Acute and chronic mitochondrial respiratory chain deficiency differentially regulate lysosomal biogenesis

Mitochondria are key cellular signaling platforms, affecting fundamental processes such as cell proliferation, differentiation and death. However, it remains unclear how mitochondrial signaling affects other organelles, particularly lysosomes. Here, we demonstrate that mitochondrial respiratory chain (RC) impairments elicit a stress signaling pathway that regulates lysosomal biogenesis via the microphtalmia transcription factor family. Interestingly, the effect of mitochondrial stress over lysosomal biogenesis depends on the timeframe of the stress elicited: while RC inhibition with rotenone or uncoupling with CCCP initially triggers lysosomal biogenesis, the effect peaks after few hours and returns to baseline. Long-term RC inhibition by long-term treatment with rotenone, or patient mutations in fibroblasts and in a mouse model result in repression of lysosomal biogenesis. The induction of lysosomal biogenesis by short-term mitochondrial stress is dependent on TFEB and MITF, requires AMPK signaling and is independent of calcineurin signaling. These results reveal an integrated view of how mitochondrial signaling affects lysosomes, which is essential to fully comprehend the consequences of mitochondrial malfunction, particularly in the context of mitochondrial diseases.Open-Access-Publikationsfonds 2017peerReviewe

<i>Aats-met</i> mutants have reduced cell proliferation.

<p>(A–B) Brains of late 3^rd instar control and <i>HV/Df</i> larvae stained with Rhodamine-Phalloidin. (C–D) Wing discs of a late 3^rd instar control and mutant larvae stained with Rhodamine-Phalloidin. (E–F) Control and mutant pupae are shown. (G) Quantification of pupal length is shown. (H) Wing disc containing wild-type (outlined in yellow) and mutant clones (outlined in red) are seen. (I) Wild-type clones are significantly larger than mutant clones, quantified in 16 to 20 pairs of clones. (J–K) Cells in mutant clones in wing discs, stained with anti-Dlg, to mark the cell membrane, are similar in size to wild-type cells. (L) PH3-staining cells in mutant versus neighboring heterozygous tissue is quantified for five wing discs, indicating that there is less cell proliferation in mutant clones. Data are mean ± s.e.m. Scale bars for (A–D) and (H) are 100 microns, (E–F) are 0.3 mm, and (J–K) are 5 microns.</p

Identification/mapping of the <i>Aats-met</i> gene.

<p>(A) ERG of the control (<i>y w</i>; <i>FRT82B iso</i>). The black and white arrowheads indicate the “on” and “off” transients, respectively. The double-pointed arrow indicates the amplitude. (B–C) ERGs of homozygous <i>HV</i> clone-containing flies at 1 d and 4 wk after eclosion. (D–E) ERGs of homozygous <i>FB</i> clone-containing flies at 1 d and 4 wk after eclosion. (F) ERG of a 1-d-old <i>HV/FB</i> escaper. (G) ERG of a 3-wk-old <i>HV/FB</i> escaper. (H) ERG of a 2-wk-old <i>HV/Df</i> fly rescued with <i>actin-Gal4</i> and <i>UAS-Aats-met</i>. (I) ERG of a 2-wk-old <i>HV/Df</i> fly rescued with <i>actin-Gal4</i> and <i>UAS-HMARS2</i>. (J) ERG of a 2-wk-old otherwise wild-type fly expressing HMARS2-FLAG driven by tub-Gal4. (K) Lethal stages of homozygous and transheretozygous allelic combinations reveal an allelic series: <i>Df>PB>FB>HV</i>. (L) The Aats-met protein's predicted domains are shown (drawn to scale), with position of mutations and percentage identity compared to human MARS2 shown. (M) The <i>Drosophila Aats-met</i> gene is homologous to the mitochondrial methionyl-tRNA synthetase genes of <i>S. cerevisiae</i>, <i>C. elegans</i>, <i>M. musculus</i>, and <i>H. sapiens</i>. (N) Colocalization of the Flag-tagged human MARS2 protein with Mito-GFP in the cell body of a neuron in the ventral nerve cord, driven by the D42-Gal4 driver, is shown.</p

The human <i>MARS2</i> mutations.

