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.
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The trained model was used to assign a P[healthy] score from zero to one to single STED images.
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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 sildenafil 45 mg chewable tablets % 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. 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.
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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. 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.
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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). 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 primary antibodies against MIC60 (Abcam) in 5 % (w/v) BSA in DPBS for 1 h at RT. 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.
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Patches with insufficient fluorescence signal were assigned to the “low information” class and excluded from the evaluation.
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After washing with DPBS, the primary antibodies were detected with secondary goat sildenafil oral jelly 100mg 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). 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.
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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). 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 “ill” when it originated from LS NPCs. For the remaining patches a P[healthy]-score was predicted.
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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.
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STED images which had a proportion of “low information” patches above 90 % were excluded.
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Luminescence-based ATP levels were detected in cellular suspension (5 x 103 cells) with ViaLight™ Plus Kit (Lonza). ATP levels were determined by luminometry using Victor Multiple Plate Reader Spectrophotometer (Revvity). For normalization purposes, we employed the DNA quantification with CyQUANT Cell Proliferation Assay (Invitrogen). Results were then expressed as luminescence A.U. relative to control lines cultured in galactose.
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For live-cell assessment of cellular bioenergetics, we employed Seahorse XF96 and XFe24 extracellular flux analyzer, as previously described.25 Briefly, the day before the assay, 100,000 cells (for XF96) or 120,000 (for XFe24) were plated into each Matrigel-coated well of the XF96 well plates and incubated overnight at 37 °C with 5 % CO2. On the day of the assay, the growth medium was removed and replaced with glucose-based unbuffered media. 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.
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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. 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. 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.
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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. 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. Confocal microscopy revealed a tubular mitochondrial network in both NPC cultures, with no evident phenotypic alterations in LS NPCs.
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Columns 23 and 24 contained controls on each plate. In row 23, a combination of 5 μM FCCP (Biozol) and antimycin A (Sigma Aldrich/Merck) was used as positive control to achieve complete mitochondrial depolarization. Row 24 contained 0.05 % DMSO as negative control. Cells were then automated washed twice with 30 μl NPC medium (w/o phenol red) using the Janus MDT (Revvity). Images were directly acquired using an Operetta CLS high content imaging system with 20 x objective in confocal mode.
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TMRM intensity within the cells and nuclei count was calculated using a customized Columbus software script and downstream data processing was performed with Activity Base (IDBS). 3 x DMSO standard deviation (SD) was set as the threshold for MMP depolarization to calculate and compare hit rates using different compound concentrations. To achieve this normalization, we used the formula z = (y – min(x)) / (max(x) – min(x)) ∗ 100, where z is the final normalized value, y is the value of interest in the dataset, min(x) is the mean value of DMSO-treated LS NPCs, and max(x) is the mean value of FCCP+AA-treated LS NPCs. We next scored the normalized values (z) based on their ability to depolarize the MMP (inhibition) and classified the compounds in mild, intermediate, and strong uncouplers. For biochemical activity of complex V, approximately 1 x 107 NPCs were suspended in 0.5 ml of 10 mM ice-cold hypotonic Tris buffer (pH 7.6) and homogenized with glass/glass Dounce homogenizer with a tight pestle by 10 strokes.
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200 μl of 1.5 M sucrose was added and then samples were centrifuged at 600 g for 10 min at 4 °C. Supernatants were collected and centrifuge at 14,000 g for 10 min at 4 °C. Mitochondrial pellets were suspended in 0.2 ml of 10 mM ice-cold hypotonic Tris buffer (pH 7.6) and subjected to three freeze-thaw cycles. Protein concentration was quantified with the Bicinchoninic Acid (BCA) protein assay Kit (Thermo Fisher Scientific). Complex V activity (ATP hydrolysis) was measured at 340 nm cleared from oligomycin-insensitive ATPase activities as described43 and normalized to citrate synthase activity.135 For ATP content analysis, NPCs were grown in NPC medium and then 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 5 mM galactose as a carbon source to measure OXPHOS-derived ATP content, and then treated with DMSO or 10 μM sildenafil for 16 h. 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.
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