Figure 6. Labeling of mitochondrial NAD tracks that of total NAD, but not of total NMN.

For all panels, data representing whole cells are depicted as solid bars, whereas data from isolated mitochondria are shown with a stippled pattern. (A) Differentiated C2C12 parental and LentiCRISPR transgenic myotubes were analyzed for NAD content. The cells are as follows: ctrl- parental line with no vector; R26- vector control; 1a and 1b- two separate guide RNAs targeting NMNAT1; 2a and 2b- two separate guide RNAs targeting NMNAT2; 3a and 3b- two separate guide RNAs targeting NMNAT3. (N = 3). (B) Mitochondria isolated from differentiated C2C12 cells were held in state 2 (MirO5 respiration buffer containing 10 mM Pyruvate, 5 mM Malate) at 37°C with shaking for 30 min. They were then collected and lysed in perchloric acid immediately, or transitioned into state three by adding ADP (12.5 mM, final concentration) with or without supplementation with NMN (0.5 mM, final concentration) and maintained for 60 min at 37°C with shaking before collection. (N = 2–4). (C–D) Total ion counts for NAD and NMN in extracts from C2C12 LentiCRISPR whole cells (C) and isolated mitochondria (D) following a 4 hr incubation with isotopically-labeled NR or NAR tracer. (Single measuements). (E–F) Fractional labeling of metabolites (NAD and NMN) measured in C2C12 LentiCRISPR whole cells (E) and isolated mitochondria (F) after a 4 hr incubation with isotopically-labeled NR or NAR tracer. (Single measurements). (G) Fractions of double-labeled NAD and NMN measured in C2C12 LentiCRISPR whole cell and mitochondrial lysates following 4 hr incubation with isotope-labeled NAR (means ± SEM). (N = 3). (H) Immunoblot confirming NMNAT1 knockout in CRISPR C2C12 cell line. (*, p<0.05; **, p<0.001; 2-tailed, unpaired Student’s t-test versus R26).

Figure 6—figure supplement 1. mRNA expression of NMNAT isoforms after CRISPR targeting.

Total RNA was extracted from differentiated cells in duplicate using Trizol (Invitrogen) according to the manufacturer’s instructions. Subsequently, cDNA was synthesized using a high capacity cDNA reverse transcription kit (ABI). RT-PCR was performed using Power SYBR Green PCR master mix (ABI) on the Quantstudio 7 Flex RT-PCR system (ABI). (A–C) For all assays, the plots show gene expression values relative to the reference sample, R26 (control), and are normalized to the control gene 36B4 (Rplp0). For each NMNAT isoform, two distinct gRNAs were generated (a and b) near the 5’ end, which are separated by a short sequence (~40 bp) and the correspondingly named primers amplify a region that is just downstream of the target site. The cells are named as follows: ctrl - parental line with no vector; R26 - control; 1a and 1b - two separate guide RNAs targeting NMNAT1; 2a and 2b - two separate guide RNAs targeting NMNAT2; 3a and 3b - two separate guide RNAs targeting NMNAT3.

Figure 6—figure supplement 2. Partial digestion of mitochondrial preps with proteinase K impairs NAD synthesis, but not respiration.

Isolated murine skeletal muscle mitochondria were diluted in MiRO5 to equal concentrations (1.1 mg/mL) and were incubated on ice for 30 min with the following treatments: ctrl - diluted in MiRO5 only; PI - incubated in MiRO5 only for 30 min followed by addition of protease inhibitor (1:100 dilution; Sigma); PI + protK [0.5] - incubated with both protease inhibitor (1:100) and proteinase K (0.5 mg/mL; Roche); protK [0.25] - incubated with proteinase K (0.25 mg/mL) followed by the addition of protease inhibitor; protK [0.5] - incubated with proteinase K (0.5 mg/mL) followed by the addition of protease inhibitor. (A) Treatment with proteinase K significantly impaired the ability of the mitochondria to generate NAD from NMN. (N = 2). (B) Proteinase K-treated does not impair mitochondrial OXPHOS activity. (N = 1).

Figure 6—figure supplement 3. Overexpressing human NMNAT1 decreases NMN accumulation in NMNAT1 targeted cells and enhances the ability of mitochondrial preps to synthesize NAD from NMN.

NMNAT1 protein was restored by introduction of a viral vector containing the human form of the NMNAT1 gene in order to prevent targeting by the mouse-specific NMNAT1 gRNA. The gateway compatible lentiviral vector, pLX304 (clone ID:HsCD00434593, DNASU, Arizona), was co-transfected with the pMD2-G envelope and psPAX2 packaging vectors into 293 cells using Fugene six transfection reagent (Promega). The consequent lentivirus was collected as described and was used to infect the C2C12 CRISPR cells. (A) Expression of human NMNAT1 in C2C12 cells lacking the murine isoform and the Rosa26 control line. (B) Human NMNAT1 reduced NMN accumulation in CRISPR-targeted cells. (C) Human NMNAT1 cDNA enhanced the ability of mitochondrial preps to synthesize NAD from NMN. Three biological replicates were used for the experiments reported in panels B and C.