ZPR1 prevents R-loop accumulation, upregulates SMN2 expression and rescues spinal muscular atrophy

Abstract

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by homozygous mutation or deletion of the survival motor neuron 1 (SMN1) gene. A second copy, SMN2, is similar to SMN1 but produces ∼10% SMN protein because of a single-point mutation that causes splicing defects. Chronic low levels of SMN cause accumulation of co-transcriptional R-loops and DNA damage leading to genomic instability and neurodegeneration in SMA. Severity of SMA disease correlates inversely with SMN levels. SMN2 is a promising target to produce higher levels of SMN by enhancing its expression. Mechanisms that regulate expression of SMN genes are largely unknown. We report that zinc finger protein ZPR1 binds to RNA polymerase II, interacts in vivo with SMN locus and upregulates SMN2 expression in SMA mice and patient cells. Modulation of ZPR1 levels directly correlates and influences SMN2 expression levels in SMA patient cells. ZPR1 overexpression in vivo results in a systemic increase of SMN levels and rescues severe to moderate disease in SMA mice. ZPR1-dependent rescue improves growth and motor function and increases the lifespan of male and female SMA mice. ZPR1 reduces neurodegeneration in SMA mice and prevents degeneration of cultured primary spinal cord neurons derived from SMA mice. Further, we show that the low levels of ZPR1 associated with SMA pathogenesis cause accumulation of co-transcriptional RNA-DNA hybrids (R-loops) and DNA damage leading to genomic instability in SMA mice and patient cells. Complementation with ZPR1 elevates senataxin levels, reduces R-loop accumulation and rescues DNA damage in SMA mice, motor neurons and patient cells. In conclusion, ZPR1 is critical for preventing accumulation of co-transcriptional R-loops and DNA damage to avert genomic instability and neurodegeneration in SMA. ZPR1 enhances SMN2 expression and leads to SMN-dependent rescue of SMA. ZPR1 represents a protective modifier and a therapeutic target for developing a new method for the treatment of SMA.

Keywords: R-loops; SMN; ZPR1; neurodegeneration; spinal muscular atrophy.

Figure 1

Genetic overexpression of the Zpr1 gene improves overall growth and survival of SMA mice. Overexpression of recombinant Flag-Zpr1 gene under the control of mouse Rosa26 promoter increases growth and the lifespan of mice with SMA (SMAΔ7). (A–C) ZPR1 overexpression improves growth of mice with SMA. Overall growth (body weight in grams) curves of normal (blue), Z-SMA (purple), and SMA (orange) mice littermates. Body weights recorded every day are presented as growth curves with mean ± SEM, n = minimum 3 mice/group for combined (male and female) group and for individual male and female groups. (D–F) Box and whisker plots and median with interquartile range (IQR) (minimum and maximum). (D) Average peak body weight (median, min, max) for males, SMA (3.27, 2.64, 3.74) and Z-SMA (5.15, 4.64, 5.91). (E) Average peak body weight for females, SMA (2.42, 0.98, 3.0) and Z-SMA (3.89, 3.43, 5.87). (F) Average peak body weight for combined males and females, SMA (2.92, 0.98, 3.74) and Z-SMA (4.78, 3.43, 5.91). These data show statistically significant increase in average peak body weight (g) of Z-SMA males (5.19 ± 0.21, n = 5) compared to SMA males (3.23 ± 0.22, P = 0.0004, unpaired t-test, two tailed), which represents ∼61% increase in body weight of Z-SMA males. Z-SMA females (4.17 ± 0.43, n = 5) compared to SMA females (2.20 ± 0.46, P = 0.0182) show ∼89% increase in peak body weight. Comparison of combined (male + female) peak body weight in Z-SMA (4.684 ± 0.285, n = 10) with SMA (2.719 ± 0.308, P = 0.0003) shows an average peak body weight increase of ∼72% in Z-SMA mice. (G) ZPR1 improves initial (embryonic) growth. Combined male and female growth (g) at birth for SMA (1.30, 0.77, 1.87) and Z-SMA (1.56, 1.15, 2.36). Statistical analysis shows significant improvement in initial growth of Z-SMA (1.642 ± 0.065, n = 20, P = 0.0008) compared to SMA (1.294 ± 0.069, n = 20) mice. (H) ZPR1 improves overall postnatal growth period of Z-SMA (11.0, 10.0, 12.0) compared to SMA (7.0, 5.0, 8.0) mice that increases from 7.00 ± 0.378 days (SMA, n = 7) to 11.29 ± 0.285 days (Z-SMA, n = 7), which shows ∼60% (P = 0.0001) increase in postnatal growth period. Growth analysis was blinded with colour coding. Gender and genotypes were confirmed by PCR-based method after euthanasia of pups. (I–P) ZPR1 overexpression improves survival of SMA mice. (I–K) Kaplan-Meier survival curves of normal (blue), Z-SMA (purple), and SMA (orange) mice littermates. Dotted lines show median survival. (L–N) Box-and-whisker plots with IQR show increase in (median, min, max) survival (days) of Z-SMA mice. (L) Z-SMA males (17.0, 16.0, 23.0) compared to SMA males (6.0, 1.0, 14.0). (M) Z-SMA females (18.0, 15.0, 22.0) compared to SMA females (6.0, 1.0, 14.0). (N) Z-SMA males and females (18.0, 15.0, 23.0) compared to SMA males and females (6.0, 1.0, 14.0). These data show ∼3-fold increase in median survival of Z-SMA mice compared to SMA mice. Statistical analysis of male and female survival show increase in average survival of Z-SMA (17.93 ± 0.34 days, n = 29) (Log-rank test, P < 0.0001) compared to SMA (7.07 ± 0.78 days, n = 26) mice. Gender-based analysis show average survival of Z-SMA males (17.85 ± 0.56, n = 13) is higher than SMA males (7.38 ± 1.19, n = 13). Average survival of Z-SMA females (18.00 ± 0.43 days, n = 16) is also higher compared to SMA females (6.76 ± 1.05, n = 13). (O) Maximum survival of SMA males (M) and females (F) is 14 days and Z-SMA males is 23 days and females is 22 days. Increase in the lifespan among Z-SMA male + female (combined) is 2.53-fold (P < 0.0001, t-test) compared to SMA mice. Increase in Z-SMA versus SMA males is 2.42-fold (P < 0.0001) and Z-SMA versus SMA females is 2.66-fold (P < 0.0001). (P) Scatter plot shows increase (15.5-fold) in initial survival of Z-SMA mice compared to SMA mice. All Z-SMA males and females survived at least 16 and 15 days, respectively, compared to SMA males and females that survived at least 1 day. Survival analysis was blinded with colour coding. Gender and genotypes were confirmed using a PCR-based method after euthanasia of pups.

