Upadhyay A , Pisupati A , Jegla T , Crook M , Mickolajczyk KJ , Shorey M , Rohan LE , Billings KA , Rolls MM , Hancock WO , Hanna-Rose W .

P40 OD010440 NIH HHS , R01 GM086786 NIGMS NIH HHS

	Figure 1. NAM-induced cell death affects two cell types in pnc-1 mutants.(a) PNC-1 converts NAM to NA during salvage biosynthesis of NAD+. Loss of PNC-1 results in accumulation of NAM. Both the b uterine uv1 cells and the c dying cells in the nerve ring in pnc-1(pk9605) null mutants (strain HV560) have a similar necrotic morphology. The cell bodies swell to many times normal size (arrows), while the nuclear membrane disintegrates and the nucleus moves to the cell periphery (arrowheads). (d) DIC image and d′ corresponding fluorescence image of a pnc-1 mutant carrying the psEx279[ocr-4p::GFP] transgene. The dying cells in the region of the nerve ring (arrows) are marked by GFP. For comparison to swollen cells, multiple cells of normal morphology and size are annotated in b–d with an asterisk directly above the nucleus and in Supplementary Fig. 1. (e) Quantification of the cell deaths in pnc-1 null mutants. uv1 cell death (black bars) was scored in late L4 according to the absence of cells with the inIs179[ida-1::GFP] marker (strain HV560), and OLQ (grey bars) death was scored in late L3 or L4 according to the absence of cells with the psEx279[ocr-4p::GFP] marker (strain HV695). Actual per cent missing cells and sample size (number of cells examined) is indicated on each bar. Error bars are 99% confidence intervals (not applicable to 0%). *P<0.0001, calculated using Fisher's exact test. (f) The dendrites of the OLQ cell extend to the tip of the nose, visualized in a wild-type animal carrying the ocr-4p::GFP transgene. (g,g′) Fluorescence images of the (g) late L3 and (g′) L4 stage of the same pnc-1; psEx279[ocr-4p::GFP] animal, demonstrating that death is a degenerative process involving (g) first the blebbing of the dendrites (g′) followed by their disappearance as the cell bodies round up and begin to disappear. Note that the image in g′ is overexposed relative to g. Scale bars in all panels are 10 μm.
	Figure 2. Acute application of NAM results in rapid death of uv1 and OLQ cells.(a) 50, 200, 500 or 1,000 mM NAM or 1,000 mM NA (control) was added to individual animals and the percentage of animals with dying uv1 (black markers) or OLQ cells (grey markers) was quantified. Actual percentages and total number of animals examined (n) are indicated for each data point. Values represent the average from at least three supplementation experiments and error bars are standard deviations. For statistical analysis, P values for all pairwise comparisons were calculated using Fisher's exact test with a Bonferroni correction considering 10 total pair-wise comparisons for each experiment. Data points marked with distinct annotations (a–d) within each experiment indicate statistical differences with a Bonferroni adjusted P<0.001. (b) Cell death in response to acute NAM treatment is rapid. Each dot represents an individual animal and the position on the X-axis represents the time after NAM addition when the first evidence of cell death, nuclear disintegration, was evident. Boxes show the upper and lower quartile values, solid and dashed vertical lines indicate the median and mean of the population distribution, respectively. The dotted rhombus indicates one standard deviation, and error bars indicate the maximum and minimum of the population distribution. Two of the data points for OLQ were statistical outliers and not included in the statistical analysis.
	Figure 3. An OCR-4–OSM-9 TRPV channel mediates NAM-induced cell death.The percentage of animals with dead uv1 cells (black bars) or OLQ cells (grey bars) for each genotype supplemented with 25 mM NAM (+NAM) or in combination with pnc-1(pk9605) is reported. Actual percentages and sample sizes (n=number of animals examined) are indicated on each bar. Reported values for NAM supplemented animals are the average of at least three experiments and error bars are standard deviation. Error bars for the pnc-1 double mutant strains represent 95% confidence intervals of the proportion of the population (not applicable to values of 0 or 100%.) The wild-type strain used is N2. All alleles are loss or reduction of function with the exception of ocr-2(vs29) which is a dominant negative allele18. For statistical analysis, P values for all pairwise comparisons were calculated using Fisher's exact test followed by a Bonferroni correction considering eight (+NAM) or two (pnc-1) total pair-wise comparisons. ** Bonferroni adjusted P<0.001. * Bonferroni adjusted P=0.0018.
	Figure 4. NAM activates the OCR-4 and OSM-9 heteromeric TRPV channel.(a) Diagram of 2-electrode voltage clamp experiment and voltage ramp protocol: Step 1: cRNA is injected in the Xenopus oocyte. In 1–2 days, ion channel proteins express on the oocyte membrane. Step 2: Oocytes are impaled with two microelectrodes; e1 and e2. Command voltage (Vc) is chosen and kept constant using a feedback amplifier. Any change in oocyte membrane potential (Vm) due to ion channel activity is sensed by e1. To make Vm=Vc, a current is injected in the oocyte by e2. The injected current, which is equal and opposite to the current generated by ion channel activity, is measured. Currents were recorded in response to 2 s voltage ramps from −100 to +100 mV from a −20 mV hold. (b–d) Large NAM-activated currents were observed in oocytes co-expressing OCR-4 or OSM-9, but not expressing each subunit individually. Current size was measured at −100 mV (star), and the half maximal concentration (K1/2) for current activation by NAM was determined from the Hill plot shown in d. n indicates the Hill coefficient. The data in d show mean±s.e.m. (n=5–9 oocytes per NAM concentration), and the red curve is a Hill equation fit. Data from individual oocytes were normalized to the current amplitude observed at 1 mM NAM before comparison. (e) 5 mM NA did not elicit large currents in OSM-9–OCR-4-expressing oocytes (−0.154±0.018 μA, n=6 oocytes), but there is a slight increase in current upon exposure to 130 mM NA (−0.269±0.026 μA, n=4). Though this is a statistically significant increase (P<0.05, two-tailed t-test), 130 mM NA still elicited over 200-fold less current than 100 μM NAM (−36.5±1.0 μA, n=4).
