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. 2019 Jun;22(6):851-862.
doi: 10.1038/s41593-019-0397-0. Epub 2019 May 13.

C9ORF72-ALS/FTD-associated poly(GR) Binds Atp5a1 and Compromises Mitochondrial Function in Vivo

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Free PMC article

C9ORF72-ALS/FTD-associated poly(GR) Binds Atp5a1 and Compromises Mitochondrial Function in Vivo

So Yoen Choi et al. Nat Neurosci. .
Free PMC article

Abstract

The GGGGCC repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, it is not known which dysregulated molecular pathways are primarily responsible for disease initiation or progression. We established an inducible mouse model of poly(GR) toxicity in which (GR)80 gradually accumulates in cortical excitatory neurons. Low-level poly(GR) expression induced FTD/ALS-associated synaptic dysfunction and behavioral abnormalities, as well as age-dependent neuronal cell loss, microgliosis and DNA damage, probably caused in part by early defects in mitochondrial function. Poly(GR) bound preferentially to the mitochondrial complex V component ATP5A1 and enhanced its ubiquitination and degradation, consistent with reduced ATP5A1 protein level in both (GR)80 mouse neurons and patient brains. Moreover, inducing ectopic Atp5a1 expression in poly(GR)-expressing neurons or reducing poly(GR) level in adult mice after disease onset rescued poly(GR)-induced neurotoxicity. Thus, poly(GR)-induced mitochondrial defects are a major driver of disease initiation in C9ORF72-related ALS/FTD.

Figures

Fig. 1 |
Fig. 1 |. Age-dependent accumulation of low level of poly(GR) in CamKII;(GR)80 mice.
a, Schematic of CamKII-tTA and (GR)80 constructs. b, Relative expression levels of (GR)80 mRNA in the frontal cortex of different CamKII;(GR)80 mouse lines at 2 months of age. Line 8 = 37.4±6.27, line 16 = 4.27±0.89, line 18 = 1.00±0, n = 2 mice per line. c, Age-dependent accumulation of poly(GR) in frontal cortex neurons of line 16 CamKII;(GR)80 mice (from 3 independently repeated experiments). Scale bar, 50 μm. d, Standard curve for the mesoscale discovery immunoassay with a synthetic (GR)8 peptide. Values are mean±s.d. of two independent experiments. e, Poly(GR) levels in frontal cortex tissues of CamKII;(GR)80 mice were measured by immunoassay; 3 months = 0.44±0.04, 7 months = 1.05±0.06, 12 months = 2.22±0.64 (n = 4 mice). Values are mean±s.e.m., F(2,9) = 86.57, **P = 0.0044, ****P= 0.0001 by one-way ANOVA with Tukey’s post hoc analysis for multiple comparisons. f, Poly(GR) levels in frontal cortex tissue of 2 control brains and 3 C9ORF72-related ALS/FTD brains measured by immunoassay. Control brain sample values were subtracted. Control brains = 0± 0, C9ORF72-related ALS/FTD brains = 7.34± 2.29. g, Neuronal types with different poly(GR) expression patterns in CamKII;(GR)80 mice at 8 months of age (repeated 3 times independently with similar results). Scale bar, 5 μm. h, Pie chart showing percentage of neuronal types A-D. Over 150 cells expressing poly(GR) were quantified. ECL, electrochemiluminescence.
