAD + precursor molecules, such as NMN or nicotinamide riboside (NR), are effective at preventing or reversing many age-or disease-related declines 5-10. NAD + and the NAD + salvage pathway are vitally important to maintain bioenergetic homeostasis, the normal health and function of many different organs and tissues in the human body, with neurons and skeletal muscles being impacted greatly. In n...

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

“…[18][19][20][21][22] Recently, we showed that deletion of NAMPT in the projection neurons using conditional and inducible NAMPT knockout (cKO) mice, i.e., Thy1-YFP-NAMPT À/À cKO mice caused motor neuron degeneration, neuromuscular junction (NMJ) abnormalities, muscle atrophy, body weight loss, paralysis and eventually death. 17,23 Our results indicate that the disruptions of bioenergetics and mitochondrial homeostasis contribute to the phenotype. To further investigate the molecular mechanisms of neuronal death in Thy1-YFP-NAMPT À/À cKO mice, we conducted a combined study of metabolomics and high-throughput RNA sequencing (RNA-Seq) to quantitatively analyze metabolic and transcriptional alterations.…”

NFKB1 Gene Mutant Was Associated with Prognosis of Coronary Artery Disease and Exacerbated Endothelial Mitochondrial Fission and Dysfunction

“…The equilibrium of mitochondrial dynamics and normal mitochondrial morphology are critical for maintaining mitochondrial function [ 38 ]. Mitochondria are dynamic organelles whose morphology is directly linked to the maintenance of their functions, and the disruption of their normal shape is a hallmark of mitochondrial dysfunction [ 39 , 40 ]. Mitochondrial dynamics are regulated by the balance of fission and fusion; imbalances in fission and fusion cause mitochondrial abnormalities that lead to many diseases, including cardiovascular disease [ 41 , 42 ].…”

Nicotinamide Mononucleotide: A Promising Molecule for Therapy of Diverse Diseases by Targeting NAD+ Metabolism

“…Remarkably, NMN (400 mg/kg) treatment lessened disease severity, restored motor function, and extended the life span of Thy1-YFP-Nampt −/− cKO mice. Recently, this same team reported that loss of NAMPT in projection neurons cause detrimental effects on the function and structure of NJM, including impaired synaptic vesicle cycling, morphological changes to the motor endplate, alterations of skeletal muscle contractile responses, and noticeable sarcomere misalignment (Lundt et al, 2020). These detrimental effects were reverse by administration of NMN (400 mg/kg/day) for 14 days.…”

“…These detrimental effects were reverse by administration of NMN (400 mg/kg/day) for 14 days. However, NMN treatment did not restore skeletal muscle mitochondria morphology change caused by deletion of Nampt (Lundt et al, 2020). Thus, NMN is a potential therapeutic drug for motor neuron (MN) degenerative diseases, including ALS.…”