Fig 2: UQCRC1 p.Tyr314Ser expression in Drosophila causes age-dependent locomotor defects, loss of TH-positive neurons and presynaptic defects in NMJs. (A) Climbing and jumping assays for wild-type control and flies that expressed either the wild-type UQCRC1 (UQCRC1WT) or mutant UQCRC1 p.Tyr314Ser (UQCRC1YS314S). The number of tested flies was shown in the bar graph and assays were repeated three times for each genotype. For the climbing assay, wild-type versus UQCRC1WT was 95.38 ± 6.21% versus 93.24 ± 5.13%, F(1, 81) = 4.8, P = 0.43; UQCRC1WT versus UQCRC1YS314S was 93.24 ± 5.13% versus 72.35 ± 7.38%, F(1,84) = 5.7, P = 0.021. For the jumping assay, wild-type versus UQCRC1WT was 98.25 ± 3.51% versus 90.13 ± 4.88%, F(1,81) = 6.7, P = 0.10; UQCRC1WT versus UQCRCY314S was 90.13 ± 4.88% versus 60.79 ± 6.42%, F(1,98) = 45.2, P = 0.006; all were based on one-way ANOVA. **P < 0.01. (B–D) Whole-mount adult brains were immunostained with anti-TH (green) to label individual dopaminergic neuronal clusters in 30-day-old flies. Images are representative of (B) schematic representation of the distribution of dopaminergic neurons in the Drosophila adult brain. Dopaminergic neurons are grouped in small clusters arranged with bilateral symmetry. PPL = protocerebral posterior lateral; PPM = protocerebral posterior medial. (C) UQCRC1WT files, and (D) UQCRC1Y314S flies. Scale bar = 20 µm. (E) Quantification of neurons that stained anti-TH-positive in the PPM1/2 and PPL1 clusters in individual genotypes at 30 days old [for PPM1/2, wild-type versus UQCRC1WT was 11.75 ± 0.38 versus 10.58 ± 0.23, F(1,45) = 6.8, P = 0.23; UQCRC1WT versus UQCRC Y314S was 10.58 ± 0.23 versus 8.12 ± 0.19, F(1,57) = 27.8, P = 0.007; For PPL1, wild-type versus UQCRC1WT was 5.57 ± 0.11 versus 5.61 ± 0.14, F(1,45) = 5.3, P = 0.49; UQCRC1WT versus UQCRC1 Y314S was 5.61 ± 0.14 versus 4.08 ± 0.22, F(1,57) = 20.3, P = 0.009 by one-way ANOVA]. (F) Representative confocal microscopy images show NMJs in third-instar larvae. Left: NMJ segment 4 (NMJ4) and right: NMJ segment 6/7 (NMJ6/7) are labelled with the membrane marker HRP. Fly genotypes are: (top row) wild-type control, (middle row) wild-type human UQCRC1 (UQCRC1WT), and (bottom row) human UQCRC1 p.Tyr314Ser mutant (UQCRC1Y314S). Scale bars = 20 µm. (G) NMJ sizes were quantified as the bouton number divided by the muscle area of NMJ4 or NMJ 6/7, expressed as bouton number × 104/muscle area (µm2). Average NMJ sizes (mean ± SEM) were compared with the one-way ANOVA post hoc Tukey test; ***P < 0.001. Sample numbers (n) are shown inside the box-and-whiskers plots.

