Different molecular mechanisms involved in spontaneous and oxidative stress-induced mitochondrial fragmentation in tripeptidyl peptidase-1 (TPP-1)-deficient fibroblasts

Abstract

NCLs (neuronal ceroid lipofuscinoses) form a group of eight inherited autosomal recessive diseases characterized by the intralysosomal accumulation of autofluorescent pigments, called ceroids. Recent data suggest that the pathogenesis of NCL is associated with the appearance of fragmented mitochondria with altered functions. However, even if an impairement in the autophagic pathway has often been evoked, the molecular mechanisms leading to mitochondrial fragmentation in response to a lysosomal dysfunction are still poorly understood. In this study, we show that fibroblasts that are deficient for the TPP-1 (tripeptidyl peptidase-1), a lysosomal hydrolase encoded by the gene mutated in the LINCL (late infantile NCL, CLN2 form) also exhibit a fragmented mitochondrial network. This morphological alteration is accompanied by an increase in the expression of the protein BNIP3 (Bcl2/adenovirus E1B 19 kDa interacting protein 3) as well as a decrease in the abundance of mitofusins 1 and 2, two proteins involved in mitochondrial fusion. Using RNAi (RNA interference) and quantitative analysis of the mitochondrial morphology, we show that the inhibition of BNIP3 expression does not result in an increase in the reticulation of the mitochondrial population in LINCL cells. However, this protein seems to play a key role in cell response to mitochondrial oxidative stress as it sensitizes mitochondria to antimycin A-induced fragmentation. To our knowledge, our results bring the first evidence of a mechanism that links TPP-1 deficiency and oxidative stress-induced changes in mitochondrial morphology.

Figure 1. TPP-1 activity in control fibroblasts, LINCL cells and LINCL loaded with recombinant TPP-1

TPP-1 activity was measured in CTL and LINCL cells pre-incubated in the presence (LINCL+TPP-1) or in the absence (LINCL) of 10 nM recombinant TPP-1 for 48 h using the synthetic substrate Ala–Ala–Phe–AMC (AAF–AMC). Briefly, at the end of the incubation with the recombinant enzyme, cells were maintained for 72 h in culture with fresh medium without TPP-1. Cells were then homogenized in ice-cold isotonic sucrose and membranes were solubilized with Triton X-100. TPP-1 activity was assayed in 50 mM acetate buffer (pH 5.0) containing 5 mM 4-methylumbelliferyl derivative as substrate. The fluorescence of 4-methylumbelliferone released was measured in a spectrofluorimeter (λ_exc, 350 nm; λ_em, 460 nm). Results were calculated as arbitrary fluorescence units normalized for protein content (AFU/μg of proteins) and expressed in percentages of CTL cells as means±S.D. (n=3). **, ***: significantly different from CTL cells with P<0.01 and P<0.001 respectively. ###: significantly different from LINCL cells loaded with TPP-1 with P<0.001 as determined by an ANOVA-1 followed by a Dunnett's test.

Figure 2. Effects of a TPP-1 deficiency on the mitochondrial abundance

(A) Mitochondrial population abundance was analysed in LINCL cells pre-incubated with (LINCL+TPP-1) or without (LINCL) recombinant TPP-1. Cells were then stained with 100 nM MitoTracker Green for 30 min and fluorescence was detected by flow cytometry as described in the Material and methods section. Results are expressed as MitoTracker Green Fluorescence MCFR calculated from autofluorescence signals measured for corresponding unstained cells and represent means±S.D. (n=3). NS, non-significantly different from LINCL cells as determined by a Student's t test. (B) The abundance of HADHA, β-subunit of ATPsynthase and mt-HSP70 was determined by Western blot analysis performed on 15 μg of clear cell lysates prepared from LINCL cells pre-incubated (LINCL+TPP-1) or not (LINCL) with recombinant TPP-1. Equal protein loading was controlled by the immunodetection of β-actin or α-tubulin (n=3). (C) Quantification of the abundance of β-subunit of ATP synthase (grey columns) HADHA (white columns), and mt-HSP70 (black columns) on blots presented in (B). Results are calculated as fluorescence intensity normalized for α-tubulin or β-actin and expressed in percentages of LINCL cells as means±S.D. (n=3). *: significantly different from LINCL cells with P<0.05 as determined by a Student's t test.