	Figure 5. Stoichiometry of OSM-9–OCR-4 channels.(a) Frequency of the number of bleaching steps observed for 113 channels from oocytes expressing GFP-Kv2.1 homotetramers. The predicted step count distribution assuming 0.69 GFP detection probability (black symbols) is a best least squares fit of the data. (b) Frequency of the number of bleaching steps observed for 71 channels from oocytes expressing GFP::OSM-9–OCR-4. The inset shows example fluorescent traces for two-step (top) and one-step (below) photobleaching. (c) Stoichiometry distribution calculated from the observed one and two-step event totals in b, assuming a 0.69 GFP detection efficiency. (d) Frequency of the number of bleaching steps observed for 66 channels from oocytes expressing OSM-9–OCR-4::GFP. (e) Stoichiometry distribution calculated from one and two-step events in d, assuming a 0.69 GFP detection efficiency.
	Figure 6. NAM induced behaviours are mediated by osm-9 and ocr-4 expressing cells.(a) Images of L4 animals documenting the foraging, exaggerated nose-bending phenotype (brackets). Scale bar is 100 μm. (b) pnc-1 mutants and NAM supplemented animals have a foraging phenotype consistent with trpa-1 mutants. Error bars are 95% confidence intervals. Actual percentages and number of animals examined is indicated in each bar. P values were calculated using Fisher's exact test with a Bonferroni correction considering three pair-wise comparisons. (c) The NAM-induced Egl phenotype depends on osm-9 and ocr-4 function. Error bars are 95% confidence intervals. Actual percentages and number of animals examined is indicated in each bar. **Bonferroni adjusted P<0.001. (d) pnc-1 mutant and NAM-treated animals have a nose touch defect like that of osm-9 mutants, but ocr-4 mutants do not share this phenotype. We touched each animal ten times, calculated the percentage of touches that resulted in a positive response and reported the ratio of per cent positive responses relative to wild type. The ratio and the number of animals examined for each genotype or condition is indicated on each bar. Error bars are 95% confidence intervals. **Bonferroni adjusted P<0.001.
	Figure 7. NAM affects Drosophila chordotonal neuron dependent phenotypes.(a) Third instar larva were pretreated with different concentrations of NAM or 1 M NA as a control for 10 min then exposed to a 500 Hz tone at 90 dB and scored according to presence or absence of a startle response. Data are presented as percentage of startle responses to the stimulus. Error bars are 95% confidence intervals. Actual percentages and sample sizes are indicated above each bar. For statistical analysis, P values for six pairwise comparisons to 0 mM NAM were calculated using Fisher's exact test followed by a Bonferroni correction. **Bonferroni adjusted P<0.001. (b) Fluorescence intensity of chordotonal neurons expressing GCaMP6 (b) increases in response to mechanical stimulus (b′), increases in response to application of 1 M NAM (b′′), but fails to further increase in response to stimulus after application of NAM (b′′′). Images are individual frames from Supplementary Movie 1. An example of the region of interest (ROI) used to quantify images is outlined in b. Scale bar is 10 μm. Fiji's ‘Fire' Heat map LUT (lookup table) has been applied to images for visualization of intensity differences. (c,d) Fluorescence intensity for each individual neuron examined is normalized to average baseline values and plotted at baseline, immediately after repeated applications of stimulus, after returning to base line, after application of NAM or buffer, and after subsequent reapplications of stimulus (spacing between stimuli is 16 s),. (e) Quantification of data from c,d. The change in normalized relative fluorescence intensity in response to stimulus, to application of buffer or NAM, and to a mechanical stimulus in the presence of NAM or buffer is plotted. Error bars are standard deviations. The dotted line marks no change. **P<0.001 calculated using a two-tailed t test with Bonferroni adjustment considering three pairwise tests.
	Figure 8. Drosophila Inactive (IAV) and Nanchung (NAN) channel is activated by NAM.(a,b) An example trace of the effect of NAM on the Nan–Iav channel. Large NAM-activated currents were observed in oocytes co-expressing Nan and Iav. Currents were recorded in response to 2 s voltage ramps from −100 to +100 mV from a −20 mV hold. Current size was measured at −100 mV (star), and the half maximal concentration (K1/2) for current activation by NAM was determined from the Hill plot shown in b. Points and bars in b represent mean±s.e.m. normalized current at a given concentration of NAM (n=5–13 oocytes per NAM concentration). The Hill coefficient (n) is estimated to be about 2.1. This is consistent with the estimated Hill coefficient for the OSM-9-OCR-4 channel (4d), suggesting that the number of NAM binding sites is conserved in invertebrate TRPV channels. K1/2 appears to be about 14 μM, which implies 4–5 fold greater affinity of NAM by the Nan–Iav channel compared with the OSM-9-OCR-4 channel (4d). The number of NAM binding sites for the Nan–Iav channel is estimated to be 2, which is consistent with the number of NAM binding sites for the OSM-9-OCR-4 channel. (c) Current is not induced in Nan–Iav channel by 5 mM NA (−0.188±0.020 μA, n=3); There is a slight increase in the conduction of the Nan–Iav channel upon exposure to 130 mM NA (−0.422±0.040 μA, n=4). Though this is a statistically significant increase (P<0.01, two-tailed t-test), this high level of NA (130 mM) elicited over 50-fold less current compared with 1,000-fold less NAM.

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