Fig 3: Genotype analysis and locomotor behaviour assay of UQCRC1 p.Y314 knock-in mice. (A) Single-stranded oligodeoxynucleotide for targeting mice UQCRC1 and Sanger sequencing of UQCRC1 p.Tyr314Ser sequence in knock-in mice. Top: The wild-type target mouse UQCRC1 gene sequence (blue); Bottom: The p.Tyr314Ser substitution sequence of the single-stranded oligodeoxynucleotide (ssODN) used for targeted mutagenesis in mice. Silent mutations were included to generate BsrGI restriction enzyme recognition sequences. (B) Gene targeting strategy. Top: Region of the mouse wild-type UQCRC1 gene that encompasses exons 7–13. Middle: Exons 7–13 are shown with the c.941A>C mutation (p.Tyr314Ser). The 5′ and 3′ diagnostic probes (blue arrows) were used in the PCR genotyping analysis. The artificial restriction enzyme sites recognized by the BsrGI enzyme are shown (black ticks) with their locations (bp) in the gene. Bottom: BsrGI restriction fragments predicted to be unique to the wild-type (361bp) and targeted (259 bp) alleles. (C) PCR genotyping of tail-blood DNA from mice of two genotypes, a mutant homozygote (+/+, left) and heterozygote (+/−; right), with a molecular weight marker. DNA samples were digested with BsrGI enzyme and PCR was performed with the following primers: VF1: 5′-CCACGTGGCCATTGCAGTAGA-3′ and VR1: 5′- TCGATACTCATGGCATCACAGAC-3′. (D–G) Trace plots of the open field locomotor tests of age and gender-matched littermate wild-type (WT) control (D), UQCRC1 p.Tyr314Ser knock-in mice (E), and UQCRC1 p.Tyr314Ser knock-in mice receiving normal saline (NS) (F) and l-DOPA treatment (G) at the age of 12 months. (H–J) Parameters of wild-type and UQCRC1 p.Tyr314Ser knock-in mouse locomotor activities with and without normal saline or l-DOPA treatment. (H) Total distance travelled of UQCRC1 p.Tyr314Ser knock-in mice versus control mice was 10.9 ± 0.8 m versus 28.9 ± 2.4 m; F(1,11) = 72.3, P < 0.01 by one-way ANOVA. The total movement distance of UQCRC1 p.Tyr314Ser knock-in mouse after intraperitoneal injection of l-DOPA compared to vehicle (saline) was 18.3 ± 0.9 m versus 9.9 ± 0.8 m, F(1,10) = 6.4, P = 0.03 by one-way ANOVA. (I) Total movement time of UQCRC1 p.Tyr314Ser knock-in mice versus control mice; 142.3 ± 11.8 s versus 265.2 ± 28.7 s; F(1,11) = 21.3, P < 0.01 by one-way ANOVA. The total movement duration of UQCRC1 p.Tyr314Ser knock-in mouse after intraperitoneal injection of l-DOPA compared to vehicle (saline) was 175.9 ± 38.5 s versus 127.2 ± 33.0 s, F(1,10) = 2.1, P = 0.07 by one-way ANOVA. (J) Movement velocity of UQCRC1 p.Tyr314Ser knock-in mice versus control mice was 3.6 ± 0.3 cm/s versus 9.6 ± 0.8 cm/s; F(1,11) = 59.1, P < 0.01 by one-way ANOVA. Movement velocity of UQCRC1 p.Tyr314Ser knock-in mouse after intraperitoneal injection of l-DOPA compared to vehicle (saline) was 5.6 ± 1.7 cm/s versus 3.3 ± 0.9 cm/s, F(1,10) = 8.7, P = 0.01 by one-way ANOVA. n = 6 for each group; *P < 0.05; ** P < 0.01.

Fig 4: Striatal dopamine, substantia nigra TH-positive cells, hippocampal dentate gyrus neurons, cerebellar Purkinje cells immunostaining and sciatic nerve fibre changes of wild-type and UQCRC1 p.Tyr314Ser knock-in mice. (A) Axial series of 6-18F-fluoro-l-DOPA PET images in 12-month-old (left) wild-type littermates (WT) and (right) UQCRC1 p.Tyr314Ser knock-in mice. The bilateral striatum was drawn manually to indicate the region of interest on the PET images. (B) The standard uptake values (SUVs) in the right and left regions of interest (shown in the PET images) were calculated and compared between wild-type littermates (black box-and-whiskers) and UQCRC1 p.Tyr314Ser knock-in (red box-and-whiskers) mice. The average SUV of right region of interest in wild-type littermates and UQCRC1 p.Tyr314Ser was 1.23 ± 