Fig. 2 |
Fig. 2 |. CamKII;(GR)80 mice have age-dependent behavioral deficits and synaptic impairment.
a, Schematic of the three-chamber social interaction test. CamKII and CamKII;(GR)80 mice were allowed to habituate alone for 10 min in the 3-chambered box with empty wire cages. A stranger mouse was then placed under the wire cage in the left chamber, and a novel object was placed in the right chamber. b,c, Quantification of the 3-chamber social interaction test for CamKII and CamKII;(GR)80 mice at 3 months (b) and 6months (c) of age. In b, CamKIIS + stranger = 58.47±1.86 (n = 11 mice), CamKII;(GR)80 + stranger = 48.79 ±5.46 (n = 12 mice), CamKII + object = 41.53 ±1.86 (n = 11 mice), CamKII;(GR)80 + object = 51.21 ± 5.46 (n = 12 mice), CamKII left = 307.07±12.04 (n = 11 mice), CamKII;(GR)80 left = 269.28± 22.34 (n = 12 mice), CamKII center = 84.58±10.47 (n = 11 mice), CamKII; (GR)80 center = 87.62 ± 8.09 (n = 12 mice), CamKII right = 205.14± 16.07 (n = 11 mice), CamKII;(GR)80 right = 245.77 ±25.38 (n = 12 mice). Values are mean± s.e.m.; F(1,21) = 9.377, P = 0.1209 for CamKII + stranger versus CamKII;(GR)80 + stranger; F(1,21) = 9.377, P = 0.1209 for CamKII + object versus CamKII;(GR)80 + object; F(1,21) = 3.450, P = 0.1653 for CamKII left versus CamKII;(GR)80 left; F(1,21) = 0.22, P = 0.8251 for CamKII center versus CamKII;(GR)80 center; F(1,21) = 2.490, P = 0.2045 for CamKII right versus CamKII;(GR)80 right by two-sided Student’s t-test. In c, CamKII + stranger = 73.96±1.71 (n = 11 mice), CamKII;(GR)80 + stranger = 59.06±4.78 (n = 9 mice), CamKII + object = 26.04±1.71 (n = 11 mice), CamKII;(GR)80 + object = 43.46 ±4.78 (n = 9 mice), CamKII left = 331.6± 25.60 (n = 11 mice), CamKII;(GR)80 left = 352.66± 24.09 (n = 9 mice), CamKII center = 49.09 ±6.36 (n = 11 mice), CamKII;(GR)80 center = 62.28 ±12.26 (n = 9 mice), CamKII right = 216.8± 26.69 (n = 11 mice), CamKII;(GR)80 right = 185±27.24 (n = 9 mice). Values are mean±s.e.m.; F(1,18) = 6.570, P = 0.0019 for CamKII + stranger versus CamKII;(GR)80 + stranger; F(1,18) = 6.570, P = 0.0019 for CamKII + object versus CamKII;(GR)80 + object; F(1,18) = 1.381, P = 0.5635 for CamKII left versus CamKII;(GR)80 left; F(1,18) = 3.040, P= 0.3268 for CamKII center versus CamKII;(GR)80 center; F(1,18) = 1.17, P= 0.4187 CamKII right versus CamKII;(GR)80 right by twosided Student’s t-test. d, Time spent (left graph) and percentage of entries (right graph) in the open arm of the elevated plus maze by CamKII and CamKII;(GR)80 mice at 6months of age. Each dot represents one mouse. Left: CamKII = 7.91 ±2.38 (n = 10 mice), CamKII;(GR)80 = 1.75±1.03 (n = 9 mice), F(1,17) = 5.980, *P= 0.0358. Right: CamKII = 9.86±2.34 (n = 10 mice), CamKII;(GR)80 = 2.96±1.86 (n = 9 mice), F(1,17) = 1.76, *P=0.0362. Values are mean ± s.e.m., by two-sided Student’s t-test. e, Western blot analysis of PSD-95 expression level in the cortex of CamKII and CamKII;(GR)80 mice at 6 months of age and densitometric quantification of the western blot in e. CamKII = 100.2 ±1.62, CamKII;(GR)80 = 78.66± 4.65, values are mean ± s.e.m.; F(1,6) = 8.290, **P = 0.0047 by two-sided Student’s t-test. f, mEPSCs in layer V pyramidal neurons of prefrontal cortex. Representative traces recorded at −70 mV are selected from 12 neurons in 4 CamKII mice and 17 neurons in 4 CamKII;(GR)80 mice. g,h, Cumulative probabilities of amplitude (g) and interevent interval (h) distributions of mEPSCs. In g, CamKII = 14.38±0.91 (n = 12 cells from 4 mice), CamKII;(GR)80 = 12.50±0.40 (n = 17 cells in 4 mice), values are mean±s.e.m.; D = 0.225, P= 0.2634, by two-tailed Kolmogorv-Smirnov test. In h, CamKII = 4.696±0.529 (n = 12 cells in 4 mice), CamKII;(GR)80 = 2.528±0.216 (n = 17 cells in 4 mice). Values are mean±s.e.m. D = 0.3667, ###P = 0.0006 by two-tailed Kolmogorv-Smirnov test; t = 3.79, **P = 0.0020 by two-sided Student’s t-test.
