Two molecular pathways initiate mitochondria-dependent dopaminergic neurodegeneration in experimental Parkinson's disease

Abstract

Dysfunction of mitochondrial complex I is associated with a wide spectrum of neurodegenerative disorders, including Parkinson's disease (PD). In rodents, inhibition of complex I leads to degeneration of dopaminergic neurons of the substantia nigra pars compacta (SNpc), as seen in PD, through activation of mitochondria-dependent apoptotic molecular pathways. In this scenario, complex I blockade increases the soluble pool of cytochrome c in the mitochondrial intermembrane space through oxidative mechanisms, whereas activation of pro-cell death protein Bax is actually necessary to trigger neuronal death by permeabilizing the outer mitochondrial membrane and releasing cytochrome c into the cytosol. Activation of Bax after complex I inhibition relies on its transcriptional induction and translocation to the mitochondria. How complex I deficiency leads to Bax activation is currently unknown. Using gene-targeted mice, we show that the tumor suppressor p53 mediates Bax transcriptional induction after PD-related complex I blockade in vivo, but it does not participate in Bax mitochondrial translocation in this model, either by a transcription-independent mechanism or through the induction of BH3-only proteins Puma or Noxa. Instead, Bax mitochondrial translocation in this model relies mainly on the JNK-dependent activation of the BH3-only protein Bim. Targeting either Bax transcriptional induction or Bax mitochondrial translocation results in a marked attenuation of SNpc dopaminergic cell death caused by complex I inhibition. These results provide further insight into the pathogenesis of PD neurodegeneration and identify molecular targets of potential therapeutic significance for this disabling neurological illness.

Fig. 1.

MPTP-induced complex I inhibition causes DNA damage and p53 induction. (a) Ventral midbrain samples from MPTP-injected mice exhibit a time-dependent increase in PARP activity, evidenced by quantifying the formation of its product, ADP-ribose polymers. PARP activity starts to increase by 24 h and peaks at 48 h after MPTP injection. (b) At the peak of PARP activation, in situ PARP histochemistry (brown signal) combined with immunohistochemistry for DAT (blue-gray signal) reveals PARP activation only in DAT-positive SNpc cells, which exhibit a definite neuronal morphology. (c) At the peak of PARP activation, p53 mRNA levels are markedly increased in the ventral midbrain of MPTP-injected mice as determined by RT–PCR. ∗, P < 0.05 compared with saline-injected mice.

Fig. 2.

Loss of p53 in gene-targeted mice inhibits MPTP-induced Bax up-regulation but not Bax mitochondrial translocation. (a and b) Loss of p53 prevents MPTP-induced ventral midbrain Bax mRNA (a) and protein (b) up-regulation at its peak (2 and 4 days after the last MPTP injection, respectively). (c) In p53-deficient mice injected with MPTP, constitutive Bax is still able to translocate to mitochondria, as assessed by Western blot analysis of ventral midbrain mitochondrial protein fractions. ∗, P < 0.05 compared with saline-injected mice; #, P ≤ 0.05 compared with MPTP-injected wild-type mice.

Fig. 3.

Loss of p53 in gene-targeted mice attenuates MPTP-induced SNpc DA apoptotic cell death. (a and b) At the peak of MPTP-induced mitochondria-dependent apoptotic cell death (4 days after the last MPTP injection), cytochrome c release and caspase-3 activation are reduced in ventral midbrain cytosolic fractions of p53-deficient mice. (c) The number of MPTP-induced SNpc apoptotic neurons is decreased in the ventral midbrain of p53-deficient mice compared with similarly treated wild-type mice. Morphological criteria to identify apoptotic cells included shrinkage of the cell body, chromatin condensation, and the presence of distinct, round, well defined chromatin clumps, demonstrated on thionin staining, as in ref. . (d) p53-deficient mice exhibit a significant protection against MPTP-induced SNpc DA cell death, as determined by assessing the number of SNpc DA neurons at 21 days after the last MPTP injection. ∗, P < 0.05 compared with saline-injected mice; #, P < 0.05 compared with MPTP-injected wild-type mice.

Fig. 4.

Role of Bim in MPTP-induced SNpc DA neurodegeneration. (a) Ventral midbrain bim mRNA expression is induced in a time-dependent manner after MPTP intoxication, peaking at 24 h post-MPTP. (b) At its peak, MPTP-induced bim mRNA up-regulation is prevented in JNK-3-deficient mice. (c) At the peak of MPTP-induced bim mRNA up-regulation, mitochondrial Bim_EL protein levels are markedly increased in the ventral midbrain. (d) Loss of Bim in gene-targeted mice significantly attenuates MPTP-induced Bax mitochondrial translocation (Left) and cytochrome c release (Right), as assessed by Western blot analysis of mitochondrial and cytosolic fractions from ventral midbrain, respectively. (e and f) Bim-deficient mice exhibit a significant reduction in the number of MPTP-induced SNpc apoptotic neurons (e) and a significant protection from MPTP-induced SNpc DA cell death (f). ∗, P < 0.05 compared with saline-injected mice; #, P < 0.05 compared with MPTP-injected wild-type mice.

Fig. 5.

Proposed pathogenic scenario induced by complex I deficiency with MPTP in which DA neuronal death results from a self-amplifying cascade of deleterious events that start at the mitochondria with the alteration of oxidative phosphorylation and finish at the mitochondria with the activation of the programmed cell death machinery. In this scenario, p53 transcriptionally up-regulates Bax, whereas JNK promotes Bax mitochondrial translocation through transcriptional activation of the BH3-only protein Bim.