Journal: bioRxiv

Article Title: SLC25A46 is in contact with lysosomes and plays a role in mitochondrial cholesterol homeostasis

doi: 10.1101/2024.04.01.587618

Figure Lengend Snippet: SLC25A46 is required for starvation-induced mitochondrial hyperfusion . The mitochondrial morphology of control fibroblasts and the SLC25A46 knock-out cell line was analyzed by immunofluorescence after treatment with cycloheximide (CHX) for 2 hours, with HBSS for 4 hours, with the mTOR inhibitor Ink128 for 24 h or CCCP for 1 hour. (a) Representative images of fibroblasts decorated with anti-PRDX3 (mitochondria) in white and DAPI (nuclei) in blue. A zoomed image of the indicated area is shown on the right. Scale bars: 10 µm. Images were analyzed using MitoMAPR to describe number of mitochondrial networks (b) and the average length of mitochondrial networks (c) (N=3, n=10) per condition shown as violin plots. (d) SDS-PAGE analysis of steady-state levels of indicated proteins in control fibroblasts and SLC25A46 knock-out cells after the indicated treatments.

Article Snippet: Antibodies directed against the following proteins were used in this study: SLC25A46 (Santa Cruz; G2-SC-515823 and Proteintech; 12277-1-AP), MFN2 (Cell Signaling; 11925S), MFN1 (Cell Signaling; 14739S), OPA1 (BD bioscience, 612607) for WB, VDAC1 (Abcam; ab14734), 4E-BP (Cell Signaling; 9644S), Cav1 (BD bioscience; 610060).

Techniques: Knock-Out, Immunofluorescence, SDS Page

Journal: bioRxiv

Article Title: SLC25A46 is in contact with lysosomes and plays a role in mitochondrial cholesterol homeostasis

doi: 10.1101/2024.04.01.587618

Figure Lengend Snippet: SLC25A46 knock-out cells have differential mRNA expression for genes encoding lysosomal proteins. Transcriptomic data was analysed for control and SLC25A46 knock-out cell lines in fibroblasts and HeLa cells. (a) Principle component analysis of gene expression quantified by RNA-seq in control and SLC25A46 knock-out fibroblast cells. (b) Only transcripts that were differently expressed in both cell lines (adjusted p-value < 0.1, Log2FC > |0.3|) were chosen to be analyzed (n = 1,790) (SLC25A46-responsive subset). (c) List of Gene Ontology (GO) terms with their p- value and the number of proteins of the group.

Article Snippet: Antibodies directed against the following proteins were used in this study: SLC25A46 (Santa Cruz; G2-SC-515823 and Proteintech; 12277-1-AP), MFN2 (Cell Signaling; 11925S), MFN1 (Cell Signaling; 14739S), OPA1 (BD bioscience, 612607) for WB, VDAC1 (Abcam; ab14734), 4E-BP (Cell Signaling; 9644S), Cav1 (BD bioscience; 610060).

Techniques: Knock-Out, Expressing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: SLC25A46 is in contact with lysosomes and plays a role in mitochondrial cholesterol homeostasis

doi: 10.1101/2024.04.01.587618

Figure Lengend Snippet: (a) Immunofluorescence analysis of SLC25A46 contacts with lysosomes in fibroblasts stably overexpressing SLC25A46-GFP (green). Mitochondria were stained with MitoTracker Deep Red (white) and lysosomes were stained with LysoTracker Red (magenta). Images were captured every 1 s for a period of 1 min. Time-lapse imaging of mitochondria-lysosome contact. Orange arrows indicate SLC25A46-GFP signal that contacts a lysosome. Scale bar: 10 μm. (b) Quantification of mitochondria-lysosome contacts positive for SLC25A46. Five cells were analyzed with a total of 94 mitochondria- lysosome contacts. (c) Quantification of the percentage of lysosomes in contact with mitochondria in control fibroblasts and in SLC25A46 knock-out cell line. Seven cells per condition were analyzed with a total of 230 lysosomes in control cells and 188 lysosomes in knock-out cells. (d) Quantification of mitochondrial fission sites with a lysosome present (lysosome pos) and positive for SLC25A46 (SLC25A46-GFP pos). Seven cells were analysed with 22 fission events. Data shown as mean+SEM.

Article Snippet: Antibodies directed against the following proteins were used in this study: SLC25A46 (Santa Cruz; G2-SC-515823 and Proteintech; 12277-1-AP), MFN2 (Cell Signaling; 11925S), MFN1 (Cell Signaling; 14739S), OPA1 (BD bioscience, 612607) for WB, VDAC1 (Abcam; ab14734), 4E-BP (Cell Signaling; 9644S), Cav1 (BD bioscience; 610060).