Fig. 3 |
Fig. 3 |. Pathological features in the cortex of CamKII;(GR)80 mice.
a, Double-immunostaining for NeuN and cleaved caspase 3 in the cortex of 6-month-old mice (repeated 3 times independently with similar results). Arrowhead indicates the cell shown in the inset. Scale bars, 100 μm (5μm in the inset). Cas3, cleaved caspase 3. b, Quantification of the number of cleaved caspase 3-positive cells per cortical section. CamKII: 3 months = 8.40±1.40 (n = 3 mice), CamKII;(GR)80: 3 months = 10.67±1.81 (n = 4 mice), CamKII: 6 months = 8.46±1.28 (n = 3 mice), CamKII;(GR)80: 6 months = 19.33 ±1.41 (n = 4 mice). Values are mean±s.e.m. F(1,5) = 2.217, P = 0.3949 for CamKII: 3 months versus CamKII;(GR)80: 3 months; F(1,5) = 1.630, **P = 0.0028 by two-sided Student’s t-test. c, Quantification of the number of cleaved caspase 3-positive cells per section in the frontal cortex versus other cortical regions of 6-month-old mice. CamKII: frontal = 7.00±2.02 (n = 3 mice), CamKII;(GR)80: frontal = 23.04±1.53 (n = 4 mice), CamKII: parietal + occipital = 9.39±1.03 (n = 3 mice), CamKII;(GR)80: parietal + occipital = 16.10±2.15 (n = 4 mice). Values are mean±s.e.m. F(1,6) = 1.950, *P = 0.039, F(1,5) = 1.300, **P = 0.0013 by two-sided Student’s t-test. d, Quantification of the number of NeuN+ cells in frontal cortex region. CamKII: 3 months = 100±1.65 (n = 3 mice), CamKII;(GR)80: 3 months = 101.5±7.25 (n = 3 mice), CamKII: 6 months = 100±0.78 (n = 3 mice), CamKII;(GR)80: 6 months = 83.23±1.67 (n = 4 mice), CamKII: 9 months = 100±1.02 (n = 3 mice), CamKII;(GR)80: 9 months = 74.95±1.06 (n = 4 mice). Values are mean±s.e.m. F(1,4) = 19.25, P = 0.8470 for 3-month-old CamKII versus 3-month-old CamKII;(GR)80, F(1,5) = 6.100, ***P = 0.0005, F(1,5) = 1.441, ****P< 0.0001 by two-sided Student’s t-test. e, Immunostaining for the microglia-specific marker Ibal in the cortex of CamKII and CamKII;(GR)80 mice at 3 and 6 months of age (repeated 3 times independently with similar results). Scale bar, 50μm. f, Quantification of lbal+ cells in the cortex of CamKII (n = 3) and CamKII;(GR)80 (n = 4) mice at 3 and 6 months of age. CamKII: 3months = 244.3 ±4.34 (n = 2 mice), CamKII;(GR)80: 3 months = 249.6±4.79 (n = 4 mice), CamKII: 6 months = 240 ±5.50 (n = 3 mice), CamKII;(GR)80: 6 months = 289.9 ±7.3 (n = 4 mice). Values are mean±s.e.m. F(1,5) = 2.35, **P= 0.0039 by two-sided Student’s t-test. g, Activated Clec7a+ microglial cells (top; scale bar, 5μm) and round lbal+ microglial cells (indicated by the arrowhead and enlarged in the white square in lower panels) in the frontal cortex of 9-month-old CamKII;(GR)80 mice (repeated independently 3 times with similar results). Scale bar, 25μm. h, Double-immunostaining for γH2AX and NeuN in the cortex of 6-month-old CamKII and 4 CamKII;(GR)80 mice (repeated 3 times independently with similar results). Scale bars, 50 μm for large images and 5μm for small images. Note: γH2AX antibody gives rise to some background signal. i, Quantification of γH2AX+ and NeuN+ cells in the cortex. CamKII: 3 months = 6.74±1.94 (n = 3 mice), CamKII;(GR)80: 3 months = 7.86±0.90 (n = 3 mice), CamKII: 6 months = 6.83±0.62 (n = 3 mice), CamKII;(GR)80: 6 months = 50.1 ±2.82 (n = 4 mice). Values are mean±s.e.m. F(1,4) = 4.68, P = 0.6270 for 3-month old CamKII: versus 3-month-old CamKII;(GR)80:, F(1,5) = 27.48, ****P< 0.0001 by two-sided Student’s t-test.