<p>(A) PCR amplification products of <i>MARS2</i> encompassing a portion of the coding sequence revealed the presence of a 268 bp deletion mutation segregating in ARSAL Family E but not in Family B. This truncated product is indicated by an arrow. The normal PCR product is around 500 bp. Segregation of the deletion is shown in Family E; brothers E10 and E11 carry the mutation. Their unaffected father E9 is also a carrier. The determined genotypes for the patients shown (summarized in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001288#pbio.1001288.s012" target="_blank">Table S5</a> for all patients) are shown above the PCR bands. (B) Wild-type sequence of <i>MARS2</i> PCR products. (C) DNA sequencing of the deletion (c.681Δ268bpfx236X). (D–E) Nonrecurrent rearrangements involving the <i>MARS2</i> gene was confirmed by the oligonucleotide custom aCGH. In patients E10 and E11, the array discriminated the presence of the duplication as well as the deletion (see arrows) as depicted by the lower band detecting only one additional copy. (F) PCR amplification products of <i>MARS2</i> encompassing the coding sequence revealed the presence of a ∼300 bp insertion mutation segregating in ARSAL family members C6 and C8 but not in Family B. This larger amplification product is indicated by an arrow. The normal amplicon size is about 800 bp. C5 is the unaffected father of C6 and C8 and also carries the mutation. (G) Wild-type sequence of <i>MARS2</i>. (H) DNA sequencing of the heterozygous case C6 corresponding to the insertion revealed parts of the <i>MARS2</i> duplication mutation. Rearrangement was confirmed by oligonucleotide custom aCGH. Note that the array data of C6, a compound heterozygote (<i>Dup2/Dup2</i>), demonstrates the presence of a potentially larger duplication while not showing the 300 bp insertion, the array not having been designed to include its sequence. (I) In homozygous patient B4 (<i>Dup1/Dup1</i>), the array suggests that the duplication has identical distal and proximal breakpoint junctions with the other ARSAL cases.</p

MARS2 mRNA expression, protein levels, mitochondrial protein translation, Complex I, aconitase activity, and cell proliferation.

<p>(A) Quantification of MARS2 mRNA expression levels was performed on six ARSAL cases and two control lymphoblast cell lines. Relative expression levels were normalized to GAPDH levels. ARSAL patients expressed up to 3× higher MARS2 mRNA levels compared to controls. (B) Mitochondrial protein synthesis was measured in lymphoblasts and fibroblasts from three controls and six ARSAL patients by pulse-labeling mitochondrial translation products with ³⁵S-methionine for 1 h in the presence of emetine, followed by electrophoresis on a 15%–20% linear-gradient polyacrylamide gel. The 13 mitochondrial products are identified at the left of the figure. A generalized mitochondrial translation deficiency is observed in three of the six ARSAL patients tested. ANOVA analysis revealed significance for three of the patient's mitochondrial translation levels: Ctrl 1-B4: **, Ctrl 1-B5: n.s., Ctrl 1-P24: n.s., Ctrl 2-B4: ***, Ctrl 2-B5: n.s., Ctrl 2-P24: *, Ctrl 3-B4: ***, Ctrl 3-B5: *, Ctrl 3-P24: ***. (C) Immunoblotting analysis was performed with antibodies against the proteins indicated at the left of the panel. MARS2 was visualized using a polyclonal antibody. For case E10 carrying the heterozygous deletion (c.681Δ268bpfx236X), the truncated product is detected at the estimated size of 24 kDa (arrow); ARSAL patients (B4, EE41, P24, B5, AA35, and E10) show decreased levels of MARS2 protein at the estimated normal size of MARS2 (67 kDa). The 130 kDa LRPPRC and the 12 kDa SLIRP were used as loading controls. (D) Each patient's MARS2 protein-level intensity from the Western Blot shown in (C) was quantified using ImageJ and divided by the protein-level intensities of LRRPRC and SLIRP. The results were then graphed for the controls and the patients, respectively. (E) Respiratory chain activity for Complex I was measured from patient fibroblast-derived disrupted mitochondria. Mutant mitochondria exhibit deficiency of complex I. Data are expressed as percentage control activity (mean ± s.e.m.). (F) Quantification of native and reactivated aconitase activity for ARSAL patient and control immortalized fibroblasts. Three controls and 6 ARSAL patients were used for the analysis. (G) Quantification of the proliferation rate for the same above-mentioned fibroblasts. (H) Graph showing the average age of onset for the three different genotypes involved.</p

Schematic representation of the MARS2 region and ARSAL mutations.

