Additional file 1 of NMNAT2 supports vesicular glycolysis via NAD homeostasis to fuel fast axonal transport
Creators: name: Yang, Sen
name: Niou, Zhen-Xian
name: Enriquez, Andrea
name: LaMar, Jacob
name: Huang, Jui-Yen
name: Ling, Karen
name: Jafar-Nejad, Paymaan
name: Gilley, Jonathan
name: Coleman, Michael P.
name: Tennessen, Jason M.
name: Rangaraju, Vidhya
name: Lu, Hui-Chen
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Physical Sciences not elsewhere classified
subject: Cell Biology
subject: Neuroscience
subject: Biotechnology
subject: Geology
subject: FOS: Earth and related environmental sciences
subject: Developmental Biology

description: Additional file 1: Fig. 1-S1. NMNAT2 expression, cKO mice survival rate, and motor behavioral deficit (A) In situ hybridization reveals nmnat2 mRNAs (blue signal) in the E14.5 brain, mainly in the cortical plate (CP) and in the lateral ganglionic eminence (LGE) and medial ganglionic eminence (MGE). (B) Illustration of Nmnat2 conditional KO (cKO) mice generation as described previously [37]. The blue and magenta triangles indicate loxP sites. (C, D) Summary of embryonic and postnatal pups genotyping. For embryonic samples, the strategy was to breed Nex-Cre/+;NMNAT2f/+ with NMNAT2f/+ mice. The genotyping results conform to the expectations of Mendelian inheritance. The breeding strategy for postnatal pups was to cross Nex-Cre/+;NMNAT2f/+ with NMNAT2f/f mice. The genotyping result showed a smaller than expected number of mice with the Nex-Cre/+;NMNAT2f/f (cKO) genotype, suggesting a reduced survival rate for cKO mice after birth. (E) Overall numbers of postnatal male and female ctrl and cKO mice. (F) 3-5 months old NMNAT2 cKO mice exhibit evident hindlimb clasping behavior (ctrl N=3, cKO N=4 were scored), Mann-Whitney test was used, data represents mean ± SEM, *p=0.04. Abbreviations: Cre, Nex-Cre; f/f, NMNAT2f/f; IZ, intermediate zone; Str, striatum; SVZ, subventricular zone; VC, ventricle; VZ, ventricular. Fig. 2-S1. Loss of NMNAT2 triggers APP accumulation in the axon of cultured cortical neurons (A) Representative confocal image of APP immunocytochemical staining in ctrl and KO neuronal culture at DIV5, 8, 10, and 14. Scale bar 30 μm. Red arrows highlight APP accumulation. (B) Area of accumulated APP in cKO and ctrl mice normalized to the average ctrl value of the corresponding DIV. Image numbers used for analysis: DIV5: 10 ctrl, 9 KO; DIV8 &amp; DIV10: 20 ctrl, 20 KO; DIV14: 50 ctrl, 67 KO. DIV5-10 were from 2 independent culture experiments and DIV14 was from &gt; 3 independent experiments. Multiple Mann-Whitney test with Holm-Šídák post-hoc adjustment, KO was compared to ctrl of the same DIV, ***p=0.000202, ****p&lt;0.0001. (C) Representative confocal images of APP, TUJ1 (βIII-tubulin), MAP2, and DAPI staining in DIV10 ctrl and KO neurons. Scale bar 20 μm. (D) Zoom-in view of axon and dendrite area highlighted by the white box in (C). Scale bar 5 μm. Fig. 2-S2. Gross neurite covering the area in vitro is not affected by Nmnat2 loss (A) Representative confocal images of MAP2, TUJ1, and DAPI staining in ctrl and KO neuronal culture at DIV10. MAP2 positive area corresponding to soma and dendrites was subtracted in TUJ1 staining images to determine area corresponding to axon regions. 100 μm scale bar in left and middle panels; 25 μm scale bar in zoom-in panels. (B) Quantification of the somatic and dendritic area labeled by MAP2 and the axonal area labeled by TUJ1 (MAP2 area subtracted) in ctrl and KO neuronal culture at DIV5, 8, 10, and 14, normalized to the average ctrl values at the same DIV. Image numbers from 1-3 independent experiments: DIV5: 10 ctrl, 10 KO; DIV8: 23 ctrl, 16 KO; DIV10: 11 ctrl, 13 KO; DIV14: 5 ctrl, 6 KO. Multiple Mann-Whitney tests with Holm-Šídák correction for comparisons were used for MAP2; two-way ANOVA with Šídák's multiple comparisons test was used for TUJ1. Data represent mean ± SEM. (C) The procedure of semi-automated quantification