Fig. 4 |
Fig. 4 |. Poly(GR) induces mitochondrial dysfunction in the cortex of CamKII;(GR)80 mice.
a, Mitochondrial morphology in prefrontal cortex of 3- and 6-month-old CamKII and CamKII;(GR)80 mice analyzed by transmission electron microscopy in 3 independent experiments with similar results. Red arrowhead indicates disruption of the mitochondrial inner membrane. Scale bar, 200 nm. b, Quantification of the percentages of normal and abnormal mitochondria in the prefrontal cortex of CamKII and CamKII;(GR)80 mice at 3 and 6 months of age. The numbers of analyzed mitochondria are indicated in each column. Values are mean±s.d. **P = 0.0067, ****P<0.0001 by two-tailed chi-squared test for categorical data. c, Representative images of mitochondria in poly(GR)-expressing mouse primary cortical neurons at 14d in vitro (DIV14) from 3 independent experiments. Scale bar, 10 μm. d, The distribution of mitochondrial length in 22 control and 20 poly(GR)-expressing mouse primary cortical neurons at DIV14. e, Average mitochondrial length in mouse primary cortical neurons at DIV14. Each dot represents 1 neuron and in total 323 mitochondria in CamKII neurons and 275 mitochondria in CamKII;(GR)80 neurons were measured. CamKII = 1.58±0.08 (n = 22 neurons), CamKII;(GR)80 = 0.93±0.045 (n = 20 neurons). Values are mean± s.d. F(1,40) = 3.57, ****P< 0.0001 by two-sided Student’s t-test. f, The activities of mitochondrial complexes I and V in the frontal cortex of 6-month-old CamKII and CamKII;(GR)80 mice. CamKII: complex I =100.2 ±4.98 (n = 3 mice), CamKII;(GR)80: complex I =71.67±3.00 (n = 3 mice), CamKII: complex V = 100±13.45 (n = 4 mice), CamKII;(GR)80: complex V = 71.45±5.64 (n = 7 mice). Values are mean±s.e.m. F(1,9) = 3.250, *P = 0.0464; F(1,4) = 2.750, **P= 0.008 by two-sided Student’s t-test. g, Double immunostaining for hsp60, a mitochondrial marker, and for poly(GR) in type A poly(GR)-expressing neurons of 9-month-old CamKII;(GR)80 mice (repeated 5 times independently with similar results). Scale bar, 5μm. Enlarged squares i, ii and iii are shown on the right. Dotted circles indicate the mitochondrial location. Scale bar, 0.5 μm. h, Representative images obtained by immuno-electron microscopy using gold particles to label poly(GR) in HEK293 cells overexpressing GFP and GFP-(GR)80 (repeated 3 times independently with similar results). Red arrowheads indicate immunogold-labeled poly(GR). Scale bars, top and middle images, 200 nm; bottom images, 20 nm. i, Number of gold particles in mitochondria from each HEK293 cell overexpressing GFP and GFP-(GR)80: GFP = 8.08±1.23 (n = 25 cells), GFP-(GR)80 = 30.61 ±2.17 (n = 24 cells). Values are mean±s.d. F(1,49) = 2.770, ****P<0.0001 by two-sided Student’s t-test.
