DataSheet1_Nucleoside supplements as treatments for mitochondrial DNA depletion syndrome.docx

Introduction: In mitochondrial DNA (mtDNA) depletion syndrome (MDS), patients cannot maintain sufficient mtDNA for their energy needs. MDS presentations range from infantile encephalopathy with hepatopathy (Alpers syndrome) to adult chronic progressive external ophthalmoplegia. Most are caused by nucleotide imbalance or by defects in the mtDNA replisome. There is currently no curative treatment available. Nucleoside therapy is a promising experimental treatment for TK2 deficiency, where patients are supplemented with exogenous deoxypyrimidines. We aimed to explore the benefits of nucleoside supplementation in POLG and TWNK deficient fibroblasts.Methods: We used high-content fluorescence microscopy with software-based image analysis to assay mtDNA content and membrane potential quantitatively, using vital dyes PicoGreen and MitoTracker Red CMXRos respectively. We tested the effect of 15 combinations (A, T, G, C, AT, AC, AG, CT, CG, GT, ATC, ATG, AGC, TGC, ATGC) of deoxynucleoside supplements on mtDNA content of fibroblasts derived from four patients with MDS (POLG1, POLG2, DGUOK, TWNK) in both a replicating (10% dialysed FCS) and quiescent (0.1% dialysed FCS) state. We used qPCR to measure mtDNA content of supplemented and non-supplemented fibroblasts following mtDNA depletion using 20 µM ddC and after 14- and 21-day recovery in a quiescent state.Results: Nucleoside treatments at 200 µM that significantly increased mtDNA content also significantly reduced the number of cells remaining in culture after 7 days of treatment, as well as mitochondrial membrane potential. These toxic effects were abolished by reducing the concentration of nucleosides to 50 µM. In POLG1 and TWNK cells the combination of ATGC treatment increased mtDNA content the most after 7 days in non-replicating cells. ATGC nucleoside combination significantly increased the rate of mtDNA recovery in quiescent POLG1 cells following mtDNA depletion by ddC.Conclusion: High-content imaging enabled us to link mtDNA copy number with key read-outs linked to patient wellbeing. Elevated G increased mtDNA copy number but severely impaired fibroblast growth, potentially by inhibiting purine synthesis and/or causing replication stress. Combinations of nucleosides ATGC, T, or TC, benefited growth of cells harbouring POLG mutations. These combinations, one of which reflects a commercially available preparation, could be explored further for treatment of POLG patients.</p

() Quantification of PicoGreen staining of proportion of mosaic MDS cell cultures A–C that appeared depleted over time

The number of cells that appeared depleted of mtDNA increased over 45 days in the patients but not the controls (200 cells were counted at each time point). Numbers were significantly higher in patients than controls at all time points, other than 28 days (0.05< < 0.0016). () Quantification of the numbers of nucleoids visible by PicoGreen staining of mosaic MDS cell cultures A–C and controls [same experiment as () and ]. Nucleoid numbers drop with time in MDS cultures ( < 0.001 and <p><b>Copyright information:</b></p><p>Taken from "Depletion of mitochondrial DNA in fibroblast cultures from patients with POLG1 mutations is a consequence of catalytic mutations"</p><p></p><p>Human Molecular Genetics 2008;17(16):2496-2506.</p><p>Published online 16 May 2008</p><p>PMCID:PMC2486441.</p><p>© 2008 The Author(s)</p

() MDS cells are mosaic for expression of mtDNA and mitochondrial transcription factor A (TFAM) and have reduced mtDNA synthesis compared with controls