Techniques: Immunofluorescence, Stable Transfection, Staining, Imaging, Knock-Out

Journal: bioRxiv

Article Title: SLC25A46 is in contact with lysosomes and plays a role in mitochondrial cholesterol homeostasis

doi: 10.1101/2024.04.01.587618

Figure Lengend Snippet: SLC25A46 influences mitochondrial cholesterol homeostasis. Heatmap of differently expressed (adjusted p-value < 0.1, Log2FC > |0.3|) SLC25A46- responsive transcripts in control and SLC25A46 knock-out fibroblasts coding for proteins involved in fatty acid biosynthesis (GO:0006633) (a) and cholesterol binding (GO:0015485) (b). Log2 fold changes are centered and represented as z-scores. Total cholesterol and non- esterified (“free”) cholesterol in whole cells (c) and in sucrose-gradient isolated mitochondria (d) from control fibroblasts, SLC25A46 knock-out cells (KO) and SLC25A46 knock-out cells expressing the wildtype SLC25A46 protein (Rescue) was measured with a Cholesterol/Cholesteryl Ester Assay Kit (Abcam, ab65359).

Article Snippet: Antibodies directed against the following proteins were used in this study: SLC25A46 (Santa Cruz; G2-SC-515823 and Proteintech; 12277-1-AP), MFN2 (Cell Signaling; 11925S), MFN1 (Cell Signaling; 14739S), OPA1 (BD bioscience, 612607) for WB, VDAC1 (Abcam; ab14734), 4E-BP (Cell Signaling; 9644S), Cav1 (BD bioscience; 610060).

Techniques: Knock-Out, Binding Assay, Isolation, Expressing

Journal: bioRxiv

Article Title: SLC25A46 is in contact with lysosomes and plays a role in mitochondrial cholesterol homeostasis

doi: 10.1101/2024.04.01.587618

Figure Lengend Snippet: Immunofluorescence analysis of cholesterol trafficking in control human fibroblasts and SLC25A46 knock-out cells. Cells were pulsed for 2 min with the fluorescent cholesterol probe TopFluor® (in magenta) and fixed after the indicated time to track the probe. PRDX3 was used as a mitochondrial marker (green). A zoomed image of the boxed area is shown on the right. Scale bars: 10 μm. (b) Quantification of colocalizing TopFluor signal with mitochondria divided by the total TopFluor signal in the cell at indicated time points. n=10 per condition.

Article Snippet: Antibodies directed against the following proteins were used in this study: SLC25A46 (Santa Cruz; G2-SC-515823 and Proteintech; 12277-1-AP), MFN2 (Cell Signaling; 11925S), MFN1 (Cell Signaling; 14739S), OPA1 (BD bioscience, 612607) for WB, VDAC1 (Abcam; ab14734), 4E-BP (Cell Signaling; 9644S), Cav1 (BD bioscience; 610060).

Techniques: Immunofluorescence, Knock-Out, Marker

Journal: bioRxiv

Article Title: SLC25A46 is in contact with lysosomes and plays a role in mitochondrial cholesterol homeostasis

doi: 10.1101/2024.04.01.587618

Figure Lengend Snippet: Cholesterol rescues the mitochondrial fragmentation phenotype of the SLC25A46 knock-out cell line and interaction of SLC25A46 with Cav1. The mitochondrial morphology of control and SLC25A46 human fibroblasts was analyzed after treating the cells with 20 μM cholesterol for 4h. Representative immunofluorescent images with the mitochondrial marker PRDX3 (white) (a). Scale bars: 10 μm. Images were analyzed using MitoMAPR across max projections keeping all parameters constant (N=3, n=30) to describe number of mitochondrial networks (b) and the average length of mitochondrial networks (c) per condition shown as violin plots. SDS-PAGE analysis of control and SLC25A46 knock-out cell lines (d). VDAC was used as a loading control. (e) Venn diagram of proteins co- immunoprecipitated with SLC25A46. Two different antibodies (G2: Santa Cruz; G2-SC-515823 and Proteintech: 12277-1-AP) against the endogenous SLC25A46 protein we used for immunoprecipitation of crude mitochondria isolated from control fibroblasts. The co- immunoprecipitated proteins were identified by mass spectrometry.