Fig. 5 |
Fig. 5 |. Poly(GR) binds to ATP5A1 and decreases Atp5a1 expression level in the cortex of CamKII;(GR)S0 mice.
a, Proteins that coimmunoprecipitated with anti-GFP antibody in 4 independently repeated experiments were analyzed by western blots with an OXPHOS cocktail of antibodies, which showed that poly(GR) preferentially binds to the mitochondrial complex V protein ATP5A1 in HEK293 cells. b, Immunostaining analysis of Atp5a1 protein levels in the DIV14 primary cortical neurons (repeated 3 times independently with similar results). Scale bar, 10 μm. c, Atp5a1 intensity in primary neurons in b. Each dot represents one neuron. CamKII = 43.07±1.35 (n = 126 neurons), CamKII;(GR)80 = 26.39±0.93 (n = 135 neurons). Values are mean±s.d. F(1, 259) = 1.960, ****P< 0.0001 by two-sided Student’s t-test. d, The representative western blot analysis (of 4 independently performed experiments) of Atp5a1 level in the DIV14 primary cortical neurons. e, Western blot analysis and relative Atp5a1 expression in the frontal cortex of 6-month-old CamKII and CamKII;(GR)80 mice. CamKII = 0.54±0.05 (n = 4 mice), CamKII;(GR)80 = 0.33 ± 0.05 (n = 4 mice). Values are mean± s.e.m. F(1,6) = 1.042, *P = 0.0240 by two-sided Student’s t-test. f, Western blot analysis and ATP5A1 levels in the frontal cortex of C9ORF72-related ALS/FTD and control brains. Control brains = 1.03±0.04 (n = 3 brains), C9ORF72-related ALS/FTD brains = 0.52±0.07 (from n = 4 patients). Values are mean±s.e.m. F(1,5) = 2.810, **P = 0.0018 by two-sided Student’s t-test. g, Western blot analysis and quantification of Ndufb8 for complex I, Sdhb for complex II and Uqcrc2 for complex III in the frontal cortex of CamKII and CamKII;(GR)80 mice at 9 months of age. CamKII: Ndufb8 = 1.00 ±0.05 (n = 3 mice), CamKII;(GR)80: Ndufb8 = 1.12±0.06 (n = 3 mice), CamKII: Sdhb = 1.00±0.08 (n = 3 mice), CamKII;(GR)80: Sdhb = 0.93±0.03 (n = 3 mice), CamKII: Uqcrc2 = 1.00±0.01 (n = 3 mice), CamKII;(GR)80: Uqcrc2 = 1.02±0.01 (n = 3 mice). Values are mean±s.e.m. F(1,4) = 1.440, P = 0.1799 for Ndufb8; F(1,4) = 5.770, P=0.5024 for Sdhb; F(1,4) = 1.470, P= 0.1454 for Uqcrc2 graph by two-sided Student’s t-test. IP, immunoprecipitation; NS, not significant.