of accumulated APP by ImageJ. Fig. 2-S3. Loss of NMNAT2 after neurite outgrowth in vitro triggers axonal APP accumulation (A) The experimental timeline in NMNAT2f/f cortical neuronal culture. The NMNAT2 mRNA level was normalized to the LV-copGFP transduced ctrl group of the corresponding DIV and expressed as percentage. Numbers of culture wells from 1-2 independent batches of culture: DIV8: 2 copGFP, 2 iCre; DIV10 &amp; 14: 4 copGFP, 4 iCre; (B) Representative confocal images of APP, TUJ1 and MAP2 staining in DIV10 NMNAT2f/f neurons that received lentivirus starting at DIV5, 30 μm scale bar. Zoom-in view of axon and dendrite area highlighted by magenta and orange box respectively in the upper panel, 10 μm scale bar. (C) The area of accumulated APP was normalized to the LV-copGFP transduced neurons of the corresponding DIV as percentage. Number of images acquired from 2-3 independent experiments: DIV10: 28 copGFP, 28 iCre; DIV14: 26 copGFP, 24 iCre. Two-way ANOVA with Šídák's multiple comparisons test, iCre was compared to copGFP of the same DIV, ****p&lt;0.0001. Fig. 3-S1. NMNAT2 colocalize and co-migrate with vesicular cargos (A) Representative confocal images with maximum intensity Z stack projection showing that NMNAT2-mCherry colocalizes with APP-EGFP, SYPH-EGFP, and SNAP25-EGFP in axons of DIV8 WT cortical neurons (Scale bar, 20 μm). (B) Representative structure illumination images with maximum intensity Z stack projection showing NMNAT2-mCherry colocalizes with APP-EGFP, SYPH-EGFP, and SNAP25-EGFP in axons of DIV8 WT cortical neurons (Scale bar, 5 μm). (C) Representative confocal image shows the distribution of MitoVenus and NMNAT2-mCherry expressed in cortical axons. (Scale bar, 10 μm) (D) Representative grey value intensity plots along the axonal distance for MitoVenus and NMNAT2-mCherry or APP-GFP and NMNAT2-mCherry. (E) The ratio of NMNAT2-mCherry puncta colocalizing to APP-EGFP, SYPH-EGFP, SNAP25-EGFP, or MitoVenus quantified from confocal images. N represents the number of neurons imaged, APP (N=10), SNAP25 (N=20), SYPH (N=20), and MitoVenus (N=6). (F) Representative kymograph showing NMNAT2-mCherry comigrates with APP-EGFP in the axon of DIV8 WT neurons (Scale bar 20 μm in length, 30 s in time, 1.7 frame/second). (G) Frequency distribution of the anterograde and retrograde velocity of NMNAT2-mCherry and APP-EGFP in DIV8 distal axons. (NMNAT2 N=445, APP N=861, N represents the cargo trajectory traced in all kymographs). Fig. 3-S2. Vesicular cargo transport in the proximal axon is less affected by NMNAT2 loss at DIV8 (A) Diagram indicating the proximal axon region (&lt; 200 µm from the soma). (B) Representative kymographs for APP-EGFP from DIV8 ctrl and KO proximal axons. Scale bar, 20 µm. (C) Distribution of APP-EGFP vesicles in anterograde motion, retrograde motion, or in a stationary/dynamic pause from 2 independent culture experiments. Numbers (neurons imaged) and statistics: 23 ctrl, 27 KO; compared with unpaired t-test. (D) Velocity of antero- and retrograde transport of APP-EGFP from 2 independent culture experiments. Numbers (neurons imaged) and statistics: anterograde, 23 ctrl, 26 KO; retrograde, 22 ctrl, 26 KO. Two-way ANOVA with Šídák's multiple comparisons. (E) Representative kymographs for SNAP25-EGFP from DIV8 ctrl and KO proximal axons. Scale bar, 20 µm. (F) Distribution of APP-EGFP vesicles in anterograde motion, retrograde motion, or in a stationary/dynamic pause from 3 independent culture experiments. Numbers (neurons imaged) and statistics: 23 ctrl, 21 KO; anterograde and retrograde categories were analyzed by Mann-Whitney test, and stationary/dynamic pause category was analyzed by unpaired t-test. (G) Velocity of antero- and retrograde transport of SNAP25-EGFP from 3 independent culture experiments. Numbers (neurons imaged) and statistics: anterograde, 23 ctrl, 21 KO; retrograde, 22 ctrl, 20 KO. Two-way ANOVA with Šídák's multiple comparisons. Data represent mean ± SEM. Fig. 