Mechanistic insights into NAD synthase NMNAT chaperoning phosphorylated Tau from pathological aggregation

Tau hyper-phosphorylation and deposition into neurofibrillary tangles have been found in brains of patients with Alzheimer’s disease (AD) and other tauopathies. Molecular chaperones are involved in regulating the pathological aggregation of phosphorylated Tau (pTau) and modulating disease progression. Here, we report that nicotinamide mononucleotide adenylyltransferase (NMNAT), a well-known NAD synthase, serves as a chaperone of pTau to prevent its amyloid aggregation in vitro as well as mitigate its pathology in a fly tauopathy model. By combining NMR spectroscopy, crystallography, single-molecule and computational approaches, we revealed that NMNAT adopts its enzymatic pocket to specifically bind the phosphorylated sites of pTau, which can be competitively disrupted by the enzymatic substrates of NMNAT. Moreover, we found that NMNAT serves as a co-chaperone of Hsp90 for the specific recognition of pTau over Tau. Our work uncovers a dedicated chaperone of pTau and suggests NMNAT as a key node between NAD metabolism and Tau homeostasis in aging and neurodegeneration.

Intriguingly, in addition to its well-studied NAD synthase activity, NMNAT has been found to be able to retrieve the activity of luciferase from heat-denatured amorphous aggregation suggesting a chaperone activity of NMNAT (Ali et al., 2016;Zhai et al., 2008). However, it remains puzzling how a single domain enzyme, at the same time, fulfills a chaperone activity. Moreover, since NAD is an essential cofactor in numerous cellular processes (e.g. transcriptional regulation (D'Amours, Germain, Orth, Dixit, & Poirier, 1998;Shogren-Knaak et al., 2006;T. Zhang et al., 2009) and oxidative reactions (Lewis et al., 2014)), it is confusing whether the protective role of NMNAT in AD animal model comes directly from its potential chaperone activity or indirectly from its enzymatic activity. In this work, we investigated the chaperone activity of NMNAT against the amyloid aggregation of Tau in vitro and in the fly model. By combining multiple biophysical and computational approaches, we revealed the molecular mechanism of NMNAT as a specific chaperone of pTau. Our work provides the structural basis for how NMNAT manages its dual functions as both an enzyme and a chaperone, as well as how NMNAT specifically recognizes pTau and serves as a cochaperone of Hsp90 for pTau clearance. Our work suggests an interplay of NAD metabolism and the progression of Tau pathology in aging and neurodegeneration.

NMNAT family members exhibit a conserved chaperone activity in preventing pTau aggregation
In the preparation of pTau proteins, we used MARK2 to phosphorylate Tau23 and a 6 truncated construct¾K19 ( Figure 1A) (Gu et al., 2013). he MARK2 phosphorylation sites on Tau23 and K19 were characterized by using 2D 1 H-15 N NMR HSQC spectra.
To comprehensively investigate the activity of NMNAT on pTau aggregation, we next prepared different isoforms of NMNAT proteins from different organisms, including three isoforms of human NMNATs (hN1, hN2, and hN3), two isoforms of Drosophila NMNATs (PD and PC) and mouse NMNAT3 (mN3). We first confirmed that the NMNAT proteins contained normal enzyme activities (Figure supplement 2).
Then, we conducted the ThT fluorescence kinetic assay and transmission electron microscopy (TEM) to monitor their influences on the amyloid aggregation of pTau. The result showed that different NMNAT isoforms generally exhibited a potent chaperone activity against the amyloid aggregation of both pTau23 ( Figure 1B  results demonstrate that the chaperone activity of NMNAT against pTau aggregation is highly conserved in the NMNAT family across different organisms. Thus, NMNAT may serve as a molecular chaperone that is directly involved in the protection of pTau from pathological deposition. 7 We next sought to investigate the structural basis of the interaction between NMNAT and pTau. Although hN2 represents the most biological relevant isoform to chaperone pathological pTau, purified hN2 is unstable and prone to aggregate in vitro, which hinders further structural characterization. Alternatively, we used mN3, which is of high thermostability and a highly conserved sequence to hN2 (Figure supplement 5). We performed solution NMR spectroscopy and used mN3 to titrate 15 N-labeled pK19. The 2D 1 H-15 N HSQC spectra showed a significant overall signal broadening of pK19, which indicates a strong interaction between mN3 and pK19 ( Figure 1C). In particular, the four phosphorylated Ser (pSer) residues showed large signal attenuations and chemical shift changes upon mN3 titration ( Figure 1D and 1E). Residues adjacent to pSer also exhibited prominent signal attenuations ( Figure 1E). Especially, repeat sequence R4 that contains two pSer residues showed the largest signal attenuations with I/I 0 < 0.3 ( Figure 1E). In contrast, as we titrated non-phosphorylated K19 at the same ratio, only slight overall signal broadening was observed ( Figure 1E and figure supplement 6A). Moreover, pTau23, but not Tau23, showed significant chemical shift changes and intensity attenuations mainly on and around the pSer residues upon mN3 titration (Figure supplement 6B). These results indicate that the pSer resides of pTau are the primary binding sites of mN3.

