Resultados de investigación

(A) Structural model for ovine (Ovis aries) mitochondrial complex I (CI, Ref: 5LNK) [17] displaying the location of the NDUFS4 subunit (colored in black). UCSF ChimeraX (v1.5) [18] was used for visualization. (B) General Cas9 HITI strategy to ablate Ndufs4. Cas9 produces a double stranded DNA break at specific target sequences within the first exon of Ndufs4. Cas9 also excises the HITI donor by cleaving the same target sequence flanking the blasticidin cassette to be inserted. The excised HITI donor is ligated into the genomic site through the NHEJ pathway. The forward integration depicted in the scheme is “locked” and cannot be processed further. A reverse integration could be corrected by continuous excision/repair cycles in virtue of flanking target sites reconstitution. Grey pentagons represent Cas9/gRNA target sequences. Black lines within pentagons indicate Cas9 cleavage sites. (C) Whole cell lysates from each resultant knockout cell line were analyzed by Western blotting using antibodies against Ndufs4 or β-actin (loading control). Correctly targeted clones were named Ndufs4−/− followed by a serial number; Par, parental cell line., Peer reviewed

Increasing evidence demonstrate that the electron transfer chain plays a critical role in controlling the effector functions of macrophages. In this work, we have generated a Ndufs4−/− murine macrophage cell lines. The Ndufs4 gene, which encodes a supernumerary subunit of complex I, is a mutational hotspot in Leigh syndrome patients. Ndufs4−/− macrophages showed decreased complex I activity, altered complex I assembly, and lower levels of maximal respiration and ATP production. These mitochondrial respiration alterations were associated with a shift towards a pro-inflammatory cytokine profile after lipopolysaccharide challenge and improved ability to phagocytose Gram-negative bacteria., Peer reviewed

Parental and Ndufs4−/− RAW 264.7 cells (40,000) were plated on 6-well plates. The cells were cultured in media containing galactose in the complete absence of glucose. The number of viable cells was determined at the indicated time points. Each point represents a biological replicate. Data are shown as the mean ± SD., Peer reviewed

A representative experiment showing OCR in LPS-pretreated RAW 264.7 sublines before and after the sequential addition of oligomycin (2.6 μM), FCCP (1 μM), and a combination of rotenone (Rot) and antimycin A (AA) (1 μM)., Peer reviewed

The NAD/NADH ratio was measured through colorimetric detection in deproteinized cell extracts from parental (Par) and Ndufs4−/− RAW 264.7 cells. *, P <0.05; **, P <0.01; ***, P<0.005; ****, P<0.001. Each point represents a biological replicate. Data are shown as the mean ± SEM., Peer reviewed

Left panel, Western blot for NDUFV1 (N module) and NDUFA9 (Q module). ATP5B and Coomassie staining were used as loading controls. Right panel, schematic representation of CI. The N module contains an NADH oxidation site, while the Q module contains a ubiquinone reduction site. P module is involved in proton-pumping activity. The positions of NDUFV1 and NDUFA9 are indicated. IMM, inner mitochondrial membrane; IMS, intermembrane space., Peer reviewed

Parental and Ndufs4−/− RAW 264.7 cells (1,000) were plated on 96-well plates. The number of viable cells was determined at the indicated time points. Each point represents a biological replicate. Data are shown as the mean ± SD., Peer reviewed

(A) Representative flow cytometry histograms of MitoSOX staining (left) and graph showing relative mitoROS levels (right). (B) Representative flow cytometry histograms (left) of MitoTracker Red CMXRos staining (for MMP) and MitoTracker Green staining (for total mitochondrial mass) and graph showing the ratio of MMP over mitochondrial mass to more accurately determine the potential differences per unit of mitochondrial mass (right). *, P <0.05; **, P <0.01; ***, P<0.005; ****, P<0.001. Each point represents a biological replicate. Data are shown as the mean ± SEM., Peer reviewed

The data shown in Fig 2A were reanalyzed and CI activity is shown as CI/CII (left panel) and CI/CIV (right panel). *, P <0.05; **, P <0.01; ***, P<0.005; ****, P<0.001. Each point represents a biological replicate. Data are shown as the mean ± SEM., Peer reviewed

Increasing evidence demonstrate that the electron transfer chain plays a critical role in controlling the effector functions of macrophages. In this work, we have generated a Ndufs4−/− murine macrophage cell lines. The Ndufs4 gene, which encodes a supernumerary subunit of complex I, is a mutational hotspot in Leigh syndrome patients. Ndufs4−/− macrophages showed decreased complex I activity, altered complex I assembly, and lower levels of maximal respiration and ATP production. These mitochondrial respiration alterations were associated with a shift towards a pro-inflammatory cytokine profile after lipopolysaccharide challenge and improved ability to phagocytose Gram-negative bacteria., Peer reviewed