Fig 2: Effects of TRPC6 siRNA and hyperforin on mitochondrial dynamics, LONP1 expression, and ERK1/2 phosphorylation under certain physiological conditions. (A) Representative images of control- and TRPC6 siRNA-treated animals. TRPC6 expression is predominantly detected in the molecular layer of the dentate gyrus and DGC. TRPC6 siRNA effectively decreases TRPC6 expression. (B,C) Effects of TRPC6 siRNA and hyperforin on mitochondrial length. TRPC6 siRNA (T-siRNA) leads to mitochondrial elongation, while hyperforin (HF) facilitates mitochondrial fragmentation. (B) Quantification of mitochondrial length. Open circles indicate each individual value. Horizontal bars indicate mean value (mean ± S.E.M.; * p < 0.05 vs. control siRNA and vehicle, respectively; Student t-test; n = 7, respectively). (C) Representative double immunofluorescent images for LONP1 and mitochondria (Mito). Inserts are high magnification images (insert bar = 1.25 µm). (D,E) Effects of TRPC6 siRNA and hyperforin on expressions of TRPC6 and LONP1, and ERK1/2 phosphorylation. TRPC6 siRNA (T-siRNA) decreases protein levels of TRPC6 and LONP1, and ERK1/2 phosphorylation. Hyperforin (HF) increases LONP1 expression and ERK1/2 phosphorylation without changing TRPC6 expression. (D) Representative western blots of expressions of TRPC6 and LONP1, and ERK1/2 phosphorylation (M.W. marker, Molecular weight marker). (E) Quantification of expressions of TRPC6 and LONP1, and ERK1/2 phosphorylation based on western blot data. Open circles indicate each individual value. Horizontal bars indicate mean value (mean ± S.E.M.; * p < 0.05 vs. control siRNA and vehicle, respectively; Student t-test; n = 7, respectively).

Fig 3: POL?A is a target of LONP1 degradation in vitro. (A) Size-exclusion chromatography of the complex formed by human LONP1S855A (catalytic dead mutant) and hPOL?AWT. The mixture was incubated for 10 min, at 37°C, in the presence of 10 mM MgCl2 and 2 mM ATP before loaded on the chromatography. (B) SDS-PAGE of the LONP1 proteolysis assay of TFAM, over time. The reactions were incubated for the indicated times (top). (C) SDS-PAGE of the LONP1 proteolysis assay of isolated mPOL?AWT (left), mPOL?B (middle), and mPOL?AWT complexed with mPOL?B, over time. The reactions were incubated for the indicated times (top). In the absence of ATP (-ATP control), LONP1 does not exert proteolysis. (D) SDS-PAGE of the LONP1 proteolysis assay of mPOL?AA449T in absence (left) or presence (middle) of mPOL?B, over time. Reactions with mPOL?AWT + mPOL?B (right) were added for reference. The reactions were incubated for the indicated times (top). In the absence of ATP (-ATP control), LONP1 does not exert proteolysis. (E) Quantification of POL?A degradation over time (0–90 min) by LONP1, related to (C and D). mPOL?AWT (black) and mPOL?AA449T (blue), in absence (solid line) or presence (dashed line) of POL?B. Data are presented as mean ± SD. (n = 3). (F) SDS-PAGE of the LONP1 proteolysis assay of hPOL?AWT in absence (left) or presence (right) of hPOL?B, over time. The reactions were incubated for the indicated times (top). In the absence of ATP (-ATP control), LONP1 does not exert proteolysis. (G) SDS-PAGE of the LONP1 proteolysis assay of hPOL?AA467T in absence (left) or presence (right) of hPOL?B, over time. The reactions were incubated for the indicated times (top). In the absence of ATP (-ATP control), LONP1 does not exert proteolysis. (H) Quantification of human POL?A degradation over time (0–90 min) by LONP1, related to (F and G). hPOL?AWT (gray) and hPOL?AA467T (blue), in absence (solid line) or presence (dashed line) of POL?B. Data are presented as mean ± SD. (n = 3).