3-S3. Examples of axonal morphology overview (A1, B1) Stitched confocal image of DIV8 ctrl or KO neuron expressing SNAP25-EGFP. (A2, B2) Zoom-in of the regions of interest identified as proximal and distal axons as highlighted in magenta and dark green boxes in A1, B1. (A3) Kymograph generated from the axon segment selected and highlighted in yellow in (A2, B2). Fig. 3-S4. Mitochondrial density, morphology, and mobility in distal axons are unaffected by NMNAT2 loss at DIV8 (A) Representative images of EGFP and Mito-DsRed in the distal axon of DIV8 ctrl and KO neurons. (Scale bar 10 μm). (B) Mitochondria density (# of mitochondria per 100 μm) in distal axons of DIV8 ctrl and KO neurons from 3 independent experiments. Numbers (neurons imaged) and statistics: 38 ctrl, 40 KO; unpaired t-test was used. (C) The cumulative percentage distribution of mitochondria morphology indicated by sphericity in DIV8 ctrl and KO distal axons from 3 independent experiments. Numbers (neurons imaged) and statistics: 38 ctrl, 40 KO; Log(Y/(1-Y)) transformation for the independent variable and linear mixed model with random intercept and the slope was used; see descriptions in Star Methods. (D) Representative kymographs show the mobility of MitoDsRed in distal axons of DIV8 ctrl and KO neurons. (Scale bar, 20 µm; total recording time, 15min). (E) The velocity of anterogradely and retrogradely moving MitoDsRed in distal axons of DIV8 ctrl and KO neurons from 2 independent batches of culture. (Anterograde: ctrl N=14, KO N=16, Mann-Whitney test was used; Retrograde: ctrl N=14, KO N=14, unpaired t test was used). (F) Percentage of MitoDsRed that is anterogradely moving, retrogradely moving or in stationary/dynamic pause in distal axons of DIV8 ctrl and KO neurons from 2 independent batches of culture (ctrl N=19, KO N=21). For analyzing anterograde and stationary/dynamic pause, an unpaired t-test was used; for retrograde, the Mann-Whitney test was used. N represents number of neurons imaged. All above data represents mean ± SEM. Fig. 5-S1. pH measurement and pH correction for Syn-ATP (A) Cyto-pHluorin imaging timeline and equation used for cytosolic pH determination in presynaptic varicosities. (B) pH measured by Cyto-pHluorin in various conditions. The mean value in each group is labeled above each box and whisker. Numbers (neurons imaged) and statistics: 27 ctrl, 25 KO, and 27 KO+NAD+ under basal conditions; 18 ctrl, 25 KO, and 27 KO+NAD+ under oligomycin-treated conditions. All groups were from a single experiment. Two-way ANOVA with Tukey's multiple comparisons test, **p= 0.005. (C) Equations used for pH correction of L/F measurements from Syn-ATP. (D) Raw L/F measurements in distal axon varicosities of DIV8 ctrl neurons with or without NAD+ supplementation. Numbers (neurons imaged) and statistics: 78 ctrl and 10 ctrl+NAD+ under basal conditions; 67 ctrl and 10 ctrl+NAD+ under oligomycin-treated conditions. Ctrl was from 5 experiments, and ctrl+NAD+ was from one experiment. Kruskal-Wallis test with Dunn's multiple comparisons test, *p= 0.0171, **p= 0.0018. All above data represent mean ± SEM. Fig. 5-S2. Increase of intracellular NAD+ by exogenous supplementationThe change of intracellular NAD+ abundance over time after one-time 1 mM NAD+ supplementation into the culture medium. All groups were normalized to ctrl+NAD+ 0 h time point and expressed as a percentage. Numbers (culture wells) and statistics: 13-14 ctrl and 12- 14 KO from 4 independent experiments, Kruskal-Wallis test with Dunn's multiple comparisons test was used, *p= 0.049, **p= 0.0023 Fig. 6-S1. The NAD+ synthesizing enzymatic activity of NMNAT2 is required to rescue APP accumulation in NMNAT2 KO neurons (A) Representative confocal images of APP staining of DIV12 KO neurons with or without lentiviral transduction to overexpress wt-NMNAT2 or ED(enzyme dead)-NMNAT2, scale bar 35 µm. (B) Comparable lentiviral transduction rate for wt-NMNAT2 and ED-NMNAT2 in KO neurons with 