Alzheimer’s disease (AD) is a neurodegenerative disorder that leads to progressive memory loss and cognitive decline and represents the most common form of late-onset dementia (LaFerla et al., 2007; Masters et al., 2015). Two of the main pathological features of AD are amyloid plaques, extracellular insoluble aggregates composed of amyloid-beta fragments (Aβ), and neurofibrillary tangles, intracellular accumulation of hyperphosphorylated Tau (Ingelsson et al., 2004; LaFerla et al., 2007). Together, amyloid pathology and Tau aggregates drive the neuronal impairment that widely affects the cerebral cortex and hippocampus of AD patients (Masters et al., 2015). The effects of these plaques and tangles on cellular functions include mitochondrial dysfunction, synaptic degeneration, and autophagy dysfunction (Ingelsson et al., 2004).

Autophagy is a cellular pathway that controls proteostasis by sequestering and delivering protein aggregates and cellular organelles to lysosomes for degradation. Multiple stages of autophagy are disrupted in AD. Postmortem brain samples of AD patients show decreased levels of Beclin 1, which is essential for autophagy initiation by recruiting membranes to form autophagosomes before fusion to lysosome. Heterozygous deletion of Beclin 1 in transgenic mice expressing human amyloid precursor protein (hAPP) shows compromised neuronal autophagy and accelerated neurodegeneration due to Aβ accumulation (Pickford et al., 2008). Enhancing autophagy through overexpression of Atg8a, a homolog of mammalian LC3, in a Drosophila model of AD increases stress resistance and extends lifespan (Tsakiri et al., 2021). Targeting autophagy and improving protein quality control shows promising therapeutic potential in various neurodegenerative disorders including AD, Huntington’s disease (HD), and Parkinson’s disease (PD) (Nixon, 2013).

Activation of autophagy is involved in the neuroprotection conferred by overexpression of Nmnat (Nicotinamide mononucleotide adenylyltransferase), an evolutionarily conserved rate-limiting enzyme involved in the synthesis of NAD⁺. In a hypertensive glaucoma model, Nmnat protects against optic nerve degeneration through increasing autophagic flux in retinal ganglion cells (Kitaoka et al., 2013). Under hypoxic stress, Nmnat functions upstream of autophagy to mitigate the damage incurred by dendrites in neurons (Wen et al., 2013). We have previously shown that in a Drosophila model of HD, mutant Huntingtin (Htt) aggregation impairs autophagic pathway, while overexpressing Nmnat promotes autophagic clearance of Htt aggregates and protects against neurodegeneration (Zhu et al., 2019). Drosophila has a single Nmnat gene (dNmnat), and its loss-of-function causes post-development photoreceptor neurodegeneration (Zhai et al., 2006). Overexpression of dNmnat rescues neurodegeneration caused by aggregation of toxic proteins including Tau (Ali et al., 2012; Ma et al., 2020), Ataxin (Zhai et al., 2008; Ruan et al., 2015), and Htt (Zhu et al., 2019).

In mammals, three Nmnat genes produce three different Nmnat protein isoforms with distinct subcellular localizations: Nmnat1 in nuclear, Nmnat2 is present in the Golgi and in the cytoplasm, and Nmnat3 is mainly mitochondrial (Brazill et al., 2017). Nmnat1 is ubiquitously expressed and is one of the most studied isoforms of Nmnat. Its expression is protective against axon degeneration caused by mechanical or toxic insults. Mutations in this gene cause a recessive, early form of blindness genetically defined as Leber Congenital Amaurosis 9 (LCA9) (Falk et al., 2012). Nmnat1 knockout mice do not survive birth, while heterozygous mice develop normally without detectable neurodegeneration or axonal pathology (Conforti et al., 2011). On the other side, Nmnat1 overexpression reduces early behavioral impairment in a mouse model of tauopathy (Rossi et al., 2018) and reverses the loss of tyrosine hydroxylase (TH) neurons in the nigrostriatal pathway of the 3xTgAD mice (Jiang et al., 2021). Nmnat2 is predominantly neuronal and has the most influence over axon survival under physiological conditions: depletion of Nmnat2 causes a primary axonal phenotype (Gilley and Coleman, 2010) and mice lacking Nmnat2 have a severe axon outgrowth defect resulting in axon truncation in the peripheral and central nervous system that is incompatible with postnatal survival (Gilley et al., 2019). Reduced Nmnat2 mRNA levels are seen in AD, PD, and HD patients (Ali et al., 2016), and in a mouse model of tauopathy (Ljungberg et al., 2012). Nmnat3 is also ubiquitously expressed but predominantly present in the liver, heart, red blood cells, and skeletal muscle (Brazill et al., 2017). Nmnat3 is upregulated upon hypoxia-ischemia insult (Galindo et al., 2017). Overexpression of Nmnat3 prevents cortical and hippocampal tissue loss, while Nmnat3 knockdown causes neurodegeneration and increases excitotoxic cell death (Galindo et al., 2017).

In this report, we analyzed the effect of expressing dNmnat and human Nmnat1, 2, and 3 in suppressing the proteotoxic phenotype in a Drosophila model of Aβ pathology. We focused on the APP processing and Aβ plaque deposition in the brain and described the regulation of autophagy in Nmnat-facilitated clearance of amyloid aggregation.