Fig 4: Docking of CDDO derivatives at an allosteric binding pocket adjacent to the ATP/ADP binding site of LonP1.A, cryo-EM structure of LonP1. Left, C-terminal view clearly showing the six homohexameric subunits of the LonP1 complex. Middle, 90° anticlockwise rotation of the C-terminal view showing the amino-terminal protein substrate binding domain, the ATPase and protease domains. Right, inset showing ATP-bound between two adjacent subunits and CDDO docked at Site 1. B and C, Site 1 and Site 2, respectively are nonoverlapping pockets that bind CDDO derivatives. B, upper panel, Site 1 is more distal to the ATP/ADP binding site than Site 2. At this site, CDDO-Me, CDDO-Im and CDDO interact with two adjacent subunits (cyan and orange). Middle panel, CDDO and CDDO-anhydride also bind Site 1 and are shown separately for clarity. Lower panel, shows from a different angle that CDDO-anhydride interacts with 3 adjacent subunits at Site 1 (cyan, orange, gold) by contrast to CDDO, CDDO-Me and CDDO-Im, which interact with two subunits. C, only CDDO and CDDO-anhydride are bound at Site 2, which is more proximal to the ATP/ADP binding site than Site 1. D, locations of amino acids C576, C637 and F547 at the CDDO binding Site 1 and 2. Shown are the side chains of residues on two adjacent subunits (cyan and orange) of LonP1, which are affected by the amino acid substitutions. E, a channel is formed by a relay of inter- and intra-subunit salt bridges leading to the binding sites for CDDO derivatives and ATP. Amino acid residue R721 forms a salt-bridge with E517 of the same subunit at the opening of the channel. The missense mutation substituting R721 to glycine (R721G) encoded by the homozygous LONP1 Amish allele of CODAS syndrome is expected to collapse this channel leading to the CDDO binding sites. CDDO, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid.

Fig 5: PITRM1-/- iPSC-derived neurons show the induction of UPRmt and enhanced mitophagy.Control PITRM1+/+ (WT) and isogenic PITRM1-/- (KO) iPSCs were differentiated into cortical neurons. a Immunostaining of indicated differentiated iPSC cultures at DIV 35. Cells were stained for TBR1 (green) and ß-III-tubulin (ß-TUBIII, red). Nuclei were counterstained with DAPI (blue). Scale bar,50 µm. b Quantification of ß-TUBIII/DAPI and TBR1/ß-TUBIII-positive cells in differentiated iPSC cultures at DIV 35 (mean + SEM; n = 3). c Isogenic PITRM1+/+ and PITRM1-/- iPSC-derived neurons were stained with tetramethylrhodamine methylester (TMRM) and MitoTracker Green (MGreen) fluorescent dyes to determine mitochondrial membrane potential and mitochondrial mass, respectively. Representative images are shown. Scale bar, 10 µm. d Mitochondrial membrane potential (TMRM fluorescence) in neuronal soma and neurites (mean + SEM; *p < 0.05, two-tailed t test, n = 3). e Gene expression levels of mitochondrial stress response genes in PITRM1+/+ and PITRM1-/- iPSC-derived cortical neurons (mean + SEM; *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed t test, n = 5). f, g Representative western blots of the mitochondrial chaperones HSPA9 and HSP60 and the mitochondrial protease LONP1 in PITRM1+/+ and PITRM1-/- iPSC-derived cortical neurons. Quantification of protein levels relative to the loading control is shown in (g) (mean + SEM; *p < 0.05, two-tailed t test, n = 3). h Western blot analysis for LC3 in PITRM1+/+ and PITRM1-/- iPSC-derived neuronal cultures, untreated (-) or treated with 200 µM leupeptin and 20 mM NH4Cl for 4 h (+). i Quantification of LC3 flux normalized to WT (mean + SEM; *p < 0.05, two-tailed t test, n = 3). j Representative western blot of isolated mitochondria from PITRM1+/+ and PITRM1-/- iPSC-derived neurons probed against ubiquitin and VDAC as the loading control. k Quantification of mitochondrial protein ubiquitination levels in PITRM1+/+ and PITRM1-/- iPSC-derived neurons (mean + SEM; **p < 0.01, two-tailed t test, n = 3). I mtDNA content was measured as the mitochondrial (16S) to nuclear (RPLP0) DNA ratio by qRT-PCR (mean + SEM; *p < 0.05, two-tailed t test, n = 3).