<p>(A) Schematic representation of the chromosome 2q33.1 locus containing the mitochondrial methionyl-tRNA synthetase sequence (based on the UCSC genome browser). <i>MARS2</i> is an intronless gene located within the intronic sequence of a noncoding mRNA (BC021693). Its CpG island encompasses much of its coding sequence. Human Genome Structural Variation Project data show the insertion of a 726 bp discordant clone (ABC8_43216400 E17, Yoruba sample) containing a 276 bp LINE sequence (L2) within the coding sequence of the <i>MARS2</i> gene. DNA of this clone is depicted as a black box below the <i>MARS2</i> ideogram. Interestingly, the clone insertion fragment is located within the same distal junction breakpoint of ARSAL CNVs. (chr2: 198,280,073–198,280,860). No polymorphic CNV, structural variation, or segmental duplication have previously been reported on chromosome 2q33.1. Repeat elements are depicted as grey boxes. Using several combinations of primer pairs, genomic sequencing of carrier chromosomes allowed us to cover over 7 Kb and showed a partial deletion sequence at the 5′ region of <i>MARS2</i> and an insertion in the 3′ region. Sequencing and CGH-array data suggest that homologies among repeat elements are responsible for complex rearrangements accompanying the MARS2 duplications. (B) Illustration of the putative order and origin of the complex rearrangements found in the <i>MARS2</i> gene in ARSAL patients. The gene begins on the left (5′). The ORF is colored red and the UTRs blue. As mentioned above, the events share a common junctional sequence position, near the stop codon (black box). The presence of repetitive elements within MARS2 3′UTR and at the 5′ end is suggestive of a template-driven event (event (1) slippage or replication fork pause) that caused partial deletions or insertion (ABC8_43216400 E17, Yoruba sample) at the DNA lesion site (event (2A), (2B), or (2C)). We hypothesize that the complex genomic architecture that has similar sequence features may be able to form cruciform structures, suggesting that these events may be recurrent and stimulated by the abundance of AT-rich sequences around and within the <i>MARS2</i> gene (event (3)). The replication fork may have switched to another nearby homologous template consisting of short direct or inverted repeats (event (4)) resulting in the generation of duplication events, which could be advancing in either direction. Sequencing and CGH-array data suggest that homologies among repeat elements are responsible for the yielding of complex rearrangements accompanying the <i>MARS2</i> duplications, but we could not determine the orientation. (C) Illustration of the four predicted rearrangements of the <i>MARS2</i> region seen in ARSAL patients. The most common rearrangement is Duplication 1, in which two copies of <i>MARS2</i> are detected on each chromosome. The first one contains the entire coding and noncoding sequence, however the duplicated copy includes only the coding sequence. The brackets (//) refer to the fact that the duplication occurs at a distance from the endogenous <i>MARS2</i> gene, at least 15 Kb away, based upon our quantitative Southern data. Duplication 2 is very similar to the first one with the exception that the rearrangement includes a small deletion in the 3′UTR (caused by event 2A). The genomic structure of the third mutation (Duplication-Deletion) displays a large deletion of the <i>MARS2</i> coding region (referred by the event 2B) resulting in a truncated MARS2 protein. Quantitative experiments on both genomic and mRNA reveal a deletion rearrangement with partial duplication of the coding region of <i>MARS2</i>. A 726 bp discordant clone (ABC8_43216400 E17, Yoruba sample) containing a 276 bp LINE sequence (L2) within the coding sequence of the <i>MARS2</i> gene is reported in the UCSC track from the Human Genome Structural Variation Project data, though its impact on mRNA and protein is unknown.</p