In this study, we investigated the effect of different Nmnat isoforms on amyloid clearance in a Drosophila model of amyloid aggregation. Our model recapitulated the age-dependent processing of APP with CTF accumulation, and deposition of amyloid plaques in the brain cortex. Among different Nmnat isoforms, we identified hNmnat1 as a potent inhibitor for APP aggregation as evidenced by a remarkable reduction of the processed APP-CTF and decreased number and size of amyloid plaques in the brain. Finally, we showed an increased conversion of Atg8a-I to Atg8a-II as well as a reduction of total Atg8a and Ref(2)P in hNmnat1 overexpressing brains, indicating that hNmnat1 reduces amyloid plaque load by promoting autophagy.

Nmnat has been identified as a neuronal maintenance factor (Zhai et al., 2006). Cytoplasmic Nmnat isoforms like dNmnat in Drosophila used in this study, hNmnat2, and hNmnat3 are essential for maintaining neuronal integrity in a physiological environment and protecting against neurodegeneration in various neurodegenerative models (Brazill et al., 2017). These Nmnat isoforms can act as chaperones to interact with client proteins, facilitate protein folding and clearance, and thereby maintain proteostasis in neurons (Ali et al., 2016; Zhu et al., 2019; Ma et al., 2020). For example, Nmnat2 forms a complex with HSP90 chaperone to promote refolding of toxic tau (Ali et al., 2016) and Nmnat3 uses its NAD⁺ substrate-binding site to bind and chaperone pTau (Ma et al., 2020).

Aggregates formed from APP processing differ from other protein aggregates in that APP is processed on the plasma membrane and deposited extracellularly (LaFerla et al., 2007). As Nmnat1 is a predominantly nuclear-localized enzyme, it is unlikely for Nmnat1 to directly interact with APP or amyloid plaques. Our findings indicate that hNmnat1 promotes the autophagic clearance of unfolded or misfolded amyloid proteins, likely due to NAD⁺-dependent transcriptional or post-translational regulations. NAD⁺ is a coenzyme involved in hundreds of metabolic redox reactions, ADP-ribosylation, histone deacetylation, and calcium signaling pathways (Brazill et al., 2017). NAD⁺ can be synthesized by a salvage pathway or by a de novo pathway and Nmnat plays an essential role in both: it synthesizes NAD⁺ from nicotinamide mononucleotide (NMN) in the salvage pathway or it converts nicotinic acid mononucleotide (NaMN) to nicotinic acid adenine dinucleotide (NaAD) in the de novo pathway (Lau et al., 2009). Nmnat1 is essential for the supply of nuclear NAD⁺, a substrate for poly (ADP-ribose) polymerase (PARP)-mediated ADP-ribosylation and sirtuin (SIRT)-mediated deacetylation, both of which are critical regulators of autophagy. For example, PARP1, a ubiquitous nuclear enzyme, catalyzes PARylation of nuclear AMP-activated protein kinase (AMPK), inducing AMPK nuclear-cytosolic export for autophagosome formation and autophagy initiation (Rodríguez-Vargas et al., 2016). Upon DNA damage, PARP1 is required for autophagy induction by increasing the expression of Bnip-3, Cathepsin b and l, and Belin-1 (Muñoz-Gámez et al., 2009). We also recently demonstrated that Nmnat1 directly forms a complex with PARP1 and increases local NAD⁺ availability to promote PARylation and regulate cell survival under stress (Liu et al., 2021). In addition, nuclear SIRTs carry out multifaceted functions in autophagy. For example, SIRT1 deacetylates essential components of the autophagy machineries, including Atg5, Atg7, and Atg8, to promote autophagy initiation (Lee et al., 2008). SIRT1 also deacetylates and activates FOXO1 and FOXO3, two essential transcriptional regulators for autophagy induction (Brunet et al., 2004; Zhou et al., 2012). We recently showed that Nmnat1 upregulates SIRT1 and inhibits the activity of p53 (Liu et al., 2021), a negative regulator of autophagy (Tasdemir et al., 2008).

In addition to promoting autophagy, the nuclear NAD⁺ pool regulates the transcription of the major cleaving enzymes involved in APP processing. For example, BACE1 is tightly regulated by peroxisome proliferator-activated receptor-γ coactivator 1 (PGC)-1α, a transcriptional coactivator that enhances BACE1 ubiquitination and proteasomal degradation and ameliorates Aβ production (Gong et al., 2013; Wang et al., 2013). A decreased level of PGC-1α has been reported in the cortex of AD patients, while exogenous expression of PGC-1α can significantly inhibit Aβ plaque formation by suppressing BACE1 transcription (Katsouri et al., 2011). The transcriptional activity of PGC-1α depends on its subcellular distribution and is promoted by SIRT1-dependent nuclear translocation (Anderson et al., 2008). A previous study showed that nicotinamide riboside (NR), an NAD⁺ precursor, upregulates the expression of PGC-1α, enhances BACE1 ubiquitination and proteasomal degradation, and thereby ameliorates Aβ production (Gong et al., 2013).

Taken together, by using a Drosophila model of AD, our study demonstrated that overexpressing hNmnat1 inhibits amyloidogenic processing of APP and reduces amyloid plaque accumulation. Recently, high-throughput screens have identified small molecules to boost Nmnat expression in neurons or enhance NAD⁺ production (Ali et al., 2017; Gardell et al., 2019), highlighting the therapeutic potential of targeting Nmnat/NAD⁺ in treating AD and other proteinopathies.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

MP and RZ conceived the project. YZ, AL, and MP carried out the experiments. YZ, AL, RZ, and MP analyzed the data and wrote the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.