Figure 2

ZPR1 overexpression improves gross motor function and muscle strength of SMA mice. (A) ZPR1 improves ability to stand on paws and walk. Ability to stand on paws and walk was determined by measuring the time to fall off in an effort to walk, and was recorded in 12-day-old SMA and Z-SMA littermates. Comparison of latency to fall (s) from paws presented as a box-and-whisker plot with IQR (median, min, max) for SMA (2.49, 2.0, 3.0) and Z-SMA (31.50, 24.66, 36.00) showing all Z-SMA pups were able to stand and walk for a median time of ∼30 s, suggesting improvement in gross motor function. Statistical analysis of (mean ± SEM, n = 6 mice/group) (s) between SMA (2.55 ± 0.15) and Z-SMA (30.61 ± 1.82), P < 0.0001 (unpaired t-test, two-tailed) for 12-day-old mice shows marked improvement in the ability of Z-SMA mice to stand on paws and walk (see Supplementary Video 1). (B) Ability of mice to right was recorded for 5–18-day-old normal (blue), SMA (orange), and Z-SMA (purple) littermates with all three genotypes present in the same litter. Time-to-right (TTR) with a time limit of 30 s for test and average of three recordings per pup are plotted. Data were collected using n = 10 (normal), 8 (SMA), and 10 (Z-SMA) mice groups are presented as a scatter plot. (C) Improvement in the motor function is demonstrated by increase in the ability of mice to right faster in Z-SMA mice compared to SMA mice is shown as box-and-whisker plots (median, min, max) starting from PND10 [SMA (26.17, 5.67, 30.0) and Z-SMA (6.83, 1.0, 30.0)], PND11 [SMA (20.50, 4.33, 30.0) and Z-SMA (5.33, 2.67, 19.0)], PND12 [SMA (30.0, 30.0, 30.0) and Z-SMA (3.0, 1.33, 30.0)], PND13 [SMA (30.0, 30.0, 30.0) and Z-SMA (3.33, 1.0, 30.0)], PND14 [SMA (30.0, 30.0, 30.0) and Z-SMA (1.33, 1.33, 30.0)]. Statistical analysis of (mean ± SEM) shows significant improvement in Z-SMA mice from PND10 [SMA (20.58 ± 3.97, n = 8) and Z-SMA (9.70 ± 2.76, n = 10), t-test, P = 0.034), PND11 [SMA (18.67 ± 5.14, n = 6) and Z-SMA (6.36 ± 1.60, n = 10), P = 0.014), PND12 [SMA (30.00 ± 1.00, n = 4) and Z-SMA (8.43 ± 3.61, n = 10), P = 0.003], PND13 [SMA (30.00 ± 1.00, n = 4) and Z-SMA (9.60 ± 3.56, n = 10), P = 0.004] to PND14 [SMA (30.00 ± 1.00, n = 4) and Z-SMA (5.36 ± 3.1, n = 10), P = 0.0005]. (D) The hind-limb suspension test (HLST) shows ZPR1 improves muscle strength in mice with SMA. Littermates aged 5–8 days were hung by both hind legs on the edge of a 50 ml plastic conical tube and time (s) was recorded until fall from the edge of the tube. Latency to fall (mean ± SEM, 6 mice/group) shown as a scatter plot and (E) as scatter plots with median with IQR (median, min, max) for each time point (day), PND5 [SMA (16.0, 4.0, 26.67) and Z-SMA (20.84, 16.0, 23.67)], PND6 [SMA (10.0, 6.0, 29.67) and Z-SMA (25.67, 16.33, 30.0)], PND7 [SMA (6.5, 5.0, 14.0) and Z-SMA (16.34, 13.0, 28.0)], PND8 [SMA (4.67, 2.0, 11.70) and Z-SMA (19.67, 10.33, 25.33)]. Statistical analysis (t-test, unpaired) shows marked increase (P = 0.0301) in hanging time for Z-SMA compared to SMA with increasing age PND5 [Z-SMA (20.00 ± 1.23) and SMA (16.61 ± 3.73), P = 0.408], PND6 [Z-SMA (24.16 ± 2.11) and SMA (14.50 ± 3.97), P = 0.057], PND7 [Z-SMA (17.61 ± 2.15) and SMA (7.445 ± 1.3762), P = 0.002], PND8 [Z-SMA (18.60 ± 2.42) and SMA (5.26 ± 2.22), P = 0.005] shows gradual increase in muscle strength of Z-SMA mice compared to SMA mice.