Fig. 6 |
Fig. 6 |. Poly(GR) increases ubiquitination and degradation of ATP5A1, and Atp5a1 overexpression rescues neuronal survival of CamKII;(GR)80 neurons.
a, Western blot analysis of protein ubiquitination in the frontal cortex of CamKII mice (n = 4) and CamKII;(GR)80 mice (n = 5) at 6 months of age. b, Quantification of the western blot in a. CamKII = 1.00±0.06 (n = 4 mice), CamKII;(GR)g0 = 1.54±0.08 (n = 5 mice). Values are mean±s.e.m. F(1,7) = 1.833, **P= 0.0012 by two-sided Student’s t-test. c, Immunoprecipitated ATP5A1 from HEK293 cells expressing GFP and GFP-(GR)80 was analyzed by western blot with anti-ubiquitin (top) and anti-ATP5A1 (bottom) antibodies. This experiment was repeated independently 3 times. d, Quantification of 3 independent experiments, as in c. GFP = 1.00±0, GFP-(GR)80 = 1.46±0.15, from n = 3 experiments. Values are mean±s.e.m. t = 3.01, *P = 0.0396 by two-sided Student’s t-test. e, ATP5A1 degradation in HEK293 cells expressing GFP and GFP-(GR)80 as revealed by treatment with CHX with or without MG132 for 30h (repeated independently 3 times). f, Quantification of 4 independent experiments, as in e. GFP = 1.00 ±0, GFP + CHX = 1.15±0.19, GFP + CHX + MG132 = 1.02±0.03, GFP-(GR)80 = 0.84±0.03, GFP-(GR)80 + CHX = 0.61 ±0.07, GFP-(GR)80 + CHX + MG132 = 1.09 ±0.08 from n = 4 experiments. Values are mean±s.e.m. F(1,6) = 454,589.000, P = 0.4647 for GFP versus GFP + CHX; F(1,6) = 9,745.00, P = 0.5383 for GFP versus GFP + CHX + MG132; F(1,6) = 14,602, **P = 0.0039 for GFP versus GFP-(GR)80; F(1,6) = 3.684, *P = 0.0213 for GFP-(GR)80 versus GFP-(GR)80 + CHX; F(1,6) = 2.724, **P = 0.0082 for GFP-(GR)80 + CHX versus GFP-(GR)80 + CHX + MG132; F(1,6) = 10.03, P= 0.0604 for GFP-(GR)80 versus GFP-(GR)80 + CHX + MG132 by two-sided Student’s t-test. g, Length of mitochondria in GFP-expressing and GFP-Atp5a1-overexpressing DIV14 primary cortical neurons from CamKII and CamKII;(GR)80 mice. Each data point is the average of 20 mitochondria in neurites of 1 neuron. CamKII GFP = 2.24±0.49 (n = 20 neurons), CamKII Atp5a1 =4.11 ±1.25 (n = 17 neurons), CamKII;(GR)80 GFP = 1.38± 0.38 (n = 17 neurons), CamKII;(GR)80 Atp5a1 = 3.01 ±0.79 (n = 20 neurons). Values are mean±s.d. F(3,70) = 37.27, **P = 0.0024 for CamKII GFP versus CamKII;(GR)80 GFP; ****P<0.0001 for CamKII;(GR)80 GFP versus CamKII;(GR)80 Atp5a1 by one-way ANOVA with Tukey’s post hoc analysis for multiple comparisons. h, ATP production in GFP-expressing and GFP-Atp5a1-overexpressing primary cortical neurons at DIV14. CamKII GFP = 97.60 ±19.48, CamKII Atp5a1 =103.61 ±19.87, CamKII;(GR)80 GFP = 52.99±16.37, CamKII;(GR)80 Atp5a1 = 94.39±6.58 from 4 independent primary cultures. Values are mean±s.d. F(3,12) = 7.860, *P = 0.0111 for CamKII GFP versus CamKII;(GR)80 GFP; *P = 0.0180 for CamKII;(GR)80 GFP versus CamKII;(GR)80 Atp5a1 by one-way ANOVA