Mechanism of the interaction between mN3 and pTau
Further, to quantitatively measure the binding affinity between NMNAT and pTau, we conducted BioLayer Interferometry (BLI) analysis that is a label-free technology for measuring biomolecular interactions (Rich & Myszka, 2007). We immobilized mN3 on the biosensor tip and profiled the association and dissociation curves in the presence 8 of either pTau or Tau ( Figure 1F and figure supplement 7). As we measured, the dissociation constant (K D ) of the interaction between mN3 and pK19 is ~ 31 µM, and that of mN3 and pTau23 is ~ 9.9 µM ( Figure 1F). In contrast, the K D of mN3 and K19 is ~ 239 µM, and that of mN3 and Tau23 is ~ 58.6 µM, which are about 10-fold weaker than that of the phosphorylated counterparts ( Figure 1F and figure supplement 7).
Taken together, these results indicate that MARK2-phosphorylation can significantly enhance the interaction between NMNAT and pTau through the specific interaction between NMNAT and the phosphorylated residues of pTau.

mN3 utilizes its enzymatic substrate-binding site to bind pTau
To identify the binding site of mN3 for pTau, we firstly determined the atomic structure of mN3 at the resolution of 2.0 Å by X-ray crystallography (Table supplement 1 Next, we conducted a cross-linking mass spectrometry (CL-MS) with chemical cross-linker BS 3 to covalently link paired lysine residues in spatial proximity (Cα-Cα distance < 24 Å) as pTau and mN3 interact, and then identified the cross-linked 9 segments by mass spectrometry. The result showed 7 pairs of cross-linked segments between pK19 and mN3 with a confidence score of < 10 -7 (Table supplement 2).
Lysine residues, K95, K139 and K206, that are involved in the cross-linking of mN3 with pK19, cluster around the entrance of the enzymatic pocket of mN3 (Figure 2A).
The entrance of the enzymatic pocket feature a positively-charged patch mainly composed of residues K55, K56, R205 and K206 for the NMN and ATP binding Since the NMR results identified the segment 349 RVQ(p)SKIG(p)SLDNI 360 shared by both pK19 and pTau23 as the primary binding segment of mN3, we next sought to characterize the complex structure of mN3 and pTau. We calculated and built the complex structure by Rosetta modeling ( Figure 2E). The complex structural model showed that the phosphorylated segment is well accommodated in the ATP/NMNbinding pocket of mN3. The phosphate groups of pS352 and pS356 orientate toward the positively charged pocket of mN3 and position in the same sites as those of ATP and NMN ( Figure 2E). This structure model explains the observation that mutations of K55, K56 and R205 (as in the KKRK mutant), which are the major components of the positively charged pocket and essential for the binding of phosphate groups, specifically abolished the binding of mN3 to the phosphorylated segment 349 RVQ(p)SKIG(p)SLDNI 360 ( Figure 2C). In addition, the chemical shift perturbations of pS262 of R1 and pS324 of R3 were also abolished as titrated by the KKRK mutant of mN3 ( Figure 2C), which indicates that these pSer residues may bind to mN3 in a similar manner as those of R4.
Primary sequence alignment shows that the key positively charged residues identified for pTau binding are highly conserved in the family of NMNATs from different species (Figure supplement 5), which suggests that different NMNAT 11 proteins employ a common and conserved interface for pTau binding. Indeed, mutation of the conserved K57 and R274 residues in hN2 severely impaired its chaperone activity against pTau23 aggregation (Figure supplement 11B).
Taken together, these results indicate that NMNAT adopts a conserved pocket to bind both the enzymatic substrates and pTau. Thus, the binding of NMNAT to pTau is similar to the specific binding of enzyme and substrate.

