A LON-ClpP Proteolytic Axis Degrades Complex I to Extinguish ROS Production in Depolarized Mitochondria

Abstract

Mitochondrial dysfunction is implicated in numerous neurodegenerative disorders and in Parkinson's disease (PD) in particular. PINK1 and Parkin gene mutations are causes of autosomal recessive PD, and these respective proteins function cooperatively to degrade depolarized mitochondria (mitophagy). It is widely assumed that impaired mitophagy causes PD, as toxic reactive oxygen species (ROS)-producing mitochondria accumulate and progressively drive neurodegeneration. Instead, we report that a LON-ClpP proteolytic quality control axis extinguishes ROS in depolarized mitochondria by degrading the complex I ROS-generating domain. Complex I deficiency has also been identified in PD brain, and our study provides a compelling non-genetic mechanistic rationale to explain this observation: intact complex I depletes if mitochondrial bioenergetic capacity is robustly attenuated.

Keywords: ClpP; LON; NADH:ubiquinone oxidoreductase; complex I; mitochondria; mitochondrial proteases; mitophagy; retrograde signaling.

Graphical abstract

Figure 1

Δp Dissipation Triggers Selective Complex I Peripheral Arm Proteolysis (A and B) SH-SY5Y and HeLa cells after 4-, 8-, and 18-hr CCCP treatment (n = 5). (C and D) HSP60 and NDUFV2 immunostained HeLa cells ± 18-hr CCCP treatment (n = 3). (E and F) Native-PAGE complex I, III, and IV levels, following 0- to 8-hr CCCP or 8-hr nigericin (Nig) or valinomycin (Val) (n = 3). (G and H) Native-PAGE in-gel complex I activity in HeLa cells and quantification compared to citrate synthase activity ± 18-hr CCCP (^∗non-complex I reactive band; n = 3). (I) Activity measurements in mitochondria isolated from HeLa cells ± CCCP (n = 3). (J and K) Respiration rates in HeLa cells ± 8-hr CCCP or valinomycin pre-treatment. Oligomycin (Oligo), FCCP, and antimycin A + rotenone (Ant.A + Rot) were added as indicated (n = 4). (L and M) Native-PAGE NDUFA9 and NDUFA10 immunoblots. HeLa cells ± 8- and 18-hr CCCP. All data are presented in the figure as mean + SDM. See also Figure S1.

Figure 2

mΔ_Ψ Dissipation Is the Peripheral Arm Degradation Trigger (A and B) Native-PAGE. HeLa cells ± 8-hr cycloheximide (CHX), antimycin A + oligomycin (Anti.A + Oligo), CCCP, or valinomycin (Val) treatment (n = 3). (C and D) SH-SY5Y (left panel) and HeLa (right panel) cells ± 8-hr Oligo, Anti.A + Oligo, nigericin (Nig), Val, and CCCP (n = 3). (E and F) SH-SY5Y cells ± 18-hr Anti.A ± Oligo or rotenone (Rot) ± Oligo (n = 3). (G) Native-PAGE NDUFA9 or NDUFA10 immunoblots ± 18-hr Anti.A + Oligo, Anti.A, CCCP, or Val. (H and I) Native-PAGE in-gel complex I activity (n = 3) and citrate synthase activity (cell lysates) (n = 5). SH-SY5Y cells ± 18-hr CCCP, Anti.A, or Anti.A + Oligo (^∗non-complex I reactivity). (J and K) SH-SY5Y cells ± 18-hr Rot, piericidin A (Pier.A), CCCP, CCCP + Rot, or CCCP + Pier.A (n = 3). All data are presented in the figure as mean + SDM. See also Figure S2.

Figure 3

Activating Mitophagy Induces Mitochondrial Oxidative Stress and Triggers Peripheral Arm Degradation (A and B) Aconitase activity in HeLa cells after antimycin A (Anti.A) ± oligomycin (Oligo), rotenone (Rot) ± Oligo. Red zone indicates activity attributable to the cytosolic isoform. (n = 4–5). (C) HeLa cells ± 8-hr CCCP ± NAC (n = 3). (D and E) Anti.A-treated SH-SY5Y cells ± cycloheximide (CHX) (n = 3). (F) mRNA levels in SH-SY5Y cells ± Anti.A treatment (n = 3). (G and H) NAD(P)H autofluorescence (a.u.) in mitochondrial-GFP (mt-GFP)-expressing SH-SY5Y cells during Anti.A treatment (n = 3). (I and J) Rates (a.u.) of ROS in mt-GFP SH-SY5Y cells incubated with mitoSOX and treated with Anti.A (n = 3). (K) NADH-induced H₂O₂ production in mitochondria isolated from HeLa cells ± 8-hr CCCP (n = 3). All data in the figure are presented as mean + SDM. See also Figure S2.

Figure 4

LON and ClpP Bind Intact Complex I and Mediate Peripheral Arm Degradation (A–D) SH-SY5Y and HeLa cells ± 8-hr MG132 (n = 3) or bafilomycin (Baf) (n = 3) ± CCCP. (E and F) Control (n = 4), LON (n = 3), or ClpP (n = 3) silenced HeLa cells ± 8-hr antimycin A (Anti.A) + oligomycin (Oligo). (G and H) NDUFB6 precipitation with ClpP (n = 3) and LON (n = 3) and LON precipitation with complex I (n = 3) ± Anti.A ± rotenone. (I) Cartoon model of proteolytic quality control in depolarized mitochondria to extinguish ROS production. All data in the figure are presented as mean + SDM. See also Figure S3.