In this study, it was determined that mitochondrial dysfunction and impaired mitophagic activity were associated with the downregulation of NAMPT-NAD+-SIRT1-FOXO1/3a axis in the hippocampus of 6-month-old APP/PS1 mice. The P7C3 treatment increased mitophagy by activating NAMPT, upregulating the NAD+-SIRT1-FOXO1/3a signalling pathway, improving mitochondrial dysfunction, and ameliorating cognitive deficits in APP/PS1 mice.
In mammalian cells, the NAD+ salvage pathway is the key to maintaining NAD+ homeostasis. Therefore, in NAMPT, the first-step rate-limiting enzyme in the salvage pathway is a regulator of the NAD+ pool[6]. The concept of the NAD World provides the insight that a decrease in NAMPT is an important trigger factor for the systemic decline of NAD+ to induce pathological changes in various tissues[10]. Mounting evidence has demonstrated that NAMPT-mediated NAD+ biosynthesis declines with age in multiple tissues, such as the muscle, liver, and brain, and thus reduces the activity of NAD+-dependent enzymes, such as SIRT1[10]. Studies have suggested that the deletion of the NAMPT gene leads to neurodegeneration by inducing mitochondrial dysfunction, apoptosis, and neuroinflammation[14, 26]. In addition, previous studies suggested that the NAMPT-mediated NAD+ level was declined in animal AD models, which may be associated with Aβ toxicity[18, 19, 27]. Recently, a study suggested that the depletion of NAMPT in hippocampus neurons induced injury of mitochondria, increased intracellular Aβ and cognitive deficiency in mice, demonstrated that the decline of NAMPT level can be a critical risk factor for neurodegeneration[28].
The APP/PS1 is a transgenic mouse model that overproduces Aβ and is often used to study the mechanisms of neuropathology of AD. Amyloid plaques begin to appear in the cerebral cortex at approximately 4 months of age, and cognitive deficits appear at approximately 6 months of age in APP/PS1 mice[29]. Consistent with previous research, we found increased Aβ plaque deposition and soluble Aβ40 and Aβ42 as well as declined learning and memory, which accompanied by decreased neuronal NAMPT and its-mediated NAD+ level in the hippocampus of 6-month-old APP/PS1 mice. This further confirms that the decrease in NAD+ levels regulated by NAMPT in the hippocampus of AD mice is related to Aβ neurotoxicity.
SIRTs, such as class III NAD+-dependent histone deacetylases, regulate a variety of physiological activities. It has been found that the decreased NAMPT via its activity of NAD+ biosynthesis enzymes involved in SIRT1 is related to neurodegenerative diseases[30]. Increasing evidence suggests that elevated NAD+ levels can improve mitochondrial quantity and quality via pathways that include SIRT1-FOXO1/3a mediated mitophagy[31–33]. Recently, it has been suggested that NAMPT-mediated NAD+ levels may modify behavioral effects of cocaine through the SIRT1 signaling pathway[34]. Studies in animal models of cerebral ischemia have demonstrated that NAMPT promotes neuronal survival by inducing autophagy in a SIRT1-dependent manner[11].
The PINK1/Parkin and BNIP3 pathways are responsible for activating mitophagy by marking damaged mitochondria. The LC3 is recognized by the mitochondrial receptor proteins P62 and BNIP3 to promote phagophore completion. The P62 protein, as an adapter protein, is negatively associated with mitophagy. When the mitochondria are damaged, PINK1 accumulates in the outer membrane of the mitochondria, recruits cytoplasmic parkin to PINK1, and then binds to the phagophore LC3 by linking to P62 and lysosomal digestion. BNIP3 directly binds to LC3 to initiate mitophagy. In our study, the downregulation of the NAMPT-NAD+-SIRT1-FOXO1/3a signaling pathway and abnormal mitophagy activity (increased mitochondrial PINK1, LC3II/I, and P62; decreased Parkin and BNIP3; and reduced mitochondrial and lysosomal fusion) were observed in 6-month-old APP/PS1 mice. Furthermore, these mice showed increased numbers of damaged mitochondria and the
reactive oxygen species (ROS) product H2O2 and reduced mtDNA relative copy numbers and ATP levels, indicating that inhibition of the NAMPT-NAD+-SIRT1-FOXO1/3a axis is involved in impaired mitophagy activity and mitochondrial dysfunction in animal AD models. These results suggest that the NAMPT-NAD+-SIRT1-FOXO1/3a cascade plays an important role in regulating PINK1/Parkin and BNIP3 pathway-mediated mitophagic activity.
Furthermore, FK866, an inhibitor of NAMPT, was used to verify the relationship between the NAMPT-NAD+- SIRT1 signaling pathway and mitophagy in the hippocampi of APP/PS1 mice. FK866, a highly specific small molecule inhibitor of NAMPT, has been widely used in research[35]. Initial observations indicated that FK866 induced cellular NAD+ depletion, followed by ATP depletion and apoptotic cell death[36]. Recently, it was demonstrated that FK866 aggravates the adverse effects of FOXO3a on mitochondrial function by inhibiting NAMPT/NAD+[37]. In this study, 12-week FK866 treatments successfully inhibited NAMPT/NAD+-SIRT1-FOXO1/3a axis, PINK1/Parkin-induced and BNIP3 induced mitophagy initiation, and mitochondrion and lysosome fusion as well as induced mitochondrial dysfunction in the hippocampus of wild type mice and exacerbated above these indicators and even Aβ plaque loads in the hippocampus of APP/PS1mice. These results further indicate that NAMPT/NAD+ axis-mediated mitophagy disorders contribute to neurodegeneration in AD. Therefore, we propose that interventions that enhance NAMPT-mediated mitophagy may ameliorate AD pathology.
P7C3 was first discovered due to its neuroprotective effects [38]. In 2014, Wang et al. confirmed that the neuroprotective effect of P7C3 was related to the increased activity of the NAMPT-NAD+ axis[39]. It is worth noting that P7C3 treatment did not affect the expression level of NAMPT protein, but increased NAD+ levels because P7C3 mainly promotes the generation of NAD+ by mediating NAMPT activity[39]. A subsequent study verified that P7C3 regulates the NAMPT/NAD+ axis and plays a neuroprotective role in a variety of brain injury models[40]. Previous studies confirmed that P7C3 ameliorates cognitive deficits in TgF344-AD rats without altering amyloid deposition; however, the specific mechanism remains unclear[41]. Similarly, we also found that P7C3 failed to reduce Aβ plaque loads while improving learning and memory ability in APP/PS1 mice. However, P7C3 upregulates the NAMPT-NAD+-SIRT1-FOXO1/3a axis, increases mitophagy, and improves mitochondrial dysfunction. These results indicated that the effect of P7C3 on improving APP/PS1 mice cognitive impairment is associated with increased mitophagy activity mediated by NAMPT-NAD+-SIRT1-FOXO1/3a axis, rather than relying on Aβ pathological change.
Collectively, this study demonstrates for the first time that NAMPT regulates mitophagic activity in the hippocampus of APP/PS1 mice through the NAD+-SIRT1-FOXO1/3a axis. These results provide new insights into the molecular mechanisms underlying the neuroprotective effects of NAMPT and may be helpful in the development of AD drugs.