0.06 and 1.06 ± 0.04, F(1,6) = 7.2, P = 0.02 by one-way ANOVA; the average SUV of the left region of interest in wild-type littermates and UQCRC1 p.Tyr314Ser was 1.27 ± 0.05 and 1.09 ± 0.03, F(1,6) = 6.9, P = 0.03 by one-way ANOVA. n = 4 for each genotype. (C) Scatterplot comparison of striatal dopamine concentration measured by HPLC in wild-type littermates and UQCRC1 p.Tyr314Ser knock-in mice at the age of 12 months. UQCRC1 p.Tyr314Ser knock-in mice versus control mice; 52.9 ± 2.6 µg/ml versus 58.5 ± 4.0 µg/ml; F(1,6) = 9.7, P = 0.013 by one-way ANOVA. (D) Schematic representations of western blot. At 12 months of age, UQCRC1 p.Tyr314Ser mice presented with lower nigral and striatal TH levels than littermate controls. The relative expression level of TH to the GAPDH in wild-type littermates versus UQCRC1 p.Tyr314Ser at the age of 12 months was 0.79 ± 0.04 versus 0.58 ± 0.07, F(1,4) = 10.2, P = 0.02 by one-way ANOVA. The experiments were repeated three times. *P < 0.05. (E–J) Representative mid-brain sections show anti-TH immunostaining of dopaminergic neurons in the SNc of (E–G) wild-type and (H–J) UQCRC1 p.Tyr314Ser knock-in mice at 6, 9, and 12 months of age, as indicated. ×100 magnification. (K) Average numbers of TH-positive neurons observed in the SNc of wild-type and UQCRC1 p.Tyr314Ser knock-in mice at 6, 9, and 12 months of age, determined with stereological counting. At 6 months, wild-type littermates versus UQCRC1 p.Tyr314Ser was 16 189 ± 621 versus 16 200 ± 735; P = 0.82; At 9 months, wild-type littermates versus UQCRC1 p.Tyr314Ser was 14 980 ± 683 versus 12 389 ± 702; P = 0.08; At 12 months, wild-type littermates versus UQCRC1 p.Tyr314Ser was 13 127 ± 591 versus 10 830 ± 782; P = 0.032 by one-way ANOVA. n = 6 mice in each genotype, for all analyses. Values are mean ± SEM. *P < 0.05. (L) Representative sections showed hippocampal dentate gyrus from wild-type (WT) and UQCRC1 p.Tyr314Ser knock-in mice at the age of 12 months using Nissl staining. Gcl = granule cell layer. (M) Quantitative evaluation of the hippocampal dentate gyrus thickness in wild-type and UQCRC1 p.Tyr314Ser knock-in mice at 12 months of age is shown. Wild-type littermates versus UQCRC1 p.Tyr314Ser was 115.57 ± 3.81 µm versus 86.30 ± 2.13 µm; F(1,7) = 0.65, P = 0.02 by one-way ANOVA. n = 4 mice in each genotype, for all analyses. Values are mean ± SEM. (N) Representative sections show posterior lobes of cerebellum from wild-type and UQCRC1 p.Tyr314Ser knock-in mice at the age of 12 months immunolabelled for the GABAergic enzyme, glutamate decarboxylase 67 (GAD67). GCL = granule cell layer; ML = molecular layer; PL = Purkinje cell layer. (O) The quantification of the mean number of GAD67-positive Purkinje cells in an area of 4 × 102 mm2 for wild-type littermate controls and UQCRC1 p.Tyr314Ser knock-in mice at 12 months of age is shown. Wild-type littermates versus UQCRC1 p.Tyr314Ser was 5.74 ± 0.31 versus 5.81 ± 0.26; F(1,7) = 0.11, P = 0.75 by one-way ANOVA. n = 4 mice in each genotype, for all analyses. Values are mean ± SEM. (P) Characteristic nerve conduction recordings of normal (top), distal, and (bottom) proximal motor responses. Sensitivity (y-axis): 2 µV/division; sweep speed (x-axis): 0.25 ms/division. (Q) Representative brightfield images of semithin sections of sciatic nerves derived from wild-type littermate controls and UQCRC1 p.Tyr314Ser knock-in mice at 12 months of age. Myelinated fibres were stained with toluidine blue. (R) Quantification of diameter of myelinated nerve fibres in sciatic nerve. Wild-type littermates versus UQCRC1 p.Tyr314Ser was 8.49 ± 0.23 µm versus 7.58 ± 0.31 µm; F(1,59) = 80.17, P = 0.01 by one-way ANOVA. n = 3 mice in each genotype. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01.