Article Snippet: Antibodies directed against the following proteins were used in this study: SLC25A46 (Santa Cruz; G2-SC-515823 and Proteintech; 12277-1-AP), MFN2 (Cell Signaling; 11925S), MFN1 (Cell Signaling; 14739S), OPA1 (BD bioscience, 612607) for WB, VDAC1 (Abcam; ab14734), 4E-BP (Cell Signaling; 9644S), Cav1 (BD bioscience; 610060).

Techniques: Knock-Out, Marker, SDS Page, Immunoprecipitation, Isolation, Mass Spectrometry

Journal: bioRxiv

Article Title: SLC25A46 is in contact with lysosomes and plays a role in mitochondrial cholesterol homeostasis

doi: 10.1101/2024.04.01.587618

Figure Lengend Snippet: An altered cellular response during nutrient starvation in the SLC25A46 knock- out cells. (a) Kernel density estimation adjusted p-value distributions for differentially expression genes in control and SLC25A46 KO cells upon treatment with HBSS, as well as genes where the differential expression upon HBSS treatment was dependent on SLC25A46 expression. A higher density of low adjusted p-values is indicative of enrichment in changes in gene expression. Heatmaps of changes in levels of transcripts of lysosomal proteins (GO:0005764) (b), proteins involved in fatty acid biosynthesis (GO:0006633) (c) and in cholesterol binding (GO:0015485) (d) in control and SLC25A46 knock-out fibroblasts in normal conditions and during nutrient starvation. Genes displayed are those where HBSS-induced changes in expression were dependent on SLC25A46 in the DESeq2 model with the design “expression ∼ genotype + condition + genotype x condition” (adjusted p-value < 0.1 and Log2FC > |0.3| for the “genotype x condition” term). Log2 fold changes are centered and represented as z-scores. (e) List of Gene Ontology (GO) terms with their p-value and the number of proteins in the group. (f) Representative images of the mitochondrial morphology of control and SLC25A46 human fibroblasts after 4 hours of incubation in normal medium (DMEM) or under nutrient starvation (HBSS), either without supplementation (no treatment) or with supplementation of 20 μM cholesterol for 4h. Mitochondrial marker PRDX3 (white). Scale bars: 10 μm. (g) Images were analyzed using MitoMAPR on ROIs keeping all parameters constant to quantify number of junction per mitochondrial networks (N=3, n=30).

Article Snippet: Antibodies directed against the following proteins were used in this study: SLC25A46 (Santa Cruz; G2-SC-515823 and Proteintech; 12277-1-AP), MFN2 (Cell Signaling; 11925S), MFN1 (Cell Signaling; 14739S), OPA1 (BD bioscience, 612607) for WB, VDAC1 (Abcam; ab14734), 4E-BP (Cell Signaling; 9644S), Cav1 (BD bioscience; 610060).

Techniques: Knock-Out, Expressing, Binding Assay, Incubation, Marker

Journal: Brain

Article Title: Loss of function of SLC25A46 causes lethal congenital pontocerebellar hypoplasia

doi: 10.1093/brain/aww212

Figure Lengend Snippet: SLC25A46 localizes to the mitochondria with differential mutant protein stability. Genomic structure of SLC25A46, with 8 exons represented by open boxes and the exonic deletion demarcated by a red box along with the mutation c.1022T>C in red in relationship to other recently reported mutations: circle = missense; triangle = frameshift. Predicted membrane-spanning 2D structure of SLC25A46 with three sequence repeats (Repeat 1–3), each with two transmembrane helices linked by an elongated loop with a short alpha-helix. The homozygous nucleotide variant c.1022T>C identified in Family 1 is predicted to alter a highly conserved residue L341 in H5, represented by a red circle in the diagram as well as the alignment of protein sequences encoded by orthologues in human and other eukaryotic organisms. The homozygous 1897-nucleotide deletion identified in Family 2 is predicted to delete exon 1, which contains the start codon ATG, with the next in-frame ATG in exon 4 predicted to encode M147 in the H1-H2 linker. The four previously described missense mutations in exon 8 are predicted to alter highly conserved G249 (grey open circle) in the linker between H3 and H4, and in H5 P333 (black open circle), E335 (grey closed circle), and R340 (black closed circle). The two frameshift mutations in A are predicted to introduce premature stops in the N terminus and H5. (B) Localization to the mitochondria of SLC25A46 by immunohistochemical analysis. LAN5 cells were fixed and incubated with antibodies against the mitochondrial marker mortalin and SLC25A46. Cell nuclei were marked with DAPI (scale bar = 10 µm). (C) Mitochondrial localization of SLC25A46 by mitochondrial labelling with MitoTracker® in a transiently transfected HeLa cell overexpressing GFP-tagged SLC25A46. Note the fragmentation of the mitochondria in the cell overexpressing GFP-SLC25A46, in contrast to the typical extensive long tubular mitochondrial network in the cell below, which was not transfected and not overexpressing SLC25A46. (D) Western blotting analysis of differential SLC25A46 expression in isolated mitochondria from the indicated cell lines. TOMM40 is included as loading control. (E) Visualization under fluorescent microscopy (left) or bright field (right) of transiently transfected HEK293 cells with plasmid constructs for GFP-tagged wild-type (WT) or L341P mutant SLC25A46, demonstrating markedly diminished fluorescent signal intensity of the mutant transfected cells compared to that in cells transfected with the wild-type construct. (F) Western blot analysis of HA-tagged mutant compared to the wild-type SLC25A46 probed with anti-HA antibodies. HEK293T cells were transiently transfected with the pCDNA3 vector containing HA-tagged SLC25A46. TOMM40 is included as a blotting control. (G) Quantitative RT-PCR demonstrated comparable levels of the L341P mutant and the wild-type mRNA. (H) In vitro transcription and translation reaction was performed on the same mRNA as in G.

