Nicotinamide mononucleotide adenylyl transferases (NMNATs) are a family of highly conserved proteins indispensable for cellular homeostasis. NMNATs are classically known for their enzymatic function of catalyzing NAD+ synthesis, but also have gained a reputation as essential neuronal maintenance factors. NMNAT deficiency has been associated with various human diseases with pronounced consequences on neural tissues, underscoring the importance of the neuronal maintenance and protective roles of these proteins.
New mechanistic studies have challenged the role of NMNAT-catalyzed NAD+ production in delaying Wallerian degeneration and have specified new mechanisms of NMNAT’s chaperone function critical for neuronal health. Progress in understanding the regulation of NMNAT has uncovered a neuronal stress response with great therapeutic promise for treating various neurodegenerative conditions under question . Given the essential role for NAD+ in cellular metabolism, it is not surprising that the enzyme is required for the survival of all living organisms, from archaebacteria to humans.
The discovery of the remarkable neuroprotective function of NMNAT pro- teins sparked a burst of investigations on NMNAT in the nervous system. More recently, NMNAT mutations have been identified to cause a severe form of retinal degener- ation and NMNAT deficiency has been associated with complex neurological diseases. In this review, we focus on the function of NMNAT in the nervous system and discuss the recent advances in understanding the regulatory mechanisms of neuronal maintenance that are relevant for neuroprotective therapies against neuro- degenerative conditions. For previously published reviews that focus on the neuroprotective effects of NMNAT, particularly in axon degeneration and injury, we refer readers to [1,4–6].
Genetic links between NMNAT and diseases of the nervous system
The only known monogenetic disease associated with NMNAT proteins is Leber congenital amaurosis (LCA), one of the most common forms of inherited blindness in children. Compound heterozygous or homozygous muta- tions in NMNAT1 cause LCA9 (OMIM 608553), an auto- somal recessive condition characterized by severe early- onset and rapid progression of vision loss and retinal degeneration . To date more than 30 mutations spread across the NMNAT1 gene have been reported, including missense, nonsense, and splicing mutations (Figure 1) [8–11]. Importantly, most LCA9 patients have reported normal physical and mental health, suggesting a specific requirement of NMNAT1 for maintenance of the neural tissue in the retina .
So far no human diseases have been shown to be directly caused by mutations in NMNAT2 or NMNAT3, though several studies have signified a putative contribution of NMNAT deficiency to the progression of complex neu- rological diseases. Microarray studies have shown that NMNAT2 mRNA levels are reduced in various neurode- generative diseases, including Alzheimer’s disease (AD), Parkinson’s disease, and Huntington’s disease [12–14]. In AD, NMNAT2 transcript levels negatively correlate with cognitive dysfunction and AD pathology [15 ]. Genome- wide association studies have indicated a SNP (rs952797) located 126 kb downstream of the NMNAT3 gene that is associated with late-onset AD . Although these asso- ciation studies do not demonstrate a causal relationship,
Humans have three NMNAT genes that produce three NMNAT protein isoforms with distinct tissue expression patterns and subcellular localizations . NMNAT1 is ubiquitously expressed and is enriched in the nucleus. NMNAT2 is predominantly expressed in the brain and is localized to the cytosol and enriched in membrane com- partments. NMNAT3 is also widely expressed but is highest in liver, heart, skeletal muscles, and red blood cells . NMNAT3 is reported to have two splice variants, encoding mitochondrial localized FKSG76 and cytosolic NMNAT3v1, though the expression and function of these endogenous protein variants are still they do recommend NMNAT as an important contributor to neuronal health.
