Pluripotent stem-cell-based screening uncovers sildenafil as a mitochondrial disease therapy

Total RNA was isolated from pelleted NPCs using the NucleoSpin RNA Plus kit (Macherey Nagel) and eluted in 30 μl RNase-free H2O.

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RNA concentration was measured with NanoDrop 2000.

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The cells were incubated at 37 °C for 60 min to allow media temperature and pH to reach equilibrium before starting the measurement of mitochondrial respiration (oxygen consumption rate, OCR). Normalization to DNA content in each well of the plate was performed using the CyQUANT Cell Proliferation Assay kit. Quantitative analyses of NAD+, NADH, NADP+, and NADPH were carried out as a service in NADMED laboratory (Helsinki, Finland) from same homogenates submitted for metabolomics analysis. We used four LS NPC lines (ATP6_2, ATP6_4, ATP6_5 and ATP6_7.) and four healthy control NPC lines (CTRL_1, CTRL_2, CTRL_3 and CTRL_4) either treated for 16 h with either 0.1 % of DMSO or with 10 μM sildenafil resuspended in 0.1 % DMSO. NPCs were washed on the plate with DPBS buffer to remove protein of the culture media followed by the addition of cold extraction solvent acetonitrile:methanol:MilliQ; 40:40:20 to quench cellular metabolism.

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Obtained sample homogenates were shipped on dry ice to the measurement facility. Before analysis the homogenates were equilibrated to room temperature and centrifuged at 20,000 x g for 10 min at 4 °C to remove proteins. Next, NAD+, NADH, NADP+, and NADPH were measured individually from every cell extract using modified cyclic enzymatic reactions with colorimetric detection. For normalization of the results, protein content was measured using the pellets obtained after centrifugation of the homogenate. NPCs were switched to DMEM glucose-free (Gibco) and Neurobasal A-Medium glucose-free (Gibco) 1:1 medium supplemented with 1 mM sodium pyruvate, 1 % B27, 0.5 % N2, 1 % Pen/Strep, 1 % L-glutamine and 0.01 % MycoZap Plus-CL with low glucose (4.6 mM), and then treated with DMSO or 1 μM sildenafil for 16 h. Differential expression analysis between the two timepoints was performed using DESeq2 (Wald test, Benjamini-Hochberg p-value adjustment).

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The values depicted in the boxplots (expression, y axis) in Figures S6E and S6F are variance-stabilized counts obtained with DESeq2’s vst(),

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For each STED image with a size of 2,048 x 2,048 pixels, 49 partially overlapping patches of 512 x 512 pixels were extracted and evaluated with the model. Patches with insufficient fluorescence signal were assigned to the “low information” class and excluded from the evaluation. For the remaining patches a P[healthy]-score was predicted. The P[healthy]-score of the single patches were averaged to assign a P(healthy)-scores to each STED image of 2,048 x 2,048 pixels. STED images which had a proportion of “low information” patches above 90 % were excluded.

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After training, the classifier could reliably differentiate the two conditions on images that were not used for the training, indicating alterations of the MIC60 fluorescence signal in LS NPCs. To gain further insights, we reviewed a subset of images individually. Confocal microscopy revealed a tubular mitochondrial network in both NPC cultures, with no evident phenotypic alterations in LS NPCs. STED imaging showed a largely uniform distribution of MIC60 clusters along mitochondrial tubules in control NPCs, whereas in LS NPCs, MIC60 appeared to accumulate at the sides of mitochondrial tubules. To investigate this phenotype, we manually analyzed the MIC60 pattern across the mitochondrial tubules sildenafil citrate 120 mg in approximately 300 images. which normalized for sequencing depth and applies a transformation that is approximately log2 for large counts but reduces variance for low counts.

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A semi-automated MATLAB-based analysis was employed to assess the MIC60 distribution in LS NPCs (ATP6_2) and control NPCs (CTRL_1). After blinding, individual elongated mitochondria were manually analyzed. The mitochondrial centerline was determined by marking discrete points along the mid-axis, which were then fitted to a Bézier curve to approximate the mitochondrial central axis. The fluorescence intensity profile across the mitochondrial transverse axis was measured every 10 nm at a length of 600 nm with its center at the Bézier curve. The profiles were averaged for all selected isolated mitochondrial fragments in a single STED image. For bulk transcriptomics of brain organoids, total RNA was isolated from day 70

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Cells were grown to confluency in a 6-well plate format. Medium was aspirated and cells were washed twice with DPBS. Quickly, cells were scraped in 1 ml DPBS. Cell suspension was collected into a 1.5 ml tube and centrifuged at 8,000 x g for 5 min at 4 °C. Cell pellets were frozen in liquid nitrogen and stored at -80 °C.