4MOI (multiplicity of infection) lentivirus. Transduction rate was calculated as the percentage of HA+ APP+ cells out of APP+ cells. N=8 per group, N represents the number of images randomly sampled, unpaired t test was used. (C) The summary for the area of accumulated APP relative to WT as percentage. N=14-18 per group from 2 independent batches of culture, N represents the number of images randomly sampled. One-way ANOVA with Tukey's multiple comparisons test was used, **p= 0.0034, ****p &lt;0.0001. All above data represents mean ± SEM. Fig 6-S2. NAD+ supplementation depends on glycolysis to ameliorate APP accumulation in NMNAT2 KO neurons (A) Timeline of NAD+ supplementation, 2DG(+Methyl-pyr) treatment, and immunostaining. (B) Representative confocal images of APP staining of DIV14 ctrl or KO neurons under Veh (vehicle) or NAD+ supplementation, treated with Veh or 2DG. Scale bar, 30 µm. (C) Area of accumulated APP in DIV14 ctrl or KO neurons supplemented with Veh or NAD+ and treated with Veh or 2DG. All values were normalized to ctrl supplemented and treated with Veh. Numbers (images) and statistics: 18 ctrl+Veh, 20 KO+Veh, 23 ctrl+ 2DG, 12 KO+2DG, 19 ctrl+NAD+, 22 KO+NAD+, 21 ctrl+NAD++2DG, and 22 KO+NAD++2DG, Two-way ANOVA with Tukey's multiple comparisons test was used, ****p&lt;0.0001, ####p&lt;0.0001. Except for KO+2DG, all groups were from 2 independent experiments. All above data represent mean ± SEM. Fig 7-S1. Gender ratio, body weight, and motor behaviors in surviving NMNAT2 cKO mice lacking one or two copies of SARM1 (A) Y axis represents the percentage of postnatally alive mice of ctrl, NMNAT2 cKO; Snull/+, and NMNAT2 cKO; Snull/Snull genotypes pooled from progenies derived from various parent genotype combinations. The actual number of mice from each genotype and gender is listed on top of each bar. (B) Body weight of ctrl, NMNAT2 cKO; Snull/+, and NMNAT2 cKO; Snull/Snull mice at P16/21, and P90. Number of mice: P16/21, 61 ctrl, 34 NMNAT2 cKO; Snull/+, 24 NMNAT2 cKO; Snull/Snull; P90, 11 ctrl, 7 NMNAT2 cKO; Snull/+, 5 NMNAT2 cKO; Snull/Snull. Kruskal-Wallis test with a two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli was used for P16/21 data, ****p&lt;0.0001. One-way ANOVA with a two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli was used for P90 data, (ctrl vs. NMNAT2 cKO; Snull/+), ***, p= 0.0004, q=0.0004; (NMNAT2 cKO; Snull/+ vs. NMNAT2 cKO; Snull/Snull), **, p= 0.0039, q= 0.002. (C) Hindlimb clasping phenotype in ctrl, NMNAT2 cKO; Snull/+, and NMNAT2 cKO; Snull/Snull mice of 3-5 months old (ctrl N=13, cKO; Snull/+ N=20, cKO; Snull/Snull N=6). Kruskal-Wallis test was used, ***p&lt;0.0005, ****p&lt;0.0001. All above data represents mean± SEM. Fig. 7-S2. APP accumulation in P5 brains of NMNAT2 cKO mice lacking one or two copies of SARM1 (A) Immunohistology images showing APP accumulation in the corpus callosum, fimbria, and striatum of P5 NMNAT2 cKO; Snull/+, but not in ctrl and NMNAT2 cKO; Snull/Snull brains. (B) Normalized APP signals in P5 brains. Number of mice: 4 ctrl, 4 NMNAT2 cKO; Snull/+, 3 NMNAT2 cKO; Snull/Snull. CC, corpus callosum; Str, striatum; Fim, Fimbria. For CC, one-way ANOVA with a two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli was used (Ctrl vs. NMNAT2 cKO; Snull/+) **, p=0.0032, q= 0.0021; (NMNAT2 cKO; Snull/+ vs. NMNAT2 cKO; Snull/Snull) **, p=0.004, q=0.0021. For Str, Kruskal-Wallis test with a two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli was used, (Ctrl vs. NMNAT2 cKO; Snull/+) *, p=0.0142, q=0.0298; (NMNAT2 cKO; Snull/+ vs. NMNAT2 cKO; Snull/Snull) *, p=0.0414, q= 0.0435. For Fim, one-way ANOVA with a two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli was used, ****p&lt;0.0001. All above data represent mean ± SEM. Fig 8-S1. SARM1 knockdown validation (A) RT-qPCR quantification of sarm1 mRNA levels across ASO treatments in primary mouse cortical neurons. Cells were treated for 2 days (DIV5-7) with a control or a SARM1 