Competition between pTau and ATP/NMN for NMNAT binding
We next sought to understand how NMNAT spatially organizes its dual functions with the same pocket in a single domain. We have shown that the KKRK mutations of mN3 diminished the chaperone activity of NMNAT since it disrupts the positively charged pocket for the binding of phosphate groups. Next, we tested the influence of the KKRK mutations on the enzymatic activity of mN3 on NAD synthesis. The result showed that the KKRK mutations also eliminated the enzymatic activity of mN3, which is conceivable due to the inefficient binding of the mutant to the phosphate groups of ATP and NMN ( Figure 3A). On the other hand, we mutated H22, a key catalytic residue for NAD synthesis (Saridakis et al., 2001) that positions deep at the bottom of the substratebinding pocket (Figure supplement 12A). The result showed that the H22A mutation resulted in elimination of the enzymatic activity ( Figure 3A), which agrees with the previous study on the enzyme activity of NMNAT (Zhai et al., 2006). However, we found that the H22A mutation showed no influence on the chaperone activity of mN3 in inhibiting the amyloid aggregation of pK19 ( Figure 3B).
To examine the competition of the two activities, we used the BLI analysis and found that as the concentration of NMN increased, the binding of pK19 to mN3 was remarkably weakened ( Figure 3C). Consistently, the ThT assays showed that as the concentrations NMN or ATP decreased, the chaperone activity of mN3 on the amyloid aggregation of pK19 dramatically increased ( Figure 3D). In contrast, as we reversely added pK19 into the enzymatic reaction of NAD synthesis, no significant influence was observed ( Figure 3E).
Taken together, our data indicate that the enzymatic substrates (i.e. NMN and ATP) and the chaperone client pTau of mN3 share the same binding pocket with a partial overlap at the phosphate-binding site. While, ATP and NMN are superior to pTau on the mN3 binding.

NMNAT protects pTau from aggregation and synaptopathy in Drosophila
To assess whether the specific binding of NMNAT to pTau characterized in vitro has functional relevance in vivo, we examined the protective capability of Drosophila isoform PD in a Drosophila tauopathy model. We overexpressed human wild type (Tau WT ) or pathogenic Tau (Tau R406W ) in the visual system of Drosophila using a photoreceptor-specific driver GMR-GAL4 . The expression pattern can be easily visualized due to the highly organized parallel structure of the compound eye: the R1-R6 photoreceptors have their axons traverse the lamina cortex ( Figure 4A and 4B, magenta box) and make synaptic contacts at the lamina neuropil ( Figure 4A and 4B, red box), while R7-R8 photoreceptors extend their axons beyond lamina and 13 project to distinct layers in medullar neuropil ( Figure 4A and 4B, orange box) (Sato, Suzuki, & Nakai, 2013).
As shown in Figure 4A, aggregation of pTau probed by antibody specifically recognizing pTau Ser262 was found in both Tau WT and Tau R406W fly brain, which was efficiently suppressed by PD overexpression (Figure 4A and figure supplement 13).

mN3 mediates the recognition of Hsp90 to pTau to Hsp90
Previous studies showed that hN2 and Hsp90 co-precipitate with pTau in the brains of AD patients, and exhibit a synergistic effect in the attenuation of pTau pathology in cell models , Here, we used the SMPull (single-molecule pull-down) assay to identify the interplay between NMNAT, Hsp90 and pTau. SMPull is a powerful tool to quantitatively detect weak and transient interactions between protein complexes. As shown in Figure 5A, His 6 -tagged Hsp90 was coated on the slide, and the binding of pTau23 can be detected by the fluorescence from Alexa-647-labeled pTau23 using the total internal reflection fluorescence (TIRF) microscopy. The result showed that in the absence of mN3, binding of pTau23 to Hsp90 was only at the basal level that is similar to that of the blank slide (~ 20 fluorescent spots per imaging area), which indicates that the interaction between them is very weak and transient. While, the addition of mN3 to the Hsp90/pTau23 system significantly increased the fluorescent spots in a dosedependent manner ( Figure 5B, figure supplement 15A). In contrast, the binding of non-phosphorylated Tau23 to Hsp90 is not affected by the addition of mN3 (Figure supplement 15A and 15B). Thus, these results indicate that mN3 mediate the binding of pTau23 to Hsp90.
Furthermore, The BLI analysis showed that Hsp90 directly bound to mN3 with a K D value of ~ 1.93 µM ( Figure 5C). While, Hsp90 was not able to differentiate pTau23 from Tau23 with the binding affinity of 16.1 µM to pTau23 and 16.2 µM to Tau23 ( Figure 5D). Taken together, our data indicate that NMNAT acts as a co-chaperone to assist Hsp90 in the recognition of of pTau.

