Designing an NMNAT2-specific high-throughput screening (HTS) platform to quantify NMNAT2 protein levels in cortical neurons

The Meso Scale Discovery (MSD) detection platform potentially provides superb sensitivity with great dynamic range for detecting analytes of choice (MULTI-ARRAY® technology and see http://www.meso-scale.com/ for details)33,34. This proprietary SULFO-TAG labeling allows for emission of light upon electrochemical stimulation initiated at the electrode surfaces of multi-array plates. The decoupling of stimulation and signal leads to strong and specific MSD signals. MSD relies on an ELISA sandwich assay that utilizes a “capture” antibody to bind the target protein in analyte. This Ab-Analyte complex is then recognized by a sulfo-tagged primary antibody (detection antibody) allowing detection. Here we aimed to design an NMNAT2-MSD platform utilizing commercially available NMNAT2 antibodies to reliably measure NMNAT2 levels.

A variety of commercially available monoclonal and polyclonal antibodies targeting either mouse or human NMNAT2 protein were acquired (Table S1). To start the optimization process, we designed the NMNAT2-MSD assay with different combinations of antibodies. Our first goal was to detect NMNAT2 protein in a dose-dependent manner with strong and specific signals (Fig. 1A). Different combinations of capture and detection antibodies were assembled and their detection efficacies were evaluated. We found that the best pair of antibodies providing the desirable linear dynamic range of detection was to use the Abcam ab110040 rabbit polyclonal antibody (epitope is aa100–200 of Rat NMNAT2) as the capture antibody together with sulfotagged-Abcam ab56980 mouse monoclonal antibody (epitope is aa208–308 of human NMNAT2) as the detection antibody (Fig. 1A). The signals were greatly reduced when the antibody order was reversed (Fig. 1A). This suggests that the proper combination of capture and detection antibody allowing for optimal conformational coverage of the analyte protein is important. In addition, almost no signal was detected when NMNAT1 protein was used as analyte (Fig. S1A) or when NeuN antibody was used as the capture antibody (Fig. S1B). At the same time, the signals were saturated when a very high quantity of recombinant NMNAT2 was placed as analyte (Fig. S1C). These finding from control experiments demonstrated that NMNAT2-MSD platform established here provides sensitive NMNAT2-specific detection.

Figure 1: Development of an NMNAT2-MSD platform specifically detecting NMNAT2 with high sensitivity. (A) Ab110040 antibody as capture and sulfotagged Ab56980 antibody as the detection antibody pair, but not in reverse order provides, a strong NMNAT2-MSD signal and a linear detection with increasing concentrations of recombinant NMNAT2 protein. (B) Dose-dependent MSD signals for cell lysates of Cos-7 cells transfected with HA-NMNAT2 or mCherry. (C) Low NMNAT2-MSD signals for Cos-7 cells transfected with HA-NMNAT2 or mCherry when the order of capture and detection antibody was reversed. (D) NMNAT2-MSD signals detected from DIV 14 NMNAT2 WT, HET and KO neurons plated at the indicated densities. Full size image

To test if we could detect NMNAT2 in cultured cells, cDNAs carrying HA-tagged NMNAT2 or mCherry (as a negative control) were transfected into cos7 cells (Fig. 1B). The signal increased in a linear fashion in proportion to the total amount of protein prepared from NMNAT2-cos7 cells (Fig. 1B), while minimal signal was detected with mCherry-cos7 cell extracts. Reversing the capture and detection antibody order, again, greatly reduced the signals despite large amounts of total protein were applied (Fig. 1C).