Figure 3

ZPR1 overexpression reduces neuron degeneration, improves NMJ innervation and muscle fibre size in SMA mice. (A) ZPR1 reduces loss of spinal cord motor neurons in mice with SMA. Immunohistochemical staining of the lumbar region spinal cord sections from 7-day-old normal, SMA, and Z-SMA littermates with anti-ChAT and SMN antibodies. Scale bar = 50 µm. (B) ZPR1 increases survival of SMN-deficient spinal cord motor neurons from SMA mice. Box-and-whisker plot with median and IQR (median, min, max) shows increase in the relative number of motor neurons (20 sections/mice, 3 mice/group) in the lumbar region of the spinal cords from Z-SMA (70.50, 60.0, 86.0) compared to SMA (47.0, 36.0, 61.0). Statistical analysis using mean ± SEM and comparison between SMA (47.03 ± 2.85%, n = 3) and Z-SMA (71.55 ± 2.24%) compared with normal (non-SMA) mice (littermates) as a reference point (101.20 ± 4.03%) shows statistically significant increase (24.52 ± 3.63%) in the number of motor neurons in Z-SMA mice, SMA versus Z-SMA (P = 0.0025, t-test, unpaired) and normal versus SMA versus Z-SMA (P < 0.0001, ANOVA). (C) ZPR1 reduces muscle degeneration in mice with SMA. Immunohistochemical staining of hind limb muscles, tibialis anterior longitudinal sections with β-actin (top) and transverse sections with dystrophin (bottom) from normal, SMA, and Z-SMA littermates. Scale bar = 50 µm (top) and 25 µm (bottom). (D) Immunohistochemical staining of hind limb muscles, gastrocnemius longitudinal sections with β-actin (top) and transverse sections with dystrophin (bottom) from normal, SMA, and Z-SMA littermates. Scale bar = 50 µm (top) and 25 µm (bottom). (E) ZPR1 improves muscle fibre diameter of SMA mice. The diameter of individual muscle fibres (μm) was measuring using transverse sections of gastrocnemius muscle from normal (blue), SMA (orange), and Z-SMA (purple) littermates stained with dystrophin. Diameter was measured at three different points, including longest and shortest along the irregular circular shape of each fibre and averaged. One hundred fibres per mouse (three mice per group) were measured to determine myofibres diameter. Scatter plots with median, min, max range for normal (17.61, 14.53, 20.44), SMA (11.53, 6.16, 15.30) and Z-SMA (13.87, 8.55, 17.97) show distribution of myofibre diameters. Statistical analysis shows increase in mean diameter (µm) for Z-SMA (13.88 ± 0.91) compared to SMA (11.06 ± 0.31), P = 0.043. (F) Immunohistochemical staining of gastrocnemius skeletal muscle with antibody to neurofilament M protein (NF, green) and BTX coupled with Alexa 594 (red) from normal, SMA, and Z-SMA littermates. Scale bars = 25 µm. (G) Immunohistochemical staining of gastrocnemius skeletal muscle with antibody to synaptophysin (SYN, red) and neurofilament (NF, green) from normal, SMA, and Z-SMA littermates. Scale bars = 25 µm. Note that littermates for all three genotypes were present in the same litter.