NMNAT represents a distinct class of molecular chaperones
NMNAT proteins have shown a robust neuroprotective activity in various models of neurodegenerative diseases correlated with the decrease of amyloid protein aggregation (Ali et al., 2013;Conforti et al., 2014). In this work, we demonstrate that NMNAT functions as a molecular chaperone that selectively protect 15 pTau from amyloid aggregation. Our work uncovers the structural basis of how NMNAT specifically binds pTau and how it manages its dual functions as both an enzyme and a chaperone. As illustrated in Figure 6, as NMNAT binds its enzymatic substrates, i.e. ATP and NMN, the substrates settle deep inside the pocket with defined Such specific binding features distinct NMNAT from other well-known chaperones. NMNAT is able to bind ATP as a substrate for its adenylyltransferase activity, while its chaperone activity is independent of ATP. NMNAT utilizes the same domain, even a shared pocket, for the binding of ATP and pTau, which explains the confusing previous observation that the neuroprotective effect of NMNAT is independent of ATP hydrolysis, yet requires the integrity of the ATP binding site (Ali et al., 2016). In addition, no large conformational change occurs as NMNAT binding to pTau.
In addition to inhibit pTau aggregation, NMNAT assists Hsp90 in the selection for pTau, implying that NMNAT serves as a co-chaperone of Hsp90 for pTau clearance.
Previous studies identified CHIP protein as an important co-chaperone of Hsp90 for pTau removal (Dickey, Kamal, et al., 2007;Dickey, Patterson, et al., 2007). whereas the binding of CHIP to Tau is disrupted once Ser262/Ser356 of Tau is phosphorylated by MARK2 (Dickey, Kamal, et al., 2007). Therefore, Hsp90 may employ different cochaperones to handle different species of Tau and maintain Tau homeostasis.

NMNAT links NAD and Tau metabolism
Our study demonstrates that the unique structural property of NMNAT gives rise to the binding of pTau with a high affinity comparable to that of an enzyme-substrate binding, but distinct from the weak interaction commonly found in chaperone-client binding (Koldewey, Horowitz, & Bardwell, 2017). The co-existence of the NAD synthase and chaperone activity sharing a common surface of NMNAT indicates an evolutionary connection between NAD metabolism and Tau proteostasis. During aging and AD pathogenesis, a concomitant decrease of ATP and NMN levels and pTau accumulation have been observed (Gomes et al., 2013;Stein & Imai, 2014;Wang & Mandelkow, 2015). Our data suggest that as ATP and NMN decrease, the shared enzymatic pocket may be more available for pTau binding. Therefore, the chaperone function of NMNAT may show up as ATP and NMN decrease during aging and neurodegeneration, implying a direct link between NAD metabolism and pTau proteostasis (Figure 6). The cellular processes of NAD metabolism and Tau phosphorylation and proteostasis are complex with multiple nodes of regulation. NMNAT emerges as a critical regulator balancing the NAD-mediated active metabolic state and the amyloid-accumulating proteotoxic stress state. Such regulation would be particularly important for maintaining the structure and functional integrity of neurons.

