Figure 4. Reduction of the NAD⁺/SIRT1 pathway inhibits mitophagy and perturbs MT homeostasis in ATM-deficient neurons and worms.

(A) Electron microscopy images showing MT morphology of control transfected and ATM-KD SH-SY5Ycells after treatment with vehicle or 500 μM NR. Mitochondria likely undergoing mitophagy (yellow arrow) and damaged mitochondria (red arrow) were marked. (B) Quantification of MT length from images such as those shown in panel A. Data are the mean ± S.D. (306 - 514 mitochondria in cells from 3 separate cultures).

(C-D) Detection of mitophagy using a mt-mKeima fluorescent reporter in HeLa cells with/without NR treatment (500 μM). FCCP (30 μM for 3 h) was as positive control. Cells were imaged by confocal microscopy (C) and quantified for mitophagy (D).

(E) A proposed working model for the mechanism of how the NAD⁺/SIR-2.1 pathway promotes MT maintenance.

(F) Effects of NR (500 μM) on the induction of mitophagy in N2 and atm-1 worms. A MT toxicant paraquat (1 mM) was used as a positive control. Data are the mean ± S.E.M. (n=37- 57 muscle cells/group from 20 worms).

(G) Relative DCT-1 protein levels were determined by quantifying levels of DCT-1 associated fluorescence (mean ± S.D., n = 10).

(H) Relative mRNA levels of dct-1. Data are the mean ± S.D. from three groups of worms.

(I) Contribution of NR-induced mitophagy to the improvement of healthspan (pharyngeal pumping) in N2 and atm-1. Vehicle- or NR-supplemented worms were fed with control RNAi or RNAi targeting the designated mitophagy genes at the egg hatching stage, followed by analysis of pharyngeal pumping on D7 adult worms. *p < 0.05, _**P < 0.01, or ***p < 0.001, and n.s., not significant.

See also Figures S3-4.