Figure 4

ZPR1 overexpression increases SMN levels in neuronal and non-neuronal tissues and leads to SMN-dependent amelioration of SMA phenotype. Proteins levels of SMN, ZPR1, Flag-ZPR1 and tubulin were examined by immunoblot analysis of mouse tissues. (A) spinal cord, (B) brain, (C) heart, (D) lung, (E) liver and (F) muscle from 7-day-old normal, SMA and Z-SMA mice using automated capillary Wes™ System (ProteinSimple) and quantification was performed using Compass software. Representative capillary-blot images of proteins are shown (full-length blots are provided in Supplementary Figs 2 and 3). Quantitative data are shown as a scatter plot with median, min and max; (median, min, max) range shows relative increase in SMN levels (%) in different tissues by ZPR1 overexpression in the: (G) spinal cord [SMA (25.96, 18.65, 26.98) and Z-SMA (77.32, 60.32, 85.63)], brain [SMA (19.96, 19.79, 21.24) and Z-SMA (57.93, 56.87, 73.67)], heart [SMA (15.76, 14.62, 22.36) and Z-SMA (49.61, 49.60, 60.32)], lung [SMA (15.61, 12.58, 26.19) and Z-SMA (55.69, 43.72, 65.36)], liver [SMA (14.82, 6.13, 30.98) and Z-SMA (20.20, 17.18, 28.65)] and muscle [SMA (14.78, 10.32, 20.36) and Z-SMA (20.36, 19.32, 25.63)] and statistical analysis (unpaired t-test) of protein levels (mean ± SEM, n = 3 mice/group) shows that ZPR1 overexpression resulted in statistically significant increase of SMN levels in the spinal cord of Z-SMA (74.42 ± 7.44, P = 0.0031) compared to SMA (23.86 ± 2.62), brain of Z-SMA (62.82 ± 5.43, P = 0.0015) compared to SMA (20.33 ± 0.45), heart of Z-SMA (53.18 ± 3.57, P = 0.0012) compared to SMA (17.58 ± 2.41), lung of Z-SMA (54.92 ± 6.25, P = 0.0080) compared to SMA (18.13 ± 4.12) but not in the liver of Z-SMA (22.01 ± 3.43, P = 0.5907) compared to SMA (17.31 ± 7.27) and muscle of Z-SMA (21.77 ± 1.95, P = 0.1317) compared to SMA (15.15 ± 2.90) relative to respective normal mice tissues protein levels (100%) normalized to tubulin. (H) Quantitative analysis of increase in relative levels (%) of total ZPR1 expression in the: spinal cord [SMA (50.36, 45.63, 66.36) and Z-SMA (153.47, 146.73, 187.32)], brain [SMA (59.34, 44.50, 70.12) and Z-SMA (171.80, 165.32, 178.36)], heart [SMA (61.36, 55.48, 62.36) and Z-SMA (163.06, 147.51, 164.91)], lung [SMA (61.89, 58.50, 63.35) and Z-SMA (170.76, 124.33, 175.32)], liver [SMA (59.99, 40.32, 69.97) and Z-SMA (80.36, 54.32, 90.32)] and muscle [SMA (50.32, 37.39, 65.95) and Z-SMA (75.66, 38.25, 92.04)]. Statistical analysis of increase in levels of total ZPR1 protein using antibody against ZPR1 shows increase in ZPR1 levels in the spinal cord of Z-SMA (162.50 ± 12.56, P = 0.0015) compared to SMA (54.12 ± 6.27), brain of Z-SMA (171.80 ± 3.76, P = 0.0002) compared to SMA (57.99 ± 7.42), heart of Z-SMA (158.50 ± 5.51, P = 0.0001) compared to SMA (59.73 ± 2.14), lung of Z-SMA (156.80 ± 16.29, P = 0.0043) compared to SMA (61.25 ± 1.43), liver of Z-SMA (75.00 ± 10.73, P = 0.2574) compared to SMA (56.76 ± 8.71) and muscle of Z-SMA (68.65 ± 15.92, P = 0.3861) compared to SMA (51.22 ± 8.25) relative to respective normal mouse tissue protein levels (100%) normalized to tubulin. (I) Quantitative data are presented as a scatter plot with median, min and max range shows relative increase in SMN levels in different tissues as fold change in Z-SMA mice compared to SMA mice; spinal cord (3.11 ± 0.31, P = 0.0031), brain (3.08 ± 0.26, P = 0.0015), heart (3.02 ± 0.20, P = 0.0012), lung (3.029 ± 0.34, P = 0.0080), liver (1.27 ± 0.19, P = 0.5911) and muscle (1.43 ± 0.13, P = 0.1333) relative to respective SMA mice tissues protein levels (100% or 1.0-fold) normalized to tubulin. (J) Quantitative data are shown as a scatter plot with median, min and max range shows relative expression (%) of recombinant Flag-ZPR1 in Z-SMA mice; spinal cord (100.027 ± 3.71), brain (84.36 ± 3.05), heart (64.86 ± 2.50), lung (61.17 ± 2.62), liver (24.98 ± 3.47) and muscle (20.78 ± 0.80) relative to Z-SMA spinal cord Flag-ZPR1 levels (100%) normalized to tubulin.