Molecular functions of NMNAT necessary for neuronal maintenance and protection
LCA9 and other neurological disease phenotypes associ- ated with NMNAT deficiency are consistent with its neural maintenance function, yet the neuroprotective capacity of NMNAT had already emerged with the serendipitous discovery of the slow Wallerian degenera- tion mutant (Wlds) mouse [17,18]. In the Wlds mouse, degeneration of the distal axon following axotomy (Wallerian degeneration) is remarkably delayed by a dominant mutation causing overexpression and redistri- bution of Nmnat1 to the cytoplasmic compartment of the axon . The neuroprotective role of NMNAT is effica- cious against not only axonal injury, but also models of various neurodegenerative conditions such as toxic neuropathy, glaucoma, spinocerebellar ataxia, tauopathy, and Huntington’s disease [1,5,19–25]. Furthermore, NMNAT homologues across species, including archae- bacteria, yeast, fly, mouse, and human, exhibit a conserved cytoprotective function, even when expressed in different model organisms [1,19]. While these studies chronicle the potent and conserved protection NMNAT proteins confer against a variety of neuronal insults, the precise mechanism by which NMNAT engenders this protection has been hard to pinpoint.
NMNAT proteins have two distinct functions that can bestow neuronal maintenance and protection: NAD synthase activity and chaperone activity . NMNAT catalyzes the reversible conversion of NMN (nicotin- amide mononucleotide) to NAD+ in the final step of both the de novo biosynthesis and salvage pathways. NAD+ is a vital coenzyme important for metabolism and redox biology, as well as a substrate in multiple signaling processes . Thus, NMNAT enzymes play a straight- forward role in neuronal maintenance via balancing NAD+ consumption with production. A molecular chap- erone function has also been demonstrated by a number of NMNAT proteins tested, including Drosophila Nmnat, mouse Nmnat2, and human NMNAT3 [15,19,28]. In vitro these NMNAT proteins bind to client proteins and prevent thermal-stress induced unfolding [15,19]. Thus, NMNAT chaperones can contribute to neuronal protein homeostasis.
There is still considerable controversy surrounding the mechanism of NMNAT-mediated neuroprotection. The prevailing hypothesis is that NMNAT overexpression pro- vides continuous enzyme activity in injured neurons, thus preventing the consequent decrease in NAD+ and the accumulation of the precursor NMN . However, recent work revealed that NAD+ depletion following axon injury is due to a dramatic increase in consumption by the pro- degenerative SARM1 (sterile alpha and TIR motif-con- taining 1), but that overexpression of cytoplasmic Nmnat1 blocks SARM1-dependent NAD+ consumption without increasing NAD+ synthesis [29 ]. Thus, NMNAT-medi- ated axon protection hinges on its ability to block pro- degenerative SARM1 signaling, but this does not rely on enzymatic conversion of NMN to NAD+. One alternative is that NMNAT provides neuroprotection via an enzyme- independent function, leading to the hypothesis that NMNAT mediated protection is chaperone-dependent. Recently, it has been shown that Nmnat2’s enzyme activity is dispensable for relieving the toxic phosphorylated tau burden in a model of fronto-temporal dementia and parkinsonism 17 (FTDP-17), and that Nmnat2 complexes with the classical HSP90 chaperone, possibly to promote refolding of toxic tau [15 ]. In another study, overexpres- sion of cytoplasmic Nmnat1 partially preserved neuronal function in a model of early-onset FTDP-17 by decreasing insoluble tau aggregates, without altering phosphorylated tau . Similarly, overexpression of yeast homologs of NMNAT, NMA1 and NMA2, suppresses proteotoxicity in yeast models of polyglutamine- and a-synuclein-induced neurodegeneration also by enhancing clearance of mis- folded proteins .