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gDNA was isolated using the NucleoSpin Tissue Kit (Macherery-Nagel). DNA concentration was measured with the NanoDrop 2000 and diluted to 50 ng/μl. qPCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems) for the genes MT-ND1 and NDUVF1 in technical triplicates, including no-template control. Ct values were corrected based on the primer efficiency.136 The mtDNA/gDNA ratio was calculated with the corrected CT values using the formula: (MT-ND1/NDUFV1) ∗ 2. To examine the impact of sildenafil on the mitochondrial nanostructure, we immunolabeled LS NPCs (ATP6_2) and control NPCs (CTRL_1) treated with 1 μM sildenafil for 24 h for MIC60, a core protein of the mitochondrial contact site and cristae organizing system (MICOS). cortical brain organoids generated using the first protocol.62,134 We used five pelleted organoids per

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For immunolabelling of MIC60, cells were fixed with prewarmed (37 °C) 4 % formaldehyde in DPBS (137 mM NaCl, 2.68 mM KCl and 10 mM Na2HPO4, pH 7.4) for 5 min at RT. Fixed cells were extracted with 0.5 % (v/v) Triton X-100 in DPBS, blocked with 5 % (w/v) BSA in DPBS. Afterwards, cells were incubated with diluted sildenafil oral solution primary antibodies against MIC60 (Abcam) in 5 % (w/v) BSA in DPBS for 1 h at RT. After washing with DPBS, the primary antibodies were detected with secondary goat anti-rabbit antibodies labelled with Abberior STAR RED (Abberior). After washing with DPBS, the cells were mounted in Mowiol with 0.1 % 1,4-Diazabicyclo[2.2.2]octan (DABCO).

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Confocal and stimulated emission depletion (STED) microscopy were performed using an INFINITY platform (Abberior Instruments). For STED super-resolution microscopy, Abberior STAR RED was excited at 640 nm and depleted with 775 nm. The fluorescence was collected between 650 nm and 720 nm. Images were recorded with a pixel size of 20 nm and a dwell time of 30 μs per pixel. To detect structural differences between LS NPCs and control NPCs, we captured approximately 4,000 STED images and trained a neural network classifier on multiple biological replicates to distinguish control NPCs (P[healthy]-score = 1) from LS NPCs (P[healthy]-score = 0). sample with n=3 biological replicates per condition, grown under normal conditions (CTRL_1, CTRL_2, ATP6_4 and ATP6_7), or treated with 10 μM sildenafil for 24 h (ATP6_4 and ATP6_7).

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Total RNA was isolated using the RNeasy Mini Kit by Qiagen.

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RNA concentration as well as purity was measured at the Nanodrop Spectrophotometer ND1000 (peQlab) using ND-1000 software (V3.8.1).

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Total RNA was mixed with 1 μg of a DNA oligonucleotide pool comprising 50-nt long oligonucleotide mix covering the

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reverse complement of the entire length of each rRNA (28S rRNA, 18S rRNA, 16S rRNA, 5.8S rRNA, 5S rRNA,

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A homogeneous MIC60 distribution results in a fluorescence intensity profile with a broad plateau at the central maximum along the transversal mitochondrial axis. MIC60 accumulation at the outer edge of the mitochondria creates a central intensity dip. LS NPCs revealed a clear dip in the distribution of fluorescence intensity. For bulk transcriptomics of NPCs, we used four MT-ATP6 mutant NPC lines (ATP6_2, ATP6_4, ATP6_5 and ATP6_7.) and four healthy control NPC lines (CTRL_1, CTRL_2, CTRL_3 and CTRL_4) either treated for 16 h with 0.1 % of DMSO alone or with 10 μM sildenafil resuspended in 0.1 % DMSO. We used n=3 biological replicates per condition. 12S rRNA), incubated with 1U of RNase H (Hybridase Thermostable RNase H, Epicentre), purified using RNA Cleanup XP beads (Agencourt), DNase treated using TURBO DNase rigorous treatment protocol (Thermo Fisher Scientific) and purified again with RNA Cleanup XP beads.

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rRNA-depleted RNA samples were further fragmented and processed into strand-specific cDNA libraries using TruSeq Stranded Total LT Sample

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A deep residual neuronal network (ResNet34) was trained with STED data of DMSO-treated LS NPCs (ATP6_2) and control NPCs (CTRL_1) in a supervised setting to distinguish between both conditions. For the training, STED images were percentile normalized and divided into 512 x 512 pixel-sized regions (patches). Every patch was assigned to the class “healthy” when it originated from control NPCs and to the class sildenafil 60mg “ill” when it originated from LS NPCs. Using a mean intensity threshold, patches without a sufficient fluorescence signal were allocated to a third class (“low information”). The final training dataset consisted of equal numbers of patches from three different biological replicates and included in total 75,000 patches of the “healthy, “ill” and “low information” classes in a 0.4:0.4:0.2 ratio.

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The performance throughout the trainings process was monitored with a validation set of 11,000 patches. For every patch, the model predicts logits values corresponding to the three classes, that are then transformed with softmax function to receive a per class probability. The per-class accuracy computed from a confusion matrix was used as mean validation metric after each epoch and as an indicator for the best checkpoint. The final evaluation of the neuronal network after the training was carried out with a test set of 140,000 patches with a 0.4:0.4:0.2 ratio. The trained model was used to assign a P[healthy] score from zero to one to single STED images. Prep Kit (Illumina) and sequenced using the NovaSeq 6000 system with stranded technology, generating paired-end reads of 150 bp.

Property	Description	Value
Molecular Formula	-	C22H30N6O4S
Molecular Weight	-	474.58 g/mol
Melting Point	-	187°C
Solubility	In water	Very low
pKa	-	6.8