Article Snippet: Antibodies used in this study: SLC25A46 (Proteintech), HA (HA.11, Biolegend), β-Tubulin (TU27, Biolegend), MFN2 (D1E9, Cell Signaling), TIMM23 and calnexin (BD Biosciences), LRP130 (H-300, Santa Cruz Biotechnology), TOMM20 (FL-145, Santa Cruz Biotechnology), Mortalin (UC Davis/NIH NeuroMab Facility), PreP, TOMM40, YME1L were generated from recombinant proteins (Pacific Immunology).

Techniques: Mutagenesis, Membrane, Sequencing, Variant Assay, Residue, Introduce, Immunohistochemical staining, Incubation, Marker, Transfection, Western Blot, Expressing, Isolation, Control, Microscopy, Plasmid Preparation, Construct, Quantitative RT-PCR, In Vitro

Journal: Brain

Article Title: Loss of function of SLC25A46 causes lethal congenital pontocerebellar hypoplasia

doi: 10.1093/brain/aww212

Figure Lengend Snippet: Localization of SLC25A46 to the outer mitochondrial membrane. (A) Subcellular fractionation of LAN5 cells by differential centrifugation. SM = starting material, P10k = 10 000g pellet, P100k = 100 000g pellet, S100k = 100 000g post-supernatant. Selected antibodies detected SLC25A46, TIMM23 (mitochondria), calnexin (ER, endoplasmic reticulum), and β-tubulin (cytosol). (B) Alkaline extraction in 0.1 M Na2CO3 solution at pH11 and 12 was performed on mitochondria isolated from LAN5 cells. The samples were separated into pellet (P) and supernatant (S) fractions. 1% Triton™ X-100 (TX-100) treatment and the starting material incubated in isotonic buffer were included as controls. Selected antibodies detected SLC25A46, MFN2 (outer membrane), and LRP130 (matrix). (C) Sub-mitochondrial localization was tested by osmotic shock (OS). After lysis of mitochondria in hypotonic buffer, the pellet fraction was collected and subject to proteinase K treatment. As a control, intact mitochondria were treated with protease and the pellet from the osmotic shock fraction was treated with 1% Triton™ X-100. Selected antibodies detected SLC25A46, MFN2 (OM, outer membrane), YME1L (IM, inner membrane), and LRP130 (matrix). (D) Import of radiolabelled SLC25A46, yeast Oac1 (IM), and yeast Tom40 (OM) into HEK293T mitochondria followed by subsequent trypsin treatment as indicated and alkaline extraction. The membrane potential was disrupted by CCCP (−ΔΨ). (E) As in C, isolated mitochondria from HEK293T cells stably expressing 2 × HA-SLC245A46 wild-type (WT) or L341P were treated with proteinase K in isotonic or hypotonic buffer to determine the sub-mitochondrial localization of wild-type and mutant SLC25A46. Controls include TOMM20 (OM), YME1L (IM) and PreP (matrix). (F) As in B, alkaline extraction (pH 12) on isolated mitochondria from stable HEK239T cells expressing 2 × HA-SLC245A46 wild-type (WT) or L341P. (G) Import of radiolabelled wild-type SLC25A46 or L341P mutant into HEK293T mitochondria followed by carbonate extraction and trypsin treatment. CCCP was used to disrupt the membrane potential (−ΔΨ).

