Figure 1.
NAD depletion underlies excitotoxic neuronal death.
(A) NAD level was measured in total cellular and mitochondrial fractions in mouse primary cortical neuron cultures exposed to NMDA 30 µM for indicated times. (mean ± SEM; n = 8; *P<0.01, difference from untreated control). (B) Cell death was assessed by measurement of LDH release (mean ± SEM; n = 8 ; *P<0.01, difference from NMDA alone). (C) NAD level was measured in neurons 4 h after exposure to NMDA 30 µM alone, NMDA plus various doses of NAD, or NAD alone (mean ± SEM; n = 4; *P<0.01, difference from untreated control; **P<0.01, difference from NMDA alone).
Figure 2.
PARP-1 activation depletes cellular NAD in NMDA-treated neurons.
(A) Western blot of PAR in neurons exposed to NMDA (30 µM) for indicated times. The signal of PAR peaked 1 hr after NMDA treatment. β-actin was used as a loading control. (B) NAD level was measured in neurons 4 h after exposure to NMDA alone, NMDA plus PJ34 (100 nM), or NMDA plus DPQ (25 µM) (mean ± SEM; n = 4; *P<0.01, difference from untreated control; **P<0.01, difference from NMDA alone). (C) LDH was measured in neuron cultures of WT or PARP-1 KO mice 24 h after NMDA exposure. (mean ± SEM; n = 8; *P<0.01, difference from the relevant controls of PARP-1 KO culture). (D) NAD level was measured in WT or PARP-1 KO cultures exposed to NMDA 30 µM for indicated times (mean ± SEM; n = 8; *P<0.01, difference from the relevant controls of PARP-1 KO culture).
Figure 3.
Mitochondrial Sirt3 is markedly increased in NMDA-treated neurons following PARP-1 activation.
(A) Western blots of Sirt1 and Sirt3 in neurons exposed to NMDA (30 µM) for indicated times. (B) Western blot of Sirt3 in neurons 4 h after exposure to NMDA (30 µM) with or without DPQ (25 µM). (C) i. Western blots of mitochondrial sirtuins, Sirt3, Sirt4, and Sirt5 in WT and PARP-1 KO neurons exposed to NMDA 30 µM for indicated times. ii. The expression level of Sirt3 in WT and PARP-1 KO neurons was normalized with actin and quantified to the relevant control value (t = 0). Similar results were observed from three independent experiments (mean ± SEM; n = 3; *P<0.01, difference from control).
Figure 4.
The expression and translocation of mitochondrial Sirt3 is increased in neurons after NMDA treatment.
(A) i, Western blot of the long (L) and short (S) forms of Sirt3 in neurons treated with NMDA 30 µM at indicated times. ii,Western blot of Sirt3 in mitochondrial fraction (M) of neurons exposed to NMDA 30 µM for indicated times. Cox IV was used as a loading control. (B) i, RT PCR analysis of Sirt3 in neurons exposed to NMDA 30 µM for indicated times. ii, The mRNA level was normalized to the control (ctrl). Similar results were observed in three independent experiments (mean ± SEM; n = 3; *P<0.01, difference from control). (C) Confocal analysis of Sirt3 localization (green) in neurons with or without NMDA 30 µM for 4 hrs. Nucleus and mitochondria were detected with DAPI and MitoTracker red, respectively.
Figure 5.
Intracellular NAD depletion causes the increased expression and activation of mitochondrial Sirt3.
(A) NAD levels were measured in neurons transfected with NADase using Bioporter. NADase transfection produced a decrease in neuronal NAD content, as measured 4 hours after Bioporter tansfection (mean ± SEM; n = 4; *P<0.01, difference from control). This decrease was not observed in cultures treated with the Bioporter vehicle alone. (B) Western blot of Sirt3 in neurons with or without NADase at 4 hr after transfection.. The expression level of Sirt3 was normalized with actin and quantified to the control value (mean± SEM; n = 3; *P<0.01, difference from control). (C) Western blot of acetyl lysine in neurons exposed to NMDA 30 µM for indicated times or NAD alone. (D) The expression of Sirt3, acetyl lysine, and PAR in neurons at 4 hr after the exogenous addition of various concentrations of NAD. (E) Western blot of acetyl lysine in mitochondrial fraction (M) of neurons exposed to NMDA 30 µM, NMDA 30 µM with exogenous NAD, or NAD alone for 4 hrs.
Figure 6.
Oxidative stress by NAD depletion increased mitochondrial Sirt3 expression.
(A) Fluorescence image of the oxidized dihydroethidium (HEt) in neurons 4 h after exposure to control (media exchange), NMDA (30 µM) alone, or NMDA plus NAD (5 mM). (B) Cellular NAD level was measured in neurons 4 h after exposure to control, NMDA alone, NMDA plus trolox (100 µM), or NMDA plus or 7-NI 100 nM for 4 hrs (mean ± SEM; n = 8; *P<0.01, difference from untreated control). (C) Western blot of Sirt3 in neurons treated similarly as (B). The expression of Sirt3 was normalized to actin and quantified to the control value (mean± SEM; n = 3; *P<0.01, difference from control). (D) Western blot of Sirt3 in neurons treated with SIN-1 1 mM for indicated times.
Figure 7.
Sirt3 acts as a prosurvival factor against excitotoxic injury.
(A) Fluorescence image of mitochondrial ROS detected with CMH2xROS in neuron cultures transfected with pIRES2-zsGreen1 (control vector) or pIRES2-zsGreen1-Sirt3 at 24 hrs after exposure to NMDA 30 µM. (B) The expression of control vector alone and pIRES2 ZsGreen-1-Sirt3 in transfected neurons 24 hr after exposure to NMDA 30 µM. Cell death was analyzed by counting the pyknotic bodies (shown as DAPI) in transfected neurons (mean ± SEM; n = 8; *P<0.01, difference from the culture transfected with the control vector). (C) Cell death was analyzed by LDH release in neurons transfected with Sirt3 siRNA or negative control siRNA at 24 hrs after exposure to NMDA 30 µM (mean ± SEM; n = 8; *P<0.01, difference from NMDA alone; **P<0.01, difference from NMDA plus Sirt3 siRNA).
Figure 8.
Schematic Diagram of NMDA-induced increase in mitochondrial Sirt3.
The proposed mechanism showing the role of Sirt3 in protecting against NMDA-induced excitotoxic injury. NMDA binds to the NMDA receptor which opens the ion channel and allows for calcium (Ca2+) influx into the cell and promotes oxidative stress. Oxidative DNA damage activates PARP-1 and decreases NAD, leading to an increase in ROS production due to mitochondrial failure. The increase in ROS promotes an increase in mitochondrial Sirt3. Mitochondrial Sirt3 levels were increased in NMDA-treated neurons following PARP-1 mediated NAD depletion. Further evidence is provided to support this hypothesis through genetic modification: Overexpression of Sirt3 prevented the increase in ROS production and neuronal death, whereas knock down of Sirt3 exacerbated neuronal excitotoxic injury.