Protein expression and purification
Genes encoding mN1, mN3, Drosophila PC, PD (gift from Dr. Yanshan Fang) and genes encoding hN1, hN3 (purchased from GENEWIZ, Inc. Suzhou, China) were amplified and inserted into pET-28a vector with an N-terminal His 6 -tag and a following thrombin cleavage site. Gene encoding hN2 (purchased from Genewiz, Inc.) was cloned into pET-32M-3C derived from pET-32a (Novagen). The resulting plasmid encodes a protein with an N-terminal MBP (maltose-binding protein) and a His 6 -tag followed by NMNATs and variants were over-expressed in E. coli BL21 (DE3) cells. Cells were grown 2´YT medium at 37 °C to an OD 600 of 0.8-1. Protein expression was induced by the addition of 0.2 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG) and incubated at 16 °C for 15 hours. Cells were harvested by centrifugation at 4,000 rpm for 20 min and lysed in 50 ml lysis buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, and 2 mM phenylmethanesulfonyl fluoride (PMSF)) by a high-pressure homogenizer (800-1000 bar, 15 min). We next purified the over-expressed proteins by using HisTrap HP (5 ml) and HiLoad 16/600 Superdex 200 columns following the manufacturer's instructions (GE Healthcare). The purified proteins were finally in a buffer of 50 mM Hepes-KOH, 19 pH 8.0, 150 mM KCl, 10 mM MgCl 2, and 5% glycerol, concentrated, flash frozen in liquid nitrogen, and stored at −80 °C. The purity was assessed by SDS-PAGE. Protein concentration was determined by BCA assay (Thermo Fisher).
Human Tau23/K19 was expressed and purified on the basis of a previously described method (Barghorn, Biernat, & Mandelkow, 2005). Briefly, Tau23/K19 was purified by a HighTrap HP SP (5 ml) column (GE Healthcare), followed by a Superdex 75 gel filtration column (GE Healthcare). For 15 N-or 15 N/ 13 C-labeled proteins, protein expression was the same as that for unlabeled proteins except that the cells were grown in M9 minimal medium with 15 NH 4 Cl (1 g l −1 ) in the absence or presence of 13 C 6glucose (2 g l −1 ).
The sites and degrees of phosphorylation were quantified using 2D 1 H-15 N HSQC spectra according to previously published procedures (Eliezer et al., 2005;Schwalbe et al., 2013;Timm et al., 2003b) 20

Thioflavin T (ThT) fluorescence assay
Amyloid fibril formation of pK19 and pTau23 were monitored using an in situ ThTbinding assay. The ThT kinetics for amyloid fibrils were recorded using a Varioskan Flash Spectral Scanning Multimode Reader (Thermo Fisher Scientific) with sealed 384-microwell plates (Greiner Bio-One). Client proteins were mixed in the absence or presence of NMNATs and variants in indicated molar ratios in a buffer of 50 mM Tris-HCl, 50 mM KCl, 5% glycerol, 0.05% NaN 3 , pH 8.0, respectively. A final concentration of 50 µM ThT was added to each sample. To promote the formation of amyloid fibrils, 5% (v/v) of fibril seeds (the seeds were prepared by sonicating fibrils for 15 s) were added to pK19 and pTau23, respectively. ThT fluorescence was measured in triplicates with shaking at 600 rpm at 37 with excitation at 440 nm and emission at 485 nm.

Transmission electron microscopy (TEM)
5 µl of samples were applied to fresh glow-discharged 300-mesh copper carbon grids and stained with 3% v/v uranyl acetate. Specimens were examined by using Tecnai G2 Spirit TEM operated at an accelerating voltage of 120 kV. Images were recorded using a 4K × 4K charge-coupled device camera (BM-Eagle, FEI Tecnai).

Nuclear magnetic resonance (NMR) spectroscopy
All NMR samples were prepared in a NMR buffer of 25 mM HEPES, 40 mM KCl, 10 21 mM MgCl2, and 10% (v/v) D2O at pH 7.0. NMR experiments were collected at 298 K on Bruker Avance 600 and 900 MHz spectrometers. Both spectrometers are equipped with a cryogenic TXI probe. Backbone assignments of K19 and pK19 were accomplished according to the previously published assignments (Eliezer et al., 2005) and validated by the collected 3D HNCACB and CBCACONH spectra, respectively.
The mN3 structure was solved by molecular replacement method using Phaser (McCoy et al., 2007) in the CCP4 crystallographic suite  with the crystal structure of NMN/NaMN adenylyltransferase (1KQN) as template.
Several cycles of refinement were carried out using Phenix and Coot Vagin et al., 2004) progress in the structural refinement was evaluated by the free R-factor.The mN3 structure belong to the P21 space group with cell dimensions a = 53.7 Å, b = 80.8 Å, c = 64.5Å.