Figure 3. Effects of a TPP-1 deficiency on the expression of BNIP3

(A) Total RNA was extracted from LINCL cells pre-incubated with (LINCL+TPP-1) or without (LINCL) TPP-1, reverse transcribed and amplified by real-time RT–qPCR in the presence of BNIP3 primers and SYBR green. GAPDH was used as reference gene for data normalization. Results are expressed in percentages of the mRNA abundance determined in LINCL cells and represent means±S.D. (n=3). *: significantly different from LINCL cells with P<0.05 as determined by a Student's t test. (B) The protein abundance of BNIP3 was determined by Western blot analysis performed on 15 μg of clear cell lysates prepared from LINCL cells pre-incubated (LINCL+TPP-1) or not (LINCL) with TPP-1 (n=3). Equal protein loading was controlled by the immunodetection of α-tubulin. (C) Quantification of the Western blot presented in (B). Fluorescence intensity of the band of interest was normalized for α-tubulin and expressed as percentages of LINCL cells. Results are means±S.D. (n=3). *: significantly different from LINCL cells with P<0.05 as determined by a Student's t test.

Figure 4. Effects of a TPP-1 deficiency on mitochondrial morphology

(A) Representative confocal micrographs of mitochondrial morphology of fibroblasts pre-incubated with (CTL+FCCP) or without (CTL) 20 μM FCCP for 30 min and of LINCL cells pre-incubated with (LINCL+TPP-1) or without (LINCL) TPP-1 and stained with 100 nM Mitotracker Green. (B and C) The length (or AR) and the degree of branching of mitochondria (or FF) were determined on micrographs using the ImageJ software as described in the Material and methods section. Results are expressed in either AR (B) or FF ratio (C) and represent means±S.E.M. (n≥ 59 from four independent experiments). ###: significantly different from CTL cells with P<0.001; *, ***: statistically different from LINCL cells as determined by a Mann–Whitney Rank Sum test with P<0.05 and P<0.001, respectively.

Figure 5. Effects of BNIP3 silencing on the mitochondrial morphology of LINCL and LINCL cells that recovered TPP-1 activity

(A) Efficiency of siRNA-mediated knock down of BNIP3 in LINCL cells. The abundance of BNIP3 was determined by Western blotting analysis performed on 15 μg of clear cell lysates prepared from untransfected LINCL cells and cells transfected either with a pool of four specific siRNAs targeting BNIP3 (siBNIP3) or with a control pool of siRNAs (siCTL). Equal protein loading was controlled by the immunodetection of α-tubulin. (B) Representative confocal micrographs of mitochondrial morphology of LINCL and LINCL cells with restored TPP-1 activity, transfected either with a control pool of siRNAs (siCTL) or with a pool of four specific siRNAs targeting BNIP3 (siBNIP3) and then stained with 100 nM Mitotracker Green. The length (AR) and the degree of branching of mitochondria (FF) were determined on micrographs using the ImageJ software, as described in the Material and methods section. Results are expressed in either AR (C) or FF ratio (D) as means±S.E.M. (n≥50 from three independent experiments). *: significantly different from LINCL cells transfected with the siCTL with P<0.05, as determined by an ANOVA-2 followed by a Holm–Sidak test and by a Mann–Whitney Rank Sum test for the AR and the FF, respectively. NS, not significantly different from LINCL cells transfected with the siCTL.

Figure 6. Effects of a TPP-1 deficiency on the abundance of major regulators of mitochondrial dynamics

(A) The abundance of Opa1, Drp1, Mfn2, Mfn1 and Fis1 was determined by fluorescence Western blot analysis on 15 μg of clear cell lysates prepared from LINCL cells pre-incubated with (LINCL+TPP-1) or without (LINCL) TPP-1 (n=3). Equal protein loading was controlled by the immunodetection of α-tubulin. Asterisks denote non-specific and uncharacterized bands. (B) Quantification of the abundance of Opa1 (white columns), Drp1 (grey columns), Mfn2 (hatched lines), Mfn1 (dots) and Fis1 (black columns) on the blots shown in (A). Results are calculated as fluorescence intensity normalized for α-tubulin and expressed as a percentage of LINCL cells as means±S.D. (n=3). *: significantly different from LINCL cells with P<0.05, as determined by a Student's t test.