Fig 5: Variants of UQCRC1 alter mitochondrial respiratory chain function of SH-SY5Y cells. (A) The oxygen consumption rate (OCR; pmol/min) was measured with a Seahorse Extracellular Flux Analyzer in 8 × 105 cultured wild-type SH-SY5Y cells (blue line), UQCRC1 p.Ill311Leu (yellow line), UQCRC1 p.Tyr314Ser (orange line) and UQCRC1 concomitant c.70-1G>A and p.Ala25Glyfs*27 aberrant splicing variant knock-in SH-SY5Y cells (purple line). OCRs were measured at regular intervals with the addition of compounds to test mitochondrial function. (B) Maximal respiration is decreased in all mutant neurons compared to wild-type neurons. Each cell line was assayed in at least three independent experiments, and means were calculated. Error bars are SEM. F(1,12) = 17.55, P = 0.006, one-way ANOVA for wild-type versus UQCRC1 p.Tyr314Ser cells; F(1,13) = 10.12, P = 0.013, for wild-type versus UQCRC1 p.Ile311Leu cells; F(1,12) = 11.35, P = 0.018, for wild-type versus UQCRC1 concomitant c.70-1G>A and p.Ala25Glyfs*27 aberrant splicing variant. (C) ATP production through oxidative phosphorylation is decreased in neurons having all human UQCRC1 variants compared to wild-type neurons. F(1,12) = 18.21, P = 0.004, one-way ANOVA for wild-type versus UQCRC1 p.Tyr314Ser cells; F(1,13) = 15.73, P = 0.008, for wild-type versus UQCRC1 p.Ile311Leu cells; F(1,15) = 17.29, P = 0.009, for wild-type versus UQCRC1 concomitant c.70-1G>A and p.Ala25Glyfs*27 aberrant splicing variant. (D) Representative flow cytometry profiles of reactive oxygen species production by DCFDA fluorescence showing reactive oxygen species production in wild-type cells and UQCRC1 p.Tyr314Ser cells before (baseline, red lines) and after exposure to 6-OHDA for 3 h (blue lines). (E) Amount of reactive oxygen species produced in individual genotypes of cells with or without treatment with 6-OHDA normalized to wild-type cells without exposure to 6-OHDA is presented as mean ± SEM. Data are representative of three experiments. F(1,4) = 26.73, P = 0.007, one-way ANOVA for wild-type versus UQCRC1 p.Ile311Leu cells; F(1,4) = 6.38, P = 0.065, for UQCRC1 p.Ile311Leu versus UQCRC1 p.Tyr314Ser cells; F(1,4) = 55.03, P = 0.002, one-way ANOVA for wild-type versus UQCRC1 p.Tyr314Ser cells; F(1,4) = 10.21, P = 0.012, one-way ANOVA for wild-type versus UQCRC1 c.70-1G>A and p.Ala25Glyfs*27 aberrant splicing variant cells; F(1,4) = 6.88, P = 0.06, for wild-type versus UQCRC1 p.Ile311Leu cells after treatment with 6-OHDA; F(1,4) = 25.55, P = 0.007, one-way ANOVA for wild-type versus UQCRC1 p.Tyr314Ser cells after treatment with 6-OHDA. F(1,4) = 9.98 P = 0.016, one-way ANOVA for wild-type versus UQCRC1 c.70-1G>A and p.Ala25Glyfs*27 aberrant splicing variant cells after treatment with 6-OHDA. (F–H) The individual activity of mitochondrial respiratory chain complex I–IV was examined further in wild-type cells and UQCRC1 p.Tyr314Ser knock-in SH-SY5Y cells. Representative seahorse profiles for the individual activity of mitochondrial respiratory chain complexes I–IV are illustrated in wild-type cells (F) and UQCRC1 p.Tyr314Ser knock-in SH-SY5Y cells (G) by measuring the OCRs before (basal respiration) and after sequential additions of oligomycin (ATP synthase inhibitor), CCCP (uncoupling protonophore), and then rotenone (complex I inhibitor) and succinate (complex II inhibitor) (blue lines); duroquinol (complex III inhibitor) (red lines); and TMPD and ascorbate (complex IV inhibitors) (green lines). This strategy allowed for the determination of the contribution of each component of respiration chain complexes. (H) The percentages of OCR attributable to the activities of complexes I–IV. In each experiment, data were collected and averaged from four separate wells for each individual cell line. Each cell line was assayed in at least three independent experiments, and means were calculated. Error bars are SEM. F(1,22) = 31.22, P = 0.018, one-way ANOVA for wild-type versus UQCRC1 p.Tyr314Ser for complex III activity. *P < 0.05. **P < 0.01.