Figure 5

ZPR1 rescues molecular defects and DNA damage associated with SMA pathogenesis in SMA motor neurons and SMA mice. Protein extracts were prepared from the spinal cords isolated from 7-day-old normal, SMAΔ7 (SMA) and Z-SMA mice, and examined using an automated capillary-based western blot system. Representative capillary-blot images of proteins are shown (full-length blots are provided in Supplementary Fig. 4). (A) Immunoblot analysis of spinal cord protein extracts. (B) Quantitative immunoblot data are presented as a scatter plot with median, min and max (median, min, max) range shows relative change (%) in levels of SMN and DNA damage markers caused by in vivo ZPR1 overexpression in the spinal cords of mice, SMN [SMA (25.64, 20.65, 27.98) and Z-SMA (75.69, 62.65, 75.69)], SETX [SMA (38.95, 32.74, 49.21) and Z-SMA (82.32, 78.32, 120.33)], p-DNA-PKcs [SMA (33.65, 30.21, 45.36) and Z-SMA (83.65, 65.79, 105.96)], total DNA-PKcs [SMA (31.70, 20.69, 35.68) and Z-SMA (84.65, 67.32, 112.36)] and γH2AX [SMA (302.69, 298.63, 330.25) and Z-SMA (130.65, 85.63, 150.32)]. Statistical analysis (mean ± SEM, n = 3 mice/group) using t-test (unpaired, two-tailed) of proteins shows increase in ZPR1 (167.40 ± 9.55%, P = 0.0005) in Z-SMA compared to ZPR1 (47.99 ± 6.78%) levels in SMA mice results in increase of SMN (73.33 ± 5.61%, P = 0.0013) in Z-SMA compared to SMN (24.76 ± 2.16%) levels in SMA mice, SETX (93.66 ± 13.39%, P = 0.0199) in Z-SMA compared to SETX (40.30 ± 4.80%) levels in SMA mice, phospho-DNA-PKcs (p-DNAPKcs) (85.13 ± 11.62%, P = 0.0175) in Z-SMA compared to p-DNA-PKcs (36.41 ± 4.58%) levels in SMA mice and total DNA-PKcs (88.11 ± 13.12%, P = 0.0133) in Z-SMA compared to DNA-PKcs (29.36 ± 4.48%) levels in SMA mice. Analysis of DNA damage response marker shows decrease in γH2AX (122.20 ± 19.15%, P = 0.0009) in Z-SMA compared to γH2AX (310.5 ± 9.93%) levels in SMA mice suggesting the rescue of DNA damage in vivo by ZPR1 overexpression. (C and D) Primary spinal cord neurons were cultured from 7-day-old normal, SMA and Z-SMA mice. (C) Neurons were stained with ZPR1 and (D) R-loops (S9.6) antibodies and high magnification images of nuclei are presented. Dotted ellipses represent nuclei. Scale bar = 5.0 μm. (E) Quantitative analysis of relative levels of accumulation of nuclear R-loops in motor neurons from normal, SMA and Z-SMA mice is presented as a scatter plot with IQR (median, min, max), normal (118.0, 65.25, 118.7), SMA (734.90, 709.40, 978.9) and Z-SMA (141.8, 118.20, 306.90). Statistical analyses (mean ± SEM, n = 3 experiments, 50 neurons/experiment) using t-test (unpaired, two-tailed) of accumulation of R-loops in SMA (807.7 ± 85.90%, P = 0.0013) and normal neurons (106 ± 17.69%) shows large (∼8-fold) increase in accumulation of R-loops in SMA compared to normal mice. Comparison of R-loop accumulation between SMA (807.7 ± 85.90) and Z-SMA (189.0 ± 59.36) using t-test (P = 0.0041) and comparison between normal, SMA and Z-SMA using ANOVA (P = 0.0003) shows statistically significant decrease in in vivo R-loop accumulation by ZPR1 overexpression in Z-SMA mice. (F and G) HeLa cells were transfected with mock (Control), antisense ZPR1 oligonucleotide (As-ZPR1) and scrambled oligonucleotides (Scramble) (100 nM). (F) Knockdown of ZPR1 results in accumulation of R-loops (green) in the nucleus and