Article Snippet: Antibodies used in this study: SLC25A46 (Proteintech), HA (HA.11, Biolegend), β-Tubulin (TU27, Biolegend), MFN2 (D1E9, Cell Signaling), TIMM23 and calnexin (BD Biosciences), LRP130 (H-300, Santa Cruz Biotechnology), TOMM20 (FL-145, Santa Cruz Biotechnology), Mortalin (UC Davis/NIH NeuroMab Facility), PreP, TOMM40, YME1L were generated from recombinant proteins (Pacific Immunology).

Techniques: Membrane, Fractionation, Centrifugation, Extraction, Isolation, Incubation, Lysis, Control, Stable Transfection, Expressing, Mutagenesis

Journal: Brain

Article Title: Loss of function of SLC25A46 causes lethal congenital pontocerebellar hypoplasia

doi: 10.1093/brain/aww212

Figure Lengend Snippet: Knockdown of slc25A46 in zebrafish causes brain maldevelopment, loss of spinal motor neurons, and abnormal increase in mitochondrial length in neurites. (A) Zebrafish embryos injected with slc25A46-specific antisense morpholinos AUG (targeted against the start codon) or SPL (targeted against the splice-donor site for exon 3) compared to those injected with non-specific control demonstrate abnormal indentation at the midbrain-hindbrain junction on lateral view (red arrowhead) as well as an abnormal gap between the optic tectum on dorsal view (black arrowhead). (B) Zebrafish embryos from the ET2 line (in which GFP is specifically expressed in the caudal primary motor neurons) injected with AUG morpholino demonstrate under epifluorescent microscopy decreased abundance of spinal motor neurons (arrows) on lateral view compared to embryos injected with control morpholinos. (C) Visualization of dsRed-labelled mitochondria in GFP-labelled neurons isolated from morpholino-injected embryos. Cells were dissociated from the brain dissected from 1–2 days post-fertilization zebrafish embryos injected with different morpholinos and maintained in culture for 1–2 days. (D) Distribution of the length of mitochondria in neurites. AUG morpholino injection led to an increase in length with a rightward shift in distribution compared to the results from control morpholino injection. Co-injection of the wild-type but not the mutant slc25a46 mRNA reverses mitochondrial length. (E) Summary of mitochondrial length measurements in cultured neurons isolated from zebrafish embryos injected with different constructs. Over 100 neurons were used in each condition, with 400–800 mitochondria being measured. Both AUG and SPL morpholino injection led to an increase in the length of mitochondria, which was reversed by the coinjection of the wild-type but not the mutant slc25a46 mRNA. Box = 25–75th percentile; whisker = 5–95th percentile; line = median; circle = mean; *P < 0.0001, two-tailed Mann-Whitney U-test. MO = morpholino.

Article Snippet: Antibodies used in this study: SLC25A46 (Proteintech), HA (HA.11, Biolegend), β-Tubulin (TU27, Biolegend), MFN2 (D1E9, Cell Signaling), TIMM23 and calnexin (BD Biosciences), LRP130 (H-300, Santa Cruz Biotechnology), TOMM20 (FL-145, Santa Cruz Biotechnology), Mortalin (UC Davis/NIH NeuroMab Facility), PreP, TOMM40, YME1L were generated from recombinant proteins (Pacific Immunology).

Techniques: Knockdown, Injection, Control, Microscopy, Isolation, Mutagenesis, Cell Culture, Construct, Whisker Assay, Two Tailed Test, MANN-WHITNEY

Journal: Brain

Article Title: Loss of function of SLC25A46 causes lethal congenital pontocerebellar hypoplasia

doi: 10.1093/brain/aww212

Figure Lengend Snippet: Knockdown of slc25A46 causes an increase in mitochondrial length in neurites of cultured neurons. Serial live imaging, taken every 2 min, shown from left to right, of dsRed-labelled mitochondria in cultured GFP-labelled neurons isolated from the brain of zebrafish embryos injected with control or AUG morpholino as in Fig. 4C.

Article Snippet: Antibodies used in this study: SLC25A46 (Proteintech), HA (HA.11, Biolegend), β-Tubulin (TU27, Biolegend), MFN2 (D1E9, Cell Signaling), TIMM23 and calnexin (BD Biosciences), LRP130 (H-300, Santa Cruz Biotechnology), TOMM20 (FL-145, Santa Cruz Biotechnology), Mortalin (UC Davis/NIH NeuroMab Facility), PreP, TOMM40, YME1L were generated from recombinant proteins (Pacific Immunology).

Techniques: Knockdown, Cell Culture, Imaging, Isolation, Injection, Control