NMNAT2 is expressed in cultured cortical neurons and its abundance is greatly increased between 8 and 13 days-in-vitro (DIV)21. NMNAT2 knockout (KO) mice die at birth23 but one can prepare NMNAT2 KO cortical neurons from NMNAT2 KO embryos. These neurons survive relatively well for more than two weeks in vitro6. To test if MSD detects endogenous NMNAT2, we prepared cortical neurons from E16.5 NMNAT2-wildtype (WT), heterozygous (HET) and KO embryos harvested from pregnant NMNAT2-HET dams mated with NMNAT2-HET male mice. These neurons were plated on poly-D-lysine–coated 96 well plates with ~12,500, 25,000, 50,000 or 100,000 cortical neurons per well. The signal to background ratio values from Nmnat2 WT and HET DIV14 neurons linearly increased proportional to neuronal density (Fig. 1D). Most importantly, only minimal signal was detected with NMANT2 KO neurons while NMNAT2 MSD signal from Nmnat2 HET DIV 14 neurons are ~50% of WT neurons. In addition, treatment with different concentrations of MG132, a proteasome inhibitor known to stabilize NMNAT214,20,35, increased the NMNAT2 MSD signal in a dose-dependent fashion up to 10 μM (Fig. 2A). Together these results suggest that this NMNAT2-MSD assay format detects endogenous NMNAT2 in cortical neurons with great specificity and sensitivity.

Figure 2: The NMNAT2-MSD platform detects dynamic changes in NMNAT2 protein levels in primary cortical neuron cultures in specific manner. (A) NMNAT2-MSD signals from cortical neurons treated with the indicated concentrations of MG132. (B–D) NMNAT2-MSD signals for neurons treated with 3 or 6 hrs of MG132. Two separate plates were examined. Neurons were plated at 25,000 (B), 50,000 (C), or 100,000 (D) cells per well of 96 well plates. (E) Summary of z factors for MG132-enhancement of NMNAT2 levels as measured by MSD. Full size image

Inter-plate variability was examined with densities of 25,000, 50,000 or 100,000 neurons per well. Neurons at these different densities were treated with vehicle or 10 uM MG132 for 3 or 6 hours (Fig. 2B–D). We found that 50,000 neurons per well gave the minimal inter-plate variability (Fig. 2B–D). The Z-factor is a standard measure for determining the ability of an assay to reliable detect a “hit” (see Methods). We found that neuronal densities of 50,000 cells per well gave Z factors >0.5 (Fig. 2E). Suggesting this cell density in 96 well plate will give the most reproducible signals while reducing the number of neurons needed in each assay.

Based on the manufacturer instructions (MSD.com), the optimal formulation and concentrations of antibodies for MSD assay should be 1–2 mg/ml of protein in PBS (pH 7.4–7.9), without any azide, carrier protein, glycine, histidine, Tris or glycerol (common additives in commercially available antibody solutions). Indeed, when the capture:detection antibody pair in optimal condition was acquired from Abcam, we found NMNAT2 MSD signals were increased for ~3.5 fold (Fig. S2). The large dynamic ranges of NMNAT2-specific MSD signals gave us confidence to conduct a drug screen using cortical neurons using the MSD technology.

LOPAC library for Proof of Concept Screen

To identify pathways modulating NMNAT2 abundance, we chose the SIGMA LOPAC library. The Sigma LOPAC1280 library contains 1280 pharmacologically-active compounds. This annotated collection of small molecule modulators and approved drugs impacts most cellular processes and covers all major drug target classes. Specifically, 10 uM of individual compounds from this library were applied to DIV14 cortical neurons plated at 50 K per well of 96 well plates (Fig. S3). Neurons in 96 well plates were treated by 10 uM of each compound in two separate plates, to account for interplate variability (Fig. S3). In every plate, 6 wells were treated with DMSO (negative control) and 6 wells were treated with 10 uM of MG132 (positive reference compound). After 6 hours of treatment, neurons were lysed with MSD buffer and prepared for NMNAT2-MSD assay. Z factors were calculated to examine inter-plate variability and only the results from a plate had Z-factor >0.4 were included for data analysis. NMNAT2-MSD signals were obtained from these treated neurons. We found that the normalized NMNAT2-MSD values of these compounds obtained were tightly clustered around a fold change of 1 (Fig. 3A), further demonstrating the reliability of the assay. Our positive reference compound MG132 yielded about 2-fold increase in NMNAT2 signals (Fig. 3B). Based on the distribution of Z factors, we found minimal inter-plate variability among different plates (Fig. 3C). The hit rate, defined by the number of drugs that either negatively or positively affected NMNAT2 abundance by 1.5 fold relative to DMSO treatment in this screen, was 2.89% (37 out of 1280) and resulted in identifying 24 positive and 13 negative NMNAT2 modulators (Table S2).