formation 53BP1 foci (red) suggesting DNA double-strand breaks (DSBs). (G) ZPR1 (green) knockdown results in loss ZPR1 nuclear foci and causes accumulation of γH2AX foci (red) suggesting activation of DNA damage response in response to DNA damage caused by ZPR1 deficiency. Scale bar = 5 μm. (H–L) Cultured primary spinal cord neurons from SMA mice were infected with adenovirus (100 MOI) expressing green fluorescent protein (GFP) (Ad-GFP) and ZPR1-GFP fusion protein (Ad-ZPR1-GFP) and stained with antibodies against neuron-specific β-tubulin-III (red), SMN, SETX, p-DNA-PKcs, R-loops and γH2AX, and immunofluorescence was examined by confocal microscopy. GFP and ZPR1-GFP (green) were detected by GFP fluorescence. Axonal defects include retraction, bending, folding of axons (arrowheads) that indicate degeneration of SMN-deficient neurons. (H) Staining of neurons with SMN (cyan) and β-tubulin (red), (I) SETX (cyan) and β-tubulin (red), (J) p-DNA-PKcs (cyan) and β-tubulin (red), (K) γH2AX (cyan) and β-tubulin (red) and (L) R-loops (cyan) and β-tubulin (red). SMA neurons with ZPR1 ectopic expression (Ad-ZPR1-GFP panels) show reduction in neuron degenerative features. Nuclei were stained with DAPI (blue). Scale bar = 25 μm. Enlarged images of merged panels (H–L) are included in Supplementary Fig. 5 to show features of axonal degeneration such as loosening, bending retraction and ballooning in SMA neurons. (M) Immunoblot analysis of in vitro cultured motor neurons from SMA mice expressing GFP (Ad-GFP) and ZPR1-GFP (Ad-ZPR1-GFP) for changes in levels of SMN and DNA damage markers, SETX, p-DNA-PKcs, total DNA-PKcs and γH2AX. (N) Quantitative immunoblot data are presented as a scatter plot with median, min and max range shows relative change (%) in levels of SMN and DNA damage markers caused by in vitro ZPR1 overexpression in cultured motor neurons from SMA mice, SMN [SMA+GFP (17.06, 15.02, 29.0) and SMA+ZPR1-GFP (87.32, 79.06, 108.63)], SETX [SMA+GFP (45.62, 39.52, 65.32) and SMA+ZPR1-GFP (120.69, 105.36, 145.63)], p-DNA-PKcs [SMA+GFP (34.56, 30.65, 40.31) and SMA+ZPR1-GFP (102.36, 89.65, 109.65)], total DNA-PKcs [SMA+GFP (35.62, 30.65, 45.98) and SMA+ZPR1-GFP (115.47, 91.20, 138.97)] and γH2AX [SMA+GFP (108.90, 104.04, 118.99) and SMA+ZPR1-GFP (19.45, 14.13, 28.96)]. Statistical analysis using unpaired t-test of quantitative data (mean ± SEM, n = 3 mice/group) from spinal cord neuron immunoblots shows increase in ZPR1 levels (5.14 ± 0.44, P = 0.0010)-fold results in marked increase in levels of SMN (4.50 ± 0.43, P = 0.0019)-fold, SETX (2.47 ± 0.23, P = 0.0075)-fold, p-DNA-PKcs (2.85 ± 0.16, P = 0.0006)-fold and total DNA-PKcs (3.07 ± 0.36, P = 0.0059)-fold leading to decrease in γH2AX levels (5.04 ± 0.03, P = 0.0002)-fold. These data suggest the rescue of DNA damage in SMA spinal cord neurons (full-length blots are provided in Supplementary Fig. 6). (O) Enlarged images of nuclei of neurons stained with S9.6 antibody (R-loops, pseudocoloured orange) from SMA+Ad-GFP (SMA) and SMA+Ad-ZPR1-GFP (SMA+ZPR1) groups of SMA neurons. (P) Quantitative and statistical analysis of R-loop accumulation in cultured SMA motor neurons, SMA+GFP (103.5, 92.80, 103.7) and SMA+ZPR1-GFP (21.36, 19.65, 27.89) shows reduced accumulation (22.97 ± 2.51%, P < 0.0001) in neurons overexpressing ZPR1 (SMA+ZPR1-GFP) compared SMA+ GFP neurons.