Though the precise mechanism may vary by disease model, it is clear that NMNAT over- expression alleviates proteotoxic stress in neurodegenera- tive conditions associated with protein misfolding, consis- tent with a chaperone function. Further supporting the chaperone function of NMNAT is the identification of its endogenous ‘client’ protein in Drosophila synapses, where Nmnat maintains active zone structure by directly interacting with the active zone protein Bruchpilot (BRP) in an activity-dependent manner . Perhaps a source of resistance to this hypothesis is that enzyme- inactive mouse Nmnat proteins fail to protect axons [31– 33], although enzyme-inactive Drosophila Nmnat is suffi- cient to protect against activity-induced retinal degenera- tion, Wallerian degeneration, and axonal degeneration induced by loss of JNK (c-Jun N-terminal kinase); in these conditions NMNAT chaperone-dependent protection is perhaps less implicit since here the client for such a chaperone role is not apparent [34–36]. Continued advances in NMNAT overexpression-mediated protection in neurodegenerative models will be invaluable for identi- fying not only the underlying mechanisms of NMNAT’s protection, but also by extension, the primary triggers of neurodegeneration.
Mouse models of NMNAT1-dependent LCA recapitu- late several aspects of human disease and further delin- eate disease progression; importantly, degeneration occurs after retinal development, is observed first within the photoreceptors, and is only observed in inner retinal neurons and RPE (retinal pigment epithelia) cells in advanced stages . The sensitivity of the retina, there- fore, may reflect a twofold requirement of NMNAT: biosynthesis of NAD+ and chaperone activity for meeting the high metabolic and protein turnover demands of photoreceptors, respectively. Although LCA mutations are widely distributed across the NMNAT1 gene, most NMNAT1 mutant proteins characterized exhibit several common features. NAD+ synthase activity is reduced by most mutations in vitro, yet patient fibroblasts exhibit normal basal NAD+ levels, suggesting that enzyme deficiency is not a primary cause of disease [10,38 ]. Importantly, ectopic expression of several LCA9 NMNAT1 mutants is sufficient to protect cultured neu- rons from Wallerian degeneration, indicating that LCA9- causing mutations do not disrupt the qualitative neuro- protective properties of NMNAT proteins [38 ]. How- ever in vitro, NMNAT1 mutant proteins are more sus- ceptible to stress-induced unfolding compared to wild type protein, which may amplified in vivo to cause disease [38 ]. Interestingly, Drosophila photoreceptors are also particularly vulnerable to loss of Nmnat, but Nmnat’s enzyme activity is completely dispensable for mainte- nance of retinal health and function in this model . So while LCA9 may be primarily attributed to a loss of NMNAT1 protein stability, more studies are needed to determine whether retinal degeneration arises from a consequential loss of NAD+ synthesis and/or loss of the molecular chaperone function (Figure 1).
Regulation of NMNAT during stress and disease
Because of the essential neuronal maintenance role of NMNAT and its capacity for neuroprotection, under- standing the regulation of NMNAT levels becomes key to unlocking its therapeutic potential. It is critical that neurons maintain sufficient levels of NMNAT2; NMNAT2 loss is considered an initiating event in Wal- lerian degeneration and also appears to be consistent in progression of neurodegenerative conditions . At the protein level, NMNAT2 is constitutively degraded by the ubiquitin-proteasome system, the exact players of which are beginning to emerge. Several ubiquitin ligases, including Phr1 (Highwire in Drosophila), Skp1a, and Fbxo45, have been identified, though direct evidence of NMNAT ubiquitination is lacking [40–42]. Genetic interference of these ligases increases NMNAT levels and subsequently enhances neuronal protection. In addi- tion to protein regulation, two functional cAMP-response elements (CREs) have been identified in the Nmnat2 promoter region that influence expression at the tran- scriptional level. In an FTDP-17 tauopathy model, a pathological reduction of phospho-CREB (CRE binding protein) levels and binding with the Nmnat2 promoter resulted in decreased Nmnat2 transcript and protein .