Figure 7. Cellular distribution of Drp1/Fis1 and colocalization of these proteins as visualized by immunofluorescence confocal microscopy

(A) Representative micrographs of Drp1 and/or Fis1 immunostaining taken with a confocal microscope. LINCL cells were cultured on coverslips and were incubated with (LINCL+TPP-1) or without (LINCL) TPP-1 before being fixed, permeabilized and incubated with antibodies raised against Fis1-(red) or Drp1-(green). In merged micrographs, the appearance of the yellow fluorescence signals reflects the colocalization between both proteins. Nuclei were stained with TO-PRO-3 iodide (blue fluorescence). (B) The co-localization between Drp1 and Fis1 was quantified using the co-localization module of Leica LAS AF software. The results are expressed as the percentages of co-localization (means±S.D.; n=20). NS, non-significantly different from LINCL cells, as determined by a Student's t test.

Figure 8. Effects of a TPP-1 deficiency on the mitochondrial membrane potential, mitochondrial matrix calcium concentration and mitochondrial O₂^•− content

(A) The mitochondrial membrane potential was analysed in LINCL cells pre-incubated with (LINCL+TPP-1) or without (LINCL) recombinant TPP-1, using the specific mitochondrial membrane potential probe TMRE. Cells were stained or not (to allow autofluorescence determination from cells without dye) with 25 nM TMRE for 30 min and fluorescence was analysed by flow cytometry. Results are expressed as TMRE fluorescence MCFR as means±S.D. (n=3), ***: significantly different from LINCL cells with P<0.001 as determined by a Student's t test. (B) The mitochondrial O₂^•− content was assessed in LINCL cells pre-incubated with (LINCL+TPP-1) or without (LINCL) TPP-1 using the specific mitochondrial MitoSOX™ Red dye. Cells were stained (white and grey columns) or not (to allow autofluorescence determination from cells without dye) with 10 μM MitoSOX™ Red specific dye for 10 min and fluorescence intensities were measured using a spectrofluorimeter (λ_exc, 485 nm; λ_em, 520 nm). As a positive control, cells were incubated for 15 min with 10 μM antimycin A (grey columns) while untreated cells were maintained for 15 min without molecule (white columns). Results were normalized for protein content and expressed in relative fluorescence arbitrary unit/μg of proteins as means±S.D. (n=3). ***, ###: significantly different from untreated LINCL and LINCL+TPP-1 cells, respectively, with P<0.001 as determined by an ANOVA-2 followed by a Holm-Sidak's t test. NS, non-significantly different from LINCL cells. (C) The mitochondrial calcium concentration in LINCL cells pre-incubated with (LINCL+TPP1) or without (LINCL) recombinant TPP-1 was determined using X-Rhod-5F, a specific mitochondrial matrix calcium probe. Cells were stained or not (to allow autofluorescence determination from cells without dye) with 2 μM X-Rhod-5F fluorescent probe for 30 min and fluorescence intensities were measured in a spectrofluorimeter (λ_exc, 580 nm; λ_em, 612 nm). To test mitochondrial calcium buffering capacity, cells were incubated with 10 μM ionomycin for 10 min (grey columns), 20 min (dark grey columns) or 30 min (black columns) or left untreated (white columns). Results are expressed as Relative Fluorescence Units (RFU) normalized for DNA content determined by propidium iodide (IP) staining as means±S.D. (n=9). ***, Significantly different from LINCL cells with P<0.001, ###: significantly different from LINCL+TPP-1 cells as determined by a Mann–Whitney Rank Sum test. NS, non-significantly different from LINCL cells.

Figure 9. Effects of BNIP3 silencing on antimycin A-induced mitochondrial fragmentation in LINCL cells

(A) Representative confocal micrographs of mitochondrial morphology in LINCL and LINCL cells that recovered TPP-1 activity. At the end of the incubations, cells were transfected for 24 h with either a pool of four specific siRNAs targeting BNIP3 (siBNIP3) or with a control pool of siRNAs (siCTL), incubated with 25 μM antimycin A for 1 h and eventually loaded with 100 nM Mitotracker Green before analysis. The length (AR) and the degree of branching of mitochondria (FF) were determined using the ImageJ software, as described in the Material and methods section. Results are expressed in either AR (B) or FF ratio (C) as means±S.E.M. (n≥40 from three independent experiments). **,***: significantly different from LINCL cells transfected with the siCTL with P<0.01 or 0.001, respectively, as determined by a Mann–Whitney Rank Sum test. NS, non-significantly different. NS, non-significantly different from LINCL cells pre-incubated with TPP-1 and transfected with the siCTL.