Figure 6

Complementation with ZPR1 rescues senataxin levels and DNA damage in SMA patient cells. SMA patient primary fibroblast cell lines, GM03813 (A–D) and GM09677 (E–H) were transfected with phrGFP (GFP) or phrZPR1-GFP (ZPR1-GFP), fixed and stained with antibodies against SMN, SETX, R-loops and γH2AX. Ectopic ZPR1 expression elevates levels of SMN and SETX and reduces R-loop accumulation and rescues DNA damage in SMA patient cells. (A and E) SMN (red) and ZPR1-GFP (green), (B and F) SETX (red) and ZPR1-GFP, (C and G) R-loops (red) and ZPR1-GFP, (D and H) γH2AX (red) and ZPR1-GFP. Nuclei were stained with DAPI (blue). Scale bar = 5.0 μm. Arrows show transfected cells and asterisks indicate non-transfected cells. (I–L) Effect of ZPR1 overexpression on SMN, SETX, and γH2AX levels in patient cells GM03813 (I-J) and GM09677 (K-L) examined by IB (full-length blots are provided in Supplementary Figs 7 and 8). (I) Immunoblot analysis of patient cells (GM03813) expressing GFP and ZPR1-GFP. (J) Quantitative immunoblot data are presented as a scatter plot with IQR (median, min, max) shows relative change (%) in levels of SMN, SETX and γH2AX caused by in vitro ZPR1 overexpression in SMA patient cells (GM03813), SMN [GFP (21.28, 16.83, 27.44) and ZPR1-GFP (98.65, 85.69, 114.67)], SETX [GFP (55.69, 47.96, 68.52) and ZPR1-GFP (120.36, 102.36, 132.65)] and γH2AX [GFP (125.69, 106.59, 146.96) and ZPR1-GFP (22.65, 10.96, 30.32)]. Statistical analysis of quantitative data from GM03813 (GFP) and GM03813 (ZPR1-GFP) cells show ZPR1 overexpression (4.37 ± 0.25, P = 0.0003)-fold increases levels of SMN (4.56 ± 0.38, P = 0.0009)-fold and SETX (2.06 ± 0.15, P = 0.0049)-fold and result in marked reduction of γH2AX levels (6.0 ± 0.04, P = 0.0012)-fold in (GM03813+ZPR1-GFP) compared to control GM03813+GFP cells suggesting rescue of DNA damage in patient cells by ZPR1 complementation. (K) Immunoblot analysis of patient cells (GM09677) expressing GFP and ZPR1-GFP. (L) Quantitative immunoblot data are presented as a scatter plot with IQR (median, min, max) shows relative change (%) in levels of SMN, SETX and γH2AX caused by in vitro ZPR1 overexpression in SMA patient cells (GM09677), SMN [GFP (20.05, 17.65, 24.08) and ZPR1-GFP (102.32, 76.92, 139.52)], SETX [GFP (59.68, 38.69, 70.26) and ZPR1-GFP (106.98, 99.87, 111.36)] and γH2AX [GFP (121.8, 103.04, 151.52) and ZPR1-GFP (22.98, 14.65, 25.36)]. Statistical analysis of quantitative data from GM09677+GFP and GM09677+ZPR1-GFP cells shows (4.51 ± 0.42, P = 0.0015)-fold increase in ZPR1, which results in marked reduction of γH2AX levels (6.08 ± 0.026, P = 0.0018)-fold compared to GM09677+GFP suggesting the rescue of DNA damage that is supported by increase in levels of SMN (5.15 ± 0.88%, P = 0.0095)-fold and SETX (1.88 ± 0.059, P = 0.0061)-fold in ZPR1-GFP complemented patient cells compared to patient cells with GFP.