MALLS)
The weight-average molecular weight (

Cross-linking mass spectrometry analysis (XL-MS)
Cross-linking experiments were performed as described previously (Zhou et al., 2015).
pK19 was incubated with mN3 at 6:1 molar ratio in a buffer containing 50 mM Hepes-KOH, 150 mM KCl at pH 8.0 for 20 min at 4 . Cross-linker BS 3 (Thermo Fisher Scientific, 21585) was added at a 1:8 mass ratio and incubated at room temperature for 1 h. The reaction was quenched with 20 mM ammonium bicarbonate at room temperature for 20 min. Cross-linking products were analyzed by SDS-PAGE to assess the cross-linking efficiency. For MS analysis, proteins were precipitated with acetone; the pellet was resuspended in 8 M urea, 100 mM Tris (pH 8.5) and digested with trypsin at room temperature overnight. The resulting peptides were analyzed by online 24 nanoflow liquid chromatography tandem mass spectrometry (LC−MS/MS). And the mass spectrometry data were analyzed by pLink (Yang et al., 2012).
The extended 12-mer peptide mimic was initially placed near the putative phosphate binding site. 5,000 models were generated by using FlexPepDock protocol to simultaneously fold and dock the peptide over the receptor surface. In this fold-anddock step, we imposed the distance restraints to confine the glutamate residues of peptide mimic within the phosphate binding sites identified from the crystal structure.
The top models with favorable Rosetta energies and satisfied constrains were selected, and the phosphoserines were modeled back by replacing two phosphoserine mimic residues glutamates. The newly modeled structure was further refined by energy minimization to get rid of potential clash and maintain the identified phosphate binding site. After refinement, the top models ranked by Rosetta energies and constraints were selected for visually inspection.

Enzyme activity assay
Enzyme activity of NMNAT was measured in a continuous spectrophotometric coupled assay by monitoring the increase in absorbance of NADH at 340 nm, The reaction process is as follows (Balducci et al., 1995): Where C 0 β-NADH, the extinction coefficient of β-NADH at 340 nm, is 6.22 (Zhai et al., 2006).

Drosophila stocks and genetics
Flies were maintained on cornmeal-molasses-yeast medium at 25 , 65% humidity, 12 h light/dark cycle. The following strains were used in this study: UAS-Tau WT and UAS-Tau R406W obtained from Dr. Mel Feany

Immunohistochemical staining of fly brains
Fly brains with attached lamina were dissected and stained as previous described . Briefly, flies were dissected in cold PBS (pH 7.4) and fixed in freshly made

Confocal imaging and processing
Fly brains were imaged using an Olympus IX81 confocal microscope coupled with a 60× oil immersion objective. Images were processed using FluoView 10-ASW software (Olympus) and analyzed using Fiji/Image J (version 1.52). The intensity data were plotted as mean ± SD and statistical analyses were performed using One-way ANOVA post hoc Tukey test by Graphpad Prism (version 7.04).
All single-molecule assays were performed in the working buffer including 50 mM NaCl, 50 mM Tris, pH 8.0 and 0.1 mM TCEP at room temperature. Single-molecule imaging was conducted in the working buffer containing an oxygen scavenging system consisting of 0.8 mg/ml glucose oxidase, 0.625% glucose, 3 mM Trolox and 0.03 mg/ml catalase to minimize photobleaching. Slides were firstly coated with a mixture of 97% mPEG and 3% biotin-PEG, flow chambers were assembled using strips of double-sided tape and epoxy. Neutravidin and 20 nM biotin-NTA (Biotium) charged with NiCl2 were sequentially flowed into the flow chamber and each was incubated for 5 min in the working buffer. The immobilization of HSP90 (5 nM) was mediated by surfaced-bound Ni2+. Next, 4 nM Tau23/pTau23 and various concentrations of mN3 were added and incubated with the immobilized HSP90 for 10 min before data acquisition. An objective type total internal reflection fluorescence (TIRF) microscopy was used to acquire single-molecule data. Alexa647 labeled Tau23 or pTau23 was excited at 647 nm with a narrow band-pass filter (ET680/40 from Chroma Technology).

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Single-molecule analysis was performed using software smCamera. Mean spot per image (imaging area 2500 µm 2 ) and standard deviation were calculated from 10 different regions. A Enzyme activities of mN3 WT and mutants. The data shown correspond to mean ± s.d., with n = 3.
B Influence of the H22A mutation on the inhibition of mN3 against the amyloid aggregation of pK19. The molar ratio of pK19 to mN3 is 1:0.2. The data shown correspond to mean ± s.d., with n = 3. Values are compared using Student's t-test. NMNAT functions as both an NAD synthase involved in NAD metabolism, and a molecular chaperone involved in the clearance of pathological pTau deposition.
During aging as the level of ATP and NMN decrease, the chaperone function of NMNAT may show up to antagonize pTau aggregation.