Figure 3: Identification of NMNAT2 modulators from the Sigma-LOPAC library screen using the NMNAT2-MSD platform. (A) NMNAT2 fold change from drug treatment of wild type cortical neurons plated in 96 well plates at density of 50 K per well and treated with 10 μM of the library compound for 6 hours. Fold change is represented as change over DMSO control. (B) Summary for NMNAT2-MSD signals detected from neurons treated with DMSO (negative control) and MG132 (positive control) from all the plates included in the data analysis. (C) Z factor distribution for all the assay plates included for data analysis. Full size image

Next, we conducted Western analysis with 33 hits (limited by compound availability and economical considerations) to validate and examine the potency of these hits. During the first round of testing, doses between 0–50 uM of 8-Br-cAMP, caffeine or Bay K were applied to DIV14 neurons for 6 hours to determine their impacts on NMNAT2 levels using the NMNAT2 2G8 antibody. Linear dose-dependent increases were found in the 0–20 uM range for all three compounds (Fig. 4A). Next, we examined the efficacy of 30 additional hits on NMNAT2 levels by treating neurons with DMSO/0, 1.25, 2.5, 5, 10 or 20 uM of the testing compound for 6 hrs (Figs S4 and S5). In total, 27 of 33 hits show similar degree of NMNAT2 level changes in MSD and Western (Table S2). Among these confirmed hits, 13 compounds induced significant changes in NMNAT2 level at the conc. of ≤2.5 uM (marked with yellow highlight on Table S2).

Figure 4: Caffeine increases NMNAT2 expression in vitro and in rTg4510 tauopathy mice. (A) Western blots show NMNAT2 and GAPDH levels in DIV14 cortical neurons after 6 hrs of treatment with 8-Bromo-cAMP, caffeine or Bay-K. Summary for the dose-dependent impact of 8-Bromo-cAMP, caffeine or Bay-K on NMNAT2 levels. *Caffeine/#BayK/$8-bromo-cAMP, */#/$p < 0.05, **/##/$$p < 0.001, ***/###/$$$p < 0.0001 when compared to DMSO control for respective drugs. (B) NMNAT2 levels in the cortex of NMNAT2 WT and HET mice after saline or caffeine treatment (two independent experiments with n = 8 animals per treatment group). (C) NMNAT2 levels in the cortex of rTg4510 mice and controls after saline or caffeine treatment (repeated in two trials, with n = 6 total animals per treatment group). Bar graphs were plotted with mean ± sem; statistical significance was assessed by one-way ANOVA, *p < 0.05, **p < 0.001 (Complete original blots provided in Supplemental Figure S7). Full size image

Impaired cell viability could account for the reduced NMNAT2 MSD signals. The MTT assay, a cell viability assay, was conducted to estimate the impacts of NMNAT2 negative modulators on the viability of DIV14 cortical neurons. We found that 6 hrs treatment of aconitine, cantharidin, E64, bendamustine HCL, wortmannin, Ziprasidone or retinoic Acid did not affect neuronal viability (Fig. S6). Etoposide and gossypol treatment did reduce neuronal viability.

Caffeine is commonly encountered in multiple beverages, has been linked to reduced dementia risk36, and is known to cross the blood brain barrier and thus we chose it to evaluate its in vivo impacts as a NMNAT2 positive modulator37. To examine whether caffeine could increase NMNAT2 in the brain, 2 different doses of caffeine (20 mg/kg or 50 mg/kg) or 0.9% saline vehicle were administered i.p. twice, separated by 4 hours, to 3-month-old NMNAT2 WT and HET mice. NMNAT2 levels in the cortex of these mice were evaluated 4 hours after the second dose with Western analysis. Similar to our previous finding, we found that NMNAT2 levels in NMNAT2 HET cortex was about 50% of the WT cortex (after vehicle treatment; Fig. 4B). Caffeine treatment resulted in a dose-dependent increase in NMNAT2 levels for both NMNAT2 WT and HET cortices. These data demonstrate that acute caffeine treatment can enhance NMNAT2 levels in vivo (Fig. 4B). Our previous studies found that NMNAT2 was greatly reduced in the cortex of rTg4510 transgenic mice, a tauopathy model13. To examine whether caffeine treatment could also increase NMNAT2 levels in the cortex of rTg4510 mice, 3-month-old rTg4510 mice and littermate controls were injected with vehicle or 50 mg/kg caffeine twice separated by 4 hours (n = 3 for each treatment). We found that cortical NMNAT2 levels were significantly increased in both control and rTg4510 mice after 8 hours of caffeine exposure (Fig. 4C).