In contrast to mammals that use three NMNAT genes to produce three different NMNAT protein isoforms, Dro- sophila has only one Nmnat gene that is alternatively spliced to generate two variants: Nmnat-RA mRNA variant encodes the nuclear Nmnat-PC protein isoform and Nmnat-RB variant encodes the cytosolic Nmnat-PD isoform. In a Drosophila model of spinocerebellar ataxia 1 (SCA1), overexpression of Nmnat-PD significantly improved behavioral and morphological defects by reduc- ing neuronal mutant human Ataxin-1 (hAtx1-[82Q]) aggregates, yet surprisingly, overexpression of Nmnat- PC increased aggregate size and exacerbated neurode- generation [43 ]. Importantly, cytoplasmic targeted Nmnat-PC is also ineffective in relieving hAtx-[82Q] proteotoxicity. This study revealed a functional diver- gence underlying Drosophila Nmnat isoform-specific neuroprotection. Therefore, alternative splicing in Dro- sophila uses one gene to produce two functionally exclu- sive mRNA variants: one housekeeping variant, Nmnat- RA, that is stably expressed under normal conditions, and one stress response variant, Nmnat-RB, that can be quickly induced under stress conditions for neuronal protection (Figure 3). Taken together with the previous finding that the transcription of Drosophila Nmnat pre- mRNA is increased under various stress conditions , the observation that stress also drives post-transcriptional alternative splicing to preferentially generate Nmnat-RB sheds light on the complex neuronal stress response to achieve self-protection [43 ].
Regulation of Drosophila Nmnat under neuronal stress may provide insight into regulation of NMNAT2 in mammals in neurodegenerative conditions. Notably, the transcript level of the neuroprotective Drosophila variant Nmnat-RB is reduced in late stages of models of tauopathy and SCA1, however it is increased in the early stages, suggesting an upregulation to combat pathogenic protein aggregation during disease progression that is perhaps overwhelmed at more advanced stages of neuro- nal degeneration and cell death [43 ]. Similarly, although many studies show a decrease of NMNAT2 transcript postmortem in patients with neurodegenerative diseases, there is also evidence of increased levels at the early stages of disease. For example, in mild cognitive impairment and early stages of AD, NMNAT2 mRNA levels are higher in the frontal cortex of patients exhibit- ing high levels of oxidative stress-induced neuronal DNA damage, compared to those with lower levels of DNA damage ; at this stage the nervous system is mounting a stress response as HSP90 is also upregulated in patient brains with high oxidative damage. In a genome-wide gene-expression study of another mouse model of FTDP- 17, Nmnat2 transcript is significantly upregulated in the hippocampus during early disease progression, but is downregulated at end stages of disease when neurofibril- lary tangle burden is heavy . Here the early rise of Nmnat2 is likely a proteotoxic stress response, while the late decrease is consistent with observed downregulation of synaptic genes corresponding to synaptic loss and upregulation of inflammatory and apoptotic genes indic- ative of neuronal cell death. Since endogenous upregula- tion of NMNAT under stress conditions seems to be insufficient to maintain neural integrity in the long term, NMNAT and its transcriptional and post-transcriptional regulation can serve as a therapeutic target for pharma- cological intervention to slow the progression of neuro- degenerative diseases.
The long life of neurons, up to a century in humans, makes neuronal maintenance an important challenge. It is conceivable that compromised maintenance would result in degeneration in an age-dependent manner. The emer- gence of the chaperone function of NMNAT, an NAD+ synthase, specifically in the nervous system for neuronal maintenance, exemplifies an evolutionarily conserved
strategy of ‘repurposing’ (or ‘moonlighting’) housekeep- ing enzymes. Neurons have developed transcriptional and post-transcriptional regulatory mechanisms to bal- ance the metabolic activity and stress response role of NMNAT. Thus, understanding neuronal requirements for NMNAT the NAD+ synthase and NMNAT the chaperone under various stress and disease conditions, in addition to dissecting the regulatory mechanisms that direct the activity of NMNAT, has huge implications for neuroprotective therpapies. Furthermore, identifying druggable targets to enhance expression and/or reduce protein degradation would be a pharmacologically approachable way to achieve higher NMNAT protein levels and therefore confer neuroprotection.