Figure 7

ZPR1 overexpression upregulates SMN2 gene transcription in SMA patient cells and SMA mice. (A) ZPR1 overexpression in vivo upregulates total SMN2 transcription under SMA conditions. Total RNA was isolated from the spinal cords of 7-day-old SMA and Z-SMA littermates and examined by qPCR using specific primers for amplification of SMNΔ7 and full-length SMN transcripts generated from SMN2. The relative mRNA levels were calculated using fold-enrichment (2^−ΔΔCT) method and presented as a scatter plot with median, min and max range. Quantitative data for SMNΔ7 [SMA (1.08, 0.74, 1.16) and Z-SMA (2.96, 2.32, 3.50)] and SMN [SMA (0.98, 0.91, 1.12) and Z-SMA (2.63, 2.14, 3.18)]. ZPR1 overexpression increases full-length SMN (2.65 ± 0.30-fold, n = 3, P = 0.0058) and SMNΔ7 (2.93 ± 0.33-fold, P = 0.0060) transcript levels in Z-SMA mice compared to SMA mice. (B) SMA patient cell lines GM03813 was transfected with pcDNA3 or pcDNA3/Flag-ZPR1. The scatter plot shows quantitative data for SMNΔ7 [SMA (0.96, 0.92, 1.12) and SMA+ZPR1 (3.87, 3.64, 4.5)] and SMN [SMA (1.28, 0.65, 1.34) and SMA+ZPR1 (3.12, 2.51, 4.62)]. Statistical analysis shows ectopic ZPR1 overexpression increases full-length SMN (3.42 ± 0.62-fold, P = 0.0249) and SMNΔ7 (4.00 ± 0.25-fold, P = 0.0003) transcript levels in GM03813+ZPR1 (SMA+ZPR1) compared to control GM03813 (SMA) patient cells. (C) SMA patient cell line GM09677 was transfected with pcDNA3 or pcDNA3/Flag-ZPR1. Quantitative data for SMNΔ7 [SMA (0.94, 0.93, 1.42) and SMA+ZPR1 (3.57, 3.21, 3.98)] and SMN [SMA (1.03, 0.60, 1.59) and SMA+ZPR1 (2.86, 2.79, 3.48)]. Statistical analysis shows that ZPR1 increases full-length SMN (3.04 ± 0.21-fold, P = 0.0054) and SMNΔ7 (3.58 ± 0.22-fold, P = 0.0008) transcript levels in GM09677+ZPR1 (SMA+ZPR1) compared to control GM09677 (SMA) patient cells. (D–F) Analysis of relative abundance of SMN and SMNΔ7 transcripts in (D) SMA and Z-SMA mice, and (E) SMA patient cell line (GM03813) and (F) SMA patient cell line (GM09677) without and with ZPR1 overexpression using qPCR. (G–N) ZPR1 interacts with RNA polymerase and in vivo associates with genomic SMN locus. Modulation of ZPR1 levels influences SMN expression. (G) Immunoprecipitation with anti-ZPR1 antibody shows pulldown of RNAPII from wild-type mouse brain protein extract suggesting in vivo interaction of ZPR1 with RNAPII. (H) GST-ZPR1 pulldown of RNAPII from HeLa cell lysate shows in vitro direct interaction of ZPR1 with RNAPII. (I–J) ZPR1 interacts with genomic SMN locus in vivo. Chromatin immunoprecipitation (ChIP) was performed using antibodies against ZPR1, H3K4me3 (positive control) and FLAG (M2) antibody (negative control) and chromatin prepared from human HeLa cells. Presence of SMN genomic locus in ChIP was detected by real-time qPCR using primers in the human SMN promoter region and SMN exon 1 region. ChIP assay shows ZPR1 associates with SMN locus and result in 4.34 ± 0.38-fold (n = 3, P = 0.0004) (promoter) and 3.66 ± 0.43-fold (P = 0.0012) (SMN exon 1) regions amplifications compared to control (IgG). (K–L) Modulation of ZPR1 levels influences expression of SMN1-Luc and SMN2-Luc reporter genes. (K) Effect of ZPR1 knockdown on ectopic SMN1 and SMN2 genes expression in cultured HeLa cells. Cells were transfected with either SMN1-Luc or SMN2-Luc or empty control reporter vector (Con-Luc). Transfected cells were retransfected after 24 h with scrambled or ZPR1 antisense oligonucleotides (As-ZPR1) to knockdown the levels of ZPR1. Cells were harvested after 24 h post-second transfection for determination of luciferase activity or (M) immunoblot analysis for ZPR1 expression. To examine the effect of ZPR1 overexpression, HeLa cells were transfected with combination of two plasmids: (i) Con-Luc + pcDNA3-FlagZPR1, (ii) SMN1-Luc or SMN2-Luc + pcDNA3 (empty); and (iii) SMN1-Luc or SMN2-Luc + pcDNA3-FlagZPR1. After 30 h post-transfection, cells were harvested for determining luciferase activity or (N) immunoblot analysis for ZPR1 expression. Quantification of luciferase activity shows ZPR1 knockdown causes decreases and ZPR1 overexpression increases levels of SMN1 and SMN2 promoters driven luciferase expression. Scatter plot with median, min and max range shows modulation of luciferase activity for SMN1-Luc (106.70, 80.48, 112.9), SMN1-Luc+As-ZPR1 (26.14, 15.59, 27.23) and SMN1-Luc+Flag-ZPR1 (227.6, 180.9, 260.0). SMN2-Luc (98.41, 97.67, 103.90), SMN2-Luc+As-ZPR1 (13.72, 9.28, 20.19) and SMN2-Luc+Flag-ZPR1 (160.10, 141.6, 178.9). Statistical analysis (mean ± SEM; n = 3) shows ZPR1 knockdown reduced levels of luciferase activity of SMN1-Luc to 22.99 ± 3.71% (P = 0.001) and SMN2-Luc to 14.40 ± 3.16% (P < 0.0001) in cells treated with antisense oligonucleotides compared to control cells with scrambled oligo. ZPR1 overexpression results in increase of luciferase activity for SMN1-Luc to 222.8 ± 22.95% (P = 0.0062) and SMN2-Luc to 160.2 ± 10.76% (P = 0.014) compared to cells without ZPR1 overexpression (full-length blots are provided in Supplementary Fig. 9).

Figure 8

Graphical summary: the molecular mechanism of ZPR1-dependent rescue of spinal muscular atrophy. ZPR1 interacts with RNAPII and associates in vivo with SMA 5q13 genomic locus. ZPR1 levels are downregulated in SMA. Low levels of ZPR1 result in reduced transcription of SMN2 gene leading to chronic low levels of SMN in SMA. Low levels of SMN cause deficiency of senataxin (SETX), which results in accumulation of RNA-DNA hybrids (R-loops). Accumulation of R-loops causes DNA double-strand breaks (DSBs) and activation of DNA damage response (DDR). In dividing cells, double strand breaks are repaired by homologous recombination (HR) and NHEJ, but neurons predominantly use NHEJ that requires DNA-PKcs activity. Chronic low levels of SMN cause deficiency of DNA-PKcs that causes defects in NHEJ-mediated double strand break repair leading to genome instability and predominant degeneration of motor neurons in SMA. ZPR1 overexpression in SMA increases transcription of SMN2 gene resulting in higher levels of SMN. Increase in SMN elevates SETX and DNA-PKcs levels that result in reduction of R-loops and the rescue of DNA damage leading to neuroprotection and amelioration of severe to moderate SMA disease phenotype.