(a) Control fibroblasts co-labelled with anti-DNA antibody (green) and Mitotracker red, showing orange-yellow labelling were there two signals co-localize. (b) Patient A fibroblasts co-labelled with anti-DNA/Mitotracker. (DAPI was used to visualize nuclei blue and an asterisk denotes a cell with no anti-DNA signal). (c) Patient B fibroblasts co-labelled with anti-DNA/Mitotracker. (note: a reduced anti-DNA signal is shown by the predominantly red co-localization with Mitotracker). (d) Patient C fibroblasts co-labelled with anti-DNA/Mitotracker (arrow shows area of residual anti-DNA/mitotracker co-labelling within a depleted cell with reduced Mitotracker labelling). (e) Control fibroblasts co-labelled with anti-TFAM antibody (green) and Mitotracker red. (f) Patient A fibroblasts co-labelled with anti-TFAM/Mitotracker (asterisks show TFAM depleted cells with reduced Mitotracker labelling). (g) Patient B fibroblasts co-labelled with anti-TFAM/Mitotracker. (h) Patient C fibroblasts co-labelled with anti-TFAM/Mitotracker. (i–l) Fibroblasts pulsed with Bromodeoxyuridine (BrdU) for 220 min, and Br-DNA immuno-detected. (i) BrdU labelling of normal fibroblasts, (j) BrdU labelling of patient A cells (inset shows magnified area of cytoplasm showing weak BrdU labelling). (k) BrdU labelling of patient B cells. (l) BrdU labelling of patient C cells (note: asterisks mark ‘ρ° type’ cells with very little or no mtDNA labelling). Bar 20 µM. () Expression of mtDNA encoded cytochrome oxidase subunit I (COXI) in MDS patients is reduced, as is COX activity, but not SDH activity. Cytochrome oxidase subunit I (COXI) expression was monitored using immuno-cytochemistry. (a) COXI labelling of normal control fibroblasts. (b) COXI labelling of patient A fibroblasts (COXI depleted cells are asterisked). (c) COXI labelling of patient B fibroblasts. (d) COXI labelling of patient C fibroblasts. (e–h) Histochemical demonstration of COX activity in fibroblasts. (e) COX activity of control. (f) COX activity of patient A cells (asterisks denote cells with markedly reduced activity). (g) COX activity of patient B cells. (h) COX activity of patient C cells. (i–l) Histochemical demonstration of SDH activity in fibroblasts. (i) SDH activity of control cells. (j) SDH activity of patient A cells. (k) SDH activity of patient B cells. (l) SDH activity of patient C cells. Bars 20 µM. () Tetramethyl-rhodamine-methyl ester (TMRM)/PicoGreen co-labelling of mosaic MDS fibroblast mitochondrial membrane potential. (a) PicoGreen labelling of normal control fibroblasts. (b) Co-staining of the same cells with TMRM. (c) Co-localization of the two signals in (a) and (b). (d and e) PicoGreen/TMRM co-labelling of later passage Patient A's fibroblasts. (f) Co-localization of the two signals in (d) and (e). (g and h) PicoGreen/TMRM co-labelling of later passage Patient B's fibroblasts. (i) Co-localization of the two signals. (j and k) PicoGreen/TMRM co-staining of late passage Patient C's cells. (l) Co-localization of the two signals. (m and n) PicoGreen/TMRM co-staining of late passage Patient D's cells. (o) Co-localization of the two signals. Bars 20 µm.<p><b>Copyright information:</b></p><p>Taken from "Depletion of mitochondrial DNA in fibroblast cultures from patients with POLG1 mutations is a consequence of catalytic mutations"</p><p></p><p>Human Molecular Genetics 2008;17(16):2496-2506.</p><p>Published online 16 May 2008</p><p>PMCID:PMC2486441.</p><p>© 2008 The Author(s)</p

Brain and muscle pathology for patient 2.

<p>A brain biopsy from patient 2 showed a little neuropil vacuolation (a) and cortical gliosis (b) but no specific diagnostic features. A muscle biopsy showed scattered cytochrome oxidase (COX)-negative fibres (f—arrows) but no other myopathic features (c) and no ragged red (d) or blue (e) fibres. Scale bars = 100 μm.</p

Liver and brain pathology for Patient 1.

<p>Post-mortem liver samples from patient 1 (a and b) showed perivenular foci of enlarged hepatocytes with fine vacuolation (arrows). On lipid staining with oil red O (b) of frozen sections there was diffuse lipid deposition. Sample of the cerebral cortex from the occipital lobe showed full thickness neuronal loss with vacuolation and astrocytosis (c and d). Samples of the hippocampi (e and f) showed segmental neuronal loss, most marked from CA1 (arrow) and gliosis in a similar pattern (f-GFAP). Scale bars, a and b = 100 μm; c and d = 200 μm; e and f = 2 mm.</p

Long range PCR and Southern Blot analysis.

<p><b>A.</b> Long PCR of muscle mt DNA from the four patients. <b>1,</b> 1kb ladder; <b>2,</b> Muscle negative control; <b>3,</b> Patient 2; <b>4,</b> Patient 3; <b>5,</b> water control; <b>6,</b> Patient 4; <b>7,</b> patient 1. <b>B.</b> Depletion of mtDNA obtained from liver of patient 1. <b>1,</b> control; <b>2,</b> control; <b>3,</b> patient 1.</p

Muscle pathology for patient 3.

<p>Haematoxylin-Eosin stained section (A) showed mild variation in fibre size and several fibres with peripheral accumulation of mitochondria (arrowhead). Gomori trichrome preparation (B) accentuated ragged red fibres (arrowhead) and that for Succinic dehydrogenase (C) showed many ragged blue fibres (arrowheads). COX histochemical preparation (D) revealed frequent COX-deficient fibres (arrowheads) in keeping with mitochondrial myopathy. Scale bar = 50 μm.</p