ASO (33 or 47) at two different concentrations to test efficacy. Number of wells: 6 ctrl; 2 ASO33 at 1 uM or 5 uM, 2 ASO47 at 5 uM or 10 uM. (B) Representative western blotting detecting SARM1 and GAPDH (internal control) protein levels at DIV8 after ASO treatment since DIV6. Brain lysates of SARM1 WT and SARM1-null (Snull/Snull) mice were included for antibody specificity validation. (C) Quantification of SARM1 protein level across various ASO treatment duration, as detected by western blot. SARM1 protein level was normalized to GAPDH first, then normalized to its corresponding ctrl-ASO-treated group. Numbers of wells: 2 days Sarm1 ASO, n=3. 2 days Ctrl ASO, 3 days Ctrl ASO, 3 days Sarm1 ASO, 5 days Ctrl ASO, 5 days Sarm1 ASO, n=4. 8 days Ctrl ASO, 8 days Sarm1 ASO, 9 days Ctrl ASO, 9 days Sarm1 ASO, n=6. 7 days Ctrl ASO, 7 days Sarm1 ASO, n=8. 4 days Ctrl ASO, 4 days Sarm1 ASO, n=12. Statistics were conducted with the Kruskal-Wallis test and corrected for multiple comparisons by controlling the False Discovery Rate using the Two-stage step-up method of Benjamin, Krieger, and Yekutieli. All above data represent mean ± SEM. *, p&lt;0.05, ***, p&lt;0.001, ****, p&lt;0.0001. Fig. 8-S2. SARM1 knockdown prevents APP accumulation and axon degeneration in NMNAT2 KO neurons in vitro (A) Representative images illustrating the effects of ctrl-ASO and SARM1-ASO on APP accumulation in WT and NMNAT2 KO primary cultured cortical neurons. (B) Quantification of APP accumulation area following ASO treatment of two timelines: DIV1 to DIV14 and DIV5 to DIV14. (C) Representative images show TUJ1 axonal staining of WT and KO cells following treatment with ctrl-ASO and SARM1-ASO. (D) Percentage of the total TUJ1 area found as aggregates following control and SARM1 ASO (ASO 33) treatments at DIV1-14 and DIV5-14. (E) Representative images of DAPI staining and quantification (F) of live cells in every sampled image. Image numbers: DIV1-14, N=6 for every group, from 2 independent experiments; DIV5-14, N=9 for every group, from 3 independent experiments. 3 images were collected from each experimental batch. Data for panel D, DIV5-14 was not normally distributed, so a Kruskal-Wallis test multiple comparisons test was performed. All other data sets were analyzed using two-way ANOVA and Tukey multiple comparisons. All above data represent mean ± SEM. ***, p&lt;0.001, ****, p&lt;0.0001. Fig 8-S3. SARM1 knockdown rescues global NAD deficiency and distal axonal NAD+/NADH ratio in NMNAT2 KO neurons in vitro (A-C) Levels of NAD+ (A), NADH (B), and the calculated NAD+/NADH ratios (C), in DIV8 WT and KO cortical neurons treated with PBS, ctrl-ASO (ASO30), or SARM1-ASO (ASO33) from DIV1-8. For ASO treated groups, the raw concentration is normalized to the corresponding protein amounts, and then normalized as percentages to the average value acquired from ctrl-ASO treated WT neurons. For PBS treated groups, the raw concentration is normalized to the corresponding protein amounts, and then normalized to WT average value. N represents each well of culture lysate from 2-3 independent culture experiments, WT+PBS (N=16), KO+PBS (N=16), WT+ASO30 (N=23), KO+ASO30 (N=23), WT+ASO33 (N=23), KO+ASO33 (N=23). Kruskal-Wallis test with Dunn's multiple comparisons test was used. (D) Representative images showing signals emitted from the SoNar (NAD+/NADH) ratiometric sensor for NAD+ and NADH in distal axons of DIV 8 WT or KO cortical neurons treated with ctrl-ASO or SARM1-ASO. Scale bar, 20 mm. (E) F488/F440 ratiometric measurements of the SoNar sensor, reflecting the NAD+/NADH ratios in distal axons of DIV8 WT or KO cortical neurons treated with ctrl-ASO or SARM1-ASO since DIV1. N represents the number of neurons imaged from two independent cultures, WT+ctrl-ASO (N=17), KO+ctrl-ASO (N=21), WT+SARM1-ASO (N=20), KO+SARM1-ASO (N=23). Two-way ANOVA with Tukey's multiple comparisons test was used. All data represent mean ± SEM. *p&lt;0.05, **p&lt;0.01, ****p&lt;0.0001