Vincristine-induced neuronal death is reduced by NMNAT2 positive modulators and enhanced by NMNAT2 negative modulators

Previously, we have demonstrated that NMNAT2 is required for neurons to counter the toxicity induced by vincristine, a chemotherapy agent commonly used to treat leukemia38. After 12 or 24 hours of vincristine treatment, the MTT assay was conducted to estimate cell viability of DIV14 NMNAT2 WT, HET and KO neurons. We found that vincristine treatment resulted in significant cell death in NMNAT2 WT neuron cultures by 12 hours of treatment (Fig. 5A). NMNAT2 HET and KO neurons were significantly more sensitive to vincristine than WT neurons. NMNAT2 KO neurons were particularly vulnerable to vincristine treatment. Less than of 10% of NMNAT2 KO neurons remained viable after 24 hours of vincristine treatment. Increasing NMNAT2 using a lentiviral vector in cultured cortical neurons offered significant protection against 12 hrs of vincristine-induced toxicity (Fig. 5B). Taken together with the dose-dependent correlation of NMNAT2 level to cell viability, this assay provides an approach to evaluate whether test compounds positively or negatively modulate NMNAT2-dependent neuroprotective effects.

Figure 5: NMNAT2 protects against vincristine induced cell death. MTT assay shows reduced viability of NMNAT2 HET and KO neurons after vincristine exposure. (A) DIV14 NMNAT2 WT, HET, KO neurons were treated with DMSO or 10 uM vincristine for 12 or 24 hours. Cell viability was assessed with a MTT reduction assay. Experiment was repeated 3 times in triplicates. (B) NMNAT2 WT, HET, KO neurons were transduced with either LV-GFP or LV-NMNAT2 at DIV2. At DIV14, these neurons were treated with 10 uM vincristine for 24 hours. Experiment was repeated 3 times in triplicates. Statistical significance assessed using One-way ANOVA, followed by posthoc Tukey analysis. Full size image

We next selected 4 positive NMNAT2 modulators: L-Aspartic acid, caffeine, PD169316, and Rolipram as well as 4 negative NMNAT2 modulators: cantharidin, Ziprasidone, retinoic acid, and wortmannin to evaluate whether they modulate sensitivity to vincristine-induced toxicity in an NMNAT2-dependent manner. DIV14 cortical neurons prepared from NMNAT2 WT, HET, or KO embryos were pretreated with 10 uM of the test compound for 6 hours first and then together with 10 uM vincristine or vehicle for an additional 6 hours. We found that pretreatment with L-Aspartic acid, caffeine, and rolipram significantly reduced the impact of vincristine on the cell viability of NMNAT2 WT and HET, but not KO neurons (Fig. 6), while PD-169316 pre-treatment reduced vincristine toxicity in all genotypes. These data suggest that L-Aspartic acid, caffeine, and rolipram require NMNAT2 to protect neurons from vincristine insult, while PD0169316 provides protection in an NMNAT2-independent fashion. NMNAT2-negative modulators: Ziprasidone, cantharidin, wortmannin and retinoic acid decreased cell viability in both NMNAT2 WT and HET but not KO neurons without vincristine. Upon vincristine treatment, these NMNAT2 negative modulators further reduced the viability of NMNAT2 WT and HET neurons. In NMNAT2 KO neurons, cantharidin, wortmannin and retinoic acid did not exaggerate vincristine-induced toxicity. Taken together these results suggest that 6 out of 8 NMNAT2 modulators affect neuronal viability by up regulating or down regulating NMNAT2 levels.