with Tukey’s post hoc analysis for multiple comparisons. i,j, Number of cleaved caspase 3-positive cells (i) and pyknotic cells (j) in GFP-expressing and GFP-Atp5a1-expressing DIV14 primary cortical neurons. Values are mean±s.d., analyzed by one-way ANOVA followed by Tukey’s post hoc test. In i, CamKII GFP = 5.51 ±1.85, CamKII Atp5a1 = 9.52 ±4.74, CamKII;(GR)80 GFP = 21.77± 2.91, CamKII;(GR)80 Atp5a1 =10.85±2.85 from n = 3 independent primary cultures. F(3,9) = 16.85, ***P = 0.0005, **P = 0.0073. In j, CamKII GFP = 8.22 ±6.06, CamKII Atp5a1 = 8.12± 6.65, CamKII;(GR)80 GFP = 34.64±5.82, CamKII;(GR)80 Atp5a1 = 12.42 ±4.79 from n = 3 independent primary cultures. F(3,9) = 17.22, **P = 0.0011 for CamKII:GFP versus CamKII;(GR)80 GFP; **P = 0.0035 for CamKII;(GR)80 GFP versus CamKII;(GR)80 Atp5a1. WB, western blot.
Fig. 7 |
Fig. 7 |. Reducing (GR)80 expression in adult mice rescues pre-existing pathological features of CamKII;(GR)80 mice.
a, Schematic of doxycycline (DOX) treatment of CamKII;(GR)80 mice from 7–9 months of age. b, Poly(GR) expression in the frontal cortex of 9-month-old CamKII;(GR)80 mice with and without doxycycline treatment (repeated 3 times independently with similar results). Scale bar, 50 μm. c, Immunostaining for NeuN and cleaved caspase 3 in the cortex of 9-month-old CamKII or CamKII;(GR)80 mice with or without doxycycline treatment (repeated 3 times independently with similar results). Arrowheads indicate cells positive for cleaved caspase 3. Scale bar, 50 μm. d, Average number of cleaved caspase 3-positive (Cas3+) neurons per section in the cortex (top) or frontal cortex only (bottom) of 9-month-old CamKII and CamKII;(GR)80 mice. All values are mean± s.e.m. and analyzed by one-way ANOVA with Tukey’s post hoc analysis for multiple comparisons. Top: CamKII -DOX = 8.26±0.87 (n = 3 mice), CamKII;(GR)80 -DOX = 27.00±1.42 (n = 4 mice), CamKII;(GR)80 +DOX = 10.21 ±1.03 (n = 3 mice). F(2,8) = 75.96, ****P< 0.0001. Bottom: CamKII-DOX = 6.92 ±1.81 (n = 3 mice), CamKII;(GR)80 -DOX = 32.14± 1.73 (n = 4 mice), CamKII;(GR)80 +DOX = 10.58±1.73 (n = 3 mice). F(2,8) = 63.39, ****P< 0.0001. e, Immunostaining and quantification of γH2AX+ and NeuN+ cells in the cortex of 9-month-old CamKII and CamKII;(GR)80 mice. CamKII -DOX = 13.66± 2.39 (n = 3 mice), CamKII;(GR)80 -DOX = 66.61 ±0.43 (n = 4 mice), CamKII;(GR)80 +DOX = 30.03±2.46 (n = 3 mice). Values are mean± s.e.m. F(2,8) = 250.800, ****P< 0.0001 by one-way ANOVA with Tukey’s post hoc analysis for multiple comparisons. Scale bar, 25 μm. f, Atp5a1 protein level in the cortex of 9-month-old mice. CamKII -DOX = 0.96± 0.08, CamKII;(GR)80 -DOX = 0.48± 0.01, CamKII;(GR)80 +DOX = 0.64± 0.05 from n = 3 mice. Values are mean ± s.e.m. F(2,6) = 21.69, **P = 0.0016, *P = 0.0120 by two-sided Student’s t-test.

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